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

细胞外囊泡（EVs）是细胞释放的微粒，通过运送货物在细胞-细胞通讯中发挥重要作用。研究电动汽车的传统方法侧重于研究源自单个细胞类型的电动汽车，而忽视了电动汽车由所有细胞类型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，将通过生物工程技术修饰EV细胞，以提高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对两个关键领域的理解：1)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)利用多种来源的ev组合探索新的治疗策略。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，并提供校内和校外的机会来展示他们的发现。该项目的成果将通过出版物和会议报告进行学术传播，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，对胚胎发育和维持成年生物血管稳态至关重要。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控。最近，从细胞中分泌的囊泡被认为与血管生成过程中的一系列介质有关。细胞释放的两种主要的非凋亡囊泡，外泌体和微囊泡被归类为ev。与活细胞不同，电动汽车是亚微米的囊泡，以无细胞的方式靶向受体细胞递送其货物，包括rna、蛋白质和脂质。先前的文献已经证明，某些microrna （miRs）参与血管生成，即所谓的angiomir。本项目的目的是研究小鼠间充质干细胞和巨噬细胞的内皮细胞对体外和体内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预先工程化，分别过表达miR-31和miR-30。两种工程细胞释放的ev混合物的体外血管生成效应将被检测，包括血管内皮细胞的增殖、迁移和管形成，并与任何一种类型的ev进行比较。为了进一步探索血管功能，我们将在小鼠后肢缺血（HLI）模型中研究两种工程ev混合的血管生成作用。此外，将测试miR-31/FIH1和miR-30/Cul2信号通路在促血管生成的重要步骤HIF-1α反激活中的协同作用。这项研究的结果将是全面了解不同的EVs如何调节血管生成。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.