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

项目总结 主动脉瓣狭窄(AS)是一种以主动脉瓣纤维化和狭窄为特征的退行性心脏疾病。AS 导致跨主动脉瓣膜的壁切应力增加，使心脏更难通过泵送血液 狭窄的阀门开口。这些影响可能会导致心力衰竭和死亡。目前治疗强直性脊柱炎的唯一方法是 瓣膜置换术，这是一种侵入性且有风险的手术。我们的长期目标是提高对 纤维化的治疗，这是AS的基础。转化生长因子-β-1(转化生长因子-β-1)是一种多功能细胞因子， 在许多疾病中导致病理性器官纤维化。细胞分泌潜伏期(L)转化生长因子-β1，血浆水平升高 在人类和AS小鼠模型中，LTGF1-β1的表达与AS相关。我们展示了墙的剪力 应激可显著激活体外血小板释放的转化生长因子-β-1。然而，转化生长因子-β-1的作用机制 AS在体内的激活及其在瓣膜细胞上的信号转导仍然知之甚少。这样做的目的是 其应用是确定转化生长因子-β-1在体内激活的机制，并鉴定转化生长因子-1对哪些细胞类型的作用。 β-1信号，导致其间质转化和随后的AS进展。血小板是主要来源 血浆中的转化生长因子-β-1，因为它们含有的转化生长因子-β-1是其他类型细胞的100多倍，我们的初步研究 数据显示，血小板在物理上附着在异凝素B4阳性的瓣膜细胞上，推测是瓣膜细胞 内皮细胞(VEC)间充质转化形成胶原的肌成纤维细胞 在AS小鼠模型中。这些结果导致了我们的中心假设，即壁面切应力激活了血小板衍生的 转化生长因子-β-1，刺激异凝素B4阳性细胞(血管内皮细胞和/或巨噬细胞)经历间充质 转化，从而产生过多的胶原蛋白，导致AS进展。此外，转化生长因子-β1可能 代表了一种先前未实现的AS进展的临床指标。我们提出了以下具体目标：(1) 确定在体壁切应力是否激活了转化生长因子-β-1；以及(2)确定转化生长因子-β-1信号是否在体内 瓣膜细胞导致间充质转化和AS进展。我们建议的研究将澄清 转化生长因子-β-1体内激活的机制及转化生长因子-β信号转导间充质的细胞类型 过渡性和渐进性。我们将使用创新、敏感的技术来评估 并检测AS患者血浆中活性转化生长因子-β-1水平。此外， 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 转化生长因子-β-1的激活和信号转导。我们对强直性脊柱炎患者转化生长因子-β1激活和信号转导的临床前评估 对于使用转化生长因子-β1作为AS生物标志物的前瞻性临床研究来说，样本将是至关重要的。总的来说，我们的 拟议的研究将提供对转化生长因子-β1在AS中的作用的更全面的了解，这可能建立 转化生长因子-β-1作为药物开发的新靶点和AS等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.