201 Focal Adhesion Protein and Cytoskeletal Remodelling in Noradrenaline and Endothelin-1 Stimulated Vascular Smooth Muscle Cells

HEART(2016)

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Background Focal adhesions (FA) are dynamic transmembrane protein complexes serving as mediators of signalling between the extracellular matrix (ECM) and the actin cytoskeleton (CK). Remodelling of FAs and the CK play key roles in some of the pathological functions of vascular smooth muscle cells (VSMC) such as proliferation, migration and contraction. Focal adhesion proteins (FAPs), constituents of FAs, indirectly dictate some VSMC functions. The FAPs, paxillin, Hic-5 and vinculin’s presence within FAs is well established in VSMCs, however, their behaviour in response to biomechanical and vasoconstrictor stimuli are not. Endogenous vasoconstrictors such as Endothelin-1 (ET-1) and Noradrenaline (NA), alongside changes in ECM have individually promoted changes in the VSMC CK and FA arrangement. To contextualise these cytoskeletal changes and their consequences on vascular function, more information is needed about FAP localisation and CK arrangement in response to vasoactive stimulation alongside ECM changes.Aims To investigate actin CK remodelling and the localisation of Hic-5, vinculin and paxillin in response to changes in substrate composition and contractile stimulation.Methods Rat VSMCS (RVSMCs) were cultured on glass or type I collagen-coated glass and were stimulated with 15I¼M NA, 100 nM ET-1 or remained unstimulated. Cells were stained for actin filaments and either Hic-5, vinculin or paxillin. Changes in CK arrangement were visually assessed. The punctate regions of FAPs were quantified using image analysis software.Results When cultured on glass, Hic-5 and paxillin occupied punctate regions within RVSMC; vinculin was diffusely spread throughout the cell. The punctate regions were principally localised to the ends of actin stress fibres. Compared to unstimulated RVSMCs (punctate region mean area, 1.043I¼m²), NA caused a reduction in paxillin punctate region area of RVSMCs cultured on both glass (0.5548I¼m²; P u003c 0.0001) and collagen (0.7060I¼m²; P u003c 0.001). NA also induced cytosolic dissemination of Hic-5 without affecting punctate region area. Vinculin localisation did not change in response to NA ET-1 or collagen. ET-1 did not induce changes in paxillin or Hic-5 localisation or CK arrangement. RVSMCs cultured on glass showed a peripheral arrangement of the CK within the cell compared to those cultured on collagen, irrespective of stimulation.Conclusion The study demonstrates that NA selectively regulates FAP localisation for paxillin and Hic-5, but not vinculin. As ET-1 does not regulate FAP localisation; these results indicate that individual FA remodelling is agonist specific within VSMCs. Furthermore, ECM composition is vital in CK reorganisation in a manner independent of NA-induced FA remodelling. Accordingly, actin CK and FA reorganisation in response to altered ECM composition or vasoconstrictors may contribute to vascular remodelling in cardiovascular disease.
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