The Role of TGF-beta Activation and Signaling in Aortic Stenosis Progression

项目摘要 主动脉瓣狭窄（AS）是一种退行性心脏病，其特征是主动脉瓣纤维化和狭窄。作为 导致主动脉瓣壁剪切应力增加，使心脏更难泵血通过 狭窄的阀门开口。这些影响可能导致心力衰竭和死亡。目前治疗AS的唯一方法是 瓣膜置换术是一种侵入性和高风险的手术。我们的长期目标是提高认识， 治疗AS的基础纤维化。转化生长因子-β1（TGF-β1）是一种多功能细胞因子， 在许多疾病中诱导病理性器官纤维化。细胞分泌潜伏（L）TGF-β1，并增加血浆水平 在人类和小鼠AS模型中，LTGF-β1的表达与AS相关。我们发现壁面剪力 应激可显著激活血小板释放LTGF-β1。然而，TGF-β1的机制 体内激活及其在AS瓣膜细胞上的信号传导仍然知之甚少。的目的 应用是确定体内LTGF-β1活化的机制，并鉴定TGF-β 1所作用的细胞类型。 β1信号，导致其间质转化和随后的AS进展。血小板是主要来源 血浆中的LTGF-β1，因为它们含有的LTGF-β1是其他细胞类型的100多倍，我们的初步研究表明， 数据显示，血小板物理地附着于同种凝集素B4阳性瓣膜细胞，推测为瓣膜细胞。 内皮细胞（VEC）经历间质转化并产生胶原蛋白生成肌成纤维细胞 在AS小鼠模型中。这些结果导致了我们的中心假设，即壁切应力激活血小板源性 TGF-β1，其刺激同种凝集素B4阳性细胞（VEC和/或巨噬细胞）经历间充质 过渡，从而产生过量的胶原蛋白并导致AS进展。此外，TGF-β1可能 代表先前未实现的AS进展的临床指标。我们提出以下具体目标：（1） 确定壁切应力是否在体内激活LTGF-β1;和（2）确定TGF-β1信号转导是否在体内激活LTGF-β 1。 瓣膜细胞导致间质转化和AS进展。我们建议的研究将澄清 TGF-β1在体内活化的机制和TGF-β信号传导的细胞类型，导致间充质 过渡和AS进展。我们将使用创新的，敏感的技术来评估AS的发展， 小鼠模型，并测量AS患者血浆样品中的活性TGF-β1水平。而且 拟议的研究将探索使用两种低剂量的小分子抑制剂来缓解AS进展。 TGF-β1活化和信号传导。我们对AS患者TGF-β1活化和信号转导的临床前评估 因此，将TGF-β1作为AS生物标志物的前瞻性临床研究将是至关重要的。总体而言，我们 拟议的研究将提供一个更完整的了解TGF-β1在AS中的作用，这可能会建立 TGF-β1作为药物开发的新靶点和AS及其他纤维化疾病的潜在生物标志物。

PROJECT SUMMARY Aortic stenosis (AS) is a degenerative heart condition characterized by fibrosis and narrowing of the aortic valve. AS causes increased wall shear stress across the aortic valve, making the heart work harder to pump blood through the narrowed valve opening. These effects can cause heart failure and death. The only current treatment for AS is valve replacement, an invasive and risky procedure. Our long-term goal is to improve the understanding and treatment of fibrosis, which underlies AS. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that induces pathologic organ fibrosis in many disorders. Cells secrete latent (L)TGF-β1, and increased plasma levels of LTGF-β1 have been associated with AS in humans and in a mouse model of AS. We showed that wall shear stress can dramatically activate LTGF-β1 released from platelets in vitro. However, the mechanism of TGF-β1 activation in vivo and its signaling on valvular cells in AS remain poorly understood. The objective of this application is to determine the mechanism of LTGF-β1 activation in vivo and identify the cell types to which TGF- β1 signals, leading to their mesenchymal transition and subsequent AS progression. Platelets are the major source of plasma LTGF-β1, as they contain more than 100 times as much LTGF-β1 as other cell types, and our preliminary data show that platelets are physically attached to isolectin B4-positive valvular cells, presumably valvular endothelial cells (VEC) undergoing mesenchymal transition and giving rise to collagen-producing myofibroblasts in an AS mouse model. These results led to our central hypothesis that wall shear stress activates platelet-derived TGF-β1, which stimulates isolectin B4-positive cells (VECs and/or macrophages) to undergo mesenchymal transition, thereby producing excessive collagen and leading to AS progression. Furthermore, TGF-β1 may represent a previously unrealized clinical indicator of AS progression. We propose the following Specific Aims: (1) Determine whether wall shear stress activates LTGF-β1 in vivo; and (2) Determine whether TGF-β1 signaling in valvular cells leads to mesenchymal transition and AS progression. Our proposed studies will clarify the mechanism of TGF-β1 activation in vivo and the cell types on which TGF-β signals, leading to mesenchymal transition and AS progression. We will use innovative, sensitive techniques to evaluate AS development in mouse models and to measure active TGF-β1 levels in plasma samples from AS patients. Furthermore, the proposed research will explore the mitigation of AS progression using two low-dose small-molecule inhibitors of TGF-β1 activation and signaling. Our pre-clinical assessment of TGF-β1 activation and signaling in AS patient samples will be crucial for prospective clinical studies on the use of TGF-β1 as a biomarker of AS. Overall, our proposed studies will provide a more complete understanding of the role of TGF-β1 in AS, which may establish TGF-β1 as a novel target for drug development and a potential biomarker of AS and other fibrotic diseases.