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

项目总结： 这项提议的目标是检验这样一个假设，即转化生长因子-1从血小板释放并被剪切力激活 循环中的应激在主动脉狭窄(AS)及其并发症的病理生理学中起作用 与心力衰竭患者的左心室辅助装置(LVAD)植入相关。这将建立 根据我们以前和新的初步研究表明：1)剪切力可以激活潜在的转化生长因子1， 2)硫醇-二硫键交换参与活化过程；3)血小板转化生长因子-β1缺陷小鼠 防止因压力超负荷而发展为心脏纤维化，4)逆转的小鼠发展为AS和 与切应力相关的血浆转化生长因子-1水平升高，以及4)AS和LVAD植入的HF 与对照组相比，患者的转化生长因子-1水平升高。我们将通过整合数据来扩展我们的发现 来自生化分析、动物模型和人类患者研究。目标1是检验假设 剪切力通过促进涉及转化生长因子-1、硫醇异构酶和/或其他的硫醇-二硫键交换来激活转化生长因子-1 含有硫醇的蛋白质，如TSP1和VWF。转化生长因子β1等含硫醇蛋白的纯化 从血小板，用质谱仪进行蛋白质组学分析，以及重组蛋白质的突变研究 将被用来确定剪切诱导的硫醇-二硫键交换对转化生长因子-β1的激活负责。目标 2将检验这一假设，即转化生长因子-1从血小板释放，随后被高切变激活。 用两种小鼠模型进行活体实验：一种手术诱导的升主动脉收缩模型 高切变，以及自发发展为AS和高切变跨主动脉的可逆小鼠模型 阀门。我们将评估转化生长因子-β1的释放和激活，以及对转化生长因子-β1激活的影响。 潜伏期相关蛋白(LAPC33S)的突变，其转化生长因子1不能被剪切和在小鼠中激活 选择性地缺乏血小板PDI(PDIflx)。逆转组小鼠的转化生长因子-1水平升高，这与 具有剪切力。我们将通过育种逆转来测试血小板转化生长因子1和PDI在AS进展中的作用 携带血小板特异性PF4Cre/Tgfb1flx和PF4Cre/PDIflx的小鼠。一种转化生长因子-1中和抗体将成为 用于监测AS的进展情况，作为干预的模型。目标3将测试释放和释放的假设 通过评估AS和植入LVAD的心衰患者体内的转化生长因子-β1水平，可以发现转化生长因子-β1的激活。 与对照组相比，AS患者的转化生长因子1水平升高。转化生长因子-1水平将与AS瓣膜相关 病理生理学。瓣膜置换术后是否降低了转化生长因子-1的水平将被评估。 植入LVAD的心力衰竭患者的转化生长因子-1水平升高，并与VWF多聚体的丢失有关。我们 将通过心脏移植监测转化生长因子-1水平，并与VWF多聚体相关联以建立转化生长因子-β1 和VWF-多聚体分别作为血栓形成和出血风险的替代标志物。总而言之， 这些研究也将阐明和指导预防AS进展的潜在干预措施的发展。 作为诊断工具来检测LVAD植入术后心衰患者的早期并发症。

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.