title: The effects of matrix stiffness and RhoA on the phenotypic plasticity of smooth muscle cells in a 3-D biosynthetic hydrogel system.
abstract: Studies using 2-D cultures have shown that the mechanical properties of the extracellular matrix (ECM) influence cell migration, spreading, proliferation, and differentiation; however, cellular mechanosensing in 3-D remains under-explored. To investigate this topic, a unique biomaterial system based on poly(ethylene glycol)-conjugated fibrinogen was adapted to study phenotypic plasticity in smooth muscle cells (SMCs) as a function of ECM mechanics in 3-D. Tuning the compressive modulus between 448 and 5804 Pa modestly regulated SMC cytoskeletal assembly in 3-D, with spread cells in stiff matrices having a slightly higher degree of F-actin bundling after prolonged culture. However, vinculin expression in all 3-D conditions was qualitatively low and was not assembled into the classic focal adhesions typically seen in 2-D cultures. Given the evidence that RhoA-mediated cytoskeletal contractility represents a critical node in mechanosensing, we molecularly upregulated contractility by inducing SMCs to express constitutively active RhoA. In these cells, F-actin bundling and total vinculin expression increased, and focal adhesion-like structures began to emerge, consistent with RhoA's mechanism of action in cells cultured on 2-D substrates. Furthermore, SMC proliferation in 3-D did not depend significantly on matrix stiffness, and was reduced by constitutive activation of RhoA irrespective of ECM mechanical properties. Conversely, the expression of contractile markers globally increased with constitutive RhoA activation and depended on 3-D matrix stiffness only in cells with heightened RhoA activity. Combined, these data suggest that the synergistic effects of ECM mechanics and RhoA activity on SMC phenotype in 3-D are distinct from those in 2-D, and highlight the importance of studying the mechanical role of cell-matrix interactions in tunable 3-D environments.
authors.affiliation: Department of Chemical Engineering and Materials Science, The Henry Samueli School of Engineering, University of California, Irvine, CA 92697-2715, United States.
authors.forename: Shelly R
authors.initials: SR
authors.lastname: Peyton
authors.forename: Peter D
authors.initials: PD
authors.lastname: Kim
authors.forename: Cyrus M
authors.initials: CM
authors.lastname: Ghajar
authors.forename: Dror
authors.initials: D
authors.lastname: Seliktar
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Putnam
mesh_terms: D000199:Actins / Q000378:metabolism; D001011:Aorta / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D005109:Extracellular Matrix / Q000737:chemistry*; D005340:Fibrinogen / Q000737:chemistry / Q000494:pharmacology; D022001:Focal Adhesions / Q000187:drug effects; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D011092:Polyethylene Glycols / Q000737:chemistry / Q000494:pharmacology; D013314:Stress, Mechanical; D013997:Time Factors; D016596:Vinculin / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
chemical_list: D000199:Actins; D003432:Cross-Linking Reagents; D016596:Vinculin; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D011092:Polyethylene Glycols; D005340:Fibrinogen; D020742:rhoA GTP-Binding Protein