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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.

摘要急性肺损伤(ALI)的特征是肺血管内皮细胞(EC)屏障受损，导致内皮细胞通透性增加，肺水肿。革兰氏阴性杆菌感染构成主要阿里的事业。关于革兰氏阴性内毒素(即脂多糖)是如何诱导 EC屏障的破坏。我们发现，内毒素能激活人肺微血管内皮细胞的内肽酶，即钙蛋白酶。 (HLMVECs)和特异性的calain抑制可阻止内毒素诱导的EC屏障功能的破坏 HLMVECs和内毒素诱导的ALI肺水肿。钙蛋白酶是一类钙依赖的非溶酶体中性半胱氨酸内肽酶，通过哺乳动物细胞中底物蛋白的有限蛋白分解而起作用，包括HLMVEC。我们的初步数据显示，内毒素诱导Talin裂解成头部和杆状结构域，并 HLMVEC中Talin磷酸化和calain过表达导致Talin切割和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是通过Talin裂解或磷酸化而激活的。塔林卵裂分离从而消除自身抑制并刺激Talin头部与整合素结合，从而诱导FA激活，导致RhoA激活和MLCP抑制以及MLC磷酸化和增加肺内皮细胞通透性。通过Ser-425的磷酸化激活Talin可以通过细胞周期蛋白依赖激酶5(CDK5)。我们的数据显示，抑制Calain可以减弱内毒素诱导的CDK5活性增加和 MLCP参与Talin去磷酸化。这项提议是为了研究一种新的假设，即Calain/MLCP 协同作用调节ALI中Talin的激活(切割/磷酸化)，导致内皮屏障的破坏。我们将确定革兰氏阴性内毒素、大肠杆菌和铜绿假单胞菌是否诱导钙激活酶 CaPin导致肺微血管内皮细胞屏障破坏和Rho介导的MLCP ALI中的磷酸化/抑制和MLC的磷酸化。我们将确定是否有内毒素、大肠杆菌和P. 铜绿假单胞菌诱导Talin激活(裂解/磷酸化)和FA增强，导致肺体外和体内微血管内皮屏障的破坏。我们将调查Calain是否调节 Talin激活(切割/磷酸化)在急性肺损伤微血管内皮细胞屏障破坏中的作用内毒素、大肠埃希菌和铜绿假单胞菌。我们将评估患有革兰氏阴性脓毒症的ALI患者的血浆通过Calain-talin-FA-MLCP途径诱导HLMVEC屏障受损。这项提议很新颖，因为它将确定Calain是肺内皮细胞屏障妥协的介质，并通过调节新的Talin来发挥这一作用 ALI中的切割/磷酸化、RhoA激活和MLCP活性。对机械原理有更好的理解 Insight将提供一个框架，在该框架中可以使用新的Calain抑制和MLCP激活策略为干预和治疗ALI而开发。