Targeting Resident Cardiac Fibroblast Subpopulations for Protection Against Fibrosis

项目总结/摘要 高血压刺激心脏成纤维细胞（CF）扩张，活化和过量的细胞外基质（ECM） 生产虽然没有批准的治疗心脏纤维化，血管紧张素转换酶 抑制（ACEi）限制CF活化和ECM积累。PI实验室的最新发现 表明居民CF，一旦被认为是功能同质的，由生理上不同的 在压力超负荷的反应中分化为不同表型的群体。这个前提是 本申请是基于这些发现，其中高血压大鼠在施用前用ACEi短暂治疗。 对驻留CF进行单细胞RNA测序。ACEi预治疗改变CF亚群， 具有降低的纤维化能力的稳态CF。这种影响在治疗停止后仍然存在，表明 记忆被保留。拟议的研究将揭示CF亚群转移到 确定如何重新编程CF以显示稳态，较少纤维化的表型。在ACEi之后， 稳态CF包括最大的常驻CF亚群，并且是最少纤维化的。轨迹 分析揭示了一个网关CF亚群，它是激活CF的直接前体， 网关群集被ACEi消耗最多。网关CF由Spp 1的高表达定义， 编码蛋白质骨桥蛋白，其诱导几种促纤维化基因并代表关键靶点 候选人来维护激活的CF池。ACEi改变了表观遗传基因的表达， 染色质结构的改变可能驱动从网关到稳态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设：血管紧张素II信号转导的短暂减少改变了 CF记忆通过成纤维细胞亚群特异性重编程保护左心室（LV）纤维化 将骨桥蛋白产生通道亚群向稳态转移 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信号传导减少介导血管紧张素II受体持续变化的程度。 保护免受未来纤维化的固有CF生理学;目的2）确定染色质结构的影响 修改转移网关集群向稳态集群;和目标3）确定程度， 骨桥蛋白的减少介导向较少纤维形成表型的转变。在本申请中， 研究小组使用多学科方法，采用体内和体外方法来测试 假说.这些实验的成功完成将决定是否减少血管紧张素II 信号转导介导了稳态CF亚群的扩增，使LV对纤维化具有抗性。是 预计调控CF从门户向稳态亚群转变的关键驱动因素将是 鉴定影响：这些预期的发现将对开发CF靶向治疗产生积极影响， 治疗和预防作为心脏病基础的纤维化重塑。

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.