Abstract 499: Ca2+-dependent Tyrosine Kinase PYK2 Promotes Atherogenesis by Inducing Monocyte/Macrophage-Derived IL-1β and MCP-1 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 into cytoplasm. 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 sinus were histologically analyzed. Atherosclerotic area was evaluated with Oil-Red-O staining. Macrophage was identified with immunostaining using anti-MOMA2 antibodies. Real-time PCR for ICAM-1, VCAM-1, MCP-1 and IL-1β was performed.
Results: After 8 weeks, the ratio of atherosclerotic area to the aortic sinus area 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). After 2 weeks, the expression of ICAM-1 and VCAM1 and MCP-1 and IL-1β in the thoracic aorta in D-KO mice was inhibited (~43 to 58%, n=5, P<0.01, respectively) by real-time PCR analysis. They expressed in the CD31+ endothelial cells (EC) by immunohistochemistry, and inflammatory cells were barely detected in the region. The relative number of the MCP-1- and IL-1β-positive peripheral blood-mononuclear cells (PB-MNCs) in total PB-MNCs of D-KO was 58% lower than ApoE-KO (P<0.05, n=5 each) by the FACS analysis. In the primary-cultured PYK2-deficient PB-MNCs, oxidized LDL (oxLDL)-induced NFκB activation was markedly decreased and expressions of IL-1β and MCP-1 were decreased to 31 and 32%, respectively, compared to the wild-type cells, each n=5, P<0.01). Furthermore, ox-LDL-induced superoxide production was attenuated to 54% of the wild-type, (p<0.05) using L-012 chemiluminescence. Stimulation of PYK2-KO-endothelial cells with oxLDL showed similar abnormality.
Conclusion: PYK2 promotes atherogenesis by inducing monocyte/ macrophage-Derived IL-1β and MCP-1.