Posts Tagged ‘CHF’

More on the Performance of High Sensitivity Troponin T and with Amino Terminal Pro BNP in Diabetes

Writer and Curator: Larry H. Bernstein, MD, FCAP

This is the final up to date review of the status of hs troponin T (or I) with or without the combined use of the Brain Type Natriuretic Peptide or its Amino Terminal peptide precursor.  In addition, a new identification of the role of the Atrial Natriuretic Peptide has been reported with respect to arrythmogenic activity.  On the one hand, the diagnostic value of the NT-proBNP has been seen as disappointing, in part because of the question of what information is gained by the test in overt known congestive heart failure, and in part because of uncertainty about following the test during a short hospital stay.  At least, this is the view of this reviewer.  However, in the last several years there has been an emphasis on the value this test adds to prediction of adverse outcomes.   In addition, there has been a hidden nvariable that has much to do with the original reference values that were established for age ranges, without any consideration of pathophysiology that might affect the values within those ranges, leading one to consider values in an aging population as normal, that might well be high.  Why is this?  Aging patients are more likely to have hypertension, and also the onset of type-2 diabetes mellitus, with cardiovascular disease consequences.  Type-2 diabetes mellitus (T2DM), for instance, is associated with insulin resistance and also fat gain with generation of adipokines, but the is also a hyalinization of insulin forming beta-cells of the pancreas, and there is hyalinization of glomeruli (glomerulosclerosis) and afferent arteriolonephrosclerosis with expected decline in glomerular filtrattion rate and hypertension as well.   Of course, this is also associated with hepatosteatosis.   Nevertheless, a reference range is established that takes none of this pathophysiology into account.   While a more reasonable approach has been pointed out, there has been no followup in the literature.

On the other hand, there has been much confusion over the restandardization of a high sensitivity troponin I or T test (hs-Tn(I or T).  The reference range declines precipitously, and there is a good identification of patients who are for the most part disease free, but there is no delineation of patients who are at high risk of acute coronary syndrome with plaque rupture, vs a  host of other cardiovascular conditions.  These have no relationship to plaque rupture, but may be serious and require further evaluation.  The question then becomes whether to admit for a hospital stay, to refer to clinic after an evaluation in the ICU without admission, or to do an extensive evaluation in the emergency department overnight before release for followup.  There is still another dimension of this that has to do with prediction of outcomes using hs-Tn(s) with or without the natriuretic peptides.  Another matter that is not for discussion in this article is the underutilization of hs-CRP.  Originally used for a marker of sepsis in the 1970s, it has come to be tied in with identification of an ongoing inflammatory condition.  Therefore, the existence of a known inflammatory condition in the family of autoimmune diseases, with one exception, might make it unnecessary.

The discussion is broken into three parts:

Part 1.   New findings on the troponins.
Part 2.  The use of combined hs-Tn with a natriuretic peptide (NT-proBNP)
Part 3.  Atrial natriuretic peptide

Part 1.    New findings on the troponins.

Troponin: more lessons to learn

C Liebetrau,HM Nef,andCW.Hamm*
Germany; (GermanCentreforCardiovascularResearch),partnersite
RheinMain,BadNauheim, Germany; and UniversityofGiessen,Medizinische
European Hear tJournal editorial refers to ‘Risk stratification in patients with acute chest pain
using three high-sensitivity cardiac troponin assays’,
by P. Haafetal. troponin entered our diagnostic armamentarium 20 years ago and –
unlike any other biomarker –

  • is going through constant expansion in its application.

Troponin started out as a marker of risk in unstable angina’, then was used

  • as gold standard for risk stratification and therapy guiding in acute coronary syndrome
  •  served further to redefine myocardial infarction, and
  • has also become a risk factor in apparently healthy subjects.

The recently introduced high-sensitivity cardiac troponin (hs-cTn) assays

  • have not only expanded the potential of troponins, but
  • have also resulted in a certain amount of confusion
    • among unprepared users.

After many years troponins were accepted as the gold standard in

  • patients with chest pain by
  • classifying them into troponin-positive and
    • troponin-negative patients.

The new generation of hs-cTn assays has

  • improved the accuracy at the lower limit of detection and
  • provided incremental diagnostic information especially
    • in the early phase of myocardial infarction.

Moreover, low levels of measurable troponins

  • unrelated to ACS have been associated with
    • an adverse long-term outcome.

Several studies demonstrated that

  • these low levels of cardiac troponin measureable 
    • only by hs-Tn assays
  • are able to predict mortality in patients with ACS
  • as well as patients with assumed
    • stable coronary artery disease.

Furthermore, hs-cTn has the potential

  • to play a role in the care of patients
    • undergoing non-cardiac surgery.

The additional determination of hs-cTn

  • improves risk stratification despite
  • established risk scores providing both diagnosis and
  • for prognosis prediction in chest pain patients.

The daily clinical challenge in using the highly sensitive assays is to 

  • interpret the troponin concentrations, especially
  • in patients with concomitant diseases
    • independently from myocardial ischaemia
  • influencing cardiac troponin concentrations
    (e.g. chronic kidney disease, or stroke). 

The troponin test lost its ‘pregnancy test’ quality with the different users.
Different opinions exist on

  • the change of hs-cTn levels compared to simple ‘positive–negative’ interpretation
  • and thereby makes diagnosis finding more complex than before.

This uncertainty probably has the paradigm that

  • serial measurements of troponins are necessary, and also
    • boosted the number of diagnoses of ACS and
    • invasive diagnostic procedures in some locations.

This is more than understandable, with acute chest pain using

  • three high-sensitivity cardiac troponins with their respective baseline value
    • before the diagnosis of acute myocardial infarction (AMI) can be made.

What is a relevant change in concentrations compatible with acute myocardial necrosis and

  • what is only biological variation for the specific biomarker and assay?

Changes in serial measurements between 20% and 200% have been debated, and
the discussion is ongoing. Furthermore, it has been proposed that

  • absolute changes in cardiac troponin concentrations 
    • have a higher diagnostic accuracy for AMI
  • compared with relative changes, and

it might be helpful in distinguishing AMI from other causes of cardiac troponin elevation.

Do we obtain any helpful directives from experts and guidelines for our daily practice?
Foreseeing this dilemma, the 2011 European Society of Cardiology (ESC) Guidelines

  • on non ST-elevation ACS acted.
  • Minor elevations of  troponins were accepted as hs-cTn values in the ‘grey zone’.

This was and still is the rule, but

  • the ESC provided a general algorithm on how to manage patients with limited data.

The ‘Study Group on Biomarkers in Cardiology’ suggested

  • a rise of 50% from the baseline value at low concentrations.

However, this group of experts could also not find a substitute for the missing data

  • needed to validate the proposed recommendation.

The story is just too complex:

  • different troponin assays with
  • different epitope targets,
  • different patient populations,
  • different sampling protocols,
  • different follow-up lengths, and much more.

Therefore, any study that helps us to see better through the fog is welcome here.

Haaf et al. have now presented the results of their study of

  • different hs-cTn assays
    (hs-cTnT, Roche Diagnostics; hs-cTnI, Beckman-Coulter; and  hs-cTnI, Siemens)

    • with respect to the -outcome of patients with acute chest pain.

The authors examine 1117 consecutive patients presenting with acute chest pain.
[340 patients with ACS (30.5%)] from the Advantageous Predictors of Acute Coronary Syndrome
Evaluation (APACE) study. Blood was collected

  • directly on admission and
  • serially thereafter at 2, 3, and 6h.

Eighty-two patients (7.3%) died during the 2-year follow-up. The main finding of the study is that

  1. hs-cTnT predicts mortality more accurately than the hs-cTnI assays, 
  2. -that a single measurement is sufficient
  3. challenges causes of cardiac troponin elevation.

These results of APACE remain in contrast to recent findings from a GUSTO IV cohortof 1335 patients with ACS (Table1).

Table1 Studies investigating high sensitivity troponins for long-term prognosis

Variable                                                       APACE (n 5 1117)              GUSTO IV (n 5 1335)              PEACE (n 5 3567)


Patient cohort                                                   Unstable                            Unstable                               Stable

Blood sampling                                     On admission,1,2,3,6h                    48h after
study randomization           Before randomization

No. of patients with detection limit             883 (79.1%)                                 UKN                                      UKN

No. of patients with hs-cTnT.
99thpercentile                                        401 (35.9%)                              1015 (76%)                          395 (10.9%)

No. of patients with hs-cTnI (Abbott).
detection limit                                           UKN                                             UKN                              3567 (98.5%)

No.of patients with hs-cTnI (Abbott).
99th percentile                                          UKN                                         988(74%)                           105 (2.9%)

No. of patients with NSTEMI                     170 (15.2%)                              100 (100%)                             0 (0%)

Follow-up                                               24 months                                  12 months                            5.2 years

Non-fatal AMI                                           UKN                                              UKN                               209 (5.9%)

Mortality or primary endpoint                    82 (7.3%)                                 119(8.9%)                           203 (5.7%)


Key findings                                    cTnT better than cTnI                      cTnI ¼cTnT                   cTnI better than cTnT

Single cTn sample sufficient

AMI, acute mycordial infaction; cTn, cardiac tropononin; NSTEMI ,non-ST-elevation myocardial infarction; UKN, unknown

NSTEMI defined in the GUSTO IV trial:
  1. one or more episodes of angina lasting ≥ 5min,
  2. within 24h of admission and
  3. either a positive cardiac TnT or I test
    (above the upper limit of a normal for the local assay; during the years 1999 and 2000)
  4. or ≥ 0.5 mm of transient or persistent ST-segment depression.

the prognostic capacity of four different sensitive cardiac troponin assays were compared

  1. hs-cTnT; Roche Diagnostics,
  2. cTnI and hs-cTnI;
  3. Abbott Diagnostics, and
  4. Acc-cTnI; Beckman-Coulter.

In total, 119 patients (8.9%) died during the 1-year follow-up. Looking at their

  • receiver operating characteristic curve (ROC) analyses,
  • there were only negligible diffferences
    • in the area under the curves between the assays.

Contrasting results have also been reported in patients(n 1/4 3.623)

  • with stable coronary artery disease and preserved systolic left ventricular function

from the PEACE trial (Table1).

During a median follow-up period of 5.2 years,

  • there were 203 (5.6%) cardiovascular deaths or
  • first hospitalization for heart failure.

Concentrations of hs-cTnI (Abbott Diagnostics) at or above

  • the limit of detection of the assay were measured in 3567 patients (98.5%), but
  • concentrations of hs-cTnI at or above the gender-specific 99th percentile
    • were found in only 105 patients (2.9%).

This study revealed that

  • there was a strong and graded association
  • between increasing quartiles of hs-cTnI concentrations and
  • the risk for cardiovascular death or heart failure.

Hs-cTnI provided incremental prognostication information

  • over conventional risk markers and
  • other established cardiovascular biomarkers,
  • including hs-cTnT.

In contrast to the APACE results, only hs-cTnI, but

  • no ths-cTnT, was significantly
  • associated with the risk for AMI.

Is there a real difference between cardiac troponin T and cardiac troponin I

  • in predicting long term prognosis?

The question arises of whether there is a true clinically relevant

  • difference between cTnT and cTnI.

Given the biochemical and analytical differences,the two

  • troponins display rather similar serum profiles during AMI.

While minor biological differences between cTnT and cTnI are

  • apparently not relevant for diagnosis
  • and clinical management in the acute setting of ACS.

This is a provocative theory, but appears premature in our opinion.
Above all, the results of the current study appear

  • too inconsistent to allow such conclusions.

In the present study, hs-cTnT (Roche Diagnostics) outperformed

  • hs-cTnI (Siemens and Beckman-Coulter) in terms of
  • very long term prediction of cardiovascular death and
    • heart failure in stable patients.

We don’t know how hs-cTnI from Abbott Diagnostics

  • performs in the APACE consort.

The number of patients and endpoints provided

  • by the APACE registry are rather low.
  • The results could, therefore, be a chance finding.

It is far too early to favour one high sensitivity assay over the other. The findings need confirmation.

Implications for clinical practice

There is no doubt that high-sensitivity assays

  • are the analytical method of choice
    • in terms of risk stratification in patients with ACS.

What is new?
A single measurement of hs-cTn seems to be adequate

  • for long-term risk stratification in patients without AMI.

However, the question of which troponin might be preferable

  • for long-term risk stratification remains unanswered.

Part 2. ability of high-sensitivity cTnT and NT pro-BNP to predict cardiovascular events and death in patients with T2DM

Hillis GS; Welsh P; Chalmers J; Perkovic V; Chow CK; Li Q; Jun M; Neal B; Zoungas S; Poulter N; Mancia G; Williams B; Sattar N; Woodward M
Diabetes Care.  2014; 37(1):295-303 (ISSN: 1935-5548)


Current methods of risk stratification in patients with

  • type 2 diabetes are suboptimal.

The current study assesses the ability of

  • N-terminal pro-B-type natriuretic peptide (NT-proBNP) and
  • high-sensitivity cardiac troponin T (hs-cTnT)

to improve the prediction of cardiovascular events and death in patients with type 2 diabetes.


A nested case-cohort study was performed in 3,862 patients who participated in the Action in Diabetes and Vascular Disease:

Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial.


Seven hundred nine (18%) patients experienced a

  • major cardiovascular event

(composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke) and

  • 706 (18%) died during a median of 5 years of follow-up.

In Cox regression models, adjusting for all established risk predictors,

  • the hazard ratio for cardiovascular events for NT-proBNP was 1.95 per 1 SD increase (95% CI 1.72, 2.20) and
  • the hazard ratio for hs-cTnT was 1.50 per 1 SD increase (95% CI 1.36, 1.65). The hazard ratios for death were
    • 1.97 (95% CI 1.73, 2.24) and
    • 1.52 (95% CI 1.37, 1.67), respectively.

The addition of either marker improved 5-year risk classification for cardiovascular events
(net reclassification index in continuous model,

  • 39% for NT-proBNP and 46% for hs-cTnT).

Likewise, both markers greatly improved the accuracy with which the 5-year risk of death was predicted.
The combination of both markers provided optimal risk discrimination.


NT-proBNP and hs-cTnT appear to greatly improve the accuracy with which the

  • risk of cardiovascular events or death can be estimated in patients with type 2 diabetes.

PreMedline Identifier: 24089534

Part 3. M-Atrial Natriuretic Peptide

M-Atrial Natriuretic Peptide and Nitroglycerin in a Canine Model of Experimental Acute Hypertensive Heart Failure:
Differential Actions of 2 cGMP Activating Therapeutics.

Paul M McKie, Alessandro Cataliotti, Tomoko Ichiki, S Jeson Sangaralingham, Horng H Chen, John C Burnett
Journal of the American Heart Association 01/2014; 3(1):e000206.
Source: PubMed


Systemic hypertension is a common characteristic in

  • acute heart failure (HF).

This increasingly recognized phenotype

  • is commonly associated with renal dysfunction and
  • there is an unmet need for renal enhancing therapies.

In a canine model of HF and acute vasoconstrictive hypertension

  • we characterized and compared the cardiorenal actions of M-atrial natriuretic peptide (M-ANP),
    a novel particulate guanylyl cyclase (pGC) activator, and
  • nitroglycerin, a soluble guanylyl cyclase (sGC) activator.

HF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II
infusion. We characterized the cardiorenal and humoral actions

  • prior to,
  • during, and
  • following intravenous infusions of
  1. M-ANP (n=7),
  2. nitroglycerin (n=7),
  3. and vehicle (n=7) infusion.

Mean arterial pressure (MAP) was reduced by

  1. M-ANP (139±4 to 118±3 mm Hg, P<0.05) and
  2. nitroglycerin (137±3 to 116±4 mm Hg, P<0.05);

similar findings were recorded for

  1. pulmonary wedge pressure (PCWP) with M-ANP (12±2 to 6±2 mm Hg, P<0.05)
  2. and nitroglycerin (12±1 to 6±1 mm Hg, P<0.05).

M-ANP enhanced renal function with significant increases (P<0.05) in

  • glomerular filtration rate (38±4 to 53±5 mL/min),
  • renal blood flow (132±18 to 236±23 mL/min), and
  • natriuresis (11±4 to 689±37 mEq/min) and
  • also inhibited aldosterone activation (32±3 to 23±2 ng/dL, P<0.05), whereas

nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation.

Our results advance

the differential cardiorenal actions of

  • pGC (M-ANP) and sGC (nitroglycerin) mediated cGMP activation.

These distinct renal and aldosterone modulating actions make

M-ANP an attractive therapeutic for HF with concomitant hypertension, where

  • renal protection is a key therapeutic goal.

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The Cost to Value Conundrum in Cardiovascular Healthcare Provision

The Cost to Value Conundrum in Cardiovascular Healthcare Provision

Author: Larry H. Bernstein, MD, FCAP

I write this introduction to Volume 2 of the e-series on Cardiovascular Diseases, which curates the basic structure and physiology of the heart, the vasculature, and related structures, e.g., the kidney, with respect to:

1. Pathogenesis
2. Diagnosis
3. Treatment

Curation is an introductory portion to Volume Two, which is necessary to introduce the methodological design used to create the following articles. More needs not to be discussed about the methodology, which will become clear, if only that the content curated is changing based on success or failure of both diagnostic and treatment technology availability, as well as the systems needed to support the ongoing advances.  Curation requires:

  • meaningful selection,
  • enrichment, and
  • sharing combining sources and
  • creation of new synnthesis

Curators have to create a new perspective or idea on top of the existing media which supports the content in the original. The curator has to select from the myriad upon myriad options available, to re-share and critically view the work. A search can be overwhelming in size of the output, but the curator has to successfully pluck the best material straight out of that noise.

Part 1 is a highly important treatment that is not technological, but about the system now outdated to support our healthcare system, the most technolog-ically advanced in the world, with major problems in the availability of care related to economic disparities.  It is not about technology, per se, but about how we allocate healthcare resources, about individuals’ roles in a not full list of lifestyle maintenance options for self-care, and about the important advances emerging out of the Affordable Care Act (ACA), impacting enormously on Medicaid, which depends on state-level acceptance, on community hospital, ambulatory, and home-care or hospice restructuring, which includes the reduction of management overhead by the formation of regional healthcare alliances, the incorporation of physicians into hospital-based practices (with the hospital collecting and distributing the Part B reimbursement to the physician, with “performance-based” targets for privileges and payment – essential to the success of an Accountable Care Organization (AC)).  One problem that ACA has definitively address is the elimination of the exclusion of patients based on preconditions.  One problem that has been left unresolved is the continuing existence of private policies that meet financial capabilities of the contract to provide, but which provide little value to the “purchaser” of care.  This is a holdout that persists in for-profit managed care as an option.  A physician response to the new system of care, largely fostered by a refusal to accept Medicaid, is the formation of direct physician-patient contracted care without an intermediary.

In this respect, the problem is not simple, but is resolvable.  A proposal for improved economic stability has been prepared by Edward Ingram. A concern for American families and businesses is substantially addressed in a macroeconomic design concept, so that financial services like housing, government, and business finance, savings and pensions, boosting confidence at every level giving everyone a better chance of success in planning their personal savings and lifetime and business finances.

Part 2 is a collection of scientific articles on the current advances in cardiac care by the best trained physicians the world has known, with mastery of the most advanced vascular instrumentation for medical or surgical interventions, the latest diagnostic ultrasound and imaging tools that are becoming outdated before the useful lifetime of the capital investment has been completed.  If we tie together Part 1 and Part 2, there is ample room for considering  clinical outcomes based on individual and organizational factors for best performance. This can really only be realized with considerable improvement in information infrastructure, which has miles to go.  Why should this be?  Because for generations of IT support systems, they are historically focused on billing and have made insignificant inroads into the front-end needs of the clinical staff.

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Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker


Co-Author of the FIRST Article: Larry H. Bernstein, MD, FCAP

Reviewer and Curator of the SECOND and of the THIRD Articles: Larry H. Bernstein, MD, FCAP


Article Architecture Curator: Aviva Lev-Ari, PhD, RN

This article presents Advances in the Treatment using Subcutaneous Erythropoietin (EPO) and Intravenous Iron (Fe) for IMPROVEMENT of Severe and Resistant Congestive Heart Failure and its resultant Anemia.  The Leading Biomarker for Congestive Heart Failure is an Independent Predictor identified as an Elevated N-terminal proBNP.

NT-proBNP schematic diagram-Copy.pdf_page_1


Anemia as an Independent Predictor of Elevated N-terminal proBNP

Salman A. Haq, MD1, Mohammad E. Alam2, Larry Bernstein, MD, FCAP3,  LB Banko 1, Leonard Y. Lee, MD, FACS4, Barry I. Saul, MD, FACC5, Terrence J. Sacchi, MD, FACC6,  John F. Heitner, MD, FACC7
1Cardiology Fellow,  2  Clinical Chemistry Laboratories, 3 Program Director, Cardiothoracic Surgery, 4 Division of Cardiology,  Department of Medicine, New York Methodist Hospital-Weill Cornell, Brooklyn, NY

(Unpublished manuscript)  Poster Presentation


The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

Donald S Silverberg, MDa; Dov Wexler, MDa; David Sheps, MDa; Miriam Blum, MDa; Gad Keren, MDa; Ron Baruch, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Shoshana Steinbruch, RNa; Itzhak Shapira, MDa; Shlomo Laniado, MDa; Adrian Iaina, MDa

J Am Coll Cardiol. 2001;37(7):1775-1780. doi:10.1016/S0735-1097(01)01248-7


The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations

Donald S Silverberg, MDa; Dov Wexler, MDa; Miriam Blum, MDa; Gad Keren, MDa; David Sheps, MDa; Eyal Leibovitch, MDa; David Brosh, MDa; Shlomo Laniado, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Itzhak Shapira, MDa; Dov Gavish, MDa; Ron Baruch, MDa; Bella Koifman, MDa; Carl Kaplan, MDa; Shoshana Steinbruch, RNa; Adrian Iaina, MDa

J Am Coll Cardiol. 2000;35(7):1737-1744. doi:10.1016/S0735-1097(00)00613-6


This THREE article sequence is related by investigations occurring by me, a very skilled cardiologist and his resident, and my premedical student at New York Methodist Hospital-Weill Cornell, in Brooklyn, NY, while a study had earlier been done applying the concordant discovery, which the team in Israel had though was knowledge neglected.  There certainly was no interest in the problem of the effect of anemia on the patient with severe congestive heart failure, even though erythropoietin was used widely in patients with end-stage renal disease requiring dialysis, and also for patients with myelofibrosis.  The high cost of EPO was only one factor, the other being a guideline to maintain the Hb concentration at or near 11 g/dl – not higher.  In the first article, the authors sought to determine whether the amino terminal pro– brain type natriuretic peptide (NT-pro BNP) is affected by anemia, and to determine that they excluded all patients who had renal insufficiency and/or CHF, since these were associated with elevated NT-proBNP.  It was already well established that this pro-peptide is secreted by the heart with the action as a urinary sodium retention hormone on the kidney nephron, the result being an increase in blood volume.  Perhaps the adaptation would lead to increased stroke volume from increased venous return, but that is not conjectured.  However, at equilibrium, one would expect there to be increased red cell production to maintain the cell to plasma volume ratio, thereby, resulting in adequate oxygen exchange to the tissues.  Whether that is always possible is uncertain because any reduction in the number of functioning nephrons would make the kidney not fully responsive at the Na+ exchange level, and the NT-pro BNP would rise.  This introduces complexity into the investigation, requiring a removal of confounders to establish the effect of anemia.

The other two articles are related studies by the same group in Israel.  They surmised that there was evidence that was being ignored as to the effect of anemia, and the treatment of anemia was essential in addition to other treatments.  They carried out a randomized trial to determine just that, a benefit to treating the anemia.  But they also conjectured that an anemia with a Hb concentration below 12 g/dl has an deleterious effect on the targeted population.  Treatment by intermittent transfusions could potentially provide the added oxygen-carrying capacity, but the fractionation of blood, the potential for transfusion-transmitted disease and transfusion-reactions, combined with the need for the blood for traumatic blood loss made EPO a more favorable alternative to packed RBCs.  The proof-of-concept is told below.  Patients randomized to receive EPO at a lower than standard dose + iron did better.



In this article, Erythropoietin (EPO) and Intravenous Iron (Fe) as Therapeutics for Anemia in Severe and Resistant CHF: The Elevated N-terminal proBNP Biomarker we provides a summary of three articles on the topic and we shading new light on the role that Anemia Hb < 12 g%  plays as a Biomarker for CHF and for

  • prediction of elevated BNP, known as an indicator for the following Clinical Uses:
Clinical Use
  • Rule out congestive heart failure (CHF) in symptomatic individuals
  • Determine prognosis in individuals with CHF or other cardiac disease
  • Maximize therapy in individuals with heart failure by the use of Subcutaneous Erythropoietin (EPO) and Intravenous Iron (Fe)
Evaluation of BNP and NT-proBNP Clinical Performance
Study Sensitivity(%) Specificity(%) PPV(%) NPV(%)
Diagnose impaired LVEF3
BNP 73 77 70 79
NT-proBNP 70 73 61 80
Diagnose LV systolic dysfunction after MI2
BNP 68 69 56 79
NT-proBNP 71 69 56 80
Diagnose LV systolic dysfunction after MI12
BNP 94 40 NG 96
NT-proBNP 94 37 NG 96
Prognosis in newly diagnosed heart failure patients: prediction of mortality/survival1
BNP 98 22 42 94
NT-proBNP 95 37 47 93
Prognosis post myocardial infarction: prediction of mortality2
BNP 86 72 39 96
NT-proBNP 91 72 39 97
Prognosis post myocardial infarction: prediction of heart failure2
BNP 85 73 54 93
NT-proBNP 82 69 50 91
PPV, positive predictive value; NPV, negative predictive value; LVEF, left ventricular ejection fraction; NG, not given.
Reference Range
BNP: < 100 pg/mL13
proBNP, N-terminal: 300 pg/mL
The NT-proBNP reference range is based on EDTA plasma. Other sample types will produce higher values.
Interpretive Information
Symptomatic patients who present with a BNP or NT-proBNP level within the normal reference range are highly unlikely to have CHF. Conversely, an elevated baseline level indicates the need for further cardiac assessment and indicates the patient is at increased risk for future heart failure and mortality.BNP levels increase with age in the general population, with the highest concentrations seen in those greater than 75 years of age.14 Heart failure is unlikely in individuals with a BNP level <100 pg/mL and proBNP level ≤300 pg/mL. Heart failure is very likely in individuals with a BNP level >500 pg/mL and proBNP level ≥450 pg/mL who are <50 years of age, or ≥900 pg/mL for patients ≥50 years of age. Patients in between are either hypertensive or have mild ischemic or valvular disease and should be observed closely.15BNP is increased in CHF, left ventricular hypertrophy, acute myocardial infarction, atrial fibrillation, cardiac amyloidosis, and essential hypertension. Elevations are also observed in right ventricular dysfunction, pulmonary hypertension, acute lung injury, subarachnoid hemorrhage, hypervolemic states, chronic renal failure, and cirrhosis.NT-proBNP levels are increased in CHF, left ventricular dysfunction, myocardial infarction, valvular disease, hypertensive pregnancy, and renal failure, even after hemodialysis.Although levels of BNP and NT-proBNP are similar in normal individuals, NT-proBNP levels are substantially greater than BNP levels in patients with cardiac disease due to increased stability (half-life) of NT-proBNP in circulation. Thus, results from the two tests are not interchangeable.
  1. Cowie MR and Mendez GF. BNP and congestive heart failure. Prog Cardiovasc Dis. 2002;44:293-321.
  2. Richards AM, Nicholls MG, Yandle TG, et al. Plasma N-terminal pro-brain natriuretic peptide and adrenomedullin. New neurohormonal predictors of left ventricular function and prognosis after myocardial infarction. Circulation. 1998:97:1921-1929.
  3. Hammerer-Lercher A, Neubauer E, Muller S, et al. Head-to-head comparison of N-terminal pro-brain natriuretic peptide, brain natriuretic peptide and N-terminal pro-atrial natriuretic peptide in diagnosing left ventricular dysfunction. Clin Chim Acta. 2001;310:193-197.
  4. McDonagh TA, Robb SD, Murdoch DR, et al. Biochemical detection of left-ventricular systolic dysfunction. Lancet. 1998;351:9-13.
  5. Mukoyama Y, Nakao K, Hosoda K, et al. Brain natriuretic peptide as a novel cardiac hormone in humans: Evidence for an exquisite dual natriuretic peptide system, ANP and BNP. J Clin Invest. 1991;87:1402-1412.
  6. Hunt PJ, Richards AM, Nicholls MG, et al. Immunoreactive amino-terminal pro-brain natriuretic peptide (NT-PROBNP): a new marker of cardiac impairment. Clin Endocrinol. 1997;47:287-296.
  7. Davis M, Espiner E, Richards G, et al. Plasma brain natriuretic peptide in assessment of acute dyspnoea. Lancet. 1994;343:440-444.
  8. Kohno M, Horio T, Yokokawa K, et al. Brain natriuretic peptide as a cardiac hormone in essential hypertension. Am J Med. 1992;92:29-34.
  9. Bettencourt P, Ferreira A, Pardal-Oliveira N, et al. Clinical significance of brain natriuretic peptide in patients with postmyocardial infarction. Clin Cardiol. 2000;23:921-927.
  10. Jernberg T, Stridsberg M, Venge P, et al. N-terminal pro brain natriuretic peptide on admission for early risk stratification of patients with chest pain and no ST-segment elevation. J Am Coll Cardiol. 2002;40:437-445.
  11. Richards AM, Troughton RW. Use of natriuretic peptides to guide and monitor heart failure therapy. Clin Chem. 2012;58:62-71.
  12. Pfister R, Scholz M, Wielckens K, et al. The value of natriuretic peptides NT-pro-BNP and BNP for the assessment of left-ventricular volume and function. A prospective study of 150 patients.Dtsch Med Wochenschr. 2002;127:2605-2609.
  13. Siemens ADVIA Centaur® BNP directional insert; 2003.
  14. Redfield MM, Rodeheffer RJ, Jacobsen SJ, et al. Plasma brain natriuretic peptide concentration: impact of age and gender. J Am Coll Cardiol. 2002;40:976-982.
  15. Weber M, Hamm C. Role of B-type natriuretic peptid (BNP) and NT-proBNP in clinical routine.Heart. 2006;92:843-849.


B-type Natriuretic Peptide and proBNP, N-terminal


Anemia as an Independent Predictor of Elevated N-terminal proBNP

Salman A. Haq, MD1, Mohammad E. Alam2, Larry Bernstein, MD, FCAP3,  LB Banko 1, Leonard Y. Lee, MD, FACS4, Barry I. Saul, MD, FACC5, Terrence J. Sacchi, MD, FACC6,  John F. Heitner, MD, FACC7
1Cardiology Fellow,  2  Clinical Chemistry Laboratories, 3 Program Director, Cardiothoracic Surgery, 4 Division of Cardiology,  Department of Medicine, New York Methodist Hospital-Weill Cornell, Brooklyn, NY

(Unpublished manuscript)  Poster Presentation:

Anemia as an Independent Predictor of Elevated N-Terminal proBNP Levels in
Patients without Evidence of Heart Failure and Normal Renal Function.

Haq SA, Alam ME, Bernstein L, Banko LB, Saul BI, Lee LY, Sacchi TJ, Heitner JF.

Table 1.  Patient Characteristics

Variable No Anemia(n=138) Anemia(n=80)
Median Age (years) 63 76
Men (%) 35 33
Creatinine (mg/dl) 0.96 1.04
Hemoglobin (g/dl) 13.7 10.2
LVEF (%) 67 63
Median NT-proBNP (pg/ml) 321.6 1896.0


A series of slide showing the determination of the representation of normal NT-proBNP range
after removal of patient confounders.







N-terminal proBNP (NT-proBNP) has emerged as a primary tool for diagnosing congestive heart failure (CHF). Studies have shown that the level of

  • NT-proBNP is affected by renal insufficiency (RI) and age, independent of the diagnosis of CHF.

There is some suggestion from recent studies that

  • anemia may also independently affect NT-proBNP levels.


To assess the affect of anemia on NT-proBNP independent of CHF, RI, and age.


We evaluated 746 consecutive patients presenting to the Emergency Department (ED) with shortness of breath and underwent evaluation with serum NT-proBNP.

All patients underwent a trans-thoracic echocardiogram (TTE) and clinical evaluation for CHF. Patients were included in this study if they had a normal TTE (normal systolic function, mitral inflow pattern and left ventricular (LV) wall thickness) and no evidence of CHF based on clinical evaluation. Patients were excluded if they had RI (creatinine > 2 mg/dl) or a diagnosis of sepsis. Anemia was defined using the World Health Organization (W.H.O.) definition of

  • hemoglobin (hgb) < 13 g/dl for males and hgb < 12 g/dl for females.


Of the 746 consecutive patients, 218 patients (138 anemia, 80 no anemia) met the inclusion criteria. There was a markedly significant difference between

  • NT- proBNP levels based on the W.H.O. diagnosis of anemia.

Patients with anemia had a

  • mean NT- proBNP of 4,735 pg/ml compared to 1,230 pg/ml in patients without anemia (p=0.0001).

There was a markedly

  • significant difference in patients who had a hgb > 12 (median 295 pg/ml) when compared to
  • both patients with an hgb of 10.0 to 11.9 (median 2,102 pg/ml; p = 0.0001) and
  • those with a hgb < 10 (median 2,131 pg/ml; p = 0.001).

Linear regression analysis comparing hgb with log NT-proBNP was statistically significant (r = 0.395; p = 0.0001). MANOVA demonstrated that

  • elevated NT- proBNP levels in patients with anemia was independent of age.


This study shows that NT-proBNP is associated with anemia independent of CHF, renal insufficiency, sepsis or age.


B-type natriuretic peptide (BNP) is secreted from the myocardium in response to myocyte stretch. 1-2 BNP is released from the myocytes as a 76 aminoacid N-terminal fragment (NT-proBNP) and a 32-amino acid active hormone (BNP). 3 These peptides have emerged as a primary non-invasive modality for the diagnosis of congestive heart failure (CHF). 4- 7 In addition, these peptides have demonstrated prognostic significance in patients with invasive modality for the diagnosis of

  • congestive heart failure (CHF). 4- 7
  • heart failure 8-9,
  • stable coronary artery disease 10, and
  • in patients with acute coronary syndromes. 11-14

Studies have shown that the level of NT- proBNP is affected by

  • age and renal insufficiency (RI) independent of the diagnosis of CHF. 15,16

There is some suggestion from the literature that

  • anemia may also independently affect NT-proBNP levels. 17-20

Willis et al. demonstrated in a cohort of 209 patients without heart failure that anemia was associated with an elevated NT- proBNP. 17 Similarly, in 217 patients undergoing cardiac catheterization, blood samples were drawn from the descending aorta prior to contrast ventriculography for BNP measurements and

  • anemia was found to be an independent predictor of plasma BNP levels. 18

The objective of this study is to assess the effect of anemia on NT-proBNP independent of CHF, sepsis, age or renal insufficiency.


Patient population

The study population consisted of 746 consecutive patients presenting to the emergency room who underwent NT-proBNP evaluation for the evaluation of dyspnea. Transthoracic echocardiogram (TTE) was available on 595 patients. Patients were included in this study if they had a normal TTE, which was defined as normal systolic function (left ventricular ejection fraction [LVEF] > 45%), normal mitral inflow pattern and normal LV wall thickness. CHF was excluded based on thorough clinical evaluation by the emergency department attending and the attending medical physician. Patients with disease states that may affect the NT- proBNP levels were also excluded:

  1. left ventricular systolic dysfunction (LVEF < 45%),
  2. renal insufficiency defined as a creatinine > 2 mg/dl and
  3. sepsis (defined as positive blood cultures with two or more of the following systemic inflammatory response syndrome (SIRS) criteria: heart rate > 90 beats per minute;
  4. body temperature < 36 (96.8 °F) or > 38 °C (100.4 °F);
  5. hyperventilation (high respiratory rate) > 20 breaths per minute or, on blood gas, a PaCO2 less than 32 mm Hg;
  6. white blood cell count < 4000 cells/mm3 or > 12000 cells/mm³ (< 4 x 109 or > 12 x 109 cells/L), or greater than 10% band forms (immature white blood cells). 21

The study population was then divided into two groups, anemic and non- anemic. Anemia was defined using the world health organization (W.H.O.) definition of hemoglobin (hgb) < 13 g/dl for males and < 12 g/dl for females.The data was also analyzed by dividing the patients into three groups based on hgb levels i.e. hgb > 12, hgb 10 to 11.9 and hgb < 10.

Baseline patient data

Patient’s baseline data including age, gender, ethnicity, hemoglobin (hgb), hematocrit (hct), creatinine, NT- proBNP were recorded from the electronic medical record system in our institution. Chemistry results were performed on the Roche Modular System (Indianapolis, IN), with the NT- proBNP done by chemiluminescence assay. The hemogram was performed on the Beckman Coulter GenS. All TTE’s were performed on Sonos 5500 machine. TTE data collected included LVEF, mitral inflow pattern and LV wall thickness assessment.

Statistical analysis

The results are reported in the means with p < 0.05 as the measure of significance for difference between means. Independent Student’s t-tests were done comparing NT proBNP and anemia. Univariate ANOVAs and multivariate ANOVA (MANOVA) with post hoc tests using the Bonferroni method were used to compare NT- proBNP levels with varying ranges of hgb and age using SPSS 13.0 (SPSS, Chicago, IL). A linear regression analysis was performed using SYSTAT. Calculations included Wilks’Lamda, Pillai trace and Hotelling-Lawley trace. A GOLDMineR® plot was constructed to estimate the effects of age and anemia on NT- proBNP levels. The GOLDMineR® effects plot displays the odds-ratios for predicted NT-proBNP elevation versus the predictor values. Unlike the logistic regression, the ordinal regression, which the plot is derived from, can have polychotomous as well as dichotomous values, as is the case for NT-proBNP.


Of the 746 consecutive patients, 218 patients met the inclusion criteria (fig 1). Baseline characteristics of patients are listed in table 1. The median age for anemic patients was 76 years and 63 years for patients without anemia. One third of patients in both groups were men. The mean hemoglobin for

  • anemic patients was 10.2 g/dl as compared to 13.7 g/dl for non-anemic patients.
  • The mean LVEF of patients with anemia was 64% as compared to 67% for non-anemic patients.

Based on the WHO definition of anemia, 138 patients were determined to be anemic while 80 patients were diagnosed as non-anemic. There was a markedly  significant difference between NT-proBNP levels based on the WHO diagnosis of anemia.

Patients with anemia had a

  • mean NT-proBNP of 4,735 pg/ml compared to 1,230 pg/ml in patients without anemia (p = 0.0001).

Of the 218 patients in the study, 55 patients had a hgb of < 10 g/dl. Analysis using

  • hgb < 10 g/dl for anemia demonstrated a statistically significant difference in the NT-proBNP values.

Patients with a hgb < 10 g/dl had a mean NT- proBNP of 5,130 pg/ml

  • compared to 2,882 pg/ml in patients with a hgb of > 10 g/dl (p = 0.01)

The groups were also divided into three separate categories of hgb for subset analysis:

  • hgb > 12 g/dl,
  • hgb 10 to 11.9 g/dl and
  • hgb < 10 g/dl.

There was a markedly significant difference in

  •  the NT- ProBNP levels of patients who had a hgb > 12 g/dl (median 295 pg/ml) when
  • compared to those with a hgb range of 10.0 g/dl to 11.9 g/dl (median 2,102 pg/ml) (p = 0.0001),

and also a significant difference in

  • NT- proBNP levels of patients with a hgb > 12 g/dl (median 295 pg/ml) when
  • compared to a hgb of < 10 g/dl (median 2,131 pg/ml) (p = 0.001).

However, there was no statistically significant difference in NT-proBNP levels of patients with hgb 10 g/dl to 11.9 g/dl

  • when compared to those with a hgb of < 10 g/dl (p = 1.0).

A scatter plot comparing hgb with log NT-proBNP and fitting of a line to the data by ordinary least squares regression was significant (p = 0.0001) and demonstrated

  • a correlation between anemia and NT-proBNP levels (r = 0.395) (fig. 2).

MANOVA demonstrated that elevated NT- proBNP levels in patients with anemia was independent of age (Wilks’ Lambda [p = 0.0001]). In addition, using GOLDMineR® plots (figure 3a and 3b) with a combination of age and hb scaled as predictors of elevated NT-proBNP,

  • both age and hgb were required as independent predictors.

What about the effect of anemia? The GOLDminer analysis of ordinal regression was carried out in a database from which renal insufficiency and CHF were removed. The anemia would appear to have an independent effect on renal insufficiency. Figure 4 is a boxplot comparison of NT – proBNP, the age normalized function NKLog (NT- proBNP)/eGFR formed from taking 1000*Log(NT- proBNP) divided by the MDRD at eGFR exceeding 60 ml/min/m2 and exceeding 30 ml/min/m2. The transformed variable substantially makes the test independent of age and renal function. The boxplot shows the medians, 97.5, 75, 25 and 2.5 percentiles. There appears to be no significance in the NKLog(NT pro-BNP)/MDRD plot. Table II compares the NT-proBNP by WHO criteria at eGFR 45, 60 and 75 ml/mln/m2 using the t-test with unequal variance assumed, and the Kolmogorov-Smirnov test for nonparametric measures of significance. The significance at 60 ml/min/m2 is marginal and nonexistent at 75 ml/min/m2. This suggests that the contribution from renal function at above 60 ml/min2 can be ignored. This is consistent with the findings using the smaller, trimmed database, but there is an interaction between

  •  anemia, and
  •  eGFR at levels below 60 ml/min/m2


The findings in this study indicate that

  • anemia was associated with elevated NT-proBNP levels independent of CHF, renal insufficiency, sepsis or age.

These findings have been demonstrated with NT-proBNP in only one previous study. Wallis et al. demonstrated that after adjusting for age, sex, BMI, GFR, LVH and valvular disease;

  1. only age,
  2. valvular disease and
  3. low hemoglobin

were significantly associated with increased NT-proBNP. 18.

In our study, CHF was excluded based on both a normal TTE and a thorough clinical evaluation. In the only other study directly looking at NT- proBNP levels in anemic patients without heart failure

  • only 25% of patients had TTEs, with one patient having an LVEF of 40%. 17

BNP, the active molecule released after cleavage along with NT- proBNP, has also been studied in relation to blood hemoglobin levels. 18 In 263 patients undergoing cardiac catheterization  blood samples were drawn from the descending aorta prior to contrast ventriculography to determine the value of BNP. Anemia was present in 217 patients. Multivariate linear regression model adjusting for

  1.  age,
  2.  gender,
  3.  body mass index,
  4.  history of myocardial infarction,
  5.  estimated creatinine clearance, and
  6.  LVEF
  • found hgb to be an independent predictor of BNP levels.

In our study, patients with anemia were slightly older than those without anemia. However, both MANOVA and GOLDMineR® plot demonstrated that

  • elevated NT-proBNP levels in patients with anemia was independent of age.

Other studies have found that BNP is dependent on renal insufficiency and age. Raymond et al. randomly selected patients to complete questionnaires regarding CHF and

  1. then underwent pulse and blood pressure measurements,
  2.  electrocardiogram (ECG),
  3.  echocardiography and
  4.  blood sampling. 15

A total of 672 subjects were screened and 130 were determined to be normal, defined as

  • no CHF or ischemic heart disease,
  • normal LVEF,
  • no hypertension,
  • diabetes mellitus,
  • lung disease, and
  • not on any cardiovascular drugs.

They found

  1. older age,
  2. increasing dyspnea,
  3. high plasma creatinine and a
  4. LVEF < 45%

to be independently associated with an elevated NT-proBNP plasma level by multiple linear regression analysis. In another study, McCullough et al. evaluated the patients from the Breathing Not Properly Multinational Study

  • looking at the relationship between BNP and renal function in CHF. 16

Patients were excluded if they were on hemodialysis or had a estimated glomerular filteration rate (eGFR) of < 15 ml/min. They found that the BNP levels correlated significantly with the eGFR, especially in patients without CHF, suggesting

  1. chronic increased blood volume and
  2. increased left ventricular wall tension as a possible cause. 16

Our study was designed to exclude patients with known diseases such as CHF and renal insufficiency in order to demonstrate

  • the independent effect of anemia on elevated NT-proBNP levels.

The mechanism for elevated NT-proBNP levels in patients with anemia is unknown. Some possible mechanisms that have been reported in the literature include

  • hemodilution secondary to fluid retention in patients with CHF 18,
  • decreased oxygen carrying capacity with accompanying tissue hypoxia which
  • stimulates the cardio-renal compensatory mechanism leading to increased release of NT-proBNP. 17

The findings from our study suggest that

  •  NT-proBNP values should not be interpreted in isolation of hemoglobin levels and
  • should be integrated with other important clinical findings for the diagnosis of CHF.

Further studies are warranted

  1.  to assess the relationship between anemia and plasma natriuretic peptides, and
  2. possibly modify the NT-proBNP cutoff points for diagnosing acutely decompensated CHF in patients with anemia.


This study shows that elevated NT-proBNP levels are associated with anemia independent of

  •   CHF,
  •  renal insufficiency,
  •  sepsis and
  •  age.

NT-proBNP levels should be interpreted with caution in patients who have anemia.


1. Brunea BG, Piazza LA, de Bold AJ. BNP gene expression is specifically modulated by stretch and ET-1 in a new model of isolated rat atria.Am J Physiol  1997; 273:H2678-86.

2. Wiese S, Breyer T, Dragu A, et al. Gene expression of brain natriuretic peptide  in isolated atrial and ventricular human myocardium: influence of angiotensin II and diastolic fiber length. Circ 2000; 102:3074-79.

3. de Lemos JA, McGuire DK, Drazner MH. B-type natriuretic peptide in cardiovascular disease. Lancet 2003; 362:316-22.

4.   Dao Q, Krishnaswamy P, Kazanegra R, et al. Utility of B-type natriuretic  peptide in the diagnosis of congestive heart failure in an urgent care setting. J Am  Coll Cardiol 2001; 37:379-85.

5. Morrison LK, Harrison A, Krishnaswamy P, Kazanegra R, Clopton P, Maisel A. Utility of rapid natriuretic peptide assay in differentiating congestive heart failure from lung  disease in patients presenting with dyspnea.
J Am Coll Cardiol  2003; 39:202-09.

6.  Maisel AS, Krishnaswamy P, Nowak RM, et al.  Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure. N Engl J Med 2002; 347:161-67.

7. Januzzi JL, Camargo CA, Anwaruddin S, et al. The N-terminal Pro-BNP investigation of dyspnea in the emergency department (PRIDE) study. Am J  Cardiol 2005; 95:948-954.

8.  Tsutamoto T, Wada A, Meada K, et al.   Attenuation of compensation of  endogenous cardiac natriuretic peptide system  in chronic heart failure: prognostic role  of plasma  brain natriuretic peptide concentration in patients with chronic  symptomatic  left ventricular dysfunction.
Circulation 1997; 96(2): 509-16.

9.  Anand IS, Fisher LD, Chiang YT, et al. Changes in brain natriuretic peptide and norepinephrine over time and mortality and morbidity in the Valsartan Heart Failure Trial (Val-HEFT). Circulation 2003; 107:1278-1283.

10. Omland T, Richards AM, Wergeland R and Vik-Mo H. B-type natriuretic peptide and long term survival in patients with stable coronary artery disease.
Am J Cardiol 2005; 95:24-28.

11. Omland T, Aakvaag A, Bonarjee VV. et al. Plasma brain natriuretic peptide as an indicator of left ventricular systolic dysfunction and long term prognosis after acute myocardial infarction. Comparison with plasma atrial natriuretic peptide and N-terminal proatrial natriuretic peptide.
Circulation 1996; 93:1963-1969.

12. de Lemos JA, Morrow DA, Bently JH, et al. The prognostic value of B-type natriuretic peptide in patients with acute coronary syndromes. N Engl J Med 2001; 345:1014-1021.

13. Richards AM, Nicholls MG, Espiner EA, et al. B-type natriuretic peptides and  ejection fraction for prognosis after myocardial infarction. Circulation 2003; 107:2786-2792.

14. Sabatine MS, Morrow DA, de Lemos JA, et al.  Multimarker approach to risk  stratification in non-ST elevation acute coronary syndromes: simultaneous  assessment of troponin I, C-reactive protein and B-type natriuretic peptide.
Circulation 2002; 105:1760-1763.

15. Raymond I, Groenning BA, Hildebrandt PR, Nilsson JC, Baumann M, Trawinski   J, Pedersen F.  The influence of age, sex andother variables on the plasma level of N-terminal pro brain natriureticpeptide in a large sample of the general  population. Heart 2003; 89:745-751.

16. McCollough PA, Duc P, Omland T, McCord J, Nowak RM, Hollander JE, et al. B-type natriuretic peptide and renal function in the diagnosis of heartfailure:  an analysis from the  Breathing Not Properly Multinational Study.
Am J Kidney Dis 2003; 41:571-579.

17. Willis MS, Lee ES, Grenache DG. Effect of anemia on plasma concentrations of  NT-proBNP.
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18. Wold Knudsen C, Vik-Mo H, Omland T. Blood hemoglobin is an independent  predictor of B-type natriuretic peptide.
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19. Tsuji H, Nishino N, Kimura Y, Yamada K, Nukui M, et al. Haemoglobin level influences plasma brain natriuretic peptide concentration. Acta Cardiol 2004;59:527-31.

20. Wu AH, Omland T, Wold KC, McCord J, Nowak RM, et al. Relationship  of B-type natriuretic peptide and anemia  in patients withand without heart failure:  A substudy from the Breathing Not Properly(BNP) Multinational Study.
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22. Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, et al.  Definitions for sepsis and organ failure and guidelines for theuse of innovative therapies in sepsis.  The ACCP/SCCM Consensus Conference Committee. Chest. 1992;101(6):1644-55.

Table Legends

Table I. Clinical characteristics of the study population

Table II. Comparison of NT- proBNP means under WHO criteria at different GFR

Table I
Variable No Anemia(n=80) Anemia(n=138)
Median age (years) 63 76
    Men (%) 27 (34) 47 (34)
    Women (%) 53 (66) 91 (66)
Weight (kg) 82.9 80.1
Chest Pain 21 (26) 3 (2)
Hemoglobin (g/dl) 13.7 10.2
Hematocrit (%) 40.5 30.5
Mean Corpuscular Volume 97 87
Creatinine (mg/dl) 0.99 1.07
Median NT-proBNP (pg/ml) 321 1896
Medical History
    HTN (%) 12 (15) 51 (37)
    Prior MI (%) 11 (14) 5 (4)
    ACS (%) 16 (20) 3 (2)
    CAD (%) 2 (1) 3 (2)
     DM (%) 18 (22) 11 (8)
   Clopidogrel 58 (72) 15 (11)
   Beta Blockers 68 (85) 27 (20)
   Ace Inhibitors 45 (56) 18 (13)
   Statins 57 (71) 17 (12)
   Calcium Channel Blocker 17 (21) 8 (6)
LVEF (%) 67 64

HTN: Hypertension CAD: Coronary Artery Disease
MI: Myocardial Infarction DM: Diabetes Mellitus
ACS: Acute Coronary Syndrome LVEF: Left Ventricular Ejection Fraction

Table II
GFR WHO Mean P (F) N NPar
> 45 0 3267 0.022 (4.33) 661
1 4681
> 60* 0 2593 0.031 (5.11) 456 0.018
1 4145
> 60r 0 786 0.203 (3.63) 303 0.08
1 3880
> 75 0 2773 > 0.80 320 0.043
1 3048

*AF, valve disease and elevated troponin T included
r AF, valve disease and elevated troponin T removed


FIGURE 1. Study population flow chart. (see poster)
FIGURE 2. Relationship between proBNP and hemoglobin. (see above)
FIGURE 3. NT-proBNP levels in relation to anemia (see above)

Supplementary Material

Table based on LatentGOLD Statistical Innovations, Inc., Belmont, MA., 2000: Jeroen Vermunt & Jay Magidson)

4-Cluster Model

Number of cases                                   408
Number of parameters (Npar)             24

Chi-squared Statistics
Degrees of freedom (df)                          71                     p-value
L-squared (L²)                                    80.2033                    0.21
X-squared                                            80.8313                     0.20
Cressie-Read                                        76.6761                     0.30
BIC (based on L²)                          -346.5966
AIC3 (based on L²)                        -132.7967
CAIC (based on L²)                       -417.5966

Model for Clusters
 Intercept                Cluster1      Cluster2     Cluster3     Cluster4     Wald     p-value
————–           0.1544           0.1434        0.0115        -0.3093     1.1981     0.75
Cluster Size           0.2870          0.2838       0.2487          0.1805

< 1.5                       0.0843           0.2457       0.0006          0.0084
1.6-2.5                   0.6179            0.6458       0.0709          0.2809
2.5-3.5                  0.2848           0.1067         0.5319          0.5883
> 3.5                      0.0130           0.0018         0.3966         0.1224
> 90                     0.1341             0.7919         0.0063         0.6106
61-90                  0.6019            0.2040          0.1633         0.3713
41-60                  0.2099            0.0041          0.3317         0.0175
< 41                     0.0542            0.0001         0.4987        0.0006
under 51           0.0668           0.5646          0.0568        0.0954
51-70                 0.3462            0.3602          0.3271         0.3880
over 70             0.5870            0.0752          0.6161         0.5166
No anemia      0.7518             0.6556          0.2041         0.0998
Anemia            0.2482             0.3444          0.7959         0.9002

———          Cluster1          Cluster2      Cluster3      Cluster4
Overall           0.2870            0.2838         0.2487        0.1805

< 1.5                0.2492              0.7379           0.0013         0.0116
1.6-2.5            0.4163               0.4243           0.0427        0.1167
2.6-3.5           0.2296               0.0887          0.3723        0.3095
> 3.5              0.0328                0.0023          0.7982        0.1666
> 90              0.1001                0.5998           0.0043        0.2958
61-90           0.5198                 0.1716           0.1136         0.1950
41-60           0.3860                 0.0055          0.5847         0.0238
< 41             0.1205                  0.0002          0.8785         0.0008
< 51            0.0720                 0.7458           0.0910          0.0912
51-70         0.3036                 0.3084           0.2013          0.1867
over 70     0.3773                  0.0409          0.3633           0.2186
No anemia 0.4589              0.3957           0.1076           0.0378
Anemia     0.1342                 0.1844            0.3742           0.3073

Hemoglobin on NT proBNP 3


The effect of correction of mild anemia in severe, resistant congestive heart failure using subcutaneous erythropoietin and intravenous iron: a randomized controlled study

Donald S Silverberg, MDa; Dov Wexler, MDa; David Sheps, MDa; Miriam Blum, MDa; Gad Keren, MDa; Ron Baruch, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Shoshana Steinbruch, RNa; Itzhak Shapira, MDa; Shlomo Laniado, MDa; Adrian Iaina, MDa

J Am Coll Cardiol. 2001;37(7):1775-1780. doi:10.1016/S0735-1097(01)01248-7


This is a randomized controlled study of anemic patients with severe congestive heart failure (CHF) to assess the effect of correction of the anemia on cardiac and renal function and hospitalization.


Although mild anemia occurs frequently in patients with CHF, there is very little information about the effect of correcting it with erythropoietin (EPO) and intravenous iron.


Thirty-two patients with moderate to severe CHF (New York Heart Association [NYHA] class III to IV)
who had a left ventricular ejection fraction (LVEF) of 40% despite maximally tolerated doses of CHF medications and
  • whose hemoglobin (Hb) levels were persistently between 10.0 and 11.5 g% were randomized into two groups.
Group A (16 patients) received subcutaneous EPO and IV iron to increase the level of Hb to at least 12.5 g%. In Group B (16 patients) the anemia was not treated. The doses of all the CHF medications were maintained at the maximally tolerated levels except for oral and intravenous (IV) furosemide, whose doses were increased or decreased according to the clinical need.


Over a mean of 8.2 +/- 2.6 months,
  • four patients in Group B and none in Group A died of CHF-related illnesses.
  • The mean NYHA class improved by 42.1% in A and worsened by 11.4% in B.
  • The LVEF increased by 5.5% in A and decreased by 5.4% in B.
  • The serum creatinine did not change in A and increased by 28.6% in B.
  • The need for oral and IV furosemide decreased by 51.3% and 91.3% respectively in A and increased by 28.5% and 28.0% respectively in B.
  • The number of days spent in hospital compared with the same period of time before entering the study decreased by 79.0% in A and increased by 57.6% in B.


When anemia in CHF is treated with EPO and IV iron, a marked improvement in cardiac and patient function is seen,
  • associated with less hospitalization and renal impairment and less need for diuretics. (J Am Coll Cardiol 2001;37:1775– 80)

Anemia of any cause is known to be capable of causing congestive heart failure (CHF) (1). In patients hospitalized with CHF the 

  • mean hemoglobin (Hb) is about 12 g% (2,3),

which is considered the lower limit of normal in adults (4). Thus, anemia appears to be

common in CHF. Recently, in 142 patients in our special CHF outpatient clinic, we found that

  • as the CHF worsened, the mean Hb concentration decreased, from 13.7 g% in mild CHF (New York Heart Association [NYHA] class I) to 10.9 g% in severe CHF (NYHA 4), and
  • the prevalence of a Hb 12 g% increased from 9.1% in patients with NYHA 1 to 79.1% in those with NYHA 4 (5).
The Framingham Study has shown that anemia is an
  • independent risk factor for the production of CHF (6).
Despite this association of CHF with anemia,
  • its role is not mentioned in the 1999 U.S. guidelines for the diagnosis and treatment of CHF (7), and
  • many studies consider anemia to be only a rare contributing cause of hospitalization for CHF (8,9).
Recently, we performed a study in which the anemia of severe CHF that was resistant to maximally tolerated doses of standard medications
  • was corrected with a combination of subcutaneous (sc) erythropoietin (EPO) and intravenous iron (IV Fe) (5).
We have found this combination to be safe, effective and additive
  • in the correction of the anemia of chronic renal failure (CRF) in both
  • the predialysis period (10) and the dialysis period (11).
The IV Fe appears to be more effective than oral iron (12,13). In our previous study of CHF patients (5), the treatment resulted in
  • improved cardiac function,
  • improved NYHA functional class,
  • increased glomerular filtration rate,
  • a marked reduction in the need for diuretics and
  • a 92% reduction in the hospitalization rate
compared with a similar time period before the intervention. In the light of these positive results, a prospective randomized study was undertaken
  • to determine the effects of the correction of anemia in severe symptomatic CHF resistant to maximally tolerated CHF medication.

Abbreviations and Acronyms

CABG coronary artery bypass graft
CHF congestive heart failure
CRF chronic renal failure
EPO erythropoietin
%Fe Sat percent iron saturation
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IU international units
IV intravenous
LVEF left ventricular ejection fraction
NYHA New York Heart Association
PA pulmonary artery
sc subcutaneous
SOLVD Studies Of Left Ventricular Dysfunction


Patients.Thirty-two patients with CHF were studied. Before the study, the patients were treated for least six months in the CHF clinic with

  • maximally tolerated doses of angiotensin-converting enzyme inhibitors, the beta-blockers bisoprolol or carvedilol, aldospirone, long-acting nitrates, digoxin and oral and intravenous (IV) furosemide.

In some patients these agents could not be given because of contraindications and in others they had to be stopped because of side effects. Despite this maximal treatment

  • the patients still had severe CHF  (NYHA classIII), with  fatigue and/or shortness of breath  on even mild exertion or at rest.  All had levels of
  • Hb in the range of 10 to 11.5 g%  on at least three consecutive visits over a three-week period.
  • All had a LVEF of 40%.

Secondary causes of anemia including hypothyroidism, and folic acid and vitamin B12 deficiency were ruled out and

  • there was no clinical evidence of gastrointestinal bleeding.

The patients were randomized consecutively into two groups:

  • Group A, 16 patients, was treated with sc EPO and IV Fe to achieve a target Hb of at least 12.5 g%.
  • Group B, 16 patients, did not receive the EPO and IV Fe.

Treatment protocol for correction of anemia.

All patients in Group A received the combination of sc EPO and IV Fe. The EPO was given once a week at a starting dose of 4,000 international units (IU) per week  and
the dose was increased  to two  or  three  times a week or decreased to once every few weeks as  necessary

  • to achieve and maintain a target Hb of 12.5 g%.

The IV Fe (Venofer-Vifor International, Switzerland), a ferric sucrose product, was given in a dose of 200 mg IV in 150 ml saline over 60 min every two weeks

  • until the serum ferritin reached 400 g/l or
  • the %Fe saturation (%Fe Sat is serum iron/total iron binding capacity 100) reached 40% or
  • the Hb reached 12.5g%. 

The IV Fe was then given at longer intervals as needed to maintain these levels.


Visits to the clinic were at two- to three week intervals depending on the patient’s status. This was the same frequency of visits to the CHF clinic as before then,

  • potassium and ferritin and %Fe Sat were performed on every visit.
  • blood pressure was measured by an electronic device on every visit.
  • LVEF was measured initially and at four- to six-month intervals by MUGA radioisotope ventriculography.

This technique measures

  • the amount of blood in the ventricle at the end of systole and at the end of diastole, thus giving
  • a very accurate assessment of the ejection fraction.

It has been shown to be an accurate and reproducible method of measuring the ejection fraction (14).  Hospital records were reviewed at the end of the intervention period to compare

  • the number of days hospitalized during the study with 
  • the number of days hospitalized during a similar period 
    • when the patients were treated in the CHF clinic before the initial randomization and entry into the study.

Clinic records were reviewed to evaluate the types and doses of CHF medications used before and during the study. The mean follow-up for patients was 8.2 +/-  2.7 months (range 5 to 12 months).  The study was done with the approval of the local ethics committee.Statistical analysis.

An analysis of variance with repeated measures (over time) was performed to compare the two study groups (control vs. treatment) and

  • to assess time trend and the interactions between the two factors.
  • A separate analysis was carried out for each of the outcome parameters.
  • The Mann-Whitney test was used to compare the change in NYHA class between two groups.

All the statistical analysis was performed by SPSS (version 10).


The mean age in Group A (EPO and Fe) was 75.3 +/-  14.6 years and in group B was 72.2 +/-  9.9 years. There were 11 and 12 men in Groups A and B, respectively.
Before the study the two groups were similar in
  1. cardiac function,
  2. comorbidities,
  3. laboratory investigations and
  4. medications
  • (Tables 1, 2 and 3), except for IV furosemide (Table 3),
which was higher in the treatment group. The mean NYHA class of Group A before the study was 3.8  0.4 and was 3.5  0.5 in Group B. The contributing factors to CHF in Groups A and B, respectively, are seen in Table 1 and were similar.
Table 1. Medical Conditions and Contributing Factors to Congestive Heart Failure in the 16 Patients Treated for the Anemia and in the 16 Controls

Table 1 medical conditions heart failure anemia

Table 2. The Effect of Correction of Anemia by Intravenous Iron and Erythropoietin Therapy on Various Parameters in 16 Patients in the Treatment (A) and 16 in the Control (B) Group

Table 2 medications to treat heart failure anemia

p values are given for analysis of variance with repeated measures and for independent t tests for comparison of baseline levels between the two groups.
BP  blood pressure; Fe Sat  iron saturation; Hb  hemoglobin; IV  intravenous; NS  not stated; Std Dev.  standard deviation.

The main contributing factors to CHF were considered to be

  • ischemic heart disease (IHD) in 11 and 10 patients respectively,
  • hypertension in two and two patients,
  • valvular heart disease in twoand two patients, and
  • idiopathic cardiomyopathy in one and two patients, respectively.

A significant change after treatment was observed in the two groups in the following parameters:

  • IV furosemide,
  • days in hospital,
  • Hb,
  • ejection fraction,
  • serum creatinine and
  • serum ferritin.
In addition, the interaction between the study group and time trend was significant in all measurements except for blood pressure and %Fe Sat. This interaction indicates that
  • the change over time was significantly different in the two groups.
Table 3. The Effect of Correction of Anemia by Intravenous Iron and Erythropoietin Therapy on Various Parameters in 16 Patients in the Treatment (A) and 16 in the Control (B) Group

Table 3  CHF aneia EPO

p values are given for analysis of variance with repeated measures and for independent t tests for comparison of baseline levels between the two groups.
BP  blood pressure; Fe Sat  iron saturation; Hb  hemoglobin; IV  intravenous; NS  not stated; Std Dev.  standard deviation.

We find in the comparisons of Tables 2 and 3:

  1. before treatment the level of oral furosemide was higher in the control group (136.2 mg/day) compared with the treatment group (132.2 mg/day).
  2. after treatment, while the dose of oral furosemide of the treated patients was reduced  to 64.4 mg/day
  • the dose of the nontreated patients was increased to 175 mg/day.

The same results of improvement in the treated group and deterioration in the control group are expressed in the following parameters:

  1. IV furosemide, days in hospital,
  2. Hb,
  3. ejection fraction and
  4. serum creatinine.

The NYHA class was

  • 3.8 +/- 0.4 before treatment and 2.2 +/- 0.7 after treatment in Group A  (delta mean = – 1.6) and
  • 3.5 +/-  0.7 before treatment and 3.9 +/- 0.3 after treatment in Group B. (delta mean = 0.4)

The improvement in NYHA class was significantly higher (p < 0.0001) in the treatment group compared with the control group (Table 4).

Table 4. Changes from Baseline to Final New York Heart Association (NYHA) Class
Initial minus final

Table 4  changes from NYHA baseline  CHF anemia

The improvement in NYHA class was statistically higher (p <  0.0001) in the treatment group compared with control.

There were no deaths in Group A and four deaths in Group B.

Case 1: A 71-year-old woman with severe mitral insufficiency and severe pulmonary hypertension  (a pulmonary artery [PA] pressure of 75 mm Hg) had persistent NYHA 4 CHF  and died during mitral valve surgery  seven months after onset of the study. She was hospitalized for 21 days  in the seven months before randomization and for 28 days  during the seven months after randomization.

Case 2:

A 62-year-old man with a longstanding history of hypertension complicated by IHD, coronary artery bypass graft (CABG) and atrial fibrillation had persistent NYHA 4 CHF  and a PA pressure of 35 mm Hg,  and died from pneumonia and septic shock eight months after onset of the study. He was hospitalized for seven days in the eight months before randomization and for 21 days during the eight months  after

Case 3:
A 74-year old man with IHD, CABG, chronic obstructive pulmonary disease, a history of heavy smoking and diabetes had persistent NYHA 4 CHF and a PA pressure of  28 mm Hg, and died of pulmonary  edema and cardiogenic shock nine months after onset of the study. He was hospitalized for 14 days in the nine months before  randomization and for 41 days during the nine months after randomization.

Case 4:
A 74-year-old man with a history of IHD, CABG, diabetes, dyslipidemia, hypertension and atrial fibrillation, had persistent NYHA 4 CHF and a PA pressure of 30 mm Hg,  and died of pneumonia and septic shock   six months after onset of the study. He was hospitalized for five days in the six months before randomization and for 16 days during the nine months after randomization.


 Main findings.

The main finding of the present study is that the correction of

  • even mild anemia in patients with symptoms of very severe CHF despite being on maximally tolerated drug therapy
  • resulted in a significant improvement in their cardiac function and NYHA functional class.

There  was also a large

  • reduction in the number of days of  hospitalization compared with a similar period before the  intervention.
  • all this was achieved despite a marked reduction in the dose of oral and IV furosemide.

In the group in whom the anemia was not treated, four  patients died during the study. In all four cases

  • the CHF was unremitting and contributed to the deaths. 

In addition,  for the group as a whole, 

  • the LVEF, the NYHA class and  the renal function worsened.

There was also need for

  • increased oral and IV furosemide as well as increased  hospitalization.

Study limitations.

The major limitations of this study are

  1. the smallness of the sample size and
  2. the fact that randomization and treatment were not done in a blinded fashion.

Nevertheless, the two groups were almost identical in

  1. cardiac, renal and anemia status;
  2. in the types and doses of medication they were taking before and during the intervention and
  3. in the number of hospitalization days before the intervention.

Although the results of this study, like those of  our previous uncontrolled study (5), suggest that

  • anemia may play an important role in the mortality and morbidity of  CHF,
  • a far larger double-blinded controlled study should be carried out to verify our findings.

Anemia as a risk factor for hospitalization and death in CHF.

Our results are consistent with a recent analysis of 91,316 patients hospitalized with CHF (15). Anemia was found to be a stronger predictor of

  • the need for early rehospitalization than  was hypertension,  IHD or the presence of a previous CABG.  

A recent analysis of the Studies Of Left Ventricular Dysfunction (SOLVD) (16) showed that

  • the level of hematocrit (Hct) was an independent risk factor for mortality.

During a mean follow-up of 33 months the mortality was

  • 22%, 27% and 34% for those with a Hct of 40, 35 to 40 and 35 respectively.

The striking response of our patients to

  • correction of mild anemia suggests that the failing heart may be very susceptible to anemia.

It has, in fact, been found in both animal (17) and human studies (17–19) that

  • the damaged heart is more vulnerable to anemia and/or ischemia than is the normal heart.

These stimuli may result in a more marked reduction in cardiac function than occurs in the normal heart and may explain why,  in our study,

  • the patients were so resistant to high doses of CHF medications and
  • responded so well when the anemia was treated.

Our findings about the importance of anemia in CHF are not surprising when one considers that, in dialysis patients,

  • anemia has been shown to be associated with an increased prevalence and incidence of CHF (20) and that
  • correction of anemia in these patients is associated with improved
    • cardiac function (21,22),
    • less mortality (23,24) and
    • fewer hospitalizations (23,25).

Effect of improvement of CHF on CRF.

Congestive heart failure can cause progressive renal failure (26,27). Renal ischemia is found very early on

  • in patients with cardiac dysfunction (28,29), and
  • chronic ischemia may cause progression of renal failure (30). Indeed, the development of
  • CHF in patients with essential hypertension has been found to be one of the most powerful predictors of
  • the eventual development of end-stage renal disease (31).

Patients who develop CHF after a myocardial infarction experience a

  • fall in the glomerular filtration rate (GFR) of about 1 ml/min/month if the CHF is not treated (32).

In another recent analysis of the SOLVD study, treating the CHF with

  • both angiotensin-converting enzyme inhibitors and beta-blockers resulted in better preservation of the renal function than did
  • angiotensin-converting enzyme inhibitors alone (26),
suggesting that the more aggressive the treatment of the CHF, the better the renal function is preserved. In the present study, as in our previous one (5), we found that the deterioration of GFR was prevented with
  • successful treatment of the CHF, including correction of the anemia, whereas
  • the renal function worsened when the CHF remained severe

All these findings suggest that early detection and treatment of CHF and systolic and/or diastolic dysfunction from whatever cause could prevent

  • the deterioration not only of the cardiac function
  • but of the renal function as well.

This finding has very broad implications in the prevention of CRFbecause most patients with advanced CRF have

  • either clinical evidence of CHF or at least some degree of systolic dysfunction (33).

Systolic and/or diastolic dysfunction can occur early on in many  conditions, such as

  • essential hypertension (34),
  • renal disease of any cause (35,36) or
  • IHD, especially after a myocardial infarction (37).

The early detection and adequate treatment of this cardiac dysfunction, including correction of the anemia, could prevent this cardiorenal insufficiency. To detect cardiac dysfunction early on, one would need  at least an echocardiogram and MUGA radio-nucleotide ventriculography. These tests should probably be done not only in patients with signs and symptoms of CHF,   but in all patients where CHF or systolic  and/or diastolic dysfunction are suspected, such as those with a history of heart disease or suggestive changes of ischemia or hypertrophy on the electrocardiogram, or in patients with hypertension or renal disease.

Other positive cardiovascular effects of EPO treatment.

Another possible explanation for the improved cardiac function in this study may be the direct effect that EPO itself has on improving cardiac muscle function (38,39) and myocardial cell growth (39,40) unrelated to its  effect of the anemia. In fact EPO may be  crucial in the formation of the heart muscle in utero (40). It may also improve  endothelial function (41).  Erythropoietin may be superior to blood transfusions  not only  because adverse reactions to EPO are infrequent, but also because

  • EPO causes the production and release of young cells from the bone marrow into the blood.

These cells have an oxygen dissociation curve that is shifted to the right of the normal curve, causing the release of

  • much greater amounts of oxygen into the tissues than occurs normally (42).

On the other hand, transfused blood consists of older red cells with an oxygen dissociation curve that is

  • shifted to the left, causing the release of much less oxygen into the tissues than occurs normally (42).

The combination of IV Fe and EPO. The use of IV Fe along with EPO has been found to have an additive effect, 

  • increasing the Hb even more than would occur with EPO alone while at the same time
  • allowing the dose of EPO to be reduced (10 –13).
  • The lower dose of EPO will be cost-saving and also reduce the chances of hypertension developing (43).
 We used iron sucrose (Venofer) as our IV Fe medication because, in our experience, it is extremely well tolerated (10,11) and  
  • has not been  associated  with any serious side effects in more than 1,200 patients over six years.

Implications of treatment of anemia in CHF. The correction of anemia is not a substitute for the well-documented effective therapy of CHF but seems to be  an important, if not vital,  addition to the therapy. It is surprising, therefore,  that judging from  the  literature  on CHF,

  • such an obvious treatment for improving CHF is so rarely considered.

We believe that correction of the anemia will have an important role to play in

  • the amelioration of cardiorenal insufficiency, and that this improvement will have
  • significant economic  implications as well.


The authors thank Rina Issaky, Miriam Epstein, Hava Ehrenfeld and Hava Rapaport for their secretarial assistance.
Reprint requests and correspondence: Dr. D. S. Silverberg, Department of Nephrology, Tel Aviv Medical Center, Weizman 6, Tel Aviv, 64239, Israel.


The use of subcutaneous erythropoietin and intravenous iron for the treatment of the anemia of severe, resistant congestive heart failure improves cardiac and renal function and functional cardiac class, and markedly reduces hospitalizations

Donald S Silverberg, MDa; Dov Wexler, MDa; Miriam Blum, MDa; Gad Keren, MDa; David Sheps, MDa; Eyal Leibovitch, MDa; David Brosh, MDa; Shlomo Laniado, MDa; Doron Schwartz, MDa; Tatyana Yachnin, MDa; Itzhak Shapira, MDa; Dov Gavish, MDa; Ron Baruch, MDa; Bella Koifman, MDa; Carl Kaplan, MDa; Shoshana Steinbruch, RNa; Adrian Iaina, MDa

J Am Coll Cardiol. 2000;35(7):1737-1744. doi:10.1016/S0735-1097(00)00613-6


This study evaluated the prevalence and severity of anemia in patients with congestive heart failure (CHF) and

  • the effect of its correction on cardiac and renal function and hospitalization.


The prevalence and significance of mild anemia in patients with CHF is uncertain, and the role of erythropoietin with intravenous iron supplementation in treating this anemia is unknown.


In a retrospective study, the records of the 142 patients in our CHF clinic were reviewed to find
  • the prevalence and severity of anemia (hemoglobin [Hb]12 g).
In an intervention study, 26 of these patients, despite maximally tolerated therapy of CHF for at least six months, still had had severe CHF and were also anemic. They were treated with
  • subcutaneous erythropoietin and intravenous iron sufficient to increase the Hb to 12 g%.
The doses of the CHF medications, except for diuretics, were not changed during the intervention period.


The prevalence of anemia in the 142 patients increased with the severity of CHF,
  • reaching 79.1% in those with New York Heart Association class IV.
In the intervention study, the anemia of the 26 patients was treated for a mean of 7.2 5.5 months.
  • The mean Hb level and mean left ventricular ejection fraction increased significantly.
  • The mean number of hospitalizations fell by 91.9% compared with a similar period before the study.
  • The New York Heart Association class fell significantly,
  • as did the doses of oral and intravenous furosemide.
  • The rate of fall of the glomerular filtration rate slowed with the treatment.


Anemia is very common in CHF and its successful treatment is associated with a significant improvement in
  • cardiac function,
  • functional class,
  • renal function and
  • in a marked fall in the need for diuretics and hospitalization.
Abbreviations and Acronyms
ACE Angiotensin-converting enzyme
CHF congestive heart failure
COPD chronic obstructive pulmonary disease
CRF chronic renal failure
CVA cerebrovascular accident
EPO erythropoietin
Fe iron
g% grams Hb /100 ml blood
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IV intravenous
LVEF left ventricular ejection fraction
LVH left ventriculr hypertrophy
NYHA New York Heart Association
%Fe Sat percent iron saturation
sc subcutaneous
TNF tumor becrosis factor
ACE Angiotensin-converting enzyme
CHF congestive heart failure
COPD chronic obstructive pulmonary disease
CRF chronic renal failure
CVA cerebrovascular accident
EPO erythropoietin
Fe iron
g% grams Hb /100 ml blood
GFR glomerular filtration rate
Hb hemoglobin
Hct hematocrit
IV intravenous
LVEF left ventricular ejection fraction
LVH left ventriculr hypertrophy
NYHA New York Heart Association
%Fe Sat percent iron saturation
sc subcutaneous
TNF tumor becrosis factor

The mean hemoglobin (Hb) in patients with congestive heart failure (CHF) is about 12 g Hb per 100 ml blood (g%) (1–3), which is considered to be the lower limit of normal in adult men and postmenopausal women (4). Thus, many patients with CHF are anemic, and

  • this anemia has been shown to worsen as the severity of the CHF progresses (5,6).
Severe anemia of any cause can produce CHF, and treatment of the anemia can improve it (7). In patients with chronic renal failure (CRF) who are anemic,
  • treatment of the anemia with erythropoietin (EPO) has improved many of the abnormalities seen in CHF,
  • reducing left ventricular hypertrophy (LVH) (8 –10),
  • preventing left ventricular dilation (11) and,
    • in those with reduced cardiac function, increasing the left ventricular ejection fraction (LVEF)(8 –10),
    • the stroke volume (12) and
    • the cardiac output (12).
In view of the high prevalence of anemia in CHF, it is surprising that we could find no studies in which EPO was used in the treatment of the anemia of CHF, and the use of EPO is not included in U.S. Public Health Service guide-lines of treatment of the anemia of CHF (13). In fact, anemia has been considered
  • only a rare contributing factor to the worsening of CHF, estimated as contributing to
  • no more than 0% to 1.5% of all cases (14 –16).
Perhaps for this reason, recent guidelines for the prevention and treatment of CHF do not mention treatment of anemia at all (17). If successful treatment of anemia could improve cardiac function and patient function in CHF,
  • this would have profound implications, because,
  • despite all the advances made in the treatment of CHF, it is still a major and steadily increasing cause of hospitalizations, morbidity and mortality (18 –20).
The purpose of this study is to examine
  • the prevalence of anemia (Hb 12 g%) in patients with different levels of severity of CHF and
  • to assess the effect of correction of this anemia in severe CHF patients
  • resistant to maximally tolerated doses of CHF medication.
A combination of subcutaneous (SC) EPO and intravenous (IV) iron (Fe) was used. We have found this combination to be additive in improving the anemia of CRF (21,22).



The medical records of the 142 CHF patients being treated in our special outpatient clinic devoted to CHF were reviewed to determine the prevalence and severity of anemia and CRF in these patients. These patients were referred to the clinic either from general practice or from the various wards in the hospital.

Intervention study.

Despite at least six months of treatment in the CHF clinic,
  • 26 of the above patients had persistent, severe CHF (New York Heart Association [NYHA] class III),
  • had a Hb level of 12 g% and were on
    • angiotensin-converting enzyme [ACE] inhibitors, the 
    • alpha-beta-blocker carvedilol,
    • long-acting nitrates,
    • digoxin, 
    • aldactone and
    • oral and IV furosemide.

These 26 patients participated in an intervention study. The mean age was 71.76  8.12 years. There were 21 men and 5 women. They  all had a

  • LVEF below 35%,
  • persistent fatigue and
  • shortness

    of breath on mild to moderate exertion and often at rest, and had

  • required hospitalizations at least once during their follow-up in the CHF clinic for pulmonary edema.
In 18 of the 26 patients, the CHF was associated with ischemic heart disease either
  • alone in four patients, or
  • with hypertension in six,
  • diabetes in four,
  • the two together in three, or with
  • valvular heart disease in one.
Of the remaining eight patients,
  • four had valvular heart disease alone and
  • four had essential hypertension alone.
Secondary causes of anemia including
  • gastrointestinal blood loss (as assessed by history and by three negative stool occult blood examinations),
  • folic acid and vitamin B12 deficiency and
  • hypothyroidism were ruled out.
Routine gastrointestinal endoscopy was not carried out. The study passed an ethics committee.
Table 1. Initial Characteristics of the 142 Patients With CHF Seen in the CHF Clinic
Age, yearsMale/female,  %Associated conditionsDiabetesHypertensionDyslipidemiaSmoking

Main cardiac diagnosis
Ischemic heart disease

Dilated CMP

Valvular heart disease


LVEF,  %

Left atrial area (n 15 cm2)

Pulmonary artery pressure  (15 mm Hg)

Previous hospitalizations/year

Serum Na, mEq/liter

Serum creatinine, mg%

Hemoglobin, g%

70.1 +/- 11.1










32.5 +/- 12.2

31.3  +/- 10.3

43.1  +/-14.9

3.2  +/- 1.5

139.8  +/- 4.0

1.6   +/-  1.1

11.9   +/- 1.5

CMP  cardiomyopathy; LVEF  left ventricular ejection fraction; NYHA  New York Heart Association class.

Correction of the anemia.

All patients received the combination of SC EPO and IV Fe. The EPO was given once a week at a starting dose of 2,000 IU per week subcutaneously, and the dose was increased or decreased as necessary to achieve and maintain a target Hb of 12 g%. The IV Fe (Venofer-Vifor International, St. Gallen, Switzerland), a ferric sucrose product, was given in a dose of 200 mg IV in 150 ml saline over 60 min every week until the serum ferritin reached 400  g/liter or the percent Fe saturation (%Fe Sat: serum iron/total iron binding capacity   100) reached 40% or until the Hb reached 12 g%. The IV Fe was then given at longer intervals as needed to maintain these levels.

Medication dose.

Except for oral and IV furosemide therapy, the doses of all the other CHF medications, which were used in the maximum tolerated doses before the intervention, were kept unchanged during the intervention period.

Duration of the study.

The study lasted for a mean of 7.2  5.5 months (range four to 15 months).


Visits were at weekly intervals initially and then at two- to three-week intervals depending on the patient’s status. This was the same frequency of visits to the CHF clinic as before the intervention study.
  • A complete blood count, serum creatinine, serum ferritin and % Fe Sat were performed on every visit.
  •  An electronic device measured the blood pressure on every visit.
  • The LVEF was measured by a multiple gated ventricular angiography heart scan initially and at four- to six-month intervals.
Hospital records were reviewed to compare the number of hospitalizations during the time the patients were treated for the anemia with the number of hospitalizations
  • during a similar period of time that they were treated in the CHF clinic 
    before the anemia was treated.
Clinic records were reviewed to evaluate the types and doses of CHF medications used 
before and during the study.

Period of time that they were treated in the CHF clinic before the anemia was treated.

Clinic records were reviewed to evaluate the types and doses of CHF medications used before and during the study.  The glomerular filtration rate (GFR) was calculated from the serum creatinine by the formula: 1/serum creatinine in mg% x 100 GFR in ml/min. The rate of change of the GFR before and during the intervention period was calculated by comparing the change in GFR per month in the year before the intervention with that during the intervention.

Statistical analysis.

Mean standard deviation was calculated. One-way analysis of variance (ANOVA) was performed to compare parameter levels between the four NYHA groups. Hochberg’s method of multiple comparisons (23) was used for pair-wise comparison between two groups. A p value of less than 0.05 was considered statistically significant. In the intervention study, the significance of the difference between the initial values and those at the end of the study for the individual parameters in the 26 treated patients was assessed by paired student’s t test; p < 0.05 was considered statistically significant. All the statistical analysis was performed by the SPSS program (Version 9, Chicago, Illinois).


CHF: the whole study group.

The clinical, biochemical and hematological characteristics of the 142 patients seen in the clinic are shown in Tables 1 and 2.

  • Sixty-seven patients (47%) had severe CHF as judged by a NYHA class of IV (Table 2).
  • Seventy- nine of the 142 patients (55.6%) were anemic (Hb  12 g%).

The mean Hb level fell progressively from 13.73 +/- 0.83 g% in class I NYHA to 10.90 +/- 1.70 g% in class IV NYHA (p  0.01). The percentage of patients with Hb  12 g% increased from 9.1% in class I to 79.1% in class IV.
Fifty eight patients (40.8%) had CRF as defined as a serum creatinine  1.5 mg%. The mean serum creatinine increased from 1.18 +/_  0.38 mg% in class I NYHA, to 2.0 +/-    1.89 mg% in class IV NYHA, p  0.001. The percentage of patients with an elevated serum creatinine ( 1.5 mg%)      increased from 18.2% in class I to 58.2% in class IV.

The mean ejection fraction fell from 37.67 +/-  15.74% in class I to 27.72 +/-  9.68% (p  0.005) in class IV.

Table 2. LVEF and Biochemical and Hematological Parameters by NYHA Class in 142 Patients With CHF 
NYHA Class I II III IV  Significantly Different Pairs*

 *p  0.05 at least between the two groups by pair-wise comparison between groups.

†p  0.05 at least between the groups by ANOVA.

No. of patients





(total 142) (%)

    (7.7)    (18.3)    (26.8)    (47.2)

Hb, g%†

13.73 (0.83)

13.38 (1.26)

11.95 (1.48)

10.90 (1.70) 

1–3, 1–4, 2–3, 2–4

Serum creatinine,





1–2, 1–3, 1–4


    (0.38)     (0.29)      (0.38)     (1.89)

LVEF, %†

37.67 (15.74)

32.88 (12.41)

32.02 (10.99)

27.72 (9.68)

1–4, 2–4

Hb 12 g%,  (%)


5 (19.2) 

20 (52.6) 

53 (79.1)

Serum creatinine

    2      5     12     39

1.5 mg%,  (%) 





The intervention study: medications.

The percentage of patients receiving each CHF medication before and after the intervention period and the reasons for not receiving  them are seen in Table 3.

Table 3. Number (%) of the 26 Patients Taking Each Type of Medication Before and During the Intervention Period and the Reason Why the Medication Was Not Used

Medication    No. of Patients  (%)         Reason for Not Receiving the Medications (No. of Patients)
BP  blood pressure; CRF  chronic renal failure; IV  intravenous.

The main reason for not receiving:

  • 1) ACE inhibitors was the presence of reduced renal function;
  • 2) carvedilol was the presence of chronic obstructive pulmonary disease (COPD);
  • 3) nitrates was low blood pressure and aortic stenosis and
  • 4) aldactone was hyperkalemia.
Table 4. Mean Dose of Each Medication Initially and at the End of the Intervention Period in the 26 Patients

                                       No. of Patients                                 Initial Dose ^                 Final Dose^
Carvedilol (mg/day)                      20                                                        26.9 15.5                                   28.8 14.5
Captopril (mg/day)                          7                                                        69.6 40.0                                 70.7 40.4
Enalapril (mg/day)                        13                                                        25.7 12.5                                   26.9 12.6
Digoxin (mg/day)                          25                                                       0.10 0.07                                    0.10 0.07
Aldactone (mg/day)                       19                                                        61.2 49.2                                   59.9 47.1
Long-acting nitrates                      23                                                        53.2 13.2                                   54.1 14.4
Oral furosemide (mg/day)           26                                                      200.9 120.4                                78.3 41.3*
IV furosemide (mg/month)         26                                                      164.7 178.9                                  19.8 47.0*
*p  0.05 at least vs. before by paired Student’s t test.
^  +/-

The mean doses of the medications are shown in Table 4. 

The mean dose of oral furosemide was 200.9 +/-  120.4 mg/day before and 78.3 +/-  41.3 mg/day after the intervention (p   0.05). The dose of IV furosemide was 164.7 +/-  19.8,  178.9 mg/month before and  7.0 mg/month after the intervention (p  0.05).  

The doses of the other CHF medications were almost identical in the two periods.

Clinical results.

There were three deaths during the intervention period. An 83-year-old man died after eight months of respiratory failure after many years of COPD, a 65-year-old man at eight months of a CVA with subsequent pneumonia and septic shock and a 70-year-old man at four months of septicemia related to an empyema that developed after aortic valve replacement.
Three patients, a 76-year-old man, an 85-year-old woman and a 72-year-old man, required chronic hemodialysis after six, 16 and 18 months, respectively. The serum creatinines of these three patients at onset of the anemia treatment were 4.2, 3.5 and 3.6 mg%, respectively. All three had improvement in their NYHA status but
  • their uremia worsened as the renal function deteriorated, demanding the initiation of dialysis.

In no cases, however, was pulmonary congestion an indication for starting dialysis.

Functional results (Table 5).

During the treatment period, the NYHA class fell from a mean of 3.66 +/- 0.47 to 2.66 +/- 0.70 (p 0.05), and
  • 24 had some improvement in their functional class.
The mean LVEF increased from 27.7 +/- 4.8 to 35.4  +/- 7.6% (p 0.001), an increase of 27.8%.
Compared with a similar period of time before the onset of the anemia treatment, the mean number of hospitalizations fell from 2.72 +/-  1.21 to 0.22 +/-  0.65 per patient (p   0.05)a decrease of 91.9%.
No significant changes were found in the mean systolic/diastolic blood pressure.

Hematological results (Table 5).

  • The mean hematocrit (Hct) increased from 30.14 +/- 3.12%) to 35.9  +/- 4.22% (p < 0.001).
  • The mean Hb increased from 10.16 +/- 0.95 g%) to 12.10 +/-  1.21 g% (p <  0.001).
  • The mean serum ferritin increased from 177.07 +/-  113.80  g/liter to 346.73 +/- 207.40 g/liter (p  0.005).
  • The mean serum Fe increased from 60.4 +/- 19.0 g% to 74 +/- .80  20.7 g% (p  0.005). 
  • The mean %Fe Sat increased from 20.05   6.04% to 26.14 =/- 5.23% (p  0.005).
  • The mean dose of EPO used throughout the treatment period was 5,227  +/- 455 IU per week, and
  • the mean dose of IV Fe used was 185.1 +/- 57.1 mg per month.
In four of the patients, the target Hb of 12 g% was maintained despite stopping the EPO for at least four months.

Renal results (Table 5).

The changes in serum creatinine were not significant. The estimated creatinine clearance fell at a rate of 0.95 + 1.31 ml/min/month before the onset of treatment of the anemia and increased at a rate of 0.85 + 2.77 ml/min/month during the treatment period.
Table 5. The Hematological and Clinical Data of the 26 CHF Patients at Onset and at the End of the Intervention Period

————–                                         Initial ^                                    Final^
Hematocrit, vol%                              30.14 3.12                            35.90 4.22*
Hemoglobin, g%                                10.16 0.95                              2.10 1.21*
Serum ferritin, g/liter                    177.07 113.80                       346.73  207.40*
Serum iron, g%                                  60.4 19.0                               74.8  20.7*
% iron saturation                              20.5 6.04                               26.14 5.23*
Serum creatinine, mg%                   2.59 0.77                                 2.73 1.55
LVEF, %                                              27.7 4.8                                   35.4  7.6*
No. hospitalizations/patient          2.72 1.21                                 0.22   0.65*
Systolic BP, mm Hg                       127.1 19.4                                128.9  26.4
Diastolic BP, mm Hg                       73.9 9.9                                   74.0   12.7
NYHA (0–4)                                     3.66 0.47                                2.66 0.70*
*p  0.05 at least vs before by paired Student’s t test.     ^ +/-
BP  blood pressure; LVEF  left ventricular ejection fraction; NYHA  New York Heart Association.


The main findings in the present study are that anemia is common in CHF patients and becomes progressively more prevalent and severe as CHF progresses. In addition, for patients with resistant CHF, the treatment of the associated anemia causes a marked improvement in their

  1. functional status,
  2. ejection fraction and
  3. GFR.
        • All these changes were associated with a markedly
            • reduced need for hospitalization and
            • for oral and IV furosemide.

The effect of anemia on the ischemic myocardium.

We used the IV Fe together with EPO to avoid the Fe deficiency caused by the use of EPO alone (38,39).
The Fe deficiency will cause

  • a resistance to EPO therapy and
  • increase the need for higher and higher doses to maintain the Hb level (39,40).

These high doses will not only be expensive but may increase the blood pressure excessively (41). The IV Fe reduces the dose of EPO needed to correct the anemia, because

  • the combination of SC EPO and IV Fe has been shown to have an additive effect on correction of the anemia of CRF (21,22,39,42).

Oral Fe, however, has no such additive effect (39,42). The relatively low dose of EPO needed to control the anemia in our study may explain why

  • the blood pressure did not increase significantly in any patient.

We used Venofer, an Fe sucrose product, as our IV Fe supplement because, in our experience (21,22,43), it has very few side effects and, indeed, no side effects with its use were encountered in this study.

The Effect of Anemia Correction on Renal Function.

Congestive heart failure is often associated with some degree of CRF (1–3,27–29), and

  • this is most likely due to renal vasoconstriction and ischemia (28,29).

When the anemia is treated and the cardiac function improves,

  • an increase in renal blood flow and glomerular filtration is seen (7,28).

In the present study, renal function decreased as the CHF functional class worsened (Table 2). The rate of deterioration of renal function was slower during the intervention period. Treatment of anemia in CRF has been associated with

  • a rate of progression of the CRF that is either unchanged (30) or is slowed (31–33).

It is possible, therefore, that adequate treatment of the anemia in CHF may, in the long term, help slow down the progression of CRF.

Possible Adverse Effects of Correction of the Anemia.

There has been concern, in view of the recent Amgen study (34), that correction of the Hct to a mean 42% in hemodialysis patients might increase cardiovascular events in those receiving EPO compared with those maintained at a Hct of 30%. Although there is much uncertainty about how to interpret this study (35), there is a substantial body of evidence that shows

  • correction of the anemia up to a Hb of 12 g% (Hct 36%) in CRF on dialysis is safe and desirable (35–38), and
  • results in a reduction in mortality, morbidity and in the number and length of hospitalizations.

The same likely holds true for the anemia of CHF with or without associated CRF. Certainly, our patients’ symptoms were strikingly improved, as was their cardiac function (LVEF) and need for hospitalization and diuretics. It remains to be established

  • if correction of the anemia up to a normal Hb level of 14 g% might be necessary in order to further improve the patient’s clinical state.

The Role of Fe Deficiency and its Treatment in the Anemia of CHF.

We used the IV Fe together with EPO to avoid the Fe deficiency caused by the use of EPO alone (38,39). The Fe deficiency will cause

  • a resistance to EPO therapy and increase the need for higher and higher doses to maintain the Hb level (39,40).

These high doses will not only be expensive but may

  • increase the blood pressure excessively (41).

The IV Fe reduces the dose of EPO needed to correct the anemia, because the combination of SC EPO and IV Fe has been shown to have an additive effect on correction of the anemia of CRF (21,22,39,42). Oral Fe,  however, has no such additive effect (39,42). The relatively low dose of EPO needed to control the anemia in our study may explain

  • why the blood pressure did not increase significantly in any patient.

We used Venofer, an Fe sucrose product, as our IV Fe supplement because, in our experience (21,22,43), it has very few side effects and, indeed, no side effects with its use were encountered in this study.

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Notable Contributions to Regenerative Cardiology by Richard T. Lee – Part I


Author and Curator: Larry H Bernstein, MD, FCAP


Article Commissioner: Aviva Lev-Ari, PhD, RD



This presentation is a two part discussion of selected articles of a large body of research from Dr. Richard T. Lee, at Harvard Medical School’s Lee Laboratory and Brigham & Womens Hospital.  This work is innovative in the field of stem cell research and myocardial regeneration.  It devolves the complex cellular processes that are involved in the management of a cell transforming from a progenitor to a functional cardiomyocyte.  The cell engineering involves investigating interactions between a cell placed into the layer derived from the interstitial layer between viable cardiomyocytes.  This is only possible from a through actionable knowledge of the mechanism involved in the transformation process, which has occupied the Lee Laboratory for many years.  Part II will cover the cellular mechanisms underlying the conceptual approach to cardiac myocyte regeneration.

The Lee Laboratory uses emerging biotechnologies to discover and design new approaches to cardiovascular diseases. A central theme of the laboratory is that merging bioengineering and molecular biology approaches can yield novel approaches. Thus, the Lee Laboratory works at this interface using a broad variety of techniques in genomics, imaging, nanotechnology, physiology, cell biology, and molecular biology. The approach is to understand problems and design solutions in the laboratory and then demonstrate the effectiveness of these solutions in vivo. Ongoing projects in the laboratory include studies of cardiac regeneration, diabetic vascular disease, wound healing, heart failure, and cardiac hypertrophy.

Richard T. Lee is Professor of Medicine at Harvard Medical School and lecturer in Biological Engineering at the Massachusetts Institute of Technology. He is a 1979 graduate of Harvard College in Biochemical Sciences and received his M.D. from Cornell University Medical College in 1983.  He went on to complete his residency in 1986 and cardiology fellowship in 1989, both at Brigham and Women’s Hospital in Boston, and he obtained post-doctoral training at MIT in Bioengineering.

Dr. Lee is certified by the American Board of Internal Medicine in cardiovascular disease and is a Fellow of the American College of Cardiology. He is Leader of the Cardiovascular Program of the Harvard Stem Cell Institute.  He is a member of the Editorial Boards of the journals Circulation Research, Journal of Clinical Investigation, and Circulation, and has published over 180 peer-reviewed articles based on his research, which combines approaches in biotechnology and molecular biology to discover new avenues to manage and treat heart disease.

Regeneration of the heart

Matthew L. Steinhauser, Richard T. Lee
Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, and (2)             Harvard Stem Cell Institute, Cambridge, MA
EMBO Mol Med 2011; 3: 701–712

The death of cardiac myocytes diminishes the heart’s pump function and is a major cause of heart failure. With the exception of heart transplantation and implantation of mechanical ventricular assist devices, current therapies do not address the central problem of decreased pumping capacity owing to a depleted pool of cardiac myocytes. The field is evolving in two important directions. First, although endogenous mammalian cardiac regeneration clearly seems to decline rapidly after birth, it may still persist in adulthood. The careful elucidation of the cellular and molecular mechanisms of endogenous heart regeneration may therefore provide an opportunity for developing therapeutic interventions that amplify this process. Second, recent breakthroughs have enabled reprogramming of cells that were apparently terminally differentiated, either by dedifferentiation into pluripotent stem cells or by trans-differentiation into cardiac myocytes.
The longstanding paradigm held that the mammalian heart is a terminally differentiated organ, incapable of replenishing any myocyte attrition. During the past decade, however, studies revealed not only that mammalian cardiac myocytes retain some capacity for division (Beltrami et al, 2001), but also identified endogenous cardiac progenitor cells in the heart (Beltrami et al, 2003) or bone marrow (Orlic et al, 2001). These cells retain some potential for differentiation into the cellular components of the heart, including endothelial cells, smooth muscle cells and cardiac myocytes.
If progenitor cells residing in the adult are capable of producing new heart cells, the therapeutic delivery of such progenitors might facilitate the generation of de novo functional myocardium. In this context, cell-based therapies for the heart have been rapidly translated into the clinic to treat heart disease, but randomized clinical trials with bone marrow progenitors have shown at best modest improvements in ventricular function (Martin-Rendon et al, 2008). In short, the promise of complete cardiac regeneration has not yet been realized.  Therefore, it is worth revisiting both the foundations of cardiac regeneration and highlight recent advances that may portend future directions in the field.
We will first define the problem, that is elucidating the scope of endogenous mammalian regeneration and, by extension, the scale of the regenerative deficit. We will then summarize current regenerative approaches, including both cell-based therapies and pharmacoregenerative strategies. In this context, we will summarize the many challenges that stand in the way of cardiac regeneration, both endogenous repair processes and exogenous regenerative therapies.
The regenerative deficit of the mammalian heart is obvious when compared with organisms such as zebrafish and newts, which demonstrate a remarkable survival capacity after removal of up to 20% of the heart by transection of the ventricular apex. Pre-existing cardiac myocytes adjacent to the site appear to undergo a process of dedifferentiation, characterized by dissolution of sarcomeric structures. This is followed by incorporation of deoxyribonucleic acid (DNA) synthesis markers (e.g. nucleotide analogues) consistent with proliferation. Ultimately, newly generated cardiac myocytes are functionally integrated with the preexisting myocardium. The heart is left with little residual evidence of the injury, thus providing a natural example of complete myocardial regeneration.

Evidence for heart regeneration in mammals

During embryonic development and the early post-natal period, mice also demonstrate a remarkable regenerative capacity. Embryos heterozygous for a cardiac myocyte-specific null mutation in the x-linked holocytochrome c synthase (Hccs) gene demonstrate cardiac myocyte replacement during foetal development (Drenckhahn et al, 2008): when one of two X-chromosomes is randomly inactivated in each female somatic cell, approximately 50% of the cardiac myocytes are rendered Hccs-null and hence dysfunctional. Proliferative functional Hccs-expressing cardiac myocytes compensate for dysfunctional Hccs-null myocytes, such that, at birth, 90% of the heart is derived from myocytes containing one functional Hccs allele. However, after the first week in post-natal mice, injured myocardium is largely replaced by fibrosis and scarring.  Distinguishing whether the adult mammalian heart is incapable of cardiac myocyte replacement or whether it retains a low-level capacity for repair is therefore fundamentally important. This is the basis for an evolving view of a more plastic mammalian heart.
Arguments against the age old view of the terminally differentiated quiescent cardiac myocyte:

  1. evidence supporting cardiac myocyte plasticity relied on mathematical modelling of the myocyte population based on cytometric indices. (the measured average volume increase of cardiac myocytes was calculated to fall short of the increase predicted by the observed volumetric changes in the whole heart
  • changes in heart volume could not be explained by hypertrophy alone, and that cardiac myocyte hyperplasia contributed to changes in heart mass, but the conclusions relied on a number of assumptions about myocyte size and DNA content.
  • detecting cell cycle markers such as Ki67 or the incorporation of nucleotide analogues (e.g. iododeoxyuridine or 3H-thymidine) into newly synthesized DNA further support the notion that the mammalian heart may generate new myocytes
  • human cardiac myocytes can reenter the cell cycle, but the described rates of this phenomenon differ by more than one order of magnitude
  • experiments, made possible by nuclear arms testing in the middle of the 20th century, provide the most convincing evidence for post-natal human cardiac myocyte turnover.
  • the period of nuclear testing serves as a historical DNA labelling pulse, and the period after the test ban treaty serves as a chase.
  • the genomic DNA of cells generated during either the pulse or the chase reflect the earth’s atmospheric 14C concentration at that point in time, which allows investigators to date the age of cardiac myocytes by measuring the concentration of 14C in their nuclei
  1. Listed are a number of problems in detecting the generation of cardiac myocytes:
  • small errors may magnify projections of absolute yearly or lifetime myocyte turnover
  • mis-identification of cellular components by light microscopy
  • autofluorescence of myocardium, which complicates any method that relies on the detection of a fluorescent signal
  • confounders could also affect the 14C dating method, because it requires the isolation of cardiac myocyte nuclei by digestion and flow cytometric sorting

The heart also presents a unique challenge compared to other organs owing to the propensity of cardiac myocytes to synthesize DNA during S-phase without completing either mitosis and/or cytokinesis (Fig 1).

Figure 1. The majority of post-natal human DNA synthesis in the heart does not lead to new myocyte formation.

Cardiac myocytes can complete S-phase, followed by mitosis and cytokinesis (centre) resulting in myocyte doubling. Cardiac myocytes can also complete mitosis without cytokinesis (left), resulting in a binucleated cell. Cardiac myocytes can also undergo chromosomal replication without completing either mitosis or cytokinesis (right), resulting in polyploidy nuclei. By the completion of post-natal development, the majority of human myocyte nuclei contain ~4n chromosomal copies.

During early post-natal development, for example, the majority of rodent cardiac myocytes and an estimated 25–57% of human cardiac myocytes become binucleated. By adulthood, most cardiac myocyte nuclei have also become polyploid with at least one or two additional rounds of chromosomal replication.

The ploidy state of cardiac myocytes may increase with myocardial hypertrophy or injury, which could be mistaken for myocyte division. Conversely, hearts that have been unloaded by implantation of a ventricular assist device may have a lower percentage of polyploid myocytes, because more 2n cardiac myocytes are being generated. These aspects of cardiac myocyte biology inevitably represent potential confounders that must be considered in any quantification of cardiac myocyte formation.

Defining the cellular source of new cardiac myocytes

The majority of reports suggest some endogenous capacity for cardiac myocyte renewal, which has generated a broad focus on finding the cellular source of newly generated cardiac myocytes.  Newly generated adult mammalian cardiac myocytes may arise from an endogenous pool of progenitor cells after injury. The Lee Laboratory developed a genetic lineage mapping approach to quantify progenitor-dependent cardiac myocyte turnover (Fig 2) (Hsieh et al, 2007). In the bitransgenic MerCreMer/ZEG inducible cardiac myocyte reporter mouse, mature cardiac myocytes undergo an irreversible genetic switch from constitutive 3-galactosidase expression to green fluorescent protein (GFP) expression upon tamoxifen pulse. During a chase period, we evaluated the effect of myocardial injury on the proportion of GFP+ or 3-gal+ cardiac myocytes. Pressure overload or myocardial infarction resulted in a significant reduction in the percentage of GFP+ cardiac myocytes and a corresponding increase in the percentage of B-gal+ cardiac myocytes, consistent with repletion of the myocyte pool by B-gal— expressing progenitors. This approach cannot directly identify the molecular identity or anatomic location of the progenitor pool.
One approach to characterizing the molecular phenotype of cardiac progenitors is to study cardiac embryologic develop-ment, guided by the assumption that developmental paradigms are recapitulated during post-natal repair. When examined through a developmental lens, an increasingly detailed picture emerges of the soluble and transcriptional signals that guide the cardiogenic programme from gastrulation (formation of distinct germ layers) through the ultimate maturation of cardiac myocytes. The induction of mesoderm posterior (MESP)-1 expression by brachyury-expressing primitive mesodermal cells is a proximal require¬ment for the ultimate production of differentiated heart cells. As the developing embryo grows beyond the germ layer phase, its developing heart receives cells from distinct anatomic progeni¬tor sources: the 1st and 2nd heart fields provide the majority of the myocardium, with some contribution from epicardial progenitors.
Also, Certain fields may be preferentially marked by specific transcription factors;

  • the first heart field by T-box transcription factor 5 (Tbx5)
  • the second heart field by
    • Lim-homeodomain protein Islet1 (Isl1)
    • and epicardial progenitors by Wilms tumour-1 (WT1) or
    • T-box transcription factor 18 (Tbx18)
    • identified by embryonic lineage tracing or analysis of gene silencing include
      • homeobox protein nkx2.5
      • myocyte enhancer factor 2C (Mef2c)
      • GATA4
      • there is no consensus yet about the molecular identity of post-natal mammalian cardiac progenitor cells or ‘adult cardiac stem cells’

Figure 2. Lineage-mapping in the adult heart.

Left: Theoretical progenitor lineage-mapping is depicted. Progenitors would be selectively marked by fluorescent protein expression. After injury, the appearance of fluorescently labelled cardiac myocytes would support the concept that these progenitors were contributing to new myocyte formation. Right: Differentiated cell (cardiac myocyte) lineage-mapping. Upon treatment of the MerCreMer-ZEG mouse with OH-tamoxifen, approximately 80% of the cardiac myocytes undergo a permanent switch from I3-galactosidase to GFP expression. The dilution of the GFPþ cardiac myocyte pool after injury is consistent with repletion by I3-galþ progenitors.

A number of laboratories have identified cell populations within the post-natal mouse, which fulfil some criteria of cardiac progenitors:

  • expression of a developmentally important gene (isl-1(Laugwitz et al, 2005))
  • specific cell surface receptor profile (c-kit (Beltrami et al, 2003)
  • or sca-1 (Oh et al, 2003))
  • capacity to actively exclude Hoechst dye (so-called side population cells (Martin et al, 2004)) or based on the outgrowth of typical spherical colonies in tissue culture 

In general, the label of ‘cardiac stem cell’ results from the observation of self-propagation and cardiac myocyte transdifferentiation when exposed to cardiogenic conditions in vitro or when delivered in vivo after injury. However, the field will benefit from careful in vivo lineage tracing studies—without ex vivo culture steps—to study if and how a given cell type contributes to cardiac myocyte replenishment during either normal homeostasis or after injury (Fig 2). The lack of such publications to date owes in part to the lack of specificity of many stem cell markers (Fig 3).

Figure 3. Possible recapitulation of developmental paradigms by endogenous post-natal cardiac stem cells.

Between mesodermal development and the emergence of cardiac myocytes, cardiovascular progenitors express a number of markers that have also been detected in the various post-natal cardiac stem cell (CSC) preparations. Expression as measured by messenger RNA (mRNA) or protein expression is denoted with (þ). Absent expression is denoted by (-). Blank1/4 untested.

Moving towards a regenerative therapy

The therapeutic challenge is considerable: a typical large myocardial infarction that leads to heart failure will kill around 1 billion cardiac myocytes,  roughly a quarter of the heart’s myocytes. A possible therapeutic approach would coax an endogenous stem cell population or an exogenously delivered cell-based therapy to replace lost cardiac myocytes in a coordinated fashion. Amongst the myriad of potential cell-based therapies, no clear winning strategy has so far emerged (Segers & Lee, 2008).

Bone marrow derived progenitors

Conflicting studies sparked excitement and also uncertainty about a possible adult cardiogenic progenitor originating outside of the heart. A post-mortem examination of male heart transplant patients who had received female donor hearts found that approximately 10% of -sarcomeric actin-positive cardiac myocytes had Y-chromosomes, and two cases in which a bone marrow cell population with a higher density of the cell surface receptor c-kit, showed repopulation of murine cardiac myocytes after experimental myocardial infarction. A number of studies that followed failed to demonstrate similar rates of chimerism in transplanted hearts or potency of bone marrow stem cell.  However, some therapeutic effect was observed even in studies with no detectable transdifferentiation.

Figure 4. The challenge of regenerating the heart.

Both exogenously delivered cell therapies and progenitors in the endogenous niche encounter a similar hostile environment after myocardial injury, often including inadequate blood supply (ischemia), inflammation and fibrosis/scarring. Regenerative pathways may be activated by as yet unknown paracrine pathways, responsible for recruiting progenitors from the niche, stimulating proliferation and coaxing differentiation.  Cell-based therapy using autologous bone marrow
progenitors was rapidly translated into the clinic to treat human ischemic heart disease. A number of randomized trials, using bone marrow mononuclear cells have been performed and most studies demonstrated modest cell therapy-mediated improvements in ventricular function.

Pluripotent stem cells

Embryonic stem (ES) cells represent the prototypical stem cells with the hallmarks of clonogenicity, self-renewal and pluripotency. The potency of these cells also represents a real safety concern, given their tendency to form teratomas. One approach to overcoming this prohibitive safety problem has been to generate pluripotent-derived progenitors that have already committed to a cardiogenic pathway. As a proof-of-principle example of such a strategy, cells with an expression profile of Oct4, stage-specific embryonic antigen 1 (SSEA-1) and MESP1 demonstrated some regenerative potency when delivered therapeutically in a primate infarct model, without detectable teratoma formation. One could envision a similar strategy using cardiogenic intermediates that express any of the previously mentioned transcription factors associated with cardiac progenitors or cell surface markers such as the receptor for vascular endothelial growth factor (Flk1/KDR). Yet, such a strategy should still demonstrate both substantial preclinical efficacy without tumorigenicity before human translation. If such criteria are met, ES-derived therapies have the potential of providing ‘off-the-shelf’ cardiac myocytes to treat acute myocardial infarctions or chronic heart failure.
A second approach, which may also obviate the risk of teratomas, is to generate a pure population of ES-derived cardiac myocytes for therapeutic delivery either as a cell suspension or after ex vivo tissue engineering. There has already been enormous progress during the past decade in defining the factors and transcription signals to differentiate cardiac myocytes from ES-cells. As discussed in greater detail, cardiac myocyte development is dictated by the time and dose-dependent exposure to a series of growth factors from the wingless-type MMTV integration site (Wnt), fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and nodal families. Several laboratories have successfully generated ES-derived preparations with more than 50% of functional cardiac myocytes.  The most realistic future for such technical advances may be as an unlimited source of cardiac myocytes for engineering myocardial grafts.
The generation of induced pluripotent stem (iPS) cells may overcome two important limitations of ES cells: ethical concerns about harvesting ES cells from embryos and graft rejection

  • iPS cells can be custom-engineered from a patient’s stromal cells for autologous transplantation.
  • immunogenecity in syngeneically transplanted iPS cells, suggests that the immune system cannot yet be discounted in the development of iPS-based therapies

The initial protocols for iPS cell generation involved retro-viral-mediated expression of four stem-cell genes.
But virally reprogrammed cells may harbour an associated risk of neoplastic conversion. Alternative reprogramming strategies, such as the use of small molecules (Shi et al, 2008) or non-viral gene modifying approaches (Warren et al, 2010) will probably be a necessary component of any future therapeutic strategies. However, the most important lesson from these landmark studies may be the remarkable plasticity of even the most terminally differentiated cells when exposed to the right combination of signals.

Tissue engineering

Historically, the greatest challenge in tissue engineering has been an adequate supply of oxygen and nutrients for metabolically active tissues such as the heart. One approach has been to engineer thin cardiac sheets, which can then be stacked for in vivo delivery. Although these layered sheets demonstrate some degree of electromechanical coordination and neovascularization in vivo, it is not clear yet if such an approach can be optimized to yield full-thickness myocardium with an adequate blood supply. The addition of non-myocyte cellular components, such as fibroblasts and endothelial cells, leads to the formation of primitive vascular structures within engineered grafts, but the electro-mechanical properties are not sufficient for normal functionality.

Circumventing cell-based therapy with pharmacoregeneration?

A short-term goal may be to exploit paracrine signalling to amplify the existent endogenous regenerative response. Recent cell transplantation experiments conducted in our laboratory, using an inducible cre-based genetic lineage mapping approach, tested the hypothesis that cell-based therapies might exert proregenerative effects via a paracrine mechanism (Loffredo et al, 2011) (Fig 5).  Consistent with some prior studies, we found no evidence for transdifferentiation of exogenously delivered bone marrow cells into cardiac myocytes. However, we did find increased generation of cardiac myocytes from endogenous progenitors in mice, which were administered c-kit+ bone marrow cells but not mesenchymal stem cells. This finding suggests paracrine signalling between exogenously delivered cells and endogenous resident progenitors. It provides a rationale for therapeutic interventions aimed at activating progenitors or recruiting them from their niche to the injury site.

Figure 5. Proposed of action for cell-based therapies.

In theory, exogenously delivered cells may directly differentiate into endothelial cells, smooth muscle cells and cardiac myocytes. They may also release paracrine factors which may result in non-regenerative effects, such as immunomodulation, angiogenesis or cardioprotection. Recent work from our laboratory suggests that a dominant mechanism achieved with bone marrow progenitor therapy may be via the activation of endogenous progenitor recruitment (Loffredo et al, 2011).

Controlling the mitotic activity of mononucleated cardiac myocytes may provide an alternative approach to replenishing cardiac myocytes. A major concern with systemic growth factor therapy, however, is the potential for mitogenic effects that may impact other organs. Thus, the future of pharmacologic regeneration may lie in the local delivery of engineered proteins and small molecules that target 

Future directions

In this review, we have described the current status of research on cardiac regeneration, highlighting important recent discoveries and ongoing controversies. The initial hope that a cell progenitor would emerge with the capacity to regenerate the injured mammalian heart in the same manner that bone marrow may be reconstituted has not been realized.
Cardiac myocyte regeneration may lie in the local delivery of engineered proteins and small molecules that target specific survival, growth and differentiation pathways.

Pending issues

Dissect the mechanistic differences between adult mammals with limited regenerative capacity and organisms, such as neonatal mice, zebrafish and newts, that demonstrate unambiguous cardiac myocyte regeneration. Understanding these differences may reveal new pathways that can be therapeutically targeted to achieve more robust regeneration.

Complete molecular and functional characterization of endogenous cardiac myocyte progenitors. Multiple laboratories have isolated progenitors from the heart with different molecular characteristics. What are the in vivo functional roles of these progenitors? Do the observed molecular differences between these isolated cells represent functionally distinct cell types?

Identify paracrine signalling pathways responsible for activation and recruitment of endogenous cardiac myocyte progenitors. This may facilitate a pharmacoregenerative therapy, in which treatment with a protein or small molecule would hold the promise of amplifying endogenous regeneration.

Refine reprogramming strategies to more efficiently produce mature cardiac myocytes, both in vitro and in vivo. The ultimate bioengineering goal is to produce a pure population of mature, fully functional cardiac myocytes for ex vivo tissue engineering (or) to control the proliferation and differentiation of endogenous cell populations or exogenously delivered cell therapies such that scar tissue is replaced by myocardium. These different strategies are unified by an underlying requirement to understand the fundamental pathways involved in cardiac myocyte differentiation and maturation.

There is reason for optimism for a regenerative medicine approach to heart failure, given the intense research efforts and the capacity of higher organisms, including the neonatal mouse, to regenerate myocardium. Perhaps the most important issue in this field is identifying which findings are consistently supported by multiple experimental approaches. Ultimately, the findings that are easily reproduced by most or all laboratories will most likely benefit patients.

Selected references

Hsieh et al, 2007.  Hsieh PC, Segers VF, Davis ME, MacGillivray C, Gannon J, Molkentin JD, Robbins J, Lee RT (2007) Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med 13: 970¬974
Laugwitz et al, 2005.  Laugwitz KL, Moretti A, Lam J, Gruber P, Chen Y, Woodard S, Lin LZ, Cai CL, Lu MM, Reth M et al (2005) Postnatal isl1þ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 433: 647-653
Beltrami et al, 2003.  Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K et al (2003) Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 114: 763¬776
Oh et al, 2003. Oh H, Bradfute SB, Gallardo TD, Nakamura T, Gaussin V, Mishina Y, Pocius J, Michael LH, Behringer RR, Garry DJ et al (2003) Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci USA 100: 12313-12318
Segers & Lee, 2008.  Segers VF, Lee RT (2008) Stem-cell therapy for cardiac disease. Nature 451:937-942
Loffredo et al, 2011.  Loffredo FS, Steinhauser ML, Gannon J, Lee RT (2011) Bone marrow-derived cell therapy stimulates endogenous cardiomyocyte progenitors and promotes cardiac repair. Cell Stem Cell 8: 389-398.
Shi et al, 2008.  Shi Y, Desponts C, Do JT, Hahm HS, Scholer HR, Ding S (2008) Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 3: 568-574.
Warren et al, 2010.  Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A et al (2010) Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7: 618-630.

Mammalian Heart Renewal by Preexisting Cardiomyocytes

SE Senyo, ML Steinhauser, CL Pizzimenti, VK. Yang, Lei Cai, Mei Wang, …,and Richard T. Lee
Cardiovascular and Genetics Divisions, Brigham and Women’s Hospital and Harvard Medical School,
INSERM, Orsay (Fr), Institut Curie, Laboratoire de Microscopie Ionique, Orsay (Fr), National Resource for Imaging Mass Spectrometry, Harvard Stem Cell Institute
Nature. 2013 January 17; 493(7432): 433–436.

Although recent studies have revealed that heart cells are generated in adult mammals, the frequency and source of new heart cells is unclear. Some studies suggest a high rate of stem cell activity with differentiation of progenitors to cardiomyocytes. Other studies suggest that new cardiomyocytes are born at a very low rate, and that they may be derived from division of pre-existing cardiomyocytes. Thus, the origin of cardiomyocytes in adult mammals remains unknown. Here we combined two different pulse-chase approaches — genetic fate-mapping with stable isotope labeling and Multi-isotope Imaging Mass Spectrometry (MIMS). We show that genesis of cardiomyocytes occurs at a low rate by division of pre-existing cardiomyocytes during normal aging, a process that increases by four-fold adjacent to areas of myocardial injury. Cell cycle activity during normal aging and after injury led to polyploidy and multinucleation, but also to new diploid, mononucleated cardiomyocytes. These data reveal pre-existing cardiomyocytes as the dominant source of cardiomyocyte replacement in normal mammalian myocardial homeostasis as well as after myocardial injury.

Despite intensive research, fundamental aspects of the mammalian heart’s capacity for self-renewal are actively debated. Estimates of cardiomyocyte turnover range from less than 1% per year to more than 40% per year. Turnover has been reported to either decrease or increase with age, while the source of new cardiomyocytes has been attributed to both division of existing myocytes and to progenitors residing within the heart or in exogenous niches such as bone marrow. Controversy persists regarding the plasticity of the adult heart in part due to methodological challenges associated with studying slowly replenished tissues. Toxicity attributed to radiolabeled thymidine and halogenated nucleotide analogues limits the duration of labeling and may produce direct biological effects. The challenge of measuring cardiomyocyte turnover is further compounded by the faster rate of turnover of cardiac stromal cells relative to cardiomyocytes.

We used Multi-isotope Imaging Mass Spectrometry (MIMS) to study cardiomyocyte turnover and to determine whether new cardiomyocytes are derived from preexisting myocytes or from a progenitor pool (Fig 1a). MIMS uses ion microscopy and mass spectrometry to generate high resolution quantitative mass images and localize stable isotope reporters in domains smaller than one micron cubed15,16,17. MIMS generates 14N quantitative mass images by measuring the atomic composition of the sample surface with a lateral resolution of under 50nm and a depth resolution of a few atomic layers. Cardiomyocyte cell borders and intracellular organelles were easily resolved (Fig 1b). Regions of interest can be analyzed at higher resolution, demonstrating cardiomyocyte-specific subcellular ultrastructure, including sarcomeres (Fig 1c, Supplemental Fig 1a). In all subsequent analyses, cardiomyocyte nuclei were identified by their location within sarcomere-containing cells, distinguishing them from adjacent stromal cells.
An immense advantage of MIMS is the detection of nonradioactive stable isotope tracers. As an integral part of animate and inanimate matter, they do not alter biochemical reactions and are not harmful to the organism18. MIMS localizes stable isotope tracers by simultaneously quantifying multiple masses from each analyzed domain; this enables the generation of a quantitative ratio image of two stable isotopes of the same element15. The incorporation of a tracer tagged with the rare stable isotope of nitrogen (15N) is detectable with high precision by an increase in 15N:14N above the natural ratio (0.37%). Nuclear incorporation of 15N-thymidine is evident in cells having divided during a one-week labeling period, as observed in the small intestinal epithelium, which turns over completely in 3–5 days16 (Fig 1d); in contrast, 15N-thymidine labeled cells are rarely observed in the heart (Fig 1e) after 1 week of labeling. In subsequent studies, small intestine was used as a positive control to confirm label delivery.
To evaluate for an age-related change in cell cycle activity, we administered 15N-thymidine for 8 weeks to three age groups of C57BL6 mice starting at day-4 (neonate), at 10-weeks (young adult) and at 22-months (old adult) (Supplemental Fig 2). We then performed MIMS analysis (Fig 2a, b, Supplemental Fig 3). In the neonatal group, 56% (±3% s.e.m., n=3 mice) of cardiomyocytes demonstrated 15N nuclear labeling, consistent with the well-accepted occurrence of cardiomyocyte DNA synthesis during post-natal development19. We observed a marked decline in the frequency of 15N-labeled cardiomyocyte nuclei (15N+CM) in the young adult (neonate= 1.00%15N+CM/day ±0.05 s.e.m. vs young adult=0.015% 15N+CM/ day ±0.003 s.e.m., n=3 mice/group, p<0.001) (Fig 2a, c; Supplemental Fig 3). We found a further reduction in cardiomyocyte DNA synthesis in old mice (young adult=0.015%15N+CM/day ±0.003 s.e.m. vs. old adult=0.007 %15N+CM/day ±0.002 s.e.m., n=3/group, p<0.05) (Fig 2c). The observed pattern of nuclear 15N-labeling in cardiomyocytes is consistent with the known chromatin distribution pattern in cardiomyocytes20 (Supplemental Fig 1b) and was measured at levels that could not be explained by DNA repair (Supplemental Fig 4). Extrapolating DNA synthesis measured in cardiomyocytes over 8 weeks yields a yearly rate of 5.5% in the young adult and 2.6% in the old mice. Given that cardiomyocytes are known to undergo DNA replication without completing the cell cycle19,21,22, these calculations represent the upper limit of cardiomyocyte generation under normal homeostatic conditions, indicating a low rate of cardiogenesis.
To test whether cell cycle activity occurred in preexisting cardiomyocytes or was dependent on a progenitor pool, we performed 15N-thymidine labeling of double-transgenic MerCreMer/ZEG mice, previously developed for genetic lineage mapping (Fig 3a)23,24. MerCreMer/ZEG cardiomyocytes irreversibly express green fluorescent protein (GFP) after treatment with 4OH-tamoxifen, allowing pulse labeling of existing cardiomyocytes with a reproducible efficiency of approximately 80%. Although some have reported rare GFP expression by non-cardiomyocytes with this approach25, we did not detect GFP expression in interstitial cells isolated from MerCreMer/ZEG hearts nor did we detect GFP expression by Sca1 or ckit-expressing progenitors in histological sections (Supplemental Fig 5). Thus, during a chase period, cardiomyocytes generated from progenitors should be GFP−, whereas cardiomyocytes arising from preexisting cardiomyocytes should express GFP at a frequency similar to the background rate induced by 4OH-tamoxifen. We administered 4OH-tamoxifen for two weeks to 8 wk-old mice (n=4); during a subsequent 10-week chase, mice received 15N-thymidine via osmotic minipump.

We next used MIMS and genetic fate mapping to study myocardial injury. Cardiomyocyte GFP labeling was induced in MerCreMer/ZEG mice with 4OH-tamoxifen. Mice then underwent experimental myocardial infarction or sham surgery followed by continuous labeling with 15N-thymidine for 8wks. The frequency of 15N-labeled cardiomyocytes in sham-operated mice was similar to prior experiments in unoperated mice (yearly projected rates: sham=6.8%; unoperated=4.4%), but increased significantly adjacent to infarcted myocardium (total 15N+ cardiomyocyte nuclei: MI=23.0% vs sham=1.1%, Fig 4a–b, Supplemental Fig 8). We examined GFP expression, nucleation and ploidy status of 15N-labeled cardiomyocytes and surrounding unlabeled cardiomyocytes. We found a significant dilution of the GFP+ cardiomyocyte pool at the border region as previously shown23,24 (67% vs. 79%, p<0.05, Table 2, Supplemental Fig 9); however, 15N+ myocytes demonstrated a similar frequency of GFP expression compared to unlabeled myocytes (71% vs. 67%, Fisher’s exact=n.s.), suggesting that DNA synthesis was primarily occurring in pre-existing cardiomyocytes. Of 15N-labeled cardiomyocytes, approximately 14% were mononucleated and diploid consistent with division of pre-existing cardiomyocytes (Supplemental Fig 6, 7). We observed higher DNA content (>2N) in the remaining cardiomyocytes as expected with compensatory hypertrophy after injury. Thus, in the 8wks after myocardial infarction, approximately 3.2% of the cardiomyocytes adjacent to the infarct had unambiguously undergone division (total 15N+ × mononucleated diploid fraction = 23% × 0.14 = 3.2%). The low rate of cardiomyocyte cell cycle completion is further supported by the absence of detectable Aurora B Kinase, a transiently expressed cytokinesis marker, which was detected in rapidly proliferating small intestinal cells but not in cardiomyocytes (Supplemental Fig 10). We also considered the possibility that a subset of 15N+ myocytes that were multinucleated and/or polyploid resulted from division followed by additional rounds of DNA synthesis without division. However, quantitative analysis of the 15N+ population did not identify a subpopulation that had accumulated additional 15N-label as would be expected in such a scenario (Supplemental Fig 11). Together, these data suggest that adult cardiomyocytes retain some capacity to reenter the cell cycle, but that the majority of DNA synthesis after injury occurs in preexisting cardiomyocytes without completion of cell division.
If dilution of the GFP+ cardiomyocyte pool cannot be attributed to division and differentiation of endogenous progenitors, do these data exclude a role for progenitors in the adult mammalian heart? These data could be explained by preferential loss of GFP+ cardiomyocytes after injury, a process that we have previously considered but for which we have not found supporting evidence23. Such an explanation excludes a role for endogenous progenitors in cardiac repair and would be consistent with data emerging from lower vertebrates8,26 and the neonatal mouse27 in which preexisting cardiomyocytes are the cellular source for cardiomyocyte repletion. A second possibility to explain the dilution of the GFP+ cardiomyocyte pool is that injury stimulates progenitor differentiation without division; inevitably, this would lead to exhaustion of the progenitor pool, which if true could explain the limited regenerative potential of the adult mammalian heart.

In summary, this study demonstrates birth of cardiomyocytes from preexisting cardiomyocytes at a projected rate of approximately 0.76%/year (15N+ annual rate × mononucleated diploid fraction = 4.4% × 0.17) in the young adult mouse under normal homeostatic conditions, a rate that declines with age but increases by approximately four-fold after myocardial injury in the border region. This study shows that cardiac progenitors do not play a significant role in myocardial homeostasis in mammals and suggests that their role after injury is also limited.

Engineering insulin-like growth factor-1 for local delivery

T Tokunou, R Miller, P Patwari, ME Davis, VFM Segers, AJ Grodzinsky, and RT Lee
Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA and Biological Engineering, MIT, Cambridge, MA
FASEB J. 2008 June ; 22(6): 1886–1893.

Insulin-like growth factor-1 (IGF-1) is a small protein that promotes cell survival and growth, often acting over long distances. Although for decades IGF-1 has been considered to have therapeutic potential, systemic side effects of IGF-1 are significant, and local delivery of IGF-1 for tissue repair has been a long-standing challenge. In this study, we designed and purified a novel protein, heparin-binding IGF-1 (Xp-HB-IGF-1), which is a fusion protein of native IGF-1 with the heparin-binding domain of heparin-binding epidermal growth factor-like growth factor. Xp-HB-IGF-1 bound selectively to heparin as well as the cell surfaces of 3T3 fibroblasts, neonatal cardiac myocytes and differentiating ES cells. Xp-HB-IGF-1 activated the IGF-1 receptor and Akt with identical kinetics and dose response, indicating no compromise of biological activity due to the heparin-binding domain. Because cartilage is a proteoglycan-rich environment and IGF-1 is a known stimulus for chondrocyte biosynthesis, we then studied the effectiveness of Xp-HB-IGF-1 in cartilage. Xp-HB-IGF-1 was selectively retained by cartilage explants and led to sustained chondrocyte proteoglycan biosynthesis compared to IGF-1. These data show that the strategy of engineering a “long-distance” growth factor like IGF-1 for local delivery may be useful for tissue repair and minimizing systemic effects.

INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) is a growth factor well known as an important mediator of cell growth and differentiation. IGF-1 stimulates several signaling pathways through the tyrosine kinase IGF-1 receptor, including phosphatidylinositol (PI) 3-kinase and mitogen-activated protein kinases (MAPKs). PI3-kinase has many downstream targets, including the kinase Akt, and activation of Akt promotes survival, proliferation, and growth.
IGF-1 has been extensively studied for its therapeutic potential in tissue repair and regeneration. IGF-1 is a small and highly diffusible protein that can act over long distances. However, systemic administration of IGF-1 has significant side effects as well as the potential to promote diabetic retinopathy and cancer. Therefore, local delivery of IGF-1 has been a longstanding challenge. Here, we describe the design of a new protein, formed by fusion of IGF-1 with the heparin-binding (HB) domain of heparin-binding epidermal growth factor-like growth factor (HB-EGF). HB-EGF binds selectively to glycosaminoglycans through its highly positively charged heparin-binding domain.

Thus, we hypothesized that engineering IGF-1 to bind to glycosaminoglycans could provide selective delivery of IGF-1 to cell surfaces or to specific tissues. We demonstrate that this heparin-binding IGF-1 (Xp-HB-IGF-1) can bind to cell surfaces as well as the proteoglycan-rich tissue of cartilage; furthermore, Xp-HB-IGF-1 prolongs the stimulation of chondrocyte biosynthesis, demonstrating its potential for tissue specific repair.

Purification of Xp-HB-IGF-1

Figure 1A—C shows the constructs for Xp-HB-IGF-1 and the control Xp-IGF-1 fusion proteins.

IGF-1 has 3 disulfide bonds and includes 70 amino acids. The IGF-1 fusion proteins both contain polyhistidine tags for protein purification and Xpress tags for protein detection. The expected molecular masses of Xp-HB-IGF-1 and Xp-IGF-1 are 14,018 and 11,548 Da, respectively. Xp-HB-IGF-1 has the HB domain on the N terminus of IGF-1. The HB domain has 21 amino acids and includes 12 positively charged amino acids. Final purification of the new fusion proteins after refolding was performed with RP-HPLC (Fig. 1D, E). Identification of the correctly folded protein was performed as described previously and confirmed with bioactivity assays. These 3 IGF-1s (Xp-HB-IGF-1, Xp-IGF-1, and unmodified IGF-1) yielded similar intensities.

Xp-HB-IGF-1 binds to heparin and cell surfaces

1. Xp-HB-IGF-1 binds selectively to heparin compared with Xp-IGF-1 (Fig. 2A).
2. Xp-HB-IGF-1 bound to 3T3 fibroblast cells when treated with 10 and 100 nM concentrations.
3. Xp-HB-IGF-1 binds with neonatal cardiac myocytes, with clear selective binding of Xp-HB-IGF-1 (Fig 2C)
4. These results are consistent with binding of this HB domain to heparan sulfate in the submicromolar range
5. Xp-HB-IGF-1 was readily detected on the surfaces of ES cells in embryoid bodies — which contain multiple cell types.
6. There is more Xpress epitope tag in Xp-HB-IGF-1 group than the Xp-IGF-1 group, suggesting that Xp-HB-IGF-1 binds with heparan sulfate on the cell surface.

Xp-HB-IGF-1 bioactivity

Bioassays for IGF-1 receptor phosphorylation and Akt activation were performed. Control IGF-1, Xp-HB-IGF-1, and Xp-IGF-1 all activated the IGF-1 receptor of neonatal cardiac myocytes dose-dependently and induced Akt phosphorylation identically (Fig. 3A), and they  activated Akt with a similar time course (Fig. 3B), indicating — addition of the heparin-binding domain does not interfere with the bioactivity of IGF-1.

  1. Xp-HB-IGF-1 transport in cartilage
  2. Cartilage is a proteoglycan-rich tissue, and chondrocytes respond to IGF-1 with increased extracellular matrix synthesis (19). Because prolonged local stimulation of IGF-1 signaling could thus be beneficial for cartilage repair, we studied the ability of Xp-HB-IGF-1 to bind to cartilage.
  3. Xp-HB-IGF-1 is selectively retained by cartilage, while Xp-IGF-1 is rapidly lost.
  4. Xp-HB-IGF-1 can bind to cartilage after chondroitin sulfate digestion

To explore the possibility of nonspecific binding of Xp-HB-IGF-1 to glycosaminoglycans other than heparan sulfate, we studied the binding of Xp-HB-IGF-1 after chondroitinase ABC digestion.
Xp-HB-IGF-1 retention is not mediated by the pool of chondroitin sulfated proteoglycans in the cartilage matrix.

  1. Xp-HB-IGF-1 increases chondrocyte biosynthesis
  2. Xp-HB-IGF-1, which is selectively retained in the cartilage, stimulates chondrocyte biosynthesis over a more sustained period.


In this study, we describe a novel IGF-1 protein, Xp-HB-IGF-1, which binds to proteoglycan-rich tissue and cell surfaces but has the same bioactivity as IGF-1. Our data indicate that Xp-HB-IGF-1 can activate the IGF-1 receptor and Akt and thus that the heparin-binding domain does not interfere with interactions of IGF-1 and its receptor. IGF-1 has four domains: B domain (aa 1–29), C domain (aa 30 – 41), A domain (aa 42–62) and D domain (aa 63–70), with the C domain playing the most important role in binding to the IGF-1 receptor. Replacement of the entire C domain causes a 30-fold decrease in affinity for the IGF-1 receptor. Thus, the addition of the heparin-binding domain to the N terminus of IGF-1 was not anticipated to interfere with interactions with the IGF-1 C domain.
Both extracellular matrix and cell surfaces are rich in proteoglycans and can serve as reservoirs for proteoglycan-binding growth factors. A classic example is the fibroblast growth factor-2 (FGF-2) system, where a low-affinity, high-capacity pool of proteoglycan receptors serves as a reservoir of FGF-2 for its high-affinity receptor. Our experiments suggest that Xp-HB-IGF-1 could function in some circumstances in a similar manner, since Xp-HB-IGF-1 is selectively retained on cell surfaces. Many growth factors are known to interact with heparan sulfate, including HB-EGF (10-12), FGF-2 (26), vascular endothelial growth factor-A (VEGF-A), transforming growth factor beta (TGF-β) (28), platelet-derived growth factors (PDGFs), and hepatocyte growth factor (HGF). However, other proteins such as nerve growth factor (NGF), which induces differentiation and reduces apoptosis of neurons, does not have the heparin-binding domain. Thus, the strategy of engineering growth factors for selective matrix or cell surface binding could be used for other growth factors.
IGF-1 can also bind with extracellular matrix via IGF binding proteins (IGFBPs); in the circulation, at least 99% of IGF-1 is bound to IGFBPs (IGFBP-1 to −6). Further experiments are necessary to determine whether addition of a heparin-binding domain to IGF-1 changes interactions with IGFBPs and whether this changes its biological activity.
IGF-1 can promote the synthesis of cartilage extracellular matrix and inhibit cartilage degradation (19); however, a practical mode of IGF-1 delivery to cartilage has yet to be developed (33). Heparan sulfate proteoglycans are prevalent in the pericellular matrix of cartilage, particularly as chains on perlecan and syndecan-2, and are known to bind other ligands such as FGF-2 (34). Our experiments suggest that Xp-HB-IGF-1 protein can bind with matrix and increase local, long-term bioavailability to chondrocytes and thus may improve cartilage repair.

Selected References

Hameed M, Orrell RW, Cobbold M, Goldspink G, Harridge SD. Expression of IGF-I splice variants in young and old human skeletal muscle after high resistance exercise. J. Physiol 2003;547:247–254. [PubMed: 12562960]
Shavlakadze T, Winn N, Rosenthal N, Grounds MD. Reconciling data from transgenic mice that overexpress IGF-I specifically in skeletal muscle. Growth Horm. IGF Res 2005;15:4–18. [PubMed: 15701567]
Milner SJ, Francis GL, Wallace JC, Magee BA, Ballard FJ. Mutations in the B-domain of insulin-like growth factor-I influence the oxidative folding to yield products with modified biological properties. Biochem. J 1995;308(Pt 3):865–871. [PubMed: 8948444]
Milner SJ, Carver JA, Ballard FJ, Francis GL. Probing the disulfide folding pathway of insulin-like growth factor-I. Biotechnol. Bioeng 1999;62:693–703. [PubMed: 9951525]
Bonassar LJ, Grodzinsky AJ, Srinivasan A, Davila SG, Trippel SB. Mechanical and physicochemical regulation of the action of insulin-like growth factor-I on articular cartilage. Arch. Biochem. Biophys 2000;379:57–63. [PubMed: 10864441]
Denley A, Cosgrove LJ, Booker GW, Wallace JC, Forbes BE. Molecular interactions of the IGF system. Cytokine Growth Factor Rev 2005;16:421–439. [PubMed: 15936977]
Musaro A, Dobrowolny G, Rosenthal N. The neuroprotective effects of a locally acting IGF-1 isoform. Exp. Gerontol 2007;42:76–80. [PubMed: 16782294]
Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. Acta 1986;883:173–177. [PubMed: 3091074]
Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DM. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 1991;64:841– 848. [PubMed: 1847668]
Martin P. Wound healing—aiming for perfect skin regeneration. Science 1997;276:75–81. [PubMed: 9082989]

Figure 1.  Construction and purification of a new Xp-HB-IGF-1 fusion protein.

Figure 1.  Construction and purification of a new Xp-HB-IGF-1 fusion protein.

A) The heparin binding domain of HB-EGF was inserted N-terminal to IGF-1 to generate the fusion protein. The construct included the hexahistidine and Xpress tags from the pTrcHis vector for purification and detection. B) The resulting amino acid sequence of HB-IGF-1. C) Schematic for the structure of HB-IGF-1. Red circles: positively charged amino acids; blue circles: negatively charged amino acids; yellow circles: cysteines. The arrow shows the HB domain. In this figure the epitope tags are not shown. D, E) Representative reverse-phase high-performance liquid chromatography (RP-HPLC) elution profiles with single peaks containing correctly folded protein. Readings of optical density at 214 nm are in blue; readings at 280 nm are in red; elution is by acetonitrile (ACN) gradient. F) After RP-HPLC, Coomassie blue staining and Western blot analysis demonstrate isolation of single bands containing Xpress-tagged protein. The right panel shows that the Western blot analysis of IGF-1, and the two engineered IGF-1 proteins yield similar results using an anti-IGF-1 antibody.

Protein Therapeutics for Cardiac Regeneration after Myocardial Infarction

Vincent F.M. Segers and Richard T. Lee
Provasculon Inc, 14 Cambridge Center, and Harvard Stem Cell Institute and the Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA
J Cardiovasc Transl Res. 2010 October ; 3(5): 469–477.   http://dx.doi./10.1007/s12265-010-9207-5.

Although most medicines have historically been small molecules, many newly approved drugs are derived from proteins. Protein therapies have been developed for treatment of diseases in almost every organ system, including the heart. Great excitement has now arisen in the field of regenerative medicine, particularly for cardiac regeneration after myocardial infarction. Every year, millions of people suffer from acute myocardial infarction, but the adult mammalian myocardium has limited regeneration potential. Regeneration of the heart after myocardium infarction is therefore an exciting target for protein therapeutics.  

In this review, we discuss different classes of proteins that have therapeutic potential to regenerate the heart after myocardial infarction. Protein candidates have been described that induce angiogenesis, including fibroblast growth factors and vascular endothelial growth factors, although thus far clinical development has been disappointing. Chemotactic factors that attract stem cells, e.g. hepatocyte growth factor and stromal cell derived factor-1, may also be useful. Finally, neuregulins and periostin are proteins that induce cell cycle reentry of cardiomyocytes, and growth factors like IGF-1 can induce growth and differentiation of stem cells. As our knowledge of the biology of regenerative processes and the role of specific proteins in these processes increases, the use of proteins as regenerative drugs could develop as a cardiac therapy.
Keywords: protein therapeutics; myocardial infarction; regeneration; heart failure

The current standard of care for MI is early reperfusion of the occluded vessel with angioplasty or thrombolysis to reverse ischemia and increase the number of surviving myocytes. Efforts to decrease delays between onset of symptoms and reperfusion have resulted in decreased morbidity and mortality, but the maximal benefit of early reperfusion has reached a point close to practical limits. Besides early reperfusion therapy, ACE inhibitors and beta-blockers are used to prevent remodeling after MI and progression to heart failure. Both ACE inhibitors and beta-blockers improve long term survival but no therapies besides cardiac transplantation are currently available that restore cardiac function.
In the last decade, a large number of pre-clinical and clinical studies have been published on the potential use of stem cells for cardiac regeneration after MI. Different stem cell types have been shown to improve cardiac function in animal studies and can induce a small but potentially significant increase in ejection fraction in clinical studies. Stem cell therapy is a promising treatment option for heart failure, but numerous technical challenges and gaps in our understanding of stem cell behavior may limit translation to the clinic.
With the advent of biotechnology, protein and peptide drugs are becoming increasingly important in modern medicine. Drugs based on naturally-occurring proteins have the advantage of efficacy based on a mechanism of action refined by millions of years of biological evolution. Though promising as therapeutics, proteins might behave differently when used at pharmacological instead of physiological concentrations with an increase in adverse effects on other organs. Proteins used as therapeutics have been modified in different ways to limit immunogenicity and rapid degradation in plasma and tissues.
We discuss four different classes of proteins that could potentially benefit patients with MI (Figure 1); all of these proteins have been shown to improve cardiac function in animal models of MI or heart failure. They include angiogenic growth factors, proteins that increase recruitment of progenitor cells to the heart, proteins that induce mitosis of existing myocytes, and proteins that increase differentiation and growth of stem cells and myocytes. As more is learned about cardiac regeneration and why mammals lack sufficient myocardial regeneration, more proteins are likely to be added to this list of candidates.

A decade of extensive research on cardiac stem cell biology revealed 1 protein (G-CSF) that can be used to mobilize hematopoietic stem cells and just 2 proteins with chemotactic properties on stem cells: SDF-1 on endothelial progenitor cells and HGF on cardiac stem cells. Another protein that has been identified as a stem cell attractant is monocyte chemotactic protein-3 which attracts mesenchymal stem cells [42]. It is unknown if local administration of MCP-3 improves cardiac function. Identification of new stem cell chemotactic proteins is important because it could lead to the development of new and feasible therapeutics for treatment of MI and heart failure. At the same time, the true regenerative potential of most stem cells remains highly controversial; indicating that even if a chemotactic factor attracting stem cells to the heart is identified, formation of functional myocardial is still a challenging task.

Proteins like periostin and neuregulin which stimulate mitosis of surviving myocytes can partially restore the damage inflicted by MI. However, some requirements have to be met before this will result in a viable therapy. An inherent selectivity for myocytes would also allow for systemic delivery as opposed to the use of more complicated local delivery methods. An important factor to consider is the duration of the signal necessary to induce mitosis in a significant number of myocytes. A protein that induces cell cycle reentry in a significant fraction of myocytes with a single pulse has more therapeutical potential than a protein that needs sustained or repeated delivery. Ideally, pro-mitotic proteins will be not only specific for myocytes in general but might also be specific for myocytes in the border zone of the MI. This has drawbacks, among which is that formation of new myocytes, either by stem cell differentiation or by myocyte mitosis, carries an increased risk of ventricular arrhythmias.

Figure 1. Regeneration of the heart by 4 different classes of proteins

Figure 1. Regeneration of the heart by 4 different classes of proteins

See text for details. A) FGF and VEGF increase angiogenesis. B) G-CSF mobilizes bone marrow hematopoietic stem cells and SDF-1 attracts endothelial progenitor cells. HGF attracts cardiac stem cells. C) Neuregulin and periostin can induce division of adult cardiomyocytes. D) IGF-1 induces maturation and differentiation of cardiac stem cells.

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Phrenic Nerve Stimulation in Patients with Cheyne-Stokes Respiration and Congestive Heart Failure

Writer: Larry H Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN

Transvenous Phrenic Nerve Stimulation in Patients With Cheyne-Stokes Respiration and Congestive Heart Failure:A Safety and Proof-of-Concept Study

Xi-Long Zhang, MD; Ning Ding; Hong Wang; Ralph Augostini; Bing Yang; Di Xu; Weizhu Ju; Xiaofeng Hou; Xinli Li; Buqing Ni, PhD; Kejiang Cao; Isaac George; Jie Wang, MD, PhD; Shi-Jiang Zhang
Chest. 2012; 142(4):927-934. doi:10.1378/chest.11-1899
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Background:  Cheyne-Stokes respiration (CSR), which often occurs in patients with congestive heart failure (CHF), may be a predictor for poor outcome. Phrenic nerve stimulation (PNS) may interrupt CSR in patients with CHF. We report the clinical use of transvenous PNS in patients with CHF and CSR.

Methods:  Nineteen patients with CHF and CSR were enrolled. A single stimulation lead was placed at the junction between the superior vena cava and brachiocephalic vein or in the left-side pericardiophrenic vein. PNS stimulation was performed using Eupnea System device (RespiCardia Inc). Respiratory properties were assessed before and during PNS. PNS was assessed at a maximum of 10 mA.

Results:  Successful stimulation capture was achieved in 16 patients. Failure to capture occurred in three patients because of dislocation of leads. No adverse events were seen under maximum normal stimulation parameters for an overnight study. When PNS was applied following a series of central sleep apneic events, a trend toward stabilization of breathing and heart rate as well as improvement in oxygen saturation was seen. Compared with pre-PNS, during PNS there was a significant decrease in apnea-hypopnea index (33.8 ± 9.3 vs 8.1 ± 2.3, P = .00), an increase in mean and minimal oxygen saturation as measured by pulse oximetry (89.7% ± 1.6% vs 94.3% ± 0.9% and 80.3% ± 3.7% vs 88.5% ± 3.3%, respectively, all P = .00) and end-tidal CO2 (38.0 ± 4.3 mm Hg vs 40.3 ± 3.1 mm Hg, P = .02), but no significant difference in sleep efficiency (74.6% ± 4.1% vs 73.7% ± 5.4%, P = .36).

Conclusions:  The preliminary results showed that in a small group of patients with CHF and CSR, 1 night of unilateral transvenous PNS improved indices of CSR and was not associated with adverse events.

Trial registry:; No.: NCT00909259; URL:

Transvenous phrenic nerve stimulation in patients with Cheyne-Stokes respiration and congestive heart failure: a safety and proof-of-concept study

Zhang Xi-Long; Ding N, Wang H, Augostini R, Yang B.
CHEST 2012; 142(4):927–934
Trial registry:; No.: NCT00909259; URL:


Cheyne-Stokes respiration (CSR), a condition characterized by a cyclic pattern of waxing and waning ventilation interposed by central apneas or hypopneas, may affect up to 40% of patients with congestive heart failure (CHF).  Whether CSR is related to significantly increased morbidity and mortality 2 or has no impact on long-term survival in patients with CHF is controversial. Nevertheless, several investigators have reported that CSR might be an independent prognostic index of poor outcome in patients with CHF, so that Cheyne-Stokes respiration (CSR), which often occurs in patients with congestive heart failure (CHF), may be a predictor for poor outcome. CSR in patients with CHF is believed to be associated with a hypersensitivity to arterial CO 2 during sleep. The key pathophysiologic mechanism leading to all forms of periodic breathing is the oscillation of blood CO 2 level below and above the apneic threshold.  Clinically, these pathophysiologic changes may translate to sleep fragmentation, excessive daytime sleepiness, reduced exercise capacity, and, possibly, ventricular arrhythmias.
Current treatment options for CSR include medications, positive airway pressure devices such as adapt servo-ventilation, or oxygen therapy. Although all these therapies have shown benefi t in some patients, none has shown a consistent benefi t of suffi cient clinical magnitude to reduce mortality and morbidity. In the current study, we explored the initial feasibility, safety, and possible effects of unilateral, transvenous, synchronized PNS on CSR in 19 patients with CHF . This novel treatment resulted in a marked reduction of minute ventilation and possible improvement of CSR. The authors here suggest that phrenic nerve stimulation (PNS) may interrupt CSR in patients with CHF.

Study Population

 Nineteen patients with CHF and CSR were enrolled.  All study patients (N 5 19) had received a diagnosis of CSR and chronic CHF and were hospitalized in The First Affiliated Hospital of Nanjing Medical University (Nanjing, China). Of them, 12 with rheumatic cardiac valve disease were waiting forcardiac surgery, and seven (fi ve with dilated cardiomyopathy and two with hypertensive heart disease) were enrolled from the cardiology ward because of severe heart failure.
The inclusion criteria were aimed at identifying patients with symptoms or a diagnosed condition indicative of CSR who would tolerate the testing procedure. The patients continued on their standard medical regimen during participation, and in the case of an adverse event, medical treatment was at the discretion of the investigator. The inclusion criteria were as follows: (1) both patient and direct family member willingness to sign a Patient Ethics Committee-approved informed consent, (2) age > 18 years, (3) index CSR of > 15 times/h, (4) history of CHF with a left ventricle ejection fraction < 45%, and (5) ability to tolerate the study procedure and remain clinically stable for the duration of the study. Exclusion criteria were as follows: (1) baseline oxygen saturation <  90% on a stable FiO2 ; (2) evidence of phrenic nerve palsy; (3) temperature > 38.0°C; (4) inability to place stimulation lead (eg, coagulopathy, distorted anatomy, etc); (5) current enrollment in another clinical study that may confound the results of the present study; (6) no informed consent; (7) pregnancy or of childbearing potential without a negative pregnancy test within 10 days of the study procedure; (8) pacemaker, implantable cardioverter defibrillator, or cardiac resynchronization device; (9) severe COPD; (10) a history of myocardial infarction within 6 months prior to the study; and (11) unstable angina.

Study Design

 This short-term, prospective, open-label, nonrandomized feasibility study consisted of a treatment-only cohort in which each patient served as his or her own control. After patients were screened and enrolled in the study, PNS leads were placed through an interventional procedure for observation of 1 night only. During the 1-night study, we examined whether PNS caused pain, arousal during sleep, arrhythmia, changes in BP, and changes in either normal breathing or sleep apnea. We also examined the impact of PNS on central, obstructive, and mixed sleep apnea. Alterations in sleep apnea and hypopnea events were compared before and during PNS. “Before stimulation” was defined as the number of sleep apnea and hypopnea events occurring during a segment of 10 min just before delivery of PNS and served as the control for the effects of PNS. The total number of the 10-min segments before PNS, the total number of sleep apnea and hypopnea events occurred during the sum of the 10-min time were calculated,  then averaged (total number of sleep apnea and hypopnea events/total hours of the 10-min segments from all patients) and presented as the apnea-hypopnea index (AHI) for statistical analysis. AHI during PNS were also calculated and compared with AHI prior to PNS.

Sleep Study and Monitored Parameters

 All participants underwent a nocturnal, in-laboratory polysomnography (Embla S4500 PSG Amplifi er; Natus Medical Inc) and were monitored for at least 7 h overnight. The standard polysomnography recorded the EEG, bilateral electrooculograms, submental  electromyogram, ECG, chest and abdominal movement using respiratory effort bands, body position, nasal airflow using a pressure sensor, and oxygen saturation as measured by pulse oximetry (Sp o 2 ).
EEG, sleep staging, and arousals were monitored and scored using 30 epochs according to the method of Rechtschaffen and Kales. Classification of apnea and hypopnea was described by standard methodologies. CSR was identified as a special kind of CSA behaving as a cyclic pattern of periods of hyperventilation with waxing and waning tidal volumes alternating with periods of central hypopnea/apnea .

Lead Placement and PNS

A single stimulation lead was placed at the junction between the superior vena cava and brachiocephalic vein or in the left-side pericardiophrenic vein. PNS stimulation was performed using Eupnea System device (RespiCardia Inc). Respiratory properties were assessed before and during PNS. PNS was assessed at a maximum of 10 mA.


Successful stimulation capture was achieved in 16 patients. Failure to capture occurred in three patients because of dislocation of leads. No adverse events were seen under maximum normal stimulation parameters for an overnight study.  No new arrhythmias, muscle contractions, arterial BP variations, pain, or unpleasant sensations were observed once PNS was delivered during sleep for these patients. It was confirmed that the catheter could be secured adequately to obtain consistent predictable stimulation thresholds without arousal from sleep. During occurrence of CSR, intermittent PNS signals were first confirmed to be successfully captured in 16 patients. When PNS was applied following a series of central sleep apneic events, a trend toward stabilization of breathing and heart rate.  An improvement in oxygen saturation and elevation of end-tidal CO2 was observed as longer continuous stimulation was performed. The period of stable breathing lasted as long as 10 to 20 min in some patients after stimulation.  They found that when electric connection to the nerve was consistent, stimulation resulted in a reduced hyperventilation followed by the reduction or elimination of CSR.
Compared with pre-PNS, during PNS there was a significant decrease in apnea-hypopnea index (33.8 ± 9.3 vs 8.1 ± 2.3, P = .00), an increase in mean and minimal oxygen saturation as measured by pulse oximetry (89.7% ± 1.6% vs 94.3% ± 0.9% and 80.3% ± 3.7% vs 88.5% ± 3.3%, respectively, all P = .00) and end-tidal CO2 (38.0 ± 4.3 mm Hg vs 40.3 ± 3.1 mm Hg, P = .02), but no significant difference in sleep efficiency (74.6% ± 4.1% vs 73.7% ± 5.4%, P = .36).


CSR is characterized by cyclical oscillations of respiration and apnea. The incidence of CSR ranges from 10% to 20% in patients with stable CHF and up to 40% to 50% of all patients with New York Heart Association functional class III?IV CHF.  Nocturnal breathing alterations in patients with CHF are believed to be due to hypersensitivity to CO 2 during sleep. Breathing is controlled by a negative feedback system in which an increase in Pa co 2 stimulates breathing, whereas a decrease in Pa co 2 inhibits breathing. Normally, Pa co 2 is maintained within a narrow range. Patients with CHF and CSA have a more brisk ventilatory response to CO 2 than those without CSA.
The preliminary results showed that in a small group of patients with CHF and CSR, 1 night of unilateral transvenous PNS improved indices of CSR and was not associated with adverse events.
The study was performed using temporary catheters or leads in the right-side brachiocephalic vein, SVC, or left-side pericardiophrenic vein to transvenously stimulate the hemidiaphragm through either the leftside or the right-side phrenic nerve. To consistently stimulate the phrenic nerve using acceptable and safe current levels ( < 10 mA), the stimulation electrode needs to be within 2 to 5 mm from the phrenic nerve.  This type of stimulation caused significantly improved respiratory parameters in patients with CHF and further support that oscillation of CO 2 level in the blood below and above the apneic threshold is a central mechanism leading to the CSR pattern of breathing. Stabilization of CO 2 levels through PNS produced a regular breathing pattern, improvement in oxygen saturation, and fewer apneic events.
Dr.  Isaac George: contributed to data evaluation and drafting of the manuscript.

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Endothelial Function and Cardiovascular Disease

Pathologist and AuthorLarry H Bernstein, MD, FCAP 


This discussion is a continuation of a series on Nitric Oxide, vascular relaxation, vascular integrity, and systemic organ dysfunctions related to inflammatory and circulatory disorders. In some of these, the relationships are more clear than others, and in other cases the vascular disorders are aligned with serious metabolic disturbances. This article, in particular centers on the regulation of NO production, NO synthase, and elaborates more on the assymetrical dimethylarginine (ADMA) inhibition brought up in a previous comment, and cardiovascular disease, including:

Recall, though, that in SIRS leading to septic shock, that there is a difference between the pulmonary circulation, the systemic circulation and the portal circulation in these events. The comment calls attention to:
Böger RH. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, explains the ‘L-arginine paradox’ and acts as a novel cardiovascular risk factor. J Nutr 2004; 134: 2842S–7S.

This observer points out that ADMA inhibits vascular NO production at concentrations found in pathophysiological conditions (i.e., 3–15 μmol/l); ADMA also causes local vasoconstriction when it is infused intra-arterially. ADMA is increased in the plasma of humans with hypercholesterolemia, atherosclerosis, hypertension, chronic renal failure, and chronic heart failure.

Increased ADMA levels are associated with reduced NO synthesis as assessed by impaired endothelium-dependent vasodilation. We’ll go into that more with respect to therapeutic targets – including exercise, sauna, and possibly diet, as well as medical drugs.

It is remarkable how far we have come since the epic discovery of 17th century physician, William Harvey, by observing the action of the heart in small animals and fishes, proved that heart receives and expels blood during each cycle, and argued for the circulation in man. This was a huge lead into renaissance medicine. What would he think now?

Key Words: eNOS, NO, endothelin, ROS, oxidative stress, blood flow, vascular resistance, cardiovascular disease, chronic renal disease, hypertension, diabetes, atherosclerosis, MI, exercise, nutrition, traditional chinese medicine, statistical modeling for targeted therapy.

Endothelial Function
The endothelium plays a crucial role in the maintenance of vascular tone and structure by means of eNOS, producing the endothelium-derived vasoactive mediator nitric oxide (NO), an endogenous messenger molecule formed in healthy vascular endothelium from the amino acid precursor L-arginine. Nitric oxide synthases (NOS) are the enzymes responsible for nitric oxide (NO) generation. The generation and actions of NO under physiological and pathophysiological conditions are exquisitely regulated and extend to almost every cell type and function within the circulation. While the molecule mediates many physiological functions, an excessive presence of NO is toxic to cells.

The enzyme NOS, constitutively or inductively, catalyses the production of NO in several biological systems. NO is derived not only from NOS isoforms but also from NOS-independent sources. In mammals, to date, three distinct NOS isoforms have been identified:

  1. neuronal NOS (nNOS),
  2. inducible NOS (iNOS), and
  3. endothelial NOS (eNOS).

The molecular structure, enzymology and pharmacology of these enzymes have been well defined, and reveal critical roles for the NOS system in a variety of important physiological processes. The role of NO and NOS in regulating vascular physiology, through neuro-hormonal, renal and other non-vascular pathways, as well as direct effects on arterial smooth muscle, appear to be more intricate than was originally thought.

Vallance et al. described the presence of asymmetric dimethylarginine (ADMA) as an endogenous inhibitor of eNOS in 1992. Since then, the role of this molecule in the regulation of eNOS has attracted increasing attention.
Endothelins are 21-amino acid peptides, which are active in almost all tissues in the body. They are potent vasoconstrictors, mediators of cardiac, renal, endocrine and immune functions and play a role in bronchoconstriction, neurotransmitter regulation, activation of inflammatory cells, cell proliferation and differentiation.

Endothelins were first characterised by Yanagisawa et al. (1988). The three known endothelins ET-1, -2 and -3 are structurally similar to sarafotoxins from snake venoms. ET-1 is the major isoform generated in blood vessels and appears to be the isoform of most importance in the cardiovascular system with a major role in the maintenance of vascular tone.

The systemic vascular response to hypoxia is vasodilation. However, reports suggest that the potent vasoconstrictor endothelin-1 (ET-1) is released from the vasculature during hypoxia. ET-1 is reported to augment superoxide anion generation and may counteract nitric oxide (NO) vasodilation. Moreover, ET-1 was proposed to contribute to increased vascular resistance in heart failure by increasing the production of asymmetric dimethylarginine (ADMA).

A study investigated the role of ET-1, the NO pathway, the potassium channels and radical oxygen species in hypoxia-induced vasodilation of large coronary arteries and found NO contributes to hypoxic vasodilation, probably through K channel opening, which is reversed by addition of ET-1 and enhanced by endothelin receptor antagonism. These latter findings suggest that endothelin receptor activation counteracts hypoxic vasodilation.

Endothelial dysfunction
Patients with Raynaud’s Phemonenon had abnormal vasoconstrictor responses to cold pressor tests (CPT) that were similar in primary and secondary RP. There were no differences in median flow-mediated and nitroglycerin mediated dilation or CPT of the brachial artery in the 2 populations. Patients with secondary RP were characterized by abnormalities in microvascular responses to reactive hyperemia, with a reduction in area under the curve adjusted for baseline perfusion, but not in time to peak response or peak perfusion ratio.

Plasma ET-1, ADMA, VCAM-1, and MCP-1 levels were significantly elevated in secondary RP compared with primary RP. There was a significant negative correlation between ET-1 and ADMA values and measures of microvascular perfusion but not macrovascular endothelial function. Secondary RP is characterized by elevations in plasma ET-1 and ADMA levels that may contribute to alterations in cutaneous microvascular function.

ADMA inhibits vascular NO production within the concentration range found in patients with vascular disease. ADMA also causes local vasoconstriction when infused intra-arterially, and increases systemic vascular resistance and impairs renal function when infused systemically. Several recent studies have supplied evidence to support a pathophysiological role of ADMA in the pathogenesis of vascular dysfunction and cardiovascular disease. High ADMA levels were found to be associated with carotid artery intima-media-thickness in a study with 116 clinically healthy human subjects. Taking this observation further, another study performed with hemodialysis patients reported that ADMA prospectively predicted the progression of intimal thickening during one year of follow-up.

In a nested, case-control study involving 150 middle-aged, non-smoking men, high ADMA levels were associated with a 3.9-fold elevated risk for acute coronary events. Clinical and experimental evidence suggests elevation of ADMA can cause a relative L-arginine deficiency, even in the presence of “normal” L-arginine levels. As ADMA is a competitive inhibitor of eNOS, its inhibitory action can be overcome by increasing the concentration of the substrate, L-arginine. Elevated ADMA concentration is one possible explanation for endothelial dysfunction and decreased NO production in these diseases.
Metabolic Regulation of L-arginine and NO Synthesis 
Methylation of arginine residues within proteins or polypeptides occurs through N-methyltransferases, which utilize S-adenosylmethionine as a methyl donor. After proteolysis of these proteins or polypeptides, free ADMA is present in the cytoplasm. ADMA can also be detected in circulating blood plasma. ADMA acts as an inhibitor of eNOS by competing with the substrate of this enzyme, L-arginine. The ensuing reduction in nitric oxide synthesis causes vascular endothelial dysfunction and, subsequently, atherosclerosis. ADMA is eliminated from the body via urinary excretion and via metabolism by the enzyme DDAH to citrulline and dimethylamine.
Supplementation with L-arginine in animals with experimentally-induced vascular dysfunction atherosclerosis improves endothelium-dependent vasodilation. Moreover, L-arginine supplementation results in enhanced endothelium-dependent inhibition of platelet aggregation, inhibition of monocyte adhesion, and reduced vascular smooth muscle proliferation. One mechanism that explains the occurrence of endothelial dysfunction is the presence of elevated blood levels of asymmetric dimethylarginine (ADMA) – an L-arginine analogue that inhibits NO formation and thereby can impair vascular function. Supplementation with L-arginine has been shown to restore vascular function and to improve the clinical symptoms of various diseases associated with vascular dysfunction.

Beneficial Effects of L-Arginine

  • Angina
  • Congestive Heart Failure
  • Hypertension
  • Erectile dysfunction
  • Sickle Cell Disease and Pulmonary Hypertension

The ratio of L-arginine to ADMA is considered to be the most accurate measure of eNOS substrate availability. This ratio will increase during L-arginine supplementation, regardless of initial ADMA concentration. Due to the pharmacokinetics of oral L-arginine and the positive results from preliminary studies, it appears supplementation with a sustained-release L-arginine preparation will achieve positive therapeutic results at lower dosing levels.

Many prospective clinical trials have shown that the association between elevated ADMA levels and major cardiovascular events and total mortality is robust and extends to diverse patient populations. However, we need to define more clearly in the future who will profit from ADMA determination, in order to use this novel risk marker as a more specific diagnostic tool.
Elimination of ADMA by way of DDAH
Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both LNMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. These investigators ask whether ADMA is the underlying issue related to the pathogenesis of the vascular disorder.
They identified the structure of human DDAH-1 and probed the function of DDAH-1 both by deleting the Ddah1 gene in mice and by using DDAH-specific inhibitors that is shown by crystallography, bind to the active site of human DDAH-1. The loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. The results suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.
Methylarginines are formed when arginine residues in proteins are methylated by the action of protein arginine methyltransferases (PRMTs), and free methylarginines are liberated following proteolysis. Clear demonstration of an effect of endogenous ADMA and L-NMMA on cardiovascular physiology would be of importance, not only because of the implications for disease, but also because it would expose a link between post-translational modification of proteins and signaling through a proteolytic product of these modified proteins.
Which is it? ADMA or DDHA: Intrusion of a Genetic alteration.
The study showed that loss of DDAH expression or activity causes endothelial dysfunction, we believe that DDAH inhibition could potentially be used therapeutically to limit excessive NO production, which can have pathological effects. They then showed treated cultured isolated blood vessels with lipopolysaccharide (LPS) induced expression of the inducible isoform of NO synthase (iNOS) and generated high levels of NO, which were blocked by the iNOS-selective inhibitor 1400W and by DDAH inhibitors. Treatment of isolated blood vessels with DDAH inhibitors significantly increased ADMA accumulation in the culture medium. Treatment of isolated blood vessels with bacterial LPS led to the expected hyporeactivity to the contractile effects of phenylephrine, which was reversed by treatment with a DDAH inhibitor. The effect of the DDAH inhibitor was large and stereospecific, and was reversed by the addition of L-arginine.
In conclusion, genetic and chemical-biology approaches provide compelling evidence that loss of DDAH-1 function results in increased ADMA concentrations and thereby disrupts vascular NO signaling. A broader implication of this study is that post-translational methylation of arginine residues in proteins may have downstream effects by affecting NO signaling upon hydrolysis and release of the free methylated amino acid. This signaling pathway seems to have been highly conserved through evolution.

The crucial role of nitric oxide (NO) for normal endothelial function is well known. In many conditions associated with increased risk of cardiovascular diseases such as hypercholesterolemia, hypertension, abdominal obesity, diabetes and smoking, NO biosynthesis is dysregulated, leading to endothelial dysfunction. The growing evidence from animal and human studies indicates that endogenous inhibitors of endothelial NO synthase such as asymmetric dimethylarginine (ADMA) and NG-monomethyl-L-arginine (L-NMMA) are associated with the endothelial dysfunction and potentially regulate NO synthase.

Nitric Oxide Synthase

Asymmetric dimethylarginine (ADMA) is one of three known endogenously produced circulating methylarginines (i.e. ADMA, NG-monomethyl-L-arginine (L-NMMA) and symmetrically methylated NG, NG-dimethyl-L-arginine). ADMA is formed by the action of protein arginine methyltransferases that methylate arginine residues in proteins and after which free ADMA is released. ADMA and L-NMMA can competitively inhibit NO elaboration by displacing L-arginine from NO synthase (NOS). The amount of methylarginines is related to overall metabolic activity and the protein turnover rate of cells. Although methylarginines are excreted partly by the kidneys, the major route of elimination of ADMA in humans is metabolism by the dimethylarginine dimethylaminohydrolase enzymes[ dimethylarginine dimethylaminohydrolase-1 and -2 (DDAH)] enzymes. Inhibition of DDAH leads to the accumulation of ADMA and consequently to inhibition of NO-mediated endothelium dependent relaxation of blood vessels.
The potential role of ADMA in angina pectoris has been evaluated by Piatti and co-workers, who reported ADMA levels to be higher in patients with cardiac syndrome X (angina pectoris with normal coronary arteriograms) than in controls. According to preliminary results from the CARDIAC (Coronary Artery Risk Determination investigating the influence of ADMA Concentration) study, patients with coronary heart disease (n 816) had a higher median ADMA plasma concentration than age and sex matched controls (median 0.91 vs. 0.70 mol/l; p 0.0001). Further, in a prospective Chinese study, a high plasma ADMA level independently predicted subsequent cardiovascular adverse events (cardiovascular death, myocardial infarction, and repeated revascularization of a target vessel).

Protein detoxification pathway.

Protein detoxification pathway. (Photo credit: Wikipedia)

There are only few published findings concerning variations in human DDAH. However, polymorphisms in other genes potentially related to risk factors for endothelial dysfunction and cardiovascular events have been studied. Reduced NO synthesis has been implicated in the development of atherosclerosis. For example, there are some functionally important variants of the NOS that could affect individual vulnerability to atherosclerosis by changing the amount of NO generated by the endothelium.
There are probably several functional variations in genes coding DDAH enzymes in different populations. Some of them could confer protection against the harmful effects of elevated ADMA and others impair enzyme function causing accumulation of ADMA in cytosol and/or blood.
In a study of 16 men with either low or high plasma ADMA concentrations were screened to identify DDAH polymorphisms that could potentially be associated with increased susceptibility to cardiovascular diseases. In that study a novel functional mutation of DDAH-1 was identified; the mutation carriers had a significantly elevated risk for cardiovascular disease and a tendency to develop hypertension. These results confirmed the clinical role of DDAH enzymes in ADMA metabolism. Furthermore, it is possible that more common variants of DDAH genes contribute more widely to increased cardiovascular risk.
We found a rare variation in the DDAH-1 gene, which is associated with elevated plasma concentrations of ADMA in heterozygous mutation carriers. There was also an increased prevalence of CHD and a tendency to hypertension among individuals with this DDAH-1 mutation. These observations highlight the importance of ADMA as a possible risk factor and emphasize the essential role of DDAH in regulating ADMA levels.

ADMA Elevation and Coronary Artery Disease
Endothelial dysfunction may be considered as a systemic disorder and involves different vascular beds. Coronary endothelial dysfunction (CED) precedes the development of coronary. Endothelial dysfunction is characterized by a reduction in endogenous nitric oxide (NO) activity, which may be accompanied by elevated plasma asymmetric dimethylarginine (ADMA) levels. ADMA is a novel endogenous competitive inhibitor of NO synthase (NOS), an independent marker for cardiovascular risk.

English: Structure of asymmetric dimethylargin...

English: Structure of asymmetric dimethylarginine; ADMA; N,N-Dimethylarginine Deutsch: Asymmetrisches Dimethylarginin; N,N-Dimethyl-L-arginin; Guanidin-N,N-dimethylarginin (Photo credit: Wikipedia)

In a small study fifty-six men without obstructive coronary artery disease (CAD) who underwent coronary endothelial function testing were studied. Men with CED had significant impairment of erectile function (P ¼ 0.008) and significantly higher ADMA levels (0.50+0.06 vs. 0.45+0.07 ng/mL, P ¼ 0.017) compared with men with normal endothelial function. Erectile function positively correlated with coronary endothelial function. This correlation was independent of age, body mass index, high-density lipoprotein, C-reactive protein, homeostasis model assessment of insulin resistance index, and smoking status, suggesting that CED is independently associated with ED and plasma ADMA concentration in men with early coronary atherosclerosis.

ADMA and Chronic Renal Failure in Hepatorenal Syndrome
The concentration of SDMA was significantly higher in the patients with HRS compared to the patients without HRS and it was also higher than the values obtained from the healthy participants (1.76 ± 0.3 μmol/L; 1.01 ± 0.32 and 0.520 ± 0.18 μmol/L, respectively; p < 0.01). The concentrations of ADMA were higher in the cirrhotic patients with HRS than in those without this serious complication of cirrhosis. The concentration of ADMA in all the examined cirrhotic patients was higher than those obtained from healthy volunteers (1.35 ± 0.27 μmol/L, 1.05 ± 0.35 μmol/L and 0.76 ± 0.21 μmol/L, respectively). In the patients with terminal alcoholic liver cirrhosis, the concentrations
of ADMA and SDMA correlated with the progress of cirrhosis as well as with the development of cirrhosis complications. In the patients with HRS there was a positive correlation between creatinine and SDMA in plasma (r2 = 0.0756, p < 0.001) which was not found between creatinine and ADMA. The results demonstrate that the increase in SDMA concentration is proportionate to the progression of chronic damage of the liver and kidneys. Increased ADMA concentration can be a causative agent of renal insufficiency in patients with cirrhosis.

In patients with cirrhosis, ADMA, as well as SDMA could be markers for kidney insufficiency development. Accumulation of ADMA in plasma causes kidney
vasoconstriction and thereby retention of SDMA. Considering that ADMA has several damaging effects, it can be concluded that modulation of the activity of enzyme which participates in ADMA catabolism may represent a new therapeutic goal which is intended to reduce the progress of liver and kidney damage and thus the development of HRS.

ADMA Therapeutic Targets
Elevated plasma concentrations of the endogenous nitric oxide synthase
inhibitor asymmetric dimethylarginine (ADMA) are found in various clinical settings, including

  • renal failure,
  • coronary heart disease,
  • hypertension,
  • diabetes and
  • preeclampsia.

In healthy people acute infusion of ADMA promotes vascular dysfunction,
and in mice chronic infusion of ADMA promotes progression of atherosclerosis.
Thus, ADMA may not only be a marker but also an active player in cardiovascular disease, which makes it a potential target for therapeutic interventions.

This review provides a summary and critical discussion of the presently available data concerning the effects on plasma ADMA levels of cardiovascular drugs, hypoglycemic agents, hormone replacement therapy, antioxidants, and vitamin supplementation.
We assess the evidence that the beneficial effects of drug therapies on vascular function can be attributed to modification of ADMA levels. To develop more specific ADMA-lowering therapies, mechanisms leading to elevation of plasma ADMA concentrations in cardiovascular disease need to be better understood.

ADMA is formed endogenously by degradation of proteins containing arginine residues that have been methylated by S-adenosylmethionine-dependent methyltransferases (PRMTs). There are two major routes of elimination: renal excretion and enzymatic degradation by the dimethylarginine dimethylaminohydrolases (DDAH-1 and -2).

Oxidative stress causing upregulation of PRMT expression and/or attenuation of DDAH activity has been suggested as a mechanism and possible drug target in clinical conditions associated with elevation of ADMA. As impairment of DDAH activity or capacity is associated with substantial increases in plasma ADMA concentrations, DDAH is likely to emerge as a prime target for specific therapeutic interventions.

Cardiovascular diseases (CVD) in diabetic patients have endothelial dysfunction as a key pathogenetic event. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), plays a pivotal role in endothelial dysfunction. Different natural polyphenols have been shown to preserve endothelial function and prevent CVD. Another study assessed the effect of silibinin, a widely used flavonolignan from milk thistle, on ADMA levels and endothelial dysfunction in db/db mice.

Plasma and aorta ADMA levels were higher in db/db than in control lean mice. Silibinin administration markedly decreased plasma ADMA; consistently, aorta ADMA was reduced in silibinin-treated animals. Plasma and aorta ADMA levels exhibited a positive correlation, whereas liver ADMA was inversely correlated with both plasma and aorta ADMA concentrations. Endothelium-(NO)-dependent vasodilatation to ACh was impaired in db/db mice and was restored in the silibinin group, in accordance with the observed reduction of plasma and vascular levels of ADMA. Endothelium-independent vasodilatation to SNP was not modified by silibinin administration.

Endothelin Inhibitors
Endothelins are potent vasoconstrictors and pressor peptides and are important mediators of cardiac, renal andendocrine functions. Increased ET-1 levels in disease states such as congestive heart failure, pulmonary hypertension, acute myocardial infarction, and renal failure suggest the endothelin system as an attractive target for pharmacotherapy. A non-peptidic, selective, competitive endothelin receptor antagonist with an affinity for the ETA receptor in the subnanomolar range was administered by continuous intravenous infusion to beagle dogs, rats, and Goettingen minipigs. It caused mild arteriopathy characterised by segmental degeneration in the media of mid- to large-size coronary arteries in the heart of dog, but not rat or minipig.

The lesions only occurred in the atrium and ventricle. Frequency and severity of the vascular lesions was not sex or dose related. No effects were noted in blood vessels in other organs or tissue. Plasma concentrations at steady state, and overall exposure in terms of AUC(0–24h) were higher in minipig and rat than the dog but did not cause cardiac arteriopathy. These findings concur with those caused by other endothelin anatagonists, vasodilators and positive inotropic: vasodilating drugs such as potassium channel openers, phosphodiesterase inhibitors and peripheral vasodilators.

Results by echocardiography indicate treatment-related local vasodilatation in the coronary arteries. These data suggest that the coronary arteriopathy may be the result of exaggerated pharmacology. Sustained vasodilatation in the coronary vascular bed may alter flow dynamics and lead to increased shear stress and tension on the coronary wall with subsequent microscopic trauma. In our experience with a number of endothelin receptor antagonists, the cardiac arteriopathy was only noted in studies with multiple daily or continuous intravenous infusion inviting speculation that sustained high plasma levels are needed for development of the lesions.

Up-regulation of vascular endothelin type B (ETB) receptors is implicated in the
pathogenesis of cardiovascular disease. Culture of intact arteries has been shown to induce similar receptor alterations and has therefore been suggested as a suitable method for, ex vivo, in detail delineation of the regulation of endothelin receptors. We hypothesize that mitogen-activated kinases (MAPK) and protein kinase C (PKC) are involved in the regulation of endothelin ETB receptors in human internal mammary arteries.

The endothelin-1-induced contraction (after endothelin ETB receptor desensitization) and the endothelin ETA receptor mRNA expression levels were not altered by culture. The sarafotoxin 6c contraction, endothelin ETB receptor protein and mRNA expression levels were increased. This increase was antagonized by;

PKC inhibitors (10 μM bisindolylmaleimide I and 10 μM Ro-32-0432), and
inhibitors of the p38, extracellular signal related kinases 1 and 2 (ERK1/2) and C-jun terminal kinase (JNK) MAPK pathways
Endothelin Receptor Antagonist Tezosentan
The effects of changes in the mean (Sm) and pulsatile (Sp) components of arterial wall shear stress on arterial dilatation of the iliac artery of the anaesthetized dog were examined in the absence and presence of the endothelin receptor antagonist tezosentan (10 mg kg_1 I.V.; Ro 61-0612; [5-isopropylpyridine-2-sulphonic acid 6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2-(2-1H-tetrazol-5-ylpyridin-4-yl)-pyrimidin-4-ylamide]).

Changes in shear stress were brought about by varying local peripheral resistance and stroke volume using a distal infusion of acetylcholine and stimulation of the left ansa subclavia. An increase in Sm from 1.81 ± 0.3 to 7.29 ± 0.7 N m_2 (means ± S.E.M.) before tezosentan caused an endothelium-dependent arterial dilatation which was unaffected by administration of tezosentan for a similar increase in Sm from 1.34 ± 0.6 to 5.76 ± 1.4 N m_2 (means ± S.E.M.).

In contrast, increasing the Sp from 7.1 ± 0.8 to a maximum of 11.5 ± 1.1 N m_2 (means ± S.E.M.) before tezosentan reduced arterial diameter significantly. Importantly, after administration of tezosentan subsequent increases in Sp caused arterial dilatation for the same increase in Sp achieved prior to tezosentan, increasing from a baseline of 4.23 ± 0.4 to a maximum of 9.03 ± 0.9 N m_2 (means ± S.E.M.; P < 0.001). The results of this study provide the first in vivo evidence that pulsatile shear stress is a stimulus for the release of endothelin from the vascular endothelium.

Exercise and Diet
Vascular endotheliumis affected by plasma asymmetric dimethylarginine (ADMA), and it is induced by inflammatory cytokines of tumour necrosis factor (TNF)-a in vitro. Would a tight glycemic control restore endothelial function in patients with type-2 diabetes mellitus (DM) with modulation of TNF-a and/or reduction of ADMA level? In 24 patients with type-2 DM, the flow-mediated, endothelium-dependent dilation (FMD: %) of brachial arteries during reactive hyperaemia was determined by a high-resolution ultrasound method. Blood samples for glucose, cholesterol, TNF-a, and ADMA analyses were also collected from these patients after fasting. No significant glycemic or FMD changes were observed in 10 patients receiving the conventional therapy.

In 14 patients who were hospitalized and intensively treated, there was a significant decrease in glucose level after the treatment [from 190+55 to 117+21 (mean+SD) mg/dL, P , 0.01]. After the intensive control of glucose level, FMD increased significantly (from 2.5+0.9 to 7.2+3.0%), accompanied by a significant (P , 0.01) decrease in TNF-a (from 29+16 to 11+9 pg/dL) and ADMA (from 4.8+1.5 to 3.5+1.1 mM/L) levels. The changes in FMD after treatment correlated inversely with those in TNF-a (R ¼ 20.711, P , 0.01) and ADMA (R ¼ 20.717, P , 0.01) levels.
The exaggerated blood pressure response to exercise (EBPR) is an independent predictor of hypertension. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide inhibitor and higher plasma levels of ADMA are related to increased cardiovascular risk. The aim of this study is to identify the relationship between ADMA and EBPR.

A total of 66 patients (36 with EBPR and 30 as controls) were enrolled in the study. EBPR is defined as blood pressure (BP) measurements ≥200/100 mmHg during the treadmill test. All the subjects underwent 24-h ambulatory BP monitoring. L-arginine and ADMA levels were measured using a high performance lipid chromatography technique.

The serum ADMA levels were increased in the EBPR group compared to the healthy controls (4.0±1.4 vs 2.6±1.1 μmol/L respectively, P=0.001), but L-arginine levels were similar in the 2 groups (P=0.19). The serum ADMA levels were detected as an independent predictor of EBPR (odds ratio 2.28; 95% confidence interval 1.22–4.24; P=0.002). Serum ADMA levels might play a role in EBPR to exercise.

Endothelial dysfunction occurs early in atherosclerosis in response to cardiovascular risk factors. The occurrence of endothelial dysfunction is primarily the result of reduced nitric oxide (NO) bioavailabilty. It represents an independent predictor of cardiovascular events and predicts the prognosis of the patient. Therefore, endothelial function has been identified as a target for therapeutic intervention. Regular exercise training is a nonpharmacological option to improve endothelial dysfunction in patients with cardiovascular disease by increasing NO bioavailability.

Peripheral Arterial Disease (PAD) is a cause of significant morbidity and mortality in the Western world. risk factor modification and endovascular and surgical revascularisation are the main treatment options at present. However, a significant number of patients still require major amputation. There is evidence that nitric oxide (NO) and its endogenous inhibitor asymmetric dimethylarginine (ADMA) play significant roles in the pathophysiology of PAD.

This paper reviews experimental work implicating the ADMA-DDAH-NO pathway in PAD, focusing on both the vascular dysfunction and both the vascular dysfunction and effects within the ischaemic muscle, and examines the potential of manipulating this pathway as a novel adjunct therapy in PAD.

In patients with CHF, the peripheral vascular resistance is increased via activation of the neurohormonal system, namely by autonomous sympathetic nervous system, rennin -angiotensin- aldosterone system (RAAS), and endothelin system. The vascular endothelial function in patients with CHF, mainly represented by the endothelium-dependent vasodilation, is altered.

Such alteration leads to increased vascular tone and remodeling of the blood vessels, reducing the peripheral blood flow. Hence, the amount of oxygen for the skeletal muscles is compromised, with progressive exercise intolerance. The vascular endothelial dysfunction in the CHF is mainly due to the decrease of the nitric oxide production induced by the reduced gene expression of eNOS and increased oxidative stress.

The endothelium-dependent vasodilation alteration has been virtually reported in all cardiovascular diseases. Using sauna bath as therapeutic option for CHF is not very recent, since in the 1950’s the first studies with CHF patients were conducted and the potential beneficial effect of sauna was suggested. However, some time later the studies emphasized especially its risks and recommended caution in its use for cardiac patients.

Frequently, sports medicine physicians are invited to evaluate the impact of the sauna on diseases and on health in general. Sauna can be beneficial or dangerous depending on its use. In the past few years the sauna is considered beneficial for the cardiovascular diseases’ patients, as the heart failure and lifestyle-related diseases, mainly by improving the peripheral endothelial function through the increase in cardiac output and peripheral vasodilation.

It is widely known that the vasodilators, such as angiotensin converting enzyme inhibitors, improve the CHF and increase the peripheral perfusion. Since the endothelial function is altered in CHF, the endothelium is considered as a new therapeutic target in heart failure. Hence, the angiotensin converting enzyme inhibitors and physical training improve the endothelial function in CHF patients. One of the proposed mechanisms for the alteration of the endothelium-dependent vasodilation would be through the decrease of the NO production in the peripheral vessels in CHF patients. The decrease of peripheral perfusion would decrease the shear stress. The shear stress is an important stimulus for NO production and eNOS expression. On the other hand, the heat increases the cardiac output and improves the peripheral perfusion in CHF patients. Consequently, with the cardiac output improvement in CHF patients, an increase of the shear stress, NO production and eNOS expression are expected.

Sauna bath
The sauna bath represents a heat load of 300-600 W/m2 of body surface area. The skin temperature rapidly increases to ± 40o-41oC and the thermoregulatory mechanisms are triggered. Evaporative heat transfer by sweating is the only effective body heat loss channel in dry sauna. The sweating begins rapidly and reaches its maximum level in ± 15 min. The total sweat secretion represents a heat loss of about 200 W/m2 of the body surface area. The body cannot compensate for the heat load and causing elevation of internal temperature. The skin circulation increases substantially. The skin blood flow, in the thermo-neutral condition (± 20oC) and in rest corresponding to ± 5-10% of the cardiac output, can reach ± 50-70% of the cardiac output.

Thermal therapy in 60oC produced systemic arterial, pulmonary arterial and venous vasodilation, reduced the preload and afterload and improved the cardiac output and the peripheral perfusion, clinical symptoms, life quality, and cardiac arrhythmias in CHF patients. In infants with severe CHF secondary to ventricular septal defect, the sauna therapy decreased the systemic vascular resistance and increased the cardiac output. The sauna benefits in CHF patients are possibly caused by the improvement of the vascular endothelial function and normalization of the neurohormonal system .

Ikeda et al. discovered that the observed improvements in the sauna therapy are due to the eNOS expression increase in the arterial endothelium. They later showed that the thermal therapy with sauna improves the survival of the TO-2 cardiomyopathic hamsters with CHF and, more recently, showed that the repetitive therapy with sauna increases the eNOS expression and the nitric oxide production in artery endothelium of TO-2 cardiomyopathic hamsters with CHF.
Whether n-3 polyunsaturated fatty acid (PUFA) supplementation and/or diet intervention might have beneficial influence on endothelial function was assessed using plasma levels of ADMA and L-arginine. A male population (n = 563, age 70 ± 6 yrs) with long-standing hyperlipidemia, characterized as high risk individuals in 1970–72, was included, randomly allocated to receive placebo n-3 PUFA capsules (corn oil) and no dietary advice (control group), dietary advice (Mediterranean type), n-3 PUFA capsules, or dietary advice and n-3 PUFA combined and followed for 3 years. Fasting blood samples were drawn at baseline and the end of the study.

Compliance with both intervention regimens were demonstrated by changes in serum fatty acids and by recordings from a food frequency questionnaire. No influence of either regimens on ADMA levels were obtained. However, n-3 PUFA supplementation was accompanied by a significant increase in L-arginine levels, different from the decrease observed in the placebo group (p < 0.05). In individuals with low body mass index (<26 kg/m2), the decrease in L-arginine on placebo was strengthened (p = 0.01), and the L-arginine/ADMA ratio was also significantly reduced (p = 0.04). In this rather large randomized intervention study, ADMA levels were not influenced by n-3 PUFA supplementation or dietary counselling. n-3 PUFA did, however, counteract the age related reduction in L-arginine seen on placebo, especially in lean individuals, which might be considered as an improvement of endothelial function.

Traditional Chinese Medicine

Traditional Chinese Medicine (TCM) involves a broad range of empirical testing and refinement and plays an important role in the health maintenance for people all over the world. However, due to the complexity of Chinese herbs, a full understanding of TCM’s action mechanisms is still unavailable despite plenty of successful applications of TCM in the treatment of various diseases, including especially cardiovascular diseases (CVD), one of the leading causes of death.

An integrated system of TCM has been constructed to uncover the underlying action mechanisms of TCM by incorporating the chemical predictors, target predictors and network construction approaches from three representative Chinese herbs, i.e., Ligusticum chuanxiong Hort., Dalbergia odorifera T. Chen and Corydalis yanhusuo WT Wang widely used in CVD treatment, by combined use of drug absorption, distribution, metabolism and excretion (ADME) screening and network pharmacology techniques. These studies have generated 64 bioactive ingredients and identified 54 protein targets closely associated with CVD, to clarify some of the common conceptions in TCM, and provide clues to modernize such specific herbal medicines.

Ligusticum chuanxiong Hort., Dalbergia odorifera T. Chen and Corydalis yanhusuo WT Wang
Twenty-two of 194 ingredients in Ligusticum chuanxiong demonstrate good bioavailability (60%) after oral administration. Interestingly, as the most abundant bioactive compound of Chuanxiong, Ligustilide (M120) only has an adequate OB of 50.10%, although it significantly inhibits the vasoconstrictions induced by norepinephrine bitartrate (NE) and calcium chloride (CaCl2). Indeed, this compound can be metabolized to butylidenephthalide, senkyunolide I (M156), and senkyunolide H (M155) in vivo.

The three natural ingredients produce various pharmacological activities in cerebral blood vessels, the general circulatory system and immune system including spasmolysis contraction effects, inhibitory effects of platelet aggregation and anti-proliferative activity, and thus improve the therapeutic effect on patients. Cnidilide (M93, OB = 77.55%) and spathulenol (M169, OB = 82.37%) also closely correlate with the smooth muscle relaxant action, and thereby have the strongest spasmolytic activity. Carotol (M8) and Ferulic acid (M105) with an OB of 149.03% and 86.56%, respectively, demonstrate better bioavailability compared with cnidilide and spathulenol, which show strong antifungal, antioxidant and anti-inflammatory activity.

The pharmacological activity of ferulic acid results in the improvement of blood fluidity and the inhibition of platelet aggregation, which may offer beneficial effects against cancer, CVD, diabetes and Alzheimer’s disease. As for 3-n-butylphthalide (M85, OB = 71.28%), this compound is not only able to inhibit platelet aggregation, but also decreases the brain infarct volume and enhances microcirculation, thus benefiting patients with ischemic stroke. Platelet aggregation represents a multistep adhesion process involving distinct receptors and adhesive ligands, with the contribution of individual receptor-ligand interactions to the aggregation process depending on the prevailing blood flow conditions, implying that the rheological (blood flow) conditions are an important impact factor for platelet aggregation. Moreover, thrombosis, the pathological formation of platelet aggregates and one of the biggest risk factors for CVD, occludes blood flow causing stroke and heart attack. This explains why the traditional Chinese herb Ligusticum chuanxiong that inhibits platelet aggregates forming and promotes blood circulation can be used in treatment of CVD.

Twenty-six percent (24 of 93) of the ingredients in Dalbergia odorifera meet the OB > 60% criterion irrespective of the pharmacological activity. Relatively high bioavailability values were predicted for the mainly basic compounds odoriflavene (M275, OB = 84.49%), dalbergin (M247, OB = 78.57%), sativanone (M281, OB = 73.01%), liquiritigenin (M262, OB = 67.19%), isoliquiritigenin (M259, OB = 61.38%) and butein (M241, OB = 78.38%). Interestingly, all of the six ingredients show obvious anti-inflammatory property. Butein, liquiritigenin and isoliquiritigenin inhibit cell inflammatory responses by suppressing the NF-κB activation induced by various inflammatory agents and carcinogens, and by decreasing the NF-κB reporter activity. Inflammation occurs with CVD, and Dalbergia odorifera, one of the most potent anti-cardiovascular and anti-cerebrovascular agents, exerts great anti-inflammatory activity.

Corydalis yanhusuo has gained ever-increasing popularity in today’s world because of its therapeutic effects for the treatment of cardiac arrhythmia disease, gastric and duodenal ulcer and menorrhalgia. In our work, 21% (15 of 73) of chemicals in this Chinese herb display good OB (60% or even high), and the four main effective ingredients are natural alkaloid agents.

Dehydrocorydaline blocks the release of noradrenaline from the adrenergic nerve terminals in both the Taenia caecum and pulmonary artery, and thereby inhibits the relaxation or contraction of adrenergic neurons. As for dehydrocavidine with an OB of 47.59%, this alkaloid exhibits a significant spasmolytic effect, which acts via relaxing smooth muscle.

In recent years, CVD has been at the top list of the most serious health problems. Many different types of therapeutic targets have already been identified for the management and prevention of CVD, such as endothelin and others. The key question asked is

  • what the interactions of the active ingredients of the Chinese herbs are with their protein targets in a systematic manner and
  • how do the corresponding targets change under differential perturbation of the chemicals?

The study used an unbiased approach to probe the proteins that bind to the small molecules of interest in CVD on the basis of the Random Forest (RF) and Support Vector Machine (SVM) methods combining the chemical, genomic and pharmacological information for drug targeting and discovery on a large scale. Applied to 64 ingredients derived from the three traditional Chinese medicines Dalbergia odorifera, Ligusticum chuanxiong and Corydalis yanhusuo, which show good OB, 261 ligand-target interactions have been constructed, 221 of which are enzymes, receptors, and ion channels. This indicates that chemicals with multiple relative targets are responsible for the high interconnectedness of the ligand-target interactions. The promiscuity of drugs has restrained the advance in recent TCM, because they were thought to be undesirable in favor of more target-specific drugs.

Target Identification and Validation
To validate the reliability of these target proteins, the researchers performed a docking analysis to select the ligand-protein interactions with a binding free energies of ≤−5.0 kcal/mol, which leads to the sharp reduction of the interaction number from 5982 to 760. These drug target candidates were subsequently subject to PharmGkb (available online:; accessed on 1 December 2011), a comprehensive disease-target database, to investigate whether they were related to CVD or not, and finally, 54 proteins were collected and retained.

Fourty-two proteins (76%) were identified as the targets of Ligusticum chuanxiong, such as dihydrofolate reductase (P150), an androgen receptor (P210) and angiotensin-converting enzyme (P209) that were involved in the development of CVD. Of the proteins, seven and two were recognized as those of Dalbergia odorifera and Corydalis yanhusuo, respectively. For Dalbergia odorifera, this Chinese herb has 48 potential protein targets, 13 of which have at least one link to other drugs.

The three herbs share 29 common targets, accounting for 52.7% of the total number. Indeed, as one of the most important doctrines of TCM
abstracted from direct experience and perception, “multiple herbal drugs for one disease” has played an undeniable role. These studies explored the targets of the three Chinese herbs, indicating that these drugs target the same targets simultaneously and exhibit similar pharmacological effects on CVD. This is consistent with the theory of “multiple herbal drugs for one disease”.

The three Chinese herbs possess specific targets. The therapeutic efficacy of a TCM depends on multiple components, targets and pathways. The complexity becomes a huge obstacle for the development and innovation of TCM. For example, the Chinese herb Ligusticum chuanxiong identifies the protein caspase-3 (P184), a cysteinyl aspartate-specific protease, as one of its specific targets, and exhibits inhibitory effects on the activity of this protease. In fact, connective tissue growth factor enables the activation of caspase-3 to induce apoptosis in human aortic vascular smooth muscle cells.

Thus, modulation of the activity of caspase-3 with Ligusticum chuanxiong suggests an efficient therapeutic approach to CVD. The Chinese herb Dalbergia odorifera has the α-2A adrenergic receptor (P216) as its specific target and probably blocks the release of this receptor, and thus influences its action. As for Corydalisyanhusuo, the protein tyrosine-protein kinase JAK2 (P9) is the only specific target of this Chinese herb. The results indicate different specific targets possessed by the three Chinese herbs.

Ligand-Candidate Target and Ligand-Potential Target Networks
Previous studies have already reported the relationships of the small molecules with CVD, which indicates the reliability of our results [45,46]. Regarding the candidate targets, we have found that prostaglandin G/H synthase 2 (P46) and prostaglandin G/H synthase 1 (P47) possess the largest number of connected ingredients. Following are nitric-oxide synthase, endothelial (P66) and tyrosine-protein phosphatase non-receptor type 1 (P8), which have 62 and 61 linked chemicals, respectively.
The 29 targets shared by the three traditional Chinese herbs exhibit a high degree of correlations with CVD, which further verifies their effectiveness for the treatment of CVD. These results provide a clear view of the relationships of the target proteins with CVD and other related diseases, which actually link the Chinese herbs and the diseases via the protein targets. This result further explains the theory of “multiple herbal drugs for one disease” based on molecular pharmacology.

Target-Pathway Network
Cells communicate with each other using a “language” of chemical signals. The cell grows, divides,or dies according to the signals it receives. Signals are generally transferred from the outside of the cell. Specialized proteins are used to pass the signal—a process known as signal transduction. Cells have a number of overlapping pathways to transmit signals to multiple targets. Ligand binding in many of the signaling proteins in the pathway can change the cellular communication and finally affect cell growth and proliferation. The authors extracted nine signal pathways closely associated with CVD in PharmGkb (available online:; accessed on 1 December 2011).

As the main components in the VEGF system, proto-oncogene tyrosine-protein kinase Src, eNOS, and hsp90-α is also recognized as common targets of Dalbergia odorifera, Ligusticum chuanxiong and Corydalis yanhusuo, which are efficient for the treatment of CVD. This implies that the candidate drugs can target different target proteins involved in the same or different signal pathways, and thereby have potential effects on the whole signal system.

Target Prediction
In search of the candidate targets, the model that efficiently integrates the chemical, genomic and pharmacological information for drug targeting and discovery on a large scale is based on the two powerful methods Random Forest (RF) and Support Vector Machine (SVM). The model is supported by a large pharmacological database of 6511 drugs and 3999 targets extracted from the DrugBank database (available online:; accessed on 1 June 2011), and shows an impressive performance of prediction for drug-target interaction, with a concordance of 85.83%, a sensitivity of 79.62% and a specificity of 92.76%. the candidate targets were selected according to the criteria that the possibility of interacting with potential candidate targets was higher than 0.6 for the RF model and 0.7 for the SVM model. The obtained candidate targets were finally reserved and were further predicted for their targets.

Target Validation
Molecular docking analysis was carried out using the AutoDock software (available online:; accessed on 1 February 2012). This approach performs the docking of the small, flexible ligand to a set of grids describing the target protein. During the docking process, the protein was considered as rigid and the molecules as flexible. The crystal structures of the candidate targets were downloaded from the RCSB Protein Data Bank (available online:; accessed on 1 December 2011), and the proteins without crystal structures were performed based on homology modeling using the Swiss-Model Automated Protein Modelling Server (available online:; accessed on 1 February 2012).

TCM is a heritage that is thousands of years old and is still used by millions of people all over the world—even after the development of modern scientific medicine. Chinese herbal combinations generally include one or more plants and even animal products.

The study identified 54 protein targets, which are closely associated with CVD for the three Chinese herbs, of which 29 are common targets (52.7%), which clarifies the mechanism of efficiency of the herbs for the treatment of CVD.

Activation of NFkB

Extracellular stimuli for NFkB activation and NFkB regulated genes
Extracellular stimuli                       Regulated genes
TNFa                                         Growth factors (G/M-CSF)
Interleukin 1                            G/M CSF, M CSF, G CSF
ROS                                              Cell adhesion molecules
UV light                            ICAM-1, VCAM, E-Selectin, P-selectin
Ischaemia                                   Cytokines
Lipopolysaccharide               TNFa, IL-1, IL-2, IL-6, interferon
Bacteria                                        Transcription regulators
Viruses                                         P53, IkB, c-rel, c-myc
Amyloid                                      Antiapoptotic proteins
Glutamate                              TRAF-1, TRAF-2, c-IAP1, c-IAP2
Reactive oxygen species (ROS) are toxic and in conditions of a dysbalance between their overproduction and the diminished activity of various antioxidant enzymes and other molecules induce cellular injury termed oxidative stress. ROS are often related to a number of diseases like atherosclerosis. However, the mechanism is not clear at all. Latest years of research have brought the idea of connection between ROS and NFkB. And indeed, in vitro studies showed a rapid activation of NFkB after exposure of certain cell types to ROS. Today, no specific receptor for ROS has been found, thus, the details of the ROS induced activation of NFkB are missing.

Natural occurring agents which actions are still a matter of debate in the theory and nouvelle small molecular derivates activate or inhibit the transcriptional factor. Synthetic oligo and polypeptide inhibitors of NFkB can penetrate the cell membrane and directly act on the Rel proteins. The most sophisticated approaches towards inhibiting the activation and translocation of NFkB into the nucleus represent gene deliveries, using plasmids or adenoviruses containing genes for various super repressors—modified IkB proteins, or so called NFkB decoys, which interact with activated NFkB and thus, inhibit the interaction between the transcription factor and nuclear DNA enhancers.

A simplified scheme of the activation of NFkB by the degradation of IkB. IkB is phosphorylated by IKK and ubiquinatated by the ubiquitine ligase system (ULS). IkB is further degradated by the 26S proteasome (26S).Activated NFkB can pass the nuclear membrane and interact with kB binding sequences in enhancers of NFkB regulated genes. LPS, lipopolysaccharide; ROS, reactive oxygen species; FasL, Fas ligand; TRAF, TNFa receptor associated factor; NIK, NFkB inducing kinase; MEKK, mitogen activated protein kinase/extracellular signal regulated kinases kinases.

The medicine of this century is a medicine of molecules, the diagnostic procedure and the therapy moves further from the “clinical picture” to the use of achievements in molecular biology and genetics. However, sober scepticism and awareness are indicated. Especially the role of NFkB in multiple signal transducing pathways and the tissue dependent variability of responses to alternations in NFkB pathway may be the reasons for unwanted side effects of the therapy that are after in vitro or in vivo experiments hardly to expect in the clinical use.

Therapeutic Targets
Modern drug discovery is primarily based on the search and subsequent testing of drug candidates acting on a preselected therapeutic target. Progress in genomics, protein structure, proteomics, and disease mechanisms has led to a growing interest in an effort for finding new targets and more effective exploration of existing targets. The number of reported targets of marketed and investigational drugs has significantly increased in the past 8 years. There are 1535 targets collected in the therapeutic target database.
Knowledge of these targets is helpful for molecular dissection of the mechanism of action of drugs and for predicting features that guide new drug design and the
search for new targets. This article summarizes the progress of target exploration and investigates the characteristics of the currently explored targets to analyze their sequence, structure, family representation, pathway association, tissue distribution, and genome location features for finding clues useful for searching for new targets. Possible “rules” to guide the search for druggable proteins and the feasibility of using a statistical learning method for predicting druggable proteins directly from their sequences are discussed.

Current Trends in Exploration of Therapeutic Targets
There are 395 identifiable targets described in 1606 patents. Of these targets, 264 have been found in more than one patent and 50 appear in more than 10 patents. The number of patents associated with a target can be considered to partly correlate with the level of effort and intensity of interest currently being directed to it. Approximately one third of the patents with an identifiable target were approved in the past year. This suggests that the effort for the exploration of these targets is ongoing, and there has been steady progress in the discovery of new investigational agents directed to these targets.

Various degrees of progress have been made toward discovery and testing of agents directed at these targets. However, for some of these targets, many difficulties remain to be resolved before viable drugs can be derived. The appearance of a high number of patents associated with these targets partly reflects the intensity of efforts for finding effective drug candidates against these targets.

There are 62 targets being explored for the design of subtype-specific drugs, which represents 15.7% of the 395 identifiable targets in U.S. patents approved in 2000 through 2004. Compared with the 11 targets of FDA approved subtype-specific drugs during the same period, a significantly larger number of targets are being explored for the design of subtype-specific drugs.

What Constitutes a Therapeutic Target?
The majority of clinical drugs achieve their effect by binding to a cavity and regulating the activity, of its protein target. Specific structural and physicochemical properties, such as the “rule of five” (Lipinski et al., 2001), are required for these drugs to have sufficient levels of efficacy, bioavailability, and safety, which define target sites to which drug-like molecules can bind. In most cases, these sites exist out of functional necessity, and their structural architectures accommodate target-specific drugs that minimally interact with other functionally important but structurally similar sites.
These constraints limit the types of proteins that can be bound by drug-like molecules, leading to the introduction of the concept of druggable proteins (Hopkins and Groom, 2002; Hardy and Peet, 2004). Druggable proteins do not necessarily become therapeutic targets (Hopkins and Groom, 2002); only those that play key roles in diseases can be explored as potential targets.

 Prediction of Druggable Proteins by a Statistical Learning Method

Currently, the support vector machine (SVM) method seems to be the most accurate statistical learning method for protein predictions. SVM is based on the structural risk minimization principle from statistical learning theory. Known proteins are divided into druggable and nondruggable classes; each of these proteins is represented by their sequence-derived physicochemical features.

These features are then used by the SVM to construct a hyperplane in a higher dimensional hyperspace that maximally separates druggable proteins and nondruggable ones. By projecting the sequence of a new protein onto this hyperspace, it can be determined whether this protein is druggable from its location with respect to the hyperplane. It is a druggable protein if it is located on the side of druggable class.

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