Angiomyogenesis with HIF-1a responsive microRNAs

摘要：干细胞通过多因子机制促进实验动物模型和心肌梗死患者的功能恢复。我们假设用缺氧诱导因子-11 （HIF-11）应答microRNAs （HRMs）重编程的干细胞会促进梗死心脏的血管生成。从维持谱系承诺到血管生成旁分泌活性和分化潜力，MicroRNAs （mirnas）决定了具有离散mirnas谱的干细胞的固有特性。因此，通过操纵miRs对干细胞进行重编程可以提高它们的旁分泌活性和血管生成潜能。我们已经发现，在短暂缺氧/再氧化（a/ R）间歇循环的重编程过程中，间充质干细胞（MSCs）中的一些HRMs下调或上调，同时激活HIF-11。改变最显著的HRMs是miR-210和miR-107，它们显著影响MSCs的存活和血管生成旁分泌行为（初步结果）。本研究的总体目标是阐明HRMs及其靶基因在重编程过程中介导干细胞旁分泌活性、血管生成分化和存活等功能的机制。我们的建议旨在验证三个假设。假设1:HRMs调节干细胞血管生成旁分泌活性。与假设1相关的具体目标将定义重编程间充质干细胞（RePMSCs）中离散HRMs组的表达谱，特别强调它们的血管生成旁分泌活性。假设2:hrm是重编程干细胞功能结果的机制决定因素。相关的具体目标将涉及体外血管生成模型和急性冠状动脉结扎的实验动物模型，以表明重编程在HRMs的机制参与下增强了干细胞的血管生成潜力。体内研究的终点将包括血管密度分析、成熟指数、缺血性心脏区域血流改善和整体心功能的保存。还将对缺血心脏移植RePMSCs和直接注射HRMs进行比较。假设3:HRMs机制参与干细胞存活。我们已经证明HRMs能显著提高干细胞存活率。我们的初步研究表明，在A/R重复循环处理的干细胞中，有4种不同的抗凋亡基因上调。我们将阐明每一个已确定的HRMs靶基因在干细胞存活中的作用。我们还将证实，移植RePMSCs后梗死心脏中HRMs的变化是心脏修复的一个完整的新机制。综上所述，我们认为干细胞通过间歇性的A/R循环和HRMs的机制参与重编程，将促进其在梗死心脏中的旁分泌、血管生成和细胞保护反应。这些研究将促进基于供体干细胞中HRMs操作的新型治疗方法的发展。

DESCRIPTION (provided by applicant): Angiomyogenesis with HIF-11 responsive microRNAs Summary: Stem cells promote functional recovery in experimental animal models and patients with myocardial infarction through multifactorial mechanisms. We hypothesize that stem cells reprogrammed with hypoxia inducible factor-11 (HIF-11) responsive microRNAs (HRMs) will promote angiomyogenesis in the infarcted heart. Ranging from maintenance of lineage commitment to angiogenic paracrine activity and differentiation potential, MicroRNAs (miRs) determine the inherent properties of stem cells which possess a discrete miRs profile. Hence, reprogramming of stem cells by manipulation of miRs can improve their paracrine activity and angiomyogenic potential. We have identified a number of HRMs which are down- or upregulated in mesenchymal stem cells (MSCs) during reprogramming by intermittent cycles of brief anoxia/re-oxygenation (A/R) in parallel with activation of HIF-11. The most significantly altered HRMs were miR-210 and miR-107 which markedly influenced the survival and angiogenic paracrine behavior of MSCs (Preliminary results). The overall goal of this proposal is to elucidate the mechanistic involvement of HRMs and their target genes during reprogramming which mediate stem cell functions of paracrine activity, angiomyogenic differentiation and survival. Our proposal is aimed to validate three hypotheses. Hypothesis-1: HRMs regulate angiogenic paracrine activity in stem cells. The specific aims pertinent to hypothesis-1 will define the expression profile of the discrete set of HRMs in the reprogrammed MSCs (RePMSCs) with special emphasis on their angiogenic paracrine activity. Hypothesis-2: HRMs are mechanistic determinants of functional consequences of the reprogrammed stem cells. The relevant specific aim will involve in vitro models of angiogenesis and experimental animal model of acute coronary artery ligation to show that the reprogramming accentuate the angiogenic potential of stem cells with the mechanistic participation of HRMs. The end-points of in vivo studies will include blood vessel density analysis, maturation index and improved regional blood flow in the ischemic heart and preservation of global cardiac function. A comparison between RePMSCs engraftment and direct injection of HRMs to the ischemic heart will also be carried out. Hypothesis-3: HRMs mechanistically participate in stem cell survival. We have already shown that HRMs significantly improve stem cell survival. Our preliminary studies showed that 4 different anti-apoptotic genes were up-regulated in the stem cells treated with repeated cycles of A/R. We will elucidate the role of each one of the identified target genes of HRMs in stem cell survival. We will also establish that changes in HRMs in the infarcted heart after RePMSCs engraftment constitute an integral and novel mechanism of cardiac repair. Put together, we propose that stem cells reprogrammed by intermittent cycles of A/R with mechanistic involvement of HRMs, will promote their paracrine, angiomyogenic and cytoprotective responses in the infarcted heart. These studies will facilitate the development of novel therapeutic approaches based on manipulation of HRMs in donor stem cells. PUBLIC HEALTH RELEVANCE: This proposal is designed to investigate angiomyogenic and paracrine behavior of stem cells reprogrammed by manipulation of HIF-11 responsive microRNAs (HRMs) using our novel approach of treating the cells with intermittent multiple short cycles of anoxia/reoxygenation. We anticipate improved angiomyogenic response in the infarcted heart after treatment with reprogrammed stem cells. Our proposal will be the first to elucidate the mechanistic participation of HRMs in paracrine behavior, angiomyogenic potential and cytoprotection.