Background-Diabetes mellitus impairs endothelial cell (EC) function and postischemic reparative neovascularization by molecular mechanisms that are not fully understood. microRNAs negatively regulate the expression of target genes mainly by interaction in their 3Ј untranslated region. Methods and Results-We found that microRNA-503 (miR-503) expression in ECs is upregulated in culture conditions mimicking diabetes mellitus (high D-glucose) and ischemia-associated starvation (low growth factors). Under normal culture conditions, lentivirus-mediated miR-503-forced expression inhibited EC proliferation, migration, and network formation on Matrigel (comparisons versus lentivirus.GFP control). Conversely, blocking miR-503 activity by either adenovirus-mediated transfer of a miR-503 decoy (Ad.decoymiR-503) or by antimiR-503 (antisense oligonucleotide) improved the functional capacities of ECs cultured under high D-glucose/low growth factors. We identified CCNE1 and cdc25A as direct miR-503 targets which are downregulated by high glucose/low growth factors in ECs. Next, we obtained evidence that miR-503 expression is increased in ischemic limb muscles of streptozotocin-diabetic mice and in ECs enriched from these muscles. Moreover, Ad.decoymiR-503 delivery to the ischemic adductor of diabetic mice corrected diabetes mellitus-induced impairment of postischemic angiogenesis and blood flow recovery. We finally investigated miR-503 and target gene expression in muscular specimens from the amputated ischemic legs of diabetic patients. As controls, calf biopsies of nondiabetic and nonischemic patients undergoing saphenous vein stripping were used. In diabetic muscles, miR-503 expression was remarkably higher, and it inversely correlated with cdc25 protein expression. Plasma miR-503 levels were also elevated in the diabetic individuals. Conclusions-Our Editorial see p 236 Clinical Perspective on p 291Because of their incapacity to regulate glucose influx, endothelial cells (ECs) represent an important target for diabetes mellitus-induced damage. In particular, it is well established that ECs cultured in high glucose show delayed replication, 3,4 abnormal cell cycling, 5 and increased apoptosis. 6 Progression through the cell cycle is a tightly regulated process that includes multiple checkpoints. An orderly ex- The present study is the first to provide evidence for a role of miRNAs in diabetes mellitus-induced endothelial defects contributing to impaired postischemic angiogenesis. In fact, here we show that in vitro culture conditions mimicking diabetes mellitus and ischemia upregulate miR-503 in ECs and that, in vivo, diabetes mellitus increases miR-503 expression in ECs from ischemic limb muscles. We also show that increased miR-503 is responsible for repressed cdc25A and CCNE1 expression in ECs cultured under conditions mimicking diabetes mellitus and ischemia. Moreover, miR-503-forced expression inhibited EC proliferation, migration, and network formation on Matrigel and it additionally reduced vascular smooth muscle ce...

Objective-MicroRNAs (miRNAs) are small noncoding RNAs that have the capacity to control protein production through binding "seed" sequences within a target mRNA. Each miRNA is capable of potentially controlling hundreds of genes. The regulation of miRNAs in the lung during the development of pulmonary arterial hypertension (PAH) is unknown. Methods and Results-We screened lung miRNA profiles in a longitudinal and crossover design during the development of PAH caused by chronic hypoxia or monocrotaline in rats. We identified reduced expression of Dicer, involved in miRNA processing, during the onset of PAH after hypoxia. MiR-22, miR-30, and let-7f were downregulated, whereas miR-322 and miR-451 were upregulated significantly during the development of PAH in both models. Differences were observed between monocrotaline and chronic hypoxia. For example, miR-21 and let-7a were significantly reduced only in monocrotaline-treated rats. MiRNAs that were significantly regulated were validated by quantitative polymerase chain reaction. By using in vitro studies, we demonstrated that hypoxia and growth factors implicated in PAH induced similar changes in miRNA expression. Furthermore, we confirmed miR-21 downregulation in human lung tissue and serum from patients with idiopathic PAH. Conclusion-Defined miRNAs are regulated during the development of PAH in rats. Therefore, miRNAs may contribute to the pathogenesis of PAH and represent a novel opportunity for therapeutic intervention. Key Words: pulmonary hypertension Ⅲ small RNA molecules Ⅲ gene regulation P ulmonary arterial hypertension (PAH) is a complex disorder characterized by the obstructive remodeling of pulmonary arteries, leading to a progressive elevation in pulmonary arterial pressure (PAP) and subsequent right-sided heart failure and death. 1 Familial PAH is associated in 80% of cases with diverse heterozygous mutations in the gene-encoding bone morphogenetic protein receptor 2 (BMPR-II) 2 and can be associated with mutations in the activin-receptor kinaselike 1 gene. 3 The cause of the variable phenotypic expression of PAH among carriers of mutated BMPR-II genes is unclear, and is likely related to environmental and genetic modifiers. Although BMPR-II-related pathways are considered pivotal, many other mediator pathways participate in the pathogenesis of PAH and are being actively investigated, both independently and in combination. For example, the involvement of serotonin in the development of experimental PAH has been recently reported. 4,5 Indeed, important interactions between the serotonin and BMP pathways have recently been described. 6 Rats exposed to hypoxia or injected with the toxin monocrotaline develop pulmonary arterial changes correlated with the development of PAH, including remodeling and elevating PAP.MicroRNAs (miRNAs) are small noncoding transcripts of 16 to 29 nucleotide RNAs that regulate gene expression posttranscriptionally by targeting mRNAs. Animal miRNAs are processed from longer primary transcripts (primary miRNAs) that can contain ...