Background-Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. Methods and Results-Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2-associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. Conclusions-Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesisand is suitable for therapeutic intervention in the setting of ischemic heart disease. (Circulation. 2011;124:720-730.)Key Words: myocardial infarction Ⅲ microRNAs Ⅲ angiogenesis Ⅲ antagomir Ⅲ gene expression Ⅲ heart failure M yocardial infarction (MI) is a leading cause of morbidity and mortality worldwide. MI leads to scar formation and left ventricular remodeling, including cardiac dilatation, contractile dysfunction, cardiomyocyte hypertrophy, and fibrosis. 1 Tissue hypoxia triggers endothelial apoptosis, and insufficient capillary density further contributes to an increase of infarct size and left ventricular dysfunction. [2][3][4] Clinical Perspective on p 730MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules that regulate a substantial fraction of the genome by binding to the 3Ј untranslated region (3ЈUTR) of frequently coordinately acting target messenger RNAs. 5 MiRNAs have been identified as valuable therapeutic targets in a variety of diseases, including cardiovascular disease. 6 -12 Inhibition of miRNA processing by genetic knockdown of Dicer expression impairs endothelial functions and angiogenesis. [13][14][15] Certain miRNAs are important regulators of endothelial function, especially angiogenesis. 7,13-17 A subset of miRNAs is regulated by tissue oxygen levels, and miR-24 is activated by hypoxic conditions via the hypoxia-inducible factor 1 (HIF-1). 18 Although miR-24 is expressed in a variety Received April 19, 2011; accepted June 7, 2011 Table I). The small RNA...

Objective— MicroRNAs are a class of small ribonucleotides regulating gene/protein targets by transcript degradation or translational inhibition. Transforming growth factor-β (TGF-β) is involved in cardiac fibrosis partly by stimulation of endothelial-to-mesenchymal transition (EndMT). Here, we investigated whether microRNA (miR)-21, a microRNA enriched in fibroblasts and involved in general fibrosis, has a role in cardiac EndMT. Methods and Results— TGF-β treatment of endothelial cells significantly increased miR-21 expression and induced EndMT characterized by suppression of endothelial and increase of fibroblast markers. Overexpression of miR-21 alone also stimulated EndMT. Importantly, miR-21 blockade by transfection of specific microRNA inhibitors partly prevented TGF-β-induced EndMT. Mechanistically, miR-21 silenced phosphatase and tensin homolog in endothelial cells, resulting in activation of the Akt-pathway. Akt inhibition partly restored TGF-β-mediated loss of endothelial markers during EndMT. In vivo, pressure overload of the left ventricle led to increased expression of miR-21 in sorted cardiac endothelial cells, which displayed molecular and phenotypic signs of EndMT. This was attenuated by treatment of mice subjected to left ventricular pressure overload with an antagomir against miR-21. Conclusion— TGF-β-mediated EndMT is regulated at least in part by miR-21 via the phosphatase and tensin homolog/Akt pathway. In vivo, antifibrotic effects of miR-21 antagonism are partly mediated by blocking EndMT under stress conditions.

MicroRNA-21 (miR-21) expression is activated in multiple types of cancers, such as breast, liver, brain, prostate, myometrial cancers but also in cardiovascular diseases. MiR-21 regulates a plethora of target proteins which are involved in cellular survival, apoptosis and cell invasiveness. MiR-21 regulation is complex due to an own promoter that is target for various transcription factors and hormones. The consistent miR-21 overexpression under pathophysiological conditions points to miR-21 as a valuable tool for new therapeutic strategies. In this review, we present and analyze current data about miR-21 expression in various pathologies ranging from cancer to cardiovascular disease. Further, miR-21 regulatory mechanisms and miR-21 downstream targets are discussed. Finally, we highlight the particular role of miR-21 as a therapeutic target in various diseases.

MicroRNAs (miRs) are small non- coding RNA molecules controlling a plethora of biological processes such as development, cellular survival and senescence. We here determined miRs differentially regulated during cardiac postnatal development and aging. Cardiac function, morphology and miR expression profiles were determined in neonatal, 4 weeks, 6 months and 19 months old normotensive male healthy C57/Bl6N mice. MiR-22 was most prominently upregulated during cardiac aging. Cardiac expression of its bioinformatically predicted target mimecan (osteoglycin, OGN) was gradually decreased with advanced age. Luciferase reporter assays validated mimecan as a bona fide miR-22 target. Both, miR-22 and its target mimecan were co- expressed in cardiac fibroblasts and smooth muscle cells. Functionally, miR-22 overexpression induced cellular senescence and promoted migratory activity of cardiac fibroblasts. Small interference RNA-mediated silencing of mimecan in cardiac fibroblasts mimicked the miR-22-mediated effects. Rescue experiments revealed that the effects of miR-22 on cardiac fibroblasts were only partially mediated by mimecan. In conclusion, miR-22 upregulation in the aging heart contributed at least partly to accelerated cardiac fibroblast senescence and increased migratory activity. Our results suggest an involvement of miR-22 in age-associated cardiac changes, such as cardiac fibrosis.Electronic supplementary materialThe online version of this article (doi:10.1007/s11357-012-9407-9) contains supplementary material, which is available to authorized users.

Rationale: The endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) is increased in patients with coronary artery disease and may regulate function of circulating angiogenic progenitor cells (APCs) by small regulatory RNAs. Objectives: To study the role of microRNAs in ADMA-mediated impairment of APCs. Methods and Results: By using microarray analyses, we established microRNA expression profiles of human APCs.We used ADMA to induce APC dysfunction and found 16 deregulated microRNAs. We focused on miR-21, which was 3-fold upregulated by ADMA treatment. Overexpression of miR-21 in human APCs impaired migratory capacity. To identify regulated miR-21 targets, we used proteome analysis, using difference in-gel electrophoresis followed by mass spectrometric analysis of regulated proteins. We found that transfection of miR-21 precursors significantly repressed superoxide dismutase 2 in APCs, which resulted in increased intracellular reactive oxygen species concentration and impaired nitric oxide bioavailability. MiR-21 further repressed sprouty-2, leading to Erk Map kinase-dependent reactive oxygen species formation and APC migratory defects. Small interference RNA-mediated superoxide dismutase 2 or sprouty-2 reduction also increased reactive oxygen species formation and impaired APC migratory capacity. ADMA-mediated reactive oxygen species formation and APC dysfunction was rescued by miR-21 blockade. APCs from patients with coronary artery disease and high ADMA plasma levels displayed >4-fold elevated miR-21 levels, low superoxide dismutase 2 expression, and impaired migratory capacity, which could be normalized by miR-21 antagonism. Conclusions: We identified a novel miR-21-dependent mechanism of ADMA-mediated APC dysfunction. MiR-21 antagonism therefore emerges as an interesting strategy to improve dysfunctional APCs in patients with coronary artery disease. (Circ Res. 2010;107:138-143.)Key Words: endothelial progenitor cells Ⅲ microRNAs Ⅲ ADMA Ⅲ coronary artery disease Ⅲ oxidative stress T he endogenous nitric oxide synthase (NOS) inhibitor asymmetrical dimethylarginine (ADMA) is a major risk factor in patients with coronary artery disease (CAD). [1][2][3] Functionally, ADMA induces dysfunction of circulating angiogenic progenitor cells (APCs) (early outgrowth endothelial progenitor cells or circulating angiogenic cells) 4 and impairs neovascularization 5 by direct inhibition of the endothelial NOS. 6 NO plays a crucial role in mobilization, differentiation, and function of APCs, 7,8 whereas oxidative stress impairs APC function. 9,10 MicroRNAs (miRNAs) are a class of highly conserved, noncoding short RNA molecules that regulate a large portion of the genome. MiRNAs play a crucial role in cardiac biology, and miRNA dysregulation is often found in cardiovascular diseases. 11 Knockdown of the miRNA-processing enzyme Dicer in endothelial cells reduces the formation of capillary-like structures by profound dysregulation of angiogenesis-related genes, 12 and Dicer-deficient mice die from imp...

These data suggest that desflurane-induced APC is mediated in part by activation of mitochondrial large-conductance K(Ca) channels, and that activation of these channels by desflurane is mediated by PKA.

Abstract-Both known estrogen receptors, ER␣ and ER␤, are expressed in blood vessels. To gain further insight into the role of ER␣ in a functional setting, we investigated the effect of the novel highly selective ER␣ agonist Cpd1471 on vascular reactivity in ovariectomized spontaneously hypertensive rats (SHR). After ovariectomy or sham operation, 12-week-old female SHR received either 17␤-estradiol (E2, 2 g/kg body wt per day), the selective ER␣ agonist Cpd1471 (30 g/kg body wt per day), or placebo. Acetylcholine-induced endothelium-dependent vasorelaxation was significantly blunted in aortas from ovariectomized rats (R max , 53%Ϯ3% versus sham, 79%Ϯ2%; PϽ0.001). Treatment with E2 or Cpd1471 significantly augmented acetylcholine-induced relaxation in ovariectomized rats (R max , 70%Ϯ2%; resp, 73%Ϯ2%). Endothelium-independent relaxation induced by sodium nitroprusside was not different among the four groups. The contractile response induced by the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine, an index of basal NO formation, was significantly lower in ovariectomized rats compared with sham-operated animals (53Ϯ2% versus 77%Ϯ5%; PϽ0.01) and was normalized by both E2 (70%Ϯ2%) and Cpd1471 (70%Ϯ3%). Aortic endothelial NO synthase (eNOS) expression and phosphorylation of the vasodilator-stimulated phosphoprotein, an index of NO/cGMP-signaling, was reduced in ovariectomized SHR and normalized by E2 and Cpd1471. In SHR after ovariectomy, endothelium-dependent NO-mediated vasorelaxation and eNOS expression are attenuated. The novel selective ER␣ agonist Cpd1471 prevented these pathophysiological changes to a similar extent as E2. Thus, the pharmacological principle of selective ER␣ activation mediates positive vascular effects. Key Words: estrogen Ⅲ endothelium Ⅲ nitric oxide Ⅲ nitric oxide synthase Ⅲ rats, spontaneously hypertensive G ender differences in the risk for cardiovascular diseases are well recognized, with premenopausal women exhibiting a lower risk than age-matched men. The advantage of women over men in cardiovascular morbidity disappears after menopause, suggesting that estrogen plays an important role in cardiovascular health. 1 Estrogens are known to exert beneficial effects on the vascular wall. Long-term estrogen treatment improves endothelial dysfunction, a major contributor to the pathophysiology of cardiovascular disease, through upregulation of endothelial cell genes, such as endothelial nitric oxide synthase (eNOS). 2-4 Furthermore, estrogen has rapid nongenomic effects on the vascular endothelium, including activation of nitric oxide (NO) synthesis. 5,6 However, despite the positive effects on vascular function in animal models 7-9 and humans, 10 -14 estrogen replacement therapy with 17␤-estradiol or mixtures of equine estrogens as in the Heart and Estrogen/progestin Replacement Study (HERS) has failed to protect from cardiovascular diseases in large controlled clinical trials. [15][16][17][18] Therefore, in recent years, research has focused on selective estrogen receptor modulation as...

AimsHyperaldosteronism is associated with vascular injury and increased cardiovascular events. Bone marrow-derived endothelial progenitor cells (EPCs) play an important role in endothelial repair and vascular homeostasis. We hypothesized that hyperaldosteronism impairs EPC function and vascularization capacity in mice and humans.Methods and resultsWe characterized the effects of aldosterone and mineralocorticoid receptor (MR) blockade on EPC number and function as well as vascularization capacity and endothelial function. Treatment of human EPC with aldosterone induced translocation of the MR and impaired multiple cellular functions of EPC, such as differentiation, migration, and proliferation in vitro. Impaired EPC function was rescued by pharmacological blockade or genetic ablation of the MR. Aldosterone protein kinase A (PKA) dependently increased reactive oxygen species formation in EPC. Aldosterone infusion in mice impaired EPC function, EPC homing to vascular structures and vascularization capacity in a MR-dependent but blood pressure-independent manner. Endothelial progenitor cells from patients with primary hyperaldosteronism compared with controls of similar age displayed reduced migratory potential. Impaired EPC function was associated with endothelial dysfunction. MR blockade in patients with hyperaldosteronism improved EPC function and arterial stiffness.ConclusionEndothelial progenitor cells express a MR that mediates functional impairment by PKA-dependent increase of reactive oxygen species. Normalization of EPC function may represent a novel mechanism contributing to the beneficial effects of MR blockade in cardiovascular disease prevention and treatment.