Abstract 1683: Ca2+/Redox-Sensitive Tyrosine Kinase PYK2 Promotes Atherogenesis through OxLDL/ROS/p21-Dependent Premature Senescence and Cytokine Induction in Endothelium

Abstract

Background: Calcium/redox-sensitive tyrosine kinase PYK2 plays crucial roles in cell migration. Analysis of PYK2-deficient macrophages has been reported that their directional migration was impaired due to dysfunction of PI3K, small GTPase Rho, and Ca (2+)-mobilization. Since monocyte/macrophage plays crucial role in atherogenesis, we newly generated the PYK2-knock-out mice and investigated the role of PYK2 in atherogenesis. Methods: PYK2−/− mice (P-KO) were crossbred with ApoE−/− mice (E-KO), and PYK2−/−/ApoE−/− double knockout (D-KO) mice were established. Four-week-old mice were fed with high-fat-diet for 8 weeks, and the thoracic aorta and aortic root were histologically analyzed. Then we studied the mechanisms of the difference in atherogenesis. Results: At the 8th week, atherosclerotic area of thoracic aorta in D-KO was less than E-KO (~54%, P < 0.01, n = 15, respectively). Numbers of infiltrating macrophage in D-KO mice was also reduced (~52%, P < 0.01, n = 15). We also verified that no significant difference in atherosclerotic area between E-KO recipient with D-KO bone marrow and D-KO recipient with E-KO bone marrow. At the 7th day, the expression of VCAM1, and MCP-1 in the thoracic aorta in D-KO mice was inhibited (~43 to 58%, n = 5, P < 0.01, respectively). Inflammatory cells were barely detected in the region at that time. TNF-alpha and MCP-1 mRNA of peripheral blood-leukocytes in D-KO was also 38% lower (P < 0.05, n = 5 each). In the primary-cultured P-KO endothelial cells, TNF-alpha-induced expressions of VCAM-1 and MCP-1 was decreased to 31 and 32% respectively, compared to the wild-type (WT) (each n = 5, P < 0.01). Lysophosphatidylcholine (LPC), the component of Oxidized LDL, -induced Reactive Oxygen Species (ROS)-production was attenuated to 54% of the WT (p < 0.05) using DCF staining, and the high cholesterol diet induced ROS-production in endothelium in D-KO were decreased by 8-OHDG staining. Senescence-associated-beta-gal-stained area and ROS-dependent expression of p21 in the thoracic aorta in D-KO-mice was diminished. In P-KO ECs, LPC-induced p21 expression via Ets-1 or STAT1 was also decreased markedly. Conclusion: PYK2 promotes atherogenesis by inducing ROS/p21-dependent premature senescence and cytokine induction in endothelium.