The non-selective Rho-kinase inhibitors Y-27632 and Y-33075 decrease contraction but increase migration in murine and human hepatic stellate cells

PLOS ONE RESEARCH ARTICLE The non-selective Rho-kinase inhibitors Y-27632 and Y-33075 decrease contraction but increase migration in murine and human hepatic stellate cells Nadine Bachtler1, Sandra Torres ID1, Cristina Ortiz1, Robert Schierwagen1, Olaf Tyc ID1, Christoph Hieber1, Marie-Luise Berres2, Caroline Meier1, Nico Kraus ID1, Stefan Zeuzem1, Bart Nijmeijer3, Sebas Pronk ID3, Jonel Trebicka ID1,4*, Sabine Klein1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Internal Medicine I, Goethe University Frankfurt, Frankfurt, Germany, 2 Department of Medicine III, University Hospital RWTH Aachen, Aachen, Germany, 3 LinXis BV, Amsterdam, The Netherlands, 4 European Foundation for the Study of Chronic Liver Failure, Barcelona, Spain * Abstract OPEN ACCESS Citation: Bachtler N, Torres S, Ortiz C, Schierwagen R, Tyc O, Hieber C, et al. (2023) The non-selective Rho-kinase inhibitors Y-27632 and Y-33075 decrease contraction but increase migration in murine and human hepatic stellate cells. PLoS ONE 18(1): e0270288. https://doi.org/10.1371/journal. pone.0270288 Editor: Michael F. Olson, Ryerson University, CANADA Background The Rho-kinase ROCK II plays a major role in the activation of hepatic stellate cells (HSC), which are the key profibrotic and contractile cells contributing to the development of chronic liver disease. Inhibition of ROCK II ultimately blocks the phosphorylation of the myosin light chain (MLC) and thus inhibits stress fibre assembly and cell contraction. We investigated the effects of the ROCK inhibitors Y-33075 as well as Y-27632 in murine and human hepatic stellate cells. Received: January 22, 2022 Accepted: June 7, 2022 Methods Published: January 31, 2023 Primary isolated HSC from FVB/NJ mice and the immortalized human HSC line TWNT-4 were culture-activated and incubated with Y-27632 and Y-33075 (10nM to 10μM) for 24h. Protein expression levels were analyzed by Western Blots and transcriptional levels of profibrotic markers and proliferative markers were evaluated using real-time qPCR. Migration was investigated by wound-healing assay. Proliferation was assessed by BrdU assay. Contraction of HSC was measured using 3D collagen matrices after incubation with Y-27632 or Y-33075 in different doses. Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0270288 Copyright: © 2023 Bachtler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Results Both Rho-kinase inhibitors, Y-27632 and Y-33075, reduced contraction, fibrogenesis and proliferation in activated primary mouse HSC (FVB/NJ) and human HSC line (TWNT-4) significantly. Y-33075 demonstrated a 10-times increased potency compared to Y-27632. Surprisingly, both inhibitors mediated a substantial and unexpected increase in migration of HSC in FVB/NJ. PLOS ONE | https://doi.org/10.1371/journal.pone.0270288 January 31, 2023 1 / 15 PLOS ONE Funding: This study was supported by the German Research Foundation (DFG) project ID 403224013 – SFB 1382 (A09), by the German Federal Ministry of Education and Research (BMBF) for the DEEPHCC project and by the Hessian Ministry of Higher Education, Research and the Arts (HMWK) for the ENABLE cluster project and by Eurostars (Grant ID 12350). The MICROB-PREDICT (project ID 825694), DECISION (project ID 847949), GALAXY (project ID 668031), LIVERHOPE (project ID 731875) and IHMCSA (project ID 964590) projects have received funding from the European Union’s Horizon 2020 research and innovation program. The manuscript reflects only the authors’ views, and the European Commission is not responsible for any use that may be made of the information it contains. The funders had no influence on study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: αSMA, α-smooth muscle actin; BDL, bile duct ligation; BrdU, Bromodesoxyuridine; Col1a1, collagen 1a1; ECL, enhanced chemiluminescence; ET-1, Endothelin-1; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GEF, guanine exchange factor; GPCR, G-protein coupled receptor; HSC, hepatic stellate cel; KD, knock down; MLC, myosin light chain; MLCP, myosin light chain phosphatase; PCNA, proliferating cell nuclear antigen; PDGF, platelet-derived growth factor; PHT, portal hypertension; qPCR, quantitative polymerase chain reaction; ROCK, Rho-kinase; RT-qPCR, real-time quantitative polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEM, standard error of the mean; TBST, Trisbuffered saline with Tween20; TGFβ, transforming growth factor β. Effect of Rho-kinase inhibitors in hepatic stellate cells Conclusion ROCK inhibition by the tested compounds decreased contraction but increased migration. Y-33075 proved more potent than Y27632 in the inhibition of contraction of HSCs and should be further evaluated in chronic liver disease. Introduction Chronic liver disease is a major health problem affecting approximately 1.5 billion people worldwide and leading to 2 million deaths a year [1, 2]. Liver fibrosis is becoming more prevalent. It has various etiologies but invariably progresses towards a uniform end stage for which no treatment options exist [1, 3]. HSCs play a crucial role in the development of liver fibrosis. Due to (chronic) liver injury HSCs are activated and consequently transdifferentiate into a myofibroblast-like phenotype [4, 5]. In contrast to quiescent HSCs, these activated cells exhibit an increase in migration as well as proliferation, and produce large amounts of extracellular matrix proteins (collagen) and pro-fibrotic cytokines (e.g. transforming growth factor β, TGFβ; platelet-derived growth factor, PDGF) thus driving fibrosis [6, 7]. The accumulation of collagen in the liver, as well as the exaggerated contractile response of these myofibroblasts to vasoconstrictors cause an increased intrahepatic vascular resistance which results in the development of portal hypertension (PHT) in liver fibrosis [8]. The Rho-kinase is crucial in regulating cytoskeletal proteins and thus has a decisive effect on activation, contraction and fibrogenesis in HSCs [9, 10]. The Rho family of small G proteins comprises 20 members. RhoA, which is one of the small G proteins, is responsible for regulating the actin cytoskeleton and for mechanical forces [11]. Activated by guanine (...truncated)