Functional Analysis of the Coronary Artery associated gene SVEP1

Background: A low frequency missense variant in Sushi, von Willebrand factor type A, EGF and pentraxin domain containing protein1 (SVEP1) is associated with coronary artery disease (CAD) but the contributory mechanisms are currently unknown. We investigated the role of SVEP1 in CAD relevant disease processes.

Methods and Results: Expression profiling revealed an up-regulation of SVEP1 during monocyte to macrophage differentiation, leading us to investigate the role of SVEP1 in inflammatory processes contributing to the pathology of CAD. We used THP-1 cells as an experimental model of monocyte- macrophage behaviour and developed assays to quantify CAD relevant inflammatory mechanisms such as monocyte adhesion, migration, macrophage differentiation and phagocytosis. Knockout of SVEP1 significantly reduced THP-1 macrophage spreading and migration. The spreading phenotype was partially rescued in the SVEP1 knockout THP-1 cells using a fragment of recombinant SVEP1 corresponding to a reported integrin α9 binding domain.

We confirmed SVEP1 interacts with integrin α9 and demonstrated a novel interaction between SVEP1 and integrin α4. We measured activity of integrin second messenger signalling proteins and observed increased FAK and phospho- FAK as well as reduced levels of total and phospho-paxillin, and RAC and Rho levels in SVEP1 THP-1 knockout monocytes. Additionally, treatment of THP-1 cells with the integrin α9/β1 and integrin α4/β1 inhibitor BOP, the RAC inhibitor EHOP, and the ROCK inhibitor Y-27362, reduced differentiation induced spreading. Y-27362 also inhibited migration. We partially rescued the SVEP1 knockout spreading phenotype using an activator of RHO/RAC/CDC42.

Conclusion: SVEP1 binds to integrin α9/β1 and integrin α4/β1 and contributes to migration and differentiation induced spreading of THP-1 cells by regulating integrin signalling second messenger proteins and RHO ad RAC expression. Our results show that SVEP1, via integrin α9 or α4, is a new regulator of monocyte macrophage behaviour and may increase CAD risk through altered inflammatory cell behaviour.