MAM Proteins in Lung Vascular Injury

急性肺损伤(ALI)/急性呼吸窘迫综合征(ARDS)是一种危及生命的疾病，它影响 每年约有20万美国人接受重症监护，入院率为10%-15%，死亡率为25%-40%。 败血症是人类ALI的一个突出的肺外原因。近50%的严重脓毒症患者 发展ALI/ARDS。目前治疗ALI/ARDS的所有疗法仍然依赖于支持性护理；因此，迫切需要 开发安全有效的治疗ALI/ARDS的新策略。因为内皮细胞(EC) 屏障功能障碍是脓毒症ALI的早期和关键组成部分，对其机制有更好的了解 提高EC的通透性是开发有效治疗ALI的关键。这项建议的目标是了解如何 内质网介导的脓毒症患者内质网与线粒体的异常通讯 分子伴侣Bip/GRP78和线粒体(Mito)分子伴侣Mortalin/GRP75促进EC屏障功能障碍 是为了让阿里。我们的假设是BiP/GRP78和GRP78之间的密切相互作用和功能合作 线粒体相关内质网膜上Mortalin/GRP75的表达增加 细胞内钙离子和内质网-线粒体接触部位引起线粒体钙超载和炎症体激活， 导致败血症时肺血管损伤。我们进一步假设靶向Bip/GRP78和Mortalin/GRP75 联合应用可能被证明是对抗败血症所致的ALI和死亡率的一种更好的治疗方法。这个 这一建议是基于我们的新发现，即Bip/GRP78和Mortalin/GRP75是钙信号的关键调节因子 EC中的ER-Mito接触部位和沉默Bip/GRP78或Mortalin/GRP75均可阻止EC屏障 败血症患者的血浆和其他临床相关的促水肿激动剂引起的干扰 凝血酶和内毒素。脓毒症肺组织诱导Bip/GRP78，静息肺高表达Bip/GRP78 血管内皮细胞足以引起小鼠肺损伤。此外，分别抑制Bip/GRP78或Mortalin/GRP75 减轻小鼠的ALI。重要的是，联合抑制Bip/GRP78或Mortalin/GRP75要有效得多 (需要减少约5倍剂量的Bip/GRP78或Mortalin/GRP75抑制剂)以提高脓毒症小鼠的存活率。 该提案将解决以下目标。目标1将确定Bip/GRP78和Mortalin/GRP75的作用 在调节细胞内钙离子以引起EC通透性方面。目标2将确定BiP/GRP78的作用和 Mortalin/GRP75增加ER-Mito接触位点和线粒体钙摄取导致线粒体 功能障碍和炎性小体激活导致EC通透性。目标3将确定(I) 内皮Bip/GRP78和Mortalin/GRP75在引起肺损伤中的作用，以及(Ii)评估血管内皮细胞Bip/GRP78和Mortalin/GRP75的治疗潜力 联合抑制BiP/GRP78和Mortalin/GRP75抑制脓毒症所致的ALI和死亡率。这些研究将 对ER-Mito通讯及其在ALI发病机制中的相关性提供有价值的见解 导致开发安全有效的治疗策略，依赖于联合抑制 BiP/GRP78和Mortalin/GRP75用于控制ALI和提高脓毒症患者的存活率。

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a life-threatening condition which affects ~200,000 Americans each year with 10-15% of intensive care admissions and a mortality rate of ~ 25-40%. Sepsis is a prominent extrapulmonary cause of ALI in humans. Nearly 50% of patients with severe sepsis develop ALI/ARDS. All current therapies for ALI/ARDS still rely on supportive care; thus, there is an urgent need to develop new treatment strategies for ALI/ARDS that are safe and effective. Because endothelial cell (EC) barrier dysfunction is an early and critical component of ALI in sepsis, a better understanding of the mechanisms of EC permeability is key to developing effective therapy for ALI. The goal of this proposal is to understand how aberrant communication between endoplasmic reticulum (ER) and mitochondrion in sepsis, mediated by ER chaperone BiP/GRP78 and mitochondrial (MITO) chaperone Mortalin/GRP75, promotes EC barrier dysfunction to cause ALI. Our hypothesis is that a close interaction and functional cooperation between BiP/GRP78 and Mortalin/GRP75 at the MAMs (mitochondrial associated endoplasmic reticulum membranes) serves to increase cytosolic Ca2+ and ER-MITO contact sites to cause mitochondrial Ca2+ overload and inflammasome activation, leading to lung vascular injury in sepsis. We further hypothesize that targeting BiP/GRP78 and Mortalin/GRP75 in combination may prove a superior therapeutic approach against sepsis-induced ALI and mortality. The proposal is based on our novel findings that BiP/GRP78 and Mortalin/GRP75 are key regulators of Ca2+ signaling and ER-MITO contact sites in EC and silencing BiP/GRP78 or Mortalin/GRP75 each prevents EC barrier disruption caused by plasma from septic patients and other clinically relevant edemagenic agonists such as thrombin and LPS. BiP/GRP78 is induced in septic lung and overexpressing BiP/GRP78 in the resting lung endothelium is sufficient to cause lung injury in mice. Moreover, inhibiting BiP/GRP78 or Mortalin/GRP75 each mitigates ALI in mice. Importantly, combined inhibition of BiP/GRP78 or Mortalin/GRP75 is far more effective (requires ~5-fold less dose of BiP/GRP78 or Mortalin/GRP75 inhibitors) in improving survival in mice with sepsis. The proposal will address the following aims. Aim 1 will determine the role of BiP/GRP78 and Mortalin/GRP75 in regulating cytosolic Ca2+ to cause EC permeability. Aim 2 will determine the role of BiP/GRP78 and Mortalin/GRP75 in increasing ER-MITO contact sites and mitochondrial Ca2+ uptake to cause mitochondrial dysfunction and inflammasome activation leading to EC permeability. Aim 3 will determine (i) the contribution of endothelial BiP/GRP78 and Mortalin/GRP75 in causing lung injury, and (ii) assess the therapeutic potential of inhibiting BiP/GRP78 and Mortalin/GRP75 together against sepsis-induced ALI and mortality. These studies will provide valuable insight into the ER-MITO communication and its relevance in the pathogenesis of ALI and may lead to development of a safe and efficacious therapeutic strategy that relies on combined inhibition of BiP/GRP78 and Mortalin/GRP75 to control ALI and improve survival in sepsis.