Influence of Clinically Relevant Mechanical Forces on Vascular Smooth Muscle Cells Under Chronic High Glucose: An In Vitro Dynamic Disease Model

Abstract

Background: In this study, we subjected vascular smooth muscle cells (VSMC) to acute and chronic high glucose conditions under physiologically relevant levels of cyclic strain and low wall shear forces to compare phenotypic modulation and thus conceptualize a dynamic-disease test model which captures cellular response more accurately in comparison with static cultures.

Methods: P2-P6 rat aortic smooth muscle cells were seeded on type I collagen-coated silicone membranes and subjected to 0-7% cyclic strain at 1 Hz and 0.3 dynes/cm(2) shear stress from flow for 24 hr under acute (25 mM d-glucose, 84 hr) and chronic high glucose conditions (25 mM d-glucose, 3-4 weeks). Samples were analyzed for cell proliferation, percent apoptosis, cellular hypertrophy, and expression levels of smooth muscle contractile state-associated markers with 0.05 level of significance.

Results: Concomitant application of cyclic strain and flow shear resulted in an overall increase in proliferation of VSMCs under both acute and chronic high glucose conditions as compared with normal glucose control (P < 0.0001). Application of both cyclic strain and cyclic strain shear resulted in a significant increase in percent apoptosis with chronic high glucose treatment in comparison with both normal glucose controls (P < 0.0001) and acute high glucose (P < 0.0001). Cellular hypertrophy as estimated by measuring cell area and aspect ratio revealed a significantly altered morphology due to concomitant loading under chronic high glucose conditions with significantly higher cell area (P < 0.0001) and lower aspect ratio (P < 0.0001) indicative of a relatively rounded morphology as compared with normal glucose controls. Western blot analysis demonstrated reduced expression of SM α-actin (P < 0.0001), calponin (P < 0.0001), and SM22α (P = 0.0008) for concomitant loading under chronic high glucose treatment as compared with normal glucose controls.

Conclusions: Concomitant application of cyclic strain and low wall shear stress resulted in greater phenotypic modulation of VSMCs due to chronic high glucose treatment as compared with normal glucose controls, thus implicating cellular-response differences which may impact progression of in-stent restenosis in diabetic patients with poorly controlled hyperglycemia. Similarity of VSMC response from our study to existing preclinical models of diabetes and reports of altered phenotype of VSMCs isolated from diabetic patients substantiate the relevance of our dynamic disease test model.