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 ...

Background-Pulmonary arterial hypertension (PAH) is a hyperproliferative vascular disorder observed predominantly in women. Estrogen is a potent mitogen in human pulmonary artery smooth muscle cells and contributes to PAH in vivo; however, the mechanisms attributed to this causation remain obscure. Curiously, heightened expression of the estrogenmetabolizing enzyme cytochrome P450 1B1 (CYP1B1) is reported in idiopathic PAH and murine models of PAH. Methods and Results-Here, we investigated the putative pathogenic role of CYP1B1 in PAH. Quantitative reverse transcriptionpolymerase chain reaction, immunoblotting, and in situ analysis revealed that pulmonary CYP1B1 is increased in hypoxic PAH, hypoxicϩSU5416 PAH, and human PAH and is highly expressed within the pulmonary vascular wall. PAH was assessed in mice via measurement of right ventricular hypertrophy, pulmonary vascular remodeling, and right ventricular systolic pressure. Hypoxic PAH was attenuated in CYP1B1 Ϫ/Ϫ mice, and the potent CYP1B1 inhibitor 2,3Ј,4,5Ј-tetramethoxystilbene (TMS; 3 mg ⅐ kg Ϫ1 ⅐ d

Rationale The pathogenesis of PAH remains unclear. The four microRNAs representing the miR-143 and miR-145 stem loops are genomically clustered. Objective To elucidate the transcriptional regulation of the miR-143/145 cluster, and the role of miR-143 in PAH. Methods and Results We identified the promoter region that regulates miR-143/145 miRNA expression in pulmonary artery smooth muscle cells (PASMCs). We mapped PAH-related signalling pathways, including estrogens receptor (ER), liver X factor/retinoic X receptor (LXR/RXR), TGF-β (Smads), and hypoxia (HRE) that regulated levels of all pri-miR stem loop transcription and resulting miRNA expression. We observed that miR-143-3p is selectively upregulated compared to miR-143-5p during PASMC migration. Modulation of miR-143 in PASMCs significantly altered cell migration and apoptosis. In addition, we found high abundance of miR-143-3p in PASMCs-derived exosomes. Using assays with pulmonary arterial endothelial cells (PAECs) we demonstrated a paracrine pro-migratory and pro-angiogenic effect of miR-143-3p enriched exosomes from PASMC. Quantitative PCR and in situ hybridisation showed elevated expression of miR-143 in calf models of PAH as well as in samples from PAH patients. Moreover, in contrast to our previous findings that had not supported a therapeutic role in vivo, we now demonstrate a protective role for miR-143 in experimental PH in vivo in miR-143−/− and antimiR143-3p-treated mice exposed to chronic hypoxia in both preventative and reversal settings. Conclusions miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while inhibition of miR-143-3p blocked experimental PH. Taken together these findings confirm an important role for the miR-143/145 cluster in PAH pathobiology.

Chronic kidney disease coexists with metabolic syndrome and this relationship may be apparent before overt manifestations of cardiovascular disease. To investigate early stages of the natural history of associations between renal function and metabolic syndrome, we phenotyped 1572 young (mean age=18.4 years), apparently healthy men for metabolic risk factors and estimated their creatinine clearance based on the Cockcroft-Gault equation. High metabolic risk (clustering of at least three metabolic risk factors) was revealed in 8.7% (137) of the subjects and was associated with a 6.9-fold increase in the odds of glomerular hyperfiltration (95% confidence interval (CI): 3.9-11.5) when compared to reference (from none to two metabolic risk factors). Overweight, elevated blood pressure, and low high-density lipoprotein (HDL) cholesterol increased the multivariate-adjusted odds ratio of glomerular hyperfiltration to 6.6 (95% CI: 3.8-11.6), 1.8 (95% CI: 1.0-3.0), and 2.5 (95% CI: 1.5-4.3), respectively. Systolic and diastolic blood pressures clustered together with leptin in the factor analysis and this blood pressure-adiposity component correlated with estimated creatinine clearance (r=0.329, P<0.0001) and explained on its own 10.2% of the variance in the estimated renal function. Our data reveal the silent epidemics of metabolic risk among young, apparently healthy men. Furthermore, the results indicate that high metabolic risk is associated with glomerular hyperfiltration before overt manifestations of cardiovascular disease.

Rationale: Despite improved understanding of the underlying genetics, pulmonary arterial hypertension (PAH) remains a severe disease. Extensive remodeling of small pulmonary arteries, including proliferation of pulmonary artery smooth muscle cells (PASMCs), characterizes PAH. MicroRNAs (miRNAs) are noncoding RNAs that have been shown to play a role in vascular remodeling. Objective:We assessed the role of miR-145 in PAH. Methods and Results:We localized miR-145 in mouse lung to smooth muscle. Using quantitative PCR, we demonstrated increased expression of miR-145 in wild-type mice exposed to hypoxia. PAH was evaluated in miR-145 knockout and mice treated with anti-miRs via measurement of systolic right ventricular pressure, right ventricular hypertrophy, and percentage of remodeled pulmonary arteries. miR-145 deficiency and anti-miR-mediated reduction resulted in significant protection from the development of PAH. In contrast, miR-143 anti-miR had no effect. Furthermore, we observed upregulation of miR-145 in lung tissue of patients with idiopathic and heritable PAH compared with unaffected control subjects and demonstrated expression of miR-145 in SMC of remodeled vessels from such patients. Finally, we show elevated levels of miR-145 expression in primary PASMCs cultured from patients with BMPR2 mutations and also in the lungs of BMPR2-deficient mice. Key Words: pulmonary hypertension Ⅲ hypoxia Ⅲ molecular biology Ⅲ smooth muscle cells Ⅲ microRNA Ⅲ smooth muscle differentiation Ⅲ remodeling P ulmonary arterial hypertension (PAH) is a disease of the small pulmonary arteries (PAs), characterized by an increase in PA pressure and vascular remodeling leading to a progressive increase in pulmonary vascular resistance. 1 The consequence of vascular obliteration is right heart failure and high mortality. 2,3 Germline mutations in the gene coding for the bone morphogenetic protein (BMP) type-2 receptor (BMPR2), a receptor for the transforming growth factor (TGF)-␤ superfamily, have been identified in approximately 70% of patients with the heritable form of PAH (hPAH). 4 Moreover, BMPR2 expression is markedly reduced in PAH cases in the absence of mutations in this gene (idiopathic PAH, iPAH). In pulmonary artery smooth muscle cells (PASMCs), mutations in BMPR2 are associated with an abnormal growth response to BMPs and TGF-␤. 5 In pulmonary artery endothelial cells (PAECs), these mutations increase the susceptibility of cells to apoptosis. 4,6 The absence of BMPR2 mutations in some families and in the majority of iPAH cases suggests that further pathological mechanisms still need to be identified. One of the main histopathologic features common to all forms of PAH is the accumulation of cells expressing smooth muscle specific ␣-actin (SMA) in Original received February 23, 2012; revision received June 11, 2012; accepted June 11, 2012. In May 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.0 days. MicroRNAs (miRNAs) are a class of ...

Astrocytes undergo major phenotypic changes in response to injury and disease that directly influence repair in the CNS, but the mechanisms involved are poorly understood. Previously, we have shown that neurosphere-derived rat astrocytes plated on poly-L-lysine (PLL-astrocytes) support myelination in dissociated rat spinal cord cultures (myelinating cultures). It is hypothesized that astrocyte reactivity can affect myelination, so we have exploited this culture system to ascertain how two distinct astrocyte phenotypes influence myelination. Astrocytes plated on tenascin C (TnC-astrocytes), a method to induce quiescence, resulted in less myelinated fibers in the myelinating cultures when compared with PLL-astrocytes. In contrast, treatment of myelinating cultures plated on PLL-astrocytes with ciliary neurotrophic factor (CNTF), a cytokine known to induce an activated astrocyte phenotype, promoted myelination. CNTF could also reverse the effect of quiescent astrocytes on myelination. A combination of microarray gene expression analysis and quantitative real-time PCR identified CXCL10 as a potential candidate for the reduction in myelination in cultures on TnC-astrocytes. The effect of TnC-astrocytes on myelination was eliminated by neutralizing CXCL10 antibodies. Conversely, CXCL10 protein inhibited myelination on PLL-astrocytes. Furthermore, CXCL10 treatment of purified oligodendrocyte precursor cells did not affect proliferation, differentiation, or process extension compared with untreated controls, suggesting a role in glial/axonal ensheathment. These data demonstrate a direct correlation of astrocyte phenotypes with their ability to support myelination. This observation has important implications with respect to the development of therapeutic strategies to promote CNS remyelination in demyelinating diseases.

Despite the increasing importance of long noncoding RNA in physiology and disease, their role in endothelial biology remains poorly understood. Growing evidence has highlighted them to be essential regulators of human embryonic stem cell differentiation. SENCR, a vascular-enriched long noncoding RNA, overlaps the Friend Leukemia Integration virus 1 (FLI1) gene, a regulator of endothelial development. Therefore, we wanted to test the hypothesis that SENCR may contribute to mesodermal and endothelial commitment as well as in endothelial function. We thus developed new differentiation protocols allowing generation of endothelial cells from human embryonic stem cells using both directed and hemogenic routes. The expression of SENCR was markedly regulated during endothelial commitment using both protocols. SENCR did not control the pluripotency of pluripotent cells; however its overexpression significantly potentiated early mesodermal and endothelial commitment. In human umbilical endothelial cell (HUVEC), SENCR induced proliferation, migration, and angiogenesis. SENCR expression was altered in vascular tissue and cells derived from patients with critical limb ischemia and premature coronary artery disease compared to controls. Here, we showed that SENCR contributes to the regulation of endothelial differentiation from pluripotent cells and controls the angiogenic capacity of HUVEC. These data give novel insight into the regulatory processes involved in endothelial development and function.

iFR correlates weakly with FFR and is not independent of hyperemia. iFR cannot be recommended for clinical decision making in patients with coronary artery disease.