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Posts Tagged ‘Endothelial progenitor cell’


Circulating Endothelial Progenitors Cells (cEPCs) as Biomarkers

Article Curator: Larry H. Bernstein, MD, FCAP

and

Topic Curator: Aviva Lev-Ari, PhD, RN

Circulating progenitor cells have gained much interest rapidly in the past year primarily in identification of damaged tissue that has turnover of cells that are identifiable in the circulation.  This has to require a sensitivity for identification at one or two logs lower than circulating hematopoietic cells.  I mention this untested view only because cells of the circulation are detected routinely by automated hematology instruments like those of Beckman-Coulter and Siemens, with graphical presentation of results.  The Sysmex also reports immature granulocytes that are a small percent of the neutrophil count.  In the evaluation of leukemias, flow cytometry has been used for years, but require a preparative step.  Cell types have been identified by acidic and basic dye stains to identify basophilic, acidophilic and neutrophilic granulocyte series, and by size of the cell population, and nuclear features, differentiating mature and nucleated red cells, the granulocyte series, monocytes and lymphocytes, as well as platelets (aggregation gives an underestimate of platelet count).  But to detect cancer cells or damaged endothelial cells, the number of cells in the circulation requires and antibody to the surface with a visualizable ligand attached to an antibody for identification.  Visualization could be by a fluorophor, or perhaps a luciferase reaction.  Here are two articles that identify circulating endothelial cells, making them suitable for biomarkers of cardiovascular injury.  Whether they can detect early predictive ischemia, or frank AMI needs investigation.  The concept of piecemeal necrosis in the heart may be applicable to cardiomyocyte injury that is found unexpectedly at autopsy as “silent infarct”.

Circulating endothelial progenitors–cells as biomarkers

Rosenzweig, Anthony
N Engl J Med. 2005 Sep 8;353(10):1055-7

Comment on

Circulating endothelial progenitor cells and cardiovascular outcomes

[N Engl J Med. 2005]  PMID: 16148292 [PubMed – indexed for MEDLINE]

Endothelial injury and dysfunction are thought to be critical events in the  pathogenesis of atherosclerosis. Thus,

  • understanding the mechanisms that  maintain and restore endothelial function
    • may have important clinical  implications.

A series of clinical and basic studies prompted by the discovery 

  • of bone marrow derived endothelial progenitor cells1 have
  • provided insights into these processes and
    • opened a door to the development of new therapeutic approaches.

Growing evidence suggests that bone marrow derived endothelial progenitor cells circulate in the blood and

  • play an important role in the formation of new blood vessels as well as
  • contribute to vascular homeostasis in the adult.

Circulating endothelial progenitor cells were initially identified

  • through their expression of CD34
    (a surface marker common to hematopoietic stem cells and mature endothelial cells)
  • and vascular endothelial cell growth-factor receptor 2
    (VEGFR2 or kinase-domain related [KDR] receptor),

but not of other markers seen on fully differentiated endothelial cells.1

Subsequent studies have also used other identifiers, such as

  • the stem-cell marker CD133, and
  • functional assays, including
    • the ability to form endothelial colonies.

Endothelial progenitor cells defined in these ways probably represent

  • a heterogeneous population, which,
  • in combination with the lack of a consensual definition,

complicates the interpretation of work in this field.

Nevertheless, numerous studies in animals have shown that endothelial  progenitor cells can integrate into new and existing blood vessels.2,3,4
Intravenous injection of cytokine-mobilized human endothelial progenitor cells

  • improved myocardial neoangiogenesis and
  • the recovery of functioning in a rat model of infarction.3

Repeated injection of bone marrow derived cells in a mouse model of atherosclerosis

  • reduced the rate of plaque formation without altering serum lipids levels, and
  • donor endothelial progenitor cells could subsequently be identified in the recipient’s blood vessels.4

Previous clinical studies have shown that

  • traditional risk factors for coronary atherosclerosis
  • are associated with low levels of circulating endothelial progenitor cells,5 whereas
  • protective interventions, including statin therapy6 and exercise,7
    • appear to increase the supply of these cells.

Hill et al. found that even in healthy volunteers,

  • levels of endothelial progenitor cells were inversely correlated with the Framingham risk score and
  • actually appeared to predict vascular function better than the Framingham risk score.5

Together, these data suggest that circulating endothelial progenitor cells may participate

  • not only in forming new blood vessels
  • but also in maintaining the integrity and function of vascular  endothelium,

thereby mitigating disease processes such as atherosclerosis.

In this issue of the Journal, Werner and colleagues have further advanced our understanding of the clinical implications of endothelial progenitor cells.8 Endothelial progenitor cells were quantitated in 519 patients with coronary artery disease who

  • were followed for one year after undergoing catheterization.

Patients with higher levels of endothelial progenitor cells had

  • a reduced risk of death from cardiovascular causes and of
  • the composite end point of major cardiovascular events.

These relationships were preserved even

  • after adjustment for traditional risk factors and prognostic variables.

A similar relationship was seen

  • whether endothelial progenitor cells were  identified by virtue of expression
    either of CD34 and KDR or of CD133 or
  • because of their ability to form endothelial colonies,

further strengthening the authors’ conclusions. Repeated catheterization was not performed in this  cohort, so

  • we do not know whether the reduction in clinical events reflected a slowed progression of atherosclerosis or some other clinical effect.

A  dissociation between anatomical measures of atherosclerosis and clinical events has been well documented in other settings.

Although this study is consistent with prior work suggesting that circulating endothelial progenitor cells may play a protective role in vascular homeostasis, other explanations

  • for the association between endothelial progenitor number and outcome remain possible.

Changes in the number of endothelial progenitor cells and

  • in clinical events might reflect a common underlying etiology,
      • rather than a causal relation.

For example, a defect in the production of nitric oxide, which plays an important role

  • in both the mobilization of endothelial progenitor cells9 and blood-vessel function, might account for both observations.

Similarly, the number of endothelial progenitor cells

  • may mirror a person’s regenerative capacity more broadly and
  • predict clinical events on that basis.

Even if endothelial progenitor cells are mechanistically linked to clinical cardiovascular events,

  • such clinical studies do not distinguish between the possibility
  • that the protection is mediated through the integration of endothelial  progenitor cells into blood vessels and

its possible mediation by other  mechanisms, such as the

  • paracrine benefits of endothelial progenitor  cell secreted products.

Although such questions will undoubtedly continue to provide fertile ground  for fundamental investigation,

  • the report by Werner and colleagues has more  immediate clinical implications.

First, it suggests that circulating cell  populations may represent a new class of biomarkers

  • that naturally integrate  diverse genetic and environmental effects,
  • thereby providing robust  physiological and prognostic insights.

Second, in the context of coronary  disease, the study shows that

  • the number of endothelial progenitor cells is an independent predictor of hard clinical outcomes.

As with other biomarkers, a demonstration of clinical usefulness will ultimately require

  • the examination of other patient populations, as well as
  • a demonstration that clinical therapy can be guided and enhanced by this information.

Finally, the increased risk associated with reduced levels of endothelial progenitor cells

  • supports the growing interest in the therapeutic potential of enhancing the level of these cells.

The most dramatic extension of this line of reasoning involves

transferring  bone marrow or peripheral blood cells that are likely to include endothelial  progenitor cells to patients with coronary artery disease. Although it would be premature to judge the clinical success of these strategies, early trials, including one randomized (though incompletely blinded) trial, have suggested

  • at least short-term functional benefits of intracoronary infusion of bone marrow cells after acute infarction.10

Trials are planned to address more definitively the potential benefits of such cells

  • in the settings of acute infarction and chronic ischemic cardiomyopathy.

Such efforts would be aided substantially by the identification of specific markers as well as

  • an improved understanding of the role of subtypes of endothelial progenitor cells and
  • of the mechanisms by which they work.

Ironically, the data presented by Werner and colleagues in combination with work showing

  • the impaired functioning of endothelial progenitor cells in high-risk patients5 suggest
  • that the patients most in need of endothelial progenitor cells may be
      • those who are least able to donate them for autologous transplantation.

Whether these limitations can be overcome through

  • ex vivo expansion or  genetic modification of endothelial progenitor cells is unclear.

In addition to possible cell-based therapies, work on endothelial progenitor cells provides yet another rationale

  • for redoubling efforts to comply with established therapeutic guidelines,
  • including lifestyle modifications and the use of statin therapy,
      • both of which appear to enhance the number of circulating endothelial progenitor cells.

Whether there will be a downside to enhancing the number and function of  endothelial progenitor cells remains unclear,

  • although obvious concerns  include exacerbating conditions that are characterized by adverse vessel  formation,
    • such as diabetic retinopathy and tumor angiogenesis.

Small studies have suggested an association between high levels of circulating endothelial progenitor cells and the risk of certain cancers, such as multiple myeloma.11 Moreover, studies in animals show that

  • bone marrow derived endothelial progenitors participate in tumor angiogenesis, thereby
      • enhancing tumor growth.12

In the study by Werner and colleagues,

  • the number of deaths from cardiovascular causes among patients with high levels of endothelial progenitor cells
  • was substantially lower than that among patients with lower levels of these cells,
  • without a reduction in the risk of death overall.8

Although this finding could raise the specter of a counterbalancing adverse effect of endothelial progenitor cells,

  • there was no apparent pattern in the deaths due to other causes,
  • and no deaths from cancer were noted in this population.

It is possible that as we learn more about the biology of endothelial progenitor cells, there may be opportunities

  • to target vessel formation more specifically.

In addition, therapeutic strategies

  • tailored to individualized risk will undoubtedly help in practice.

For example, in the study by Werner et al.,

  • patients in the group with the lowest baseline levels of endothelial progenitor cells
  • had a risk of death from cardiovascular causes of 8.3 percent during one year of follow-up,
  • suggesting that the benefits of enhancing the function and number of endothelial progenitor cells
      • may well outweigh the risks in such high-risk populations.

Additional studies will be necessary to address these questions definitively. Larger studies

  • of longer duration performed in different cohorts will be required to determine fully
    • the clinical usefulness of endothelial progenitor cells as a biomarker.

Rigorous interventional studies will indicate

  • whether levels of endothelial progenitor cells can be used to guide therapy and
  • whether cell transfer has a role in augmenting the levels of these cells.

Basic-science studies should help guide these clinical efforts by

  • further defining the desirable subpopulations of endothelial progenitor cells and
  • the mechanisms by which they mediate their effects.

By establishing a connection between circulating endothelial progenitor cells and hard clinical end points, Werner and colleagues

  • provide a potent stimulus for clinical and basic studies to address these important issues.

Source Information

From the Program in Cardiovascular Gene Therapy, Massachusetts General  Hospital, and Harvard Medical School ― both in Boston.

References

Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor  endothelial cells for angiogenesis. Science 1997;275:964-967.

Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced  mobilization of bone marrow-derived endothelial progenitor cells for  neovascularization. Nat Med 1999;5:434-438.

Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001; 7: 430-436.

Rauscher FM, Goldschmidt-Clermont PJ, Davis BH, et al. Aging, progenitor cell exhaustion, and atherosclerosis. Circulation 2003; 108: 457-463.

Hill JM, Zalos G, Halcox JPJ, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348:593-600.

Vasa M, Fichtlscherer S, Adler K, et al. Increase in circulating endothelial  progenitor cells by statin therapy in patients with stable coronary artery  disease. Circulation 2001; 103: 2885-2890.

Laufs U, Werner N, Link A, et al. Physical training increases endothelial  progenitor cells, inhibits neointima formation, and enhances angiogenesis.  Circulation 2004; 109: 220-226.

Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 2005; 353: 999-1007.

Aicher A, Heeschen C, Mildner-Rihm C, et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med  2003; 9: 1370-1376.

Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow  cell transfer after myocardial infarction: the BOOST randomised controlled  clinical trial. Lancet 2004; 364: 141-148.

Zhang H, Vakil V, Braunstein M, et al. Circulating endothelial progenitor cells in multiple myeloma: implications and significance. Blood 2005; 105: 3286-3294.

Lyden D, Hattori K, Dias S, et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 2001;7:1194-1201.

Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction.

Kelly Bethel, Madelyn S Luttgen, Samir Damani, Anand Kolatkar, Rachelle Lamy, Mohsen Sabouri-Ghomi, Sarah Topol, Eric J Topol, Peter Kuhn

Physical Biology (Impact Factor: 2.62). 01/2014; 11(1):016002. http://dx.doi.org/10.1088/1478-3975/11/1/016002
Source: PubMed

Elevated levels of circulating endothelial cells (CECs) occur in response to various pathological conditions including myocardial infarction (MI). Here, we adapted

  • a fluid phase biopsy technology platform that successfully detects circulating tumor cells in the blood of cancer patients (HD-CTC assay),
  • to create a high-definition circulating endothelial cell (HD-CEC) assay for the detection and characterization of CECs.

Peripheral blood samples were collected from 79 MI patients, 25 healthy controls and six patients undergoing vascular surgery (VS). CECs were defined

  • by positive staining for DAPI, CD146 and von Willebrand Factor
  • and negative staining for CD45.

In addition, CECs exhibited distinct morphological features that

  • enable differentiation from surrounding white blood cells.
  1. CECs were found both as individual cells and as aggregates.
  2. CEC numbers were higher in MI patients compared with healthy controls.
  3. VS patients had lower CEC counts when compared with MI patients

but were not different from healthy controls.

Both HD-CEC and CellSearch® assays could discriminate

  • MI patients from healthy controls with comparable accuracy

but the HD-CEC assay exhibited

  • higher specificity while maintaining high sensitivity.

Our HD-CEC assay may be used as a robust diagnostic biomarker in MI patients.

MicroRNA function in endothelial cells

Solving the mystery of an unknown target gene using microRNA Target Site Blockers
Dr. Virginie Mattot
Dr. Virgine Mattot works in the team “Angiogenesis, endothelium activation and Cancer” directed by Dr. Fabrice Soncin at the Institut de Biologie de Lille in France where she studies the roles played by microRNAs in endothelial cells during physiological and pathological processes such as angiogenesis or endothelium activation. She has been using Target Site Blockers to investigate the role of microRNAs on putative targets which functions are yet unknown.
What is the main focus of the research conducted in your lab?
We are studying endothelial cell functions with a particular interest
  • in angiogenesis and endothelium activation during physiological and tumoral vascular development.
How did your research lead to the study of microRNAs?
A few years ago, we identified in my team
  • a new endothelial cell-specific gene which harbors a microRNA in its intronic sequence.

We have since been working on understanding

  • the functions of both this new gene and
  • its intronic microRNA in endothelial cells

What is the aim of your current project?

While we were searching for the functions of the intronic microRNA,
  • we identified an unknown gene as a putative target.
The aim of my project was to investigate if this unknown gene was actually a genuine target and
  • if regulation of this gene by the microRNA was involved in endothelial cell function.
We had already characterized the endothelial cell phenotype associated with the inhibition of our intronic microRNA.
We then used miRCURY LNA™ Target Site Blockers to demonstrate
  • that the expression of this unknown gene is actually controlled by this microRNA.
Further, we also demonstrated that the microRNA regulates
  • specific endothelial cell properties through regulation of this unknown gene.
How did you perform the experiments and analyze the results?
LNA™ enhanced target site blockers (TSB) for our microRNA were designed by Exiqon.
We transfected the TSBs into endothelial cells using our standard procedure and
  • analysed the induced phenotype.
As a control for these experiments, a mutated version of the TSB was designed by Exiqon and
  • transfected into endothelial cells.
We first verified that this TSB was functional by
  • analyzing the expression of the miRNA target
      • against which the TSB was directed in transfected cells.
Finally, we showed that the TSB induced similar phenotypes as those found when we inhibited the microRNA in the same cells. 
What were some specific challenges in your experiments and how did you overcome them?
The fact that the target gene for our microRNA was unknown was a major challenge. Without specific available tools, like antibodies,
  • it becomes difficult to demonstrate the effect of the microRNA on the gene in question and
  • to show that the unknown gene is indeed responsible for the functions of the microRNA.
However through the use of specific target site blockers, we were able to demonstrate
  • that this unknown gene was associated with the phenotype observed
    • when the microRNA was inhibited in endothelial cells.
How do you feel about your results so far?
We are very pleased with the results of the TSB experiments and
  • altogether these results demonstrate that our miRNA of interest
  • is functional in endothelial cells
    • through the regulation of a target gene with a previously unknown role.
What do you find to be the main benefits/advantage of the LNA™ microRNA target site blockers from Exiqon?
Target Site Blockers are efficient tools to demonstrate the

  • specific involvement of putative microRNA targets
  • in the function played by this microRNA.
The use of LNA™ allows the design of short oligonucleotides that are very specific and easy to work with. 
What would be your advice to colleagues about getting started with microRNA functional analysis?
In order to address the role played by a microRNA,
  • it is essential to perform both gain and loss of functions experiments.
What are the next steps in the current project and how do you plan to perform them?
We plan to use microRNA inhibitor libraries to identify
  • more microRNAs specifically involved in the processes that we currently study.
When and where will be hear /read more about your studies?
We are currently in the process of submitting a manuscript regarding the function of my microRNA of interest.
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Accurate Identification and Treatment of Emergent Cardiac Events


Accurate Identification and Treatment of Emergent Cardiac Events

Author: Larry H Bernstein, MD, FCAP
In the immediately preceding article, I discussed the difficulties in predicting long-term safety for developing drugs, and the cost of failure in early identification.

It is not the same scale of issue as for the patient emergently presenting to the ED. Despite enormous efforts to reduce the development of and the complications of acute ischemia related cardiac events, the accurate diagnosis of the patient presenting to the emergency room is still, as always, reliant on clinical history, physical examination, effective use of the laboratory, and increasingly helpful imaging technology. The main issue that we have a consensus agreement that PLAQUE RUPTURE is not the only basis for a cardiac ischemic event. The introduction of  high sensitivity troponin tests has made it no less difficult after throwing out the receiver-operator characteristic curve (ROC) and assuming that any amount of cardiac troponin released from the heart is pathognomonic of an acute ischemic event.  This has resulted in a consensus agreement that

  • ctn measurement at a coefficient of variant (CV) measurement in excess of 2 Std dev of the upper limit of normal is a “red flag”
  • signaling AMI? or other cardiomyopathic disorder

This is the catch.  The ROC curve established AMI in ctn(s) that were accurate for NSTEMI – (and probably not needed with STEMI or new Q-wave, not previously seen) –

  1. ST-depression
  2. T-wave inversion
    • in the presence of other findings
    • suspicious for AMI

Wouldn’t it be nice if it was like seeing a robin on your lawn after a harsh winter?  Life isn’t like that.  When acute illness hits the patient may well present with ambiguous findings.   We are accustomed to relying on

  1. clinical history
  2. family history
  3. co-morbidities, eg., diabetes, obesity, limited activity?, diet?
    1. stroke and/or peripheral vascular disease
    2. hypertension and/or renal vascular disease
    3. aortic atherosclerosis or valvular heart disease
      • these are evidence, and they make up syndromic classes
  4. Electrocardiogram – 12 lead EKG (as above)
  5. Laboratory tests
    1. isoenzyme MB of creatine kinase (CK)… which declines after 12-18 hours
    2. isoenzyme-1 of LD if the time of appearance is > day-1 after initial symptoms (no longer used)
    3. cardiac troponin cTnI or cTnT
      • genome testing
      • advanced analysis of EKG

This may result in more consults for cardiologists, but it lays the ground for better evaluation of the patient, in the long run.  When you look at the amount of information that has to be presented to the physician, there is serious need for improvement in the electronic medical record to benefit the patient and the caregivers.  Recently, we have a publication on a new test that has been evaluated, closely related to the C-reactive protein (CRP), a test that has generated much discussion over the effect of treatment for patients who have elevated CRP in the absence of increased LDL cholesterol, diabetes, or obvious atherosclerotic comorbidities.  The serum pentraxin 3 test is related to cell mediated immunity, and an evaluation has been published in the Journal of Investigative Medicine.

Journal of Investigative Medicine Feb 2013; 61 (2): 278–285.
http://dx.doi.org/10.231/JIM.0b013e31827c2971

Serum Pentraxin 3 Levels Are Associated With the Complexity and Severity of Coronary Artery Disease in Patients With Stable Angina Pectoris
Karakas, Mehmet Fatih MD*; Buyukkaya, Eyup MD*; Kurt, Mustafa MD*; et al.
From the Departments of Cardiology and,Clinical Biochemistry, Mustafa Kemal University, Tayfur Ata Sokmen Medical School, Hatay, Turkey.
Reprints: Mehmet Fatih Karakas, MD, Antakya 31005, Turkey. E-mail: mfkarakas@hotmail.com.

Abstract
Background: Atherosclerosis is a complex inflammatory process. Although pentraxin 3 (PTX-3), a newly identified inflammatory marker, was associated with adverse outcomes in stable angina pectoris,

  • an association between PTX-3 and the complexity of coronary artery disease (CAD) has not been reported.

The aim of the present study is to assess

  • the association between the level of PTX-3 and
  • the complexity and severity of CAD assessed with
  • SYNTAX and Gensini scores in patients with stable angina pectoris.

Methods: The study population is 2 groups:

  • 161 patients with anginal symptoms and evidence of ischemia
    • who underwent coronary angiography and
  • 50 age- and sex- matched control subjects without evidence of ischemia .

Patients were grouped into 3 groups according to the complexity and severity of coronary lesions

  • assessed by the SYNTAX score (30 patients with a SYNTAX score of 0 were excluded).

Serum PTX-3 and high-sensitivity C-reactive protein (hs-CRP) levels were measured in both groups.

Results: The PTX-3 levels demonstrated

  • an increase from low to high SYNTAX groups (r = 0.72, P < 0.001).

Whereas the low SYNTAX group had statistically significantly higher PTX-3 levels when compared with the control group (0.50 ± 0.01 vs 0.24 ± 0.01 ng/mL, P < 0.001),

  • the hs-CRP levels were not different (0.81 ± 0.42 vs 0.86 ± 0.53 mg/dL, P = 0.96).
  • but  the intermediate SYNTAX group had higher hs-CRP levels compared with the low SYNTAX group (1.3 ± 0.66 vs 0.86 ± 0.53 mg/dL, P = 0.002).

Serum PTX-3 levels and hs-CRP levels were both correlated with the SYNTAX scores and Gensini scores (for SYNTAX: r = 0.87 [P < 0.001] and r = 0.36 [P = 0.01]; for Gensini: r = 0.75 [P < 0.001] and r = 0.27 [P = 0.002], respectively), and

  • according to the results of univariate and multivariate analyses, for “intermediate and high” SYNTAX scores, age, diabetes mellitus, low-density lipoprotein cholesterol, hs-CRP, and PTX-3
  • were found to be independent predictors, whereas
  • for the presence of “high” SYNTAX score only PTX-3 was found to be an independent predictor.
  • The receiver operating characteristic curve analysis further revealed that the PTX-3 level was
    • a strong indicator of high SYNTAX score with an area under the curve of 0.91 (95% confidence interval, 0.86–0.96).

Conclusions: Pentraxin 3, a novel inflammatory marker, was more tightly associated with the complexity and severity of CAD than hs-CRP and

    • it was found to be an independent predictor for high SYNTAX score.

The association between atherosclerosis and inflammation has been more understood during recent years. Currently, atherosclerosis is considered as a complex inflammatory process in which

    • leukocytes and inflammatory markers are involved.1

Several inflammatory markers

  1.  high-sensitivity C-reactive protein (hs-CRP),
  2. fibrinogen, and
  3. complement C3…. are associated with cardiovascular events.1–5

Pentraxin 3 (PTX-3), that resembles CRP both in structure and function,1 is produced both by

  • hematopoietic cells such as macrophages, dendritic cells, neutrophils, and by
  • nonhematopoietic cells such as fibroblasts and vascular endothelial cells.2

Plasma PTX-3 levels may be elevated in patients with

  1. vasculitis,6
  2. acute myocardial infarction,7,8 and
  3. systemic inflammation or sepsis,9
  4. psoriasis,
  5. unstable angina pectoris, and
  6. heart failure.10–13

Dubin et al14 reported that PTX-3 levels are associated with with adverse outcomes in stable angina pectoris (SAP). Despite reports about the association of PTX-3 and coronary artery disease (CAD),

an association between the level of PTX-3 and the complexity and severity of CAD is not established.15,16 Thus, the aim of this study was

  • to assess the association between the level of PTX-3 and the complexity and severity of CAD assessed with SYNTAX and Gensini scores in SAP patients.

MATERIALS AND METHODS

Of 211 patients were prospectively recruited,  161 SAP patients with evidence of ischemia (positive treadmill or myocardial perfusion scan) underwent coronary angiography for suspected CAD, and 50 age- and sex- matched outpatient subjects with a negative treadmill or myocardial perfusion scan test were taken as the control group. Patients were excluded if they had

  •  acute coronary syndrome
  • history of previous myocardial infarction;
  • coronary artery bypass grafting or percutaneous coronary intervention;
  • secondary hypertension (HT);
  • renal failure;
  • hepatic failure;
  • chronic obstructive lung disease and/or
  • manifest heart disease, such as
    • cardiac failure (left ventricular ejection fraction <50%),
    • atrial fibrillation, and
    • moderate to severe cardiac valve disease; and
    • SYNTAX score of zero

Similarly, patients were excluded with

  • infection,
  • acute stress, or chronic systemic inflammatory disease and
  • those who had been receiving medications affecting the number of leukocytes .

Thirty patients were excluded from the study because the coronary angiograms revealed normal coronary arteries (SYNTAX score of 0). All the participants included in the study were informed about the study, and they voluntarily consented to participate. The Serum PTX-3 level was measured on blood samples collected after 12-hour fast just prior to coronary angiography and kept at −80°C until the assays were performed. PTX3 was measured by enzyme immunoassay (EIA) using quantitative kit (human PTX-3/TSG-14 immunoassay, DPTX30; R&D Systems, Inc, Minneapolis, MN). The intra-assay and interassay coefficients of variation ranged from 3.8% to 4.4% and 4.1% to 6.1%, respectively (minimum detectable concentration, 0.025 ng/mL). High-sensitivity CRP was measured in serum by EIA (Immage hs-CRP EIA Kit; Beckman Coulter Inc, Brea, CA). Transthoracic echocardiography was performed, and biplane Simpson’s ejection fraction (%) was calculated before coronary angiography. Hypertension was defined as having at least 2 blood pressure measurements greater than 140/90 mm Hg or using antihypertensive drugs, whereas diabetes mellitus (DM) was defined as having at least 2 fasting blood sugar measurements greater than 126 mg/dL or using antidiabetic drugs. Smoking was categorized into current smokers and nonsmokers. Nonsmokers included ex-smokers who had quit smoking for at least 6 months before the study. Body mass index (BMI) values were calculated based on the height and weight of each patient. Medications used before the coronary angiography were noted. The study was approved by the local ethics committee.
SYNTAX and Gensini Scores
To grade the complexity of CAD, the SYNTAX score was used. Each coronary lesion with a stenosis diameter of 50% or greater in vessels of 1.5 mm or greater was scored. Parameters used in the SYNTAX scoring are shown in Table 1. The latest online updated version (2.11) was used in the calculation of the SYNTAX scores (www.syntaxscore.com).17 The SYNTAX score was classified as follows:

  1. low SYNTAX score (≤22),
  2. intermediate SYNTAX score (23–32)
  3. high SYNTAX score (≥33).

Table 1   http://images.journals.lww.com/jinvestigativemed/LargeThumb.00042871-201302000-00007.TT1.jpeg

The severity of CAD was determined by the Gensini score, which

  • measures the extent of coronary stenosis according to degree and location.18

In the Gensini scoring system,

  • larger segments are more heavily weighted ranging from 0.5 to 5.0
    • left main coronary artery × 5;
    • proximal segment of the left anterior descending coronary artery [LAD] × 2.5;
    • proximal segment of the circumflex artery × 2.5;
    • midsegment of the LAD × 1.5;
    • right coronary artery distal segment of the LAD,
    • posterolateral artery, and obtuse marginal artery × 1;
    • and others × 0.5.

The narrowing of the coronary artery lumen is rated

  1. 2 for 0% to 25% stenosis,
  2. 4 for 26% to 50%,
  3. 8 for 51% to 75%,
  4. 16 for 76% to 90%,
  5. 32 for 91% to 99%,
  6. 64 for 100%.

The Gensini index is the sum of the total weights for each segment. All angiographic variables of the SYNTAX and Gensini score were computed by

  • 2 experienced cardiologists who were blinded to the procedural data and clinical outcomes.

The final decision was reached by consensus when a conflict occurred.The number of diseased vessels with

  • greater than 50% luminal stenosis was scored from 1 to 3 (namely, 1-, 2-, or 3-vessel disease), and
  • a lesion greater than 50% in the left main coronary artery was regarded as a 2-vessel disease.

Statistical Analyses

Statistical analyses were conducted with SPSS 17 (SPSS Inc, Chicago, IL) software package program.
Continuous variables were expressed as mean ± SD or median ± interquartile range values, whereas categorical variables were presented as percentages.
The differences between normally distributed numeric variables were evaluated by Student t test or 1-way analysis of variance, whereas

  • non–normally distributed variables were analyzed by Mann-Whitney U test or Kruskal-Wallis variance analysis as appropriate.

χ2 Test was used for the comparison of categorical variables. Pearson test was used for correlation analysis.
To determine the independent predictors of “intermediate and high” SYNTAX scores and only “high” SYNTAX scores,

  • 2 different sets of univariate and multivariate analyses were performed
    • (in the first model SYNTAX cutoff was 22, whereas
    • in the second model SYNTAX cutoff was 33).

The standardized parameters that were found to have a significance (P < 0.10) in the univariate analysis were evaluated by stepwise logistic regression analysis.
Ninety-five percent confidence interval (CI) and odds ratio (OR) per SD increase were presented together. Interobserver and intraobserver variability for SYNTAX scores

  • was done by Bland-Altman analysis.

An exploratory evaluation of additional cut points was performed using the receiver operating characteristic (ROC) curve analysis.
All the P values were 2-sided, and a P < 0.05 was considered as statistically significant.
RESULTS
Baseline Characteristics
In total, 181 patients (50.2 ± 6.5 years, 52.5% were composed of males) were included in the study. Baseline clinical, angiographic, and laboratory characteristics of the patients
relative to SYNTAX score groups are shown in Table 2. Age, sex, HT, DM, BMI, and medication were not different between the groups. Baseline clinical and laboratory characteristics
of patients according to PTX-3 quartiles are shown in Table 3. The Bland-Altman analysis revealed that the degrees of intraobserver and interobserver variability for SYNTAX score
and Gensini score readings were 5% and 6% for SYNTAX and 8% and 9% for Gensini,
respectively.
Table 2   http://images.journals.lww.com/jinvestigativemed/Original.00042871-201302000-00007.TT2.jpeg
Table 3   http://images.journals.lww.com/jinvestigativemed/Original.00042871-201302000-00007.TT3.jpeg

The PTX-3 levels demonstrated an increase from the low SYNTAX group to the high SYNTAX group (r = 0.87, P < 0.001).
The low SYNTAX group had statistically significantly higher PTX-3 levels when compared with the control group (0.50 ± 0.01 vs 0.24 ± 0.01 ng/mL, P < 0.001); similarly,
the PTX-3 levels were higher in the high SYNTAX group than in both

  • the intermediate SYNTAX group (0.84 ± 0.08 vs 0.55 ± 0.01 ng/mL, P < 0.001) and
  • the low SYNTAX group (0.84 ± 0.08 vs 0.50 ± 0.01 ng/mL, P < 0.001).
  • there was no difference in levels of PTX-3 between the low and the intermediate SYNTAX group (0.50 ± 0.01 vs 0.55 ± 0.01 ng/mL, P = 0.09).

On the other hand, there was no difference in levels of hs-CRP between the control and the low SYNTAX group (0.81 ± 0.42 vs 0.86 ± 0.53 mg/dL, P = 0.96).
The intermediate SYNTAX group had statistically significantly higher hs-CRP levels

  • compared with the low SYNTAX group (1.3 ± 0.66 vs 0.86 ± 0.53 mg/dL, P = 0.002);
  • the hs-CRP levels were not different between the high SYNTAX group
    • and the intermediate SYNTAX group. (1.3 ± 0.66 vs 1.3 ± 0.43 mg/dL, P = 0.99).

Univariate correlation analysis revealed a positive correlation between serum PTX-3 levels and hs-CRP levels with

  • the SYNTAX and Gensini scores
    • for SYNTAX: r = 0.87 [P < 0.001] and r = 0.36 [P = 0.01];
    • for Gensini: r = 0.75 [P < 0.001] and r = 0.27 [P = 0.002],  (Fig. 1).

In addition to that, the Gensini and SYNTAX scores are found to be well correlated with each other (r = 0.80, P < 0.001).
When the SYNTAX score was taken as continuous variable, multivariate linear regression analysis revealed that

  • the SYNTAX score was correlated with PTX-3 and hs-CRP (for PTX-3: β = 0.84 [P < 0.001]; hs-CRP: β =0.08 [P = 0.032]).

Figure 1   http://images.journals.lww.com/jinvestigativemed/Original.00042871-201302000-00007.FF1.jpeg

For determining the predictors of intermediate and high SYNTAX scores and only-high SYNTAX scores,

  • 2 different sets of univariate and multivariate analyses were performed among the patients who underwent coronary angiography.

For predicting the intermediate and high SYNTAX scores, the SYNTAX score was dichotomized into

  • high (score ≥22) and
  • low (<22) groups,

whereas for predicting the only-high SYNTAX scores, the SYNTAX score was dichotomized into

  • 2 groups with a score of 33 or greater and a score of less than 33.

In the first multivariate analysis (where SYNTAX cutoff was 22), the parameters showing significance in the univariate analysis

  • age,
  • sex,
  • HT,
  • DM,
  • low-density lipoprotein cholesterol [LDL-C],
  • hs-CRP,
  • PTX-3

were evaluated by multivariate analysis to determine the

  • independent predictors of intermediate and high SYNTAX scores.

In the univariate analysis, higher values of

  • age (OR, 1.5 [95% CI, 1.1–2.0]; P = 0.01),
  • LDL-C (OR, 1.3 [95% CI, 0.98–1.8]; P = 0.068),
  • hs-CRP (OR, 2.6 [95% CI, 1.8–3.8]; P < 0.001), and
  • PTX-3 (OR, 13.6 [95% CI, 6.4–28.9]; P < 0.001)
    • were associated with higher SYNTAX scores,
  • HT (OR, 0.44 [95% CI, 0.24–0.80]; P = 0.008) and
  • DM (OR, 0.48 [95% CI, 0.25–0.91]; P = 0.02)
    • were associated with lower SYNTAX scores.

In the multivariate analysis – age, DM, LDL-C, hs-CRP, and PTX-3 – were found to be

  • independent predictors of “intermediate to high” SYNTAX score (Table 4).

Increased

  • age (OR, 2.5 [95% CI, 1.3–4.8]; P = 0.007),
  • LDL-C (OR, 2.8 [95% CI, 1.5–5.2]; P = 0.001),
  • hs-CRP (OR, 3.3 [95% CI, 1.8–6.1]; P < 0.001), and
  • PTX-3 (OR, 35.4 [95% CI, 10.1–123.6]; P < 0.001)
    • were associated with increased SYNTAX scores,

whereas DM (OR, 0.08 [95% CI, 0.02–0.33]; P < 0.001) was associated with lower SYNTAX score (Table 4).

In the second univariate and multivariate analyses (where SYNTAX cutoff was 33),

  • the parameters that showed significance in the univariate analysis were age, LDL-C, glucose, hs-CRP, and PTX-3.
  • In the univariate analysis, increased
    • age (OR, 1.5 [95% CI, 1.0–2.3]; P = 0.05),
    • LDL-C (OR, 1.5 [95% CI, 0.97–2.2]; P = 0.07),
    • hs-CRP (OR, 1.4 [95% CI, 0.97–2.1]; P = 0.072), and
    • PTX-3 (OR, 18.5 [95% CI, 6.6–51.8]; P < 0.001)
      • were found to be associated with increased SYNTAX scores.

When these parameters were evaluated with multivariate analysis, only PTX-3 (OR, 18.4 [95% CI, 6.2–54.2]; P < 0.001)

    • was found to be an independent predictor for high SYNTAX score (Table 4).

Table 4   http://images.journals.lww.com/jinvestigativemed/Original.00042871-201302000-00007.TT4.jpeg

The ROC curve analysis further revealed that the PTX-3 level was a strong indicator of high SYNTAX score with

  • an area under the curve (AUC) of 0.91 (95% CI, 0.86–0.96) (Fig. 2).

The optimal cutoff of PTX-3 for the high SYNTAX score was 0.75 ng/mL.
Sensitivity, specificity, positive predictive value, and negative predictive value to identify high SYNTAX score for the PTX-3 level

  • were 90%, 84%, 97%, and 60%, respectively.
  • the ROC curve analysis of PTX-3 for intermediate-high SYNTAX score revealed that the AUC value was 0.82 (95% CI, 0.75–0.89).

The optimal threshold of PTX-3 level that

  • maximized the combined specificity and sensitivity to predict
    • intermediate to high SYNTAX score was 0.73 ng/mL.

For the cutoff value of 0.73 ng/mL, sensitivity, specificity, positive predictive value, and negative predictive value

  • to identify intermediate-high SYNTAX score were 56%, 98%, 97%, and 56%, respectively.

Figure 2   http://images.journals.lww.com/jinvestigativemed/Original.00042871-201302000-00007.FF2.jpeg

In the ROC analysis of hs-CRP for high SYNTAX scores, the AUC value was found to be 0.68 (95% CI, 0.59–0.77; P < 0.001).
The optimal threshold of hs-CRP that maximized the combined specificity and sensitivity to predict for high SYNTAX scores was 0.89 mg/dL.
Similarly, the ROC analysis of hs-CRP for the intermediate-high SYNTAX scores revealed an AUC of 0.74 (95% CI, 0.65–0.83; P = 0.001).
The cutoff value of hs-CRP to predict the intermediate-high SYNTAX scores with a maximized sensitivity and specificity was 0.66 mg/dL.
DISCUSSION
In this particular study, we investigated the relationship between the serum PTX-3 level and the severity of CAD

  • assessed by SYNTAX and Gensini scores in patients with SAP.

The PTX-3, was significantly higher than control group in the patients with CAD, and the serum PTX-3 levels

  • were associated with the SYNTAX and Gensini scores.

When compared with the hs-CRP, the PTX-3 was found to be more tightly associated with the complexity and severity of CAD in the patients with SAP.
Pentraxin 3, an acute-phase reactant that is functionally and structurally similar to CRP,1 is produced both by different kinds of cells such as

  • macrophages, dendritic cells, neutrophils, fibroblasts, and vascular endothelial cells.2
  • Pentraxin 3 is released following the inflammatory stimuli19; therefore, it may reflect the local inflammatory status in tissues.20

Serum PTX-3 levels were shown to be elevated in patients with

  • vasculitis,6 acute myocardial infarction,7,8 and systemic inflammation or sepsis,9 psoriasis, unstable angina pectoris, and heart failure.10–13

Higher PTX3 levels were reported to be associated with worse cardiovascular outcomes

  1. after acute coronary syndromes,8,21
  2. in the elderly people without known cardiovascular disease22 and
  3. associated with overall mortality in patients with stable coronary disease,
  4. independent of systemic inflammation.14

There are 2 reports investigating the association of PTX-3 level and the atherosclerotic burden.15,16 In one of these reports,

  • Knoflach et al.15 took B-mode ultrasonography as the atherosclerosis index.

They did not provide any information about coronary anatomy, and in the other report, Soeki et al.16 evaluated 40 patients who

  • underwent coronary angiography and measured their Gensini scores.

However, in none of the studies were the SYNTAX score and Gensini score used together to assess the degree of coronary atherosclerotic burden.
To our knowledge, this is the first report that showed the association of PTX-3 levels with the complexity and severity of CAD assessed by

  • SYNTAX and Gensini scores in patients with stable coronary disease.

Chronic low-grade inflammation has been thought to play a major role in the pathogenesis of atherosclerosis.23,24 Previous studies have reported that

  • levels of inflammatory markers such as hs-CRP, interleukin 6, and so on were increased in atherosclerosis.25

In the present study, both the SYNTAX and the Gensini scores were found to be correlated with serum PTX-3 and hs-CRP levels,

  • which in turn might reflect the degree of inflammation.

The SYNTAX score is an important tool in the classification of complex CAD26 and can give predictive information about short- and long-term outcomes

  • in patients with stable CAD who undergo percutaneous coronary intervention.27–30

Although the SYNTAX score is currently used for assessing the angiographic complexity of CAD rather than the severity of coronary atherosclerotic burden,

  • because more complex lesions tend to have more atherosclerotic burden,
  • the SYNTAX scores may also reflect the severity of coronary atherosclerotic burden.

The Gensini score, a well-known and widely used scoring system to evaluate the severity of CAD,18 was measured and

  • found to be well correlated with the SYNTAX score,
    • which supports the idea that angiographically more complex lesions tend to have more atherosclerotic burden.

When compared with the hs-CRP,

  • the PTX-3 seems to be more tightly associated with coronary disease burden (r = 0.36 vs r = 0.87).

We found out that the serum PTX-3 levels were higher than those in the control group, even in the low SYNTAX group.
On the other side, the serum hs-CRP levels were not different in the control and the low SYNTAX groups.
It was reported that the leukocytes mainly found in the coronary artery lumen are the neutrophils.31
It is also known that PTX-3 is stored in specific granules of neutrophils and released in response to inflammatory signals.32
The reason why serum PTX-3 levels seem more tightly associated with the coronary disease burden

  • when compared with serum hs-CRP levels may be the association of the
  • on-site presence of neutrophils and local inflammatory signal–triggered release of  PTX-3.

On the other hand, some human studies revealed that PTX-3 was produced more in areas of atherosclerosis and may contribute to its pathogenesis.31
Some other studies suggested that PTX-3 may be part of a protective mechanism in

  • vascular repair via inhibiting fibroblast growth factor 2 or some other growth factors responsible for smooth muscle proliferation.33,34

But still, the exact role of PTX-3 in the pathophysiology of atherosclerosis seems to be obscure for the time being. It is well established that atherosclerosis
has an inflammatory background in most of the cases. In addition to that, high blood CRP level is known as an indicator of future cardiovascular disease risk
even in healthy individuals.35 According to the results of univariate and multivariate analyses, for intermediate and high SYNTAX scores,

  1. age, DM, LDL-C, hs-CRP, and PTX-3 were found to be independent predictors, whereas for the presence of
  2. high SYNTAX score, only PTX-3 was found to be an independent predictor.

Because of the tighter association with atherosclerotic burden and the on-site vascular presence,

    • PTX-3 may be a promising candidate marker for vascular inflammation and future cardiovascular events.

LIMITATIONS
The major limitation of the current study is the number of patients included. It would be better to include more patients to increase the statistical power.

Besides, the SYNTAX and Gensini scores give us an idea about the complexity and severity of coronary atherosclerosis; however,
with coronary angiography alone, it is not possible to understand the extent of coronary plaque. In addition to that, the coronary anatomy of the
control group was not known, which was another limitation. Our selected population was free of other confounders of systemic inflammation, and
we did not have data about inflammatory markers other than hs-CRP, such as interleukin 6, tumor necrosis factor α, and so on, which may be accepted
as a limitation. Another limitation of the current study is that because there was no long-term follow-up of the patients, it did not provide any prognostic
data in terms of future cardiovascular events.
CONCLUSIONS
Pentraxin 3, a novel inflammatory marker, is associated with the complexity and severity of the CAD assessed by the SYNTAX and the Gensini scores in patients with SAP and seems to be more tightly associated with coronary atherosclerotic burden than hs-CRP.

REFERENCES

1. Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352: 1685–1695.
2. Brown DW, Giles WH, Croft JB. White blood cell count: an independent predictor of coronary heart disease mortality among a national cohort. J Clin Epidemiol. 2001; 54: 316–322.
3. Kannel WB, Anderson K, Wilson PW. White blood cell count and cardiovascular disease. Insights from the Framingham Study. JAMA. 1992; 267: 1253–1256.
4. Muscari A, Bozzoli C, Puddu GM, et al.. Association of serum C3 levels with the risk of myocardial infarction. Am J Med. 1995; 98: 357–364.
5. Ridker PM, Cushman M, Stampfer MJ, et al.. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med. 1997; 336: 973–979.
6. Fazzini F, Peri G, Doni A, et al.. PTX3 in small-vessel vasculitides: an independent indicator of disease activity produced at sites of inflammation. Arthritis Rheum. 2001; 44: 2841–2850.
7. Peri G, Introna M, Corradi D, et al.. PTX3, A prototypical long pentraxin, is an early indicator of acute myocardial infarction in humans. Circulation. 2000; 102: 636–641.
8. Latini R, Maggioni AP, Peri G, et al.. Prognostic significance of the long pentraxin PTX3 in acute myocardial infarction. Circulation. 2004; 110: 2349–2354.
9. Muller B, Peri G, Doni A, et al.. Circulating levels of the long pentraxin PTX3 correlate with severity of infection in critically ill patients. Crit Care Med. 2001; 29: 1404–1407.
10. Bevelacqua V, Libra M, Mazzarino MC, et al.. Long pentraxin 3: a marker of inflammation in untreated psoriatic patients. Int J Mol Med. 2006; 18: 415–423.
11. Inoue K, Sugiyama A, Reid PC, et al.. Establishment of a high sensitivity plasma assay for human pentraxin3 as a marker for unstable angina pectoris. Arterioscler Thromb Vasc Biol. 2007; 27: 161–167.
12. Suzuki S, Takeishi Y, Niizeki T, et al.. Pentraxin 3, a new marker for vascular inflammation, predicts adverse clinical outcomes in patients with heart failure. Am Heart J. 2008; 155: 75–81.
13. Matsubara J, Sugiyama S, Nozaki T, et al.. Pentraxin 3 is a new inflammatory marker correlated with left ventricular diastolic dysfunction and heart failure with normal ejection fraction. J Am Coll Cardiol. 2011; 57: 861–869.
14. Dubin R, Li Y, Ix JH, et al.. Associations of pentraxin-3 with cardiovascular events, incident heart failure, and mortality among persons with coronary heart disease: data from the Heart and Soul Study. Am Heart J. 2012; 163: 274–279.
16. Soeki T, Niki T, Kusunose K, et al.. Elevated concentrations of pentraxin 3 are associated with coronary plaque vulnerability. J Cardiol. 2011; 58: 151–157.
17. SYNTAX working group. SYNTAX score calculator. Available at http://www.syntaxscore.com. Accessed May 20, 2012.
18. Gensini GG. A more meaningful scoring system for determining the severity of coronary heart disease. Am J Cardiol. 1983; 51: 606.
20. Mantovani A, Garlanda C, Bottazzi B, et al.. The long pentraxin PTX3 in vascular pathology. Vascul Pharmacol. 2006; 45: 326–330.
21. Matsui S, Ishii J, Kitagawa F, et al.. Pentraxin 3 in unstable angina and non-ST-segment elevation myocardial infarction. Atherosclerosis. 2010; 210: 220–225.
22. Jenny NS, Arnold AM, Kuller LH, et al.. Associations of pentraxin 3 with cardiovascular disease and all-cause death: the Cardiovascular Health Study. Arterioscler Thromb Vasc Biol. 2009; 29: 594–599.
26. Serruys PW, Morice MC, Kappetein AP, et al.. Percutaneous coronary intervention versus coronary-artery bypass grafting for severe coronary artery disease. N Engl J Med. 2009; 360: 961–972.
27. van Gaal WJ, Ponnuthurai FA, Selvanayagam J, et al.. The SYNTAX score predicts peri-procedural myocardial necrosis during percutaneous coronary intervention. Int J Cardiol. 2009; 135: 60–65.
28. Lemesle G, Bonello L, de Labriolle A, et al.. Prognostic value of the SYNTAX score in patients undergoing coronary artery bypass grafting for three-vessel coronary artery disease. Catheter Cardiovasc Interv. 2009; 73: 612–617.
29. Capodanno D, Di Salvo ME, Cincotta G, et al.. Usefulness of the SYNTAX score for predicting clinical outcome after percutaneous coronary intervention of unprotected left main coronary artery disease. Circ Cardiovasc Interv. 2009; 2: 302–308.
30. Kim YH, Park DW, Kim WJ, et al.. Validation of SYNTAX (Synergy between PCI with Taxus and Cardiac Surgery) score for prediction of outcomes after unprotected left main coronary revascularization. JACC Cardiovasc Interv. 2010; 3: 612–623.
32. Jaillon S, Peri G, Delneste Y, et al.. The humoral pattern recognition receptor PTX3 is stored in neutrophil granules and localizes in extracellular traps. J Exp Med. 2007; 204: 793–804.
33. Inforzato A, Baldock C, Jowitt TA, et al.. The angiogenic inhibitor long pentraxin PTX3 forms an asymmetric octamer with two binding sites for FGF2. J Biol Chem. 2010; 285: 17681–17692.
34. Camozzi M, Zacchigna S, Rusnati M, et al.. Pentraxin 3 inhibits fibroblast growth factor 2–dependent activation of smooth muscle cells in vitro and neointima formation in vivo. Arterioscler Thromb Vasc Biol. 2005; 25: 1837–1842.
35. Koenig W, Sund M, Frohlich M, et al.. C-Reactive protein, a sensitive marker of inflammation, predicts future risk of coronary heart disease in initially healthy middle-aged men: results from the MONICA (Monitoring Trends and Determinants in Cardiovascular Disease) Augsburg Cohort Study, 1984 to 1992. Circulation. 1999; 99: 237–242.
Keywords:  pentraxin 3; coronary artery disease; SYNTAX score; hs-CRP; inflammation

This is not the only recent finding that adds to the ability to evaluate these patients.  An as yet unpublished paper, expected to be published soon reports on

QRS fragmentation as a Prognostic test in Acute Coronary Syndrome,  and this reviewer expects the work to have a high impact.  The authors state that
QRS complex fragmentation is a promising bed-side test for assessment of prognosis in those patients.  Presence of fragmented QRS in surface ECG during ACS

  • represents myocardial scar or fibrosis and reflect severity of coronary lesions and a correlation between fQRS and depression of Lv function is established.

There are still other indicators that need to be considered, such as the mean arterial blood pressure.

There has been review and revisions of the guidelines for treatment of UA/NSTEMI within the last year, with differences being resolved among the Europeans and US.

Guidelines Updated for Unstable Angina/Non-ST Elevation Myocardial Infarction
According to the current study by Jneid and colleagues, new evidence is available on the management of unstable angina. This report replaces the 2007 American College of Cardiology Foundation/American Heart Association (ACC/AHA) Guidelines for the Management of Patients With Unstable Angina/Non–ST-Elevation Myocardial Infarction (UA/NSTEMI) that were updated by the 2011 guidelines.

This guideline was reviewed by

  • 2 official reviewers each nominated by the ACCF and the AHA, as well as
  • 1 or 2 reviewers each from the American College of Emergency Physicians; the Society for Cardiovascular Angiography and Interventions; and the Society of Thoracic Surgeons; and
  • 29 individual content reviewers, including members of the ACCF Interventional Scientific Council.

The recommendations in this focused update are considered current

  • until they are superseded in another focused update or the full-text guideline is revised, and are official policy of both the ACCF and the AHA.

STUDY SYNOPSIS AND PERSPECTIVE
American cardiology societies have caught up with the European Society of Cardiology by

  • issuing their second update to the UA/NSTEMI guidelines in 18 months,
  • with the 2012 focused update replacing the 2011 guidelines [1].

The new recommendations include ticagrelor (Brilinta) as one of the options for antiplatelet therapy alongside prasugrel (Effient) and clopidogrel, bringing them in line with European.
The European guidance, however, gave precedence to the new antiplatelets over clopidogrel, whereas the American update “places ticagrelor on an equal footing with the other two antiplatelets available
this is the main reason for the update,” lead author Dr Hani Jneid (Baylor College of Medicine, Houston, TX), told heartwire . “Doctors now have a choice for second-line therapy after aspirin, depending on

  • the patient’s clinical scenario,
  • physician preference, and cost,”
    • now that clopidogrel is available generically.

The US decision to recommend

  • first prasugrel–in its 2011 update to the UA/NSTEMI guidelines–and
  • now ticagrelor as equivalent antiplatelet therapy choices to clopidogrel after aspirin
    • puts it somewhat at odds with the Europeans,
    • who reserve clopidogrel use for those who cannot take the newer agents.

The reason for the Americans differing stance is that because while they are faster acting and more potent–

  • the cost-effectiveness of the new agents is not known.
  • it isn’t clear how the efficacy observed in pivotal clinical trials of these agents is going to translate into real-world benefit,
  • and issues such as bleeding with prasugrel and compliance with a twice-daily drug such as ticagrelor remain concerns.

Bulk of 2012 Update on How to Use Ticagrelor
The 2012 ACCF/AHA focused update for the management of UA/NSTEMI stresses that

  • all patients at medium/high risk should receive dual antiplatelet therapy on admission,
  • with aspirin being first-line, indefinite therapy.

The bulk of the update centers on how to use ticagrelor which–

  • like prasugrel or clopidogrel–
  • can be added to aspirin for up to 12 months (or longer, at the discretion of the treating clinician).

Jneid notes it’s important to remember that prasugrel can only be used in the cath lab

  • in patients undergoing percutaneous coronary intervention (PCI),
  • whereas ticagrelor, like clopidogrel, can be used in medically managed or PCI patients.

And he emphasizes that, in line with the FDA’s black-box warning on ticagrelor,

The 81-mg aspirin dose is also considered a reasonable option in preference to a higher maintenance dose of 325 mg in

  • any acute coronary syndrome (ACS) patient following PCI, he adds, as
  • this strategy is believed to result in equal efficacy and lower bleeding risk.

With regard to how long antiplatelet therapy should be stopped before planned cardiac surgery, the recommendation is

  • five days for ticagrelor–the same as that advised for clopidogrel.
  • and seven days prior to surgery for prasugrel.

Jneid also highlights other important recommendations from the 2011 focused update carried over to 2012:

It is “reasonable” to proceed with cardiac catheterization and revascularization within

  • 12–24 hours of admission in initially stable, very high-risk patients with ACS.

An invasive strategy is “reasonable” in patients with

  • mild and moderate chronic kidney disease.

In those with diabetes hospitalized with ACS, insulin use should target glucose levels <180 mg/dL,

  • a less-intensive reduction than previously recommended.

Platelet function or genotype testing for clopidogrel resistance are both considered “reasonable”

  • if clinicians think the results will alter management,
  • but Jneid acknowledged that “there is not much evidence to support these assays” .

Committee Encourages Participation in Registries
Jneid observes that unstable angina and NSTEMI are “very common” conditions that carry a high risk of death and recurrent heart attacks,

  • which is why “the AHA and ACCF constantly update their guidelines so that physicians can provide patients with
  • the most appropriate, aggressive therapy with the goal of improving health and survival.”

To this end, he notes that the writing panel encourages

  • clinicians and hospitals to participate in quality-of-care registries designed
  • to track and measure outcomes, complications, and
  • adherence to evidence-based medicines.

Conflicts of interest for the writing committee are listed in the paper.

References

Jneid H, Anderson JL, Wright SR, et al. 2012 ACCF/AHA focused update on the guideline for the management of patients with unstable angina/non-ST elevation myocardial infarction (Updating the 2007 guideline and replacing the 2011 focused update): A report of the ACCF/AHA.
Circulation 2012;      Available at: http://circ.ahajournals.org/  http://dx.doi.org/10.1161/CIR0b013e3182566fleo
source   http://www.medscape.org

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Micrograph of an artery that supplies the hear...

Micrograph of an artery that supplies the heart with significant atherosclerosis and marked luminal narrowing. Tissue has been stained using Masson’s trichrome. (Photo credit: Wikipedia)

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Positioning a Therapeutic Concept for Endogenous Augmentation of cEPCs — Therapeutic Indications for Macrovascular Disease: Coronary, Cerebrovascular and Peripheral

Author and Investigator Initiated Study: Aviva Lev-Ari, PhD, RN

 

Macrovascular Disease – Therapeutic Potential of cEPCs: Promise for CV Risk Reduction

  • Introduction
  • Biomarker Discovery – a comprehensive Post on this topic is forthcoming
  • What are our Contributions in the Domain of Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk
  • Postulates of Multiple Indications for the Method Presented: Positioning of a Therapeutic Concept for Endogenous Augmentation of cEPCs — Potential Therapeutic Indications for ElectEagle
  • A Three Component Method for Endogenous Augmentation of cEPCs — Macrovascular Diseases – Therapeutic Potential of cEPCs
  • The Promise of the Proposed Pharmacotherapy as a Method of CVD Risk Reduction
  • Emergence of Clinical Trial Results on Genous R stent — Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth – (HEALING II)
  • Conclusions
  • References

Key words: coronary artery disease, circulating Endothelial Progenitor Cells (cEPCs), Endothelial Progenitor Cells (EPC), genetic engineering, CVD, CAD, CHF, myocardial infarction, neovascularization, vascular repair, “multimarker biomarker”, macrovascular disease, Endogenous Augmentation of cEPCs, Primary Endpoint, Secondary Endpoint.

Abbreviations used: ED, endothelial dysfunction; CAD, coronary artery disease; CVD, cardiovascular disease; cEPCs, circulating Endothelial Progenitor Cells; EPC, Endothelial Progenitor Cells; CHF, congestive heart failure; MI, myocardial infarction; MNC, mononuclear cells; VEGF, vascular endothelial growth factor; BMMNCs, bone marrow-derived mononuclear cells; G-CSF, granulocyte colony-stimulating factor; SDF, stromal derived factor; PB-MNCs, peripheral blood-mononuclear cells; EF, ejection fraction; PO2, partial pressure of oxygen; BMS, bare-metal stent; CABG, coronary artery bypass graft; DES, drug-eluting stent; GP, glycoprotein; LAD, left anterior descending; LCx, left circumflex; MI, myocardial infarction; RCA, right coronary artery; S/P , status-post stent implantation; MACE, Major Adverse Cardiac Events; TLR, target lesion revascularization; TVR, target vessel revascularization; TVF, target lesion vessel failure; eNOS, endothelial Nitric Oxide Synthase 

Introduction

Cardinal to the study of reendothelialization and neovascularization is the mechanism of action (MOA) of EPCs. It requires exact biological phenotype of the true EPC and its MOA on the endothelium. Is the EPC autocrine or paracrine in its functional role? It is critical to understand this biological unknown for planning therapeutic approaches. Patients with unstable angina and no evidence of cardiac necrosis exhibited increased cEPCs. Systemic inflammation and recognized growth factors may play a role in peripheral mobilization of EPCs in patients with unstable anginal syndromes. Proportion of cEPCs in coronary ischemia, acute or chronic and its potential for restoring left ventricular dysfunction is still experimental. EC injury facilitates an accelerated development of atherosclerotic plaque which triggers cardiovascular risk factors where the magnitude of the endothelial dysfunction predicts the level of risk for a macrovascular event (George, 2004).

Diminished level of cEPCs is associated with risk factors for CVD implicating impaired endothelial repair as a contributor to a dynamic state of endothelial dysfunction. cEPCs is further reduced if multiple risk factors for CVD are present. Endothelial dysfunction is associated with cEPCs counts. It is only if cEPCs counts are low then endothelial dysfunction (ED) emerges. In the case of ED, the cells were more senescent compared with an age group without CVD and the risk factors involved with it. Impaired repair capacity due to reduced availability of cEPCs enhances the exposure to risk factors when injury occurs due to endothelial denudation, ischemic tissue, neointima build up and remodeling.

Mobilization and EPC-mediated neovascularization is critically regulated. Statins and physical exercise are stimulatory while risk factors for CAD are inhibitory in the modulation function of the level of cEPCs. Recruitment of cEPCs requires a coordinated sequence of adhesive and signaling events including adhesion and migration by integrins, chemoattraction of SDF-1/CXCR4 and differentiation of EC.

Bone-marrow derived cells in the circulating blood have an endothelial phenotype and peripheral blood can be cultured to generate ECs. cEPCs provide both diagnostic and prognostic information on CVD. EPCs are analyzed by their phenotypic markers, as discerned by fluorescence-activated cell sorting (FACS) analysis as well as by their functional capability to produce colonies in culture conditions.

Kiernan (2006) identifies the two classes of therapeutic applications of cEPCs: (a) induction of angiogenesis and (b) large vessel repair. Transplantation of autologous EPCs over-expressing eNOS in injured vessels enhances the vasculoprotective properties of the reconstituted endothelium, leading to inhibition of neointimal hyperplasia. This cell-based gene therapy strategy may be useful in treatment of vascular disease. Stents coated in CD34 antibody which binds to the CD34 antigen of cEPCs have the capability to promote re-endothelialisation in minutes to hours. This mechanism seeks to restore the normal biology of the vessel wall rather than perpetuate the wall disruption as drug eluting stents are found recently to be implicated to cause both restenosis and thrombosis (Tung et al., 2006). Thus, cEPCs are of cardinal importance in healing cardiovascular injury. Identification of augmentation methods which are endogenous in nature, are systemic rather than local, as cell-based therapy is, and therefore, it will deliver systemic protective measures against atherosclerosis delaying angioplasty and potentially avoiding cell implantation or vascular engrafting.

Biomarker Discovery – a comprehensive Post on this topic is forthcoming

A comprehensive review of “Traditional” vs. “Novel” risk markers for cardiovascular disease was recently undertaken by Folsom et al., (2006) and the Editorial to this article by Lloyd-Jones and Tian (2006). Among the “Traditional” Risk Markers, they list: Age, Race, Sex, Total/HDL levels, Smoking Status, Diabetes, Systolic BP and Use of antihypertensive  drugs. The list of “Novel” Risk Markers is impressively longer and includes: CRP, Lp-PLA2, E-Selectin, Fibrinogen, PAI-1, Vitamin B6, D-dimer, ICAM-1, Homocysteine, IL-6, HSV-1 Antibody, CMV Antibody and Folate.

Only two risk factors make the top five list following the data adjustment to Age and /or All the Traditional Risk Factors, respectively, I would conclude that only the following two are of paramount importance for clinical application and drug therapy design.

Risk Factor RANKING

Risk Factor RANKING if

Data Adjusted to

AGE

Risk Factor RANKING if

Data Adjusted to

All “Traditional” Risk Factors

1 Chlamydia Intracellular adhesion molecule
2 Lp-PLA2 lipoprotein-associatedphospholipase A2 Cytomegalovirus
3 Tisshe Plasminogen Activator D-Dimer
4 Tissue inhibitor of Metalloproteinase1 IL-6
5 Intracellular adhesion molecule Tissue inhibitor of Metalloproteinase1

In light of these results, chiefly edified by Folsom et al., (2006)  conclusion that: “Based on the totality of evidence, however, CRP level does not emerge as a clinically useful addition to basic risk factor assessment for identifying patients at risk of a first CHD event.” (Folsom, 2006, 1372).

What are our Contributions in the Domain of

Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

(a) This is the first paper to look at cEPCs from two academic schools of thought.  One, represented by the review article of Dzau et al., Hypertension, 2005 with 122 references which treats cEPCs from two perspectives: Vascular Biology and Molecular Cardiology. The other, is the review article by Lapidot & Petit, Experimental Hematology, 2002 with 86 references which treats cEPCs as stem cells and covers the research in Immunology and in Hematology, cEPCs is circulating in our blood, it is a stem cell! The overlap between the references N=122 in Dzau and N=86 in Lapidot & Petit is zero. These two schools do not cite the findings of the other school. That happens when both schools (Vascular Biology/Molecular Cardiology) and (Immunology/Hematology), BOTH schools are researching the same biologic phenomenon, i.e., one circulating EPC. We are the first to put together in one paper the two schools in the context of cEPCs. The pathophysiology of cECs, cEPCs and Trans-Endothelium Cell Migration in one location.

(b) Table of content of Part I yielded a theoretical treatment of cEPCs not in existence anywhere.  We defined for the first time that the Clinical Frontier for cEPCs is of quadruple nature: (Vascular Biology/Molecular Cardiology) PLUS (Immunology/Hematology). We made the statement that the Clinical Frontier has 20 Future Fast Acting Therapy modality currently under research. We cited the limitation of exogenous methods for augmentation of cEPCs as a scientifically derived justification for our selection of an endogenous augmentation method.

Upon selection of the endogenous method, we specified three components:

–   inhibition of ET-1

–   induction of eNOS

–   stimulation of PPAR-gamma

The proposed combination drug therapy yielded a new multimarker biomarker for reduction of CVD risk for macrovascular events, called the ElectEagle Version I. We specified the potential indications for the ElectEagle Version I method in terms of cardiovascular disease and co-morbidity with other endothelial dysfunction derived disease.

Method name:            ElectEagle

E.L.E.C.T.

E – Efficient

L – Ligands of cEPCs

E – Elective and Individualized Diagnosis and Therapy

C – Cardiovascular diseases & secondary sequalea

T – Treatment adjustable by three agents

E.A.G.L.E.

E – Endogenous

A – Augmentation

G – Gamma-PPAReceptor

L – Ligand occupied ETA and ETA-ETB – binding Nitric Oxide

E – EPCs fast generator

ElectEaglestands for an Efficient Ligands of cEPCs Elective and Individualized Diagnosis and Therapy for Cardiovascular diseases & secondary vascular sequalea, using Treatment adjustable by three agents. It is a method for Endogenous Augmentation of circulating EPCs by using Gamma-PPAR agonists, inhibitors of Ligand occupied ETA and ETA-ETB and agonist for binding Nitric Oxide and induce eNOS.

A Three Component Method for Endogenous Augmentation of cEPCs — Macrovascular Diseases – Therapeutic Potential of cEPCs

Observations on Intellectual Property Development For an Unrecognized Future Fast Acting Therapy for Patients at High Risk for Macrovascular events

ElectEagle represents a discovery of a novel “multimarker biomarker” for cardiovascular disease that innovates on four counts.

First, it proposes new therapeutic indications for acceptable drugs.

Second, it defines a specific combination of therapeutic agents, thus, it put forth a proprietary drug combination.

Third, it targets receptor systems that have not been addressed in the context of cEPCs augmentation methods. Chiefly, modulation of the following three-targeted receptor systems: (a) inhibition of ET-1, ETA and ETA-ETB receptors by antagonists (b) induction of eNOS, by agonists and NO stimulation and (c) upregulation of PPAReceptor-gamma by agonists (TZD). While (b) and (c) are implicated as having favorable effects of cEPCs count, each exerting its effect by a different pathway, it is suggested in this project that (a) might be identify to be the more powerful of the three markers. Our method, ElectEagleis the FIRST to postulate the following: (1) time concentration dependence on eNOS reuptake (2) dose concentration dependence on NO production (3) time and dose concentration dependence for ET-1, ETA and ETA-ETB inhibition, and (4) dose concentration dependence on PPAReceptor-gamma. Points First, Second and Third are covered in Part II where a special focus is placed on ET-1, ETA and ETA-ETB receptors.

Fourth, ElectEagle proposes a platform with triple modes of delivery and use of the test, as described in Part III. The triple modes are as follows: (A) an automated platform from a centralized lab with integration to Lab’s information management system. (B) a point-of-care testing device with appropriate display of test results (small benchtop analyzers in PCP office). (C) a device used for home monitoring of analytes (the hand-held device facilitates rapid read of scores and their translation to drug concentration of each of the three therapeutic agents, with computation of the three drug concentrations done by the device. Thus, it offers quicker optimization of treatment.  ElectEagle is the FIRST to propose a CVD patient kit, hand-held device, which calculates on demand an adjustable therapeutic regimen as a function of cEPCs count biomarker. In this regard, a similarity to the pump, in management of blood sugar in DM patients, exists. Since there is a high co-morbidity between DM and CVD, our methods, ElectEagle may eventually become a targeted therapy for the DM Type 2 population.

Postulates of Multiple Indications for the Method Presented: Positioning of a Therapeutic Concept for Endogenous Augmentation of cEPCs — Potential Therapeutic Indications for ElectEagle

ElectEagle can become the drug therapy of choice for the following indications:

  •      CAD patients
  •      Endothelial Dysfunction in DM patients with or without Erectile Dysfunction
  •      Atherosclerosis patients: Arteries and or veins
  •      pre-stenting treatment phase
  •      post-stenting treatment phase
  •      if stent is a Bare Metal stent (BMS)
  •      if stent is Drug Eluting stent (DES)
  •      if stent is EPC antibody coated (the ElectEagle method increase cEPCs generation in vitro) so availability of cEPCs is increased
  •      post CABG patients (the ElectEagle enhances healing by endogenous augmentation of cEPCs)
  •      target sub segments of CVD patients on blood thinner drugs (the ElectEagle does not require treatment with antiplatelet agents, it is suitable for all patients on Coumadin. This population have a counter indication for antiplatelet agents which is a follow up treatment after stent implantation for 30 days, with stent-eluting long term regimen of antiplatelet agents, 6 months and in some cases indefinitely (Tung, 2006).
  •      ElectEagle is based on systemic therapeutics (versus the localized stent solution requiring multiple and even overlapping stents)
  •      ElectEagle will be having potential in three contexts

(a) Coronary disease

(b) Periphery vascular disease

(c) Cerebrovascular

Comparative analysis of endogenous and exogenous cEPCs augmentation methods:

A. Endogenous augmentation method properties:

  •         temporal – while drug therapy in use – drug action is interruptible
  •         time concentration on eNOS reuptake
  •         dose concentration on NO production
  •         time and dose concentration manner for ETB inhibition
  •         dose concentration on PPAR-gamma

B.  Cell-based and other exogenous methods

  • permanent colonization till apoptosis if no repeated attempts of re-transfer,
  • re-implantation as the protocol usually has several stages

The Promise of the Proposed Pharmacotherapy as a Method of CVD Risk Reduction

It is expected that ElectEagle will be resulting in potential delay of stenting implantation. Patients that are target for stenting may benefit form ElectEagle that will facilitate and accelerate healing after the stent is in place. EPC antibody coated stents will work if and only if the patient has more that just low cEPCs, most patient undergoing stenting tend to have low level of cEPC. The ElectEagle method can be coupled with that type of new stents, called Genous, now in clinical trials (HEALING II, III). These stents enhance the body ability in mobilization of cEPCs, only. However, if the initial population of cEPCs is low, an endogenous fast acting cell augmentation method is needed for pretreatment before the PCI procedure with Genous is scheduled.

Emergence of Clinical Trial Results on Genous R stent — Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth – (HEALING II)

Latest publications on HEALING II – Clinical Trial of EPC coated stent

Genous R stent
n=63
Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth – II

S Silber et al; 12 Month Outcomes of the e-HEALING (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth) Worldwide Registry; EuroIntervention 2011;6:819-825

P Damman et al; Coronary Stenting With the Genous Bio-engineered R stent in Elderly Patients – 12-month Outcomes From the e-HEALING Registry; Circulation Journal 2011;75(11):2590-2597

P Damman et al; Twelve-month Outcomes After Coronary Stenting With the Genous Bio-Engineered R Stent in Diabetic Patients from the e-HEALING Registry; Journal of Interventional Cardiology 2011;24(4):285-94 

J Aoki et al; Endothelial progenitor cell capture by stents coated with antibody against CD34: the HEALING-FIM (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth-First In Man) Registry.J.Am.Coll.Cardiol. 2005 May 17;45(10):1574-9

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Cardiovascular Outcomes: Function of circulating Endothelial Progenitor Cells (cEPCs): Exploring Pharmaco-therapy targeted at Endogenous Augmentation of cEPCs

Author and Curator: Aviva Lev-Ari, PhD, RN

UPDATED on 1/25/2018

The secret to building a strong heart lies in blood vessels, Stanford researcher find

Curiously, blood flow through those missing vessels – and the oxygen it provides – is only part of the story. In a follow-up experiment, the researchers grew heart muscles in a dish along with endothelial cells that had not yet formed into blood vessels. The team found that when those endothelial cells produced no Ino80, the heart muscle didn’t develop properly. Apparently, Red-Horse said, “endothelial cells are producing something that’s a growth factor” for cardiac muscle cells. “The next step is to identify that factor.”

https://news.stanford.edu/2018/01/25/secret-building-strong-heart-blood-vessels/

This is a post in Clinical Cardiology Frontiers:

  • Resident-Cell-based Therapy and
  • Molecular Cardiology

An Overview of the State of  Science on Circulating Endothelial Progenitor Cells (cEPCs) and Cardiovascular Outcomes: Exploring Pharmaco-therapy targeted at Endogenous augmentation of cEPCs

 

Werner (2005) reported that after 12 months a total of 43 participants died, 23 from cardiovascular(CV) causes. A first major cardiovascular event occurred in 214 patients in 519 patients with coronary artery disease as confirmed on angiography. Endothelial progenitor cells (EPCs) derived from bone marrow are believed to support the integrity of the vascular endothelium. His study identified that the number and function of endothelial progenitor cells correlate inversely with cardiovascular risk factors, but the prognostic value associated with circulating endothelial progenitor cells has not been defined. The level of circulating CD34+KDR+endothelial progenitor cells predicts the occurrence of cardiovascular events and death from cardiovascular causes and may help to identify patients at increased cardiovascular risk. The number of endothelial progenitor cells positive for CD34 and kinase insert domain receptor (KDR) was determined with the use of flow cytometry, they evaluated the association between baseline levels of endothelial progenitor cells and death from cardiovascular causes, the occurrence of a first major cardiovascular event (myocardial infarction, hospitalization, revascularization, or death from cardiovascular causes), revascularization, hospitalization, and death from all causes (italics added).

Werner (2005) reported that after 12 months a total of 43 participants died, 23 from cardiovascular(CV) causes. A first major cardiovascular event occurred in 214 patients in 519 patients with coronary artery disease as confirmed on angiography. Endothelial progenitor cells (EPCs) derived from bone marrow are believed to support the integrity of the vascular endothelium. His study identified that the number and function of endothelial progenitor cells correlate inversely with cardiovascular risk factors, but the prognostic value associated with circulating endothelial progenitor cells has not been defined. The level of circulating CD34+KDR+endothelial progenitor cells predicts the occurrence of cardiovascular events and death from cardiovascular causes and may help to identify patients at increased cardiovascular risk. The number of endothelial progenitor cells positive for CD34 and kinase insert domain receptor (KDR) was determined with the use of flow cytometry, they evaluated the association between baseline levels of endothelial progenitor cells and death from cardiovascular causes, the occurrence of a first major cardiovascular event (myocardial infarction, hospitalization, revascularization, or death from cardiovascular causes), revascularization, hospitalization, and death from all causes (italics added).

In light of the inverse correlation found between CV risk and enumeration and function of cEPCs, this study proposes a pharmaco-therapeutic method to enhance the cell count by a method of endogenous augmentation as presented in Part II and called ElectEagle.

 

Phenotypic Identification of Circulating Endothelial Progenitor Cells (cEPCs)

 

In the current state of science on cEPCs, the definition of these cells is ambiguous, as found in Fadini et al. (2004) letter to the Editors of Heart. On this subject, additional five letters were addressed to NEJM Editor in 2005, in reference to Werner et al. (2005) article Correspondence titled: Circulation Endothelial Progenitor Cells. Werner et al. (2005) was in fact the stimulant for this project which takes his result as a starting point and carries the research into pharmaco-therapy and device design for diagnostic based on a quantitative model derived from Werner’s data.

In the article in Heart, peripheral blood CD34+ cells are defined as endothelial progenitor cells. In Fadini’s concern he notes that cEPCs represent a subset of peripheral blood mononuclear cells (PBMNCs) expressing immature surface markers common to hematopoietic stem cells and endothelial lineage markers. By contrast CD34 represents a marker of immature staminal cells that may be used to characterize EPCs together with other surface antigens, but that identifies not only EPCs. Peripheral blood CD34+ cells form a very heterogeneous pool containing also CD45+ cells (lymphatic precursors), CD14+ cells (monocyte/macrophage lineage precursors) and other non-hematopoietic cells not belonging to the endothelial lineage.

Fadini’s concern is supported by George et al. (2006), who concluded that current methods for quantitatively assessing numbers of circulating EPC are not correlated. George’s findings may suggest that CD34/KDR is more appropriate for the definition of circulating EPC, whereas CFU (colony forming Unit) numbers are more likely to reflect their ability to proliferate. Fadini’s research supports the percentage of EPCs among the CD34+ pool vary widely from patient to patient and, in the same patient, under different pathophysiological conditions, indicating possible peripheral differentiation rather than bone-marrow mobilization. His observation is supported by Lapidot & Petit (2002) and Hur et al., (2004).

Furthermore, CD133 is considered the best surface marker to define, identify and isolate circulating EPCs. Even if the exact phenotype of EPCs has not been clearly established, additional markers reflecting endothelial commitment, including Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2 or KDR), Platelet-Endothelial Cells Adhesion Molecule-1 (PECAM-1 or CD31), Vascular Endothelial-Cadherin, von Willebrand Factor, c-kit, Tie-2 and VEGFR-1, are required. Using flow cytometry less than 0,001% of PBMNCs is identified as EPCs, but two or three markers are needed to avoid unspecific count. Thus minimal requirement to identify EPCs should be the parallel use of CD34 (or CD133) and KDR expression, as supported by George (2006).

Thus, he suggests that PBMNCs-derived CD34+ cells may not be used to identify EPCs. Fadini suggests that if we consider that EPCs or CD34+ cells stimulate angiogenesis in a paracrine way by means of producing growth factors, then, it would be more appropriate to call them “Circulating Angiogenic Cells” (CACs) as already proposed. EPCs reduction and endothelial dysfunction as observed by circulating cells and vascular wall cells of diabetic patients are exposed to high oxidative stress, thus increased apoptosis or reduced peripheral differentiation are likely to explain low EPCs counts. Several other clinical conditions characterized by poor endothelial function, like diabetes mellitus, obesity, hypertension, autoimmune disorders (such as Systemic Lupus Erythematosus), chronic renal diseases, etc., all are likely to be influenced by EPCs reduction (Fadini, 2004).

The five letters to NEJM Editor, Kim et al (2005) are of great scientific merit and of great interest to this project. We are developing new intellectual property (IP) in several forms in Part II and Part III. The IP in Part III is actually using Werner et al. (2005a) data, for our method called ElectEagle. Therefore, here, Werner’s (2005b) points will be considered as his reply to the five letter correspondence and no discussion of the content of the five letters is presented.

  •           Werner & Nickenig (2005b) disagree with Bertolini et al. that CD34+KDR+ cells are mature circulating endothelial cells. Endothelial cells are predominantly identified by the presence of CD146 (and results of Boos et al., as described in their letter).
  •           In Werner et al (2005a), CD146+ circulating endothelial cells were not predictive of cardiovascular outcomes, which indicates that CD34+KDR+ cells differ substantially from circulating endothelial cells (unpublished data). It is an accepted standard to identify circulating endothelial progenitor cells by the presence of CD34 and KDR.
  •           To confirm the results, Werner et al (2005a) measured CD133+ endothelial progenitor cells and obtained similar results, which appear in the online Supplementary Appendix to our article.
  •           Drs. Ott and Taylor suggest calculating the absolute number of endothelial progenitor cells with the use of peripheral-blood mononuclear cells or lymphocytes. However, absolute cell counts measured by flow cytometry can be determined only with the use of enumeration systems (e.g., flow count beads).
  •           At present, we cannot think of a major advantage to measuring the absolute number of endothelial progenitor cells. The method provided allows a single measurement that is easy to perform, highly predictive, and transferable to other laboratories.
  •           Dr. Kim and Dr. Leu and colleagues address  the role of endothelial progenitor cells in acute coronary syndromes and acute myocardial infarction. Only one study has investigated the mobilization of CD34+KDR+ endothelial progenitor cells in myocardial infarction, whereas other studies have measured CD34+ cells or non–endothelial progenitor cell subfractions.
  •           None of the studies have systematically looked at the time course directly after acute myocardial infarction, owing to the fact that the exact onset of myocardial infarction is difficult to determine. Treatment of myocardial infarction requires the administration of multiple drugs that may influence the number of endothelial progenitor cells. Therefore, current data on progenitor cells in myocardial infarction are questionable.
  •           In order to elucidate the mobilization of endothelial progenitor cells after myocardial infarction, Werner et al (2005a) measured the number of CD34+KDR+ cells in patients undergoing transcoronary ablation of septal hypertrophy (unpublished data). Preliminary results indicate that directly after myocardial infarction, the number of endothelial progenitor cells decreases as a result of consumption of cells within the ischemic region. The increase in cells described previously may be due to medical treatment.
  •           No patient who was included in the study had had a recent ischemic event, so misclassification of patients was not an issue.
  •           Werner et al. (2005a) did not find an association between high sensitivity CRP measures and the number of endothelial progenitor cells. To their knowledge, there are no data available on the association between endothelial progenitor cells and inflammatory markers in a similar population of patients. Data that are available come from in vitro, animal, and small-scale studies investigating the role of endothelial progenitor cells in acute coronary syndromes. Since their study population consisted mainly of patients with stable coronary artery disease, this may explain the lack of an association.

Circulating Endothelial Cells (cECs) and Circulating Endothelial Progenitor Cells (cEPCs)

Vascular endothelial cells (EC) respond to numerous pathophysiological stimuli such as growth factors, cytokines, lipoproteins, and oxidative stress. Prolonged or unregulated activation of these cells often results in a loss of EC integrity and, thus, dysfunction—a process that can be assessed by the use of specific plasma markers such as von Willebrand factor (vWf), tissue plasminogen activator, soluble EC protein C receptor, soluble E selectin, and soluble thrombomodulin, as well as physiological techniques such as flow-mediated dilatation (FMD). Indeed, endothelial perturbation in cancer may well contribute to an increased risk of thrombosis in these patients. (Goon et al., 2006)

The presence of circulating endothelial cells (cECs) has recently been recognized as a useful marker of vascular damage. Usually absent in the blood of healthy individuals, cECs counts are elevated in diseases hallmarked by the presence of vascular insult, such as sickle cell anemia, acute myocardial infarction, Cytomegalovirus (CMV) infection, endotoxemia, and neoplastic processes. Current opinion suggests toxemia, that cECs are cells driven from the intima after vascular insult, and are thus the consequence—rather than the initiator—of a particular pathology (Goon et al., 2006).

A related circulating cell population are endothelial progenitor cells (cEPCs), which originate from the bone marrow, rather than from vessel walls. Seen in small numbers in healthy individuals, their numbers tend to increase following vascular injury. So far, experiments have established the ability of EPCs to form colonies in vitro, suggesting a role in both angio-genesis and in the, maintenance of existing vessel walls.

CEC are generally accepted as cells expressing endothelial markers [e.g., vWf, CD146, and vascular endothelial cadherin (VE-cadherin)] in the absence of hematopoietic (CD45 and CD14) and progenitor (CD133) markers. Interestingly, the progenitor marker CD34 is also present on mature cECs. Although CD146 is widely regarded as the principal marker for cECs (mature cell form), it has also been described in trophoblasts, mesenchymal stem cells, periodontal and malignant (prostatic cancer and melanoma) tissues, and activated lymphocytes (Goon et al., 2006).

Optimal Method for cECs and cEPCs Quantification (Cell Count) Remains Unknown

Together with EPCs, cECs only represent between 0.01% and 0.0001% of mononuclear cells in normal peripheral blood (Khan et al. (2005), making it very difficult to accurately quantify their numbers. To do this, it is often necessary to employ cell enrichment techniques combined with specific cell marker labeling.  The immunobead capture method (immunomagnetic beads bearing CD146 antibodies) developed by George et al. (1992) is the most widely used. Immunobeads have been successfully employed by other investigators, albeit with modifications [e.g., addition of EDTA and albumin to minimize cECs autoaggregation; drying cECs on a glass slide before counting (this enables storage at room temperature and secondary labeling); use of UEA-1 (an EC-specific stain); addition of an Fc receptor blocking agent, and double labeling for further analyses (e.g., for CD31 and CD34)]. After cell separation, either fluorescence microscopy, immunocyto-chemistry, or flow cytometry is used to confirm the endothelial chemistry, phenotype of the cells. Other methods used to concentrate mononuclear cell suspensions include standard and density (Lymphoprep, Axis-Shield, Oslo, Norway; Percoll, Sigma, St. Louis, MO; Ficoll, Sigma) centrifugation and mononuclear cell culturing on fibronectin-coated plates. The main alter- alternative to the immunobead method is flow cytometry” (Goon, 2006).

Werner et al. (2005a) used the following method for Flow Cytometry — For fluorescence-activated cell-sorting analysis, mononuclear cells were resuspended in 100 µl of a fluorescence-activated cell-sorting buffer containing phosphate-buffered saline, 0.1 percent bovine albumin, and aprotinin (20 µl per milliliter). Immunofluorescent cell staining was performed with the use of the fluorescent conjugated antibody CD34–fluorescein isothiocyanate (FITC) (10 µl; Becton Dickinson), KDR (kinase insert domain receptor), and CD133–phycoerythrin (PE) (10 µl; Miltenyi). For the identification of KDR+ cells, indirect immunolabeling was performed with the use of a biotinylated goat mononuclear antibody against the extracellular domain of human KDR (R&D Systems). IgG2a–FITC–PE antibody (Becton Dickinson) served as a negative control. For staining of KDR, extensive blocking was required with the use of human immunoglobulin (polyglobulin, 10 percent; Bayer) and goat serum (Sigma-Aldrich). Cell fluorescence was measured immediately after staining, and data were analyzed with the use of CellQuest software (FACS Calibur, Becton Dickinson). Units of all measured components are absolute cell counts obtained after the measurement of 10,000 events in the lymphocyte gate. To assess the reproducibility of the measurements, two separate blood samples were obtained, on days 0 and 7, from 10 subjects. The intraclass correlation between the two probes was 0.94. Probes were measured at the same time of day, with identical instrument settings, by two investigators. For each patient, a corresponding negative control with IgG2a–FITC–PE antibody was obtained.

Colony-Forming Units of Endothelial Cells (Werner et al. 2005a)

In an endothelial basal medium (CellSystems) with supplements, 1×107 mononuclear cells were seeded on human fibronectin–coated plates (Sigma-Aldrich). After 48 hours, 1×106 nonadherent cells were transferred into new fibronectin-coated wells to avoid contamination with mature endothelial cells and nonprogenitor cells.22 After seven days in vitro, endothelial colony-forming units in at least three wells were counted by two independent investigators. Colony-forming units of endothelial cells are expressed as absolute numbers of colonies per well. (Werner, et al. 2005)

George et al. (2006) reports using the following method while performing an analysis of several methods used for cEPCs assessment and correlated them with humoral factors known to influence their numbers:

Peripheral blood mononuclear cells were obtained and stained for FACS analysis with antibodies to CD34, CD45, CD133, and KDR and the remaining cells grown under endothelial cell conditions for assessment of colony-forming unit (CFU) numbers and adhesive properties. Levels of circulating vascular endothelial growth factor (VEGF), erythropoietin (EPO), and C-reactive protein (CRP) were determined and correlated with each of the EPC markers.

Pathophysiology of cECs

The endothelium can be viewed as a membrane-like layer lining the circulatory system, its primary function being the maintenance of vessel wall permeability and integrity. The EC layer is relatively quiescent, with an estimated cell turnover period of between 47 and 23,000 days, as shown by labeling studies. Proliferation seems to occur mainly at sites of vasculature branching and turbulent flow. cECs are thought to have ‘‘sloughed off’’ vessel walls, indicating severe endothelial damage. Thus, unsurprisingly, cECs have been shown to correlate with various endothelial dysfunction and inflammatory markers.

Although not fully understood, it would appear that cECs detachment from the endothelium involves multiple factors, such as mechanical injury, alteration of endothelial cellular adhesion molecules (such as integrin alphaVbeta3), defective binding to anchoring matrix proteins (such as fibronectin, laminin, or type IV collagen), and cellular apoptosis with decreased survival of cytoskeletal proteins. The net effect is a reduced interaction between the EC and basement membrane proteins, with subsequent cellular detachment (Goon et al. 2006).

 

Pathophysiology of cEPCs

In Science 1997, Asahara et al. was the first to isolate EPC in human peripheral blood, using anti-CD34 monoclonal antibodies. With the use of CD133, an antigen specifically identifying primitive stem cells, a novel means to precisely delineate mature (cECs) from immature (cEPCs) EC forms was possible (Asahara et al. 1997), although this antigen is only present in human EPCs and cannot be applied to mouse EPCs (Rafii et al. 2003). To detect cEPCs in peripheral blood, Flow Cytometry and culture have become the principal methods employed. Other markers used include vWf, VE-cadherin, vascular endothelial growth factor receptor-2 (VEGFR-KDR) and binding by lectins and acetylated low-density lipoproteins (Peichev et al. 2000, Rafii et al. 2003).

cEPCs are potentially crucial for neovascularization and may be recruited from the bone marrow after tissue ischemia, vascular insult, or tumor growth (Rafii et al. 2003). They possess the ability to migrate, colonize, proliferate, and, ultimately, differentiate into endothelial lineage cells. These cells have yet to acquire mature ECs characteristics while appearing to contribute to vascular homeostasis.

cEPCs have been isolated previously from human umbilical cord blood, adult bone marrow, human fetal liver cells, and cytokine-mobilized peripheral blood, and an increase in cEPCs follows in vivo administration of the angiogenic growth factor VEGF. When incubated with VEGF, fibroblast growth factor-2 (FGF-2), and insulin-like growth factor, CD133+ cells differentiated into mature-type adherent EC, expressing endothelial-specific cell markers (vWf and VE-VE cadherin) and abolishing CD133 expression (Goon, 2006). Generation of endothelial outgrowths that are positive for CD146, vWf (mature endothelial growth markers), and CD36 (a representative scavenger receptor marker as well as a microvascular marker) markers from circulating mononuclear cells (of donor genotype in bone marrow transplant patients), strongly suggests the viability and proliferative potential of cEPCs.

cEPCs recruitment and mobilization have been positively correlated with increased levels of angiogenic growth factors such as VEGF which induces the proliferation, differentiation, and chemotaxis of cEPCs, and is essential for hematopoiesis, angiogenesis, and, ultimately, survival.  cEPCs influence cells mainly by interactions with VEGFR-1 and VEGFR-2, both being receptors expressed on hematopoietic stem cells (HSC) and cEPCs. In another study, granulocyte colony-colony stimulating factor also increased the number of CD34 stimulating CD34+ cells, potentially stimulating neovascularization in areas of is- ischemic myocardium Other angiogenic growth factors stimulating cEPCs mobilization include angiopoietin-1, FGF, SDF-1, PlGF, and (in mice) macrophage colony-stimulating factor. After mobilization, cEPCs appear to “home in” and become incorporated into sites of vascular injury and ischemia, with evidence of improvement in the function and viability of tissue (e.g., after acute myocardial infarction) (Kocher et al. 2001).

Chemotactic agents responsible for this process include VEGF and SDF-1, but others may also be involved. In the clinical setting, moderate exercise of patients with stable coronary artery diseases leads to a significant increase in cEPCs (Laufs et al. 2004). Furthermore, cEPCs and HSC introduced into the circulation of acute and chronic cardiovascular disease patients through injection have shown vascular encouraging preliminary results, with evidence of improved cardiovascular function and tissue perfusion Tse et al. (2003); as of yet, there are no randomized control trial!

Recent reports suggest that cECs and cEPCs enumeration can be used to monitor antiangiogenesis drug therapy with some success. This exciting prospect needs to be fully corroborated in a clinical setting. In addition, cECs and cEPCs monitoring would need to be efficient, specific, robust, and reproducible. Therefore, it is vital to reach a general consensus regarding definitions and techniques for cECs and cEPCs quantification, in order to validate further reports that have implications for future clinical trials involving these markers (Goon, 2006).

In 2002, matrix metalloproteinase-9 (MMP-9) was identified as the molecular key to the release of EPCs from the bone-marrow compartment via cleavage of membrane kit ligand (Heissig et al., 2002). MMP-9 activity has also been shown to be upregulated by SDF-1alpha, VEGF, and hypoxia. Hypoxia is a potent stimulus for neovascularization, ischemia-induced growth, EPC trafficking and upregulation, vascular malformations and malignant endothelial cell tumors. This include activation of two upstream mediators of vasculogenesis, SDF-1 alpha and MMP-9, during the proliferative phase via EPC mediated vasculogenesis when these stem cells may rapidly proliferate in the ischemic tissue resulting in growth.

How a mobilized population of progenitor cells homes to ischemic tissue under repair was examined using bone-marrow transplantation studies. Following this procedure, reconstitution is regulated by chemokine ligand-receptor pair, stromal-cell derived factor 1 (SDF-1) and CXCR4 (Lapidot & Petit, 2002 reporting their discovery of 1999). EPCs express CXCR4, CXCR4/SDF-1 signal for EPC homing to peripheral sites of neovascularization. EPC SDF-1alpha expression was increased in proportion to reduced oxygen tension and this correlated with EPC localization in the most ischemic tissue sections (Kleinman, et al. 2005).

Table 1: Humoral factors known to influence eCPCs numbers

CD34 CD45 CD133 KDR CD34/KDRMost appropriate Definition of cEPCs CD34/CD133/KDR CD34/CD133 Adhesive properpies
VEGF level corr Positive correlation
EPO
CRP
CFU Colony forming unit numbersReflects cEPCs ability to proliferate No corr No corr Negative correlation Positive correlation
Adhesive properties No corr No corr No corr No corr
CD34
CD45
CD133
KDR
CD34/CD133/KDR Positive correlation
CD34/CD133 No corr

 SOURCE:

Table 1 is constructed from data in George et al. (2005),(2006) who concluded that current methods for quantitatively assessing numbers of circulating EPC are not correlated. VEGF serum levels are associated only with CD34/KDR and CD34/ CD133/KDR, whereas CFU numbers correlate with EPC functional properties. These findings may suggest that CD34/KDR is more appropriate for the definition of circulating EPC, whereas CFU numbers are more likely to reflect their ability to proliferate.

 

Trans-Endothelium Cell Migration

Lapidot and Petit in a recent review of the stem cell mobilization research field concluded that the following are the seminal processes at work in the facilitation of transendothelium cell migration. These processes could mediate stem cell release and remodeling of the bone marrow microenvironment, followed by stem cell migration via the circulation, homing back to the bone marrow and repopulation of damaged/restructured sites in an organ as part of the continuous replenishment of the blood with new immature and maturing cells while maintaining undifferentiated stem cells (Lapidot and Petit, 2002).

  •       Regulation of hematopoietic stem cell release, migration, and homing to the bone marrow, as well as the mechanism of different mobilization pathways, involve a complex interplay between adhesion molecules, chemokines, cytokines, proteolytic enzymes, stromal cells, and hematopoietic cells, the mechanism is not fully understood;
  •       The chemokine, stromal derived factor-1 (SDF-1)and its receptor CXCR4 play a major role in stem cell mobilization, including granulocyte colony-stimulating factor (G-CSF) and G-CSF with the chemotherapeutic agents cyclophosphamide Cy-induced mobilization, as well as in stem cell homing to the bone marrow and anchorage (i.e., activation of adhesion interactions in order to retain stem cells within the organ).
  •       They suggested that in addition to SDF-1 degradation and inactivation within the bone marrow by proteolytic enzymes such as neutrophil elastase, which is essential for optimal stem cell mobilization, interactions between this chemokine and its receptor are also needed for stem cell release and mobilization. For example, they suggested that IL-8, which is secreted in response to SDF-1 stimulation, and MMP-2 and MMP-9, which are mostly secreted by neutrophils but are also secreted by immature human CD34+ progenitor cells in response to stimulation with this chemokine, can also lead to migration away from the bone marrow across the endothelium into the circulation also in the absence of or against a gradient of SDF-1 under shear flow forces within the extravascular space of the bone marrow (Cinamon et al., 2001).
  •        They suggest that in order to maintain stem cells in the circulation low levels of surface CXCR4 are required and may be achieved by factors in the blood plasma such as proteolytic enzymes that can also cleave CXCR4 in addition to SDF-1 . Valenzuela-Fernandez (2002). Furthermore, increase in the levels of CXCR4 expression on the surface of stem cells in the circulation will mediate their homing and reengraftment of the bone marrow as part of homeostatic regulation of leukocyte trafficking as well as steady-state hematopoiesis and stem cell self-renewal, which go hand in hand with bone destruction and bone remodeling. However, this hypothesis is also an oversimplification of a much more complex and dynamic situation with physiological steady-state homeostatic as well as stress-induced mobilization situations in which the mechanisms and mode of regulation are still poorly understood.
  •        A significant number of studies in the past few years have revealed insights into regulation of hematopoietic stem cell release, migration, and homing as well as the mechanism of different mobilization pathways. However, the exact sequence of events involving many different molecules is still not clear. More importantly, in addition to results from clinical mobilization protocols using Cy and G-CSF, which demonstrate a role for SDF-1 and CXCR4 interactions in immature human CD34+ cell mobilization, two recent reports also demonstrate a role for these interactions in autologous and allogenic CD34+ cell homing and repopulation.
  •        Taken together, these results suggest that stem cell homing and release or mobilization are mirror images utilizing a similar mechanism and suggest manipulation of SDF-1/CXCR4 interactions in order to improve stem cell mobilization or to target migration of transplanted cells to specific organs. These results strongly support the idea that increasing the migration potential of immature human CD34+ cells prior to transplantation, either by short term stimulation with SCF and IL-6 and/or by cotransplantation with accessory cells. It could also increase homing and repopulation in transplanted patients, leading to improved treatment efficiencies and cure rates in clinical protocols.

 

Prospects and Limitations of Exogenous methods for cEPCs Augmentation

ElectEagle represents a conceptual formulation for several strategies to increase cEPCs number endogenously.

Additional posts on this Scientific Web Site on related topics are:

Lev-Ari, A., (2012S). Endothelial Dysfunction, Diminished Availability of cEPCs, Increasing CVD Risk for Macrovascular Disease – Therapeutic Potential of cEPCs

https://pharmaceuticalintelligence.com/2012/08/27/endothelial-dysfunction-diminished-availability-of-cepcs-increasing-cvd-risk-for-macrovascular-disease-therapeutic-potential-of-cepcs/

Lev-Ari, A., (2012T). Vascular Medicine and Biology: CLASSIFICATION OF FAST ACTING THERAPY FOR PATIENTS AT HIGH RISK FOR MACROVASCULAR EVENTS Macrovascular Disease – Therapeutic Potential of cEPCs

https://pharmaceuticalintelligence.com/2012/08/24/vascular-medicine-and-biology-classification-of-fast-acting-therapy-for-patients-at-high-risk-for-macrovascular-events-macrovascular-disease-therapeutic-potential-of-cepcs/

Below, this method is contrasted with exogenous methods involving the cell-based vascular therapy approaches currently applied for angiogenesis (mature cell-derived generation of new vessels), vasculogenesis (EPC-dependent generation of new vessels), neovascularization (ischemic tissue) and re-endothelialization (injured blood vessel.)  The majority of these methods are exogenous involving implantation or transplantation of various kinds: genetically engineered vein grafts, vascular bioprosthesis, retroviral transduction of genetic modifications to over-express a therapeutic gene(s).Despite the hurdles quoted below, the outlook for EPC-based therapy for cardiovascular disease is promising.Among the remaining outstanding issues in this fast growing research discipline, Dzau et al., chart a perspective for future research directions (Dzau et al. 2005)

“Despite the encouraging results regarding the therapeutic potential of EPCs, several issues currently stand in the way of their wide clinical application. Strategies need to be developed to enhance the number of EPCs to allow the harvesting of adequate number for therapeutic application. The limited ability to expand PB-MNC–derived EPCs in culture to yield sufficient number for clinical application indicates that alternative sources of cells (i.e., chord blood) or strategies to increase their number endogenously need to be explored. We believe that further characterization of the biology of EPCs, the nature of the mobilizing, migratory and homing signals, and the mechanisms of differentiation and incorporation into the target tissues need to be identified and further characterized. Strategies to improve retention and survival of the transplanted cells need to be developed as well. The issues of the timing of cell administration, the appropriate clinical condition, the optimal cell number, and, most importantly, the safety of cell transplantation must be defined. There is urgent need to standardize the protocols for isolation, cultivation, and therapeutic application for cell-based therapy. Finally, large-scale randomized, controlled, multi-centric trials will be essential to evaluate the long-term safety and efficacy of EPC therapy for treatment of tissue ischemia and vessel repair amid concerns of potential side effects such as neovascularization of occult neoplasias and the development of age- and diabetes-related vasculopathies. Despite these hurdles, the outlook for EPC-based therapies for tissue ischemia and blood vessel repair appears promising. Genetic engineering of EPC may provide an important strategy to enhance EPC mobilization, survival, engraftment, and function, thereby rendering these cells efficient therapeutic modalities for cardiovascular diseases.” (italics added).

In the Brief Review in Hypertension, Dzau et al., list several serious potential problems with therapeutic use of EPCs (Dzau et al. 2005)

“Although the preclinical and clinical studies reviewed here generally lend support to the therapeutic potential of autologous EPCs in the treatment of tissue ischemia and repair of injured blood vessels, the clinical application of EPCs is limited by several factors. First, the scarcity of cEPCs makes it difficulty to expand sufficient number of cells for therapeutic application without incurring the risk of cell senescence and change in phenotype (Asahara, et al., 1997, 1999). Furthermore, EPCs from patients with cardiovascular diseases display varying degrees of functional impairment (Vasa et al., 2001a, 2001b), (Hill et al., 2003), (Heeschen et al., 2004), (George et al., 2003), (Loomans et al., 2004), (Tepper et al., 2002). Aging and diabetes markedly reduce the availability and impair the function of EPCs (Hill et al., 2003), (Loomans et al., 2004), (Tepper et al., 2002), (Schatteman et al., 2000), (Scheubel et al., 2003), (Edelberg et al., 2002). Because older and diabetic patients are the most vulnerable populations for cardiovascular diseases, this severely restricts the ability to treat with autologous EPCs the patients who theoretically need them most.

The purity and developmental stage of the cells used for transplantation are important factors. Yoon et al reported recently that injection of total bone marrow cells into the heart of infarcted rats could potentially lead to severe intramyocardial calcifications (Yoon et al., 2004). In contrast, animals receiving the same number of clonally expanded bone marrow cells did not show myocardial calcification. Thus, this finding brings attention to the potential risks of transplanting unselected bone marrow cells and cautions against their premature use in the clinical setting.

Exogenous mobilization of bone marrow with hematopoietic growth factors and other endothelial cell growth factors may recruit progenitor cells to sites of occult neoplasia, leading to vascularization of dormant tumors. In addition, mobilization could potentially accelerate progression of atherosclerotic plaque by recruiting inflammatory and vascular smooth muscle cell progenitor cells into the plaque, contributing to neointima hyperplasia and transplant arteriopathy (Caplice et al., 2003), (Sata et al., 2002). Increased rate of in-stent restenosis led recently to the cancellation of the MAGIC clinical trial using G-CSF for endogenous mobilization of progenitor cells in patients with myocardial infarction.120 Finally, there has been one study that has shown evidence that EPC may themselves contribute to allograft vasculopathy by promoting neovascularization of the plaque(Hu et al., 2003). However, another study failed to show evidence that EPCs contribute significantly to transplant arteriosclerosis (Hillebrands et al., 2003).”

In accordance with this account is the latest review of EPC as therapeutic vectors in CV disorders covering experimental models and human trials (Ben-Shoshan and George, 2006).

The conceptual formulation for several strategies to increase cEPCs number endogenously presented in this investigation is complementary to methods currently applied or are still in clinical trials, as reviewed by Dzau et al. (2005). However, our approach, ElectEagle, involves endogenous augmentation of cEPCs by development of a concept-based protocol for therapeutic treatment using three components:

  •                Inhibition of ET1, ETB
  •                Induction of NO production and stimulation of eNOS
  •                Treatment Regimen with PPAR-gamma agonists (TZD) 

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Macrovascular Disease – Therapeutic Potential of cEPCs: Reduction Methods for CV Risk

Curator: Aviva Lev-Ari, PhD, RN

An Investigation of the Potential of circulating Endothelial Progenitor Cells (cEPCs) as a Therapeutic Target for Pharmacological Therapy Design for Cardiovascular Risk Reduction: A New Multimarker Biomarker Discovery

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