Background/Purpose: Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by activation of the immune system, vascular dysfunction and tissue fibrosis. Vascular dysfunction in SSc is one of the most prominent features of the disease that generally manifest as Raynaud’s phenomenon and vascular wall proliferation resulting in progressive fibro-proliferative vasculopathy seen in all involved organs. There is evidence for increased vascular smooth muscle cell (vSMC) proliferation, migration and resistance to apoptosis in SSc. In this study, we wished to understand the role of cofilin signaling pathways involvement in SSc-vSMC dysfunction. Cofilin is a member of the actin associated proteins that accelerates actin depolymerization at pointed ends and severs long actin filaments. It plays a crucial role in cell migration by promoting the rapid turnover of actin filaments through severing filamentous actin (F-actin) and depolymerizing actin filaments from the pointed end. Cofilin is inactivated by phosphorylation through LIM-Kinases (LIMK), and activated by dephosphorylation mediated by Slingshot phosphatases (SSH-1).

Methods: We isolated vSMC from 4mm punch skin biopsies from 3 patients with diffuse cutaneous SSc (dcSSc) and 3 healthy controls. We examined the expression levels of P-cofilin, total cofilin, P-LIMK and Total LIMK by western blot analysis. We used insulin at a concentration of 5nM, which activates LIMK by phosphorylation, to evaluate the effect of cofilin on vSMC migration that was examined by the scratch test assay.

Results: We demonstrate significant increase in SSc-vSMC migration compared to control cells that was noted at 2 hours and continued for 48 hours, with an average increase migration of 2.1 fold at 24 hours. The expression ratio of P-cofilin to total cofilin was 3.7 and 0.3 in control-vSMC and SSc-vSMC, respectively (P<0.01). We also noted decreased ratio of phosphorylation of LIMK in SSc-vSMC (0.21) compared to control vSMC (0.55, P <0.01). The addition of insulin increased the ratio of phosphorylation of LIMK by 2 folds at 24 hours, which was associated with 6 fold increase in phosphorylation of cofilin. Moreover, in vitro treatment of vSMC with insulin decreased SSc-vSMC migration potential and reverted SSc-vSMC migration to a pattern similar to control vSMC.

Conclusion: We provide an experimental evidence for the role of cofilin in increased migration of SSc-vSMC, which contribute to the abnormal phenotype of SSc-vSMC. We report activation of cofilin in SSc-vSMC as shown by decrease P-cofilin in association with inactivation of LIMK by decreased P-LIMK levels in the cells. Furthermore, the addition of insulin activates LIMK by phosphorylation, which in turn inactivates cofilin. Indeed, the addition of insulin to SSc-vSMC cultures resulted in reduced SSc-vSMC migration to a level comparable to control vSMC migration rate.

« Back to 2018 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-role-of-cofilin-an-actin-associated-protein-in-activation-of-systemic-sclerosis-vascular-smooth-muscle-cells/