title: Role of Rho-Associated Coiled-Coil Forming Kinase Isoforms in Regulation of Stiffness-Induced Myofibroblast Differentiation in Lung Fibrosis.
abstract: Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho-associated coiled-coil forming kinase (ROCK) has been shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in a fibrotic state. There are two known ROCK isoforms in humans, ROCK1 and ROCK2, but the specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a gelatin methacryloyl hydrogel-based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix, but not soft matrix, can induce myofibroblast differentiation with high smooth muscle actin isoform (αSMA) expression. Furthermore, our data confirmed that the inhibition of ROCK signaling by a pharmacological inhibitor (i.e., Y27632) attenuates stiffness-induced αSMA expression and fiber assembly in myofibroblasts. To assess the role of ROCK isoforms in this process, we used short interfering RNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix, as opposed to soft matrix, where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fiber assembly. Moreover, complete loss of αSMA fiber assembly was seen only in the absence of both ROCK isoforms, suggesting that both isoforms are implicated in this process. Overall, our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices.
authors.affiliation: 1 Division of Immunology, School of Life Sciences, Faculty of Medicine and Health Sciences, Queen's Medical Centre, and.
authors.forename: Su S
authors.initials: SS
authors.lastname: Htwe
authors.affiliation: 2 Biomaterials Innovation Research Centre, Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, Massachusetts.
authors.forename: Byung H
authors.initials: BH
authors.lastname: Cha
authors.affiliation: 2 Biomaterials Innovation Research Centre, Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, Massachusetts.
authors.forename: Kan
authors.initials: K
authors.lastname: Yue
authors.affiliation: 2 Biomaterials Innovation Research Centre, Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Cambridge, Massachusetts.
authors.forename: Ali
authors.initials: A
authors.lastname: Khademhosseini
authors.affiliation: 3 Division of Respiratory Medicine, School of Medicine, University of Nottingham, Nottingham, United Kingdom; and.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Knox
authors.affiliation: 1 Division of Immunology, School of Life Sciences, Faculty of Medicine and Health Sciences, Queen's Medical Centre, and.
authors.forename: Amir M
authors.identifier: 0000-0003-3160-8759
authors.initials: AM
authors.lastname: Ghaemmaghami
mesh_terms: D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D001696:Biomechanical Phenomena / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D005780:Gelatin / Q000494:pharmacology; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D020100:Hydrogels / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008689:Methacrylates / Q000494:pharmacology; D058628:Myofibroblasts / Q000201:enzymology* / Q000473:pathology*; D058105:Polymerization / Q000187:drug effects; D011658:Pulmonary Fibrosis / Q000201:enzymology* / Q000473:pathology*; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D013314:Stress, Mechanical; D013379:Substrate Specificity / Q000187:drug effects; D054457:Tissue Scaffolds / Q000737:chemistry; D015534:Trans-Activators / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D000577:Amides; D020100:Hydrogels; D007527:Isoenzymes; C431575:MRTFA protein, human; D008689:Methacrylates; D011725:Pyridines; D015534:Trans-Activators; C108830:Y 27632; C008384:methacrylic acid; D005780:Gelatin; C515851:ROCK1 protein, human; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases
keywords: ROCK; extracellular matrix; fibrosis; myofibroblast; stiffness