Angiotensin II Induces Human Monocyte Migration and Pyk2/ Paxillin Phosphorylation

Abstract

12 Angiotensin II (AII) has been shown to enhance the development of atherosclerotic lesions. Transendothelial migration of monocytes is an early critical step in the atherosclerotic process. To elucidate mechanisms by which AII promotes atherogenesis, we investigated its effects on human monocyte migration. AII induced migration of human peripheral blood monocytes (HPBM) and human THP-1 monocytes at concentrations between 0.01-1μmol/L, with 3.3-fold induction in HPBM and 4.8-fold in THP-1cells at 1μmol/L AII (both p< 0.01 vs. unstimulated cells). To identify cell signalling pathways involved in AII-directed migration, we blocked extracellular regulated protein kinase (ERK1/2) - MAPK activation with PD98059 (PD) and p38-MAPK activity with SB203580 (SB). Pretreatment of HPBM with PD (30μmol/L) and SB (10μmol/L) blocked AII (1μmol/L) stimulated migration by 75.6 +/- 3.1% (p< 0.01) and 52.8 +/- 2.6% (p< 0.01), respectively. AII (1μmol/L) directed migration of THP-1 monocytes was inhibited by 67.7 +/- 15.9% (PD 30μmol/L, p< 0.01) and 47.2 +/- 12.3% (SB 10μmol/L, p<0.05). Addition of the AII receptor type 1 (AT1R) antagonist Losartan (LOS) completely supressed AII induced monocyte migration, demonstrating an AT1R mediated mechanism (HPBM: LOS 100μmol/L 83.1 +/- 6.5% inhibition (p< 0.05)/ THP-1: LOS 100μmol/L 91.3+/- 21.5% inhibition (p<0.01)). Cell movement requires the phosphorylation of cytoskeleton-associated tyrosine kinases and formation of focal adhesion complexes. Western immunblotting with phosphospecific antibodies showed that AII (1μmol/L) induced the rapid tyrosine phosphorylation of PYK2 , a calcium-dependent tyrosine kinase, in THP-1 cells by 3.6-fold peaking after 10min. Phosphorylation of the focal adhesion related protein paxillin was enhanced in THP-1 cells by AII (1μmol/L) with a 4.3-fold induction after 2 min. This study demonstrates a potential proatherogenic mechanism of AII by stimulating human monocyte migration through an AT1R-ERK1/2-p38-dependent process, which might be, in part, mediated by AII induced PYK2/Paxillin phosphorylation.