INTRODUCTION
Arterial diseases including hypertension and coronary artery disease (CAD) display a degree of dysregulation in the contractile behaviour of the smooth muscle. Vascular tone is regulated by the relative contractile state of vascular smooth muscle cells (VSMCs)1,2. VSMC contraction provides force generation through the phosphorylation of myosin light chain kinase (MLCK) which facilitates interaction between actin and myosin filaments. MLCK is directly phosphorylated by calcium-bound calmodulin. Increases in intracellular calcium concentrations ([Ca2+]i) occur through PKC-mediated activation of L-type voltage-gated calcium channels (VGCCs) leading to an influx of extracellular Ca2+ ions, or via activation of Gαq-Protein Coupled Receptors (GPCRs) leading to PLCβ-mediated Ca2+ ion release from the sarcoplasmic reticulum. Whilst these central signalling pathways controlling contraction are widely characterised, modulation of these pathways remains ill-defined.
Several integrins can directly modulate vascular smooth muscle cell contraction by regulating calcium influx through VGCCs3-7. Within the airway, integrin α9β1 has been specifically identified as preventing exaggerated airway smooth muscle contraction, where conditional knockout of the α9 subunit in airway smooth muscle causes a spontaneous increase in pulmonary resistance in response to multiple GPCR agonists8. SVEP1, a high affinity ligand for integrin α9β19, is a 390 kDa secreted extracellular matrix (ECM) protein comprised of sushi (complement control protein (CCP)), von Willebrand factor type A, epidermal growth factor-like, and pentraxin domains10. The function of SVEP1 remains unclear but it has reported roles in cell adhesion9-12, lymphatic vessel formation13,14, epidermal differentiation15, and cancer progression16,17. A low frequency coding variant rs111245230 (p.D2702G) within SVEP1 associates with elevated blood pressure (BP)18 and CAD19. rs111245230 is situated adjacent to the binding motif through which SVEP1 binds to integrin α9β19. Genetic variants associated with reduced expression of ITGA9 , which encodes the α9 subunit of integrin α9β1, have also been reported to associate with increased BP20,21. Neither SVEP1 nor integrin α9β1 have a reported role in vasoregulation, however direct activation of integrin α4β1, with which integrin α9β1 forms an integrin subfamily22, can induce VSMC contraction4.
The role of SVEP1 in cardiovascular disease is only now beginning to be realised with two recent in vivo studies showing SVEP1 to be primarily expressed within blood vessels23 and SVEP1 knockout affecting atherosclerosis development23,24. Due to the genetic association between variants in SVEP1 andITGA9 with BP, the role of integrin α9β1 in airway contraction, and SVEP1 expression, we examined the role of these proteins in regulating VSMC contractility.