Nikos Werner

Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis.

Background—The molecular mechanisms by which physical training improves peripheral and coronary artery disease are poorly understood. Bone marrow–derived endothelial progenitor cells (EPCs) are thought to exert beneficial effects on atherosclerosis, angiogenesis, and vascular repair. Methods and Results—To study the effect of physical activity on the bone marrow, EPCs were quantified by fluorescence-activated cell sorter analysis in mice randomized to running wheels (5.1±0.8 km/d, n=12 to 16 per group) or no running wheel. Numbers of EPCs circulating in the peripheral blood of trained mice were enhanced to 267±19%, 289±22%, and 280±25% of control levels after 7, 14, and 28 days, respectively, accompanied by a similar increase of EPCs in the bone marrow and EPCs expanded from spleen-derived mononuclear cells. eNOS−/− mice and wild-type mice treated with NG-nitro-l-arginine methyl ester showed lower EPC numbers at baseline and a significantly attenuated increase of EPC in response to physical activity. Exercise NO dependently increased serum levels of vascular endothelial growth factor and reduced the rate of apoptosis in spleen-derived EPCs. Running inhibited neointima formation after carotid artery injury by 22±2%. Neoangiogenesis, as assessed in a subcutaneous disc model, was increased by 41±16% compared with controls. In patients with stable coronary artery disease (n=19), moderate exercise training for 28 days led to a significant increase in circulating EPCs and reduced EPC apoptosis. Conclusions—Physical activity increases the production and circulating numbers of EPCs via a partially NO-dependent, antiapoptotic effect that could potentially underlie exercise-related beneficial effects on cardiovascular diseases.

Bone Marrow–Derived Progenitor Cells Modulate Vascular Reendothelialization and Neointimal Formation: Effect of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibition

Objective—Atherosclerosis and restenosis after vascular injury are both characterized by endothelial dysfunction, apoptosis, inappropriate endothelialization, and neointimal formation. Bone marrow–derived endothelial progenitor cells have been implicated in neovascularization, resulting in adult blood vessel formation. Despite the anticipated stem cell plasticity, the role of bone marrow–derived endothelial progenitor cells has not been clarified in vascular lesion development. Methods and Results—We investigated vascular lesion formation in mice after transplantation of bone marrow transfected by means of retrovirus with enhanced green fluorescent protein. Carotid artery injury was induced, resulting in neointimal formation. Fluorescence microscopy and immunohistological analysis revealed that bone marrow–derived progenitor cells are involved in reendothelialization of the vascular lesions. Treatment with rosuvastatin (20 mg/kg body wt per day), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, enhanced the circulating pool of endothelial progenitor cells, propagated the advent of bone marrow–derived endothelial cells in the injured vessel wall, and, thereby, accelerated reendothelialization and significantly decreased neointimal formation. Conclusions—Vascular lesion development initiated by endothelial cell damage is moderated by bone marrow–derived progenitor cells. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibition promotes bone marrow–dependent reendothelialization and diminishes vascular lesion development. These findings may help to establish novel pathophysiological concepts and therapeutic strategies in the treatment of various cardiovascular diseases.

Estrogen Increases Bone Marrow–Derived Endothelial Progenitor Cell Production and Diminishes Neointima Formation

Background Estrogens improve endothelial function and accelerate reendothelialization after vascular injury via largely unknown mechanisms. Bone marrow‐derived endothelial progenitor cells (EPCs) are thought to positively influence endothelialization, vascular repair, and angiogenesis. Methods and Results In mice subjected to sham operation, ovariectomy, or ovariectomy and estrogen replacement treatment, estrogen deficiency significantly decreased EPCs circulating in the peripheral blood and residing in the bone marrow, as well as EPCs that were in vitro expanded from spleen‐derived mononuclear cells. These effects were completely prevented by estrogen replacement. Human women with increased estrogen plasma concentrations also displayed profoundly increased levels of circulating EPCs. Estrogens increase EPC numbers through a decreased apoptosis rate, which is mediated via a caspase‐8‐dependent pathway. Estrogen deficiency increased neointima formation after carotid artery injury in mice, but this effect was diminished by estrogen replacement therapy. In mice transplanted with green fluorescent protein‐positive bone marrow, reendothelialization of injured vessel segments by bone marrow‐derived cells was decreased during estrogen deficiency and increased in response to estrogen treatment. Conclusions Estrogens increase numbers of EPCs by antiapoptotic effects leading to accelerated vascular repair and decreased neointima formation. (Circulation. 2003;107:3059‐3065.)

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.

Risk and Fate of Cerebral Embolism After Transfemoral Aortic Valve Implantation : A Prospective Pilot Study With Diffusion-Weighted Magnetic Resonance Imaging

OBJECTIVES The aim of this study was prospective investigation of silent and clinically apparent cerebral embolic events and neurological impairment after transfemoral aortic valve implantation (TAVI). BACKGROUND TAVI is a novel therapeutic approach for multimorbid patients with severe aortic stenosis. We investigated peri-interventional cerebral embolism with diffusion-weighted magnetic resonance imaging (DW-MRI) and its relationship to clinical and serologic parameters of brain injury. METHODS Cerebral DW-MRI was performed before, directly, and 3 months after TAVI with the current third-generation self-expanding CoreValve (Medtronic, Minneapolis, Minnesota) prosthesis. At the timepoints of the serial MRI studies, focal neurological impairment was assessed according to the National Institutes of Health Stroke Scale (NIHSS), and serum concentration of neuron-specific enolase (NSE), a marker of the volume of brain tissue involved in an ischemic event, were determined. RESULTS Thirty patients were enrolled; 22 completed the imaging protocol. Three patients (10%) had new neurological findings after TAVI, of whom only 1 (3.6%) had a permanent neurological impairment. Of the 22 TAVI patients with complete imaging data, 16 (72.7%) had 75 new cerebral lesions after TAVI presumed to be embolic. The NIHSS and NSE were not correlated with DW-MRI lesions. CONCLUSIONS The incidence of clinically silent peri-interventional cerebral embolic lesions after TAVI is high. However, in this cohort of 30 patients, the incidence of persistent neurological impairment was low. (Incidence and Severity of Silent and Apparent Cerebral Embolism After Conventional and Minimal-invasive Transfemoral Aortic Valve Replacement; NCT00883285).

Aortic regurgitation index defines severity of peri-prosthetic regurgitation and predicts outcome in patients after transcatheter aortic valve implantation.

OBJECTIVES The aim of this study was to provide a simple, reproducible, and point-of-care assessment of peri-prosthetic aortic regurgitation (periAR) during transcatheter aortic valve implantation (TAVI) and to decipher the impact of this peri-procedural parameter on outcome. BACKGROUND Because periAR after TAVI might be associated with adverse outcome, precise quantification of periAR is of paramount importance but remains technically challenging. METHODS The severity of periAR was prospectively evaluated in 146 patients treated with the Medtronic CoreValve (Minneapolis, Minnesota) prosthesis by echocardiography, angiography, and measurement of the aortic regurgitation (AR) index, which is calculated as ratio of the gradient between diastolic blood pressure (DBP) and left ventricular end-diastolic pressure (LVEDP) to systolic blood pressure (SBP): [(DBP - LVEDP)/SBP] × 100. RESULTS After TAVI, 53 patients (36.3%) showed no signs of periAR and 71 patients (48.6%) showed only mild periAR, whereas 18 patients (12.3%) and 4 patients (2.7%) suffered from moderate and severe periAR, respectively. The AR index decreased stepwise from 31.7 ± 10.4 in patients without periAR, to 28.0 ± 8.5 with mild periAR, 19.6 ± 7.6 with moderate periAR, and 7.6 ± 2.6 with severe periAR (p < 0.001), respectively. Patients with AR index <25 had a significantly increased 1-year mortality risk compared with patients with AR index ≥25 (46.0% vs. 16.7%; p < 0.001). The AR index provided additional prognostic information beyond the echocardiographically assessed severity of periAR and independently predicted 1-year mortality (hazard ratio: 2.9, 95% confidence interval: 1.3 to 6.4; p = 0.009). CONCLUSIONS The assessment of the AR index allows a precise judgment of periAR, independently predicts 1-year mortality after TAVI, and provides additional prognostic information that is complementary to the echocardiographically assessed severity of periAR.

Circulating CD31+/Annexin V+ Apoptotic Microparticles Correlate With Coronary Endothelial Function in Patients With Coronary Artery Disease

Objective—Endothelial dysfunction predicts morbidity and mortality in patients at cardiovascular risk. Endothelial function may be decisively influenced by the degree of endothelial cell apoptosis. Methods and Results—To test this hypothesis in humans, endothelial-dependent vasodilatation was invasively assessed in 50 patients with coronary artery disease (CAD) by quantitative coronary angiography during intracoronary acetylcholine infusion. Flow cytometry was used to assess endothelial cell apoptosis by quantification of circulating CD31+/annexin V+ apoptotic microparticles in peripheral blood. Increased apoptotic microparticle counts positively correlated with impairment of coronary endothelial function. Multivariate analysis revealed that increased apoptotic microparticle counts predict severe endothelial dysfunction independent of classical risk factors, such as hypertension, hypercholesterolemia, smoking, diabetes, age, or sex. Conclusions—In patients with CAD, endothelial-dependent vasodilatation closely relies on the degree of endothelial cell apoptosis, which is readily measurable by circulating CD31+/annexin V+ apoptotic microparticles. These findings possibly provide new options for risk assessment and may have implications for future treatment strategies of CAD.

Renal Function as Predictor of Mortality in Patients After Percutaneous Transcatheter Aortic Valve Implantation

OBJECTIVES The aim of this study was to determine the influence of baseline renal function and periprocedural acute kidney injury (AKI) on prognosis after transcatheter aortic valve implantation (TAVI). BACKGROUND Evidence is growing that renal function is a major predictor of mortality in patients after TAVI. METHODS TAVI was performed with the 18-F CoreValve prosthesis via transfemoral access. All-cause mortality was determined 30 days and 1 year after TAVI in 77 patients with a mean Society of Thoracic Surgeons mortality score of 9.3 ± 6.1% and a mean logistic European System for Cardiac Operative Risk Evaluation of 31.2 ± 17.6%. RESULTS Overall procedural success rate was 98% with 1 periprocedural death. The 30-day mortality was 10%, and 1-year mortality was 26%. The mortality risk increased stepwise across quartiles of baseline serum creatinine. An AKI occurred in 20 of 77 patients: 12 patients (60%) with AKI died during follow-up. The incidence of AKI was related to peripheral arterial disease (65% vs. 39%; p = 0.04), the occurrence of a systemic inflammatory response syndrome (60% vs. 21%, p = 0.002), and post-procedural peri-prosthetic regurgitation ≥2+ (35% vs. 9%, p = 0.02). Impaired renal function at baseline reflected by serum creatinine ≥1.58 mg/dl (hazard ratio: 3.9, 95% confidence interval: 1.6 to 9.5; p = 0.002) and the occurrence of AKI (hazard ratio: 5.9, 95% confidence interval: 2.4 to 14.5, p < 0.001) that was not related to the amount of contrast dye were strong predictors of 1-year mortality after TAVI. CONCLUSIONS Impaired renal function at baseline and the occurrence of periprocedural AKI, independent whether renal function returns to baseline or not, are strong predictors of 30-day and 1-year mortality after TAVI.

Improvement of Endothelial Function by Systemic Transfusion of Vascular Progenitor Cells

Endothelial dysfunction is characterized by abnormalities in vasoreactivity and is a marker of the extent of atherosclerosis. Cellular repair by circulating progenitor cells of ongoing vascular injury may be essential for vascular integrity and function and may limit abnormalities in vasoreactivity. Apolipoprotein E–deficient (apoE−/−) mice were splenectomized and treated with high-cholesterol diet for 5 weeks, resulting in marked impairment of endothelium-dependent vasodilation of aortic segments as compared with wild-type mice. Intravenous transfusion of 2×107 spleen-derived mononuclear cells (MNCs) isolated from wild-type mice on 3 consecutive days restored endothelium-dependent vasodilation in the apoE−/− mice, as measured 7, 14, and 45 days after transfusion. Histological analyses of aortic tissue identified fluorescent-labeled, exogenously applied progenitor cells that expressed the endothelial cell marker CD31 in the endothelial cell layer of atherosclerotic lesions. Progenitor cell treatment led to increased vascular nitric oxide synthase activity. Transfusion of either in vitro-differentiated Dil-Ac-LDL/lectin-positive endothelial progenitor cells, CD11b-positive (monocyte marker), CD45R-positive (B-cell marker), or Sca-1–positive (stem cell marker) MNC subpopulations significantly improved endothelium-dependent vasodilation, although these treatments were not as effective as transfusion of total MNCs. Depletion of MNCs of either CD11b-positive, CD45R-positive, or Sca-1–positive cells resulted in significant attenuation of endothelium-dependent vasodilation as compared with nondepleted MNCs; however, vasoreactivity was still significantly improved as compared with saline-treated apoE−/− mice. Intravenous transfusion of spleen-derived MNCs improves endothelium-dependent vasodilation in atherosclerotic apoE−/− mice, indicating an important role of circulating progenitor cells for the repair of ongoing vascular injury. More than 1 subpopulation of the MNC fraction seems to be involved in this effect.

Endothelial Microparticle–Mediated Transfer of MicroRNA-126 Promotes Vascular Endothelial Cell Repair via SPRED1 and Is Abrogated in Glucose-Damaged Endothelial Microparticles

Background— Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. Methods and Results— Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Conclusions— Endothelial microparticles promote vascular endothelial repair by delivering functional miR-126 into recipient cells. In pathological hyperglycemic conditions, EMP-mediated miR-126–induced EC repair is altered.