Abstract 11468: Myo-Fibroblast Commitment of Human Aortic Valve Interstitial Cells is Induced by Substrate Stiffness and is Associated With Discrete Cellular Rigidity
Background: Aortic valve calcification (AoV) is a widely diffuse pathology caused by aortic valve interstitial cells (aVICs) conversion into myofibroblasts and bone-depositing cells. In the present study, we investigated the role of substrate elasticity in human aVICs differentiation into pathologic phenotypes using controlled stiffness substrates and force measurements by atomic force microscopy (AFM).
Methods and Results: To culture aVICs onto substrates with different elasticity, Polyacrylamide gels (PAAg) were produced by adjusting acrylamide concentration and Acryl/Bis-Acrylamide ratio, and thereafter functionalized with Collagen I. The PAAg elastic module (0.8±0.08; 9.4±3.6; 20.5±1.64; 58.9±10.1 KPa, mean±SD) was validated by AFM. Human aVICs were derived from patients undergoing aortic valve substitution. After expansion and characterization, they were seeded onto PAAg at a 2000 cells/cm2 density, and cultured for 48 hrs. Cells were then fixed and either stained for immunofluorescence or mechanically assessed by direct measurement of cell rigidity by AFM. The cytoskeleton organization showed an increase in polymerized αSMA onto stiffer gels, suggesting conversion of these cells into myofibroblasts, cells typically participating in AoV stenosis. Computer-assisted cell morphometry showed that cells had an increasingly spread shape on substrates with mid-high/high vs. low/mid-low elasticity (cell perimeter, μm: 566±64.8 [20.5KPa] and 692.3±83.3 [58.9KPa] vs. 293.8±42.2 [0.8KPa] and 320.1±38.8 [9.4KPa]; mean±SE; P<0.01, 1-way ANOVA; n>6). Assessment of cellular rigidity (evaluated via punctual AFM nano-indentation measurements using cantilevers endowed with a terminal microsphere) showed, finally a striking difference between rigidity of cells plated onto stiffer vs. more compliant matrices (3.93 folds increase on [58.9KPa] vs. [0.8KPa] gels; n>100; P<0.0001 unpaired t-test).
Conclusions: These results suggest that elevated substrate stiffness is sufficient to convert human aVICs into pro-inflammatory cells. This establishes, for the first time, a direct relationship between mechanical compliance of the substrate, biophysical characteristics and pro-pathologic evolution of human AoV-resident cells.
- © 2013 by American Heart Association, Inc.