Functional Involvement of IntegrinB4/ITGB4 and Kindlin/FERMT2 in Focal Adhesion Dynamic Remodeling in ARDS

摘要： 新冠肺炎大流行中观察到的急性呼吸窘迫综合征死亡率上升突显了过度 机械通气促进肺血管通透性致死性增加所产生的机械应力 保佑。整合素β4基因和kindlin-2基因是内皮细胞必不可少的黏附分子 细胞(EC)焦点粘连(FA)是机械传感的关键结构，对相互之间的双向信号传递至关重要。 EC细胞骨架和细胞-基质界面，以及EC屏障调节。动静压产生的力学基础。 炎症性EC屏障功能障碍和随后的屏障修复期间的NAMIC FA协调是一件有趣的事情- 仍未解决的根本性问题。我们推测FA结构的协调控制需要 整合素β4和Kindlin-2与关键的PPG细胞骨架效应因子(nmMLCK， 皮质素、DOCK1、层脂素、巴西林)，以在EC屏障反应(PE- 外周细胞骨架重塑、脂肪板层形成、缝隙闭合)。ITGB4是一种独特的机械传感， 层粘连蛋白结合整合素，而kindlin-2是一个多结构域机械传感接头FA蛋白，它招募结构- 结构和信号分子与细胞骨架重排协同作用于FA。我们推测这些欧共体 反应受到ITGB4和Kindlin-2翻译后修饰(PTM)和编码Poll-2的高度影响- 同形现象(SNPs)。由于活性氧物种(ROS)是FA动态和损失的重要刺激 EC屏障完整性，核心B，SA#1将表征三个ROS感应转录因子的作用 (NRF2、HIF1α/HIF2α)、ITGB4/FERMT2SNPs和DNA甲基化在遗传/表观遗传调控中的作用 ITGB4/FERMT2的表达及其对唯一ITGB4选择性剪接、阻隔基因产生的影响 调控变异体，整合素β4E(ITGB4E)，我们发现它参与了机械传感和EC屏障 监管。与Core D一起，SA#2将进行深入的结构/功能研究，包括3D活细胞成像 突变型ITGB4和Kindlin-2融合蛋白(SNPs，PTMS)研究ITGB4/kindlin-2在空间上的功能 特定的EC细胞骨架重排驱动EC屏障的破坏和屏障的修复。SA#3将检查 片状脂体中ITGB4/kindlin-2与已知和新的FA结合细胞骨架相互作用的功能 合作伙伴，包括Cortactin、DOCK1、lamelLipodin和与nmMLCK的高度新颖的相互作用(项目1)。 最后，利用经典的大鼠和猪的内毒素/VILI模型(核心C)，SA#4将评估 SMurf抑制剂A01或SRI-38832增强酪氨酸偶联细胞中Kindlin-2的表达 脂质体(TySIPosome，项目4)。项目3研究将确定结构/功能和分子 ITGB4和kindlin-2动态调控FA的基础，并对其功能相关性产生了重要的见解 这一独特的FA信号轴在恢复EC屏障功能中发挥着重要作用。我们的高度翻译性方法还将 提供可行的EC屏障调节策略，以恢复受损肺循环的完整性 同时对ITGB4/FERMT2变异在ARDS健康差异中的参与提供了见解。

ABSTRACT: The elevated ARDS mortality observed in the COVID-19 pandemic has highlighted the contribution of excessive mechanical stress produced by mechanical ventilation in promoting lethal increases in lung vascular permea- bility. Integrin β4 (ITGB4 gene) and kindlin-2 (FERMT2 gene) are essential adhesion molecules in endothelial cell (EC) focal adhesions (FAs), structures critical for mechano-sensing, for bidirectional signaling between the EC cytoskeleton and the cell-matrix interface, and for EC barrier regulation. The mechanistic basis for dy- namic FA coordination during inflammatory EC barrier dysfunction and subsequent barrier restoration is a fun- damental question that remains unresolved. We speculate that coordinate control of FA structures requires the dynamic interactions of integrin β4 (ITGB4) and kindlin-2 with key PPG cytoskeletal effectors (nmMLCK, cortactin, Dock1, lamellipodin, paxillin) to efficiently assemble functional FAs during EC barrier responses (pe- ripheral cytoskeletal remodeling, lamellipodial formation, gap closure). ITGB4 is a unique mechano-sensing, laminin-binding integrin, and kindlin-2 is a multi-domain mechano-sensing adapter FA protein that recruits struc- tural and signaling molecules to FAs in concert with cytoskeletal rearrangement. We speculate that these EC responses are highly influenced by ITGB4 and kindlin-2 post-translational modifications (PTMs) and coding pol- ymorphisms (SNPs). As reactive oxygen species (ROS) is an important stimulus for FA dynamics and loss of EC barrier integrity, with Core B, SA #1 will characterize the role of three ROS-sensing transcription factors (NRF2, HIF1α/HIF2α), ITGB4/FERMT2 SNPs and DNA methylation in genetic/epigenetic regulation of ITGB4/FERMT2 expression and the influence on generation of the unique ITGB4 alternatively-spliced, barrier- regulatory variant, Integrin β4E (ITGB4E), that we identified as involved in mechano-sensing and EC barrier regulation. With Core D, SA #2 will conduct in depth structure/function studies including 3D live cell imaging of mutant ITGB4 and kindlin-2 fusion proteins (SNPs, PTMs) to characterize ITGB4/kindlin-2 function in spatially- specific EC cytoskeletal rearrangements driving EC barrier-disruption and barrier-restoration. SA #3 will examine the functionality of ITGB4/kindlin-2 interactions within lamellipodia with known and novel FA-binding cytoskeletal partners, including cortactin, Dock1, lamellipodin and highly novel interactions with nmMLCK (Project #1). Finally, utilizing elegant rat and porcine models of LPS/VILI (Core C), SA #4 will assess the therapeutic utility of the SMURF inhibitor A01, or SRI-38832 to augment kindlin-2 expression as cargo in TySIPonate-conjugated liposomes (TySIPosomes, Project #4). Project #3 studies will determine the structure/function and molecular basis for the dynamic FA control by ITGB4 and kindlin-2 and yield important insights into functional relevance of this unique FA signaling axis in restoration of EC barrier function. Our highly translational approaches will also provide actionable EC barrier-regulatory strategies that restore the integrity of the injured pulmonary circulation while yielding insights into ITGB4/FERMT2 variant participation in ARDS health disparities.