Abstract
The importance of microRNA (miRNA) to vascular biology is becoming increasingly evident; however, the function of a significant number of miRNA remains to be determined. In particular, the effect of growth factor regulation of miRNAs on endothelial cell morphogenesis is incomplete. Thus, we aimed to identify miRNAs regulated by pro-angiogenic vascular endothelial growth factor (VEGF) and determine the effects of VEGF-regulated miRNAs and their targets on processes important for angiogenesis. Human umbilical vein endothelial cells (HUVECs) were thus stimulated with VEGF and miRNA levels assessed using microarrays. We found that VEGF altered expression of many miRNA, and for this study focused on one of the most significantly down-regulated miRNA in HUVECs following VEGF treatment, miR-30b. Using specific miRNA mimics, we found that overexpression of miR-30b inhibited capillary morphogenesis in vitro, while depletion of endogenous miR-30b resulted in increased capillary morphogenesis indicating the potential significance of down-regulation of miR-30b as a pro-angiogenic response to VEGF stimulation. MiR-30b overexpression in HUVEC regulated transforming growth factor beta 2 (TGFβ2) production, which led to increased phosphorylation of Smad2, indicating activation of an autocrine TGFβ signaling pathway. Up-regulation of TGFβ2 by miR-30b overexpression was found to be dependent on ATF2 activation, a transcription factor known to regulate TGFβ2 expression, as miR-30b overexpressing cells exhibited increased levels of phosphorylated ATF2 and depletion of ATF2 inhibited miR-30b-induced TGFβ2 expression. However, miR-30b effects on ATF2 were indirect and found to be via targeting of the known ATF2 repressor protein JDP2 whose mRNA levels were indirectly correlated with miR-30b levels. Increased secretion of TGFβ2 from HUVEC was shown to mediate the inhibitory effects of miR-30b on capillary morphogenesis as treatment with a neutralizing antibody to TGFβ2 restored capillary morphogenesis to normal levels in miR-30b overexpressing cells. These results support that the regulation of miR-30b by VEGF in HUVEC is important for capillary morphogenesis, as increased miR-30b expression inhibits capillary morphogenesis through enhanced expression of TGFβ2.
Highlights
Angiogenesis is the process of new blood vessel growth from pre-existing vessels
We examined vascular endothelial growth factor (VEGF) regulation of miRNA expression in human umbilical vein endothelial cells (HUVEC) in order to identify candidate miRNAs that may function in response to VEGF-promotion of angiogenesis
Furthering our understanding of VEGF-mediated miRNA expression in angiogenic processes, we have identified miR-30b as a VEGF regulated miRNA with a negative regulatory role in capillary morphogenesis of HUVEC
Summary
Angiogenesis is the process of new blood vessel growth from pre-existing vessels. It is a complex tightly regulated process that involves a number of coordinated steps for vessel formation to occur. Expression profiling of human umbilical vein endothelial cells (HUVEC) [6] and subsequently other endothelial cell types [7] has provided insight into the importance of individual miRNA expression patterns to endothelial cell biology. Since those initial studies, roles for individual miRNAs in angiogenic processes are increasingly being identified with both pro- [8,9,10,11,12,13] and anti-angiogenic [14,15,16,17] effects being observed. A better understanding of the roles of individual specific miRNAs is vitally important for determining the feasibility of manipulating such miRNAs for therapeutic purposes to combat pathological angiogenesis
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