The role of extracellular pressure and stiffness in podosome regulation

Abstract

Atherosclerosis is a subtype of cardiovascular disease with pathological vascular wall remodelling, extracellular stiffness changes, and is often associated with hypertension. Vascular smooth muscle cells (VSMCs) are a major component of the vascular wall. VSMC dysregulation and phenotypic switch are associated with atherosclerosis progression. Hallmarks of vascular smooth muscle cell phenotypic switch include actin cytoskeleton remodelling and the formation of podosomes. Podosomes are actin­based membrane protrusions and can serve as extracellular matrix anchors, mechanosensors, force transducer, and presumably have roles in cell migration, matrix degradation, and cell invasion. This study is aimed to test whether pathological levels of pressure and stiffness are sufficient to induce VSMC phenotypical switch using podosome formation as the hallmark. To test this hypothesis, A7r5 aortic SMC were cultured on Polydimethylsiloxane (PDMS) coated cover slips at physiological and pathological stiffnesses and stimulated with physiological and pathological pressure by an external pressure stimulator. Using this approach, we have shown that pathological high pressure and soft stiffness had synergistically promoted podosome formation, matrix degradation, and cellular stiffness reduction in VSMCs. To further characterize the role of substratum stiffness in VSMC podosome formation, VSMCs were first cultured on PDMS­coated cover slips, stimulated by the PKC pathway activator Phorbol 12,13­ Dibutyrate (PDBu), and were proceed to podosome quantification, podosome lifetime measurement, and matrix degradation assay. Results have demonstrated an elevation in podosome formation, accelerated podosome dynamics, and an increase in matrix degradation efficiency. To elucidate the underlying molecular mechanisms, the presence of common actin binding and regulatory proteins at podosomes were first tested by immunostatining. Results have highlighted the clear reciprocal relationship between substratum stiffnesses and the abundance of active non­phosphorylated cofilin at podosomes. Biochemical western blot assay had shown an inverse relationship between substratum stiffnesses and cytosolic cofilin activity. Besides, expression of constitutively active cofilin mutant was sufficient to accelerate podosomal actin dynamics. Altogether these data had demonstrated the novel role of cofilin in regulating podosomal actin dynamics in a stiffness­dependent manner. Additionally, the level of the cofilin suppressor vphospho­LIMK was directly proportional to substratum stiffness, showing the involvement of the LIMK pathway in stiffness­dependent cofilin activity regulation. Lastly, chemical suppression of the cofilin activator Slingshot family of phosphatases have significantly cancelled out the positive effects on podosome formation from pathological pressure and stiffness stimuli, indicating cofilin as the novel and necessary mediator of the synergy between pressure and stiffness signals. To summarize, in the field of cell biology and molecular biology, these data altogether have demonstrated the central role of cofilin as the hub mediating the signals from stiffness­ and pressure­driven signalling cascades to podosomal actin regulation. In pathological aspect, these data have provided new insights into the molecular mechanisms of mechanically induced vascular smooth muscle cell phenotypic switch and podosome formation and have paved a way for further investigations about the possible roles of VSMC podosomes in atherosclerosis progression.