Redirecting valvular myofibroblasts into dormant fibroblasts through light-mediated reduction in substrate modulus.
Fibroblasts residing in connective tissues throughout the body are responsible for extracellular matrix (ECM) homeostasis and repair. In response to tissue damage, they activate to become myofibroblasts, which have organized contractile cytoskeletons and produce a myriad of proteins for ECM remodeling. However, persistence of myofibroblasts can lead to fibrosis with excessive collagen deposition and tissue stiffening. Thus, understanding which signals regulate de-activation of myofibroblasts during normal tissue repair is critical. Substrate modulus has recently been shown to regulate fibrogenic properties, proliferation and apoptosis of fibroblasts isolated from different organs. However, few studies track the cellular responses of fibroblasts to dynamic changes in the microenvironmental modulus. Here, we utilized a light-responsive hydrogel system to probe the fate of valvular myofibroblasts when the Young's modulus of the substrate was reduced from ~32 kPa, mimicking pre-calcified diseased tissue, to ~7 kPa, mimicking healthy cardiac valve fibrosa. After softening the substrata, valvular myofibroblasts de-activated with decreases in α-smooth muscle actin (α-SMA) stress fibers and proliferation, indicating a dormant fibroblast state. Gene signatures of myofibroblasts (including α-SMA and connective tissue growth factor (CTGF)) were significantly down-regulated to fibroblast levels within 6 hours of in situ substrate elasticity reduction while a general fibroblast gene vimentin was not changed. Additionally, the de-activated fibroblasts were in a reversible state and could be re-activated to enter cell cycle by growth stimulation and to express fibrogenic genes, such as CTGF, collagen 1A1 and fibronectin 1, in response to TGF-β1. Our data suggest that lowering substrate modulus can serve as a cue to down-regulate the valvular myofibroblast phenotype resulting in a predominantly quiescent fibroblast population. These results provide insight in designing hydrogel substrates with physiologically relevant stiffness to dynamically redirect cell fate in vitro.
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado, United States of America.
Huan
H
Wang
Sarah M
SM
Haeger
April M
AM
Kloxin
Leslie A
LA
Leinwand
Kristi S
KS
Anseth
D000179:Acrylates / Q000737:chemistry; D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001021:Aortic Valve / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D015415:Biomarkers / Q000378:metabolism; D040761:Biomimetic Materials / Q000737:chemistry*; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D002454:Cell Differentiation / Q000187:drug effects / Q000528:radiation effects; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism; D055119:Elastic Modulus / Q000528:radiation effects*; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000528:radiation effects; D020100:Hydrogels; D008027:Light; D058628:Myofibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D009842:Oligopeptides / Q000138:chemical synthesis; D011092:Polyethylene Glycols / Q000737:chemistry; D061251:Primary Cell Culture; D013552:Swine; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
D000179:Acrylates; D000199:Actins; D015415:Biomarkers; D024042:Collagen Type I; D005353:Fibronectins; D020100:Hydrogels; D009842:Oligopeptides; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D011092:Polyethylene Glycols