Sodium has long been labeled the blood-pressure bogeyman. But are we giving salt a fair shake? A new study published in the American Journal of…

Source: time.com

See on Scoop.itCardiovascular Disease: PHARMACO-THERAPY

Got high cholesterol? Lower your cholesterol naturally with these 7 heart-healthy foods.

Source: www.forbes.com

See on Scoop.itCardiovascular Disease: PHARMACO-THERAPY

All-cause mortality higher in those who spend a lot of time in chairs, irrespective of exercise.

Source: www.medpagetoday.com

See on Scoop.itCardiovascular and vascular imaging

Calculating your heart age could change your life. People who calculated theirs were found in a 2014 study to have more success sticking with healthy life changes than those who were given a plain old risk score.

Source: www.prevention.com

See on Scoop.itCardiovascular and vascular imaging

Heart-Lung-Kidney: Essential Ties

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


Heart-Lung-Kidney: Essential Ties


The basic functioning of the heart, and the kidney have been covered in depth elsewhere, and pulmonary function less, except in this series.  The relationship between them on the basis of endocrine, signaling, and metabolic balance is the focus in this piece.

Other elated articles can be found in http://pharmaceuticalintelligence.com:

The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide – Part I

Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II

Stroke and Bleeding in Atrial Fibrillation with Chronic Kidney Disease

Risks of Hypoglycemia in Diabetics with Chronic Kidney Disease (CKD)

Acute Lung Injury

Neonatal Pathophysiology

Altitude Adaptation

Action of Hormones on the Circulation

Innervation of Heart and Heart Rate

Neural Activity Regulating Endocrine Response

Adrenal Cortex

Thyroid Function and Disorders

Highlights in the History of Physiology

The Evolution of Clinical Chemistry in the 20th Century

Complex Models of Signaling: Therapeutic Implications

Cholesterol and Regulation of Liver Synthetic Pathways

A Brief Curation of Proteomics, Metabolomics, and Metabolism

Natriuretic Peptides in Evaluating Dyspnea and Congestive Heart Failure

Omega-3 fatty acids, depleting the source, and protein insufficiency in renal disease

Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases

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

Diagnostic Value of Cardiac Biomarkers

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

The Young Surgeon and The Retired Pathologist: On Science, Medicine and HealthCare Policy – Best writers Among the WRITERS

Renal Function Biomarker, β-trace protein (BTP) as a Novel Biomarker for Cardiac Risk Diagnosis in Patients with Atrial Fibrillation

Leptin signaling in mediating the cardiac hypertrophy associated with obesity

The Role of Tight Junction Proteins in Water and Electrolyte Transport

Selective Ion Conduction

Translational Research on the Mechanism of Water and Electrolyte Movements into the Cell

Landscape of Cardiac Biomarkers for Improved Clinical Utilization

Calcium-Channel Blocker, Calcium as Neurotransmitter Sensor and Calcium Release-related Contractile Dysfunction (Ryanopathy)

Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage

The Cardio-Renal Syndrome (CRS) in Heart Failure (HF)


Sodium homeostasis

Icariin attenuates angiotensin IIinduced hypertrophy and apoptosis in H9c2 cardiomyocytes by inhibiting reactive oxygen speciesdependent JNK and p38 pathways

H Zhou, Y Yuan, Y Liu, Wei Deng, Jing Zong, Zhou‑Yan Bian, Jia Dai and Qi‑Zhu Tang
Exper and Therapeutic Med 7: 1116-1122, 2014

Icariin, the major active component isolated from plants of the Epimedium family, has been reported to have potential protective effects on the cardiovascular system. However, it is not known whether icariin has a direct effect on angiotensin II (Ang II)‑induced cardiomyocyte enlargement and apoptosis. In the present study, embryonic rat heart‑derived H9c2 cells were stimulated by Ang II, with or without icariin administration. Icariin treatment was found to attenuate the Ang II‑induced increase in mRNA expression levels of hypertrophic markers, including atrial natriuretic peptide and B‑type natriuretic peptide, in a concentration‑dependent manner. The cell surface area of Ang II‑treated H9c2 cells also decreased with icariin administration. Furthermore, icariin repressed Ang II‑induced cell apoptosis and protein expression levels of Bax and cleaved‑caspase 3, while the expression of Bcl‑2 was increased by icariin. In addition, 2′,7’‑dichlorofluorescein diacetate incubation revealed that icariin inhibited the production of intracellular reactive oxygen species (ROS), which were stimulated by Ang II. Phosphorylation of c‑Jun N‑terminal kinase (JNK) and p38 in Ang II‑treated H9c2 cells was blocked by icariin. Therefore, the results of the present study indicated that icariin protected H9c2 cardiomyocytes from Ang II‑induced hypertrophy and apoptosis by inhibiting the ROS‑dependent JNK and p38 pathways.

Short-term add-on therapy with angiotensin receptor blocker for end-stage inotrope-dependent heart failure patients: B-type natriuretic peptide reduction in a randomized clinical trial

Marcelo E. Ochiai, ECO Brancalhao, RSN Puig, KRN Vieira, et al.
Clinics. 2014; 69(5):308-313

OBJECTIVE: We aimed to evaluate angiotensin receptor blocker add-on therapy in patients with low cardiac output during decompensated heart failure. METHODS: We selected patients with decompensated heart failure, low cardiac output, dobutamine dependence, and an ejection fraction ,0.45 who were receiving an angiotensin-converting enzyme inhibitor. The patients were randomized to losartan or placebo and underwent invasive hemodynamic and B-type natriuretic peptide measurements at baseline and on the seventh day after intervention. ClinicalTrials.gov: NCT01857999. RESULTS: We studied 10 patients in the losartan group and 11 patients in the placebo group. The patient characteristics were as follows: age 52.7 years, ejection fraction 21.3%, dobutamine infusion 8.5 mcg/kg.min, indexed systemic vascular resistance 1918.0 dynes.sec/cm5.m2, cardiac index 2.8 L/min.m2, and B-type natriuretic peptide 1,403 pg/mL. After 7 days of intervention, there was a 37.4% reduction in the B-type natriuretic peptide levels in the losartan group compared with an 11.9% increase in the placebo group (mean difference, – 49.1%; 95% confidence interval: -88.1 to -9.8%, p = 0.018). No significant difference was observed in the hemodynamic measurements. CONCLUSION: Short-term add-on therapy with losartan reduced B-type natriuretic peptide levels in patients hospitalized for decompensated severe heart failure and low cardiac output with inotrope dependence.

Development of a Novel Heart Failure Risk Tool: The Barcelona Bio-Heart Failure Risk Calculator (BCN Bio-HF Calculator)

Josep Lupon, Marta de Antonio, Joan Vila, Judith Penafiel, et al.
PLoS ONE 9(1): e85466. http://dx.doi.org:/10.1371/journal.pone.0085466

Background: A combination of clinical and routine laboratory data with biomarkers reflecting different pathophysiological pathways may help to refine risk stratification in heart failure (HF). A novel calculator (BCN Bio-HF calculator) incorporating N-terminal pro B-type natriuretic peptide (NT-proBNP, a marker of myocardial stretch), high-sensitivity cardiac troponin T (hs-cTnT, a marker of myocyte injury), and high-sensitivity soluble ST2 (ST2), (reflective of myocardial fibrosis and remodeling) was developed. Methods: Model performance was evaluated using discrimination, calibration, and reclassi-fication tools for 1-, 2-, and 3-year mortality. Ten-fold cross-validation with 1000 bootstrapping was used. Results: The BCN Bio-HF calculator was derived from 864 consecutive outpatients (72% men) with mean age 68.2612 years (73%/27% New York Heart Association (NYHA) class I-II/III-IV, LVEF 36%, ischemic etiology 52.2%) and followed for a median of 3.4 years (305 deaths). After an initial evaluation of 23 variables, eight independent models were developed. The variables included in these models were age, sex, NYHA functional class, left ventricular ejection fraction, serum sodium, estimated glomerular filtration rate, hemoglobin, loop diuretic dose, β-blocker, Angiotensin converting enzyme inhibitor/Angiotensin-2 receptor blocker and statin treatments, and hs-cTnT, ST2, and NT-proBNP levels. The calculator may run with the availability of none, one, two, or the three biomarkers. The calculated risk of death was significantly changed by additive biomarker data. The average C-statistic in cross-validation analysis was 0.79. Conclusions: A new HF risk-calculator that incorporates available biomarkers reflecting different pathophysiological pathways better allowed individual prediction of death at 1, 2, and 3 years.

TNF and angiotensin type 1 receptors interact in the brain control of blood pressure in heart failure

Tymoteusz Zera, Marcin Ufnal, Ewa Szczepanska-Sadowska
Cytokine 71 (2015) 272–277

Accumulating evidence suggests that the brain renin-angiotensin system and proinflammatory cytokines, such as TNF-α, play a key role in the neuro-hormonal activation in chronic heart failure (HF). In this study we tested the involvement of TNF-α and angiotensin type 1 receptors (AT1Rs) in the central control of the cardiovascular system in HF rats. Methods: we carried out the study on male Sprague–Dawley rats subjected to the left coronary artery ligation (HF rats) or to sham surgery (sham-operated rats). The rats were pretreated for four weeks with intracerebroventricular (ICV) infusion of either saline (0.25 µl/h) or TNF-α inhibitor etanercept (0.25 µg/0.25 µl/h). At the end of the pretreatment period, we measured mean arterial blood pressure (MABP) and heart rate (HR) at baseline and during 60 min of ICV administration of either saline (5 µl/h) or AT1Rs antagonist losartan (10 µg/5 µl/h). After the experiments, we measured the left ventricle end-diastolic pressure (LVEDP) and the size of myocardial scar. Results: MABP and HR of sham-operated and HF rats were not affected by pretreatments with etanercept or saline alone. In sham-operated rats the ICV infusion of losartan did not affect MABP either in saline or in etanercept pretreated rats. In contrast, in HF rats the ICV infusion of losartan significantly decreased MABP in rats pretreated with saline, but not in those pretreated with etanercept. LVEDP was significantly elevated in HF rats but not in sham-operated ones. Surface of the infarct scar exceeded 30% of the left ventricle in HF groups, whereas sham-operated rats did not manifest evidence of cardiac scarring. Conclusions: our study provides evidence that in rats with post-infarction heart failure the regulation of blood pressure by AT1Rs depends on centrally acting endogenous TNF-α.

Statins in heart failure—With preserved and reduced ejection fraction. An update

Dimitris Tousoulis , E Oikonomou, G Siasos, C Stefanadis
Pharmacology & Therapeutics 141 (2014) 79–91

HMG-CoA reductase inhibitors or statins beyond their lipid lowering properties and mevalonate inhibition exert also their actions through a multiplicity of mechanisms. In heart failure (HF) the inhibition of isoprenoid intermediates and small GTPases, which control cellular function such as cell shape, secretion and proliferation, is of clinical significance. Statins share also the peroxisome proliferator-activated receptor pathway and inactivate extracellular-signal-regulated kinase phosphorylation suppressing inflammatory cascade. By down-regulating Rho/Rho kinase signaling pathways, statins increase the stability of eNOS mRNA and induce activation of eNOS through phosphatidylinositol 3-kinase/Akt/eNOS pathway restoring endothelial function. Statins change also myocardial action potential plateau by modulation of Kv1.5 and Kv4.3 channel activity and inhibit sympathetic nerve activity suppressing arrhythmogenesis. Less documented evidence proposes also that statins have antihypertrophic effects – through p21ras/mitogen activated protein kinase pathway – which modulate synthesis of matrix metalloproteinases and procollagen 1 expression affecting interstitial fibrosis and diastolic dysfunction. Clinical studies have partly confirmed the experimental findings and despite current guidelines new evidence supports the notion that statins can be beneficial in some cases of HF. In subjects with diastolic HF, moderately impaired systolic function, low B-type natriuretic peptide levels, exacerbated inflammatory response and mild interstitial fibrosis evidence supports that statins can favorably affect the outcome. Under the lights of this evidence in this review article we discuss the current knowledge on the mechanisms of statins’ actions and we link current experimental and clinical data to further understand the possible impact of statins’ treatment on HF syndrome.

Since 1980 when the first 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor or statin was introduced in clinical practice, statins have been extensively used in the treatment of patients with dyslipidemia as well as of those with coronary artery disease (CAD). Importantly, large scale trials and metanalysis have documented their significant benefits in terms of primary and secondary CAD prevention which out-weigh any potential side effects. Statins’ benefits extend, according to recent studies, even in patients with normal or low cholesterol levels and beyond their lipid lowering effects, indicating their multiple protective mechanisms.

Heart failure (HF) is a complex syndrome with different definitions and its diagnosis is based on a combination of symptoms, clinical signs and imaging or laboratory data. different categorization schemes have been used dividing HF in acute or chronic, in systolic or diastolic, and in ischemic or dilated simply reflecting the complexity of the syndrome and the multiplicity of the pathophysiologic mechanisms implicated in the disease development and progression. In addition to the diverse pathophysiology of HF the syndrome is also characterized by high morbidity and mortality. Recent treatment advantages such as angiotensin converting enzyme inhibitors and beta blockers have not yet proven their clinical benefit in subjects with diastolic HF.

As the most common cause of HF is CAD and statins have proven their benefits in a wide spectrum of diseases directly or indirectly associated with atherosclerotic cardiovascular disease, HMG-CoA reductase inhibitors have been tested in subjects with HF. Interestingly, non-randomized, observational and retrospective early studies in subjects with HF of ischemic and non-ischemic etiology have suggested that statins are associated with improved outcomes. Thereafter, two large scale randomized control trials failed to demonstrate any benefits in mortality of HF patients treated with rosuvastatin and subsequently current HF guidelines do not include recommendations for statin use except from when they are indicated for comorbidities, such as established CAD.

Statins inhibit HMG-CoA reductase. This enzyme catalyzes the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to L-mevalonic acid, which is the rate-limiting step in the cholesterol synthesis pathway. Inhibition of the mevalonate pathway and of cholesterol synthesis triggers an increase in LDL receptor activity by stimulating production of mRNA for LDL receptor in liver. The induction of LDL receptors is responsible for the observed increase in plasma clearance of LDL cholesterol. CAD is the cause of approximately two-thirds of cases of systolic HF. The beneficial effects of statins-induced LDL reduction are well established in patients with atherosclerosis and CAD. Nevertheless, the results from statin treatment, even in ischemic HF cases, are not straightforward and several mechanisms have been proposed for this paradox.

multiplicity of HMG CoA reductase inhibitors mechanisms and their effects

multiplicity of HMG CoA reductase inhibitors mechanisms and their effects

The figure demonstrates the multiplicity of HMG CoA reductase inhibitors mechanisms and their effects. ↓: decrease; ↑ increase; FPP: farnesyl pyrophosphate: GGPP: geranylgeranyl pyrophosphate; Ras, Rac, Rho; small GTPases; eNOS: endothelial nitric oxide synthase; ATP: adenosine triphosphate; PI-3 kinase: phosphatidylinositol 3-kinase; AMPK: AMP activated protein kinase; GTP: Guanosine triphosphate; NADPH: Nicotinamide adenine dinucleotide phosphate; ERK: extracellular-signal-regulated kinase; Shadow box represents adverse mechanism and actions of HGM CoA reductase inhibitors.

The anti-inflammatory effects of HMG CoA reductase inhibitors in atherosclerosis have been early recognized. Statins also have a potent anti-inflammatory effect in HF models. Importantly, there is a link between inflammation and HF pathogenesis and is now widely accepted that pro-inflammatory cytokines cause systolic dysfunction, myocardial hypertrophy, activate a fetal gene program in cardiac myocytes, disturb extracellular matrix structure, cause cardiac cachexia etc. In addition, data from the Vesnarinone trial (VEST) in 384 patients with HF demonstrate a decline in survival with increasing TNFα levels confirming the notion that circulating cytokines are associated with adverse prognosis of HF patients.

The proposed, by the aforementioned mechanisms, anti-inflammatory effects of statins have been confirmed experimentally. Indeed, in a rat HF model with preserved ejection fraction (EF), treatment with rosuvastatin resulted in a significant additional improvement in HF and cardiac remodeling, partly due to decreased myocardial inflammation. In rats after acute myocardial infarction simvastatin treatment for 4 weeks beneficially modified the levels of TNFα, interleukin (IL)-1, 6 and 10 in the infarct regions. Importantly, in 446 patients with systolic HF, followed up for a period of 24 months, statins’ treatment was associated with a decrease in serum levels of C-reactive protein (CRP), IL-6 and tumor necrosis factor-alpha receptor II. Recently, in a randomized study of 22 subjects with ischemic HF short term atorvastatin treatment achieved a significant decrease in serum levels of intracellular adhesion molecule-1.

Taken together we can conclude that HMG CoA reductase inhibitors can modify inflammatory status by modulation of PRAP and ERK pathways by down regulating Toll like receptor 4 mRNA expressions and LDL oxidation and by reducing soluble lipoprotein-associated phospholipase A2 mass and activity. Importantly, the theoretical anti-inflammatory properties were confirmed in experimental and clinical HF models.

Endothelial dysfunction contributes to the pathogenesis of HF and can enhance adverse left ventricle (LV) remodeling and increase afterload in subjects with HF. Interestingly, statins have been constantly associated with improved endothelial function in subjects with a variety of cardiovascular diseases. Endothelium derived nitric oxide (NO) is an important determinant of endothelial function and HMG-CoA reductase inhibitors can up regulate endothelial NO synthase (eNOS) by different mechanisms.

Statins induce down regulation of Rho/Rho kinase signaling pathways, increasing the stability of eNOS mRNA and its expression . In addition, in human endothelial cells the Rho-kinase inhibitor, hydroxyfasudil leads to the activation of the phosphatidylinositol 3-kinase/Akt/eNOS pathway. Statins also induce activation of eNOS through the rapid activation of the serine–threonine protein kinase Akt. The beneficial effects of Akt activation are not limited to eNOS phoshorylation but extend to the promotion of new blood vessels growth. HMG CoA reductase inhibitors can further affect endothelial function through their effect on caveolin-1. Caveolin-1 binds to eNOS inhibiting NO production. Incubation of endothelial cells with atorvastatin promotes NO production by decreasing caveolin-1 expression, regardless of the level of extracellular LDL-cholesterol. These effects were reversed with mevalonate highlighting the therapeutic potential of inhibiting cholesterol synthesis in peripheral cells to correct NO-dependent endothelial dysfunction associated with hypercholesterolemia and possibly other diseases.

Although the experimentally confirmed benefits of HMG CoA reductase inhibitors in diastolic dysfunction and left ventricle stiffness, few data exist concerning the underlying mechanisms. As diastolic dysfunction precedes myocardial hypertrophy the anti-hypertrophic pathways mentioned in the previous section (inhibition of RhoA/Ras/ERK, PRAPγ pathways, inhibition of a large G(h) protein-coupled pathway etc.), may also contribute to the restoration of diastolic function. Moreover, in angiotensin II induced diastolic dysfunction in hypertensive mice, pravastatin not only improved diastolic function but also down-regulated collagen I, transforming growth factor-beta, matrix metalloproteinases (MMPs)-2 and -3, atrial natriuretic factor, IL-6 TNFα, Rho kinase 1 gene expression, and upregulated eNOS gene expression. These findings suggest the potential involvement of Rho kinase 1 in the beneficial effects of pravastatin in diastolic HF. Taken together data suggest that HMG CoA reductase inhibitors might be beneficial in patients with diastolic HF, a hypothesis that remains to be confirmed by clinical studies. Nevertheless, mechanistic studies have not fully explored the pathways affecting diastolic function and most data until now are indirect. Therefore efforts should be focus on the underline mechanisms affecting collagen synthesis, MMPs activity extracellular matrix synthesis and overall diastolic function in HF subjects under statin treatment.

Statins through inhibition of small GTPases can modulate MMPs activity in several cell types such as endothelial cells and human macrophages. In rat and human cardiac fibroblasts, stimulated with either transforming growth factor β1 or angiotensin II, atorvastatin reduced collagen synthesis and α1-procollagen mRNA as well as gene expression of the profibrotic peptide connective tissue growth factor 4. This antifibrotic action may contribute to the anti-remodelling effect of statins. In mouse cardiac fibroblasts treated with angiotensin II, the combination of pravastatin and pioglitazone blocked angiotensin II p38 MAPK and p44/42 MAPK activation and procollagen expression-1.

Several studies have documented the impact of statin treatment on arrhythmia potential. The arrhythmic protective effects of statins can be attributed not only to anti-inflammatory properties but also to changes in myocardial action potential plateau by modulation of Kv1.5 and Kv4.3 channel activity. Atorvastatin and simvastatin block Kv1.5 and Kv4.3 channels shifting the inactivation curve to more negative potentials following a complex mechanism that does not imply the binding of the drug to the channel pore. Moreover, in hypertrophied neonatal rat ventricular myocytes simvastatin alleviated the reduction of Kv4.3 expression, I(to) currents in subepicardial myocardium from the hypertrophied left ventricle. Furthermore, pravastatin in an animal model attenuated reperfusion induced lethal ventricular arrhythmias by inhibition of calcium overload.

Taking together experimental and cellular evidence supporting an effect of statin treatment in myocardial contractility is spare and for the time being we cannot definitively conclude on the clinical impact of HMG CoA reductase inhibitors in myocardial systolic performance.

Half of the cases of HF are attributed to diastolic dysfunction and the prognosis of HF with preserved EF is as ominous as the prognosis of HF with systolic dysfunction. Unfortunately, no treatment has yet been shown, convincingly, to reduce morbidity and mortality in patients with HF and preserved EF, while this group of patients is usually excluded from large prospective randomized trials and accordingly few data exist for the role of statins in this heterogeneous population.

As there is substantially lack of evidence concerning the effects of HMG CoA reductase inhibitors in subjects with HF and preserved EF the first indirect hypothesis was extrapolated from observational prospective studies in subjects with ischemic heart disease and no evidence of congestive HF. Indeed, in a cohort of 430 consecutive patients with ischemic heart disease and a mean EF of 57% Okura et al. observed that subjects under HMG CoA reductase inhibitors treatment had decreased E/E′ ratio—corresponding to a better diastolic function—and a significantly higher survival rate (Okura et al., 2007). According to the authors those beneficially effects can be attributed to improved endothelial function and vasodilatory response to reactive hyperemia, attenuation of myocardial hypertrophy, and interstitial fibrosis.

Despite the positive results from mechanistic and experimental studies clinical studies have failed to confirm a definitive role of HMG CoA reductase inhibitors in HF. Nevertheless, by extrapolating experimental and mechanistic data in clinical settings we further understand how HMG-CoA reductase inhibitors can beneficially affect subgroups of HF subjects such as those with preserved EF, low B-type natriuretic peptide levels, exacerbated inflammatory response and limited interstitial fibrosis. Nevertheless, as a definitive mechanism is lacking, there is uncertainty about the decisive mode of action and further mechanistic studies are needed to reveal how HMG-CoA reductase inhibitors act in HF substrate.

Pro- A-Type Natriuretic Peptide, Proadrenomedullin, and N-Terminal Pro-B-Type Natriuretic Peptide Used in a Multimarker Strategy in Primary Health Care in Risk Assessment of Patients with Symptoms of Heart Failure

Urban Alehagen, Ulf Dahlstr€Om,  Jens F. Rehfeld, And Jens P. Goetze
J Cardiac Fail 2013; 19(1):31-39. http://dx.doi.org/10.1016/j.cardfail.2012.11.002

Use of new biomarkers in the handling of heart failure patients has been advocated in the literature, but most often in hospital-based populations. Therefore, we wanted to evaluate whether plasma measurement of N-terminal pro-B-type natriuretic peptide (NT-proBNP), midregional pro-A-type  atriuretic peptide (MR-proANP), and midregional proadrenomedullin (MR-proADM), individually or combined, gives prognostic information regarding cardiovascular and all-cause mortality that could motivate use in elderly patients presenting with symptoms suggestive of heart failure in primary health care. Methods and Results: The study included 470 elderly patients (mean age 73 years) with symptoms of heart failure in primary health care. All participants underwent clinical examination, 2-dimenstional echocardiography, and plasma measurement of the 3 propeptides and were followed for 13 years. All mortality was registered during the follow-up period. The 4th quartiles of the biomarkers were applied as cutoff values. NT-proBNP exhibited the strongest prognostic information with 4-fold increased risk for cardiovascular mortality within 5 years. For all-cause mortality MR-proADM exhibited almost 2-fold and NTproBNP 3-fold increased risk within 5 years. In the 5e13-year perspective, NT-proBNP and MR-proANP showed significant and independent cardiovascular prognostic information. NT-proBNP and MR-proADM showed significant prognostic information regarding all-cause mortality during the same time. In those with ejection fraction (EF) !40%, MR-proADM exhibited almost 5-fold increased risk of cardiovascular mortality with 5 years, whereas in those with EF O50% NT-proBNP exhibited 3-fold increased risk if analyzed as the only biomarker in the model. If instead the biomarkers were all below the cutoff value, the patients had a highly reduced mortality risk, which also could influence the handling of patients. Conclusions: The 3 biomarkers could be integrated in a multimarker strategy for use in primary health care.

Novel Biomarkers in Heart Failure with Preserved Ejection Fraction

Kevin S. Shah, Alan S. Maisel
Heart Failure Clin 10 (2014) 471–479


  • Heart failure with preserved ejection fraction (HFPEF) is a common subtype of congestive heart failure for which therapies to improve morbidity and mortality have been limited thus far.
  • Numerous biomarkers have emerged over the past decade demonstrating prognostic significance in HFPEF, including natriuretic peptides, galectin-3, soluble ST2, and high-sensitivity troponins.
  • These markers reflect the multiple mechanisms implicated in the pathogenesis of HFPEF, and future research will likely use these markers to not only help determine heart failure phenotypes but also target specific therapies.

Heart failure (HF) is a global epidemic, defined as an abnormality of cardiac function leading to the inability to deliver oxygen at a rate adequate to meet the requirements of tissues. It is truly a clinical syndrome of symptoms and signs resulting from this cardiac abnormality. Over the past decade, further characterization into 2 entities has occurred: HF with preserved ejection fraction (HFPEF) and HF with reduced ejection fraction (HFREF). HFPEF, previously termed diastolic HF, encompasses the syndrome of HF with a preserved ejection fraction. Cutoffs for this ejection fraction typically are from 45% to 50%. The prevalence of HF is upward of 1% to 2% of the adult population, with an increased prevalence found in elderly and female patients. Multiple studies have shown that the prevalence of HFPEF is actually comparable with the number of patients with HFREF. As expected, most deaths from HFPEF are cardiovascular, comprising 51% to 70% of mortality.

The pathophysiology of HFPEF is controversial and remains poorly understood. Originally, HFPEF was thought to be a primary manifestation of diastolic dysfunction of the left ventricle. However, patients with HFREF are known to also commonly have impaired ventricular relaxation. The primary mechanism of left ventricular (LV) dysfunction is based on structural remodeling and endothelial dysfunction, lending itself to LV stiffness, and increased left atrial pressure. This pressure change is what drives pulmonary venous congestion and subsequent symptomatology. The ventricular stiffness commonly seen in HFPEF is attributed to multiple mechanisms, including fibrosis, excessive collagen deposition, cardiomyocyte stiffness, and slow LV relaxation.

The natriuretic peptides (NPs) are the cornerstone biomarker in congestive HF (CHF). Many of the details of the role of NPs are covered in an article – Florea VG, Anand IS. Biomarkers. Heart Fail Clin 2012;8(2):207–24. The Breathing Not Properly trial originally helped establish the role of B-type natriuretic peptide (BNP) in the diagnosis of CHF. BNP and the N-terminal prohormone BNP (NT-proBNP) have been shown in numerous trials to be an excellent tool for ruling out CHF as a cause of acute dyspnea. Aside from a strong negative predictive value, NPs correlate with HF severity, prognostication, outpatient CHF management, and screening. When attempting to use NPs specifically to distinguish between HFPEF and HFREF, results have shown that NPs do not have a particular cutoff, but are typically elevated in HFPEF in comparison with patients without HF. These levels of NPs in HFPEF are typically lower than levels in patients with HFREF.

Although the role of novel renal biomarkers has not been fully explored specifically in HFPEF, they likely have an impactful role in the assessment and management of acute kidney injury (AKI) and the cardiorenal syndrome. Two biomarkers are briefly discussed here: neutrophil gelatinase-associated lipocalin (NGAL) and cystatin C. NGAL is a 25-kDa protein in the lipocalin family of proteins with a role in inflammation and immune modulation.

The future of biomarkers and their utility in HF is very promising, starting with the potential for using biomarkers as end points in trials. Biomarkers serve as surrogates for various pathophysiologic mechanisms, and there are potential benefits in using them as trial end points. Advantages include the ability to obtain quick and early data, as well as possibly better understand the nature of the disease. However, the counterargument against using biomarkers as trial end points includes whether treatment effects on a biomarker reliably predict effects on a clinically meaningful end point.
Reduced cGMP signaling activates NF-κB in hypertrophied hearts of mice lacking natriuretic peptide receptor-A

Elangovan Vellaichamy, Naveen K. Sommana, Kailash N. Pandey
Biochemical and Biophysical Research Communications 327 (2005) 106–111

Mice lacking natriuretic peptide receptor-A (NPRA) develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for cardiac hypertrophic growth in the absence of NPRA signaling are not yet known. We sought to determine the activation of nuclear factor-κB (NF-κB) in Npr1 (coding for NPRA) gene-knockout (Npr1-/-) mice exhibiting cardiac hypertrophy and fibrosis. NF-κB binding activity was 4-fold greater in the nuclear extract of Npr1-/-mutant mice hearts as compared with wild-type (Npr1+/+) mice hearts. In parallel, inhibitory κB kinase-b activity and IκB-α protein phosphorylation were also increased 3- and 4-fold, respectively, in hypertrophied hearts of mutant mice. cGMP levels were significantly reduced 5-fold in plasma and 10-fold in ventricular tissues of mutant mice hearts  relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates NF-κB binding activity associated with hypertrophic growth of mutant mice hearts.

Regulation of guanylyl cyclase/natriuretic peptide receptor-A gene expression

Renu Garg, Kailash N. Pandey
Peptides 26 (2005) 1009–1023

Natriuretic peptide receptor-A (NPRA) is the biological receptor of the peptide hormones atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The level and activity of this receptor determines the biological effects of ANP and BNP in different tissues mainly directed towards the maintenance of salt and water homeostasis. The core transcriptional machinery of the TATA-less Npr1 gene, which encodes NPRA, consists of three SP1 binding sites and the inverted CCAAT box. This promoter region of Npr1 gene has been shown to contain several putative binding sites for the known transcription factors, but the functional significance of most of these regulatory sequences is yet to be elucidated. The present review discusses the current knowledge of the functional significance of the promoter region of Npr1 gene and its transcriptional regulation by a number of factors including different hormones, growth factors, changes in extracellular osmolarity, and certain physiological and patho-physiological conditions.

Atrial natriuretic peptide (ANP), a member of natriuretic peptide family is a polypeptide consisting of 28 amino acids and was discovered as a potent vasodilator and diuretic hormone produced in granules of the atrium. The natriuretic peptide family consists of the peptide hormones ANP, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), each of which is derived from a separate gene. ANP and BNP are cardiac derived peptides, which are secreted and up-regulated in myocardium in response to different patho-physiological stimuli, while CNP is an endothelium-derived mediator that plays an important paracrine role in the vasculature. All of these natriuretic peptides elicit a number of vascular, renal, and endocrine effects mainly directed towards the maintenance of blood pressure and extracellular fluid volume by binding to their specific cell surface receptors. ANP exerts its effects at a number of sites including the kidney, where it produces natriuretic and diuretic responses; the adrenal gland, where it inhibits aldosterone synthesis and secretion; vascular smooth muscle cells, where it produces vasorelaxation; the endothelial cells, where it may regulate vascular permeability; gonadal cells, where it affects synthesis of androgen and estradiol. Each of these target sites of ANP activity has been shown to possess specific high affinity receptors for ANP. To date, three different subtypes of natriuretic peptide receptors have been characterized, purified, and cloned, i.e. natriuretic peptide receptors A, B, and C also designated as NPRA, NPRB, and NPRC, respectively. ANP and BNP specifically bind to NPRA, which contains guanylyl cyclase catalytic activity and produces intracellular secondary messenger cGMP in response to hormone binding.

NPRA is considered the biological receptor of ANP and BNP because most of the physiological effects of these hormones are triggered by generation of cGMP or its cell permeable analogs. Recent studies with mice lacking the Npr1 gene, demonstrated that genetic disruption of NPRA increases the blood pressure and causes hypertension in these animals. On the other hand, the effect of ANP was found to be increased linearly in Npr1 gene-duplicated mice
in a manner consistent with gene copy number. All this clearly indicates that the level of NPRA expression determines the extent of the biological effects of ANP and BNP. But the intervention strategies aimed at controlling NPRA expression are limited by the paucity of studies in this area. The cDNA and gene encoding NPRA designated as Npr1 has been cloned and characterized in mouse, rat, bull frog, euryhaline eel, and medaka fish. The primary structure of this gene is essentially same in all the different species and contains 22 exons interrupted by 21 introns.  The Npr1 gene sequence has been found to be interspersed with a number of repetitive elements including (SINES), (MER2), and tandem repeat elements in all the different species.

Although the Npr1 gene transcriptional regulation is only poorly understood, the activity and expression of NPRA assessed primarily through ANP stimulated cGMP accumulation are found to be regulated by a number of factors including auto-regulation by natriuretic peptides themselves, other hormones such as endothelin, glucocorticoids, and angiotensin II (ANG II), growth factors, changes in extracellular ion composition, and certain physiological and patho-physiological conditions.

The core molecular machinery of the TATA-less Npr1 gene consisting of SP1 binding sites and the inverted CCAAT box has been authenticated to be indeed functional in rat promoter element. It has been shown that the molecular machinery that regulates the basal expression of Npr1 gene consists of three SP1 binding sites in conjunction with an inverted CCAAT box present in the proximal promoter region. Mutation in any of these SP1 binding sites which
are located within 350 bp upstream of transcription start site in rat Npr1 promoter inhibited SP1 and SP3 binding and decreased the promoter activity by 50–75%, while simultaneous mutation of all the three led to a >90% reduction in promoter activity. The proximal SP1 binding site was much more effective than the distal sites in inducing the expression implying that the proximity to the core transcriptional machinery contributes to the magnitude of the observed effect. The over-expression of either SP1 or SP3 resulted in the induction of the wild type Npr1 promoter, confirming that these transcription factors serve as positive regulators of the Npr1 gene expression.

A number of natriuretic peptides such as ANP, BNP, CNP, and urodilatin (i.e. ANP95–126) can down-regulate ligand dependent NPRA activity after as little as 2 h prior exposure to the ligand, which remains suppressed until 48 h of exposure in cultured cells. The early reduction of NPRA activity is independent of changes in Npr1 gene expression as the pretreatment of cultured cells with actinomycin D (an inhibitor of transcription) for 1 h failed to block the response to ANP implying that ligand acts, at least early on, through a post transcriptional mechanism in reducing NPRA activity. The sustained reduction of NPRA activity, on the other hand, has been shown in fact due to reduction in NPRA mRNA levels (∼50%) by treatment with 100nM ANP for 48 h. This reduction could also be affected by treatment of cultured cells with 8-Br-cGMP with similar kinetic response and was amplified by phosphodiesterase inhibitors, but was not shared by NPRC-selective ligand cANF, suggesting that the down regulation of Npr1 gene expression is mediated by elevations of intracellular cGMP involving either NPRA or NPRB. .. The cGMP regulatory region was pinpointed to position−1372 to−1354 bp from the transcription start site of Npr1 by gel shift assays and footprinting analysis, which indicated its interaction with transcriptional factor(s). Further cross-competition experiments with mutated oligonucleotides led to the definition of a consensus sequence (−1372 bp AaAtRKaNTTCaAcAKTY −1354 bp) for the novel cGMP-RE, which is conserved in the human (75% identity) and mouse (95% identity) Npr1 promoters. The combination of these transcriptional and post-transcriptional ligand-dependent regulatory mechanisms provides the cells with greater flexibility in both initiating and maintaining the suppression of NPRA activity.

The peptide hormone Ang II is an important component of renin-angiotensin system (RAS) and exerts its biological effects such as blood pressure regulation, vasoconstriction, and cell proliferation in many tissues including the kidney, adrenal glands, brain, and vasculature. The two vasoactive peptide hormones, Ang II (vasoconstrictive) and ANP (vasodilatory), interact and mutually antagonize the biological effects of each other at various levels. ANP has been shown to inhibit Ang II-induced contraction of isolated glomeruli and cultured mesangial cells, as well as Ang II-stimulated activation of protein kinase C and mitogen activated protein kinase in vascular smooth muscle cells in a cGMP-dependent manner. Inversely, Ang II has been shown to down-regulate guanylyl cyclase activity of the biological receptor of ANP, NPRA, by activating protein kinase C and/or by stimulating protein tyrosine phosphatase activity, thereby inhibiting the ANP stimulated cGMP accumulation. Ang II also reduces the ANP dependent cGMP levels by stimulating cGMP hydrolysis, apparently
via a calcium dependent cGMP phosphodiesterase.

Endothelin is a vasoconstrictor peptide that was originally isolated from porcine endothelial cells. It is produced as three isoforms (ET1-3) that bind to two receptor subtypes (ETA and ETB). ET is produced in the kidney and subject to regulation by a number of local and systemic factors including immune cytokines and extracellular tonicity. Since, endothelin is avidly expressed in the nephron segment, where NPRA is up-regulated by osmotic stimulus, it was investigated whether endothelin plays a role in the control of basal or osmotically regulated Npr1 gene expression in these cells. The endogenous endothelin and not the exogeneously administered endothelin inhibit the basal but not osmotically stimulated expression of Npr1. The type A (BQ610) and type B (IRL 1038) endothelin receptor antagonists increased the level of NPRA mRNA by two to three-fold, whereas co-administration of exogenous endothelin resulted in partial reversal of this stimulatory effect of receptor antagonists. The increase in extracellular tonicity reduces the endothelin mRNA accumulation (∼15% of control levels) in inner medullary collecting duct cells but this reduction is not found to be linked to the stimulation of NPRA activity/expression in response to osmotic stress.

Glucocorticoids influence the cardiovascular system and induce a rapid increase in blood pressure. Glucocorticoids are known to regulate
transcription in many systems, possibly by interacting with glucocorticoid responsive elements and associated chromatin proteins. These have been shown to affect the atrial endocrine system by regulating both the synthesis and secretion of ANP in vitro and in vivo. Thus, it seems plausible that glucocorticoid may also interact with the atrial endocrine system by modulating ANP receptor levels. The stimulation of vascular smooth muscle cells from rat mesenteric artery with dexa-methasone (a highly specific synthetic glucocorticoid agonist) caused an increase in NPRA mRNA levels in a time dependent manner which reached a plateau after 48 h of glucocorticoid administration. This mRNA increase was mimicked by cortisol and inhibited by glucocorticoid receptor antagonists RU38486. Also cGMP generated by NPRA in dexamethasone treated cells was higher than in control cells and this production was mimicked by cortisol and blocked by RU 38486. These results suggest that glucocorticoids exert a positive effect on NPRA transcription in rat mesenteric arteries.

Previous studies have shown that guanylyl cyclase activity of NPRA is either activated, or inhibited by an increase in extracellular tonicity. Though none of these studies were definitive in terms of elucidating the mechanisms involved, they suggested that the activation predominates with longer exposure (∼24 h), while the inhibition with short-term exposure (minutes) to the osmotic stimulus. More recently, the mechanism(s) underlying the activation of NPRA expression by osmotic stimulus has been investigated. The NaCl (75 mM) or sucrose (150 mM), but not osmotically inert solute, urea (150 mM) increased NPRA activity, gene expression, and promoter activity after as early as 4 h reaching a maximum at 24 h in inner medullary collecting duct cells. The osmotic stimulus also activated extracellular signal regulated kinase (ERK), c-Jun-NH2-terminal kinase (JNK), and p38 mitogen activated protein kinase- (p38 MAPK-β). The inhibition of p38 MAPK-βwith SB20580 completely  blocked the osmotic stimulation of receptor activity and expression, and caused a dose-dependent reduction in promoter activity, whereas inhibition of ERK with PD98059 had no effect.

The expression of NPRB, the biological receptor of CNP, has been shown to be regulated by a number of factors including natriuretic peptide ligands, intracellular cAMP levels, water deprivation, TGF-1, dexamethasone treatment, as well as renal sodium status, as its mRNA levels were upregulated in the renal cortex of sodium depleted animals. NPRB expression has also been found to be regulated by alternative splicing. Three isoforms of NPRB have been identified of which NPRB1 is the full length form and responds maximally to CNP, NPRB2 isoform contains a 25 amino acid deletion in protein kinase homology domain and NPRB3 contains a partial extracellular ligand binding domain and fails to bind the ligand. The relative expression levels of the three isoforms vary across different tissues. Since, the smaller splice variants of NPRB act as dominant negative isoforms by blocking formation of active NPRB1 homodimers, these isoforms might play important role in the tissue specific regulation of receptor, NPRB.

The NPRC expression has also been found to be down-regulated by its ligands and their secondary messenger, cGMP, hormones, growth factors, dietary salt supplementation, β-adrenergic blocker, and physiological as well as patho-physiological conditions. On the other hand, NPRC expression gets augmented by TGF-β1, 1,25-dihydroxy VitaminD3 and during conditions like chronic heart failure.

Hypertension is the leading cause of human deaths in today’s world. The natriuretic peptide system plays a well defined role in the regulation of blood pressure and fluid volume. The cellular and physiological effects of natriuretic peptides (ANP, BNP, and CNP) are mediated by their specific receptors NPRA, NPRB, and NPRC. The transcriptional regulation of these receptors has been studied since their identification, but still remains poorly understood. Better understanding and the elucidation of different molecular mechanisms responsible for the regulation of NPRA expression would provide us the framework to develop the therapeutic strategies to manipulate the expression levels of this receptor and to control the biological actions of ANP and BNP during different patho-physiological conditions.

Inhibition of Heat Shock Protein 90 (Hsp90) in Proliferating Endothelial Cells Uncouples Endothelial Nitric Oxide Synthase Activity

Jingsong Ou, Zhijun Ou, AW Ackerman, KT Oldham, & KA Pritchard, Jr.
Free Radical Biol Med 2003; 34(2):269–276
PII S0891-5849(02)01299-6

Dual increases in nitric oxide (•NO) and superoxide anion (O2•-) production are one of the hallmarks of endothelial cell proliferation. Increased expression of endothelial nitric oxide synthase (eNOS) has been shown to play an important role in maintaining high levels of •NO generation to offset the increase in O2•- that occurs during proliferation. Although recent reports indicate that heat shock protein 90 (hsp90) associates with eNOS to increase •NO generation, the role of hsp90 association with eNOS during endothelial cell proliferation remains unknown. In this report, we examine the effects of endothelial cell proliferation on eNOS expression, hsp90 association with eNOS, and the mechanisms governing eNOS generation of •NO and O2•-. Western analysis revealed that endothelial cells not only increased eNOS expression during proliferation but also hsp90 interactions with the enzyme. Pretreatment of cultures with radicicol (RAD, 20 µM), a specific inhibitor that does not redox cycle, decreased A23187-stimulated •NO production and increased Lω-nitroargininemethylester (L-NAME)-inhibitable O2•-generation. In contrast, A23187 stimulation of controls in the presence of L-NAME increased O2•- generation, confirming that during proliferation eNOS generates •NO. Our findings demonstrate that hsp90 plays an important role in maintaining •NO generation during proliferation. Inhibition of hsp90 in vascular endothelium provides a convenient mechanism for uncoupling eNOS activity to inhibit •NO production. This study provides new understanding of the mechanisms by which ansamycin antibiotics inhibit endothelial cell proliferation. Such information may be useful in the development and design of new antineoplastic agents in the future.

Natriuretic Peptides, Ejection Fraction, and Prognosis – Parsing the Phenotypes of Heart Failure

James L. Januzzi, JR
J Amer Coll Cardiol 2013; 61(14): 1507-9

Since the first pivotal studies introduced the natriuretic peptides as biomarkers for the diagnosis of heart failure (HF), use of both B-type natriuretic peptide (BNP) and its N-terminal equivalent (NT-proBNP) has grown not only for this indication, but also for establishing HF prognosis as well. Indeed, a vast array of studies has established the natriuretic peptides as the biomarker gold standard to prognosticate risk for a wide array of relevant complications in HF (ranging from ventricular arrhythmias to pump failure). In these studies, the prognostic information provided by BNP and NT-proBNP in HF was independent of a number of relevant covariates, including left ventricular ejection fraction (LVEF).

It has been known for quite a while that patients with heart failure and preserved ejection fraction (HFpEF) typically have lower natriuretic peptide values than do those with heart failure and reduced ejection fraction (HFrEF). A conundrum is thus present: whereas both BNP and NTproBNP tend to be lower in HFpEF, when these peptides are elevated in this setting, they remain prognostic; this intriguing circumstance has been relatively poorly studied. It is in this setting that van Veldhuisen et al. examined the impact of LVEF on the prognostic merits of BNP in the COACH (Coordinating Study Evaluating Outcomes of Advising and Counseling in Heart Failure) study in the present issue of the Journal. The investigators found—as expected—that BNP levels were lower in HFpEF, but for a given BNP concentration, prognosis of those with HFpEF in COACH was just as poor as those with HFrEF at matched BNP values. Stated differently, a high BNP in a patient with HFpEF imparted similar prognostic information as it would in someone with HFrEF. Actually, whereas LVEF was not obviously prognostically impactful, when considered across the range of ventricular function, an elevated BNP concentration in the most normal range of LVEF seemed to be associated with a higher risk than at the lower ranges of pump function. Although it is previously established that BNP or NT-proBNP are prognostic independently of LVEF, the well-executed analysis by van Veldhuisen et al. (van Veldhuisen DJ, Linssen GCM, Jaarsma T, et al. B-type natriuretic peptide and prognosis in heart failure patients with preserved and reduced ejection fraction. J Am Coll Cardiol 2013;61:1498–506.) allows for a more in-depth examination of this phenomenon and raises some important questions.

Phenotypic Definition of the Patient With Heart Failure

Phenotypic Definition of the Patient With Heart Failure

Phenotypic Definition of the Patient With Heart Failure

Natriuretic Peptides in Heart Failure with Preserved Ejection Fraction

Mark Richards, James L. Januzzi Jr, Richard W. Troughton
Heart Failure Clin 10 (2014) 453–470


  • Threshold values of B-type natriuretic peptide (BNP) and N-terminal prohormone B-type natriuretic peptide (NT-proBNP) validated for diagnosis of undifferentiated acutely decompensated heart failure (ADHF) remain useful in patients with heart failure with preserved ejection fraction (HFPEF), with minor loss of diagnostic performance.
  • BNP and NT-proBNP measured on admission with ADHF are powerfully predictive of in-hospital mortality in both HFPEF and heart failure with reduced EF (HFREF), with similar or greater risk in HFPEF as in HFREF associated with any given level of either peptide.
  • In stable treated heart failure, plasma natriuretic peptide concentrations often fall below cut-point values used for the diagnosis of ADHF in the emergency department; in HFPEF, levels average approximately half those in HFREF.
  • BNP and NT-proBNP are powerful independent prognostic markers in both chronic HFREF and chronic HFPEF, and the risk of important clinical adverse outcomes for a given peptide level is similar regardless of left ventricular ejection fraction.
  • Serial measurement of BNP or NT-proBNP to monitor status and guide treatment in chronic heart failure may be more applicable in HFREF than in HFPEF.


The bioactivity of atrial NP (ANP) and B-type NP (BNP) encompasses short-term and longterm hemodynamic, renal, neurohormonal, and trophic effects. The relationship between cardiac hemodynamic load, plasma concentrations of ANP and BNP, and the cardioprotective profile of NP bioactivity have led to investigation of both biomarker and therapeutic potential of

NPs in HF.

PlasmaBNPandNT-proBNP thresholds (100pg/mL and 300 pg/mL, respectively) used in the diagnosis of undifferentiated ADHF retain good diagnostic

performance for acute HFPEF


Plasma NPs are related to multiple echo indicators of cardiac structure and function in both HFREF and HFPEF.
Box 1

Causes of increased plasma cardiac natriuretic peptides


Heart failure, acute and chronic

Acute coronary syndromes

Atrial fibrillation

Valvular heart disease




Left ventricular hypertrophy



Female sex

Renal impairment

Pulmonary embolism

Pneumonia (severe)

Obstructive sleep apnea

Critical illness

Bacterial sepsis

Severe burns

Cancer chemotherapy

Toxic and metabolic insults


BNP and NT-proBNP fall below ADHF thresholds in stable HFREF in approximately 50% and 20% of cases, respectively. Levels in stable HFPEF are even lower, approximately half those in HFREF.
Whereas BNPs have 90% sensitivity for asymptomatic LVEF of less than 40% in the community (a precursor state for HFREF), they offer no clear guide to the presence of early community based HFPEF.
Guidelines recommend BNP and NT-proBNP as adjuncts to the diagnosis of acute and chronic HF and for risk stratification. Refinements for application to HFPEF are needed.
The prognostic power of NPs is similar in HFREF and HFPEF. Defined levels of BNP and NT-proBNP correlate with similar short-term and long-term risks of important clinical adverse outcomes in both HFREF and HFPEF.
Diagnostic algorithm for suspected heart failure presenting either acutely or nonacutely

Diagnostic algorithm for suspected heart failure presenting either acutely or nonacutely

Diagnostic algorithm for suspected heart failure presenting either acutely or nonacutely. a In the acute setting, mid-regional pro–atrial natriuretic peptide may also be used (cutoff point 120 pmol/L; ie, <120 pmol/L 5 heart failure unlikely). b Other causes of elevated natriuretic peptide levels in the acute setting are an acute coronary syndrome, atrial or ventricular arrhythmias, pulmonary embolism, and severe chronic obstructive pulmonary disease with elevated right heart pressures, renal failure, and sepsis. Other causes of an elevated natriuretic level in the nonacute setting are old age (>75 years), atrial arrhythmias, left ventricular hypertrophy, chronic obstructive pulmonary disease, and chronic kidney disease. c Exclusion cutoff points for natriuretic peptides are chosen to minimize the false-negative rate while reducing unnecessary referrals for echocardiography. d Treatment may reduce natriuretic peptide concentration, and natriuretic peptide concentrations may not be markedly elevated in patients with heart failure with preserved ejection fraction. BNP, B-type natriuretic peptide; ECG, electrocardiogram; NT-proBNP, N-terminal prohormone of B-type natriuretic peptide. (From McMurray JJ, Adamopoulos S, Anker SD, et al. The task force for the diagnosis and treatment of acute and chronic heart failure 2012 of the European Society of Cardiology. ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012. Eur Heart J 2012;33:1787–847; with permission.)

Natriuretic Peptide Receptor-A Negatively Regulates Mitogen-Activated Protein Kinase and Proliferation of Mesangial Cells: Role of cGMP-Dependent Protein Kinase

Kailash N. Pandey, Houng T. Nguyen, Ming Li, and John W. Boyle
Biochem Biophys Res Commun 271, 374–379 (2000)

peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (NPRA) on mitogen-activated protein kinase/extracellular signal-regulated kinase 2 (MAPK/ERK2) activity in rat mesangial cells overexpressing NPRA. Agonist hormones such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), angiotensin II (ANG II), and endothelin-1 (ET-1) stimulated 2.5- to 3.5-fold immunoreactive MAPK/ERK2 activity in these cells. ANP inhibited agonist-stimulated activity of MAPK/ERK2 by 65–75% in cells overexpressing NPRA, whereas in vector transfected cells, its inhibitory effect was only 18–20%. NPRA antagonist A71915 and KT5823, a specific inhibitor of cGMP-dependent protein kinase (PKG) completely reversed the inhibitory effect of ANP on MAPK/ERK2 activity. ANP also inhibited the PDGF stimulated [3H]thymidine uptake by almost 70% in cells overexpressing NPRA, as compared with only 20–25% inhibition in vector-transfected cells. These
results demonstrate that ANP/NPRA system negatively regulates MAPK/ERK2 activity and proliferation of mesangial cells in a PKG-dependent manner.


Regulation of lipoprotein lipase by Angptl4

Wieneke Dijk and Sander Kersten
Trends in Endocrin and Metab, Mar2014; 25(3):146-155

Triglyceride (TG)-rich chylomicrons and very low density lipoproteins (VLDL) distribute fatty acids (FA) to various tissues by interacting with the enzyme lipoprotein lipase (LPL). The protein angiopoietin-like 4 (Angptl4) is under sensitive transcriptional control by FA and the FA-activated peroxisome proliferator activated receptors (PPARs), and its tissue expression largely overlaps with that of LPL. Growing evidence indicates that Angptl4 mediates the physiological fluctuations in LPL activity, including the decrease in
adipose tissue LPL activity during fasting. This review focuses on the major ambiguities concerning the mechanism of LPL inhibition by Angptl4, as well as on the physiological role of Angptl4 in lipid metabolism, highlighting its function in a variety of tissues, and uses this information to make suggestions for further research.

Box 1. LPL and TG metabolism

LPL belongs to a family of lipases that also includes hepatic lipase, pancreatic lipase, and endothelial lipase. Because LPL is essential in the lipolytic processing of chylomicrons and VLDL, LPL is primarily expressed in tissues that either require large amounts of FA as fuel or are responsible for TG storage, which include heart, skeletal muscle, and adipose tissue. Upon production by the underlying parenchymal cells, LPL is released into the subendothelial space and is transported to the luminal side of the capillary endothelium by the GPI-anchored protein GPIHBP1, which after transport continues to anchor LPL to the capillary endothelium. The essential role for LPL in the clearance of plasma TG is well-demonstrated by the severe hypertriglyceridemia of patients carrying homozygous mutations in the LPL gene. Generalized deletion of LPL in mice results in severe hypertriglycer-idemia, resulting in the premature death of pups within 24 h after birth. Analogous to the deletion of LPL, the mislocalization of LPL to the subendothelial spaces due the absence or misfolding of GPIHBP1 also results in severe chylomicronemia and hypertriglyceridemia. The LPL enzyme is catalytically active as a non-covalent head-to-tail dimer with a catalytic N-terminal domain and a non-catalytic C terminal domain. Folding of LPL into its dimer conformation occurs in the endoplasmic reticulum, chaperoned by lipase maturation factor 1, calreticulin, and calnexin. In its active 3D conformation, the catalytic site of LPL is postulated to be covered by a lid, which can be opened by the binding of chylomicrons and VLDL to the C terminus. The active LPL dimers rapidly exchange subunits, indicating that a dynamic equilibrium exists between LPL dimers and dimerization-competent monomers. Dimerization-competent monomers have, however, not yet been isolated, and it is unclear whether this monomer is catalytically active. The enzymatic activity of LPL is lost when the LPL dimer is converted into inactive, folded monomers. This conversion to inactive monomers is mainly regulated via post-translational mechanisms and is dependent on nutritional state. Enzymatic activity of inactive monomers can be regained in vitro by the addition of calcium, indicating that inactivation of LPL is a reversible process.

One of the key questions is whether (patho)physiological variations in LPL activity are mediated via regulation of Angptl4 cleavage and/or oligomerization, and which factors are involved in modulating Angptl4 in vivo. Recent biochemical evidence suggests that FA may be able to promote dissociation of oligomers, which, by destabilizing the protein, would impair its ability to inhibit LPL. Destabilization of Angptl4 by FA is, however, seemingly at odds with the marked stimulatory effect of FA on Angptl4 production observed in vitro and in vivo.

The currently accepted molecular model for the inhibition of LPL by Angptl4 is that Angptl4 stimulates the conversion of catalytically active LPL dimers into inactive monomers – following in vitro studies showing that coincubation of LPL and Angptl4 increases the abundance of LPL monomers. Subsequent studies revealed that the proportion of LPL dimers is reduced in post-heparin plasma of mice that overexpress Angptl4 in favor of LPL monomers, providing in vivo support for the dimer-to monomer conversion. The elucidation of the purported biochemical mechanism has strengthened the status of Angptl4 as a LPL inhibitor, but several questions related to the in vivo mechanism remain unanswered. Whereas the original in vitro experiments favored the hypothesis that Angptl4 enzymatically and irreversibly catalyzes the LPL dimer-to-monomer conversion, an in vivo study of Angptl4 transgenic mice suggested that Angptl4 is physically bound to LPL monomers, thereby driving the LPL dimer–monomer equilibrium towards inactive monomers. The latter study also revealed that the relative decrease in post-heparin plasma LPL activity upon Angptl4 overexpression is much more pronounced than the relative decrease in heparin-releasable LPL dimers, pointing to an additional or alternative mechanism. In support, a recently published study suggests that Angptl4, instead of acting as a catalyst, functions as a conventional, non-competitive inhibitor that binds to LPL to prevent the hydrolysis of substrate LPL and Angptl4 are regulated by changes in nutritional state in a tissue-specific manner, reflecting the different functions of these tissues and the corresponding variations in physiological requirements for lipids. Below, we discuss current knowledge on the regulation of Angptl4 and LPL in response to various physiological stimuli and address the importance of Angptl4 in lipid uptake. An overview of the role of Angptl4 in physiological regulation of lipid metabolism is presented in Figure 2.

model for mechanisms of lipoprotein lipase (LPL) inhibition by Angptl4.

model for mechanisms of lipoprotein lipase (LPL) inhibition by Angptl4.

Figure 1. Hypothetical model for mechanisms of lipoprotein lipase (LPL) inhibition by Angptl4. Angiopoietin-like 4 (Angptl4) and LPL are expressed in the parenchymal cells of muscle, heart, and adipose tissue. Following secretion of LPL and Angptl4 into the subendothelial space, transport of LPL to the capillary lumen is mediated by two mechanisms. The principal transport mechanism (1) relies on GPIHBP1 [glycosylphosphatidylinositol (GPI)-anchored high density lipoprotein-binding protein] picking up LPL from the subendothelial space and transporting it to the capillary lumen. This action by GPIHBP1 is opposed by Angptl4, which is bound to extracellular matrix (ECM) proteins and which retains and inhibits LPL. In the presence of GPIHBP1, high expression levels of Angptl4 are needed to overcome the competition with GPIHBP1. Angptl4 secreted into the capillary lumen, primarily as N-terminal truncation fragment generated by cleavage by proprotein convertases (PCs), inhibits LPL activity on the endothelium by promoting the irreversible conversion of LPL dimers into inactive monomers and/or via a reversible mechanism that requires binding of Angptl4 to LPL. The second transport mechanism involves a so far unidentified carrier and can be disrupted by Angptl4. In the absence of GPIHBP1, Angptl4 fully retains LPL in the subendothelial space (a). The additional loss of Angptl4 liberates LPL and allows it to be transported to the endothelial surface via the unidentified carrier (b). This model suggests that Angptl4 and LPL start interacting before arrival in the capillary lumen, either in the parenchymal cells or in the subendothelial space. Abbreviation: HSPG, heparan sulfate proteoglycan.

Regulation and role of angiopoietin-like 4 (Angptl4)

Regulation and role of angiopoietin-like 4 (Angptl4)

Figure 2. Regulation and role of angiopoietin-like 4 (Angptl4) in lipid metabolism. Angptl4 is expressed in parenchymal cells of white adipose tissue (WAT), liver, intestine, heart and muscle, as well as in macrophages, where it is subject to cell- and tissue-specific regulation. Angptl4 is a sensitive target of peroxisome proliferator-activated receptor (PPAR) transcription factors in several tissues. In WAT the expression of Angptl4 is induced during fasting and by the transcription factors PPARg, glucocorticoid receptor (GR), and hypoxia inducible factor 1a (HIF1a). In WAT Angptl4 stimulates lipolysis of stored triglycerides (TG) and inhibits lipoprotein lipase (LPL) activity. Expression of Angptl4 in liver is stimulated by PPARa, PPARd, and GR. Because the liver does not express LPL, Angptl4 is mainly released into the blood, affecting LPL activity in peripheral tissues. Angptl4 may also impact upon hepatic lipase activity in liver. Expression of Angptl4 in heart and skeletal muscle is potently induced by fatty acids (FA) via PPARd activation. Angptl4 inhibits LPL activities in cardiac and likely skeletal muscle. FA also stimulate Angptl4 expression in macrophages via PPARd, leading to local inhibition of LPL activity. We hypothesize that macrophage LPL enables uptake of remnant particles containing lipid antigens, which are subsequently presented to natural killer T cells. In the intestine, FA stimulate Angptl4 expression via one of the PPARs. Angptl4 produced by enterocytes may be released towards the lumen and inhibit pancreatic lipase activity. Angptl4 produced by enteroendocrine cells is released towards the blood and may inhibit LPL in distant tissues.

Box 2. Outstanding questions

  1. What is the importance of Angptl4 cleavage and oligomerization to Angptl4 function in vivo?
  2. What is the precise biochemical mechanism behind the inhibition of LPL activity by Angptl4?
  3. At which cellular location(s) does the inhibition of LPL by Angptl4 occur and, if at multiple locations, what is the relative contribution of both tissue-produced Angptl4 compared to circulating Angptl4 with respect to inhibition of tissue LPL activity.
  4. What is the interplay between GPIHBP1 and Angptl4 in the regulation of LPL activity?
  5. What is the protein structure of Angptl4 and LPL?
  6. Does Angptl4 also regulate LPL activity in brown adipose tissue and skeletal muscle and, if so, how is the expression of Angptl4 regulated in these tissues?
  7. What is the potential of Angptl4 as a biomarker in the context of disorders of lipid metabolism?

In the past decade, angiopoietin-like proteins have been demonstrated to regulate plasma TG levels powerfully in mice and humans. The elucidation of these proteins as inhibitors of LPL activity has led to a paradigm shift in how clearance of circulating TG and thereby tissue uptake of FA are regulated. Most of our understanding of angiopoietin-like proteins has resulted from detailed study of Angptl4.

A major portion of the physiological variation in LPL activity in various tissues can be attributed to regulation of Angptl4 production. We predict that Angptl4 will turn out to be equally important for governing LPL activity in muscle during exercise, in brown adipose tissue during cold, and in several tissues during fasting.

Besides the increasing recognition of the pivotal role of Angptl4 in lipid metabolism as an inhibitor of LPL, major insight has been gained into the molecular mechanism of action of Angptl4. Key questions remain, however, especially related to the interaction between LPL, GPIHBP1, and Angptl4 on the endothelium and in the subendothelial space. Several points of interest have been highlighted throughout the text; these include the elucidation of the molecular structure for LPL and Angptl4 by X-ray crystallography and the clarification of in vivo Angptl4 cleavage and oligomerization.

Native Low-Density Lipoprotein Induces Endothelial Nitric Oxide Synthase Dysfunction: Role of Heat Shock Protein 90 And Caveolin-1

Kirkwood A. Pritchard, Jr., Allan W. Ackerman, Jingsong Ou, et al.
Free Radical Biol & Med 2002; 33(1):52–62 PII S0891-5849(02)00851-1

Although native LDL (n-LDL) is well recognized for inducing endothelial cell (EC) dysfunction, the mechanisms remain unclear. One hypothesis is n-LDL increases caveolin-1 (Cav-1), which decreases nitric oxide (•NO) production by binding endothelial nitric oxide synthase (eNOS) in an inactive state. Another is n-LDL increases superoxide anion (O2•-), which inactivates •NO. To test these hypotheses, EC were incubated with n-LDL and then analyzed for •NO, O2•-, phospho-eNOS (S1179), eNOS, Cav-1, calmodulin (CaM), and heat shock protein 90 (hsp90). n-LDL increased NOx by more than 4-fold while having little effect on A23187-stimulated nitrite production. In contrast, n-LDL decreased cGMP under basal and A23187-stimulated conditions and increased O2•-by a mechanism that could be inhibited by L-nitroargininemethylester (L-NAME) and BAPTA/AM. n-LDL increased phospho-eNOS by 149%, eNOS by [1]34%, and Cav-1 by 28%, and decreased the association of hsp90 with eNOS by 49%. n-LDL did not appear to alter eNOS distribution between membrane fractions (-85%) and cytosol (-15%). Only 3–6% of eNOS in membrane fractions was associated with Cav-1. These data support the hypothesis that n-LDL increases O2•-, which scavenges •NO, and suggest that n-LDL uncouples eNOS activity by decreasing the association of hsp90 as an initial step in signaling eNOS to generate O2•-.

In conclusion, n-LDL decreases the association of hsp90 with eNOS, increases phospho-eNOS levels, and increases eNOS-dependent O2•-generation. These findings suggest that activation of eNOS without adequate levels of hsp90 may signal eNOS to switch from •NO to O2•-generation. Such changes in eNOS radical product generation may play an important role in impairing endothelial and vascular function.

New insights into IGF-1 signaling in the heart

Rodrigo Troncoso, C Ibarra, JM Vicencio, E Jaimovich, and S Lavandero
Trends in Endocrin and Metab, Mar 2014; 25(3):128-131

Insulin-like growth factor 1 (IGF-1) signaling regulates contractility, metabolism, hypertrophy, autophagy, senescence, and apoptosis in the heart. IGF-1 deficiency is associated with an increased risk of cardiovascular disease, whereas cardiac activation of IGF-1 receptor (IGF-1R) protects from the detrimental effects of a high-fat diet and myocardial infarction. IGF-1R activates multiple pathways through its intrinsic tyrosine kinase activity and through coupling to heterotrimeric G protein. These pathways involve classic second messengers, phosphorylation cascades, lipid signaling, Ca2+ transients, and gene expression. In addition, IGF-1R triggers signaling in different subcellular locations including the plasma membrane, perinuclear T tubules, and also in internalized vesicles. In this review, we provide a fresh and updated view of the complex IGF-1 scenario in the heart, including a critical focus on therapeutic strategies.

The hormone insulin-like growth factor 1 (IGF-1) is a small peptide of 7.6 kDa, which is composed of 70 amino acids and shares 50% homology with insulin. IGF-1 plays key roles in regulating proliferation, differentiation, metabolism, and cell survival. It is mainly synthesized and secreted by the liver in response to hypothalamic growth hormone (GH); its plasma concentration is finely regulated (Box 1). However, other tissues also produce IGF-1, which acts locally as an autocrine and paracrine hormone. IGF-1 exhibits pleiotropic effects in many organs and is also involved in the development of several pathologies.

Box 1. IGF-1 synthesis and biodisponibility

Insulin-like growth factor 1 (IGF-1) is a 70 amino acid peptide

hormone with endocrine, paracrine, and autocrine effects. It shares

>60% structure homology with IGF-2 and 50% with pro-insulin. IGF-

1 is mainly synthesized in the liver in response to hypothalamic

growth hormone (GH). In the peripheral circulation it exerts negative

feedback on the somatotrophic axis suppressing pituitary GH

release. IGF-1 can also be generated in almost all tissues, but liver

synthesis accounts for nearly 75% of circulating IGF-1 levels. As a

hormone with a wide range of physiological roles, IGF-1 circulating

levels must be strictly controlled. Around 98% of circulating IGF-1 is

bound to insulin-like growth factor binding protein (IGFBP). Six

forms of high affinity IGFBP have been described, with IGFBP3

binding approximately 90% of circulating IGF-1. Also, IGFBP1–6 and

their fragments have significant intrinsic biological activity independent

of IGF-1 interaction.

Canonical and noncanonical IGF-1 signaling pathways Activation of IGF-1R requires the sequential phosphorylation of three conserved tyrosine residues within the activation loop of the catalytic domain. From these phosphorylated motifs, tyrosine 950 contained in an NPXY motif provides a docking site for the recruitment of adaptor proteins, such as insulin receptor substrate-1 (IRS-1) and Shc, as an obligatory step to initiate signaling cascades. Two canonical pathways are activated by IGF-1R in cardiomyocytes – the phosphatidylinositol-3 kinase (PI3K)/Akt pathway and the extracellular signal-regulated kinase (ERK) pathway. Both pathways have been extensively studied, and their involvement in the pro-hypertrophic and pro-survival actions in cardiomyocytes is well established. Interestingly, a noncanonical signaling mechanism for IGF-1R in cardiomyocytes has been described in several recent studies. These studies show that some of the effects of IGF-1 are inhibited by the heterotrimeric Gi protein blocker Pertussis toxin (PTX) in several cell lines, suggesting that IGF-1R is a dual-activity receptor that triggers tyrosine-kinase-dependent responses as well as Gi-protein-dependent pathways. This duality has been reported in cultured neonatal cardiomyocytes; IGF-1R can activate ERK and Akt but also phospholipase C (PLC), which increases inositol 1,4,5 triphosphate (InsP3; IP3) leading to nuclear Ca2+ signals.

The cardiac effects of IGF-1 are mediated by activation of the plasma membrane IGF-1R, which belongs to the receptor tyrosine kinase (RTK) family. IGF-1R comprises a α2β2 heterotetrameric complex of approximately 400 kDa. Structurally, IGF-1R has two extracellular a-subunits that contain the ligand-binding sites. Each α-subunit couples to one of two membrane-spanning β-subunits, which contain an intracellular domain with intrinsic tyrosine kinase activity. Both subunits of IGF-1R are the product of one single gene, which is synthesized as a 180 kDa precursor. The immature IGF-1R full peptide is further glycosylated, dimerized, and proteolytically processed for assembly of the mature receptor isoforms a and b. In neonatal and adult rat cardiomyocytes, the IGF-1R precursor peptide and the processed α and β receptor subunits have been detected. Binding of IGF-1 to its receptor initiates a complex signaling cascade in cardiomyocytes.

Figure 1. not shown. Canonical and noncanonical signaling pathways activated by insulin-like growth factor 1 (IGF-1) in cardiomyocytes. Binding of IGF-1 to plasma membrane IGF-1 receptor (IGF-1R) leads to receptor autophosphorylation in the intracellular β-subunits. Docking of Grβ2 to the phosphorylated IGF-1Rβ subunits leads to extracellular signal-regulated kinase (ERK) phosphorylation through the Ras/Raf/Mitogen-activated protein kinase (MEK) axis. Phosphorylated ERK can translocate to the nucleus to control gene expression. Phosphorylated β-subunits also provide docking sites for insulin receptor substrate-1 (IRS-1), which mediates phosphatidylinositol-3 kinase (PI3K) activation and Akt phosphorylation. Downstream targets of activated Akt are mechanistic target of rapamycin (mTOR), which suppresses autophagy and promotes protein synthesis by activating S6K and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Akt also phosphorylates and inactivates Bad, thus inhibiting apoptosis. IGF-1R activation also promotes its interaction with a Pertussis-toxin-sensitive heterotrimeric Gi protein, which mediates the activation of phospholipase C (PLC) and hydrolysis of plasma membrane phosphatidylinositol 4,5 biphosphate (PIP2) to form inositol 1,4,5 triphosphate (InsP3; IP3) which activates InsP3 receptors located at the endoplasmin reticulum (ER)/nuclear envelope Ca2+ store, producing nucleoplasmic and cytoplasmic Ca2+ increases. The former is involved in the regulation of specific target genes and the latter promotes mitochondrial Ca2+ uptake, which increases mitochondrial respiration and metabolism, further preventing apoptosis and regulating autophagy. Canonical signaling pathways include the ERK and Akt axes, and are shown in red, whereas the noncanonical G protein pathway is shown in blue. Both pathways interact as Ca2+ contributes to ERK activation and additionally both Akt and ERK can compensate each other’s activation. Abbreviations: MEK, Mitogen-activated protein kinase; mTOR, mechanistic target of rapamycin; 4EBP1, eukaryotic translation initiation factor 4E binding protein 1; PIP2, phosphatidylinositol 4,5 biphosphate.

Figure 2. not shown. Classical versus proposed models of nuclear Ca2+ signaling in cardiomyocytes. The insulin-like growth factor 1 receptor (IGF-1R) can specifically regulate nuclear Ca2+ signaling independently of the role of Ca2+ on excitation–contraction coupling. On the classic model, inositol 1,4,5 triphosphate (InsP3; IP3) produced after IGF-1R activation travels from the peripheral plasma membrane to the nucleus, where it activates InsP3 receptors. In this model InsP3 bypasses its receptors present on the sarcoplasmic reticulum, which would lead to cytosolic Ca2+ signals. The novel model that we propose is based on recent findings, where the IGF-1R signaling complex is present in T-tubule invaginations toward the nucleus. In these compartments, IGF-1R activation leads to locally restricted InsP3 production that allows nuclear Ca2+ signals to regulate gene expression of genes associated with the development of cardiomyocyte hypertrophy. Abbreviations: RyR, ryanodine receptor; ECC, excitation–contraction coupling; PLC, phospholipase C; DHPR, dihydropyridine receptor.

The beneficial roles of IGF-1 in the cardiovascular system largely explain the interest in the development of new IGF-1-based treatments for cardiovascular disease. So far the FDA has approved two drugs for the treatment of IGF-1 deficiency: mecasermin (Increlex1), a human recombinant IGF-1 analog; and mecasermin rinfabate (IPLEX1), a binary protein complex of human recombinant IGF-1 and human recombinant IGBP-3. The safety of a chronic systemic IGF-1 therapy is open to question because it could promote severe adverse effects, such as an increased risk of cancer. To avoid these problems, several researchers have selectively overexpressed IGF-1 and IGF-1R in the heart.

Box 2. Outstanding questions

Insulin-like growth factor 1 (IGF-1) is an old friend of the heart. Despite the well-known protective effects of IGF-1 on cardiac function and the antiapoptotic effects of this peptide, novel evidence opens new questions to this longstanding relationship.

·       How do the multiple signaling pathways triggered by IGF-1 receptor (IGF-1R) interact with each other?

·       What lies further than extracellular signal-regulated kinase (ERK)/Akt/Ca2+ activation toward heart function?

·       Do these signaling pathways regulate cardiac fibroblast or endothelial cell function?

·       Which are the specific downstream signaling pathways of the different pools of IGF-1R and their role in regulating cardiomyocyte survival, hypertrophy, metabolism, proliferation?

·       What drives IGF-1R to such specific subcellular compartments?

·       What is the relevance of the hybrid IGF-1R/insulin receptors on cardiovascular disease?

·       Does a crosstalk exist between insulin receptor and IGF-1R in the heart under physiological and pathological conditions?

·       Is one pathway more beneficial than the other?

·       Will stem cell therapy of cardiac progenitors be able to provide concrete treatment opportunities?

·       Is IGF-1 a key regulator of this outcome?

Abundant evidence supports the key physiological roles of IGF-1 in the heart. In cardiomyocytes, IGF-1 activates multiple downstream signaling pathways for controlling cell death, metabolism, autophagy, differentiation, transcription, and protein synthesis (Figure 1). Of great interest are the findings that the entire IGF-1R complex is strategically located in perinuclear sarcolemmal invaginations that locally control nuclear Ca2+ signaling and transcriptional upregulation (Figure 2). This novel evidence changesmthe classical paradigm of IGF-1 signaling and adds a new level of complexity that may be relevant for other signaling receptors in the heart: interorganelle communication between plasma membrane invaginations and the nucleus.
The strategic localization of IGF-1R in these structures and the association with heterotrimeric G proteins may explain the differences in the phenotypic response induced by IGF-1 and others agonists, like endothelin-1 and angiotensin II, that also signal through intracellular Ca2+. By activating a noncanonical, selective mechanism of nuclear Ca2+ release, IGF-1 can regulate the expression of a specific set of cardiac genes via the generation of a particular signal-encoding pattern, leading to adaptive cardiac hypertrophy, antiapoptotic effects, and metabolic adaptation.

Pulmonary Hypertension in Heart Failure with Preserved Ejection Fraction – any Pathophysiological Role of Mitral Regurgitation

Marco Guazzi

read with interest the study by Lam et al. (1) as an important contribution to the pathophysiological and clinical impact of pulmonary hypertension (PH) in hypertensive patients with heart failure and preserved left ventricular ejection fraction (HFpEF). Recent guidelines on arterial PH recognize HFpEF as a growing cause of left-sided PH, but a definitive appreciation of its true prevalence and prognostic relevance is lacking. The present study provides some new important information on this subject.

It is noteworthy that HFpEF was associated, in a high rate of cases (83%), with a typical hemodynamic pattern of precapillary PH, and a strong correlation was found between pulmonary artery systolic pressure and pulmonary capillary wedge pressure. Most important, pulmonary artery systolic pressure, rather than other echocardiography-derived measures of diastolic dysfunction, was the only significant multivariate predictor of mortality, a finding that was confirmed even when combined comorbid diseases potentially contributing to PH development, such as chronic obstructive pulmonary disease, were taken into account.

In patients with systolic heart failure, a major determinant of PH development is mitral regurgitation. Whether mitral regurgitation could be a putative factor in the pathogenesis of PH in HFpEF patients remains an open and intriguing question.

Accordingly, it would be of interest if the authors could provide some details on how many HFpEF patients exhibited mitral regurgitation, especially in comparison with control hypertensive patients without HFpEF.

Lam CSP, Roger VL, Rodeheffer RJ, Borlaug BA, Enders FT, Redfield MM. Pulmonary hypertension in heart failure with preserved ejection fraction: a community-based study. J Am Coll Cardiol 2009; 53:1119–23.

Midregion Prohormone Adrenomedullin and Prognosis in Patients Presenting with Acute Dyspnea Results from the BACH (Biomarkers in Acute Heart Failure) Trial

Alan Maisel, MD, Christian Mueller, Richard M. Nowak,W. Frank Peacock, et al.
J Am Coll Cardiol 2011; 58(10):1057–67

Objectives The aim of this study was to determine the prognostic utility of midregion proadrenomedullin (MR-proADM) in all patients, cardiac and noncardiac, presenting with acute shortness of breath.
The recently published BACH (Biomarkers in Acute Heart Failure) study demonstrated that MR-proADM had superior accuracy for predicting 90-day mortality compared with B-type natriuretic peptide (area under the curve: 0.674 vs. 0.606, respectively, p < 0.001) in acute heart failure.
Methods The BACH trial was a prospective, 15-center, international study of 1,641 patients presenting to the emergency department with dyspnea. Using this dataset, the prognostic accuracy of MR-proADM was evaluated in all patients enrolled for predicting 90-day mortality with respect to other biomarkers, the added value in addition to clinical variables, as well as the added value of additional measurements during hospital admission.
Results Compared with B-type natriuretic peptide or troponin, MR-proADM was superior for predicting 90-day all-cause mortality in patients presenting with acute dyspnea (c index = 0.755, p < 0.0001). Furthermore, MR-proADM added significantly to all clinical variables (all adjusted hazard ratios: HR=3.28), and it was also superior to all other biomarkers. MRproADM added significantly to the best clinical model (bootstrap-corrected c index increase: 0.775 to 0.807; adjusted standardized hazard ratio: 2.59; 95% confidence interval: 1.91 to 3.50; p < 0.0001). Within the model, MR-proADM was the biggest contributor to the predictive performance, with a net reclassification improvement of 8.9%. Serial evaluation of MR-proADM performed in patients admitted provided a significant added value compared with a model with admission values only (p< 0.0005). More than one-third of patients originally at high risk could be identified by the biomarker evaluation at discharge as low-risk patients. Conclusions MR-proADM identifies patients with high 90-day mortality and adds prognostic value to natriuretic peptides in patients presenting with acute shortness of breath. Serial measurement of this biomarker may also prove useful for monitoring, although further studies will be required. (Biomarkers in Acute Heart Failure [BACH]; NCT00537628)

Invasive Hemodynamic Characterization of Heart Failure with Preserved Ejection Fraction

Mads J. Andersen, Barry A. Borlaug
Heart Failure Clin 10 (2014) 435–444


  • Invasive hemodynamic assessment in heart failure with preserved ejection fraction (HFpEF) was originally a primary research tool to advance the understanding of the pathophysiology of HFpEF.
  • The role of invasive hemodynamic assessment in HFpEF is expanding to the diagnostic arena where invasive assessment offers a robust, sensitive, and specific way to diagnose or exclude HFpEF in patients with unexplained dyspnea and normal ejection fraction.
  • In future years, invasive hemodynamic profiling may more rigorously phenotype patients to individualized therapy and, potentially, deliver novel device-based structural interventions.

The circulatory system serves to deliver substrates to the body via the bloodstream while removing the byproducts of cellular metabolism. Hemodynamics broadly refers to the study of the forces involved in the circulation of blood, which are governed by to the physical properties of the heart and vasculature and their dynamic regulation by the autonomic nervous system.

Afterload represents the forces opposing ventricular ejection and can be quantified by systolic left ventricular (LV) wall stress and aortic input impedance or its individual components (resistance, compliance, characteristic impedance). Wall stress is inconvenient because it depends on heart size and geometry, whereas impedance is cumbersome because it is a frequency-domain parameter that cannot be easily coupled with time-domain measures of ventricular function. Effective arterial elastance (Ea), defined by the ratio of LV end-systolic pressure (ESP) to stroke volume, provides a robust measure of total arterial load. Ea is not a directly measured parameter but, instead, a net or lumped stiffness of the vasculature that incorporates both mean and oscillatory components of afterload (Fig. 1). Preload reflects the degree of myofiber stretch before the onset of contraction, which, in turn, dictates the force and velocity of contraction according to the Frank-Starling principle. In everyday practice, preload is often conceptualized as equivalent to LV filling pressures. However, in fact, preload is most accurately reflected by the LV volume at end-diastole volume (EDV). Filling pressures are related to EDV by the LV diastolic chamber stiffness, which differs in healthy volunteers and subjects with HFpEF.

Fig. 1. Not shown. Ventricular-arterial coupling in the pressure-volume plane. Pressure volume loop at steady state is shown in dark black. The area subtended by the loop (shaded) represents the stroke work. Stroke volume is the difference between end-diastolic volume (EDV) and end-systolic volume (ESV). Ea is defined by the negative slope connecting the ESP and ESV coordinates with EDV and pressure = 0. With acute preload reduction (dotted line loops) there is progressive reduction in EDV, ESV, and ESP. The linear slope of the endsystolic pressure volume relationship (ESPVR) is LV end-systolic elastance (Ees). The curvilinear slope of the end diastolic pressure–volume relationship (EDVPR) is derived by fitting pressure volume coordinates measured during diastasis to the equation shown. The exponential power or stiffness constant (b) obtained is a measure of LV diastolic stiffness. (Adapted from Borlaug BA, Kass DA. Invasive hemodynamic assessment in heart failure. Heart Fail Clin 2009;5(2):217–28; with permission.)

Fig. 3. Not shown. Left ventricular diastolic reserve in HFpEF. In the normal healthy adult, the rate of LV pressure decay during isovolumic contraction (t) is rapid and increases markedly during exercise in association with a reduction in LVmin, allowing for suction of blood into the LV, with no increase in left atrial pressure or LV end-diastolic pressure (LVEDP) despite an increase in LV end-diastolic volume and marked shortening of the cycle length. In HFpEF, relaxation is prolonged at baseline (increased t) with inadequate hastening (shortening of t) during exercise, contributing to an inability to reduce LVmin and, consequently, a complete lack of suction effects. LV filling then completely depends on left atrial hypertension, which develops in tandem with marked elevation in LVEDP. (Data from Borlaug BA, Jaber WA, Ommen SR, et al. Diastolic relaxation and compliance reserve during dynamic exercise in heart failure with preserved ejection fraction. Heart 2011;97(12):964–9.)

Fig. 4. Preload and filling pressures in HFpEF. (A) Cumulative distribution plot shows that acute changes in stroke volume with nitroprusside infusion are lower in HFpEF (black) compared with HFrEF (red). Because afterload (Ea) is lowered, any acute reduction in SV must be related to reduction in preload volume (EDV) and nearly 40% of HFpEF patients experienced stroke volume reduction with nitroprusside, despite high filling pressures (PCWP 20–25 mm Hg), indicating increased reliance on high pressures to achieve adequate EDV. *p<0.0001 compared with HFrEF. (B) LVEDP in a healthy adult (blue) and in a HFpEF patient with increased LV diastolic stiffness (green). At the same preload (EDV), pressure is more than twofold higher in HFpEF. In contrast, at the same LV diastolic pressure (15 mm Hg), LV volume is much lower in HFpEF, indicating decreased LV diastolic capacitance. V15, volume at end-diastolic pressure = 15 mm Hg; LVEDP. (Adapted from Schwartzenberg S, Redfield MM, From AM, et al. Effects of vasodilation in heart failure with preserved or reduced ejection fraction implications of distinct pathophysiologies on response to therapy. J Am Coll Cardiol 2012;59(5):442–51; with permission.)

Updated Clinical Classification of Pulmonary Hypertension

Gérald Simonneau, Ivan M. Robbins, Maurice Beghetti, et al.
J Am Coll of Cardiol   2009; 54(1), Suppl S

The aim of a clinical classification of pulmonary hypertension (PH) is to group together different manifestations of disease sharing similarities in pathophysiologic mechanisms, clinical presentation, and therapeutic approaches. In 2003, during the 3rd World Symposium on Pulmonary Hypertension, the clinical classification of PH initially adopted in 1998 during the 2nd World Symposium was slightly modified. During the 4th World Symposium held in 2008, it was decided to maintain the general architecture and philosophy of the previous clinical classifications. The modifications adopted during this meeting principally concern Group 1, pulmonary arterial hypertension (PAH). This subgroup includes patients with PAH with a family history or patients with idiopathic PAH with germline mutations (e.g., bone morphogenetic protein receptor-2, activin receptor-like kinase type 1, and endoglin). In the new classification, schistosomiasis and chronic hemolytic anemia appear as separate entities in the subgroup of PAH associated with identified diseases. Finally, it was decided to place pulmonary venoocclusive disease and pulmonary capillary hemangiomatosis in a separate group, distinct from but very close to Group 1 (now called Group 1=). Thus, Group 1 of PAH is now more homogeneous. (J Am Coll Cardiol 2009; 54: S43–54)
Updated Evidence-Based Treatment Algorithm in Pulmonary Arterial Hypertension

Robyn J. Barst,  J. Simon R. Gibbs, Hossein A. Ghofrani, et al.
J Am Coll Cardiol 2009; 54(1), Suppl S,

Uncontrolled and controlled clinical trials with different compounds and procedures are reviewed to define the risk benefit profiles for therapeutic options in pulmonary arterial hypertension (PAH). A grading system for the level of evidence of treatments based on the controlled clinical trials performed with each compound is used to propose an evidence-based treatment algorithm. The algorithm includes drugs approved by regulatory agencies for the treatment of PAH and/or drugs available for other indications. The different treatments have been evaluated mainly in idiopathic PAH, heritable PAH, and in PAH associated with the scleroderma spectrum of diseases or with anorexigen use. Extrapolation of these recommendations to other PAH subgroups should be done with caution. Oral anticoagulation is proposed for most patients; diuretic treatment and supplemental oxygen are indicated in cases of fluid retention and hypoxemia, respectively. High doses of calcium-channel blockers are indicated only in the minority of patients who respond to acute vasoreactivity testing. Nonresponders to acute vasoreactivity testing or responders who remain in World Health Organization (WHO) functional class III, should be considered candidates for treatment with either an oral phosphodiesterase-5 inhibitor or an oral endothelin-receptor antagonist. Continuous intravenous administration of epoprostenol remains the treatment of choice in WHO functional class IV patients. Combination therapy is recommended for patients treated with PAH monotherapy who remain in WHO functional class III. Atrial septostomy and lung transplantation are indicated for refractory patients or where medical treatment is unavailable. (J Am Coll Cardiol 2009;54:S78–84)

Inhibition and down-regulation of gene transcription and guanylyl cyclase activity of NPRA by angiotensin II involving protein kinase C

Kiran K. Arise, Kailash N. Pandey
Biochem and Biophys Res Commun 349 (2006) 131–135

The objective of this study was to investigate the role of protein kinase C (PKC) in the angiotensin II (Ang II)-dependent repression of Npr1 (coding for natriuretic peptide receptor-A, NPRA) gene transcription. Mouse mesangial cells (MMCs) were transfected with Npr1 gene promoter-luciferase construct and treated with Ang II and PKC agonist or antagonist. The results showed that the treatment of MMCs with 10 nM Ang II produced a 60% reduction in the promoter activity of Npr1 gene. MMCs treated with 10 nM Ang II exhibited 55% reduction in NPRA mRNA levels, and subsequent stimulation with 100 nM ANP resulted in 50% reduction in guanylyl cyclase (GC) activity. Furthermore, the treatment of MMCs with Ang II in the presence of PKC agonist phorbol ester (100 nM) produced an almost 75% reduction in NPRA mRNA and 70% reduction in the intracellular accumulation of cGMP levels. PKC antagonist staurosporine completely reversed the effect of Ang II and phorbol ester. This is the first report to demonstrate that ANG II-dependent transcriptional repression of Npr1 gene promoter activity and down-regulation of GC activity of translated protein, NPRA is regulated by PKC pathways.

Transcriptional regulation of guanylyl cyclase/natriuretic peptide receptor-A gene

Prerna Kumar, Kiran K. Arise, Kailash N. Pandey
peptides 27 (2006) 1762–1769

Activation of natriuretic peptide receptor-A (NPRA) produces the second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. In the present study, we have examined the role of trans-acting factor Ets-1 in transcriptional regulation of Npr1 gene (coding for NPRA).Using deletional analysis of the Npr1 promoter, we have defined a 400 base pair (bp) region as the core promoter, which contains consensus binding sites for transcription factors including: Ets-1, Lyf-1, and GATA-1/2. Over-expression of Ets-1 in mouse mesangial cells (MMCs) enhanced Npr1 gene transcription by 12-fold. However, overexpression of GATA-1 or Lyf-1 repressed Npr1 basal promoter activity by 50% and 80%, respectively. The constructs having a mutant Ets-1 binding site or lacking this site failed to respond to Ets-1 activation of Npr1 gene transcription. Collectively, the present results demonstrate that Ets-1 greatly stimulates Npr1 gene promoter activity, implicating its critical role in the regulation and function of NPRA at the molecular level.

Several agents that are known to upregulate Ets-1 transcription, include RA, TNF-alpha, VEGF, and TPA. Ets-1 is upregulated at exposure to agonists such as serum in vitro and is expressed in injured vasculature. MAPK-mediated phosphorylation positively regulates the transcriptional activation functions of Ets-1 by recruiting CBP/p300. Not much is known about Ets-1 expression or regulation in mesangial cells. A temporal increase of mesangial cell Ets-1 expression has been reported which correlates with mesangial cell activation
in mesangioproliferative glomerulonephritis suggesting involvement of PDGF-B. There might be a possibility that during glomerulonephritis increased Ets-1 expression upregulates Npr1 gene as a protective mechanism. Npr1 gene has been shown to negatively regulate mitogen-activated protein kinase and proliferation of mesangial cells.

In conclusion, our results demonstrate that the precise control of Npr1 gene transcriptional activity is achieved through a synergy of activators and repressors in which Ets-1 plays an integral role as a transcriptional activator. Comparatively, Lyf-1 and GATA-1 act as repressors, inhibiting and regulating the transcriptional activity of Npr1 gene promoter. The present findings suggest that Ets-1 plays a critical role in enhancing Npr1 gene transcription and may have an important influence in hypertension and cardiovascular homeostasis at the molecular level.

Krüppel-like transcription factor 11 (KLF11) overexpression inhibits cardiac hypertrophy and fibrosis in mice

Yue Zheng, Ye Kong, Feng Li
Biochem and Biophys Res Commun 443 (2014) 683–688

The Krüppel-like factors (KLFs) belong to a subclass of Cys2/His2 zinc-finger DNA-binding proteins. The KLF family member KLF11 is originally identified as a transforming growth factor b (TGF-b)-inducible gene and is one of the most studied in this family. KLF11 is expressed ubiquitously and participates  in diabetes and regulates hepatic lipid metabolism. However, the role of KLF11 in cardiovascular system is largely unknown. Here in this study, we reported that KLF11 expression is down-regulated in failing human hearts and hypertrophic murine hearts. To evaluate the roles of KLF11 in cardiac hypertrophy, we generated cardiac-specific KLF11 transgenic mice. KLF11 transgenic mice do not show any difference from their littermates at baseline. However, cardiac-specific KLF11 overexpression protects mice from TAC-induced cardiac hypertrophy, with reduced radios of heart weight (HW)/body weight (BW), lung weight/BW and HW/tibia length, decreased left ventricular wall thickness and increased fractional shortening. We also observe lower expression of hypertrophic fetal genes in TAC-challenged KLF11 transgenic mice compared with WT mice. In addition, KLF11 reduces cardiac fibrosis in mice underwent hypertrophy. The expression of fibrosis markers are also down-regulated when KLF11 is overexpressed in TAC-challenged mice. Taken together, our findings identify a novel anti-hypertrophic and anti-fibrotic role of KLF11, and KLF11 activator may serve as candidate drug for heart failure patients.

Acute Lung Injury

Acute Lung Injury

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


Acute Lung Injury


Acute lung injury is a serious phenomenon only recognized as having significant relevance to allogeneic blood transfusion in the last 15 years.  It is not limited to transfusion events, and is also related to SIRS and sepsis.  It is simulated in experimental models by lipoprotein, such as endotoxin.  It occurs in the pretransfused surgical patient, or in the medical patient as well.  Why it was not recognized earlier is a matter of conjecture.  The significant reduction in immune modulated blood type incompatibility reactions in Western countries is a factor.  The other factor is that the lipoprotein antigenic fractions involved are associated with component transfusions other than stored red cells. The following discussion will elaborate on what is increasingly recognized as a relevant issue in medicine today.
Transfusion Related Reaction

In medicinetransfusion related acute lung injury (TRALI) is a serious blood transfusion complication characterized by the acute onset of non-cardiogenic pulmonary edema following transfusion of blood products.[1]

Although the incidence of TRALI has decreased with modified transfusion practices, it is still the leading cause of transfusion-related fatalities in the United States from fiscal year 2008 through fiscal year 2012.

Transfusion Related Acute Lung Injury



Micrograph of diffuse alveolar damage, the histologic correlate of TRALI. H&E stain. Very high magnification micrograph of hyaline membranes, as seen in diffuse alveolar damage (DAD), the histologic correlate of acute respiratory distress syndrome (ARDS), transfusion related acute lung injury (TRALI), acute interstitial pneumonia (AIP).

TRALI is defined as an acute lung injury that is temporally related to a blood transfusion; specifically, it occurs within the first six hours following a transfusion.[3]

It is typically associated with plasma components such as platelets and Fresh Frozen Plasma, though cases have been reported with packed red blood cells since there is some residual plasma in the packed cells. The blood component transfused is not part of the case definition. Transfusion-related acute lung injury (TRALI) is an uncommon syndrome that is due to the presence of leukocyte antibodies in transfused plasma. TRALI is believed to occur in approximately one in every 5000 transfusions. Leukoagglutination and pooling of granulocytes in the recipient’s lungs may occur, with release of the contents of leukocyte granules, and resulting injury to cellular membranes, endothelial surfaces, and potentially to lung parenchyma. In most cases leukoagglutination results in mild dyspnea and pulmonary infiltrates within about 6 hours of transfusion, and spontaneously resolves;

Occasionally more severe lung injury occurs as a result of this phenomenon and Acute Respiratory Distress Syndrome (ARDS) results. Leukocyte filters may prevent TRALI for those patients whose lung injury is due to leukoagglutination of the donor white blood cells, but because most TRALI is due to donor antibodies to leukocytes, filters are not helpful in TRALI prevention. Transfused plasma (from any component source) may also contain antibodies that cross-react with platelets in the recipient, producing usually mild forms of posttransfusion purpura or platelet aggregation after transfusion.

Another nonspecific form of immunologic transfusion complication is mild to moderate immunosuppression consequent to transfusion. This effect of transfusion is not completely understood, but appears to be more common with cellular transfusion and may result in both desirable and undesirable effects. Mild immunosuppression may benefit organ transplant recipients and patients with autoimmune diseases; however, neonates and other already immunosuppressed hosts may be more vulnerable to infection, and cancer patients may possibly have worse outcomes postoperatively.




Perioperative transfusion-related acute lung injury: The Canadian Blood Services experience

Asim Alam, Mary Huang, Qi-Long Yi, Yulia Lin, Barbara Hannach
Transfusion and Apheresis Science 50 (2014) 392–398

Purpose: Transfusion-related acute lung injury (TRALI) is a devastating transfusion-associated adverse event. There is a paucity of data on the incidence and characteristics of TRALI cases that occur perioperatively. We classified suspected perioperative TRALI cases reported to Canadian Blood Services between 2001 and 2012, and compared them to non-perioperative cases to elucidate factors that may be associated with an increased risk of developing TRALI in the perioperative setting. Methods: All suspected TRALI cases reported to Canadian Blood Services (CBS) since 2001 were reviewed by two experts or, from 2006 to 2012, the CBS TRALI Medical Review Group (TMRG). These cases were classified based on the Canadian Consensus Conference (CCC) definitions and detailed in a database. Two additional reviewers further categorized them as occurring within 72 h from the onset of surgery (perioperative) or not in that period (non-perioperative). Various demographic and characteristic variables of each case were collected and compared between groups. Results: Between 2001 and 2012, a total of 469 suspected TRALI cases were reported to Canadian Blood Services; 303 were determined to be within the TRALI diagnosis spectrum. Of those, 112 (38%) were identified as occurring during the perioperative period. Patients who underwent cardiac surgery requiring cardiopulmonary bypass (25.0%), general surgery (18.0%) and orthopedics patients (12.5%) represented the three largest surgical groups. Perioperative TRALI cases comprised more men (53.6% vs. 41.4%, p = 0.04) than non-perioperative patients. Perioperative TRALI patients more often required supplemental O2 (14.3% vs. 3.1%, p = 0.0003), mechanical ventilation (18.8% vs. 3.1%), or were in the ICU (14.3% vs. 3.7%, p = 0.0043) prior to the onset of TRALI compared to non-perioperative TRALI patients. The surgical patients were transfused on average more components than non-perioperative patients (6.0 [SD = 8.3] vs. 3.6 [5.2] products per patient, p = 0.0002). Perioperative TRALI patients were transfused more plasma (152 vs. 105, p = 0.013) and cryoprecipitate (51 vs. 23, p < 0.01) than non-perioperative TRALI patients. There was no difference between donor antibody test results between the groups. Conclusion: CBS data has provided insight into the nature of TRALI cases that occur perioperatively; this  group represents a large proportion of TRALI cases.


Transfusion-related acute lung injury: a clinical review

Alexander P J Vlaar, Nicole P Juffermans
Lancet 2013; 382: 984–94

Three decades ago, transfusion-related acute lung injury (TRALI) was considered a rare complication of transfusion medicine. Nowadays, the US Food and Drug Administration acknowledge the syndrome as the leading cause of transfusion-related mortality. Understanding of the pathogenesis of TRALI has resulted in the design of preventive strategies from a blood-bank perspective. A major breakthrough in efforts to reduce the incidence of TRALI has been to exclude female donors of products with high plasma volume, resulting in a decrease of roughly two-thirds in incidence. However, this strategy has not completely eradicated the complication. In the past few years, research has identified patient-related risk factors for the onset of TRALI, which have empowered physicians to take an individualized approach to patients who need transfusion.

Development of an international consensus definition has aided TRALI research, yielding a higher incidence in specific patient populations than previously acknowledged Patients suffering from a clinical disorder such as sepsis are increasingly recognized as being at risk for development of TRALI. Thereby, from a diagnosis by exclusion, TRALI has become the leading cause of transfusion-related mortality. However, the syndrome is still under diagnosed and under-reported in some countries.

Although blood transfusion can be life-saving, it can also be a life-threatening intervention. Physicians use blood transfusion on a daily basis. Increased awareness of the risks of this procedure is needed, because management of patient-tailored transfusion could reduce the risk of TRALI. Such an individualized approach is now possible as insight into TRALI risk factors evolves. Furthermore, proper reporting of TRALI could prevent recurrence.

Absence of an international definition for TRALI previously contributed to underdiagnosis. As such, a consensus panel, and the US National Heart, Lung and Blood Institute Working Group in 2004, formulated a case definition of TRALI based on clinical and radiological parameters. The definition is derived from the widely used definition of acute lung injury (panel 1). Suspected TRALI is defined as fulfilment of the definition of acute lung injury within 6 h of transfusion in the absence of another risk factor (panel 1).

Although this definition seems to be straightforward, the characteristics of TRALI are indistinguishable from acute lung injury due to other causes, such as sepsis or lung contusion. Therefore, this definition would rule out the possibility of diagnosing TRALI in a patient with an underlying risk factor for acute lung injury who has also received a transfusion. To identify such cases, the term possible TRALI was developed.

Although the TRALI definition is an international consensus definition, surveillance systems in some countries, including the USA, France and the Netherlands, use an alternative in which imputability is scored. Imputability aims to identify the likelihood that transfusion is the causal factor. Imputability scores mostly imply that other causes of acute lung injury can be ruled out, so that diagnosis of TRALI is by exclusion. However, observational and animal studies suggest that risk factors for TRALI include other disorders, such as sepsis. Therefore, an imputability definition would result in underdiagnosis of TRALI. The consensus definition accommodates the uncertainty of the association of acute lung injury to the transfusion in possible TRALI. The conventional definition of TRALI uses a timeframe of 6 h in which acute lung injury needs to develop after a blood transfusion. In critically ill patients, transfusion increases the risk (odds ratio 2·13, 95% CI 1·75–2·52) for development of acute lung injury 6–72 h after transfusion.  However, whether the pathogenesis of delayed TRALI is similar to that of TRALI is unclear.

A two-hit hypothesis has been proposed for TRALI. The first hit is underlying patient factors, resulting in adherence of primed neutrophils to the pulmonary endothelium. The second hit is caused by mediators in the blood transfusion that activate the endothelial cells and pulmonary neutrophils, resulting in capillary leakage and subsequent pulmonary edema. The second hit can be antibody-mediated or non-antibody-mediated.

Panel 1: Definition of transfusion-related acute lung injury (TRALI)

Suspected TRALI

  • Acute onset within 6 h of blood transfusion
    • PaO2/FIO2<300 mm Hg, or worsening of P to F ratio
    • Bilateral infi ltrative changes on chest radiograph
    • No sign of hydrostatic pulmonary oedema (pulmonary arterial occlusion
    pressure ≤18 mm Hg or central venous pressure ≤15 mm Hg)
    • No other risk factor for acute lung injury

Possible TRALI
Same as for suspected TRALI, but another risk factor present for acute lung injury

Delayed TRALI
Same as for (possible) TRALI and onset within 6–72 h of blood transfusion

Pathophysiology of two-hit mediated transfusion-related acute lung injury (TRALI).  The pre-phase of the syndrome consists of a fi rst hit, which is mainly systemic. This first hit is the underlying disorder of the patient (eg, sepsis or pneumonia) causing neutrophil attraction to the capillary of the lung. Neutrophils are attracted to the lung by release of cytokines and chemokines from upregulated lung endothelium. Loose binding by L-selectin takes place. Firm adhesion is mediated by E-selectin and platelet-derived P-selectin and intracellular adhesion molecules (ICAM-1). In the acute phase of the syndrome, a second hit caused by mediators in the blood transfusion takes place. This hit results in activation of inflammation and coagulation in the pulmonary compartment. Neutrophils adhere to the injured capillary endothelium and marginate through the interstitium into the air space, which is filled with protein-rich edema fluid. In the air space, cytokines interleukin-1, -6, and -8, (IL-1, IL-6, and IL-8, respectively) are secreted, which act locally to stimulate chemotaxis and activate neutrophils resulting in formation of the elastase-α1-antitrypsin (EA) complex. Neutrophils can release oxidants, proteases, and other proinflammatory molecules, such as platelet-activating factor (PAF), and form neutrophil extracellular traps (NETs). Furthermore, activation of the coagulation system happens, shown by an increase in thrombin-antithrombin complexes (TATc), as does a decrease in activity of the fibrinolysis system, shown by a reduction in plasminogen activator activity. The influx of protein-rich edema fluid into the alveolus leads to the inactivation of surfactant, which contributes to the clinical picture of acute respiratory distress in the onset of TRALI. PAI-1 = plasminogen activator inhibitor-1.

Antibody-mediated TRALI is caused by passive transfusion of HLA or human neutrophil antigen (HNA) and corresponding antibodies from the donor directed against antigens of the recipient. Neutrophil activation occurs directly by binding of the antibody to the neutrophil surface (HNA antibodies) or indirectly, mainly by binding to the endothelial cells with activation of the neutrophil (HLA class I antibodies) or to monocytes with subsequent activation of the neutrophil (HLA class II antibodies). The antibody titer and the volume of antibody containing plasma both increase the risk for onset of TRALI. Although the role of donor HLA and HNA antibodies from transfused blood is widely accepted, not all TRALI cases are antibody mediated. In many patients, antibodies cannot be detected. Furthermore, many blood products containing antibodies do not lead to TRALI. This finding has led to development of an alternative hypothesis for the onset of TRALI, termed non-antibody-mediated TRALI.

Non-antibody-mediated TRALI is caused by accumulation of proinflammatory mediators during storage of blood products, and possibly by ageing of the erythrocytes and platelets themselves. Although most preclinical studies have noted a positive correlation between storage time of cell-containing blood products and TRALI, the mechanism is controversial. Two mechanisms have been suggested, including either plasma or the aged cells. In a small-case study and animal experiments, accumulation of bioactive lipids and soluble CD40 ligand (sCD40L) in the plasma layer of cell-containing blood products has been associated with TRALI. Bioactive lipids are thought to cause neutrophil activation through the G-protein coupled receptor on the neutrophil.

The two-hit model suggests that patients in a poor clinical state are at risk for development of TRALI. However, cases have been described of antibody-mediated TRALI developing in fairly healthy recipients. To explain this discrepancy, a threshold model has been suggested in which a threshold must be overcome to induce a TRALI reaction. The threshold is dependent both on the predisposition of the patient (first hit) and the quantity of antibodies in the transfusion (second hit). A large quantity of antibody that matches the recipient’s antigen can cause severe TRALI in a recipient with no predisposition.

Threshold model of antibody-mediated transfusion-related acute lung injury (TRALI). A specific threshold must be overcome to induce a TRALI reaction. To overcome a threshold, several factors act together: the activation status of the pulmonary neutrophils at the time of transfusion, the strength of the neutrophil-priming activity of transfused mediators (A), and the clinical status of the patient (B).

Panel 2: Clinical characteristics of transfusion-related acute lung injury (TRALI) and transfusion-associated circulatory overload (TACO)

• Dyspnea
• Fever
• Usually hypotension
• Hypoxia
• Leukopenia
• Thrombocytopenia
• Pulmonary edema on chest x-ray
• Normal left ventricular function*
• Normal pulmonary artery occlusion pressure

• Dyspnea
• Usually hypertension
• Hypoxia
• Pulmonary edema on chest radiographs
• Normal or decreased left ventricular function
• Increased pulmonary artery occlusion pressure
• Raised brain natriuretic peptide

Restrictive transfusion policy

The most effective prevention is a restrictive transfusion strategy. In a randomised clinical trial in critically ill patients, a restrictive transfusion policy for red blood cells was associated with a decrease in incidence of acute lung injury compared with a liberal strategy (7·7% vs 11·4%), suggesting that some of these patients might have had TRALI. The restrictive threshold was well tolerated and has greatly helped in guidance of red blood cell transfusion in the intensive-care unit.

Patient-tailored transfusion policy

Transfusion cannot be avoided altogether. A multivariate analysis in patients in intensive care showed that patient related risk factors contributed more to the onset of TRALI than did transfusion-related risk factors, suggesting that development of a TRALI reaction is dependent more on host factors then on factors in the blood product. Therefore, a patient-tailored approach aimed at reducing TRALI risk factors could be effective to alleviate the risk of TRALI.

Despite limitations of diagnostic tests, TRALI incidence seems to be high in at-risk patient populations. Therefore, TRALI is an underestimated health-care problem. Preventive measures, such as mainly male donor strategies, have been successful in reducing risk of TRALI. Identification of risk factors further improves the risk–benefit assessment of a blood transfusion. Efforts to further decrease the risk of TRALI needs increased awareness of this syndrome among physicians.


Transfusion-related acute lung injury: Current understanding and preventive strategies

A.P.J. Vlaar
Transfusion Clinique et Biologique 19 (2012) 117–124

Transfusion-related acute lung injury (TRALI) is the most serious complication of transfusion medicine. TRALI is defined as the onset of acute hypoxia within 6 hours of a blood transfusion in the absence of hydrostatic pulmonary edema. The past decades have resulted in a better understanding of the pathogenesis of this potentially life-threating syndrome. The present notion is that the onset of TRALI follows a threshold model in which both patient and transfusion factors are essential. The transfusion factors can be divided into immune and non-immune mediated TRALI. Immune-mediated TRALI is caused by the passive transfer of human neutrophil antibodies (HNA) or human leukocyte antibodies (HLA) present in the blood product reacting with a matching antigen in the recipient. Non-immune mediated TRALI is caused by the transfusion of stored cell-containing blood products. Although the mechanisms behind immune-mediated TRALI are reasonably well understood, this is not the case for non-immune mediated TRALI. The increased understanding of pathways involved in the onset of immune-mediated TRALI has led to the design of preventive strategies. Preventive strategies are aimed at reducing the risk to exposure of HLA and HNA to the recipient of the transfusion. These strategies include exclusion of “at risk” donors and pooling of high plasma volume products and have shown to reduce the TRALI incidence effectively.

Studies show that, in at risk patient populations, up to 8% of transfused patients may develop TRALI. Since the syndrome TRALI has been recognized, evidence on the pathogenesis of TRALI has been accumulating. The present notion is that the onset of TRALI follows a threshold model in which both patient and transfusion factors are essential in the development of TRALI. The transfusion factors can be divided into immune and non-immune mediated TRALI. Immune-mediated TRALI is caused by the passive transfer of human neutrophil antibodies (HNA) or human leukocyte antibodies (HLA) present in the blood product, reacting with a matching antigen in the recipient. Non-immune mediated TRALI is caused by the transfusion of stored cell-containing blood products. In recent years, many countries have successfully implemented preventive strategies resulting in a decrease of the incidence of TRALI.

Definition of transfusion-related acute lung injury (TRALI).

  • Acute onset within 6 hours after a blood transfusion
  • PaO2/FiO2 < 300 mmHg
  • Bilateral infiltrative changes on the chest X-ray
  • No sign of hydrostatic pulmonary edema (PAOP < 18 mmHg or CVP < 15 mmHg)
  • No other risk factor for acute lung injury present

Possible TRALI

  • Other risk factor for acute lung injury present

PAOP: pulmonary arterial occlusion pressure; CVP: central venous pressure

The first landmark report creating the basis for the understanding of the pathogenesis of TRALI was published by Popovsky et al. in 1983. They provided evidence on the association between the presence of leucocyte antibodies in the donor serum and onset of acute lung injury in the recipient of the transfusion. It was also recognized that multiparous blood donors whose plasma contained these antibodies represented a potential transfusion hazard. It was this research group that was the first to identify TRALI as a distinct clinical entity. Subsequently, many other authors reported on the association between the presence of HLA or HNA antibodies in donor blood and the onset of TRALI in the recipient.

Although the role of transfused blood donor HLA and HNA antibodies was widely accepted to be involved in the onset of TRALI, not all cases could be explained by this theory. A significant part of reported TRALI cases have no detectable antibodies. Also, many antibody-containing blood products fail to produce TRALI.

The alternative hypothesis proposed by the group of Silliman posed that TRALI is a “two hit” event. The “first hit” is the underlying condition of the patient, resulting in priming of the pulmonary neutrophil. The “second hit” is the transfusion of a blood product causing activation of the neutrophils in the pulmonary compartment, causing pulmonary edema finally resulting in TRALI. The transfusion factors causing the “second hit” are divided in two groups; immune and non-immune mediated TRALI.

The “second hit” is the transfusion itself and is either immune or non-immune mediated TRALI. The mechanisms behind immune-mediated TRALI are widely accepted and proven in both pre-clinical and clinical studies.  The mechanisms involved in non-immune mediated TRALI are less clear.

The role of stored cell-containing blood products in the onset of non-immune TRALI has extensively been studied in preclinical and clinical studies. Although most of the pre-clinical studies find a positive correlation between the transfusion of stored cell-containing blood products in the presence of a “first hit” and the onset of TRALI, the mechanism behind the onset is controversial.

TRALI management consists mainly of preventing future adverse reactions and providing proper incidence estimates. All suspected TRALI cases should be reported to the blood bank for immunologic work-up as it is impossible to distinguish immune-mediated TRALI from non-immune mediated TRALI at bedside. Immunologic work-up includes testing of incompatibility by cross-matching donor plasma against recipient’s leucocytes. A donor with antibodies which are incompatible with the patient is excluded from further donation of blood for transfusion products. Furthermore, it is important to stress that the absence of a positive serologic work-up does not exclude the diagnosis of TRALI. TRALI is a clinical diagnosis and the immunologic work-up can be supportive but is not part of the diagnosis of TRALI. the two-event hypothesis and threshold hypothesis do not exclude the role of antibodies in the occurrence of TRALI in the presence of an inflammatory condition. Thus any patient fulfilling the TRALI definition (including possible TRALI) should be reported to the blood bank for an immunologic work-up of the recipient and the implicated donors on the presence of HLA and HNA antibodies.

Prevention of immune-mediated TRALI is achieved by exclusion of donors proven to have HLA or HNA antibodies in their plasma present or donors “at risk” to have these antibodies present.

  1. Exclusion of HLA or HNA positive donors
  2. Exclusion of donors “at risk” of being HLA or HNA positive
    Female donors – more specifically, multiparous donors
  3. Testing donors for HLA or HNA antibodies
  4. Multiple plasma pooling
    solvent/detergent plasma is produced from multiple donations, leading to an at least 500-fold dilution of a single plasma unit;
    neither HNA nor HLA antibodies are detectable in solvent/detergent fresh frozen plasma.
  5. To prevent non-immune mediated TRALI, the use of fresh blood only has been suggested

Strategies to prevent the onset of TRALI include the exclusion of female plasma donors and the pooling of plasma products. These strategies have already been implemented in some countries resulting in a reduction of the incidence of TRALI.
Transfusion-related immunomodulation (TRIM): An update

Eleftherios C. Vamvakas, Morris A. Blajchman
Blood Reviews (2007) 21, 327–348

Allogeneic blood transfusion (ABT)-related immunomodulation (TRIM) encompasses the laboratory immune aberrations that occur after ABT and their established or purported clinical effects. TRIM is a real biologic phenomenon resulting in at least one established beneficial clinical effect in humans, but the existence of deleterious clinical TRIM effects has not yet been confirmed. Initially, TRIM encompassed effects attributable to ABT by immunomodulatory mechanisms (e.g., cancer recurrence, postoperative infection, or virus activation). More recently, TRIM has also included effects attributable to ABT by pro-inflammatory mechanisms (e.g., multiple-organ failure or mortality). TRIM effects may be mediated by: (1) allogeneic mononuclear cells; (2) white-blood-cell (WBC)-derived soluble mediators; and/or (3) soluble HLA peptides circulating in allogeneic plasma. This review categorizes the available randomized controlled trials based on the inference(s) that they permit about possible mediator(s) of TRIM, and examines the strength of the evidence available for relying on WBC reduction or autologous transfusion to prevent TRIM effects.

Allogeneic blood transfusion (ABT) may either cause alloimmunization or induce tolerance in recipients. ABTs introduce a multitude of foreign antigens into the recipient, including HLA-DR antigens found on the donor’s dendritic antigen presenting cells (APCs). The presence or absence of recipient HLA-DR antigens on the donor’s white blood cells (WBCs) plays a decisive role as to whether alloimmunization or immune suppression will ensue following ABT. In general, allogeneic transfusions sharing at least one HLA-DR antigen with the recipient induce tolerance, while fully HLA-DR-mismatched transfusions lead to alloimmunization.

In addition to the degree of HLA-DR compatibility between donor and recipient, the immunogenicity of cellular or soluble HLA antigens associated with transfused blood components depends on the viability of the donor dendritic APCs and the presence of co-stimulatory signals for the presentation of the donor antigens to the recipient’s T cells. Nonviable APCs and/or the absence of the requisite co-stimulatory signals result in T-cell unreponsiveness.  Thus, when a multitude of antigens is introduced into the host by an ABT, the host response to some of these antigens is often decreased, and immune tolerance ensues. ABT has been shown to cause decreased helper T-cell count, decreased helper/suppressor T-lymphocyte ratio, decreased lymphocyte response to mitogens, decreased natural killer (NK) cell function, reduction in delayed-type hypersensitivity, defective antigen presentation, suppression of lymphocyte blastogenesis, decreased cytokine (IL-2, interferon-c) production, decreased monocyte/macrophage phagocytic function, and increased production of antiidiotypic and anticlonotypic antibodies.

All these laboratory immune aberrations that indicate immune suppression and occur in transfused patients could potentially be associated with clinically-manifest ABT effects. Thus a variety of beneficial or deleterious clinical effects, potentially attributable to ABT-related immunosuppression, have been described over the last 30 years. The constellation of all such ABT-associated laboratory and clinical findings is known as ABT-related immunomodulation (TRIM). Initially, TRIM encompassed effects attributable to ABT by means of immunologic mechanisms only; however more recently, the term has been used more broadly, to encompass additional effects that could be related to ABT by means of ‘‘proinflammatory’’ rather than ‘‘immunomodulatory’’ mechanisms.

Over 30 years ago, it was reported that pre-transplant ABTs could improve renal-allograft survival in patients who had undergone renal transplantation.  This beneficial immunosuppressive effect of ABT has been confirmed by animal data, observational clinical studies, and clinical experience worldwide, although it has not been proven in randomized controlled trials (RCTs). Before the advent of the AIDS pandemic, it had become standard policy in many renal units to deliberately expose patients on transplant waiting lists to one or more red blood cell (RBC) transfusions.

All the available data considered together indicate that TRIM is most likely a real biologic phenomenon, which results in at least one established beneficial clinical effect in humans, although the available evidence has not yet confirmed  the existence and/or magnitude of the deleterious clinical TRIM effects. In fact, the debate over the existence of such deleterious clinical TRIM effects has been long and sometimes acrimonious.

Many studies tended to indicate that patients receiving perioperative transfusion (compared with those not needing transfusion) almost always had a higher risk of developing postoperative bacterial infection. The studies also indicated that patients receiving ABT differed from those not receiving a transfusion in several prognostic factors that predisposed to adverse clinical outcomes.

The specific constituent(s) of allogeneic blood that mediate(s) either or both the immunomodulatory and the pro-inflammatory effect(s) of ABT remain
(s) unknown, and the published literature suggests that these TRIM effects
may be mediated by: (1) allogeneic mononuclear cells; (2) soluble biologic response modifiers released in a time dependent manner from WBC granules or membranes into the supernatant fluid of RBC or platelet concentrates
during storage; and/or  (3) soluble HLA class I peptides that circulate in allogeneic plasma. If each of these mediators do cause TRIM effects, ABT effects mediated by allogeneic mononuclear cells would be expected to be preventable by WBC reduction (performed either before or after storage of cellular blood components), as well as by autologous transfusion. The ABT effects mediated by soluble HLA peptides circulating in allogeneic plasma would be expected to be preventable only by autologous transfusion.


  1. Enhanced survival of renal allografts
  2. Reduced recurrence rate of Crohn’s disease


  1. Increased recurrence rate of resected malignancies
  2. Increased incidence of postoperative bacterial infections
  3. Activation of endogenous CMV or HIV infection
  4. Increased short-term (up to 3-month) mortality

Possible mechanisms and mediators of TRIM effects

Although the mechanisms of TRIM have been debated extensively, the exact mechanism(s) of this phenomenon has yet to be elucidated. A number of putative mechanisms have been postulated. The three major mechanisms accounting for much of the experimental data include:

  • clonal deletion,
  • induction of anergy, and
  • immune suppression.

Conceptually, clonal deletion refers to the inactivation and removal of alloreactive lymphocytes that would, for example, cause the rejection of an allograft; anergy implies immunologic nonresponsiveness; and immune suppression suggests that the responding cell is being inhibited of doing so by a cellular mechanism or by a cytokine. Antiidiotypic antibodies, which are predominantly of the VH6 gene family, have also been demonstrated in the sera of ABT recipients and in patients with long-term functioning renal allografts.

To date, no RCT has enrolled patients with sarcomas—tumors whose growth is stimulated by TGF-β—or patients with tumors for which the immune response plays a major role. (These would include skin tumors—such as melanomas, keratoacanthomas, squamous and basal-cell carcinomas—and certain virus-induced tumors—notably Kaposi’s sarcoma and certain lymphomas.) Instead, the 3 available RCTs of ABT and cancer recurrence enrolled patients with colorectal cancer—a tumor that is not sufficiently antigenic to render an impairment of host immunity capable of facilitating tumor growth, and a tumor whose cells have not been shown to be stimulated by TGF-β.

Fig not shown. Randomized controlled trials (RCTs) investigating the association of WBC-containing allogeneic blood transfusion (ABT) with cancer recurrence. For each RCT, the figure shows the odds ratio (OR) of cancer recurrence in recipients of non-WBC-reduced allogeneic versus autologous or WBC-reduced allogeneic RBCs, as calculated from an intention-to-treat analysis. A deleterious effect of ABT (and thus a benefit from autologous transfusion or WBC reduction) exists when the OR is greater than 1 as well as statistically significant. (In the figure, each OR is surrounded by its 95% confidence interval [CI]; if the 95% CI of the OR includes the null value of 1, the TRIM effect is not statistically significant [p > 0.05]).

Fig not shown. Randomized controlled trials (RCTs) investigating the association of WBC-containing allogeneic blood transfusions with postoperative infection (n = 17). For each RCT, the figure shows the odds ratio (OR) of postoperative infection in recipients of non-WBC reduced allogeneic versus autologous or WBC-reduced allogeneic RBCs, as calculated from an intention-to-treat analysis. A deleterious effect of ABT (and thus a benefit from autologous transfusion or WBC reduction) exists when the OR is greater than 1 as well as statistically significant. (In the figure, each OR is surrounded by its 95% confidence interval [CI]; if the 95% CI of the OR includes the null value of 1, the TRIM effect is not statistically significant [p > 0.05]).

The totality of the evidence from RCTs does not demonstrate a TRIM effect manifest across all clinical settings and transfused RBC products. Instead, WBC-containing ABT is associated with an increased risk of short-term (up to 3-month post transfusion) mortality from all causes combined specifically in cardiac surgery. The additional deleterious TRIM effect detected by the latest meta-analysis (i.e., the effect on postoperative infection prevented by poststorage filtration) contradicts current theories about the pathogenesis of TRIM, because it is not accompanied by a similar or larger effect prevented by prestorage filtration.

Thus, only in cardiac surgery (Fig. 5 – not shown) are the findings of RCTs pertaining to a deleterious TRIM effect consistent. Even in this setting, however, the reasons for the excess deaths attributed to WBC containing ABT remain elusive. The initial hypothesis suggested that WBC-containing ABT may predispose to MOF which, in turn, may predispose to mortality. However, hitherto, no cardiac-surgery RCT has demonstrated an association between WBC-containing ABT and MOF, and no other cause of death specifically attributed to WBC-containing ABT has been proposed.

The TRIM effect seen in cardiac surgery deserves further study to pinpoint the cause(s) of the excess deaths, but-now that the majority of transfusions in Western Europe and North America are WBC reduced- the undertaking of further RCTs comparing recipients of non-WBC-reduced versus WBC reduced allogeneic RBCs in cardiac surgery is unlikely. For countries that have not yet converted to universal WBC reduction, whether to opt for WBC reduction of all cellular blood components transfused in cardiac surgery-in the absence of information on the specific cause(s) of death ascribed to WBC-containing ABT-is a policy decision that will have to be made based on the hitherto available data.


Regulation of alveolar fluid clearance and ENaC expression in lung by exogenous angiotensin II

Jia Denga, Dao-xin Wanga, Wang Deng, Chang-yi Li, Jin Tong, Hilary Ma
Respiratory Physiology & Neurobiology 181 (2012) 53– 61

Angiotensin II (Ang II) has been demonstrated as a pro-inflammatory effect in acute lung injury, but studies of the effect of Ang II on the formation of pulmonary edema and alveolar filling remains unclear. Therefore, in this study the regulation of alveolar fluid clearance (AFC) and the expression of epithelial sodium channel (ENaC) by exogenous Ang II was verified. SD rats were anesthetized and were given Ang II with increasing doses (1, 10 and 100 [1]g/kg per min) via osmotic minipumps, whereas control rats received only saline vehicle. AT1 receptor antagonist ZD7155 (10 mg/kg) and inhibitor of cAMP degeneration rolipram (1 mg/kg) were injected intraperitoneally 30 min before administration of Ang II. The lungs were isolated for measurement of alveolar fluid clearance. The mRNA and protein expression of ENaC were detected by RT-PCR and Western blot. Exposure to higher doses of Ang II reduced AFC in a dose-dependent manner and resulted in a non-coordinate regulation of α-ENaC vs the regulation of β- and ϒ-ENaC, however Ang II type 1 (AT1) receptor antagonist ZD7155 prevented the Ang II-induced inhibition of fluid clearance and dysregulation of ENaC expression. In addition, exposure to inhibitor of cAMP degradation rolipram blunted the Ang II-induced inhibition of fluid clearance. These results indicate that through activation of AT1 receptor, exogenous Ang II promotes pulmonary edema and alveolar filling by inhibition of alveolar fluid clearance via downregulation of cAMP level and dysregulation of ENaC expression.

Effects of angiotensin II (Ang II) receptor antagonists and rolipram  on AFC

Effects of angiotensin II (Ang II) receptor antagonists and rolipram on AFC

Effects of angiotensin II (Ang II) receptor antagonists and rolipram on rat alveolar fluid clearance (AFC). Then AFC was measured 1 h after fluid instillation (4 mL/kg). Amiloride (100 [1]M), Ang II (10−7 M), ZD7155 (10−6 M), and rolipram (10−5 M) were added to the instillate as indicated (n = 10 per group). Mean values ± SEM. p < 0.01 vs control. p < 0.01 vs Ang II + ZD7155.
p < 0.05 vs amiloride. p < 0.05 vs Ang II.

Effects of angiotensin II (Ang II) on cyclic adenosine monophosphate (cAMP)

Effects of angiotensin II (Ang II) on cyclic adenosine monophosphate (cAMP)

Effects of angiotensin II (Ang II) on cyclic adenosine monophosphate (cAMP) concentration in lung. Rats were given saline or Ang II (1, 10 and 100 µg/kg per min) for 6 h, and cAMP in lung was determined by RIA (n = 30 per group). Mean values ± SEM. p < 0.01 vs control. p < 0.05 vs 10 µg/kg Ang II.

Histological examination of lung

Histological examination of lung

Histological examination of lung. Rats were given saline or Ang II (10 µg/kg per min) by osmotic minipump for 6 h. ZD7155 (10 mg/kg) was injected intraperitoneally 30 min before administration of Ang II. Shown are representative lung specimens obtained from the control (A), Ang II (B) and Ang II + ZD7155 (C) groups. All photographs are at 100× magnification. Interstitial edema and inflammatory cell infiltration were seen in Ang II group, but reduced in Ang II + ZD7155 group.
The present results demonstrate that Ang II infusion is associated with pulmonary edema and alveolar filling. Three important findings were observed:

(1) high doses of Ang II led to reduction of alveolar fluid clearance, and this effect was blunted by an AT1 receptor antagonist.
(2) Ang II infusion increased the abundance of α-ENaC, whereas decreased the abundance ofβ and ϒ-ENaC, and these effects were reversed in response to an AT1 receptor antagonist.
(3) Ang II infusion decreased cAMP concentration in lung tissue, and an inhibitor of cAMP degradation prevented inhibition of alveolar fluid clearance by Ang II, but had no effect on the dysregulation of ENaC.

Our data indicate that Ang II results in pulmonary edema by inhibition of alveolar fluid clearance via down-regulation of cellular cAMP level and dysregulation of the abundance of ENaC, whereas these effects are prevented by an AT1 receptor antagonist.

The renin-angiotensin system is a major regulator of body fluid and sodium balance, predominantly through the actions of its main effector Ang II. Several previous experimental studies demonstrated that plasma Ang II levels vary in both physiological and pathological conditions. In the kidney, Ang II added to the peritubular perfusion has a biphasic action with stimulation of sodium reabsorption at low doses (10−12–10−10M) and inhibition at high doses (10−7–10−6M) (Harris and Young, 1977). In vitro, Ang II also exerts a dose-dependent dual action on intestinal absorption (Levens, 1985). The evidence shows that the effect of Ang II on sodium and water absorption is dose-dependent. Our results showed that low intravenous doses of Ang II (<1 µg/kg per min) had no effect on alveolar fluid clearance which represents the sodium and water reabsorption in alveoli. However, with high intravenous doses, Ang II decreased alveolar fluid clearance. This finding suggests that the effect of Ang II on fluid absorption in lung is also dose-dependent.


Rat models of acute lung injury: Exhaled nitric oxide as a sensitive,noninvasive real-time biomarker of prognosis and efficacy of intervention

Fangfang Liu, Wenli Lib, Jürgen Pauluhn, Hubert Trübel, Chen Wang
Toxicology 310 (2013) 104– 114

Exhaled nitric oxide (eNO) has received increased attention in clinical settings because this technique is easy to use with instant readout. However, despite the simplicity of eNO in humans, this endpoint has not frequently been used in experimental rat models of septic (endotoxemia) or irritant acute lung injury (ALI). The focus of this study is to adapt this method to rats for studying ALI-related lung disease and whether it can serve as instant, non-invasive biomarker of ALI to study lung toxicity and pharmacological efficacy. Measurements were made in a dynamic flow of sheath air containing the exhaled breath from spontaneously breathing, conscious rats placed into a head-out volume plethysmograph. The quantity of eNO in exhaled breath was adjusted (normalized) to the physiological variables (breathing frequency, concentration of exhaled carbon dioxide) mirroring pulmonary perfusion and ventilation. eNO was examined on the instillation/inhalation exposure day and first post-exposure day in Wistar rats intratracheally instilled with lipopolysaccharide (LPS) or single inhalation exposure to chlorine or phosgene gas. eNO was also examined in a Brown Norway rat asthma model using the asthmagen toluene diisocyanate (TDI). The diagnostic sensitivity of adjusted eNO was superior to the measurements not accounting forthe normalization of physiological variables. In all bioassays – whether septic, airway or alveolar irritant or allergic, the adjusted eNO was significantly increased when compared to the concurrent control. The maximum increase of the adjusted eNO occurred following exposure to the airway irritant chlorine. The specificity of adjustment was experimentally verified by decreased eNO following inhalation dosing ofthe non-selective nitric oxide synthase inhibitor amoni-guanidine. In summary, the diagnostic sensitivity of eNO can readily be applied to spontaneously breathing, conscious rats without any intervention or anesthesia. Measurements are definitely improved by accounting for the disease-related changes inexhaled CO2and breathing frequency. Accordingly, adjusted eNO appears to be a promising methodological improvement for utilizing eNO in inhalation toxicology and pharmacological disease models
with fewer animals.


Role of p38 MAP Kinase in the Development of Acute Lung Injury

J Arcaroli, Ho-Kee Yum, J Kupfner, JS Park, Kuang-Yao Yang, and E Abraham
Clinical Immunology 2001; 101(2):211–219

Acute lung injury (ALI) is characterized by an intense pulmonary inflammatory response, in which neutrophils play a central role. The p38 mitogen-activated protein kinase pathway is involved in the regulation of stress-induced cellular functions and appears to be important in modulating neutrophil activation, particularly in response to endotoxin. Although p38 has potent effects on neutrophil functions under in vitro conditions, there is relatively little information concerning the role of p38 in affecting neutrophil driven inflammatory responses in vivo. To examine this issue, we treated mice with the p38 inhibitor SB203580 and then examined parameters of neutrophil activation and acute lung injury after hemorrhage or endotoxemia. Although p38 was activated in lung neutrophils after hemorrhage or endotoxemia, inhibition of p38 did not decrease neutrophil accumulation in the lungs or the development of lung edema under these conditions. Similarly, the increased production of proinflammatory cytokines and activation of NF-kB in lung neutrophils induced by hemorrhage or endotoxemia was not diminished by p38 inhibition. These results indicate that p38 does not have a central role
in the development of ALI after either hemorrhage or endotoxemia.


The coagulation system and pulmonary endothelial function in acute lung injury

James H. Finigan
Microvascular Research 77 (2009) 35–38

Acute lung injury (ALI) is a disease marked by diffuse endothelial injury and increased capillary permeability. The coagulation system is a major participant in ALI and activation of coagulation is both a consequence and contributor to ongoing lung injury. Increased coagulation and depressed fibrinolysis result in diffuse alveolar fibrin deposition which serves to amplify pulmonary inflammation. In addition, existing evidence demonstrates a direct role for different components of coagulation on vascular endothelial barrier function. In particular, the pro-coagulant protein thrombin disrupts the endothelial actin cytoskeleton resulting in increased endothelial leak. In contrast, the anti-coagulant activated protein C (APC) confers a barrier protective actin configuration and enhances the vascular barrier in vitro and in vivo. However, recent studies suggest a complex landscape with receptor cross-talk, temporal heterogeneity and pro-coagulant/anticoagulant protein interactions. In this article, the major signaling pathways governing endothelial permeability in lung injury are reviewed with a particular focus on the role that endothelial proteins, such as thrombin and APC, which play on the vascular barrier function.

Acute lung injury (ALI) is a devastating illness with an annual incidence of approximately 200,000 and a mortality of 40%. Most commonly seen in the setting of sepsis, ALI is a complex inflammatory syndrome marked by increased vascular permeability resulting in tissue edema and organ dysfunction. The vascular endothelium is a key target and critical participant in the pathogenesis of sepsis-induced organ dysfunction and disruption of the endothelial barrier is central to the pathophysiology of both sepsis and ALI. Sepsis and acute lung injury (ALI) are syndromes marked by diffuse inflammation with a key feature being endothelial cell barrier disruption and increased vascular permeability resulting in widespread organ dysfunction. The endothelial cytoskeleton has been identified as a critical regulator of vascular barrier integrity with a current model of endothelial barrier regulation suggesting a balance between barrier-disrupting cellular contractile forces and barrier-protective cell–cell and cell–matrix forces. These competing forces exert their opposing effects via manipulation of the actin-based endothelial cytoskeleton and associated endothelial regulatory proteins. Endothelial cells generate tension via an actomyosin motor, and focally distributed changes in tension/relaxation can be accomplished by spatially-defined regulation of the phosphorylation of the regulatory 20 kDa myosin light chain (MLC) catalyzed by the Ca2+/calmodulin (CaM)-dependent enzyme myosin light chain kinase (MLCK).

Thrombin is the proto-typical coagulation protein with direct effects on the endothelial barrier via alterations in the cytoskeleton. In the coagulation cascade, thrombin converts fibrinogen to fibrin in the final step of thrombus formation and also activated platelets. In addition, this multifunctional protease is present at sites of vascular inflammation and induces barrier dysfunction. Through its receptor, protease-activated receptor-1 (PAR1), thrombin initiates a series of events which includes MLC phosphorylation, dramatic cytoskeletal reorganization and stress fiber formation, increased cellular contractility, paracellular gap formation, and enhanced fluid and protein transport. Similarly, thrombin exposure results in increased pulmonary edema in vivo, a finding which is also seen after treatment with a PAR1 activating peptide and attenuated in PAR1 knockout mice.

Disruptions in the coagulation system have long been recognized to be an integral part of inflammation, sepsis and ALI. In 1969, Saldeen demonstrated that thrombin infusion produced canine respiratory insufficiency which was linked pathologically to emboli in the pulmonary microcirculation, a condition he labeled the “Microembolism Syndrome” (Saldeen, 1979). Elemental to the pathophysiology of sepsis and ALI is a shift towards a pro-coagulant state. Bronchoalveolar (BAL) fluid from patients with ALI reflects this increase in procoagulant activity with elevated levels of fibrinopeptide A, factor VII and d-dimer. Concomitantly, there is a decrease in fibrinolytic activity, as shown by depressed BAL levels of urokinase and increased levels of the fibrinolysis inhibitors plasminogen activator inhibitor (PAI) and α2-antiplasmin.

Given that APC is a vascular endothelial protein which interacts with other coagulation proteins such as thrombin, it seems logical that it might have an effect on endothelial integrity. In cultured human pulmonary endothelial cells, while thrombin results in decreased electrical resistance, a reflection of increased permeability, pre- or post-exposure to physiologic concentrations of APC significantly attenuates this thrombin-induced drop in resistance. These APC-mediated alterations in barrier function are associated with MLC phosphorylation as well as activation of the endothelial protein Rac, and cytoskeletal re-arrangement in a barrier protective configuration all findings very reminiscent of the barrier protective signaling induced by the bioactive lipid, S1P. Interestingly, APC appears to activate sphingosine kinase and mediate its barrier protective effects through PI3 kinase and AKT-dependent ligation of the S1P receptor, S1P1. Moreover, the endothelial barrier-protective effects of APC have been observed in other tissues including brain and kidney. The barrier protection in these beds appears independent of any anti-coagulant effect of APC and is associated with decreased endothelial apoptosis.

Recently, the endothelial protein C receptor (EPCR) has been identified as a crucial participant in the protein C pathway. Structurally similar to the major histocompatibility class I/CD1 family of molecules, EPCR binds protein C, presenting it to the thrombin/TM complex, thereby increasing the activation of protein C by ∼20 fold. Importantly, APC can also bind EPCR, and while the bound form of APC loses its extra-cellular anti-coagulant activity, increasing evidence indicates that much, if not all, of APC intra-cellular signaling requires EPCR. APC-mediated increases in endothelial phosphor-MLC and activated Rac are all EPCR-dependent and APC-induced endothelial barrier protection requires ligation of EPCR.

Sepsis and ALI are significant causes of morbidity and mortality in the intensive care unit and are marked by zealous activation of the coagulation system. While this could conceivably confer certain benefits, such as enclosing and spatially controlling an infection, it is clear that this pro-coagulant environment participates in the pathophysiology of ALI, particularly via exacerbating endothelial damage and augmenting endothelial permeability. However, the biology of coagulation in ALI is incompletely understood and trials of new therapies specifically targeting coagulation in patients with ALI have been disappointing. Despite this, recent advances in the knowledge of the dynamic interplay between inflammation and coagulation in ALI as well as endothelial receptor-ligand binding and receptor cross talk have stimulated promising research and identified novel therapeutic targets for patients with ALI.


Phosphatidylserine-expressing cell by-products in transfusion: A pro-inflammatory or an anti-inflammatory effect?

  1. Saas, F. Angelot, L. Bardiaux, E. Seilles, F. Garnache-Ottou, S. Perruche
    Transfusion Clinique et Biologique 19 (2012) 90–97

Labile blood products contain phosphatidylserine-expressing cell dusts, including apoptotic cells and microparticles. These cell by-products are produced during blood product process or storage and derived from the cells of interest that exert a therapeutic effect (red blood cells or platelets). Alternatively, phosphatidylserine-expressing cell dusts may also derived from contaminating cells, such as leukocytes, or may be already present in plasma, such as platelet-derived microparticles. These cell by-products present in labile blood products can be responsible for transfusion induced immunomodulation leading to either transfusion-related acute lung injury (TRALI) or increased occurrence of post-transfusion infections or cancer relapse. In this review, we report data from the literature and our laboratory dealing with interactions between antigen-presenting cells and phosphatidylserine-expressing cell dusts, including apoptotic leukocytes and blood cell-derived microparticles. Then, we discuss how these phosphatidylserine-expressing cell by-products may influence transfusion.

Potential consequences of phosphatidylserine-expressing cell by-products in transfusion

Potential consequences of phosphatidylserine-expressing cell by-products in transfusion

Potential consequences of phosphatidylserine-expressing cell by-products in transfusion. Interactions of phosphatidylserine-expressing cell dusts (apoptotic cells or microparticles) may lead to antigen-presenting cell activation or inhibition. Antigen-presenting cell activation may trigger inflammation and be involved in transfusion-related acute lung injury (TRALI), while antigen-presenting cell inhibition may exert transient immunosuppression or tolerance. Blood product process or storage may influence the generation of phosphatidylserine-expressing cell dusts. PtdSer: phosphatidylserine; APC: antigen-presenting cell.

Several publications report the presence of phosphatidylserine-expressing cell by-products in blood products. These cell by-products may be generated during the blood product process, such as filtration, or during storage (either cold storage for red blood cells or between 20–24 ◦C for platelets). Alternatively, they may be limited by filtration. Phosphatidylserine-expressing cell by-products can be apoptotic cells. Apoptotic cells have been found in different blood products: red blood cell units and platelet concentrates. These apoptotic cells correspond to dying cells of interest: red blood cells or platelets, both enucleated cells that can undergo apoptosis.

Immunomodulatory effects of apoptotic leukocytes

Immunomodulatory effects of apoptotic leukocytes

Immunomodulatory effects of apoptotic leukocytes. Early during the apoptotic program, phosphatidylserine-exposure occurs leading to apoptotic cell removal by macrophages or conventional dendritic cells. This uptake by antigen-presenting cells induces the production of anti-inflammatory factors and concomitantly inhibits the synthesis of inflammatory cytokines. These antigen-presenting cells are refractory to TLR activation. This leads to a transient immunosuppressive microenvironment. If antigen-presenting cells from this microenvironment migrate to secondary lymphoid organs, naive T cells are converted into inducible regulatory T cells. This leads to tolerance against apoptotic cell-derived antigens. M[1]: macrophage; cDC: conventional dendritic cells; PtdSer: phosphatidylserine; Treg: regulatory T cells; Th1: helper T cells; HGF: hepatocyte growth factor; IL-: interleukin; NO: nitrite oxide; PGE-2: prostaglandin-E2; TGF: transforming growth factor; TNF: tumor necrosis factor; TLR: Toll-like receptor.

Implication of phosphatidylserine in the inhibition of both inflammation and specific immune responses has been further demonstrated using  phosphatidylserine-expressing liposomes and is sustained by the following observations:

  • phosphatidylserine-dependent ingestion of apoptotic cells induces TGF-β secretion and resolution of lung inflammation;
  • inhibition of phosphatidylserine recognition through annexin-V enhances the immunogenicity of irradiated tumor cells in vivo;
  • masking of phosphatidylserine inhibits apoptotic cell engulfment and induces autoantibody production in mice.

Based on data from our group and Peter Henson’s group, some authors have speculated that apoptotic leukocytes present in blood products may be responsible for transfusion-related immunosuppression.

The first consequences of phosphatidylserine-expressing apoptotic cells in blood products may be a transient immunosuppression−responsible for an increase in infection rate and of cancer relapse−or tolerance induction− as observed after donor-specific transfusion − when Treg have been generated. However, apoptotic leukocytes become secondarily necrotic in the absence of phagocytes. This may certainly occur in blood product bags. Necrotic cells, through the release of damage-associated molecular patterns, may become immunogenic. The same process may occur for platelets. Necrotic platelets may represent the procoagulant form of platelets. Thus, hemostatic activation of platelets or their by-products may link thrombosis and inflammation to amplify lung microvascular damage during nonimmune TRALI.

What are the next steps to answer the question on the role of phosphatidylserine-expressing cell dusts in the modulation of immune responses after transfusion?

The next steps are to characterize or identify factors involved in the triggering of inflammation or its inhibition and produced during blood product storage or process. Several factors influence the immune responses against dying cells. We can speculate on some factors, including:

  • the number of phosphatidylserine-expressing cell byproducts contained per blood product, as the immunogenicity of apoptotic cells may be proportional to their number;
  • the occurrence of secondary necrosis and so the passive release of intracellular damage-associated molecular patterns that overpasses the inhibitory signals delivered by phosphatidylserine. One of these damage associated molecular patterns can be the heme released from stored red blood cells which signals via TLR4;
  • the size of cell by-products and especially microparticles, since these latter exert different functions according to their size. Moreover, antigen-presenting cells, such as plasmacytoid dendritic cells, respond only to lower size synthetic particles. This may explain the different responses observed between “amateur” phagocytes (plasmacytoid dendritic cells) versus professional phagocytes (conventional dendritic cells/macrophages) after incubation with microparticles. The size of cell by-products diminishes during plasma filtration, as assessed by dynamic light scattering from 101 to 464 nm in unfiltered fresh-frozen plasma versus 21 to 182 nm after 0.2 µm filtration process;
  • expression of the recently described phosphatidylserine receptors on different antigen-presenting cell subsets may also explain the different responses between plasmacytoid dendritic cells versus conventional dendritic cells/macrophages and may impact on the overall immune response.


Peroxisome proliferator-activated receptors and inflammation

Leonardo A. Moraes, Laura Piqueras, David Bishop-Bailey
Pharmacology & Therapeutics 110 (2006) 371 – 385

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptors family. PPARs are a family of 3 ligand-activated transcription factors: PPARa (NR1C1), PPARh/y (NUC1; NR1C2), and PPARg (NR1C3). PPARα, -h/y, and -ϒ are encoded by different genes but show substantial amino acid similarity, especially within the DNA and ligand binding domains. All PPARs act as heterodimers with the 9-cis-retinoic acid receptors (retinoid X receptor; RXRs) and play important roles in the regulation of metabolic pathways, including those of lipid of biosynthesis and glucose metabolism, as well as in a variety of cell differentiation, proliferation, and apoptosis pathways. Recently, there has been a great deal of interest in the involvement of PPARs in inflammatory processes. PPAR ligands, in particular those of PPARα and PPARϒ, inhibit the activation of inflammatory gene expression and can negatively interfere with proinflammatory transcription factor signaling pathways in vascular and inflammatory cells. Furthermore, PPAR levels are differentially regulated in a variety of inflammatory disorders in man, where ligands appear to be promising new therapies.

Fig. not shown.  Structure and transcriptional activation of PPARs. (A) Generic schematic of the structure of the PPAR family of nuclear receptors. Indicated are the N–C terminal regions subdivided in to 4 domains: the A/B, N terminal domain [also called the activation function (AF)-1 domain]; C, the DNA binding domain; D, the F hinge_region; and E, the ligand binding domain (AF-2). (B) Generic scheme for the activation of a PPAR receptor as a transcription factor. PPAR activation leads to heterodimerization with RXR and an accumulation in the nucleus. Ligand activation of PPAR results in a change from a repressed binding protein complex which may contain histone deacetylases (HDAC), the nuclear receptor corepressor (NCo-R), and the silencing mediator of retinoid and thyroid signaling (SMRT) to an activation complex that may contain the histone acetylases, steroid receptor co-activator-1 (SRC-1), the PPAR binding protein (PBP), cAMP response element binding protein (CBP/p300), TATA box binding proteins, and RNA polymerase (RNA pol) III. The activated PPAR–RXR heterodimer complex binds to DNA sequences called PPAR response elements (PPRE) in target genes initiation their transcription.

Although the nature of true endogenous PPAR ligands are still not known (Bishop-Bailey & Wray, 2003), PPARs can be activated by a wide variety of F endogenous or pharmacological ligands. PPARα activators include a variety of endogenously present fatty acids, LTB4 and hydroxyeicosatetraenoic acids (HETEs), and clinically used drugs, such as the fibrates, a class of first-line drugs in the treatment of dyslipidemia. Similarly, PPARg can be activated by a number of ligands, including docosahexaenoic acid, linoleic acid, the anti-diabetic glitazones, used as insulin sensitizers, and a number of lipids, including oxidized LDL, azoyle-PAF, and eicosanoids, such as 5,8,11,14-eicosatetraynoic acid and the prostanoids PGA1, PGA2, PGD2, and its dehydration products of the PGJ series of cyclopentanones (e.g., 15 deoxy-D12,14-PGJ2). Dyslipidemia and insulin-dependent diabetes are commonly found existing together as part of the metabolic X syndrome.

Because PPARa and PPARg ligands independently are useful clinical drugs in the treatment of these respective disorders, synthetic dual PPARα/ϒ ligands have recently been developed and show a combined clinical efficacy. PPAR h/y activators include fatty acids and prostacyclin and synthetic compounds L-165,041, GW501516, compound F and L-783,483. Unlike PPARα or-ϒ, there are no PPAR h/y drugs in the clinic, although ligands are in phase II clinical trials for dyslipidemia (http://www.science.gsk.com/pipeline). Indeed, part of the challenge in determining the function of PPARh/y has been the identification and availability of new ligands with more potency and selectivity for use as pharmacological tools.

Fig. not shown. Mechanisms of the anti-inflammatory effects of PPARα. PPARα ligands inhibit the activities of NF-nB, AP-1, and T-bet within cells. In sites of local inflammation, tissue and endothelial cell activity is inhibited, and expressions of adhesion molecules (ICAM-1 and VCAM-1), pro-inflammatory cytokines (IL-1, -6, -8, -12, and TNFα), vasoactive mediators (inducible cyclo-oxygenase, inducible nitric oxide synthase, and endothelin-1; COX-2, iNOS, and ET-1), and proteases (MMP-9) are decreased. The inflammatory responses in leukocytes are also diminished. Monocyte/macrophage activity is decreased, and lipid metabolizing pathways increased, T- and B-lymphocyte proliferation and differentiation are inhibited, and T-lymphocyte and eosinophil chemotaxis reduced. Bold italic text indicates positive regulation by the PPAR, all other text indicates a negative regulation.

Fig. not shown. Mechanisms of the anti-inflammatory effects of PPAR h/y. PPAR h/y ligands inhibit the activities of NF-nB and release the suppressor BCL-6 from PPAR h/y. In sites of local inflammation, endothelial cell adhesion molecule (VCAM-1) and chemokine (MCP-1) are reduced. PPAR h/y and its endogenous ligand(s) are induced during the inflammatory response in keratinocytes, which then promotes cell survival (integrin-linked kinase—Akt pathway) and wound healing. The inflammatory responses in monocyte/ macrophages are modulated. In the absence of ligand, PPAR h/y sequesters BCL-6 and induces MCP-1, MCP-3, and IL-1h. When PPAR h/y ligand is given, BCL-6 is released and MCP-1, -3, and IL-1h levels are reduced. Bold italic text indicates positive regulation by the PPAR, all other text indicates a negative regulation.

Fig. not shown. Mechanisms of the anti-inflammatory effects of PPARg. PPARg ligands can inhibit the activities of NF-nB, AP-1, STAT-1, N-FAT, Erg-1, Jun, and GATA-3 within cells. In sites of local inflammation, tissue and endothelial cell activity is inhibited, and expression of adhesion molecules (ICAM-1), proinflammatory cytokines (IL-8, -12, and TNFα), chemokines (MCP-1, MCP-3, IP-10, Mig, and I-TAC), vasoactive mediators (inducible nitric oxide synthase and endothelin-1; iNOS and ET-1), and proteases (MMP-9) are decreased. The inflammatory responses in leukocytes are also diminished. Monocyte/ macrophage activity is decreased, T- and B-lymphocyte proliferation and differentiation are inhibited, and T-lymphocyte and eosinophil chemotaxis reduced. Platelet activity is inhibited and dendritic cell production of IL-12, and expression of CCL3, CCL5, and CD80 is reduced, so pro-inflammatory TH1 lymphocytes maturation is inhibited. Bold italic text indicates positive regulation by the PPAR, all other text indicates a negative regulation.

The PPARs are one of the most intensely studied members of the nuclear receptor gene family, and since their initial discovery just over decade ago, the PPARs have attracted an increasing amount of experimental and clinical research by investigators from different scientific areas. PPARs through their central roles in regulating energy homeostasis regulate physiological function in many cell types, tissues, and organ systems. Many disease states from carcinogenesis to inflammation have been linked to abnormalities in the function of PPAR-regulated transcription factors. PPARs are expressed or regulate pathophysiology of diverse human disorders including atherosclerosis, inflammation, obesity, diabetes, and the immune response. PPARs have beneficial effects in many inflammatory conditions, where they regulate cytokine production, adhesion molecule expression, fibrinolysis cell proliferation, apoptosis, and differentiation. Further studies and development of novel PPAR ligands and their selective modulators may lead to novel therapeutic agents in the many conditions associated with inflammatory processes.


Regulators of endothelial and epithelial barrier integrity and function in acute lung injury

Rudolf Lucas, Alexander D. Verin, Stephen M. Black, John D. Catravas
Biochemical Pharmacology 77 (2009) 1763–1772

Pulmonary permeability edema is a major complication of acute lung injury (ALI), severe pneumonia and ARDS. This pathology can be accompanied by

(1) a reduction of alveolar liquid clearance capacity, caused by an inhibition of the expression of crucial sodium transporters, such as the epithelial sodium channel (ENaC) and the Na+-K+-ATPase,
(2) an epithelial and endothelial hyperpermeability and
(3) a disruption of the epithelial and endothelial barriers, caused by increased apoptosis or necrosis.

Since, apart from ventilation strategies, no standard treatment exists for permeability edema, the following chapters will review a selection of novel approaches aiming to improve these parameters in the capillary endothelium and the alveolar epithelium.

Apoptosis is an essential physiological process for the selective elimination of cells. However, the dysregulation of apoptotic pathways is thought to play an important role in the pathogenesis of ALI. Both delayed neutrophil apoptosis and enhanced endothelial/epithelial cell apoptosis have been identified in ALI/ARDS. In the case of neutrophils, which contribute significantly to ALI/ ARDS, studies in both animals and ARDS patients suggest that apoptosis is inhibited during the early stages (<2 h) of inflammation.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily, that includes receptors for steroid hormones, thyroid hormones, retinoic acid, and fat-soluble vitamins. Since their discovery in 1990, increasing data has been published on the role of PPARs in diverse processes, including lipid and glucose metabolism, diabetes and obesity, atherosclerosis, cellular proliferation and differentiation, neurological diseases, inflammation and immunity. PPARs have both gene-dependent and gene-independent effects. Gene-dependent functions involve the formation of heterodimers with the retinoid X-receptor. Activation by PPAR ligands results in the binding of the heterodimer to peroxisome proliferator response elements, located in the promoter regions of PPAR-regulated genes. Gene independent effects involve the direct binding of PPARs to transcription factors, such as NF-kB, which then alters their binding to DNA promoter elements. PPARs can also bind and sequester various cofactors for transcription factors, and thus further alter gene expression. Importantly, the precise effects of PPARs vary greatly between cell types. To date, three subtypes of PPAR have been identified: α, β, and ϒ. There is increasing data suggesting that PPAR signaling may play an important role in the pathobiology of systemic vascular disease. However, there is less data implicating PPAR signaling in diseases of the lung.

A role for PPARs in the control of inflammation was first evidenced for PPARα, where mice deficient in PPARα exhibited an increased duration of ear-swelling in response to the proinflammatory mediator, LTB4. More recently, a number of studies in mice and in humans have shown that PPAR agonists exhibit anti-inflammatory effects under a wide range of conditions. There are two main mechanisms by which PPARs exert their anti-inflammatory effect. The first involves complex formation, and the inhibition of transcription factors that positively regulate the transcription of pro-inflammatory genes. These include nuclear factor-kB (NF-kB), signal transducers and activators of transcription (STATs), nuclear factor of activated T cells (NF-AT), CAAT/enhancer binding protein (C/EBP) and activator protein 1 (AP-1). These transcription factors are the main mediators of the major proinflammatory cytokines, chemokines, and adhesion molecules involved in inflammation. The second PPAR-mediated anti-inflammatory pathway is mediated by the sequestration of rate limiting, but essential, co-activators or co-repressors.

Recent studies have shown that PPAR signaling can attenuate the airway inflammation induced by LPS in the mouse. It was shown that mice treated with the PPARα agonist, fenofibrate, had decreases in both inflammatory cell infiltration and inflammatory mediators. Conversely, PPARα -/- mice have been shown to have a greater number of neutrophils and macrophages, and increased levels of inflammatory mediators in bronchoalveolar lavage fluids (BALF). Other PPAR agonists, such as rosiglitazone or SB 21994 have also been shown to reduce LPS-mediated ALI in the mouse lung. PPARϒ signaling has also been shown to be protective in regulating pulmonary inflammation associated with fluorescein isothiocyanate (FITC)-induced lung injury, with the PPARϒ ligand pioglitazone decreasing neutrophil infiltration. Collectively, these data suggest that therapeutic agents that activate either or both PPARα and PPARϒ could be beneficial for the treatment of ALI.

Permeability edema is characterized by a reduced alveolar liquid clearance capacity, combined with an endothelial hyperpermeability. Various signaling pathways, such as those involving reactive oxygen species (ROS), Rho GTPases and tyrosine phosphorylation of junctional proteins, converge to regulate junctional permeability, either by affecting the stability of junctional proteins or by modulating their interactions. The regulation of junctional permeability is mainly mediated by dynamic interactions between the proteins of the adherens junctions and the actin cytoskeleton. Actin-mediated endothelial cell contraction is the result of myosin light chain (MLC) phosphorylation by MLC kinase (MLCK) in a Ca2+/calmodulin-dependent manner. RhoA additionally potentiates MLC phosphorylation, by inhibiting MLC phosphatase activity through its downstream effector Rho kinase (ROCK). As such, actin/myosin-driven contraction will generate a contractile force that pulls VE-cadherin inward. This contraction will force VE-cadherin to dissociate from its adjacent partner, as such producing interendothelial gaps.

Vascular endothelial cells can be regulated by nucleotides released from platelets. During vascular injury, broken cells are also the source of the extracellular nucleotides. Furthermore, endothelium may provide a local source of ATP within vascular beds. Primary cultures of human endothelial cells derived from multiple blood vessels release ATP constitutively and exclusively across the apical membrane under basal conditions. Hypotonic challenge or the calcium agonists (ionomycin and thapsigargin) stimulate ATP release in a reversible and regulated manner. Enhanced release of pharmacologically relevant amounts of ATP was observed in endothelial cells under such stimuli as shear stress, lipopolysaccharide (LPS), and ATP itself. Pearson and Gordon demonstrated that incubation of aortic endothelial and smooth muscle cells with thrombin resulted in the specific release of ATP, which was converted to ADP by vascular hydrolases. Yang et al. showed that endothelial cells isolated from guinea pig heart release nucleotides in response to bradykinin, acetylcholine, serotonin and ADP. Nucleotide action is mediated by cell surface purinoreceptors. Once released from endothelial cells, ATP may act in the blood vessel lumen at P2 receptors on nearby endothelium downstream from the site of release. ATP is also degraded rapidly and its metabolites have also been recognized as signaling molecules, which can initiate additional receptor-mediated functions. These include ADP and the final hydrolysis product adenosine.

Signal transduction pathways implicated in ATP-mediated endothelial barrier enhancement

Signal transduction pathways implicated in ATP-mediated endothelial barrier enhancement

Signal transduction pathways implicated in ATP-mediated endothelial barrier enhancement

During the course of ALI, the alveolar space, as well as the interstitium, are sites of intense inflammation, leading to the local production of pro-inflammatory cytokines, such as IL-1β, TGF-β and TNF. The latter pleiotropic cytokine is a 51 kDa homotrimeric protein, binding to two types of receptors, i.e. TNF-R1 and TNF-R2 and which is mainly produced by activated macrophages and T cells. Soluble TNF, as well as the soluble TNF receptors 1 and 2, are generated upon cleavage of membrane TNF or of the membrane associated receptors, respectively, by the enzyme TNF-α convertase (TACE). TNF-R1, but not TNF-R2, contains a death domain, which signals apoptosis upon the formation of the Death Inducing Signaling Complex (DISC). In spite of its lack of a death domain, TNF-R2 can nevertheless be implicated in apoptosis induction, since its activation causes degradation of TNF Receptor Associated Factor 2 (TRAF2), an inhibitor of the TNF-R1-induced DISC formation. Moreover, apoptosis induction of lung microvascular endothelial cells by TNF was shown to require activation of both TNF receptors. TNF-R2 was also shown to be important for ICAM-1 upregulation in endothelial cells in vitro and in vivo, an activity important in the sequestration of leukocytes in the microvessels. Moreover, lung microvascular endothelial cells isolated from ARDS patients express significantly higher levels of TNF-R2 and of ICAM-1 than cells isolated from patients who had undergone a lobectomy for lung carcinoma, used as controls. These findings therefore suggest that ICAM-1 and TNF-R2 may have a particular involvement in the pathogenesis of acute lung injury.

Dichotomous activity of TNF in alveolar liquid clearance and barrier protection

Dichotomous activity of TNF in alveolar liquid clearance and barrier protection

Dichotomous activity of TNF in alveolar liquid clearance and barrier protection during ALI. TNF, which is induced during ALI, causes a downregulation of ENaC expression in type II alveolar epithelial cells, upon activating TNF-R1. Moreover, TNF increases permeability, by means of interfering with tight junctions (TJ) in both alveolar epithelial (AEC) and capillary endothelial cells (MVEC). ROS, the generation of which is frequently increased during ALI, were also shown to downregulate ENaC and Na+-K+-ATPase expression and moreover also lead to decreased endothelial barrier integrity. The TIP peptide, mimicking the lectin-like domain of TNF, is able to increase sodium uptake in alveolar epithelial cells and to restore endothelial barrier integrity, as such providing a significant protection against the development of permeability edema (red lines: inhibition, green arrows: activation).

Proposed mechanism of action for the anti-inflammatory and barrier-protective actions of hsp90 inhibitors.

Proposed mechanism of action for the anti-inflammatory and barrier-protective actions of hsp90 inhibitors.

Proposed mechanism of action for the anti-inflammatory and barrier-protective actions of hsp90 inhibitors.

Permeability edema represents a life-threatening complication of acute lung injury, severe pneumonia and ARDS, characterized by a combined dysregulation of pulmonary epithelial and endothelial apoptosis, endothelial barrier integrity and alveolar liquid clearance capacity. As such, it is likely that several of these parameters have to be targeted in order to obtain a successful therapy. This review focuses on a selection of recently discovered substances and mechanisms that might improve ALI therapy. As such, we have discussed the inhibition of apoptosis and necrosis occurring during ALI, by means of the restoration of Zn2+ homeostasis. PPARα and ϒ agonists can represent therapeutically  promising molecules, since they inhibit transcription factors as well as essential co-activators involved in the activation of pro-inflammatory cytokines, chemokines and adhesion molecules, all of which are implicated in ALI. Apart from inducing a potent inhibition of inflammation upon interfering with NF-kB activation, hsp90 inhibitors were shown to prevent and restore endothelial barrier integrity. These agents are able to significantly improve survival and lung function during LPS-induced ALI. A restoration of endothelial barrier integrity during ALI can also be obtained upon increasing extracellular levels of ATP or adenosine, which activate the purinoreceptors P2Y and P1A2, respectively, leading to a decrease in myosin light chain phosphorylation and an increase in MLC phosphatase 1 activity. The pro-inflammatory cytokine TNF is involved in endothelial apoptosis and hyperpermeability, as well as in the reduction of alveolar liquid clearance, upon activating its receptors. However, apart from its receptor binding sites, TNF harbors a lectin-like domain, which can be mimicked by the TIP peptide. This peptide has been shown to increase alveolar liquid clearance and moreover induces endothelial barrier protection. As such, TNF can be considered as a moonlighting cytokine, combining both positive and negative activities for permeability edema generation within one molecule.


The protective effect of CDDO-Me on lipopolysaccharide-induced acute lung injury in mice

Tong Chen, Yi Moua, Jiani Tan, LinlinWei, Yixue Qiao, Tingting Wei, et al.
International Immunopharmacology 25 (2015) 55–64

ALI is a clinical syndrome characterized by a disruption of epithelial integrity, neutrophil accumulation, noncardiogenic pulmonary edema, severe hypoxemia and an intense pulmonary inflammatory response with a wide array of increasing severity of lung parenchymal injury. Previous studies have shown that lots of pathogenesis contribute to ALI, such as oxidant/antioxidant dysfunction, dysregulation of inflammatory/anti-inflammatory pathway, upregulation of chemokine production and adhesion molecules. However, to date there is no effective medicine to control ALI. Lipopolysaccharide (LPS) is a main component of the outer membrane of Gram negative bacteria. It has been reported to activate toll like receptors 4 (TLR4) and to stimulate the release of inflammatory mediators inducing ALI-like symptoms. Intratracheal administration of LPS has been used to construct animal models of ALI.

The biological importance of naturally occurring triterpenoids has long been recognized. Oleanolic acid, exhibiting modest biological activities, has been marketed in China as an oral drug for the treatment of liver disorders in humans. Among its derivatives, bardoxolonemethyl (2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methylester) CDDO-Me, had completed a successful phase I clinical trial for the treatment of cancer and started a phase II trial for the treatment of patients with pulmonary arterial hypertension. For its broad spectrum antiproliferative and anti-tumorigenic activities, CDDO-Me has also been reported to possess a number of pharmacological activities such as antioxidant, anti-tumor and anti-inflammatory effects. However, the mechanisms by which CDDO-Me exerted its anti-inflammatory effects on macrophage were insufficiently elucidated. More importantly, there is no available report to evaluate its therapeutic effect on acute lung injury.

CDDO-Me, initiated in a phase II clinical trial, is a potential useful therapeutic agent for cancer and inflammatory dysfunctions, whereas the therapeutic efficacy of CDDO-Me on LPS-induced acute lung injury (ALI) has not been reported as yet. The purpose of the present study was to explore the protective effect of CDDO-Me on LPS-induced ALI in mice and to investigate its possible mechanism. BalB/c mice received CDDO-Me (0.5 mg/kg, 2 mg/kg) or dexamethasone (5 mg/kg) intraperitoneally 1 h before LPS stimulation and were sacrificed 6 h later. W/D ratio, lung MPO activity, number of total cells and neutrophils, pulmonary histopathology, IL-6, IL-1β, and TNF-α in the BALF were assessed. Furthermore, we estimated iNOS, IL-6, IL-1β, and TNF-α mRNA expression and NO production as well as the activation of the three main MAPKs, AkT, IκB-α and p65. Pretreatment with CDDO-Me significantly ameliorated W/D ratio, lung MPO activity, inflammatory cell infiltration, and inflammatory cytokine production in BALF from the in vivo study. Additionally, CDDO-Me had beneficial effects on the intervention for pathogenesis process at molecular, protein and transcriptional levels in vitro. These analytical results provided evidence that CDDO-Me could be a potential therapeutic candidate for treating LPS-induced ALI.

Effects of CDDO-Me on LPS-mediated lung changes

Effects of CDDO-Me on LPS-mediated lung histopathologic changes in lung tissues. (A) The lung section from the control mice; (B) the lung section from the mice administered with LPS (8 mg/kg); (C) the lung section from the mice administered with dexamethasone (5 mg/kg) and LPS (8 mg/kg); (D) the lung section from the mice administered with CDDO-Me (0.5mg/kg) and LPS (8mg/kg); (E) the lung section from the mice administered with CDDO-Me (2mg/kg) and LPS (8mg/kg); (hematoxylin and eosin staining, magnification 200×). Control group: the green arrow indicated alveolar wall, no hyperemia. All the other groups: The black arrow indicated the inflammatory cell infiltration; the green arrow indicated alveolar wall hyperemia.


The impact of cardiac dysfunction on acute respiratory distress syndrome and mortality in mechanically ventilated patients with severe sepsis and septic shock: An observational study

Brian M. Fuller, Nicholas M. Mohr, Thomas J. Graetz, et al.
Journal of Critical Care 30 (2015) 65–70

Purpose: Acute respiratory distress syndrome (ARDS) is associated with significant mortality and morbidity in survivors. Treatment is only supportive, therefore elucidating modifiable factors that could prevent ARDS could have a profound impact on outcome. The impact that sepsis-associated cardiac dysfunction has on ARDS is not known. Materials and Methods: In this retrospective observational cohort study of mechanically ventilated patients with severe sepsis and septic shock, 122 patients were assessed for the impact of sepsis-associated cardiac dysfunction on incidence of ARDS (primary outcome) and mortality. Results: Sepsis-associated cardiac dysfunction occurred in 44 patients (36.1%). There was no association of sepsis-associated cardiac dysfunction with ARDS incidence (p= 0.59) or mortality, and no association with outcomes in patients that did progress to ARDS after admission. Multivariable logistic regression demonstrated that higher BMI was associated with progression to ARDS (adjusted OR 11.84, 95% CI 1.24 to 113.0, p= 0.02). Conclusions: Cardiac dysfunction in mechanically ventilated patients with sepsis did not impact ARDS incidence, clinical outcome in ARDS patients, or mortality. This contrasts against previous investigations demonstrating an influence of nonpulmonary organ dysfunction on outcome in ARDS. Given the frequency of ARDS as a sequela of sepsis, the impact of cardiac dysfunction on outcome should be further studied.


Suppression of NF-κβ pathway by crocetin contributes to attenuation of lipopolysaccharide-induced acute lung injury in mice

Ruhui Yang, Lina Yang, Xiangchun Shen, Wenyuan Cheng, et al.
European Journal of Pharmacology 674 (2012) 391–396

Crocetin, a carotenoid compound, has been shown to reduce expression of inflammation and inhibit the production of reactive oxygen species. In the present study, the effect of crocetin on acute lung injury induced by lipopolysaccharide (LPS) was investigated in vivo. In the mouse model, pretreatment with crocetin at dosages of 50 and 100 mg/kg reduced the LPS-induced lung edema and histological changes, increased LPS-impaired superoxide dismutase (SOD) activity, and decreased lung myeloperoxidase (MPO) activity. Furthermore, treatment with crocetin significantly attenuated LPS-induced mRNA and the protein expressions of interleukin-6 (IL-6), macrophage chemoattractant protein-1 (MCP-1), and tumour necrosis factor-α (TNF-α) in lung tissue. In addition, crocetin at different dosages reduced phospho-IκB expression and NF-κB activity in LPS-induced lung tissue alteration. These results indicate that crocetin can provide protection against LPS-induced acute lung injury in mice.


Sauchinone, a lignan from Saururus chinensis, attenuates neutrophil pro-inflammatory activity and acute lung injury

Hui-Jing Han, Mei Li, Jong-Keun Son, Chang-Seob Seo, et al.
International Immunopharmacology 17 (2013) 471–477

Previous studies have shown that sauchinone modulates the expression of inflammatory mediators through mitogen-activated protein kinase (MAPK) pathways in various cell types. However, little information exists about the effect of sauchinone on neutrophils, which play a crucial role in inflammatory process such as acute lung injury (ALI). We found that sauchinone decreased the phosphorylation of p38 MAPK in lipopolysaccharide (LPS)-stimulated murine bone marrow neutrophils, but not ERK1/2 and JNK. Exposure of LPS-stimulated neutrophils to sauchinone or SB203580, a p38 inhibitor, diminished production of tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2 compared to neutrophils cultured with LPS. Treatment with sauchinone decreased the level of phosphorylated ribosomal protein S6 (rpS6) in LPS-stimulated neutrophils. Systemic administration of sauchinone to mice led to reduced levels of phosphorylation of p38 and rpS6 in mice lungs given LPS, decreased TNF-α and MIP-2 production in bronchoalveolar lavage fluid, and also diminished the severity of LPS-induced lung injury, as determined by reduced neutrophil accumulation in the lungs, wet/dry weight ratio, and histological analysis. These results suggest that sauchinone diminishes LPS-induced neutrophil activation and ALI.

In the present study, the systemic administration of sauchinone decreased the phosphorylation of p38 MAPK and rpS6 in mice lungs subjected to LPS and diminished the severity of LPS-induced ALI. Neutrophils play an important role in acute inflammatory processes, such as ALI, which was demonstrated by various experimental models. Previous reports suggested that p38 MAPK inhibition of murine neutrophils could lead to the loss of chemotaxis toward MIP-2, as well as the loss of TNF-αandMIP-2 production in response to LPS, and also attenuated neutrophil accumulation in LPS-induced ALI models. Therefore, the beneficial effects of sauchinone on LPS-induced ALI are likely associated with decreases in the production of pro-inflammatory mediators by neutrophils, consistent with our in vitro experiments. However, we cannot exclude that the effects of sauchinone on reducing the release of TNF-α and MIP-2 in mice lungs subjected to LPS, with the resultant prevention of ALI, could be affected by various pulmonary cell populations, such as alveolar macrophages. Also, the inhibitory effects of sauchinone on NF-κB activation through various pulmonary cell populations (Supplemental Fig. S2), in addition to p38MAPK activity in mouse lungs given LPS, might enhance the anti-inflammatory action of sauchinone in mouse lungs subjected to LPS. In conclusion, we found that sauchinone significantly diminished the release of inflammatory mediators in isolated neutrophils and lungs subjected to LPS. The anti-inflammatory action of sauchinone was associated with the prevention of p38 MAPK and rpS6 activation. These findings suggest that sauchinone may be an appropriate pharmacological candidate for the treatment of ALI as well as other neutrophil driven acute inflammatory diseases.
Supplementary data to this article can be found online at


Protective effect of dexmedetomidine in a rat model of α-naphthylthiourea- induced acute lung injury

Volkan Hancı, Gamze Yurdakan, Serhan Yurtlu, et al.
J Surg Res 178 (2012):424-430

Background: We assessed the effects of dexmedetomidine in a rat model of a-naphthylthiourea (ANTU)einduced acute lung injury.  Methods: Forty Wistar Albino male rats weighing 200e240 g were divided into 5 groups (n = 8 each), including a control group. Thus, there were one ANTU group and three dexmedetomidine groups (10-, 50-, and 100-mg/kg treatment groups), plus a control group. The control group provided the normal base values. The rats in the ANTU group were given 10 mg/kg of ANTU intraperitoneally and the three treatment groups received 10, 50, or 100 mg/kg of dexmedetomidine intraperitoneally 30 min before ANTU application. The rat body weight (BW), pleural effusion (PE), and lung weight (LW) of each group were measured 4 h after ANTU administration. The histopathologic changes were evaluated using hematoxylin-eosin staining. Results: The mean PE, LW, LW/BW, and PE/BW measurements in the ANTU group were significantly greater than in the control groups and all dexmedeto-midine treatment groups (P < 0.05). There were also significant decreases in the mean PE, LW, LW/BW and PE/BW values in the dexmedetomidine 50-mg/kg group compared with those in the ANTU group (P < 0.01). The inflammation, hemorrhage, and edema scores in the ANTU group were significantly greater than those in the control or dexmedetomidine 50-mg/kg group (P < 0.01). Conclusion: Dexmedetomidine treatment has demonstrated  a potential benefit by preventing ANTU-induced acute lung injury in an experimental rat model. Dexmedetomidine could have a potential protective effect on acute lung injury in intensive care patients.


Protective effects of Isofraxidin against lipopolysaccharide-induced acute lung injury in mice

Xiaofeng Niu, YuWang, Weifeng Li, Qingli Mu, et al.
International Immunopharmacology 24 (2015) 432–439

Acute lung injury (ALI) is a life-threatening disease characterized by serious lung inflammation and increased capillary permeability, which presents a high mortality worldwide. Isofraxidin (IF), a Coumarin compound isolated from the natural medicinal plants such as Sarcandra glabra and Acanthopanax senticosus, has been reported to have definite anti-bacterial, anti-oxidant, and anti-inflammatory activities. However, the effects of IF against lipopoly-saccharide-induced ALI have not been clarified. The aim of the present study is to explore the protective effects and potential mechanism of IF against LPS-induced ALI in mice. In this study, We found that pretreatment with IF significantly lowered LPS-induced mortality and lung wet-to-dry weight (W/D) ratio and reduced the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) in serum and bronchoalveolar lavage fluid (BALF). We also found that total cells, neutrophils and macrophages in BALF,MPO activity in lung tissues were markedly decreased. Besides, IF obviously inhibited lung histopathological changes and cyclooxygenase-2 (COX-2) protein expression. These results suggest that IF has a protective effect against LPS induced ALI, and the protective effect of IF seems to result from the inhibition of COX-2 protein expression in the lung, which regulates the production of PGE2.

Ingestion of LPS stimulates vascular permeability, promotes inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from blood into lung tissues and activates numerous inflammatory cells such as neutrophils and macrophages. In macrophages, LPS challenge induces the transcription of gene encoding pro-inflammatory protein, which leads to cytokine release and synthesis of enzymes, such as cyclo-oxygenase-2 (COX-2). COX-2 usually can’t be found in normal tissues, but widely induced by pro-inflammatory stimuli, such as cytokines, endotoxins, and growth factors. COX-2 plays a vital role in the regulation of inflammatory process by modulating the production of prostaglandin E2 (PGE2). PGE2, induced by cytokines and other initiator, is an inflammatory mediator which is produced in the regulation of COX-2. Previous researches demonstrated that inhibition of COX-2 produced a dramatically anti-inflammatory effect with little gastrointestinal toxicity. Therefore, inhibition of COX-2 protein expression has far-reaching significance in the treatment of ALI.

effects of IF on LPS-induced mortality in ALI mice

effects of IF on LPS-induced mortality in ALI mice

The effects of IF on LPS-induced mortality in ALI mice (n = 12/group). IF (5, 10, 15 mg/kg, i.p.) or DEX (5 mg/kg, i.p.) were given to mice 1 h prior to LPS challenge. The mortalities were observed at 0, 12, 24, 36, 48, 60, and 72 h. ###P = 0.001 when compared with the control group; *P = 0.05, **P = 0.01, and ***P = 0.001 when compared with the LPS group.


Protective effects of intranasal curcumin on paraquot induced acute lung injury (ALI) in mice

Namitosh Tyagi, Asha Kumaria, D. Dash, Rashmi Singh
Environment  Toxicol  & Pharmacol  38 (2014) 913–921

Paraquot (PQ) is widely and commonly used as herbicide and has been reported to be hazardous as it causes lung injury. However, molecular mechanism underlying lung toxicity caused by PQ has not been elucidated. Curcumin, a known anti-inflammatory molecule derived from rhizomes of Curcuma longa has variety of pharmacological activities including free-radical scavenging properties but the protective effects of curcumin on PQ-induced acute lung injury (ALI) have not been studied. In this study, we aimed to study the effects of curcumin on ALI caused by PQ in male parke’s strain mice which were challenged acutely byPQ (50 mg/kg, i.p.) with or without curcumin an hour before (5 mg/kg, i.n.) PQ intoxication. Lung specimens and the bronchoalveolar lavage fluid (BALF) were isolated for pathological and biochemical analysis after 48 h of PQ exposure. Curcumin administration has significantly enhanced superoxide dismutase (SOD) and catalase activities. Lung wet/dry weight ratio, malondialdehyde (MDA) and lactate dehydrogenase (LDH) content, total cell number and myeloperoxidase (MPO) levels in BALF as well as neutrophil infiltration were attenuated by curcumin. Pathological studies also revealed that intranasal curcumin alleviate PQ-induced pulmonary damage and pro-inflammatory cytokine levels like tumor necrosis factor-α (TNF-α) and nitric oxide (NO). These results suggest that intranasal curcumin may directly target lungs and curcumin inhalers may prove to be effective in PQ-induced ALI treatment in near future.


Phillyrin attenuates LPS-induced pulmonary inflammation via suppression of MAPK and NF-κB activation in acute lung injury mice

Wei-ting Zhong, Yi-chun Wu, Xian-xing Xie, Xuan Zhou, et al.
Fitoterapia 90 (2013) 132–139

Phillyrin (Phil) is one of the main chemical constituents of Forsythia suspensa (Thunb.), which has shown to be an important traditional Chinese medicine. We tested the hypothesis that Phil modulates pulmonary inflammation in an ALI model induced by LPS. Male BALB/c mice were pretreated with or without Phil before respiratory administration with LPS, and pretreated with dexamethasone as a control. Cytokine release (TNF-α, IL-1β, and IL-6) and amounts of inflammatory cell in bronchoalveolar lavage fluid (BALF) were detected by ELISA and cell counting separately. Pathologic changes, including neutrophil infiltration, interstitial edema, hemorrhage, hyaline membrane formation, necrosis, and congestion during acute lung injury in mice were evaluated via pathological section with HE staining. To further investigate the mechanism of Phil anti-inflammatory effects, activation of MAPK and NF-κB pathways was tested by western blot assay. Phil pretreatment significantly attenuated LPS-induced pulmonary histopathologic changes, alveolar hemorrhage, and neutrophil infiltration. The lung wet-to-dry weight ratios, as the index of pulmonary edema, were markedly decreased by Phil retreatment. In addition, Phil decreased the production of the proinflammatory cytokines including (TNF-α, IL-1β, and IL-6) and the concentration of myeloperoxidase (MPO) in lung tissues. Phil pretreatment also significantly suppressed LPS-induced activation of MAPK and NF-κB pathways in lung tissues. Taken together, the results suggest that Phil may have a protective effect on LPS-induced ALI, and it potentially contributes to the suppression of the activation of MAPK and NF-κB pathways. Phil may be a new preventive agent of ALI in the clinical setting.

A mass of studies have been reported basically on alleviating LPS-induced acute lung injury in models. Phillyrin (Fig. 1), a lignin, is one of the main chemical constituents of Forsythia suspensa (Thunb.), which is an important traditional Chinese medicine (“Lianqiao” in Chinese), and has long been used for gonorrhea, erysipelas, inflammation, pyrexia and ulcer. Previous studies indicated that Phil significantly inhibited NO production in LPS-activated macrophage cells. But there is not much evidence showing the anti-inflammatory properties of phillyrin. In the present study, we sought to investigate the effects of phillyrin on LPS-induced pulmonary inflammation in mice.

Fig. not shown. A: Effects of Phil on histopathological changes in lung tissues in LPS-induced ALI mice. Mice were given an intragastric administration of Phil (10 and 20 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. Then mice were anesthetized and lung tissue samples were collected at 6 h after LPS challenge for histological evaluation. These representative histological changes of the lung were obtained from mice of different groups (hematoxylin and eosin staining, original magnification 200×, Scale bar: 50 μm). B: Effects of Phil on LPS-induced lung morphology. The slides were histopathologically evaluated using a semi-quantitative scoring method. Lung injury was graded from 0 (normal) to 4 (severe) in four categories: congestion, edema, interstitial inflammation and inflammatory cell infiltration. The total lung injury score was calculated by adding up the individual scores of each category. The values presented are the means ± S.E.M. (n = 4–6 in each group). ##P b 0.01 vs. the control group, **P b 0.01 vs. the LPS group. Cont: control group; LPS: LPS group; Phil + LPS: Phil + LPS group; Dex + LPS: Dex + LPS group.

In summary, the present study indicated that Phil has a protective effect on LPS-induced acute lung injury. Phil significantly attenuated histopathological changes initiated by LPS via reducing over inflammatory responses. We also demonstrated that MAPK and NF-κB signaling pathways are the important targets of Phil to perform its actions. Phil acts by preventing NF-κB translocation to the nucleus or inhibiting the activation of MAPKs directly or indirectly, which is to be investigated in further studies. All these results suggest that Phil may be a new therapeutic agent for the prevention of inflammation during acute lung injury.





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