{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,1]],"date-time":"2026-11-01T04:58:08Z","timestamp":1793509088417,"version":"4.5.0"},"reference-count":49,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,7,15]],"date-time":"2022-07-15T00:00:00Z","timestamp":1657843200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100001818","name":"Research to Prevent Blindness","doi-asserted-by":"publisher","award":["Career Development Award, Unrestricted grant to SUNY Upstate Medical University Department of Ophthalmology and Visual Sciences"],"award-info":[{"award-number":["Career Development Award, Unrestricted grant to SUNY Upstate Medical University Department of Ophthalmology and Visual Sciences"]}],"id":[{"id":"10.13039\/100001818","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007126","name":"Syracuse University","doi-asserted-by":"publisher","award":["BioInspired Pilot Grant"],"award-info":[{"award-number":["BioInspired Pilot Grant"]}],"id":[{"id":"10.13039\/100007126","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Ophthalmol."],"abstract":"<jats:p>\n                    Interactions between trabecular meshwork (TM) cells and their extracellular matrix (ECM) are critical for normal outflow function in the healthy eye. Multifactorial dysregulation of the TM is the principal cause of elevated intraocular pressure that is strongly associated with glaucomatous vision loss. Key characteristics of the diseased TM are pathologic contraction and actin stress fiber assembly, contributing to overall tissue stiffening. Among first-line glaucoma medications, the Rho-associated kinase inhibitor (ROCKi) netarsudil is known to directly target the stiffened TM to improve outflow function\n                    <jats:italic>via<\/jats:italic>\n                    tissue relaxation involving focal adhesion and actin stress fiber disassembly. Yet, no\n                    <jats:italic>in vitro<\/jats:italic>\n                    studies have explored the effect of netarsudil on human TM (HTM) cell contractility and actin remodeling in a 3D ECM environment. Here, we use our bioengineered HTM cell-encapsulated ECM hydrogel to investigate the efficacy of different netarsudil-family ROCKi compounds on reversing pathologic contraction and actin stress fibers. Netarsudil and all related experimental ROCKi compounds exhibited significant ROCK1\/2 inhibitory and focal adhesion disruption activities. Furthermore, all ROCKi compounds displayed potent contraction-reversing effects on HTM hydrogels upon glaucomatous induction in a dose-dependent manner, relatively consistent with their biochemical\/cellular inhibitory activities. At their tailored EC\n                    <jats:sub>50<\/jats:sub>\n                    levels, netarsudil-family ROCKi compounds exhibited distinct effect signatures of reversing pathologic HTM hydrogel contraction and actin stress fibers, independent of the cell strain used. Netarsudil outperformed the experimental ROCKi compounds in support of its clinical status. In contrast, at uniform EC\n                    <jats:sub>50<\/jats:sub>\n                    -levels using netarsudil as reference, all ROCKi compounds performed similarly. Collectively, our data suggest that netarsudil exhibits high potency to rescue HTM cell pathobiology in a tissue-mimetic 3D ECM microenvironment, solidifying the utility of our bioengineered hydrogel model as a viable screening platform to further our understanding of TM pathophysiology in glaucoma.\n                  <\/jats:p>","DOI":"10.3389\/fopht.2022.948397","type":"journal-article","created":{"date-parts":[[2022,7,15]],"date-time":"2022-07-15T04:44:31Z","timestamp":1657860271000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":22,"title":["Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel"],"prefix":"10.3389","volume":"2","author":[{"given":"Tyler","family":"Bagu\u00e9","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayushi","family":"Singh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rajanya","family":"Ghosh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hannah","family":"Yoo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Curtis","family":"Kelly","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mitchell A.","family":"deLong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Casey C.","family":"Kopczynski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samuel","family":"Herberg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2022,7,15]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2008.11.033","article-title":"The Changing Paradigm of Outflow Resistance Generation: Towards Synergistic Models of the JCT and Inner Wall Endothelium","volume":"88","author":"Overby","year":"2009","journal-title":"Exp Eye Res"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1016\/bs.pmbts.2015.06.007","article-title":"Intraocular Pressure and the Mechanisms Involved in Resistance of the Aqueous Humor Flow in the Trabecular Meshwork Outflow Pathways","volume":"134","author":"Tamm","year":"2015","journal-title":"Prog Mol Biol Transl Sci"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2008.10.024","article-title":"Stem Cells in the Trabecular Meshwork: Present and Future Promises","volume":"88","author":"Kelley","year":"2009","journal-title":"Exp Eye Res"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMra0804630","article-title":"Primary Open-Angle Glaucoma","volume":"360","author":"Kwon","year":"2009","journal-title":"N Engl J Med"},{"key":"B5","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1016\/j.exer.2016.07.011","article-title":"Trabecular Meshwork Stiffness in Glaucoma","volume":"158","author":"Wang","year":"2017","journal-title":"Exp Eye Res"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1097\/ICU.0b013e32834ff23e","article-title":"Current Understanding of Conventional Outflow Dysfunction in Glaucoma","volume":"23","author":"Stamer","year":"2012","journal-title":"Curr Opin Ophthalmol"},{"key":"B7","doi-asserted-by":"publisher","first-page":"100897","DOI":"10.1016\/j.preteyeres.2020.100897","article-title":"Normal and Glaucomatous Outflow Regulation","volume":"82","author":"Acott","year":"2021","journal-title":"Prog Retin Eye Res"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1056\/NEJM199304153281507","article-title":"Open-Angle Glaucoma","volume":"328","author":"Quigley","year":"1993","journal-title":"N Engl J Med"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1136\/bjo.2005.081224","article-title":"The Number of People With Glaucoma Worldwide in 2010 and 2020","volume":"90","author":"Quigley","year":"2006","journal-title":"Br J Ophthalmol"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1001\/jama.2014.3192","article-title":"The Pathophysiology and Treatment of Glaucoma: A Review","volume":"311","author":"Weinreb","year":"2014","journal-title":"JAMA"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1016\/j.ophtha.2014.05.013","article-title":"Global Prevalence of Glaucoma and Projections of Glaucoma Burden Through 2040: A Systematic Review and Meta-Analysis","volume":"121","author":"Tham","year":"2014","journal-title":"Ophthalmology"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.2147\/OPTH.S32933","article-title":"Current Primary Open-Angle Glaucoma Treatments and Future Directions","volume":"6","author":"Beidoe","year":"2012","journal-title":"Clin Ophthalmol"},{"key":"B13","doi-asserted-by":"publisher","first-page":"40","DOI":"10.1089\/jop.2017.0023","article-title":"Discovery and Preclinical Development of Netarsudil, a Novel Ocular Hypotensive Agent for the Treatment of Glaucoma","volume":"34","author":"Lin","year":"2018","journal-title":"J Ocul Pharmacol Ther"},{"key":"B14","article-title":"Modulation of Aqueous Humor Outflow Facility by the Rho Kinase-Specific Inhibitor Y-27632","volume":"42","author":"Rao","year":"2001","journal-title":"Invest Ophthalmol Vis Sci"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1038\/nrd3745","article-title":"Ophthalmic Drug Discovery: Novel Targets and Mechanisms for Retinal Diseases and Glaucoma","volume":"11","author":"Zhang","year":"2012","journal-title":"Nat Rev Drug Discovery"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.2147\/OPTH.S41000","article-title":"An Emerging Treatment Option for Glaucoma: Rho Kinase Inhibitors","volume":"8","author":"Wang","year":"2014","journal-title":"Clin Ophthalmol"},{"key":"B17","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1016\/j.exer.2016.08.023","article-title":"Role of the Rho GTPase\/Rho Kinase Signaling Pathway in Pathogenesis and Treatment of Glaucoma: Bench to Bedside Research","volume":"158","author":"Rao","year":"2017","journal-title":"Exp Eye Res"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1016\/j.ophtha.2018.04.040","article-title":"Rho Kinase Inhibitors as a Novel Treatment for Glaucoma and Ocular Hypertension","volume":"125","author":"Tanna","year":"2018","journal-title":"Ophthalmology"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1097\/IJG.0b013e3182952213","article-title":"Effect of 0.04% AR-13324, a ROCK, and Norepinephrine Transporter Inhibitor, on Aqueous Humor Dynamics in Normotensive Monkey Eyes","volume":"24","author":"Wang","year":"2015","journal-title":"J Glaucoma"},{"key":"B20","doi-asserted-by":"publisher","first-page":"20","DOI":"10.1016\/j.ejphar.2016.04.002","article-title":"Visualization of Conventional Outflow Tissue Responses to Netarsudil in Living Mouse Eyes","volume":"787","author":"Li","year":"2016","journal-title":"Eur J Pharmacol"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.16-20189","article-title":"Netarsudil Increases Outflow Facility in Human Eyes Through Multiple Mechanisms","volume":"57","author":"Ren","year":"2016","journal-title":"Invest Ophthalmol Vis Sci"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1089\/jop.2017.0138","article-title":"The Effects of Netarsudil Ophthalmic Solution on Aqueous Humor Dynamics in a Randomized Study in Humans","volume":"34","author":"Kazemi","year":"2018","journal-title":"J Ocul Pharmacol Ther"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1016\/j.ajo.2021.01.019","article-title":"Netarsudil Improves Trabecular Outflow Facility in Patients With Primary Open Angle Glaucoma or Ocular Hypertension: A Phase 2 Study","volume":"226","author":"Sit","year":"2021","journal-title":"Am J Ophthalmol"},{"key":"B24","doi-asserted-by":"publisher","first-page":"3524","DOI":"10.3390\/jcm9113524","article-title":"Effects of Netarsudil on Actin-Driven Cellular Functions in Normal and Glaucomatous Trabecular Meshwork Cells: A Live Imaging Study","volume":"9","author":"Keller","year":"2020","journal-title":"J. Clin. Med"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2008.01.013","article-title":"Extracellular Matrix in the Trabecular Meshwork","volume":"86","author":"Acott","year":"2008","journal-title":"Exp Eye Res"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2009.02.007","article-title":"The Trabecular Meshwork Outflow Pathways: Structural and Functional Aspects","volume":"88","author":"Tamm","year":"2009","journal-title":"Exp Eye Res"},{"key":"B27","doi-asserted-by":"publisher","first-page":"45","DOI":"10.1167\/iovs.10-5620","article-title":"The Elastin Fiber System Between and Adjacent to Collector Channels in the Human Juxtacanalicular Tissue","volume":"52","author":"Hann","year":"2011","journal-title":"Invest Ophthalmol Vis Sci"},{"key":"B28","doi-asserted-by":"publisher","first-page":"3","DOI":"10.13188\/2334-2838.1000003","article-title":"The Juxtacanalicular Region of Ocular Trabecular Meshwork: A Tissue With a Unique Extracellular Matrix and Specialized Function","volume":"1","author":"Keller","year":"2013","journal-title":"J Ocul Biol"},{"key":"B29","doi-asserted-by":"publisher","first-page":"9","DOI":"10.13188\/2334-2838.1000017","article-title":"The Trabecular Meshwork: A Basic Review of Form and Function","volume":"2","author":"Abu-Hassan","year":"2014","journal-title":"J Ocul Biol"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3839","article-title":"A Practical Guide to Hydrogels for Cell Culture","volume":"13","author":"Caliari","year":"2016","journal-title":"Nat Methods"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2020.00033","article-title":"Is It Time to Start Transitioning From 2D to 3D Cell Culture","volume":"7","author":"Jensen","year":"2020","journal-title":"Front Mol Biosci"},{"key":"B32","doi-asserted-by":"publisher","first-page":"108472","DOI":"10.1016\/j.exer.2021.108472","article-title":"A Tissue-Engineered Human Trabecular Meshwork Hydrogel for Advanced Glaucoma Disease Modeling","volume":"205","author":"Li","year":"2021","journal-title":"Exp Eye Res"},{"key":"B33","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1080\/02713683.2022.2071943","article-title":"TGFbeta2 Regulates Human Trabecular Meshwork Cell Contractility via ERK and ROCK Pathways With Distinct Signaling Crosstalk Dependent on the Culture Substrate","author":"Li","year":"2022","journal-title":"Curr Eye Res"},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.3389\/fcell.2022.844342","article-title":"Extracellular Matrix Stiffness and TGFbeta2 Regulate YAP\/TAZ Activity in Human Trabecular Meshwork Cells","volume":"10","author":"Li","year":"2022","journal-title":"Front Cell Dev Biol"},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1007\/s004170000241","article-title":"Transforming Growth Factor-Beta 2 Levels in Aqueous Humor of Glaucomatous Eyes","volume":"239","author":"Inatani","year":"2001","journal-title":"Graefes Arch Clin Exp Ophthalmol"},{"key":"B36","article-title":"Aqueous Humor TGF-\u03b22 Levels in Patients With Open-Angle Glaucoma: A Meta-Analysis","volume":"21","author":"Agarwal","year":"2015","journal-title":"Mol Vision"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.3109\/02713689508998409","article-title":"Isolation and Culture of Human Trabecular Meshwork Cells by Extracellular Matrix Digestion","volume":"14","author":"Stamer","year":"1995","journal-title":"Curr Eye Res"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2018.03.001","article-title":"Consensus Recommendations for Trabecular Meshwork Cell Isolation, Characterization and Culture","volume":"171","author":"Keller","year":"2018","journal-title":"Exp Eye Res"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1016\/j.bmcl.2016.03.104","article-title":"Discovery of the ROCK Inhibitor Netarsudil for the Treatment of Open-Angle Glaucoma","volume":"26","author":"Sturdivant","year":"2016","journal-title":"Bioorg Med Chem Lett"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.1002\/adfm.201501489","article-title":"A Highly Elastic and Rapidly Crosslinkable Elastin-Like Polypeptide-Based Hydrogel for Biomedical Applications","volume":"25","author":"Zhang","year":"2015","journal-title":"Adv Funct Mater"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1016\/S1350-9462(99)00015-4","article-title":"The Regulation of Trabecular Meshwork and Ciliary Muscle Contractility","volume":"19","author":"Wiederholt","year":"2000","journal-title":"Prog Retin Eye Res"},{"key":"B42","doi-asserted-by":"publisher","first-page":"a018267","DOI":"10.1101\/cshperspect.a018267","article-title":"The Actin Cytoskeleton and Actin-Based Motility","volume":"10","author":"Svitkina","year":"2018","journal-title":"Cold Spring Harbor Perspect Biol"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.079509","article-title":"Deconstructing the Third Dimension: How 3D Culture Microenvironments Alter Cellular Cues","volume":"125","author":"Baker","year":"2012","journal-title":"J Cell Sci"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1016\/j.biomaterials.2016.06.061","article-title":"Dimensionality and Spreading Influence MSC YAP\/TAZ Signaling in Hydrogel Environments","volume":"103","author":"Caliari","year":"2016","journal-title":"Biomaterials"},{"key":"B45","doi-asserted-by":"publisher","DOI":"10.1016\/S1359-6446(02)02273-0","article-title":"Engineering Cellular Microenvironments to Improve Cell-Based Drug Testing","volume":"7","author":"Bhadriraju","year":"2002","journal-title":"Drug Discovery Today"},{"key":"B46","doi-asserted-by":"publisher","DOI":"10.1089\/adt.2014.573","article-title":"Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors","volume":"12","author":"Edmondson","year":"2014","journal-title":"Assay Drug Dev Technol"},{"key":"B47","doi-asserted-by":"publisher","first-page":"147","DOI":"10.3390\/bioengineering8110147","article-title":"Fundamental Biomaterial Considerations in the Development of a 3D Model Representative of Primary Open Angle Glaucoma","volume":"8","author":"Lamont","year":"2021","journal-title":"Bioengineering"},{"key":"B48","article-title":"In Vitro Localization of TIGR\/MYOC in Trabecular Meshwork Extracellular Matrix and Binding to Fibronectin","volume":"43","author":"Filla","year":"2002","journal-title":"Invest Ophthalmol Vis Sci"},{"key":"B49","doi-asserted-by":"publisher","DOI":"10.1076\/ceyr.25.6.347.14226","article-title":"Transformation of Human Trabecular Meshwork Cells With SV40 TAg Alters Promoter Utilization","volume":"25","author":"Liu","year":"2002","journal-title":"Curr Eye Res"}],"container-title":["Frontiers in Ophthalmology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fopht.2022.948397\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,7,15]],"date-time":"2022-07-15T04:44:37Z","timestamp":1657860277000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fopht.2022.948397\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,7,15]]},"references-count":49,"alternative-id":["10.3389\/fopht.2022.948397"],"URL":"https:\/\/doi.org\/10.3389\/fopht.2022.948397","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2022.05.19.492652","asserted-by":"object"}]},"ISSN":["2674-0826"],"issn-type":[{"value":"2674-0826","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,7,15]]},"article-number":"948397"}}