Survivin as a mediator of stiffness-induced cell cycle progression and proliferation of vascular smooth muscle cells

Abstract

Stiffened arteries are a pathology of atherosclerosis, hypertension, and coronary artery disease and a key risk factor for cardiovascular disease events. The increased stiffness of arteries triggers a phenotypic switch, hypermigration, and hyperproliferation of vascular smooth muscle cells (VSMCs), leading to neointimal hyperplasia and accelerated neointima formation. However, the mechanism underlying this trigger remains unknown. Our analyses of whole-transcriptome microarray data from mouse VSMCs cultured on stiff hydrogels simulating arterial pathology identified 623 genes that were significantly and differentially expressed (360 upregulated and 263 downregulated) relative to expression in VSMCs cultured on soft hydrogels. Functional enrichment and gene network analyses revealed that these stiffness-sensitive genes are linked to cell cycle progression and proliferation. Importantly, we found that survivin, an inhibitor of apoptosis protein, mediates stiffness-dependent cell cycle progression and proliferation as determined by gene network and pathway analyses, RT-qPCR, immunoblotting, and cell proliferation assays. Furthermore, we found that inhibition of cell cycle progression did not reduce survivin expression, suggesting that survivin functions as an upstream regulator of cell cycle progression and proliferation in response to ECM stiffness. Mechanistically, we found that the stiffness signal is mechanotransduced via the FAK-E2F1 signaling axis to regulate survivin expression, establishing a regulatory pathway for how the stiffness of the cellular microenvironment affects VSMC behaviors. Overall, our findings indicate that survivin is necessary for VSMC cycling and proliferation and plays a role in regulating stiffness-responsive phenotypes.

FAK and p130Cas modulate stiffness-mediated early transcription and cellular metabolism

“…To investigate the roles of substrate stiffness and FAK/p130Cas in the early transcriptional response, a whole-transcriptome analysis was performed using mRNA samples from MEFs transfected with FAK siRNA, p130Cas siRNA, or non-targeting control siRNA and cultured for 1 h on soft (3–8 kPa) or stiff (18–24 kPa) [4] polyacrylamide-based hydrogels. Immunoblotting analysis of total lysates confirmed that the siRNAs successfully downregulated their targets ( Fig.…”

“…no. 31985070, Gibco) according to previously described methods [4, 14, 16]. MEFs were transfected with siRNA for 5 h followed by immediate serum starvation in DMEM with 1 mg/ml BSA for 24 h. Cells were then trypsinized and seeded on fibronectin-coated hydrogels with DMEM containing 10% FBS.…”

“…As previously described [4, 14, 16], total cell lysates were collected from MEFs cultured on polyacrylamide hydrogels by incubating the hydrogels face down for 1 to 2 min at room temperature on 5× SDS sample buffer containing β-mercaptoethanol. Equal amounts of extracted protein were fractionated on reducing 10% SDS-polyacrylamide gels, and the fractioned proteins were subsequently transferred electrophoretically onto nitrocellulose blotting membranes.…”

“…As previously described [4,14,16], total cell lysates were collected from MEFs cultured on polyacrylamide hydrogels by incubating the hydrogels face down for 1 to 2 min at room temperature on 5× SDS sample buffer containing β-mercaptoethanol.…”

“…Extracellular matrix (ECM) is part of a dynamic microenvironment that modulates various energy-demanding cellular processes, including cell adhesion, spreading, differentiation, migration, and proliferation [1, 2, 3, 4, 5]. There is evidence that the stiffness of the ECM serves as a biomechanical signal that influences mitochondrial network structure and function [6, 7, 8].…”