Calpain-mediated lung endothelial barrier modulation in acute lung injury

钙蛋白酶介导的肺内皮屏障调节急性肺损伤

基本信息

批准号：
10617685
负责人：
YUNCHAO SU
金额：
--
依托单位：
CHARLIE NORWOOD VA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10617685
关键词：
Actins Acute Lung Injury Acute Respiratory Distress Syndrome Acute respiratory failure Affect Antibiotics Attenuated Bacterial Toxins Binding Blood Vessels Calcium Calpain Caring Caspase Catalytic Domain Complication Cyclin-Dependent Kinase 5 Cytoskeletal Modeling Cytoskeleton Data Ectopic Expression Endopeptidases Endothelial Cells Endothelium Endotoxins Escherichia coli Family Focal Adhesions Functional disorder Gram-Negative Bacteria Gram-Negative Bacterial Infections Head Human In Vitro Infection Integrins Intensive Care Intervention Knock-out Life Lipopolysaccharides Lung Malignant neoplasm of lung Mammalian Cell Mediating Mediator Military Personnel Molecular Myosin Light Chains Pathway interactions Patients Permeability Phosphorylation Plasma Plasmids Population Protein Dephosphorylation Proteins Proteolysis Pseudomonas aeruginosa Pulmonary Edema Resistance Rod Role Stress Fibers TLR4 gene Talin Testing Ubiquitination Vascular Endothelial Cell Vascular Endothelium Veterans calpain inhibitor combat gram-negative sepsis improved in vivo insight interdisciplinary approach knock-down lung microvascular endothelial cells mortality mouse model mutant myosin phosphatase novel overexpression prevent rho

项目摘要

Summary Acute lung injury (ALI) is characterized by lung vascular endothelial (EC) barrier compromise resulting in increased EC permeability and pulmonary edema. The infections of Gram negative bacteria compose the major cause for ALI. Little has been known about how Gram negative endotoxins (i.e. lipopolysaccharides, LPS) induce EC barrier disruption. We found that LPS activates endopeptidase, calpain, in human lung microvascular ECs (HLMVECs) and the specific calpain inhibition prevents LPS-induced disruption of EC barrier function of HLMVECs and LPS-induced pulmonary edema in ALI. Calpain is a family of calcium-dependent non-lysosomal neutral cysteine endopeptidases that act via limited proteolysis of substrate proteins in mammalian cells, including HLMVECs. Our preliminary data show that LPS induces talin cleavage into head and rod domain and talin phosphorylation in HLMVECs and that overexpression of calpain causes talin cleavage and RhoA activation and myosin light chain phosphatase (MLCP) phosphorylation resulting in MLCP inhibition and myosin light chain (MLC) phosphorylation. Talin is activated through talin cleavage or phosphorylation. Talin cleavage separates head from rod domain thus removing auto-inhibition and stimulating talin head binding to integrin and thus induces FA activation, leading to RhoA activation and MLCP inhibition and MLC phosphorylation and increased lung EC permeability. Talin activation through phosphorylation at Ser-425 can be through cyclin-dependent kinase 5 (CDK5). Our data show that calpain inhibition attenuates LPS-induced increase in CDK5 activity and that MLCP is involved in talin dephosphorylation. This proposal is to study a novel hypothesis that calpain/MLCP coordination regulates talin activation (cleavage/phosphorylation) leading to endothelial barrier disruption in ALI. We will determine whether Gram negative endotoxin LPS, E. coli and P. aeruginosa induce calpain activation and calpain leads to lung microvascular endothelial barrier disruption and Rho-mediated MLCP phosphorylation/inhibition and MLC phosphorylation in ALI. We will define whether LPS, E. coli and P. aeruginosa induce talin activation (cleavage/phosphorylation) and FA strengthening, leading to lung microvascular endothelial barrier disruption in vitro and in vivo. We will investigate whether calpain regulates talin activation (cleavage/phosphorylation) in lung microvascular endothelial barrier disruption in ALI induced by LPS, E. coli and P. aeruginosa. We will assess whether plasma from ALI patients with Gram negative sepsis induces HLMVEC barrier compromise via calpain-talin-FA-MLCP pathway. This proposal is novel because it will identify calpain as mediators in lung EC barrier compromise and calpain serves this role by regulating novel talin cleavage/phosphorylation, RhoA activation and MLCP activity in ALI. A better understanding of the mechanistic insight will provide a framework from which novel calpain inhibition and MLCP activation strategies can be developed for intervention and treatment of ALI.

概括急性肺损伤（ALI）的特点是肺血管内皮（EC）屏障受损，导致 EC 通透性增加和肺水肿。革兰氏阴性菌感染是主要感染引起 ALI 的原因。关于革兰氏阴性内毒素（即脂多糖，LPS）如何诱导 EC屏障破坏。我们发现 LPS 激活人肺微血管 EC 中的肽链内切酶、钙蛋白酶 (HLMVEC) 和特异性钙蛋白酶抑制可防止 LPS 诱导的 EC 屏障功能破坏 ALI 中 HLMVEC 和 LPS 诱导的肺水肿。钙蛋白酶是钙依赖性非溶酶体家族通过哺乳动物细胞中底物蛋白的有限蛋白水解起作用的中性半胱氨酸内肽酶，包括 HLMVEC。我们的初步数据表明，LPS 诱导 talin 分裂成头域和杆域，并且 HLMVEC 中的踝蛋白磷酸化以及钙蛋白酶的过度表达会导致踝蛋白裂解和 RhoA 激活和肌球蛋白轻链磷酸酶 (MLCP) 磷酸化，导致 MLCP 抑制和肌球蛋白轻链（MLC）磷酸化。 Talin 通过 Talin 裂解或磷酸化被激活。 Talin 裂解分离头从杆结构域中分离出来，从而消除了自身抑制并刺激了talin头与整合素的结合，从而诱导 FA 激活，导致 RhoA 激活、MLCP 抑制和 MLC 磷酸化，并增加肺 EC 通透性。 Talin 通过 Ser-425 磷酸化激活可以通过细胞周期蛋白依赖性激酶 5 (CDK5)。我们的数据表明，钙蛋白酶抑制减弱了 LPS 诱导的 CDK5 活性增加， MLCP参与talin去磷酸化。本提案旨在研究钙蛋白酶/MLCP 的新假设协调调节talin激活（裂解/磷酸化），导致ALI中的内皮屏障破坏。我们将确定革兰氏阴性内毒素 LPS、大肠杆菌和铜绿假单胞菌是否诱导钙蛋白酶激活钙蛋白酶导致肺微血管内皮屏障破坏和 Rho 介导的 MLCP ALI 中的磷酸化/抑制和 MLC 磷酸化。我们将定义是否 LPS、大肠杆菌和 P. 铜绿假单胞菌诱导talin激活（裂解/磷酸化）和FA强化，导致肺体外和体内微血管内皮屏障破坏。我们将研究钙蛋白酶是否调节肺微血管内皮屏障破坏中的talin激活（裂解/磷酸化） LPS、大肠杆菌和铜绿假单胞菌。我们将评估来自患有革兰氏阴性败血症的 ALI 患者的血浆是否通过钙蛋白酶-talin-FA-MLCP 途径诱导 HLMVEC 屏障受损。这个提议很新颖，因为它将确定钙蛋白酶是肺 EC 屏障受损的介质，钙蛋白酶通过调节新型 talin 发挥这一作用 ALI 中的切割/磷酸化、RhoA 激活和 MLCP 活性。更好地理解机制洞察力将提供一个框架，通过该框架可以开发新的钙蛋白酶抑制和 MLCP 激活策略开发用于干预和治疗 ALI。