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

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.

Membrane cholesterol and substrate stiffness co-ordinate to induce the remodelling of the cytoskeleton and the alteration in the biomechanics of vascular smooth muscle cells.

AIMS: Cholesterol not only deposits in foam cells at the atherosclerotic plaque, but also plays an important role as a regulator of cell migration in atherogenesis. In addition, the progression of atherosclerosis leads to arterial wall stiffening, and thus altering the micromechanical environment of vascular smooth muscle cells (VSMCs) in vivo. Our studies aim to test the hypothesis that membrane cholesterol and substrate stiffness co-ordinate to regulate VSMCs biomechanics, and thus potentially regulate VSMCs migration and atherosclerotic plaque formation. METHODS AND RESULTS: Methyl-ß-cyclodextrin was used to manipulate membrane cholesterol content in VSMCs isolated from the descending thoracic aorta of male Sprague-Dawley rats and cultured on Type I collagen-coated polyacrylamide gel substrates with varying stiffness. Atomic force microscopy (AFM) was used to determine VSMCs stiffness and integrin-fibronectin (FN) adhesion. The alignment of submembranous actin filaments was visualized with AFM and confocal microscopy. The constriction force of rat aorta was measured ex vivo using a multi-wire myograph system. Our results demonstrated that cholesterol-depletion and substrate-softening induced a significant decrease in VSMCs stiffness and adhesion to FN, as well as cytoskeletal disorganization. In addition, the contractile force of rat aorta was reduced upon cholesterol-depletion. Cholesterol-enrichment resulted in an increase in stiffness, adhesion to FN, cytoskeletal organization of VSMCs compared with the cholesterol-depleted cells, and enhanced contractile force of rat aortas compared with the cholesterol-depleted vessel rings. CONCLUSION: Cell membrane cholesterol and substrate stiffness synergistically affect VSMCs elastic modulus (E-modulus) by regulating the organization of the actin cytoskeleton. Except for the 3.5 kPa gel substrate, cholesterol-depletion decreased VSMCs-FN adhesion force, adhesion loading rate, cytoskeletal orientation, and E-modulus compared with the control VSMCs. Conversely, cholesterol-enrichment significantly increased cytoskeleton orientation, stiffness, and VSMCs-FN cell adhesion force compared with both control and cholesterol-depleted VSMCs on a soft substrate.

Functionalization of PCL-3D Electrospun Nanofibrous Scaffolds for Improved BMP2-Induced Bone Formation.

Bone morphogenic protein 2 (BMP2) is a key growth factor for bone regeneration, possessing FDA approval for orthopedic applications. BMP2 is often required in supratherapeutic doses clinically, yielding adverse side effects and substantial treatment costs. Considering the crucial role of materials for BMPs delivery and cell osteogenic differentiation, we devote to engineering an innovative bone-matrix mimicking niche to improve low dose of BMP2-induced bone formation. Our previous work describes a novel technique, named thermally induced nanofiber self-agglomeration (TISA), for generating 3D electrospun nanofibrous (NF) polycaprolactone (PCL) scaffolds. TISA process could readily blend PCL with PLA, leading to increased osteogenic capabilities in vitro, however, these bio-inert synthetic polymers produced limited BMP2-induced bone formation in vivo. We therefore hypothesize that functionalization of NF 3D PCL scaffolds with bone-like hydroxyapatite (HA) and BMP2 signaling activator phenamil will provide a favorable osteogenic niche for bone formation at low doses of BMP2. Compared to PCL-3D scaffolds, PCL/HA-3D scaffolds demonstrated synergistically enhanced osteogenic differentiation capabilities of C2C12 cells with phenamil. Importantly, in vivo studies showed this synergism was able to generate significantly increased new bone in an ectopic mouse model, suggesting PCL/HA-3D scaffolds act as a favorable synthetic extracellular matrix for bone regeneration.