Maja Mueller

Statin-Induced Improvement of Endothelial Progenitor Cell Mobilization, Myocardial Neovascularization, Left Ventricular Function, and Survival After Experimental Myocardial Infarction Requires Endothelial Nitric Oxide Synthase

Background—Endothelial nitric oxide (eNO) bioavailability is severely reduced after myocardial infarction (MI) and in heart failure. Statins enhance eNO availability by both increasing eNO production and reducing NO inactivation. We therefore studied the effect of statin treatment on eNO availability after MI and tested its role for endothelial progenitor cell mobilization, myocardial neovascularization, left ventricular (LV) dysfunction, remodeling, and survival after MI. Methods and Results—Wild-type (WT) and eNO synthase (eNOS)−/− mice with extensive anterior MI were randomized to treatment with vehicle (V) or atorvastatin (Ator, 50 mg/kg QD by gavage) for 4 weeks starting on day 1 after MI. Ator markedly improved endothelium-dependent, NO-mediated vasorelaxation; mobilization of endothelial progenitor cells; and myocardial neovascularization of the infarct border in WT mice after MI while having no effect in eNOS−/− mice. LV dysfunction and interstitial fibrosis were markedly attenuated by Ator in WT mice, whereas no effect was observed in eNOS−/− mice after MI. Importantly, Ator significantly increased the survival rate during 4 weeks after MI in WT mice (Ator versus V, 80% versus 46%; P<0.01, n=75) but not in eNOS−/− mice (43% versus 48%; NS, n=42). Conclusions—These findings suggest that increased eNO availability is required for statin-induced improvement of endothelial progenitor cell mobilization, myocardial neovascularization, LV dysfunction, interstitial fibrosis, and survival after MI. eNO bioavailability after MI likely represents an important therapeutic target in heart failure after MI and mediates beneficial effects of statin treatment after MI.

Mechanisms underlying adverse effects of HDL on eNOS-activating pathways in patients with coronary artery disease

Therapies that raise levels of HDL, which is thought to exert atheroprotective effects via effects on endothelium, are being examined for the treatment or prevention of coronary artery disease (CAD). However, the endothelial effects of HDL are highly heterogeneous, and the impact of HDL of patients with CAD on the activation of endothelial eNOS and eNOS-dependent pathways is unknown. Here we have demonstrated that, in contrast to HDL from healthy subjects, HDL from patients with stable CAD or an acute coronary syndrome (HDLCAD) does not have endothelial antiinflammatory effects and does not stimulate endothelial repair because it fails to induce endothelial NO production. Mechanistically, this was because HDLCAD activated endothelial lectin-like oxidized LDL receptor 1 (LOX-1), triggering endothelial PKCβII activation, which in turn inhibited eNOS-activating pathways and eNOS-dependent NO production. We then identified reduced HDL-associated paraoxonase 1 (PON1) activity as one molecular mechanism leading to the generation of HDL with endothelial PKCβII-activating properties, at least in part due to increased formation of malondialdehyde in HDL. Taken together, our data indicate that in patients with CAD, HDL gains endothelial LOX-1- and thereby PKCβII-activating properties due to reduced HDL-associated PON1 activity, and that this leads to inhibition of eNOS-activation and the subsequent loss of the endothelial antiinflammatory and endothelial repair-stimulating effects of HDL.

Endothelial-Vasoprotective Effects of High-Density Lipoprotein Are Impaired in Patients With Type 2 Diabetes Mellitus but Are Improved After Extended-Release Niacin Therapy

Background— High-density lipoprotein (HDL)–raising therapies are currently under intense evaluation, but the effects of HDL may be highly heterogeneous. We therefore compared the endothelial effects of HDL from healthy subjects and from patients with type 2 diabetes mellitus and low HDL (meeting the criteria for metabolic syndrome), who are frequently considered for HDL-raising therapies. Moreover, in diabetic patients, we examined the impact of extended-release (ER) niacin therapy on the endothelial effects of HDL. Methods and Results— HDL was isolated from healthy subjects (n=10) and patients with type 2 diabetes (n=33) by sequential ultracentrifugation. Effects of HDL on endothelial nitric oxide and superoxide production were characterized by electron spin resonance spectroscopy analysis. Effects of HDL on endothelium-dependent vasodilation and early endothelial progenitor cell–mediated endothelial repair were examined. Patients with diabetes were randomized to a 3-month therapy with ER niacin (1500 mg/d) or placebo, and endothelial effects of HDL were characterized. HDL from healthy subjects stimulated endothelial nitric oxide production, reduced endothelial oxidant stress, and improved endothelium-dependent vasodilation and early endothelial progenitor cell–mediated endothelial repair. In contrast, these beneficial endothelial effects of HDL were not observed in HDL from diabetic patients, which suggests markedly impaired endothelial-protective properties of HDL. ER niacin therapy improved the capacity of HDL to stimulate endothelial nitric oxide, to reduce superoxide production, and to promote endothelial progenitor cell–mediated endothelial repair. Further measurements suggested increased lipid oxidation of HDL in diabetic patients, and a reduction after ER niacin therapy. Conclusions— HDL from patients with type 2 diabetes mellitus and metabolic syndrome has substantially impaired endothelial-protective effects compared with HDL from healthy subjects. ER niacin therapy not only increases HDL plasma levels but markedly improves endothelial-protective functions of HDL in these patients, which is potentially more important. Clinical Trial Registration— clinicaltrials.gov. Identifier: NCT00346970.

Altered Activation of Endothelial Anti- and Proapoptotic Pathways by High-Density Lipoprotein from Patients with Coronary Artery Disease Role of High-Density Lipoprotein–Proteome Remodeling

Background— Endothelial dysfunction and injury are thought to play an important role in the progression of coronary artery disease (CAD). High-density lipoprotein from healthy subjects (HDLHealthy) has been proposed to exert endothelial antiapoptotic effects that may represent an important antiatherogenic property of the lipoprotein. The present study therefore aimed to compare effects of HDLCAD and HDLHealthy on the activation of endothelial anti- and proapoptotic pathways and to determine which changes of the lipoprotein are relevant for these processes. Methods and Results— HDL was isolated from patients with stable CAD (HDLsCAD), an acute coronary syndrome (HDLACS), and healthy subjects. HDLHealthy induced expression of the endothelial antiapoptotic Bcl-2 protein Bcl-xL and reduced endothelial cell apoptosis in vitro and in apolipoprotein E–deficient mice in vivo. In contrast, HDLsCAD and HDLACS did not inhibit endothelial apoptosis, failed to activate endothelial Bcl-xL, and stimulated endothelial proapoptotic pathways, in particular, p38-mitogen-activated protein kinase–mediated activation of the proapoptotic Bcl-2 protein tBid. Endothelial antiapoptotic effects of HDLHealthy were observed after inhibition of endothelial nitric oxide synthase and after delipidation, but not completely mimicked by apolipoprotein A-I or reconstituted HDL, suggesting an important role of the HDL proteome. HDL proteomics analyses and subsequent validations and functional characterizations suggested a reduced clusterin and increased apolipoprotein C-III content of HDLsCAD and HDLACS as mechanisms leading to altered effects on endothelial apoptosis. Conclusions— The present study demonstrates for the first time that HDLCAD does not activate endothelial antiapoptotic pathways, but rather stimulates potential endothelial proapoptotic pathways. HDL-proteome remodeling plays an important role for these altered functional properties of HDL. These findings provide novel insights into mechanisms leading to altered vascular effects of HDL in coronary disease.

Angiotensin II Induces Endothelial Xanthine Oxidase Activation Role for Endothelial Dysfunction in Patients With Coronary Disease

Objective—Xanthine oxidase (XO), a major source of superoxide, has been implicated in endothelial dysfunction in atherosclerosis. Mechanisms, however, leading to endothelial XO activation remain poorly defined. We tested the effect of angiotensin II (Ang II) on endothelial XO and its relevance for endothelial dysfunction in patients with coronary disease. Methods and Results—XO protein levels and XO-dependent superoxide production were determined in cultured endothelial cells in response to Ang II. In patients with coronary disease, endothelium-bound XO activity as determined by ESR spectroscopy and endothelium-dependent vasodilation were analyzed before and after 4 weeks of treatment with the AT1-receptor blocker losartan, the XO inhibitor allopurinol, or placebo. Ang II substantially increased endothelial XO protein levels and XO-dependent superoxide production in cultured endothelial cells, which was prevented by NAD(P)H-oxidase inhibition. In vivo, endothelium-bound XO activity was reduced by losartan and allopurinol, but not placebo therapy in patients with coronary disease. XO inhibition with oxypurinol improved endothelium-dependent vasodilation before, but not after losartan or allopurinol therapy. Conclusions—These findings suggest a novel mechanism whereby Ang II promotes endothelial oxidant stress, ie, by redox-sensitive XO activation. In patients with coronary disease, losartan therapy reduces endothelium-bound XO activity likely contributing to improved endothelial function.

Loss of AngiomiR-126 and 130a in Angiogenic Early Outgrowth Cells From Patients With Chronic Heart Failure Role for Impaired In Vivo Neovascularization and Cardiac Repair Capacity

Background— MicroRNAs are key regulators of angiogenic processes. Administration of angiogenic early outgrowth cells (EOCs) or CD34+ cells has been suggested to improve cardiac function after ischemic injury, in particular by promoting neovascularization. The present study therefore examines regulation of angiomiRs, microRNAs involved in angiogenesis, in angiogenic EOCs and circulating CD34+ cells from patients with chronic heart failure (CHF) and the role for their cardiac repair capacity. Methods and Results— Angiogenic EOCs and CD34+ cells were isolated from patients with CHF caused by ischemic cardiomyopathy (n=45) and healthy subjects (n=35). In flow cytometry analyses, angiogenic EOCs were largely myeloid and positive for alternatively activated M2 macrophage markers. In vivo cardiac neovascularization and functional repair capacity were examined after transplantation into nude mice with myocardial infarction. Cardiac transplantation of angiogenic EOCs from healthy subjects markedly increased neovascularization and improved cardiac function, whereas no such effect was observed after transplantation of angiogenic EOCs from patients with CHF. Real-time polymerase chain reaction analysis of 14 candidate angiomiRs, expressed in angiogenic EOCs, revealed a pronounced loss of angiomiR-126 and -130a in angiogenic EOCs from patients with CHF that was also observed in circulating CD34+ cells. Anti–miR-126 transfection markedly impaired the capacity of angiogenic EOCs from healthy subjects to improve cardiac function. miR-126 mimic transfection increased the capacity of angiogenic EOCs from patients with CHF to improve cardiac neovascularization and function. Conclusions— The present study reveals a loss of angiomiR-126 and -130a in angiogenic EOCs and circulating CD34+ cells from patients with CHF. Reduced miR-126 expression was identified as a novel mechanism limiting their capacity to improve cardiac neovascularization and function that can be targeted by miR-126 mimic transfection.Background— MicroRNAs are key regulators of angiogenic processes. Administration of angiogenic early outgrowth cells (EOCs) or CD34+ cells has been suggested to improve cardiac function after ischemic injury, in particular by promoting neovascularization. The present study therefore examines regulation of angiomiRs, microRNAs involved in angiogenesis, in angiogenic EOCs and circulating CD34+ cells from patients with chronic heart failure (CHF) and the role for their cardiac repair capacity. Methods and Results— Angiogenic EOCs and CD34+ cells were isolated from patients with CHF caused by ischemic cardiomyopathy (n=45) and healthy subjects (n=35). In flow cytometry analyses, angiogenic EOCs were largely myeloid and positive for alternatively activated M2 macrophage markers. In vivo cardiac neovascularization and functional repair capacity were examined after transplantation into nude mice with myocardial infarction. Cardiac transplantation of angiogenic EOCs from healthy subjects markedly increased neovascularization and improved cardiac function, whereas no such effect was observed after transplantation of angiogenic EOCs from patients with CHF. Real-time polymerase chain reaction analysis of 14 candidate angiomiRs, expressed in angiogenic EOCs, revealed a pronounced loss of angiomiR-126 and -130a in angiogenic EOCs from patients with CHF that was also observed in circulating CD34+ cells. Anti–miR-126 transfection markedly impaired the capacity of angiogenic EOCs from healthy subjects to improve cardiac function. miR-126 mimic transfection increased the capacity of angiogenic EOCs from patients with CHF to improve cardiac neovascularization and function. Conclusions— The present study reveals a loss of angiomiR-126 and -130a in angiogenic EOCs and circulating CD34+ cells from patients with CHF. Reduced miR-126 expression was identified as a novel mechanism limiting their capacity to improve cardiac neovascularization and function that can be targeted by miR-126 mimic transfection. # Clinical Perspective {#article-title-54}

Exercise training improves in vivo endothelial repair capacity of early endothelial progenitor cells in subjects with metabolic syndrome

Background: Endothelial dysfunction and injury are considered to contribute considerably to the development and progression of atherosclerosis. It has been suggested that intense exercise training can increase the number and angiogenic properties of early endothelial progenitor cells (EPCs). However, whether exercise training stimulates the capacity of early EPCs to promote repair of endothelial damage and potential underlying mechanisms remain to be determined. The present study was designed to evaluate the effects of moderate exercise training on in vivo endothelial repair capacity of early EPCs, and their nitric oxide and superoxide production as characterized by electron spin resonance spectroscopy analysis in subjects with metabolic syndrome. Methods and results: Twenty-four subjects with metabolic syndrome were randomized to an 8 weeks exercise training or a control group. Superoxide production and nitric oxide (NO) availability of early EPCs were characterized by using electron spin resonance (ESR) spectroscopy analysis. In vivo endothelial repair capacity of EPCs was examined by transplantation into nude mice with defined carotid endothelial injury. Endothelium-dependent, flow-mediated vasodilation was analysed using high-resolution ultrasound. Importantly, exercise training resulted in a substantially improved in vivo endothelial repair capacity of early EPCs (24.0 vs 12.7%; p < 0.05) and improved endothelium-dependent vasodilation. Nitric oxide production of EPCs was substantially increased after exercise training, but not in the control group. Moreover, exercise training reduced superoxide production of EPCs, which was not observed in the control group. Conclusions: The present study suggests for the first time that moderate exercise training increases nitric oxide production of early endothelial progenitor cells and reduces their superoxide production. Importantly, this is associated with a marked beneficial effect on the in vivo endothelial repair capacity of early EPCs in subjects with metabolic syndrome.

Altered Activation of Endothelial Anti- and Pro-Apoptotic Pathways by High-Density Lipoprotein from Patients with Coronary Artery Disease: Role of HDL-Proteome Remodeling

Background— Endothelial dysfunction and injury are thought to play an important role in the progression of coronary artery disease (CAD). High-density lipoprotein from healthy subjects (HDLHealthy) has been proposed to exert endothelial antiapoptotic effects that may represent an important antiatherogenic property of the lipoprotein. The present study therefore aimed to compare effects of HDLCAD and HDLHealthy on the activation of endothelial anti- and proapoptotic pathways and to determine which changes of the lipoprotein are relevant for these processes. Methods and Results— HDL was isolated from patients with stable CAD (HDLsCAD), an acute coronary syndrome (HDLACS), and healthy subjects. HDLHealthy induced expression of the endothelial antiapoptotic Bcl-2 protein Bcl-xL and reduced endothelial cell apoptosis in vitro and in apolipoprotein E–deficient mice in vivo. In contrast, HDLsCAD and HDLACS did not inhibit endothelial apoptosis, failed to activate endothelial Bcl-xL, and stimulated endothelial proapoptotic pathways, in particular, p38-mitogen-activated protein kinase–mediated activation of the proapoptotic Bcl-2 protein tBid. Endothelial antiapoptotic effects of HDLHealthy were observed after inhibition of endothelial nitric oxide synthase and after delipidation, but not completely mimicked by apolipoprotein A-I or reconstituted HDL, suggesting an important role of the HDL proteome. HDL proteomics analyses and subsequent validations and functional characterizations suggested a reduced clusterin and increased apolipoprotein C-III content of HDLsCAD and HDLACS as mechanisms leading to altered effects on endothelial apoptosis. Conclusions— The present study demonstrates for the first time that HDLCAD does not activate endothelial antiapoptotic pathways, but rather stimulates potential endothelial proapoptotic pathways. HDL-proteome remodeling plays an important role for these altered functional properties of HDL. These findings provide novel insights into mechanisms leading to altered vascular effects of HDL in coronary disease.

Altered Activation of Endothelial Anti- and Proapoptotic Pathways by High-Density Lipoprotein from Patients with Coronary Artery DiseaseClinical Perspective

Background— Endothelial dysfunction and injury are thought to play an important role in the progression of coronary artery disease (CAD). High-density lipoprotein from healthy subjects (HDLHealthy) has been proposed to exert endothelial antiapoptotic effects that may represent an important antiatherogenic property of the lipoprotein. The present study therefore aimed to compare effects of HDLCAD and HDLHealthy on the activation of endothelial anti- and proapoptotic pathways and to determine which changes of the lipoprotein are relevant for these processes. Methods and Results— HDL was isolated from patients with stable CAD (HDLsCAD), an acute coronary syndrome (HDLACS), and healthy subjects. HDLHealthy induced expression of the endothelial antiapoptotic Bcl-2 protein Bcl-xL and reduced endothelial cell apoptosis in vitro and in apolipoprotein E–deficient mice in vivo. In contrast, HDLsCAD and HDLACS did not inhibit endothelial apoptosis, failed to activate endothelial Bcl-xL, and stimulated endothelial proapoptotic pathways, in particular, p38-mitogen-activated protein kinase–mediated activation of the proapoptotic Bcl-2 protein tBid. Endothelial antiapoptotic effects of HDLHealthy were observed after inhibition of endothelial nitric oxide synthase and after delipidation, but not completely mimicked by apolipoprotein A-I or reconstituted HDL, suggesting an important role of the HDL proteome. HDL proteomics analyses and subsequent validations and functional characterizations suggested a reduced clusterin and increased apolipoprotein C-III content of HDLsCAD and HDLACS as mechanisms leading to altered effects on endothelial apoptosis. Conclusions— The present study demonstrates for the first time that HDLCAD does not activate endothelial antiapoptotic pathways, but rather stimulates potential endothelial proapoptotic pathways. HDL-proteome remodeling plays an important role for these altered functional properties of HDL. These findings provide novel insights into mechanisms leading to altered vascular effects of HDL in coronary disease.

Altered Activation of Endothelial Anti- and Proapoptotic Pathways by High-Density Lipoprotein from Patients with Coronary Artery DiseaseClinical Perspective: Role of High-Density Lipoprotein–Proteome Remodeling

Background— Endothelial dysfunction and injury are thought to play an important role in the progression of coronary artery disease (CAD). High-density lipoprotein from healthy subjects (HDLHealthy) has been proposed to exert endothelial antiapoptotic effects that may represent an important antiatherogenic property of the lipoprotein. The present study therefore aimed to compare effects of HDLCAD and HDLHealthy on the activation of endothelial anti- and proapoptotic pathways and to determine which changes of the lipoprotein are relevant for these processes. Methods and Results— HDL was isolated from patients with stable CAD (HDLsCAD), an acute coronary syndrome (HDLACS), and healthy subjects. HDLHealthy induced expression of the endothelial antiapoptotic Bcl-2 protein Bcl-xL and reduced endothelial cell apoptosis in vitro and in apolipoprotein E–deficient mice in vivo. In contrast, HDLsCAD and HDLACS did not inhibit endothelial apoptosis, failed to activate endothelial Bcl-xL, and stimulated endothelial proapoptotic pathways, in particular, p38-mitogen-activated protein kinase–mediated activation of the proapoptotic Bcl-2 protein tBid. Endothelial antiapoptotic effects of HDLHealthy were observed after inhibition of endothelial nitric oxide synthase and after delipidation, but not completely mimicked by apolipoprotein A-I or reconstituted HDL, suggesting an important role of the HDL proteome. HDL proteomics analyses and subsequent validations and functional characterizations suggested a reduced clusterin and increased apolipoprotein C-III content of HDLsCAD and HDLACS as mechanisms leading to altered effects on endothelial apoptosis. Conclusions— The present study demonstrates for the first time that HDLCAD does not activate endothelial antiapoptotic pathways, but rather stimulates potential endothelial proapoptotic pathways. HDL-proteome remodeling plays an important role for these altered functional properties of HDL. These findings provide novel insights into mechanisms leading to altered vascular effects of HDL in coronary disease.