Targeting Resident Cardiac Fibroblast Subpopulations for Protection Against Fibrosis

项目摘要/摘要 高血压刺激心脏成纤维细胞(CF)的扩张、激活和细胞外基质(ECM)的过剩 制作。尽管目前还没有被批准的治疗心脏纤维化的方法，但血管紧张素转换酶 血管紧张素转换酶抑制物(ACEI)限制了CF的激活和ECM的积累。猪的实验室的最新发现 证明常驻CFS，一旦被认为在功能上是同质的，由生理上不同的 在压力超载的情况下分化成不同表型的种群。这样做的前提是 应用是基于这些发现，其中高血压大鼠在服用血管紧张素转换酶抑制剂之前进行了短暂的治疗 常驻CFs上的单细胞RNA测序。ACEI的前处理使CF亚群产生 动态平衡的CFS纤维化能力降低。这种效果在治疗停止后仍然存在，表明 内存被保留。拟议的研究将揭示CF亚群转移到 确定如何重新编程CFS以显示内稳态、较少纤维化的表型。在ACEI之后， 动态平衡型CFS构成了常住CFS中最大的亚群，并且是最不容易纤维化的。弹道 分析揭示了一个网关CF亚群，它是激活的CFS的直接前驱，而这 ACEI耗尽最多的是网关集群。通过Spp1的高表达来定义网关CFs， 编码骨桥蛋白，它诱导几个促纤维化基因，并代表一个关键靶点 维护激活的CF池的候选人。血管紧张素转换酶抑制剂改变表观遗传基因的表达，表明改变 在染色质结构中可能驱动着从门户亚群到动态平衡的CF亚群的持续转变。这些 令人信服的初步结果导致了中心假说：血管紧张素II信号的一过性减少改变 Cf通过成纤维细胞亚群特异性重新编程来预防左心室(LV)纤维化的记忆 染色质结构使产生骨桥蛋白的通道亚群向内环境平衡转变 致纤维化能力低的亚群。为了检验这一假设，提出了以下具体目标： 目的1)阐明血管紧张素II信号的减少在多大程度上调节血管紧张素转换的持续性变化 防止未来纤维化的常驻CF生理学；目的2)确定染色质结构的影响 关于将网关簇向内稳态簇移动的修改；以及目标3)确定 骨桥蛋白的减少介导了向纤维化程度较低的表型的转变。在此应用程序中， 研究小组使用多学科方法，采用体内和体外方法来测试 假设。这些实验的成功完成将确定血管紧张素II的减少 信号转导了使左室抗纤维化的动态平衡CFs子集的扩张。它是 预计规范从门户向动态平衡亚群转变的关键驱动因素将是 已确认身份。影响：这些预期的发现将对开发针对CF的治疗方法产生积极影响 心脏纤维化重塑的治疗和预防。

PROJECT SUMMARY/ABSTRACT Hypertension stimulates cardiac fibroblast (CF) expansion, activation, and excess extracellular matrix (ECM) production. Although there are no approved treatments for cardiac fibrosis, angiotensin converting enzyme inhibition (ACEi) limits CF activation and ECM accumulation. Recent findings from the laboratory of the PI demonstrate that resident CFs, once considered functionally homogeneous, consist of physiologically distinct populations that differentiate to diverse phenotypes in response to pressure overload. The premise for this application is based on these findings in which hypertensive rats were transiently treated with an ACEi prior to single cell RNA sequencing on resident CFs. Pre-treatment with ACEi shifts CF subpopulations to generate homeostatic CFs with a reduced capacity for fibrosis. This effect persists after treatment is stopped, indicating memory is retained. The proposed studies will reveal the mechanisms by which CF subpopulations shift to determine how to reprogram CFs to display a homeostatic, less fibrogenic phenotype. Following ACEi, homeostatic CFs comprise the largest subpopulation of resident CFs and are the least fibrogenic. Trajectory analysis revealed a gateway CF subpopulation that is the immediate precursor to activated CFs, and this gateway cluster was the most depleted by ACEi. Gateway CFs were defined by high expression of Spp1, encoding for the protein osteopontin, which induces several pro-fibrotic genes and represents a critical target candidate to maintain the activated CF pool. ACEi altered expression of epigenetic genes, indicating changes in chromatin structure may drive the persistent shift from gateway to homeostatic CF subpopulations. These compelling preliminary results led to the central hypothesis: transient reduction in angiotensin II signaling alters CF memory to protect against left ventricle (LV) fibrosis by fibroblast subpopulation-specific reprogramming of chromatin structure to shift an osteopontin-producing gateway subpopulation toward a homeostatic subpopulation with low fibrogenic capacity. To test the hypothesis, the following specific aims are proposed: Aim 1) elucidate the degree to which reduction in angiotensin II signaling mediates the persistent shift in resident CF physiology that protects from future fibrosis; Aim 2) determine the impact of chromatin structural modification on shifting the gateway cluster toward the homeostatic cluster; and Aim 3) ascertain the degree to which reduction in osteopontin mediates the shift to a less fibrogenic phenotype. In this application, the research team uses a multidisciplinary approach employing in vivo and in vitro methodologies to test the hypothesis. Successful completion of these experiments will determine whether reduction in angiotensin II signaling mediates the expansion of a subset of homeostatic CFs that renders the LV resistant to fibrosis. It is expected that the key drivers regulating the shift from a gateway to a homeostatic subpopulation of CFs will be identified. Impact: These anticipated findings will have a positive impact in developing CF-targeted therapies for the treatment and prevention of fibrotic remodeling that underlies heart disease.