Endothelial laminin in blood brain barrier regulation

项目摘要/摘要 这项应用的长期目标是充分了解血管内皮层粘连蛋白的生物学特性。 脑血管在生理条件下的发育和功能。这与NHLBI的 最主要的目的是了解正常的生物功能。这项提案旨在调查 动态平衡状态下内皮层粘连蛋白在血脑屏障完整性中的生物学作用 条件，并探索其潜在的分子机制。在目标1中，内皮细胞的丢失 层粘连蛋白在发育早期影响血脑屏障的形成或在后期维持血脑屏障 使用在我们的研究中产生的创新内皮层粘连蛋白基因敲除小鼠系(EKO)进行研究 实验室。血脑屏障破坏的程度将使用各种不同的荧光示踪剂来确定 分子量。接下来，潜在的分子机制(细胞旁和/或跨细胞 运输)将在体外和体内的生化、超微结构和功能水平上进行探索。 由于造血细胞来源的层粘连蛋白在这些突变体中也被消融，我们将进一步研究是否 EKO小鼠血脑屏障的破坏是由于内皮细胞或造血细胞层粘连蛋白的丢失 VE-Cherin-Creert系列。在目标2中，内皮层粘连蛋白调节血脑屏障完整性的假设 将通过腺酰环化酶-2(AC2)进行检测。首先，内皮层粘连蛋白是否以及如何调控 将在体外和体内检测AC2在脑微血管内皮细胞中的表达。接下来， AC2在血脑屏障通透性和细胞旁/跨细胞转运中的作用将通过 功能损失法和功能增益法。第三，AC2对血脑屏障完整性是否有影响 依赖其腺酰环化酶活性(cAMP的产生)将使用AC2抑制剂进行研究 和/或激活剂。如果答案是肯定的，哪条cAMP下游效应/信号通路(PKA 与EPAC相比)介导AC2‘S血脑屏障的调节功能将利用PKA和/或EPAC特异性来探索 抑制剂和激活剂。这一提议的成功完成将阐明该蛋白的生物学功能 内皮层粘连蛋白在血脑屏障中的完整性及其潜在的分子机制；揭示以前 AC2在跨细胞转运(跨细胞)中的作用尚不清楚；并鉴定内皮层粘连蛋白和 AC2作为血脑屏障调节的新分子靶点，将促进创新的发展 血脑屏障紊乱的治疗。此外，这项提案还将产生创新的研究材料 (例如转基因小鼠品系和慢病毒构建体)在层粘连蛋白/基底膜领域有用， 由于其内在的复杂性和研究工具的缺乏，对它的研究还不够深入。

Project Summary/Abstract The long-term objective of this application is to fully understand the biology of endothelial laminin in cerebrovascular development and function in physiological conditions. This is in line with NHLBI’s overarching objective to understand normal biological function. This proposal aims to investigate the biological function of endothelial laminin in blood brain barrier (BBB) integrity under homeostatic conditions, and explore its underlying molecular mechanism. In Aim 1, whether loss of endothelial laminin affects BBB formation in early development or BBB maintenance at later stage will be investigated using an innovative endothelial laminin knockout mouse line (EKO) generated in our laboratory. The extent of BBB disruption will be determined using fluorescent tracers of various molecular weights. Next, the underlying molecular mechanism (paracellular and/or transcellular transport) will be explored at biochemical, ultrastructural, and functional levels both in vitro and in vivo. Since hematopoietic cell-derived laminin is also ablated in these mutants, we will further investigate if BBB breakdown in EKO mice is due to loss of laminin in endothelial cells or hematopoietic cells using the VE-Cadherin-CreERT line. In Aim 2, the hypothesis that endothelial laminin regulates BBB integrity via adenylyl cyclase-2 (AC2) will be tested. First, whether and how exactly endothelial laminin regulates AC2 expression in brain microvascular endothelial cells will be examined in vitro and in vivo. Next, the function of AC2 in BBB permeability and paracellular/transcellular transport will be investigated using both loss-of-function and gain-of-function approaches. Third, whether the effect of AC2 on BBB integrity relies on its adenylyl cyclase activity (generation of cAMP) will be investigated using AC2 inhibitors and/or activators. If the answer is yes, which cAMP-downstream effector/signaling pathway (PKA versus Epac) mediates AC2’s BBB-regulating function will be explored using PKA- and/or Epac-specific inhibitors and activators. Successful completion of this proposal will elucidate the biological function of endothelial laminin in BBB integrity and its underlying molecular mechanism; uncover a previously unrecognized role of AC2 in transcellular transport (transcytosis); and identify endothelial laminin and AC2 as novel molecular targets in BBB regulation, which will promote the development of innovative treatments for BBB disruption. In addition, this proposal will also generate innovative research materials (e.g. transgenic mouse line and lentiviral constructs) useful in the field of laminin/basement membrane, which is understudied due to its intrinsic complexity and lack of research tools.