Focal adhesion kinase regulation of lung vascular permeability and edemagenesis

项目摘要。肺微血管通透性增加，导致富含蛋白质的肺泡水肿， 慢性炎症，导致ARDS（急性呼吸窘迫综合征），急性肺损伤的致命形式 （ALI）。在上一个资助周期中，我们证明了内皮细胞（EC）特异性局灶性粘附缺失 激酶（FAK）破坏粘附连接，引起慢性肺水肿。我们还发现FAK ARDS患者肺组织中TNF-α水平明显降低。这些发现表明FAK的表达对于 内皮屏障修复和因此肺液稳态。细胞疗法可以解决炎症性肺 血管损伤的动物模型，但骨髓来源的间充质干细胞（MSC）或MSC来源的 外泌体在EC-FAK-/-小鼠中不能这样做。在研究EC-FAK在调节有效性方面的作用时， 细胞治疗在解决肺血管损伤中的作用，我们进行了基本观察，EC-FAK是 维持鞘氨醇-1-磷酸受体1（S1 PR 1）在EC中的表达所需，已知其 增强内皮屏障，防止肺损伤。因此，我们假设受损的SIPR 1合成是 负责EC-FAK-/-小鼠中有缺陷的内皮屏障修复和干细胞治疗功效的丧失 疗法有趣的是，S1 PR 1的表达和屏障功能可以在FAK耗竭的EC中被拯救， 转录因子Kruppel样因子2（KLF 2）的转导，表明FAK通过上调 KLF 2活性因此，活性KLF 2通过恢复S1 PR 1表达和功能来克服FAK耗竭。这些 这些发现导致了我们的第二个开创性观察，即EC-FAK-/-小鼠中KLF 2转录活性的丧失是由于 DNA甲基转移酶3a（DNMT 3a）的活化引起的KLF 2-DNA的表观遗传修饰。DNMT3A 将胞嘧啶转化为5-甲基胞嘧啶（5 mc），抑制基因转录。因此，DNMT 3a甲基化的 KLF 2启动子导致S1 PR 1合成和屏障修复受损。根据这些调查结果， 研究将确定FAK在损伤后诱导EC屏障修复的新作用， 修饰KLF 2，从而使S1 PR 1转录和功能。我们将使用最先进的方法， 包括EC特异性敲除小鼠、细胞和核成像以及生物物理方法， 测量细胞张力来建立这个概念。我们的具体目标是：#1：解决概念 FAK维持S1 PR 1在内皮细胞的转录是内皮细胞内源性凋亡所必需的。 屏障修复，从而解决肺血管损伤，以及#2：定义FAK的作用 DNMT 3a抑制内皮细胞KLF 2-DNA甲基化作为S1 PR 1合成的机制 KLF 2及其通路在恢复内皮屏障完整性、缓解肺血管损伤中的作用 和促进EC-FAK-/-小鼠对继发性肺损伤的耐受性。我们相信我们的研究将定义 选择性抑制DMNT 3a作为预防ARDS致死性的潜在临床靶点。

Project Summary. Increased lung microvascular permeability, resulting in protein-rich alveolar edema and chronic inflammation, causes ARDS (Acute Respiratory Distress Syndrome), the lethal form of acute lung injury (ALI). During the last funding cycle, we demonstrated that endothelial cell (EC)-specific deletion of focal adhesion kinase (FAK) disrupts adherens junctions, causing chronic pulmonary edema. We also showed that FAK is markedly reduced in lungs of ARDS patients. These findings suggest that FAK expression is critical for endothelial barrier repair and hence lung-fluid homeostasis. Cellular therapy can resolve inflammatory lung vascular injury in animal models, but bone marrow-derived mesenchymal stem cells (MSCs) or MSC-derived exosomes failed to do so in EC-FAK-/- mice. In investigating the role of EC-FAK in regulating the effectiveness of cellular therapy in resolving lung vascular injury, we made the fundamental observation that EC-FAK is required to maintain the expression of sphingosine-1-phosphate receptor1 (S1PR1) in ECs, which is known to strengthen the endothelial barrier and prevent lung injury. Thus, we postulated that impaired SIPR1 synthesis is responsible for defective endothelial barrier repair in EC-FAK-/- mice and for the loss of efficacy of stem cell therapy. Intriguingly, S1PR1 expression and barrier function could be rescued in FAK-depleted ECs following transduction of the transcription factor, Kruppel like factor 2 (KLF2), indicating that FAK functions by upregulating KLF2 activity. Active KLF2 thus overcomes FAK depletion by restoring S1PR1 expression and function. These findings led to our second seminal observation that loss of KLF2 transcriptional activity in EC-FAK-/- mice is due to epigenetic modification of KLF2-DNA caused by activation of DNA methyltransferase 3a (DNMT3a). DNMT3a converts cytosine to 5-methylcytosine (5mc) repressing gene transcription. Hence, DNMT3a methylation of the KLF2 promoter led to impaired S1PR1 synthesis and barrier repair. Based on these findings, the planned research will define the novel role of FAK in inducing EC barrier repair following injury by suppressing epigenetic modification of KLF2, thereby enabling S1PR1 transcription and function. We will use state of the art approaches, including EC specific knockout mice, cellular and nuclear imaging and biophysical approaches such as measurement of cellular tension to establish this concept. Our Specific Aims are: #1: to address the concept that FAK maintenance of S1PR1 transcription in the endothelium is required for intrinsic endothelial barrier repair and thereby for resolving lung vascular injury, and #2: to define the role of FAK suppression of endothelial KLF2-DNA methylation by DNMT3a as a mechanism for S1PR1 synthesis by KLF2 and the role of this pathway in restoring endothelial barrier integrity, resolving lung vascular injury and promoting tolerance to secondary lung injury in EC-FAK-/- mice. We believe our studies will define selective inhibition of DMNT3a as a potential clinical target for preventing the lethality of ARDS.