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）的特征是肺血管内皮（EC）屏障妥协，导致 EC渗透性和肺水肿增加。革兰氏阴性细菌的感染是主要的原因是阿里。关于革兰氏阴性内毒素（即脂多糖，LPS）如何诱导的革兰氏阴性内毒素几乎知之甚少 EC屏障破坏。我们发现LPS在人肺微血管EC中激活内肽酶Calpain （HLMVECS）和特定的钙蛋白酶抑制可防止LPS引起的EC屏障功能的破坏 HLMVEC和LPS诱导的ALI肺水肿。钙蛋白酶是一个依赖钙的非溶酶体的家族中性半胱氨酸内肽酶通过有限的蛋白质水解作用于哺乳动物细胞中底物蛋白的蛋白质，包括hlmvecs。我们的初步数据表明，LP会诱导塔林裂解到头部和杆域，以及 hlmvecs中的塔林磷酸化和钙蛋白酶的过表达会导致塔林裂解和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。塔林通过塔林裂解或磷酸化激活。塔林裂解分开从杆状域中的头部，从而消除了自动抑制和刺激塔林头与整合素的结合，从而诱导FA激活，导致RhoA激活和MLCP抑制和MLC磷酸化并增加肺部渗透性。通过在Ser-425处通过磷酸化激活的塔林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，钙蛋白酶抑制作用减弱了LPS引起的CDK5活性的增加和该MLCP参与塔林去磷酸化。该建议是研究一个新的假设，即钙蛋白酶/MLCP 协调调节塔林激活（切割/磷酸化），导致ALI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LPS，E。Coli和P.是否存在铜绿诱导塔林激活（切割/磷酸化）和FA增强，导致肺微血管内皮屏障的体外和体内破坏。我们将调查钙蛋白酶是否调节在ALI中，ALI诱导的肺微血管内皮屏障破坏中的塔林激活（裂解/磷酸化） LPS，大肠杆菌和铜绿假单胞菌。我们将评估来自革兰氏阴性败血症患者的血浆通过Calpain-Talin-FA-MLCP途径诱导HLMVEC屏障妥协。该建议是新颖的，因为它将通过调节新颖的塔林，将钙蛋白酶识别为肺部障碍妥协中的介体和钙蛋白酶的作用 ALI中的切割/磷酸化，RhoA激活和MLCP活性。更好地理解机械的洞察力将提供一个框架，新型的calpain抑制和MLCP激活策略可以是开发用于干预和治疗ALI。