Ischemic cardiac and cerebral vascular diseases continue to represent a significant and growing source of morbidity and mortality despite advances in traditional treatments. Recently, novel emerging therapeutic strategies focus on stem cells. Adipose tissue derived stem cells (ASCs), which are easily acquired and have reduced surface histocompatibility antigens increasing their allo-transplantation potential, were demonstrated to have beneficial effect on ischemic heart and limb in animals likely due to a paracrine function rather than transdifferentiation. Besides secreted soluble growth factors, cell-released microvesicles (MVs) have been recently described as a new mechanism of intercellular communication. MVs play an important role in cell biologic processes not only by specifically targeting recipient cells to deliver proteins, lipids and/or trigger downstream signalng events, but also by transferring genetic material, mRNA and microRNA. Our preliminary studies for the first time demonstrated that 1). MVs are released from human ASCs (hASCs) and promote vascular endothelial cell migration, 2). They contain RNA, mostly small RNA, 3). These small RNAs, rich in angiogenesis-regulating miRNAs, are proangiogenic. Although the importance of cognate miRNA in angiogenesis and endothelial function has been addressed, the mechanism for of angiogenic effect of miRNAs present in stem cell-released MVs is so far unknown. The goal of this proposal is to unravel the mechanistic aspects of the proangiogenic effect of hASCs-MVs in detail. We hypothesize that microRNAs in MVs secreted by hASCs is proangiogenic. In Aim 1 we will examine the angiogenic effects of miRNAs in hASCs-released MVs using two loss-of-function strategies. hASCs will be transduced with Lentivirus-based specific anti-miRNA hairpin expression construct and the angiogenic potential of the ensuing MVs on the endothelial cells will be assessed. Vascular endothelial cells will be co-transfected with specific anti-miRNA inhibitor and small RNAs isolated from hASCs-released MVs and assessed for angiogenesis. In Aim 2, we will determine the target genes of proangiogenic miRNA from hASCs-released MVs in vascular endothelial cells by profiling analysis of mRNA and proteins. The predicted target genes of miRNA from hASCs-released MVs in recipient cells will be confirmed by using a 3'UTR insertion reporter constructs. These studies will expand our currently scant knowledge of mechanistic aspects of proangiogenic miRNA present in stem cell-released MVs as well as shed light on their paracrine/endocrine properties and set the basis for their use as a novel therapeutic approach for cerebro-vascular disease.

SC2GM099629

2012-04-10

2015-03-31