Proangiogenic microRNA in Microvesicles Released from Adipose-derived Stem Cells

说明(申请人提供)：尽管传统治疗方法取得了进步，但缺血性心脏和脑血管疾病仍是发病率和死亡率的一个重要且不断增加的来源。最近，新出现的治疗策略集中在干细胞上。脂肪组织来源的干细胞(ASCs)是一种易获得的干细胞，可减少表面组织相容性抗原，增加其同种异体移植的潜能，可能通过旁分泌功能而不是转分化对动物的缺血心脏和肢体产生有益的影响。除了分泌可溶性生长因子外，细胞释放的微囊泡(MVS)最近被描述为一种新的细胞间通讯机制。MVS在细胞生物学过程中发挥着重要作用，不仅通过特异性靶向受体细胞传递蛋白质、脂类和/或触发下游信号事件，而且还通过转移遗传物质、mRNA和microRNA。我们的初步研究首次证明了这一点。人血管内皮细胞(HASCs)释放血管内皮细胞，促进血管内皮细胞迁移。它们含有RNA，主要是小RNA，3)。这些小RNA富含调节血管生成的miRNAs，是促血管生成的。虽然同源miRNA在血管生成和内皮功能中的重要性已经被阐明，但干细胞释放的MVS中存在的miRNAs的血管生成作用的机制到目前为止还不清楚。该提案的目的是详细揭示hASCs-MVS促血管生成效应的机制方面。我们假设hASCs分泌的MVS中的microRNAs是促血管生成的。在目标1中，我们将使用两种功能丧失策略来研究miRNAs在hASCs释放的MVS中的血管生成效应。将以慢病毒为基础的特异性抗miRNA发夹表达载体转导hASCs，随后的MVS在内皮细胞上的血管生成潜力将被评估。血管内皮细胞将与特定的抗miRNA抑制剂和从hASCs释放的MVS中分离的小RNA共转染，并评估血管生成。在目标2中，我们将通过对mRNA和蛋白质的图谱分析，从hASCs释放的血管内皮细胞中确定促血管生成miRNA的靶基因。从hASCs释放的受体细胞中预测的miRNA的靶基因将通过使用3‘UTR插入报告构建来确认。这些研究将扩大我们目前对干细胞释放的MVs中存在的促血管生成miRNA的机制方面的缺乏的了解，并阐明它们的旁分泌/内分泌特性，为它们作为一种新的脑血管疾病治疗方法奠定基础。 公共卫生相关性：尽管医学、介入治疗方面取得了重大进展，但全球范围内的缺血性心脏病和中风的流行迫切需要创新的治疗方法。 以及这些疾病的外科治疗。近年来，干细胞治疗策略受到越来越多的关注，尤其是从脂肪组织中容易获得干细胞。我们的项目是检查旨在支持和促进这一策略使用的分子机制。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The worldwide epidemic of ischemic heart disease and stroke urgently requires innovative treatments in spite of significant advances in medical, interventional and surgical therapy for these diseases. Recently, therapeutic strategies using stem cells have been attracting more and more attention, especially easily acquired stem cells from fat tissues. Our project is to inspect molecular mechanisms aimed to support and facilitate the use of this strategy.