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.

Keywords: atomic force microscopy; cell adhesion; cell mechanics; cholesterol; cytoskeleton; fluvastatin; vascular smooth muscle cells.

Figure 1.

VSMC cholesterol content and migration on collagen 1or fibronectin coated glass substrates. A, B, E, F. Position plots of VSMC migration on collagen 1 or fibronectin surface. C, D, G, H. Migration distance covered by VSMCs on collagen 1 or fibronectin surface. I, J. Average migration distance versus time for all cells. K. Average migration distance covered by each VSMCs experimental group. L. Displacement of each VSMCs experimental group. M. GC/MS cholesterol quantification. (n= 8 for CTRL, n=9 for Fluvastatin). Displacement is defined as the difference between the starting and ending position of a cell. For each dish, five to ten regions of interest were chosen and imaged with a 10× objective every 10 min for 24 h. For each experiment, 50 cells were tracked. All data were presented as the mean ± SD (n=150 cells from 3 independent experiments).

Figure 2.

Effect of fluvastatin-mediated cholesterol depletion on VSMC biomechanics and adhesion proteins expression. A. Schematic illustration of adhesion force measurement using AFM. ECM-coated stylus AFM probe set to contact cell surface, establish binding with receptors, and retract to dissociate the ligand-receptor binding. B. A representative AFM force curve (approach-blue, retraction-red). Black numbers indicate adhesion force of each rupture; Green numbers indicate adhesion force loading rate of each rupture; Blue numbers indicate adhesion event in this individual sampling cycle. C. Adhesion probability represented by the number of ruptures per curve. D. Average adhesion force obtained by multiplying individual rupture heights by the cantilever spring constant. E. Average adhesion force loading rate, which was defined by the product of retraction speed and slope of the force curve right before the rupture. F, H. Full cell lysate protein expression for integrins α5 and α2. G, I. Average relative protein expression for integrins α5 and α2 to β-actin average of 3 independent experiments. All AFM data are reported as the mean ± SD. (n=100 cells from 7 independent experiments). All protein expression data are presented as the mean ± SD (n=3 from 3 independent experiments conducted in triplicates).

Figure 3.

Real time VSMC stiffness measurement. A. A spherical glass bead (5 μm in diameter) attached to an AFM probe. B. Schematic illustration of overall cell E-modulus (E-map) measurement using spherical AFM probe by scanning over a 40×40 cell surface with approximately 500 pN indentation force. C. 40×40 μm (cyan square) cell surface area was scanned for E-mapping. D. 40×40 μm cell surface area was divided to 6×6 sub-regions. E, F. Representative 6×6 E-map of a control VSMC and fluvastatin-treated VSMC. G. Effect of statin on overall VSMC E-modulus. Fluvastatin-treatment significanly decreased VSMC E-modulus. All data are presented as the mean ± SD (n=58 for control and n=59 for fluvastatin-treated cells from 4 independent experiments).

Figure 4.

AFM image for live VSMC submembranous stress fibers. A, B. Representative three-dimensional stress fibers height topography of a control and fluvastatin-treated VSMCs. Alteration in stress fiber height from low to high is presented as color changes from blue to red. C, D. Representative 40×40 μm flattened AFM height images of control and fluvastatin-treated VSMCs. E, F. Representative submembranous stress fibers surface area fraction images were obtained from their respective AFM height images. The red represents the surface area of stress fiber, while the blue represents the empty background. G. The summarized area fraction of VSMC submembranous actin stress fibers. H. The summarized stress fiber surface roughness average. All data are presented as the mean ± SD (n=20 cells from 3 independent experiments).

Figure 5.

Live VSMCs submembranous stress fiber organization. A, B. Representative AFM deflection images of control and fluvastatin-treated VSMCs submembranous stress fibers. C, D. Circular histograms of stress fiber orientation frequency of control (blue) and fluvastatin (red) treated VSMCs. The histogram data were computed from the representative images in panel A and B, respectively. E, F. Normalized percentage circular histograms along the dominant orientation. G, H. Summarized percentage circular histograms of VSMCs stress fiber orientation for each experimental group. I. Circular variance of stress fiber orientation for each individual VSMC. All data are reported as the mean ± SD (n=20 cells from 3 independent experiments).

Figure 6.

The global effect of fluvastatin on VSMC actin stress fiber architecture. A, C. Representative confocal images of control and fluvastatin-treated VSMCs. Actin was stained in green and the nucleus was in blue. B, D. A pseudo color map of stress fibers orientation computed from the corresponding confocal images with various colors representing different fibers orientation angles. Cells with a clear boundary without overlay with neighboring cells were analyzed for their fiber orientation. E-H. Representative percentage circular histograms of detected stress fiber orientation frequency of 4 control cells. I-L. Representative percentage circular histograms of detected stress fiber orientation frequency of 4 fluvastatin-treated cells.

Figure 7.

Summarized overall VSMCs actin stress fiber orientation. A, B. Summarized percentage circular histograms of control (blue) and fluvastatin-treated (red) VSMCs stress fiber. C. The average normalized stress fiber orientation percentage histogram for all VSMCs, in which the dominant stress fiber orientation angle was set as zero degree for each cell. D. Circular variance of stress fiber orientation for each experimental group of VSMCs. All data for panel D is presented as the mean ± SD (n = 120 cells from 6 independent experiments)

Figure 8.

Fluvastatin-mediated native aorta cholesterol depletion and its effect on the vessel ring contractile activity. A. Cholesterol loading in control and fluvastatin-treated aortic rings (n=11 aortic rings from 4 independent experiments). B. Stiffness of control and fluvastatin-treated rat aortic rings (n= at least 6 aortic rings from 6 independent experiments). C. Average real-time contractile response of aortic rings to phenylephrine (PE) stimulation. D. Summarized aortic ring maximum constriction force (n=8 aortic rings from 8 independent experiments). All data are presented as the mean ± SD.