Ariane Fischer

MicroRNA-92a Controls Angiogenesis and Functional Recovery of Ischemic Tissues in Mice

Of Life, Limb, and a Small RNA Gene expression in mammals is controlled not only by proteins but by small noncoding RNAs called microRNAs. The involvement of these RNAs provides powerful clues about the molecular origins of human diseases and how they might be treated. Ischemic diseases arise from an inadequate blood supply. Bonauer et al. (p. 1710, published online 21 May) find that a specific microRNA that is expressed in the cells lining blood vessels (called miR-92a) functions to repress the growth of new blood vessels. MiR-92a probably acts through effects on expression of integrins, proteins involved in cell adhesion and migration. In mouse models in which an inadequate blood supply had caused damage either to heart or limb muscle, therapeutic inhibition of miR-92a led to an increase in blood vessel density in the damaged tissues and enhanced functional recovery. Inhibition of a microRNA that represses blood vessel growth enhances the recovery of tissue damaged by an inadequate blood supply. MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by binding to target messenger RNAs (mRNAs), leading to translational repression or degradation. Here, we show that the miR-17~92 cluster is highly expressed in human endothelial cells and that miR-92a, a component of this cluster, controls the growth of new blood vessels (angiogenesis). Forced overexpression of miR-92a in endothelial cells blocked angiogenesis in vitro and in vivo. In mouse models of limb ischemia and myocardial infarction, systemic administration of an antagomir designed to inhibit miR-92a led to enhanced blood vessel growth and functional recovery of damaged tissue. MiR-92a appears to target mRNAs corresponding to several proangiogenic proteins, including the integrin subunit alpha5. Thus, miR-92a may serve as a valuable therapeutic target in the setting of ischemic disease.

Circulating MicroRNAs in Patients With Coronary Artery Disease

Rationale: MicroRNAs are small RNAs that control gene expression. Besides their cell intrinsic function, recent studies reported that microRNAs are released by cultured cells and can be detected in the blood. Objective: To address the regulation of circulating microRNAs in patients with stable coronary artery disease. Methods and Results: To determine the regulation of microRNAs, we performed a microRNA profile using RNA isolated from n=8 healthy volunteers and n=8 patients with stable coronary artery disease that received state-of-the-art pharmacological treatment. Interestingly, most of the highly expressed microRNAs that were lower in the blood of patients with coronary artery disease are known to be expressed in endothelial cells (eg, miR-126 and members of the miR-17∼92 cluster). To prospectively confirm these data, we detected selected microRNAs in plasma of 36 patients with coronary artery disease and 17 healthy volunteers by quantitative PCR. Consistent with the data obtained by the profile, circulating levels of miR-126, miR-17, miR-92a, and the inflammation-associated miR-155 were significantly reduced in patients with coronary artery disease compared with healthy controls. Likewise, the smooth muscle–enriched miR-145 was significantly reduced. In contrast, cardiac muscle–enriched microRNAs (miR-133a, miR-208a) tend to be higher in patients with coronary artery disease. These results were validated in a second cohort of 31 patients with documented coronary artery disease and 14 controls. Conclusions: Circulating levels of vascular and inflammation-associated microRNAs are significantly downregulated in patients with coronary artery disease.

MicroRNA-34a regulates cardiac ageing and function

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.

Members of the microRNA-17-92 cluster exhibit a cell-intrinsic antiangiogenic function in endothelial cells

MicroRNAs are endogenously expressed small noncoding RNAs that regulate gene expression on the posttranscriptional level. The miR-17-92 cluster (encoding miR-17, -18a, -19a/b, -20a, and miR-92a) is highly expressed in tumor cells and is up-regulated by ischemia. Whereas miR-92a was recently identified as negative regulator of angiogenesis, the specific functions of the other members of the cluster are less clear. Here we demonstrate that overexpression of miR-17, -18a, -19a, and -20a significantly inhibited 3-dimensional spheroid sprouting in vitro, whereas inhibition of miR-17, -18a, and -20a augmented endothelial cell sprout formation. Inhibition of miR-17 and miR-20a in vivo using antagomirs significantly increased the number of perfused vessels in Matrigel plugs, whereas antagomirs that specifically target miR-18a and miR-19a were less effective. However, systemic inhibition of miR-17/20 did not affect tumor angiogenesis. Further mechanistic studies showed that miR-17/20 targets several proangiogenic genes. Specifically, Janus kinase 1 was shown to be a direct target of miR-17. In summary, we show that miR-17/20 exhibit a cell-intrinsic antiangiogenic activity in endothelial cells. Inhibition of miR-17/20 specifically augmented neovascularization of Matrigel plugs but did not affect tumor angiogenesis indicating a context-dependent regulation of angiogenesis by miR-17/20 in vivo.

MicroRNA-29 in Aortic Dilation: Implications for Aneurysm Formation

Rationale: Aging represents a major risk factor for coronary artery disease and aortic aneurysm formation. MicroRNAs (miRs) have emerged as key regulators of biological processes, but their role in age-associated vascular pathologies is unknown. Objective: We aim to identify miRs in the vasculature that are regulated by age and play a role in age-induced vascular pathologies. Methods and Results: Expression profiling of aortic tissue of young versus old mice identified several age-associated miRs. Among the significantly regulated miRs, the increased expression of miR-29 family members was associated with a profound downregulation of numerous extracellular matrix (ECM) components in aortas of aged mice, suggesting that this miR family contributes to ECM loss, thereby sensitizing the aorta for aneurysm formation. Indeed, miR-29 expression was significantly induced in 2 experimental models for aortic dilation: angiotensin II-treated aged mice and genetically induced aneurysms in Fibulin-4R/R mice. More importantly, miR-29b levels were profoundly increased in biopsies of human thoracic aneurysms, obtained from patients with either bicuspid (n=79) or tricuspid aortic valves (n=30). Finally, LNA-modified antisense oligonucleotide-mediated silencing of miR-29 induced ECM expression and inhibited angiotensin II-induced dilation of the aorta in mice. Conclusion: In conclusion, miR-29-mediated downregulation of ECM proteins may sensitize the aorta to the formation of aneurysms in advanced age. Inhibition of miR-29 in vivo abrogates aortic dilation in mice, suggesting that miR-29 may represent a novel molecular target to augment matrix synthesis and maintain vascular wall structural integrity.

MicroRNA-29 in aortic dilation: implications for aneurysm formation [Molecular Medicine]

Rationale: Aging represents a major risk factor for coronary artery disease and aortic aneurysm formation. MicroRNAs (miRs) have emerged as key regulators of biological processes, but their role in age-associated vascular pathologies is unknown. Objective: We aim to identify miRs in the vasculature that are regulated by age and play a role in age-induced vascular pathologies. Methods and Results: Expression profiling of aortic tissue of young versus old mice identified several age-associated miRs. Among the significantly regulated miRs, the increased expression of miR-29 family members was associated with a profound downregulation of numerous extracellular matrix (ECM) components in aortas of aged mice, suggesting that this miR family contributes to ECM loss, thereby sensitizing the aorta for aneurysm formation. Indeed, miR-29 expression was significantly induced in 2 experimental models for aortic dilation: angiotensin II-treated aged mice and genetically induced aneurysms in Fibulin-4R/R mice. More importantly, miR-29b levels were profoundly increased in biopsies of human thoracic aneurysms, obtained from patients with either bicuspid (n=79) or tricuspid aortic valves (n=30). Finally, LNA-modified antisense oligonucleotide-mediated silencing of miR-29 induced ECM expression and inhibited angiotensin II-induced dilation of the aorta in mice. Conclusion: In conclusion, miR-29-mediated downregulation of ECM proteins may sensitize the aorta to the formation of aneurysms in advanced age. Inhibition of miR-29 in vivo abrogates aortic dilation in mice, suggesting that miR-29 may represent a novel molecular target to augment matrix synthesis and maintain vascular wall structural integrity.

Inhibition of MicroRNA-92a Protects Against Ischemia/Reperfusion Injury in a Large-Animal Model

Background— MicroRNAs (miRs) are small noncoding RNAs that posttranscriptionally control gene expression. Small-animal studies suggest that miRs might offer novel therapeutic targets in cardiovascular diseases such as cardioprotection of murine hearts after myocardial infarction via miR-92a inhibitors. Because the functional benefits of miR-92a inhibitors in larger preclinical models are not known, we assessed the therapeutic efficacy of miR-92a inhibition in a porcine model of ischemia and reperfusion. Methods and Results— Pigs (n=5 per group) underwent percutaneous ischemia/reperfusion (60 min/72 h or 7 days, respectively). Locked nucleic acid–modified antisense miR-92a (LNA-92a) was applied either regionally (antegrade or retrograde) with a catheter or systemically (intravenously). LNA-92a significantly (P<0.01) reduced miR-92a expression in the infarct zone regardless of the application venue. However, catheter-based delivery, but not intravenous infusion, of LNA-92a significantly (P<0.05) reduced the infarct size compared with control LNA–treated pigs, which correlated with an improved ejection fraction and left ventricular end-diastolic pressure (P<0.05). Histochemistry revealed that LNA-92a increased capillary density but decreased leukocyte influx and cardiac cell death. Complete loss of miR-92a in mice attenuated the infarct-related myocardial dysfunction to a larger extent than cardiomyocyte-specific miR-92a deletion. In vitro, LNA-92a protected against hypoxia/reoxygenation–induced cardiomyocyte cell death. Conclusions— Regional LNA-92a delivery reduces miR-92a levels and infarct size and postischemic loss of function. LNA-92a exerts cell-protective, proangiogenic, and anti-inflammatory effects. miR-92a inhibition might be a novel therapeutic tool to preserve cardiac function after ischemia.

Inhibition of MicroRNA-17 Improves Lung and Heart Function in Experimental Pulmonary Hypertension

RATIONALE MicroRNAs (miRs) control various cellular processes in tissue homeostasis and disease by regulating gene expression on the posttranscriptional level. Recently, it was demonstrated that the expression of miR-21 and members of the miR-17-92 cluster was significantly altered in experimental pulmonary hypertension (PH). OBJECTIVES To evaluate the therapeutic efficacy and antiremodeling potential of miR inhibitors in the pathogenesis of PH. METHODS We first tested the effects of miR inhibitors (antagomirs), which were specifically designed to block miR-17 (A-17), miR-21 (A-21), and miR-92a (A-92a) in chronic hypoxia-induced PH in mice and A-17 in monocrotaline-induced PH in rats. Moreover, biological function of miR-17 was analyzed in cultured pulmonary artery smooth muscle cells. MEASUREMENTS AND MAIN RESULTS In the PH mouse model, A-17 and A-21 reduced right ventricular systolic pressure, and all antagomirs decreased pulmonary arterial muscularization. However, only A-17 reduced hypoxia-induced right ventricular hypertrophy and improved pulmonary artery acceleration time. In the monocrotaline-induced PH rat model, A-17 treatment significantly decreased right ventricular systolic pressure and total pulmonary vascular resistance index, increased pulmonary artery acceleration time, normalized cardiac output, and decreased pulmonary vascular remodeling. Among the tested miR-17 targets, the cyclin-dependent kinase inhibitor 1A (p21) was up-regulated in lungs undergoing A-17 treatment. Likewise, in human pulmonary artery smooth muscle cells, A-17 increased p21. Overexpression of miR-17 significantly reduced p21 expression and increased proliferation of smooth muscle cells. CONCLUSIONS Our data demonstrate that A-17 improves heart and lung function in experimental PH by interfering with lung vascular and right ventricular remodeling. The beneficial effects may be related to the up-regulation of p21. Thus, inhibition of miR-17 may represent a novel therapeutic concept to ameliorate disease state in PH.

Heparin Disrupts the CXCR4 / SDF-1 Axis and Impairs the Functional Capacity of Bone Marrow–Derived Mononuclear Cells Used for Cardiovascular Repair

Rationale: Cell therapy is a promising option for the treatment of acute or chronic myocardial ischemia. The intracoronary infusion of cells imposes the potential risk of cell clotting, which may be prevented by the addition of anticoagulants. However, a comprehensive analysis of the effects of anticoagulants on the function of the cells is missing. Objective: Here, we investigated the effects of heparin and the thrombin inhibitor bivalirudin on bone marrow–derived mononuclear cell (BMC) functional activity and homing capacity. Methods and Results: Heparin, but not bivalirudin profoundly and dose-dependently inhibited basal and stromal cell–derived factor 1 (SDF-1)–induced BMC migration. Incubation of BMCs with 20 U/mL heparin for 30 minutes abrogated SDF-1–induced BMC invasion (16±8% of control; P<0.01), whereas no effects on apoptosis or colony formation were observed (80±33% and 100±44% of control, respectively). Pretreatment of BMCs with heparin significantly reduced the homing of the injected cells in a mouse ear-wound model (69±10% of control; P<0.05). In contrast, bivalirudin did not inhibit in vivo homing of BMCs. Mechanistically, heparin binds to both, the chemoattractant SDF-1 and its receptor, chemokine receptor 4 (CXCR4), blocking CXCR4 internalization as well as SDF-1/CXCR4 signaling after SDF-1 stimulation. Conclusions: Heparin blocks SDF-1/CXCR4 signaling by binding to the ligand as well as the receptor, thereby interfering with migration and homing of BMCs. In contrast, the thrombin inhibitor bivalirudin did not interfere with BMC homing or SDF-1/CXCR4 signaling. These findings suggest that bivalirudin but not heparin might be recommended as an anticoagulant for intracoronary infusion of BMCs for cell therapy after cardiac ischemia.

Inhibition of miR-92a improves re-endothelialization and prevents neointima formation following vascular injury

Aims MicroRNA (miR)-92a is an important regulator of endothelial proliferation and angiogenesis after ischaemia, but the effects of miR-92a on re-endothelialization and neointimal lesion formation after vascular injury remain elusive. We tested the effects of lowering miR-92a levels using specific locked nucleic acid (LNA)-based antimiRs as well as endothelial-specific knock out of miR-92a on re-endothelialization and neointimal formation after wire-induced injury of the femoral artery in mice. Methods and results MiR-92a was significantly up-regulated in neointimal lesions following wire-induced injury. Pre-miR-92a overexpression resulted in repression of the direct miR-92a target genes integrin α5 and sirtuin1, and reduced eNOS expression in vitro. MiR-92a impaired proliferation and migration of endothelial cells but not smooth muscle cells. In vivo, systemic inhibition of miR-92a expression with LNA-modified antisense molecules resulted in a significant acceleration of re-endothelialization of the denuded vessel area. Genetic deletion of miR-92a in Tie2-expressing cells, representing mainly endothelial cells, enhanced re-endothelialization, whereas no phenotype was observed in mice lacking miR-92a expression in haematopoietic cells. The enhanced endothelial recovery was associated with reduced accumulation of leucocytes and inhibition of neointimal formation 21 days after injury and led to the de-repression of the miR-92a targets integrin α5 and sirtuin1. Conclusion Our data indicate that inhibition of endothelial miR-92a attenuates neointimal lesion formation by accelerating re-endothelialization and thus represents a putative novel mechanism to enhance the functional recovery following vascular injury.