Cellular Mechanisms of Cardiac ECM Structure and Function

慢性心力衰竭(CHF)在退伍军人管理局有一个指定的质量增强研究倡议(QUERI) 系统，以解决如何改善患有CHF的退伍军人的心血管保健。在以下方面取得成功 慢性压力超负荷(高血压、主动脉瓣狭窄)所致心力衰竭的治疗受限于 尽管我们有能力使血流动力学负荷正常化，但仍存在持续性间质纤维化。这个 拟议的研究将定义导致这一关键的临床需求的细胞机制的异常。 满足这一需求将取决于确定控制初始ECM的基本因果决定因素 血流动力学负荷正常化后间质心肌纤维化的降解和持续。 细胞外基质稳态的主要细胞调节因子被认为是心肌巨噬细胞和成纤维细胞。 我们之前的研究和初步数据导致了我们的中心假设：慢性血流动力学 超负荷导致巨噬细胞和成纤维细胞表型发生根本性变化，其特点是 蛋白水解酶稳态失调，进而阻碍细胞对卸载的反应并限制完全卸载 即使在血流动力学负荷正常化后，纤维化也会消退。为了验证这一假设，在 建立体内动物模型和体外成纤维细胞培养方法。在体内，临床上与LVPO相关的逆转 (卸载)是通过手术切除横状主动脉缩窄(UNTAC)在小鼠中产生的。联柬权力机构是 发现启动但导致心脏纤维化的不完全消退。初步数据显示， 心肌巨噬细胞的显著增加与胶原降解的启动是一致的 血流动力学卸载，但巨噬细胞的这些增加在UNTAC后的较晚时间不会持续。至 解决成纤维细胞表型的负荷依赖性变化是否是这种重塑的关键因素， 建立了模拟临床相关心肌僵硬的成纤维细胞培养体系。在体内， 心肌硬度测量表明，成纤维细胞暴露在PO中的~8kPa力中 心肌和正常心肌的~2kPa.生理上相关的，与僵硬相关的变化 在正常心肌成纤维细胞中观察到成纤维细胞的表型，而来自TAC和TAC的成纤维细胞 UNTAC心肌表现为对僵硬改变无反应的促纤维化表型。初步数据 提示金属蛋白酶组织抑制物(TIMP)-1是肝纤维化持续存在的原因之一 表型。我们强劲的初步数据产生了以下具体目标，以检验我们的核心假设： 目的1：验证持续性血流动力学超负荷逆转使心肌改变的假设 巨噬细胞表型转变为一种独特但短暂的抗纤维化(ECM降解)表型 启动(但不完成)累积的间质ECM的负荷依赖性回归。 目的2：验证体内持续增加血流动力学负荷改变心肌的假说 成纤维细胞向促纤维化、TIMP-1依赖的表型转化，即使在 血流动力学负荷是相反的。

Chronic heart failure (CHF) has a designated Quality Enhancement Research Initiative (QUERI) in the VA system to address ways to improve cardiovascular healthcare for Veteran’s suffering from CHF. Success in treatment of CHF associated with chronic pressure overload (hypertension, aortic valve stenosis) is limited by the presence of persistent interstitial fibrosis despite our ability to normalize hemodynamic load. The proposed studies will define abnormalities in cellular mechanisms that cause this critical clinical unmet need. Filling this need will depend on defining the fundamental causal determinants that control both initial ECM degradation and persistence of interstitial myocardial fibrosis following normalization of hemodynamic load. Primary cellular regulators of ECM homeostasis are postulated to be myocardial macrophages and fibroblasts. Our previous studies and preliminary data have led to our central hypothesis: Chronic hemodynamic overload causes fundamental changes in both macrophage and fibroblast phenotype, the hallmark of which is dysregulated protease homeostasis that in turn impedes cellular response to unloading and limits complete regression of fibrosis even after normalization of hemodynamic load. To test this hypothesis, innovations in in vivo animal models and in vitro fibroblast culture were developed. In vivo, a clinically relevant reversal of LVPO (unloading) was created in mice by surgical removal of the transverse aortic constriction (unTAC). UnTAC was found to initiate but lead to an incomplete regression of cardiac fibrosis. Preliminary data indicate that a significant increase in myocardial macrophages coincides with initiation of collagen degradation following hemodynamic unloading but these increases in macrophages are not sustained at later times after unTAC. To address whether load-dependent changes in fibroblast phenotype were a key factor in this remodeling, a fibroblast culture systems that mimics clinically relevant myocardial stiffness was established. In vivo, measurements of myocardial stiffness demonstrated that fibroblasts are exposed to a force of ~8 kPA in PO myocardium and ~2 kPA in normal myocardium. Physiologically relevant, stiffness-dependent changes in fibroblasts phenotype were observed in fibroblasts from normal myocardium whereas fibroblasts from TAC and unTAC myocardium exhibited a pro-fibrotic non-responsive phenotype to changes in stiffness. Preliminary data indicate that Tissue inhibitor of metalloproteinase (TIMP)-1 was a causal factor in this pro-fibrotic persistent phenotype. Our strong preliminary data gave rise to the following Specific Aims to test our central hypothesis: Aim 1: Test the hypothesis that reversal of sustained hemodynamic overload shifts myocardial macrophage phenotype to a distinct but transient anti-fibrotic (ECM-degradation) phenotype that initiates, but does not complete, a load-dependent regression of accumulated interstitial ECM. Aim 2: Test the hypothesis that sustained in vivo increases in hemodynamic load change myocardial fibroblasts to a profibrotic, TIMP-1 dependent phenotype that remains profibrotic even when hemodynamic load is reversed.