The Mechanism of Shear-Induced Release and Activation of TGF-beta1

项目摘要： 本研究的目的是验证TGF-β 1从血小板中释放并被剪切激活的假设 循环中的应力有助于主动脉瓣狭窄（AS）的病理生理学和并发症 与心力衰竭（HF）患者左心室辅助装置（LVAD）植入相关。这将构建 根据我们先前和新的初步研究，证明：1）剪切力可以激活潜伏的TGF-β 1， 2)巯基-二硫键交换有助于活化过程，3）血小板TGF-β 1缺陷的小鼠， 保护免于响应于压力超负荷而发展心脏纤维化，4）Reversa小鼠发展AS， 与剪切应力相关的血浆TGF-β 1水平增加，以及4）AS和LVAD植入的HF 与对照组相比，患者的TGF-β 1水平增加。我们将通过整合数据来扩展我们的发现 从生化分析、动物模型和人类患者研究中。目的1是检验假设， 剪切通过促进涉及TGF-β 1、硫醇异构酶和/或其它的硫醇-二硫化物交换来活化TGF-β 1 含巯基的蛋白质，例如TSP 1和vWf。TGF-β 1和其它含巯基蛋白的纯化 从血小板，蛋白质组学分析使用质谱，和突变研究的重组蛋白 将用于鉴定负责TGF-β 1活化的剪切诱导的巯基-二硫化物交换。目的 2将检验TGF-β 1从血小板中释放并随后被高剪切激活的假设， 体内使用两种小鼠模型：一种是急性诱导的升主动脉缩窄模型， 高剪切和自发发展AS和跨越主动脉的高剪切的Reversa小鼠模型 阀.我们将评估TGF-β 1的释放和激活以及对小鼠TGF-β 1激活的影响， 潜伏相关蛋白（LAPC 33 S）突变，其TGF-β 1不能被剪切激活， 选择性缺乏血小板PDI（PDIflox）。逆转小鼠的TGF-β 1水平增加， 剪切应力。我们将通过繁殖逆转录病毒来检测血小板TGF-β 1和PDI在AS进展中的作用。 血小板特异性PF 4Cre/Tgfb 1flox小鼠和PF 4Cre/PDIflox小鼠。TGF-β 1中和抗体将 用于监测AS进展，作为干预模型。目标3将检验释放和 通过评估AS和LVAD植入的HF患者中的TGF-β 1水平，体内发生TGF-β 1的活化。 与对照组相比，AS患者具有升高的TGF-β 1水平。TGF-β 1水平与AS瓣膜相关 病理生理学将评估瓣膜置换术后TGF-β 1水平是否降低。 TGF-β 1水平在植入LVAD的HF患者中升高，并与vWf多聚体的丢失相关。我们 将通过心脏移植监测TGF-β 1水平，并与vWf多聚体相关以建立TGF-β 1 和vWF-多聚体分别作为血栓形成和出血风险的替代标志物。总的来说， 这些研究也将阐明和指导预防AS进展的潜在干预措施的开发 作为检测LVAD植入后HF患者早期并发症的诊断工具。

Project Summary: The goal of this proposal is to test the hypothesis that TGF-ß1 released from platelets and activated by shear stress in the circulation contributes to the pathophysiology of aortic stenosis (AS) and the complications associated with implantation of left ventricular assist device (LVAD) in heart failure (HF) patients. This will build upon our previous and new preliminary studies demonstrating that: 1) shear force can activate latent TGF-ß1, 2) thiol-disulfide exchange contributes to the activation process, 3) mice deficient in platelet TGF-ß1 are protected from developing cardiac fibrosis in response to pressure overload, 4) Reversa mice develop AS and increased plasma TGF-ß1 levels that correlate with shear stress, and 4) both AS and LVAD-implanted HF patients have increased levels of TGF-ß1 compared to controls. We will extend our findings by integrating data from biochemical analyses, animal models, and human patient studies. Aim 1 is to test the hypothesis that shear activates TGF-ß1 by facilitating thiol-disulfide exchange involving TGF-ß1, thiol isomerases, and/or other thiol-containing proteins, such as TSP1 and vWf. Purification of TGF-ß1 and other thiol-containing proteins from platelets, proteomic analysis using mass spectrometry, and mutational studies of recombinant proteins will be employed to identify the shear-induced thiol-disulfide exchange responsible for TGF-ß1 activation. Aim 2 will test the hypothesis that TGF-ß1 is released from platelets and subsequently activated by high shear in vivo using two mouse models: a surgically-induced Ascending Aorta Constriction model to acutely simulate high shear, and a Reversa mouse model that spontaneously develops AS and high shear across the aortic valve. We will assess TGF-ß1 release and activation as well as the impact on TGF-ß1 activation in mice with a mutation in the latency-associated protein (LAPC33S), whose TGF-ß1 cannot be activated by shear and in mice selectively deficient in platelet PDI (PDIflox). Reversa mice have increased levels of TGF-ß1 that correlates with shear stress. We will test the role of platelet TGF-ß1 and PDI on AS progression by breeding Reversa mice with platelet-specific PF4Cre/Tgfb1flox and PF4Cre/PDIflox mice. A TGF-ß1 neutralizing antibody will be used to monitor AS progression as a model for intervention. Aim 3 will test the hypothesis that release and activation of TGF-ß1 occurs in vivo by assessment of TGF-ß1 levels in AS and LVAD-implanted HF patients. AS patients have elevated TGF-ß1 levels compared to controls. TGF-ß1 levels will be correlated with AS valve pathophysiology. Whether TGF-ß1 levels are decreased after valve replacement surgery will be assessed. TGF-ß1 levels are elevated in LVAD-implanted HF patients and associated with the loss of vWf multimers. We will monitor TGF-ß1 levels through heart transplantation and correlate with vWf multimers to establish TGF-ß1 and vWf-multimers as surrogate markers for the risk of thrombosis and hemorrhage, respectively. Collectively, these studies will illuminate and guide development of potential interventions to prevent AS progression as well as diagnostic tools to detect early complications in post-LVAD implantation HF patients.