Abstract 308: Ca2+-dependent Tyrosine Kinase PYK2 Promotes Atherogenesis by Inducing Monocyte and endothelial cell-Derived TNF-alpha and MCP-1 Synthesis Unmasked by Analysis of PYK2/ApoE Double Deficient Mice.
Background: Calcium-dependent tyrosine kinase PYK2 plays crucial roles in cell migration or survival. 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 (PYK2-KO) were crossbred with ApoE−/− mice (ApoE-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. Atherosclerotic area was evaluated with Oil-Red-O staining. Macrophage was identified with immunostaining by anti-MOMA2 antibodies.
Results: At the 8th week, the ratio of atherosclerotic area to total surface of thoracic aorta in D-KO was less than ApoE-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). At the 2nd week, the expression of VCAM1, MCP-1 and TNF-alpha in the thoracic aorta in D-KO mice was inhibited (~43 to 58%, n = 5, P < 0.01, respectively). They were expressed in the CD31+ endothelial cells (EC) by immunohistochemistry, and inflammatory cells were barely detected in the region at that time. MCP-1 and TNF-alpha mRNA of peripheral blood-leukocytes (PBLs) in D-KO was 38% lower than ApoE-KO (P < 0.05, n = 5 each). In the primary-cultured PYK2-deficient (P-KO) endothelial cells, LPC (lysophosphatidylcholine)-induced LOX-1 expression was markedly decreased, compared with wild type cells. TNF-alpha induced expressions of VCAM-1 and MCP-1 in P-KO was decreased to 31 and 32% respectively, compared to the wild-type cells (each n = 5, P < 0.01). LPC-induced superoxide production was attenuated to 54% of the wild-type, (p < 0.05) using DCF staining. Furthermore, at the 2nd week, senescence-associated (SA)-beta-gal-stained area and expression of p53/21 in the thoracic aorta in D-KO-mice was diminished, compared to those in P-KO-mice.
Conclusion: PYK2 promotes atherogenesis by inducing both monocyte/macrophage and endothelial cells derived MCP-1, TNF-alpha and VCAM-1.