血管基底膜对内皮细胞NFкB信号通路的调控作用及其机制研究

NF-кB signaling in endothelial cells (EC) has been increasingly recognized as a critical player in the pathogenesis of many inflammation-relevant diseases such as sepsis and atherosclerosis. EC sit atop of a layer of basement membrane (BM) comprised of multiple distinct macromolecules. Although individual component of BM can impact various EC functions, the studies of whether and how BM as a whole regulates EC functions, including NFкB signaling, are still lacking. This is mainly due to the paucity of in vitro model systems capable of recapitulating the in vivo complexity of this specialized extracellular matrix. Based on the in vivo observations that non-inflammatory EC have intact BM while inflammation-engaging capillary EC lose BM integrity, we hypothesize that BM as a whole may regulate EC NFкB signaling. Indeed, using ex vivo BM model that preserves BM integrity and authenticity, we have revealed an inhibitory effect of BM on EC NFкB signaling in our preliminary studies. In Specific Aim 1 of this project, we will systemically study the effects of BM on EC NFкB signaling. In Specific Aim 2, Cre-LoxP recombination as well as activated leukocytes will be utilized to create defective BM in vivo or in vitro respectively, in order to study the importance of BM integrity in mediating the inhibitory effects on EC NFкB signaling . Specific Aim 3 is to investigate the underlying molecular mechanisms responsible for the inhibitory effect of BM on EC NFкB signaling with special emphasis on the functions of VE-Cadherin mediated cell-cell interaction on NFкB signaling. Specific Aim 4 will address the importance of BM integrity in mediating EC inflammatory response in experimental sepsis induced by LPS. The significance of this study is several folds. First, it will unravel an uncharacterized function as well as its working mechanisms of intact BM on EC NFкB signaling, which will have great impact on our understanding the pathogenesis of many inflammation-relevant diseases. Second, this project will provide new targets for inflammation prevention and treatment. Third, this project will pave the way for discovering new functions for BM in other BM-interacting cells.

完整基底膜是否对血管内皮细胞炎症反应（特别是内皮细胞NFкB 信号通路）产生调节作用仍不清楚。为此，我们利用完整、真实的ex vivo 基底膜模型，研究其对体外培养的内皮细胞NFкB 信号的影响。我们的初步研究发现，与其它细胞外基质或被广泛应用的基底膜替代物Matrigel相比，完整基底膜具有抑制内皮细胞NFкB信号的功能，从而揭示了完整基底膜这一全新功能。基于这一发现，我们将利用该基底膜体外模型，并利用Cre-LoxP技术建立体内基底膜缺失模型，系统性研究基底膜对内皮细胞NFкB信号的调节作用及其分子机制，旨在揭示基底膜与VE-Cadherin 介导的细胞间粘附、NFкB 信号之间的关系。本研究的理论意义涉及败血症、动脉粥样硬化等众多炎症相关疾病，为炎症反应干预提供新的思路和药物作用新靶点。

众多研究表明血管内皮细胞NF-кB信号在诸如败血病、动脉粥样硬化等炎症疾患中发挥重要作用。血管内皮细胞坐落于一层由复杂大分子成分组成的基底膜之上。尽管基底膜各单一成分均能调控内皮细胞功能，但基底膜作为一个整体是否或如何调控内皮细胞功能仍不清楚，这主要是由于长期缺乏理想的体内外基底膜研究模型导致的。本研究利用来源于体内大网膜的基底膜（ex vivo）模型，首次揭示完整基底膜具有抑制内皮细胞NFkB信号的作用，而且这一作用依赖于基底膜的完整性。体外机制研究发现完整基底膜增强内皮细胞VE-Cadherin表达，进而通过调控Rho A活性和β-Catenin－CRD-BP－β-TRCP1 轴线对NFкB 炎症信号进行调控。原计划利用Cre-LoxP技术条件敲除小鼠血管基底膜Laminin1成分，以破坏基底膜的完整性，从而研究基底膜对内皮细胞NFкB信号的调控作用。但因诸多因素，未能在课题结题前及时建立LamC1 flox+／+; Tie2-CreERT2+/+转基因品系小鼠，目前该部分研究仍在进行中，将在未来的工作中完成。该研究的意义在于：首次揭示基底膜对血管内皮细胞NFкB炎症信号的调控作用及其分子机制，从全新角度阐明败血症、动脉粥样硬化等疾病的发生、发展机制，并为治疗炎症相关性疾病提供新的策略和新靶点。