MicroRNA-regulated pathways of flow-stimulated angiogenesis and vascular remodeling in vivo.

BACKGROUND: Vascular shear stress promotes endothelial cell sprouting in vitro. The impact of hemodynamic forces on microRNA (miRNA) and gene expression within growing vascular networks in vivo, however, remain poorly investigated. Arteriovenous (AV) shunts are an established model for induction of neoangiogenesis in vivo and can serve as a tool for analysis of hemodynamic effects on miRNA and gene expression profiles over time. METHODS: AV shunts were microsurgically created in rats and explanted on postoperative days 5, 10 and 15. Neoangiogenesis was confirmed by histologic analysis and micro-computed tomography. MiRNA and gene expression profiles were determined in tissue specimens from AV shunts by microarray analysis and quantitative real-time polymerase chain reaction and compared with sham-operated veins by bioinformatics analysis. Changes in protein expression within AV shunt endothelial cells were determined by immunohistochemistry. RESULTS: Samples from AV shunts exhibited a strong overexpression of proangiogenic cytokines, oxygenation-associated genes (HIF1A, HMOX1), and angiopoetic growth factors. Significant inverse correlations of the expressions of miR-223-3p, miR-130b-3p, miR-19b-3p, miR-449a-5p, and miR-511-3p which were up-regulated in AV shunts, and miR-27b-3p, miR-10b-5p, let-7b-5p, and let-7c-5p, which were down-regulated in AV shunts, with their predicted interacting targets C-X-C chemokine receptor 2 (CXCR2), interleukin-1 alpha (IL1A), ephrin receptor kinase 2 (EPHA2), synaptojanin-2 binding protein (SYNJ2BP), forkhead box C1 (FOXC1) were present. CXCL2 and IL1A overexpression in AV shunt endothelium was confirmed at the protein level by immunohistochemistry. CONCLUSIONS: Our data indicate that flow-stimulated angiogenesis is determined by an upregulation of cytokines, oxygenation associated genes and miRNA-dependent regulation of FOXC1, EPHA2 and SYNJ2BP.

Role of plasma membrane-associated AKAPs for the regulation of cardiac IK1 current by protein kinase A.

The cardiac IK1 current stabilizes the resting membrane potential of cardiomyocytes. Protein kinase A (PKA) induces an inhibition of IK1 current which strongly promotes focal arrhythmogenesis. The molecular mechanisms underlying this regulation have only partially been elucidated yet. Furthermore, the role of A-kinase anchoring proteins (AKAPs) in this regulation has not been examined to date. The objective of this project was to elucidate the molecular mechanisms underlying the inhibition of IK1 by PKA and to identify novel molecular targets for antiarrhythmic therapy downstream ß-adrenoreceptors. Patch clamp and voltage clamp experiments were used to record currents and co-immunoprecipitation, and co-localization experiments were performed to show spatial and functional coupling. Activation of PKA inhibited IK1 current in rat cardiomyocytes. This regulation was markedly attenuated by disrupting PKA-binding to AKAPs with the peptide inhibitor AKAP-IS. We observed functional and spatial coupling of the plasma membrane-associated AKAP15 and AKAP79 to Kir2.1 and Kir2.2 channel subunits, but not to Kir2.3 channels. In contrast, AKAPyotiao had no functional effect on the PKA regulation of Kir channels. AKAP15 and AKAP79 co-immunoprecipitated with and co-localized to Kir2.1 and Kir2.2 channel subunits in ventricular cardiomyocytes. In this study, we provide evidence for coupling of cardiac Kir2.1 and Kir2.2 subunits with the plasma membrane-bound AKAPs 15 and 79. Cardiac membrane-associated AKAPs are a functionally essential part of the regulatory cascade determining IK1 current function and may be novel molecular targets for antiarrhythmic therapy downstream from ß-adrenoreceptors.