. 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.
Loss of endothelial barrier repair is central to pathogenesis of acute lung injury but it has proven challenging to treat even with stem cell therapy. The proposed studies will define an approach for promoting intrinsic endothelial barrier repair based on the novel concept that focal adhesion kinase suppresses DNA methyltransferase 3a activity to induce sphingosine 1 phosphate receptor 1 transcription and restore lung-fluid homeostasis.
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