Erik B. Friedrich

CD34−/CD133+/VEGFR-2+ Endothelial Progenitor Cell Subpopulation With Potent Vasoregenerative Capacities

Our goal was to identify functionally important subpopulations within the heterogenous group of endothelial progenitor cells (EPC). Fluorescence-activated cell sorter analysis of CD133+ progenitor cells revealed the presence of CD34+ and CD34− subpopulations. CD34−/133+ progenitors differentiate into CD34+/133+ EPC, adhere more potently than these in response to SDF-1, and rapidly home to sites of limb ischemia in human volunteers. In human coronary atherectomy samples, fewer CD34−/133+ than CD34+/133+ EPC are present in stable plaques, whereas cell numbers increase with a reversion of the ratio in unstable lesions. In CD34−/133+ EPC-injected nude mice, more transplanted cells coexpressing endothelial markers home to carotid artery lesion endothelium than in CD34+/133+-injected mice. In the former, lesions were smaller and reendothelialization higher than in the latter. We identified a new CD34−/133+ EPC subpopulation, which is apparently a precursor of “classical” CD34+/133+ EPC, and functionally more potent than these with respect to homing and vascular repair.

Heart-rate reduction by If-channel inhibition with ivabradine restores collateral artery growth in hypercholesterolemic atherosclerosis

AIMS Collateral arteries protect tissue from ischaemia. Heart rate correlates with vascular events in patients with arterial obstructive disease. Here, we tested the effect of heart-rate reduction (HRR) on collateral artery growth. METHODS AND RESULTS The I(f)-channel inhibitor ivabradine reduced heart rate by 11% in wild-type and 15% in apolipoprotein E (ApoE)(-/-) mice and restored endothelium-dependent relaxation in aortic rings of ApoE(-/-) mice. Microsphere perfusion and angiographies demonstrated that ivabradine did not change hindlimb perfusion in wild-type mice but improved perfusion in ApoE(-/-) mice from 40.5 ± 15.8-60.2 ± 18.5% ligated/unligated hindlimb. Heart rate reduction (13%) with metoprolol failed to improve endothelial function and perfusion. Protein expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS, and eNOS activity were increased in collateral tissue following ivabradine treatment of ApoE(-/-) mice. Co-treatment with nitric oxide-inhibitor N (G)-nitro-L-arginine methyl ester abolished the effects of ivabradine on arteriogenesis. Following ivabradine, classical inflammatory cytokine expression was lowered in ApoE(-/-) circulating mononuclear cells and in plasma, but unaltered in collateral-containing hindlimb tissue, where numbers of perivascular macrophages also remained unchanged. However, ivabradine reduced expression of anti-arteriogenic cytokines CXCL10and CXCL11 and of smooth muscle cell markers smoothelin and desmin in ApoE(-/-) hindlimb tissue. Endothelial nitric oxide synthase and inflammatory cytokine expression were unchanged in wild-type mice. Ivabradine did not affect cytokine production in HUVECs and THP1 mononuclear cells and had no effect on the membrane potential of HUVECs in patch-clamp experiments. CONCLUSION Ivabradine-induced HRR stimulates adaptive collateral artery growth. Important contributing mechanisms include improved endothelial function, eNOS activity, and modulation of inflammatory cytokine gene expression.

Circulating microparticles as indicators of peripartum cardiomyopathy

AIMS Peripartum cardiomyopathy (PPCM) is associated with high mortality and morbidity. Endothelial damage involving cathepsin-D to form a 16 kDa prolactin (PRL) peptide is pathogenetically relevant. Inhibiting PRL peptide with bromocriptine has yielded promising results. We investigated whether microparticles (MPs) can be quantified in serum as markers for diagnosis and treatment effects in PPCM. METHODS AND RESULTS Patients with PPCM were compared with age-matched healthy post-partum women (PPCTR), healthy pregnant women (PCTR), healthy non-pregnant women (NPCTR), patients with ischaemic cardiomyopathy (ICM), patients with stable coronary artery disease (CAD) and healthy controls (HCTR). Peripartum cardiomyopathy treated with bromocriptine (PPCM-BR) and with PPCM without bromocriptine-treatment as control (PPCM-BRCTR) were compared. Microparticles were determined by flow cytometry. Endothelial MPs (EMPs) were elevated in PPCM compared with PPCTR, PCTR, and NPCTR, each P< 0.001. They were significantly elevated compared with ICM, CAD, and HCTR (P< 0.001). Pregnancy (PCTR) exhibited only slight increases vs. ICM, CAD, NPCTR, and HCTR. The increase in PPCM was due to an increase of activated but not apoptotic EMPs. Platelet-derived microparticles were highly increased in PPCM compared with ICM (P< 0.001) but 9.3 ± 4.4-fold compared with CAD (P< 0.001). In NPCTR (P< 0.001) compared with NPCTR, the increase was 5.9 ± 1.7-fold (P< 0.001). Microparticles generated from monocytes (MMPs) were increased 2.4 ± 1.8-fold in PPCM compared with PCTR (P< 0.001) and 4.8 ± 3.6-fold compared with CAD (P< 0.001), whereas leucocyte MPs (LMPs) were not significantly elevated. Endothelial microparticles were significantly reduced in PPCM treated additionally with bromocriptine compared with PPCM treated only with heart failure therapy (P< 0.001). CONCLUSION Microparticle profiles may in long-term distinguish PPCM from normal pregnancy, heart failure, and vascular diseases and might be a diagnostic marker related to the pathomechanism of PPCM.

MANAGEMENT OF END STAGE HEART FAILURE

Heart failure is a major public health problem, with a patient population of at least 10 million in Europe and approximately 5 million in North America.1–3 Because of its age-dependent increase in incidence and prevalence, heart failure is one of the leading causes of death and hospitalisation among the elderly. As a consequence of the worldwide increase in life expectancy, and due to improvements in the treatment of heart failure in recent years, the proportion of patients that reach an advanced phase of the disease, so-called end stage, refractory or terminal heart failure, is steadily growing. Patients with end stage heart failure fall into stage D of the ABCD classification of the American College of Cardiology (ACC)/American Heart Association (AHA), and class III–IV of the New York Heart Association (NYHA) functional classification; they are characterised by advanced structural heart disease and pronounced symptoms of heart failure at rest or upon minimal physical exertion, despite maximal medical treatment according to current guidelines.1–3 This patient population has a 1-year mortality rate of approximately 50% and requires special therapeutic interventions.4 Every attempt should be made to identify and correct reversible causes for a worsening of heart failure, such as poor patient compliance, myocardial ischaemia, tachy- or bradyarrhythmias, valvular regurgitation, pulmonary embolism, infection, or renal dysfunction. In this article, we describe current strategies for the treatment of end stage heart failure. Current recommendations for the pharmacological treatment of heart failure patients with NYHA class III–IV are summarised in table 1, while table 2 gives an overview of the drugs discussed in this article.1–3,5 Angiotensin-converting enzyme (ACE) inhibitors are recommended as first-line treatment in all patients with reduced left ventricular (LV) systolic function (ejection fraction (EF) ⩽35–40%) independent of clinical symptoms (NYHA I–IV), unless there are contraindications. …

ACE Inhibition in secondary prevention: are the results controversial?

SummaryResults from the HOPE and EUROPA trials showed that ACE inhibitors lower cardiovascular mortality of patients with atherosclerosis and preserved left ventricular function. However, despite apparently adequate study design, the recently conducted PEACE trial detected no benefit of an additional ACE inhibitor treatment in patients with coronary artery disease and no heart failure with respect to cardiovascular risk reduction. One of the main reasons for this discrepancy might be the lower cardiovascular baseline risk of the PEACE study population, which was more intensively treated with lipid lowering drugs and myocardial revascularization prior to enrollment than patients in HOPE or EUROPA. Another reason for the negative results of PEACE might be substance-specific differences between individual ACE inhibitors (trandolapril in PEACE, ramipril in HOPE, and perindopril in EUROPA) in their clinical efficacy to reduce cardiovascular end-points. The PEACE trial did not achieve the originally projected sample size and the addition of a soft end-point of revascularization has not been helpful. While the results from the PEACE trial suggest that low-risk patients with coronary artery disease and with preserved left ventricular function who receive intensive standard therapy including lipid lowering and coronary revascularization may not benefit from additional ACE inhibition therapy, this conclusion should be made with caution. A number of reasons, other than drug treatment efficacy, may explain the neutral results in the PEACE trial. Further studies are needed to try to resolve this issue. In the meantime, the overwhelming data still support the use of ACE inhibitors in patients with coronary artery disease with preserved left ventricular function.

Integrin-linked kinase is a central mediator in angiotensin II type 1- and chemokine receptor CXCR4 signaling in myocardial hypertrophy.

Inflammation and pro-hypertrophic signaling are important for development and progression of myocardial hypertrophy (LVH) and chronic heart failure (CHF). Here we investigated the relevance of integrin-linked kinase (ILK) for chemokine receptor CXCR4- and angiotensin II type 1-triggered signaling and its regulation and role in cardiac remodeling. Using ELISA, real-time-PCR, and Western blotting, the present study demonstrates that SDF-1 and its receptor CXCR4 are up-regulated in plasma and left ventricles, respectively, in mouse models of cardiac hypertrophy (transaortic constriction, transgenic cardiac-specific overexpression of rac1) and in human CHF in association with increased cardiac ILK-expression. In isolated cardiomyocytes, ILK is activated by CXCR4-ligation and necessary for SDF-1-triggered activation of rac1, NAD(P)H oxidase, and release of reactive oxygen species. Importantly, the pro-hypertrophic peptide angiotensin II induces ILK-activation dependent on rac1 in cardiomyocytes, where ILK is necessary for angiotensin II-mediated stimulation of hypertrophy genes and protein synthesis. We conclude that in both SDF-1- and angiotensin II-triggered signaling, ILK is a central mediator of rac1-induced oxidative stress and myocardial hypertrophy.

Differential chemokine receptor expression regulates functional specialization of endothelial progenitor cell subpopulations

Postnatal vasculogenesis is mediated by endothelial progenitor cells (EPCs) which consist of subpopulations with different functional capacities. Our goal was to profile chemokine receptor expression on relevant subsets of EPCs and to characterize their role for effector functions. CD34+/CD133+/VEGFR2+ EPCs were characterized by high expression of chemokine receptors CXCR4, CX3CR1, BLT1, and low level expression of CXCR2 and CCR2, while primordial CD34−/CD133+/VEGFR2+ EPCs express these chemokine receptors at comparably low levels. Migration assays revealed that SDF-1, fractalkine, and LTB4 significantly increase migration of CD34−/CD133+/VEGFR2+ EPCs, while SDF-1 was the only potent agonist of migration of CD34+/CD133+/VEGFR2+ EPCs. SDF-1, fractalkine, and LTB4 trigger significant increase adhesion of CD34+/CD133+/VEGFR2+ EPCs, while in CD34−/CD133+/VEGFR2+ EPCs SDF-1 and fractalkine are equipotent agonists and LTB4 triggers a smaller though still significant increase in adhesion. Differential expression of specific chemokine receptors is an important regulator in terms of migration and adhesion of biologically relevant EPC-subpopulations, which may have implications for cell therapeutic strategies for treatment of ischemic vascular disease.

Role of integrin-linked kinase for functional capacity of endothelial progenitor cells in patients with stable coronary artery disease

Number and function of endothelial progenitor cells (EPCs) are down-regulated in patients with coronary artery disease (CAD). Integrin-linked kinase (ILK) is a signal and adaptor protein that regulates survival of mature endothelial cells and vascular development. Here we show that EPC dysfunction in patients with CAD is paralleled by down-regulation of ILK while restoration of ILK expression rescues the migratory defect of CAD-EPCs. Human EPCs transduced with dominant-negative ILK (DN-ILK) display significantly reduced expression of CD34(+)/VEGFR-2(+), DiI-Ac-LDL uptake, and Ulex europaeus lectin binding. Mechanistically, DN-ILK-transfected EPCs are characterized by decreased proliferation, while proliferation is increased in wild-type ILK-transfected EPCs. These effects are paralleled by changes in cyclin D1 expression, colony forming units, and cytoskeletal rearrangement. Functionally, ILK is necessary and sufficient for SDF-1-triggered migration and adhesion in EPCs. These data extend current knowledge about the role of ILK in EPC biology and implicate ILK as a therapeutic target in CAD.

Ramipril and Telmisartan Exhibit Differential Effects in Cardiac Pressure Overload-Induced Hypertrophy Without an Additional Benefit of the Combination of Both Drugs

Objectives: We aimed to characterize different cellular effects of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin 1 (AT1) receptor blockers (ARBs) as mono- or combination therapy in cardiac pressure overload. Methods and Results: C57B1/6 mice received either the ACEI ramipril (2.5 mg/kg body weight), the ARB telmisartan (20 mg/kg body weight), or the combination. In all groups, pressure overload was induced by transverse aortic constriction (TAC). Cardiac hypertrophy (heart weight/tibia length) induced by TAC was reduced in all 3 treatment groups, with the most pronounced effect in the telmisartan group. The cardiomyocyte short-axis diameter and cardiac fibrosis were increased by TAC and similarly reduced by ACEI, ARB, and the combination therapy. The TAC-induced increase in the number of proliferating Ki67pos cardiomyocytes and noncardiomyocytes was reduced more potently by ACEI than by ARB. Four days of drug treatment induced a significant increase in Scalpos/VEGFR1pos endothelial progenitor cells (EPCs) in all animals in the treated SHAM groups. After 1 day of aortic constriction, only ramipril increased EPC numbers; after 5 weeks, telmisartan monotherapy did not change the EPC levels compared to vehicle or the combination therapy but raised it compared to ramipril. Neither TAC nor one of the therapies changed the number of cardiac capillaries per cardiomyocytes. Conclusions: ACE inhibition and AT1 receptor blockade have beneficial effects in remodeling processes during cardiac pressure overload. There are small differences between the 2 therapeutical approaches, but the combination therapy has no additional benefit.

Effect of hypoxia on integrin-mediated adhesion of endothelial progenitor cells

Homing of endothelial progenitor cells (EPCs) is crucial for neoangiogenesis, which might be negatively affected by hypoxia. We investigated the influence of hypoxia on fibronectin binding integrins for migration and cell‐matrix‐adhesion. AMP‐activated kinase (AMPK) and integrin‐linked kinase (ILK) were examined as possible effectors of hypoxia.Human EPCs were expanded on fibronectin (FN) and integrin expression was profiled by flow cytometry. Cell‐matrix‐adhesion‐ and migration‐assays on FN were performed to examine the influence of hypoxia and AMPK‐activation. Regulation of AMPK and ILK was shown by Western blot analysis. We demonstrate the presence of integrin β1, β2 and α5 on EPCs. Adhesion to FN is reduced by blocking β1 and α5 (49% and 2% of control, P < 0.05) whereas α4‐blockade has no effect. Corresponding effects were shown for migration. Hypoxia and AMPK‐activation decrease adhesion on FN. Although total AMPK‐expression remains unchanged, phospho‐AMPK increases eightfold.The EPCs require α5 for adhesion on FN. Hypoxia and AMPK‐activation decrease adhesion. As α5 is the major adhesive factor for EPCs on FN, this suggests a link between AMPK and α5‐integrins. We found novel evidence for a connection between hypoxia, AMPK‐activity and integrin activity. This might affect the fate of EPCs in ischaemic tissue.