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A7393 - Pathogenic Variants in CRIPAK Are Associated with Endothelial Dysfunction and Small Vessel Loss in Pulmonary Arterial Hypertension
Author Block: J. Tenorio1, M. Orcholski2, E. Shamskhou3, K. Yuan4, E. Granda5, P. Arias5, P. Lapunzina5, V. De Jesus Perez6; 1Cardiogenetics Unit (INGEMM) and Pulmonary and Critical Care (Stanford University), Institute of Medical and Molecular Geneticist and Stanford School of Medicine, Madrid and Stanford, CA, United States, 2Dept of Medicine, Stanford Univ, Stanford, CA, United States, 3Pulmonary and Critical Care, Stanford University, Palo Alto, CA, United States, 4Medicine, Stanford University, Palo Alto, CA, United States, 5Genetics, Institute of Medical and Molecular Geneticist, Madrid, Spain, 6Stanford Med Ctr, Palo Alto, CA, United States.
Background: Pulmonary Arterial Hypertension (PAH) is a rare disease of unclear etiology that is associated with abnormally increased pulmonary pressures and chronic right heart failure. Endothelial dysfunction is a major feature of PAH and contributes to vascular pathology in patients. Use of whole exome sequencing (WES) by our group and others has led to the discovery of gene variants linked to PAH pathobiology in patients with familial PAH but whether this approach could also be used for gene variant discovery in unrelated PAH patients is unclear. The main aim of this project was to perform a bioinformatics analysis of the whole exome sequencing (WES) data of 35 unrelated PAH patients with PAH to identify pathologic gene variants potentially involved in endothelial dysregulation in PAH. (124 words)
Material and methods: Bioinformatic and in silico analysis was applied to WES data from patients with idiopathic, heritable and methamphetamine (METH) induced PAH. Expression of WES candidate genes was assessed in pulmonary microvascular endothelial cells (PMVECs) from healthy donors and PAH patients. Functional gene analysis was carried via tube formation and scratch assays in PMVECs transfected with either nontargeting (NT) and gene specific siRNA (63 words).
Results: A frameshift mutation in CRIPAK was found in a high percentage in PAH patients compare to controls (31% vs. 2%). CRIPAK is the key protein responsible for regulation of PAK1, a major signaling mediator of VEGF responsible for promoting proliferation, migration and survival of PMVECs. Compared to healthy donors, western blots of PAH PMVECs lysates demonstrated significant reduction in CRIPAK along with concomitantly increased phospho (i.e. active) PAK1 levels. Given that PAH PMVECs exhibit impaired tube formation and motogenic responses in culture, we speculated that reduction in CRIPAK could produce a similar phenotype in healthy PMVECs. Indeed, transfection of CRIPAK siRNA (siCRIPAK) into healthy PMVECs significantly reduced angiogenic response to VEGF-A, as evidenced by reduced tube network formation and gap closure in matrigel and scratch assays, respectively. (129 words)
Conclusion: WES analysis has identified CRIPAK as a potential modifier gene in PAH. Reduced CRIPAK could contribute to PAH by reducing endothelial viability, promoting small vessel loss and accelerating vascular remodeling. (31 words)