IL10 Regulation of Fibroblast Progenitor Cell Mediated Cardiac Fibrosis

摘要 激活的心脏成纤维细胞（肌成纤维细胞; myofibroblast; myofibroblast）是过度纤维化的关键因素， 最终导致心肌僵硬和心力衰竭的发展。因此，成纤维细胞的调节 激活和相关的纤维化将是治疗心脏病的潜在治疗策略。 疾病以前的研究表明，除了常驻成纤维细胞，骨髓成纤维细胞， 祖细胞（FPCs）可能归巢于衰竭的心脏并导致过度纤维化。总目标 本申请的目的是确定BM衍生的FPCs及其旁分泌信号传导（通过 外泌体）对压力超负荷（PO）诱导的心脏纤维化的影响，并确定是否抑制外泌体的作用。 该细胞群的功能导致心脏纤维化和重塑的减少。我们有 报道PO诱导的心脏纤维化通过全身性IL 10治疗和IL 10敲除而减弱 小鼠（IL 10 KO）显示出过度的心脏纤维化。但IL 10抗纤维化作用的确切机制 并没有很好的定义。因此，我们假设“IL 10抑制成纤维细胞祖细胞（FPC）归巢， 转分化成myoFB并修饰FPC促纤维化旁分泌信号传导，从而减少心脏 纤维化和改善心脏功能”。我们将使用WT（一些具有GFP报告系统），IL 10 KO， TGFbr 1/2 KO和CCR 2 KO（RFP报告系统）小鼠。在SA 1中，通过骨髓 在移植策略中，我们将确定FPC是否在血管紧张素II诱导的心脏纤维化中起作用。 WT和IL 10敲除小鼠以及IL 10治疗是否抑制了它。 TGF β下游信号传导靶点[经典（Smad 2/3）、非经典（ERK-MAP激酶）和非经典（ERK-MAP激酶）]。 与纤维化相关的miR]及其在FPC转分化中的作用。为了确定他们的角色，我们将 感染/清除显性阴性腺病毒或激酶死亡构建体（Smad/ERK）或miR 在WT和IL 10 KO FPC中分别进行对照。在具体目标3中，我们建议研究旁分泌 FPCs对IL 10 KO小鼠中常驻心脏成纤维细胞活化和过度纤维化的调节。在 为此，我们将探索FPC（WT/IL 10 KO）通过外泌体分泌的促纤维化因子是否诱导纤维化， 心肌成纤维细胞转变为myoFB。为了在临床前环境中转化这一发现，在具体目标3中，我们 将研究移植源自离体修饰的FPC的外泌体（用于靶向 miR/蛋白）抑制Ang II诱导的纤维化。本研究的意义在于 确定IL 10在调节心脏纤维化中的新机制。我们提出的实验可以 潜在地确定基于骨髓操作的心脏修复的新治疗策略， 压力超负荷诱导的心脏损伤后的成纤维细胞祖细胞信号传导。

Abstract Activated cardiac fibroblasts (myofibroblast; myoFBs) are crucial players in excessive fibrosis, which ultimately results in myocardial stiffness and heart failure development. Thus, regulation of fibroblasts activation and associated fibrosis would be a potential therapeutic strategy in the treatment of cardiac diseases. Previous studies suggest that in addition to resident fibroblasts, bone marrow fibroblast progenitor cells (FPCs) may home to the failing heart and contribute to excessive fibrosis. The overall goal of this application is to determine the role of BM-derived FPCs and their paracrine signaling (via exosomes) on pressure overload (PO)-induced cardiac fibrosis, and to determine whether inhibition of function of this cell population results in the reduction of cardiac fibrosis and remodeling. We have reported that PO-induced cardiac fibrosis is attenuated by systemic IL10 treatment and IL10-knockout mice (IL10 KO) display exaggerated cardiac fibrosis. But the precise mechanism of anti-fibrotic role of IL10 is not well defined. Thus we hypothesized that “IL10 inhibits fibroblast progenitor cell (FPCs) homing and trans-differentiation into myoFB and modifies FPC pro-fibrotic paracrine signaling thereby reducing cardiac fibrosis and improving cardiac function”. We will use WT (some with GFP reporter system), IL10 KO, TGFbr1/2 KO and CCR2KO (RFP reporter system) mice in this study. In SA1, via bone marrow transplantation strategy, we will determine whether FPC contributes in Ang II-induced cardiac fibrosis in WT and IL10 Knock out mice and whether IL10 treatment inhibits it. In specific aim 2 we will identify the TGFb downstream signaling targets [both canonical (Smad2/3), non-canonical (ERK-MAP kinase) and miR associated with fibrosis] and their roles in FPC's trans-differentiation. To identify their role, we will infect/transfect dominant negative adenovirus or kinase-dead construct (Smad/ERK) or antagomir (miRs) with their respective control in WT and IL10KO FPCs. In specific aim 3 we propose to study the paracrine regulation of FPCs on resident cardiac fibroblast activation and exaggerated fibrosis in IL10KO mice. In this aim we will explore whether pro-fibrotic factors secreted by FPC (WT/IL10KO) via exosomes, induce cardiac fibroblast transition to myoFB. To translate this finding in preclinical settings, in specific aim 3 we will investigate whether transplantation of exosomes-derived from ex vivo-modified FPC (for target miRs/proteins) in IL10 KO mice heart inhibits Ang II-induced fibrosis. The significance of this study is to identify the novel mechanism(s) of IL10 in regulation of cardiac fibrosis. Our proposed experiments can potentially identify novel therapeutic strategies for cardiac repair based on manipulation of bone marrow fibroblast progenitor cells signaling after pressure overload-induced cardiac injury.