{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:18:47Z","timestamp":1785424727157,"version":"3.56.0"},"posted":{"date-parts":[[2025,11,8]]},"group-title":"Molecular Biology","reference-count":48,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,11,8]],"date-time":"2025-11-08T00:00:00Z","timestamp":1762560000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nd\/4.0\/"}],"funder":[{"award":["EY032974"],"award-info":[{"award-number":["EY032974"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"award":["EY034096"],"award-info":[{"award-number":["EY034096"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"award":["EY036189"],"award-info":[{"award-number":["EY036189"]}],"id":[{"id":"https:\/\/ror.org\/001d55x84","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/00pvmkr98","id-type":"ROR","asserted-by":"publisher"}]},{"award":["G2024006S"],"award-info":[{"award-number":["G2024006S"]}],"id":[{"id":"https:\/\/ror.org\/03cvfxv40","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/04drjs621","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,12,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Purpose<\/jats:title>\n                  <jats:p>\n                    Fuchs\u2019 Endothelial Corneal Dystrophy (FECD), a degenerative corneal disorder, is marked by the thickening of Descemet\u2019s membrane and a progressive loss of corneal endothelial cells, ultimately leading to vision loss. A feature associated with the disease is the reduced stiffness of Descemet\u2019s membrane. However, the effects of this change in Descemet\u2019s membrane, on corneal endothelial cell health are not well understood. To explore this, we used\n                    <jats:italic>in vitro, in vivo,<\/jats:italic>\n                    and\n                    <jats:italic>ex vivo<\/jats:italic>\n                    studies to investigate how changes in substrate stiffness and the signaling pathways associated with these changes influence corneal endothelial functions.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>\n                    For in-vitro studies, we cultured bovine corneal endothelial cells for 96 hours on stiff (32 kPa) and soft (8 kPa) substrate CytoSoft plates. By using Jess immunoassay and traditional western blotting, we evaluated changes in integrin signaling components, endothelial-to-mesenchymal transition, apoptosis, autophagy, and ubiquitin-proteasome pathway markers. Mitochondrial health and mitochondrial superoxide levels were assessed using commercial kits. We assessed the protein levels of the above-mentioned markers in the\n                    <jats:italic>\n                      Col8a2\n                      <jats:sup>Q455K\/Q455K<\/jats:sup>\n                    <\/jats:italic>\n                    FECD mouse model. To evaluate whether FAK signaling contributes to the FECD pathogenesis, we injected the\n                    <jats:italic>\n                      Col8a2\n                      <jats:sup>Q455K\/Q455K<\/jats:sup>\n                    <\/jats:italic>\n                    mice with an FAK inhibitor and assessed the corneal phenotypes.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                    We observed increased levels of phosphorylated FAK, integrins \u03b14 and \u03b15 in bovine corneal endothelial cells cultured on soft substrate. We also found upregulated endothelial-to-mesenchymal transition (EndMT) markers, mitochondrial dysfunction, and apoptosis in cells grown on soft substrate. In the\n                    <jats:italic>Col8a2<\/jats:italic>\n                    <jats:sup>Q455K\/Q455K<\/jats:sup>\n                    mouse model of FECD, there was increased pFAK Y397 levels coincident with the onset of phenotypes. Intraperitoneal injections of a pFAK inhibitor improved antioxidant protein expression and decreased EndMT; however, it did not improve FECD-associated disease progression.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion:<\/jats:title>\n                  <jats:p>In this study, we explored how changes in the physical characteristics of the Descemet\u2019s membrane impact corneal endothelial cell health. While we discovered activation of Focal adhesion kinase as a result of stiffness changes, its inhibition alone was insufficient to improve cell health in an FECD mouse model.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.11.08.687366","type":"posted-content","created":{"date-parts":[[2025,11,9]],"date-time":"2025-11-09T05:25:16Z","timestamp":1762665916000},"source":"Crossref","is-referenced-by-count":0,"title":["Decreased substrate stiffness leads to mitochondrial dysfunctions and Endothelial to Mesenchymal transition through Focal Adhesion Kinase activity in corneal endothelial cells"],"prefix":"10.64898","author":[{"given":"Sachin Anil","family":"Ghag","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5969-9109","authenticated-orcid":false,"given":"Viviane Souza de","family":"Campos","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7368-7593","authenticated-orcid":false,"given":"Subashree","family":"Murugan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7196-2010","authenticated-orcid":false,"given":"Samuel","family":"Herberg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2154-4173","authenticated-orcid":false,"given":"Rajalekshmy","family":"Shyam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025120323351422000_2025.11.08.687366v2.1","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2021.108495"},{"key":"2025120323351422000_2025.11.08.687366v2.2","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2018.11.021"},{"key":"2025120323351422000_2025.11.08.687366v2.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2022.109376"},{"key":"2025120323351422000_2025.11.08.687366v2.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.preteyeres.2023.101234"},{"key":"2025120323351422000_2025.11.08.687366v2.5","doi-asserted-by":"publisher","DOI":"10.1038\/sj.eye.6700559"},{"key":"2025120323351422000_2025.11.08.687366v2.6","doi-asserted-by":"publisher","DOI":"10.1016\/S0161-6420(82)34746-6"},{"key":"2025120323351422000_2025.11.08.687366v2.7","doi-asserted-by":"publisher","DOI":"10.1016\/j.medntd.2022.100180"},{"key":"2025120323351422000_2025.11.08.687366v2.8","doi-asserted-by":"publisher","DOI":"10.3390\/ijms16059406"},{"key":"2025120323351422000_2025.11.08.687366v2.9","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms1668"},{"key":"2025120323351422000_2025.11.08.687366v2.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.biomaterials.2019.01.010"},{"key":"2025120323351422000_2025.11.08.687366v2.11","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.65.1.38"},{"key":"2025120323351422000_2025.11.08.687366v2.12","doi-asserted-by":"publisher","DOI":"10.1038\/srep23096"},{"key":"2025120323351422000_2025.11.08.687366v2.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2009.03.012"},{"key":"2025120323351422000_2025.11.08.687366v2.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2022.109303"},{"key":"2025120323351422000_2025.11.08.687366v2.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.exer.2020.108193"},{"key":"2025120323351422000_2025.11.08.687366v2.16","doi-asserted-by":"publisher","DOI":"10.1101\/2025.04.11.648416"},{"key":"2025120323351422000_2025.11.08.687366v2.17","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/ddr473"},{"key":"2025120323351422000_2025.11.08.687366v2.18","doi-asserted-by":"publisher","DOI":"10.1038\/s41389-018-0032-z"},{"key":"2025120323351422000_2025.11.08.687366v2.19","doi-asserted-by":"publisher","DOI":"10.1038\/s41389-018-0083-1"},{"key":"2025120323351422000_2025.11.08.687366v2.20","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-019-0134-2"},{"issue":"3\u20134","key":"2025120323351422000_2025.11.08.687366v2.21","first-page":"305","article-title":"Proliferation and Differentiation of Bovine Corneal Endothelial Cells in Culture","volume":"6","year":"1982","journal-title":"Metab. 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