Background Pulmonary hypertension (PH) is driven by diverse pathogenic etiologies. Owing to their pleiotropic actions, microRNA (miRNA) are potential candidates for coordinated regulation of these disease stimuli. Methods and Results Using a network biology approach, we identify miRNA associated with multiple pathogenic pathways central to PH. Specifically, microRNA-21 (miR-21) is predicted as a PH-modifying miRNA, regulating targets integral to bone morphogenetic protein (BMP) and Rho/Rho kinase signaling as well as functional pathways associated with hypoxia, inflammation, and genetic haplo insufficiency of the BMP Receptor Type 2 (BMPRII). To validate these predictions, we have found that hypoxia and BMPRII signaling independently up-regulate miR-21 in cultured pulmonary arterial endothelial cells. In a reciprocal feedback loop, miR-21 down-regulates BMPRII expression. Furthermore, miR-21 directly represses RhoB expression and Rho kinase activity, inducing molecular changes consistent with decreased angiogenesis and vasodilation. In vivo, miR-21 is up-regulated in pulmonary tissue from several rodent models of PH and in humans with PH. Upon induction of disease in miR-21-null mice, RhoB expression and Rho-kinase activity are increased, accompanied by exaggerated manifestations of PH. Conclusions A network-based bioinformatic approach coupled with confirmatory in vivo data delineates a central regulatory role for miR-21 in PH. Furthermore, this study highlights the unique utility of network biology for identifying disease-modifying miRNA in PH.
Iron–sulfur (Fe-S) clusters are essential for mitochondrial metabolism, but their regulation in pulmonary hypertension (PH) remains enigmatic. We demonstrate that alterations of the miR-210-ISCU1/2 axis cause Fe-S deficiencies in vivo and promote PH. In pulmonary vascular cells and particularly endothelium, hypoxic induction of miR-210 and repression of the miR-210 targets ISCU1/2 down-regulated Fe-S levels. In mouse and human vascular and endothelial tissue affected by PH, miR-210 was elevated accompanied by decreased ISCU1/2 and Fe-S integrity. In mice, miR-210 repressed ISCU1/2 and promoted PH. Mice deficient in miR-210, via genetic/pharmacologic means or via an endothelial-specific manner, displayed increased ISCU1/2 and were resistant to Fe-S-dependent pathophenotypes and PH. Similar to hypoxia or miR-210 overexpression, ISCU1/2 knockdown also promoted PH. Finally, cardiopulmonary exercise testing of a woman with homozygous ISCU mutations revealed exercise-induced pulmonary vascular dysfunction. Thus, driven by acquired (hypoxia) or genetic causes, the miR-210-ISCU1/2 regulatory axis is a pathogenic lynchpin causing Fe-S deficiency and PH. These findings carry broad translational implications for defining the metabolic origins of PH and potentially other metabolic diseases sharing similar underpinnings.
Background Glutathione peroxidase-3 (GPx-3) is a selenocysteine-containing plasma protein that scavenges reactive oxygen species in the extracellular compartment. A deficiency of this enzyme has been associated with platelet-dependent thrombosis, and a promoter haplotype with reduced function has been associated with stroke risk in young individuals. Methods and Results We recently developed a genetic mouse model to assess platelet function in hemostasis and thrombosis in the setting of GPx-3 deficiency. GPx-3(−/−) mice showed an attenuated bleeding time compared with wild-type mice. Platelet aggregation studies revealed an enhanced aggregation response to the agonist ADP in GPx-3(−/−) compared to wild-type mice. We also found an increase in the plasma levels of soluble P-selectin and a decrease in plasma cyclic GMP in GPx-3(−/−) mice compared with wild-type mice. ADP was infused into the right ventricle of mice to induce platelet aggregation in the pulmonary vasculature, and produced a more robust platelet activation response in the GPx-3(−/−) mice than in wild-type mice; histological sections from the pulmonary vasculature of GPx-3(−/−) compared with wild-type mice show increased platelet-rich thrombi and a higher percentage of occluded vessels. Endothelial function studies using a cremaster muscle preparation revealed dysfunction in the GPx-3(−/−) compared to wild-type mice. Using a no-flow ischemia-reperfusion stroke model, GPx-3(−/−) mice had significantly larger cerebral infarctions compared with wild-type mice. To investigate the effect of platelet inhibition on stroke size in GPx-3 deficiency, we found that clopidogrel treatment reduced stroke size significantly in GPx-3(−/−) mice compared with vehicle-treated controls. To assess the neuroprotective role of antioxidants in this model, we found that MnTBAP treatment reduced stroke size in GPx-3(−/−) mice compared with vehicle-treated controls. Conclusions These findings demonstrate that GPx-3 deficiency results in a prothrombotic state and vascular dysfunction that promotes platelet-dependent arterial thrombosis. These data illustrate the importance of this plasma antioxidant enzyme in regulating platelet activity, endothelial function, platelet-dependent thrombosis, and vascular thrombotic propensity.
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