Statin‐mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration

Abstract

Key points This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin‐mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin‐mediated cholesterol depletion. Proof is provided that statin‐mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin‐mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. Abstract Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG‐CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin‐mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin‐mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real‐time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time‐lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin‐mediated cholesterol depletion (‐27.8%) lowered VSMC migration distance on a fibronectin (FN)‐coated surface (‐14.8%) but not on a type 1 collagen (COL1)‐coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (‐46.6%) and ex vivo aortic ring contraction force (‐40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (‐24.5%) following statin‐mediated cholesterol depletion. Altogether, it is concluded that statin‐mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function.

Impact of statins on vascular smooth muscle cells and relevance to atherosclerosis

“…Reduced arterial stiffness is considered to be beneficial for cardiovascular health; however, the role of VSMC stiffness in advanced atherosclerotic plaque requires further elucidation. Although not in an atherogenic model, Sanyour et al, (2020) demonstrated that Fluvastatin reduced VSMC stiffness in vitro in association with other pro-atherogenic phenotypic shifts. This finding creates an open question as to how Fluvastatin modulates VSMC stiffness in a setting of advanced atherosclerotic plaque and what role it plays on plaque stability.…”

“…Although a variety of stimuli are known to modulate VSMC migration in vitro; technical limitations have left us with a lack of understanding on how these modulations are related to in vivo conditions of atherosclerotic plaque progression. Using a gas chromatography/mass spectrometry technique, the investigators found that the Fluvastatin treatment led to an expected reduction in cellular cholesterol in VSMCs resulting in a reduction in VSMC migration on a fibronectin (FN)-coated surface (Sanyour et al 2020). These findings provide evidence that statin treatment may influence the morphology of the atherosclerotic cap, and in this case, in a deleterious manner.…”

“…Numerous pleiotropic effects of statins on atherosclerotic plaques have been described in ECs and immune cells; however, little consensus has been reached on the pleiotropic effects of statins on VSMCs (Oesterle et al 2017). A recent study published in the Journal of Physiology by Sanyour et al (2020) described the effect of statins on rat aortic primary VSMC using a state-of-the-art atomic force microscopy technique. In this study, rat aortic primary VSMCs were isolated, cultured and treated with 1 µM Fluvastatin, one of the statins.…”

Atomic force microscopy reveals the role of vascular smooth muscle cell elasticity in hypertension

“…All animal procedures were done under the Guide for the Care and Use of Laboratory Animals (NIH 85-23, revised 2011). Primary VSMCs from thoracic aorta of three experimental WKY and SHR rats were enzymatically isolated and cultured in Dulbecco's Modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 10 mmol/L HEPES, 2 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B and used at passages 2 to 4 (Sanyour H.J., et al, 2020).…”

“…A number of recent studies have demonstrated that changes in a cell's elastic characteristics can affect its response to the external mechanical force (Hill M.A., et al, 2016;Dhar S., et al, 2017). The single-cell mechanical property and behavior of VSMC is chiefly considered to play a crucial role in the development of vascular diseases, and atomic force microscopy (AFM) is currently the most wonderful tools for determining this interaction (Zhu W., et al, 2018;Leloup A.J.A., et al, 2019;Sanyour H.J., et al, 2020).…”