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

TGF-β1 剪切诱导释放和激活的机制

基本信息

批准号：
8837319
负责人：
Jasimuddin Ahamed
金额：
$ 3.93万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-11-07 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8837319
关键词：
Affect Animal Model Aortic Valve Stenosis Biochemical Biological Markers Blood Circulation Blood Platelets Breeding Cessation of life Cholesterol Clinical Complex Data Development Diagnostic Disease model Disulfides Elderly Event Fibrosis Functional disorder Genes Goals Heart Heart Transplantation Heart failure Hemorrhage Human Hyperlipidemia Implant In Vitro Intervention Isomerase Label Lead Left Mass Spectrum Analysis Measures Medical Modeling Molecular Molecular Conformation Molecular Weight Monitor Mus Mutagenesis Mutation Operative Surgical Procedures Pathology Patients Phosphorylation Plasma Plasminogen Activator Inhibitor 1 Platelet Activation Process Proteins Proteomics Recombinant Proteins Recombinant Transforming Growth Factor Recombinants Risk Role Signal Transduction Source Sulfhydryl Compounds Surrogate Markers Testing Thrombosis Thrombospondin 1 Transforming Growth Factor beta Transforming Growth Factors aorta constriction aortic valve ascending aorta constriction coronary fibrosis cytokine design disulfide bond human TGFB1 protein implantation in vivo latency-associated protein mouse model mutant neutralizing antibody novel novel diagnostics pressure prevent response shear stress tool valve replacement ventricular assist device von Willebrand Factor western diet

项目摘要

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.

项目概要：该提案的目的是检验以下假设：TGF-β1 从血小板中释放并通过剪切激活循环压力导致主动脉瓣狭窄 (AS) 的病理生理学及其并发症与心力衰竭（HF）患者植入左心室辅助装置（LVAD）相关。我们之前和新的初步研究表明：1) 剪切力可以激活潜在的 TGF-ß1， 2) 硫醇-二硫化物交换有助于激活过程，3) 缺乏血小板 TGF-ß1 的小鼠防止因压力超负荷而发生心脏纤维化，4) Reversa 小鼠发生 AS 和与剪切应力相关的血浆 TGF-ß1 水平增加，4) AS 和 LVAD 植入的 HF 与对照组相比，患者的 TGF-ß1 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的目标 1 是检验以下假设：剪切通过促进涉及 TGF-β1、硫醇异构酶和/或其他酶的硫醇-二硫化物交换来激活 TGF-β1 含硫醇蛋白，例如 TSP1 和 vWf 以及其他含硫醇蛋白的纯化。血小板、使用质谱的蛋白质组学分析以及重组蛋白的突变研究将用于识别剪切诱导的硫醇-二硫化物交换，该交换负责 TGF-ß1 的激活。 2 将检验以下假设：TGF-ß1 从血小板中释放，随后在高剪切作用下被激活使用两种小鼠模型进行体内实验：通过手术诱导的升主动脉缩窄模型来精确模拟高剪切力，以及自发发生主动脉AS和高剪切力的Reversa小鼠模型我们将评估 TGF-β1 的释放和激活以及对小鼠中 TGF-β1 激活的影响。潜伏期相关蛋白 (LAPC33S) 发生突变，其 TGF-ß1 不能被剪切激活，并且在小鼠体内选择性缺乏血小板 PDI (PDIFlox) 的小鼠的 TGF-ß1 水平升高，与之相关。我们将通过培育 Reversa 来测试血小板 TGF-ß1 和 PDI 对 AS 进展的作用。具有血小板特异性 PF4Cre/Tgfb1flox 的小鼠和 PF4Cre/PDIflox 小鼠将是 TGF-ß1 中和抗体。用于监测 AS 进展作为干预模型，目标 3 将检验释放和释放的假设。通过评估 AS 和植入 LVAD 的心力衰竭患者的 TGF-β1 水平，体内发生 TGF-β1 的激活。与对照组相比，AS 患者的 TGF-β1 水平升高，这与 AS 瓣膜相关。将评估瓣膜置换手术后 TGF-β1 水平是否降低。植入 LVAD 的心力衰竭患者中 TGF-β1 水平升高，并且与 vWf 多聚体的丧失相关。将通过心脏移植监测 TGF-ß1 水平，并与 vWf 多聚体相关联以建立 TGF-ß1 和 vWf-多聚体分别作为血栓形成和出血风险的替代标志物。这些研究将阐明并指导潜在干预措施的开发，以预防 AS 进展作为检测 LVAD 植入后心力衰竭患者早期并发症的诊断工具。