Excellence in Research: Bioengineered extracellular vesicles from stem cells and macrophages act synergistically in angiogenesis

细胞外小泡(EVS)是细胞释放的微粒，通过运送货物在细胞间的交流中发挥重要作用。传统的研究EV的方法侧重于研究来自单个细胞类型的EV，而忽略了EV由所有类型的细胞分泌并共存于体内各种细胞外液的可能性。该项目对不同来源的电动汽车在促进血管形成方面的协同效应进行了创新探索。此外，EV细胞将通过生物工程技术进行修饰，以增强EV的生产和促血管生成(血管形成)活性。该项目的成功完成将促进我们在两个关键领域的理解：1)来自不同细胞类型的EV在正常生理过程和病理反应中的协同作用；2)探索利用来自多种来源的EV的组合来探索新的治疗策略。此外，还设计了一项综合教育计划，以增加来自传统上在科学领域代表性不足的群体的学生的参与。该项目将为学生提供实施和分析每个目标的研究培训，并提供校内和校外展示他们的发现的机会。该项目的成果将通过出版物和会议报告以学术形式传播，并将纳入与血管形成有关的现有课程。血管生成是一个严格调控的过程，对于胚胎发育和维持成年生物体的血管稳态是必不可少的。干细胞和巨噬细胞通过细胞分化和因子分泌参与血管生成的调节。最近，细胞分泌的囊泡被认为与血管生成过程中的一系列介质有关。由细胞释放的两类主要非凋亡性囊泡--外切体和微泡被归类为EVS。与活细胞不同，EV是亚微米级的囊泡，以受体细胞为目标，以无细胞的方式运送它们的货物，包括RNA、蛋白质和脂质。以前的文献已经证明，某些microRNAs(MiRs)参与了血管生成，即所谓的AngiomiRs。本项目的目的是研究来自小鼠间充质干细胞和巨噬细胞的EVS在体外和体内对血管生成的协同作用。根据之前的报道，干细胞和巨噬细胞将被提前改造，分别过表达miR-31和miR-30。从两种类型的工程细胞中释放的EVS的混合物在体外对血管内皮细胞的血管生成作用将被检测，包括增殖、迁移和管道形成，并与任何一种类型的EVS进行比较。为了进一步探索新生血管的功能，我们将在小鼠后肢缺血(HLI)模型上研究两种工程EV的混合对血管生成的影响。此外，将测试miR-31/FIH1和miR-30/cul2信号通路在HIF-1α反式激活中的协同作用，HIF-1是促血管生成的关键步骤。这项研究的结果将是全面了解不同的电动汽车如何监管血管生成。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Extracellular vesicles (EVs) are cell-released microparticles that play essential roles in cell-cell communication by delivering their cargo. Traditional approaches to studying EVs have focused on investigating EVs derived from individual cell types, disregarding the likelihood that EVs are secreted by all cell types and coexist in various extracellular fluids in the body. This project introduces an innovative exploration of the collaborative effects of EVs from diverse sources in promoting the formation of blood vessels. Additionally, the EV cells will be modified through bioengineering techniques to enhance the production and proangiogenic (blood vessel forming) activities of EVs. Successful completion of this project will advance our understanding in two key areas: 1) the cooperative actions of EVs from different cell types in normal physiological processes and pathological responses, and 2) the exploration of novel therapeutic strategies utilizing EVs derived from multiple sources in combination. Furthermore, an integrated education plan is designed to increase the participation of students from groups traditionally underrepresented in science. This project will provide students with research training in the implementation and analysis of each objective and with intramural and extramural opportunities to present their findings. The outcomes of this project will be scholarly disseminated via publication and conference presentations and will be incorporated into existing courses related to the formation of blood vessels. Angiogenesis is a tightly regulated process essential for embryonic development and the maintenance of vascular homeostasis in adult organisms. Stem cells and macrophages are involved in the regulation of angiogenesis via cell differentiation and factor secretion. Recently, vesicles secreted from the cells have been implicated in the array of mediators in the angiogenic processes. Two primary classes of non-apoptotic vesicles, exosomes and microvesicles, released by cells are categorized as EVs. Unlike living cells, EVs are submicron vesicles that target recipient cells to deliver their cargo, including RNAs, proteins, and lipids, in a cell-free fashion. Previous literature has demonstrated that certain microRNAs (miRs) are involved in angiogenesis, so-called AngiomiRs. The objectives of this project are to investigate the synergistic effects of EVs from mouse mesenchymal stem cells and macrophages on angiogenesis in vitro and in vivo. The stem cells and macrophages will be engineered beforehand to overexpress miR-31 and miR-30, respectively, based on previous reports. The in vitro angiogenic effects of a mixture of the EVs released from the two types of engineered cells will be examined on vascular endothelial cells including proliferation, migration, and tube formation in comparison with either type of EVs. To further explore the function of neovasculature, the angiogenic effects of the mixture of the two engineered EVs will be investigated in a mouse hindlimb ischemia (HLI) model. Moreover, the synergistic roles of miR-31/FIH1 and miR-30/Cul2 signaling pathways on HIF-1α transactivation, an essential step in proangiogenesis, will be tested. The result of this study will be a comprehensive understanding of how diverse EVs regulate angiogenesis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.