Abstract
Glaucoma trabecular meshwork (GTM) cells cultured in vitro retain many characteristics of their in situ phenotype. Here, we used isobaric tandem mass tags (TMTpro) to label peptides from glaucomatous and non-glaucomatous TM (NTM) cells to identify differentially regulated proteins. Confluent NTM (n = 5) and GTM (n = 5) cells were lysed, proteins were trypsin digested, and peptides were labeled with 18-plex TMTpro. TMT-labeled peptides were fractionated on an Orbitrap Fusion mass spectrometer and data were processed using the PAW/Comet pipeline and EdgeR with Benjami–Hochberg multiple correction testing. Isobaric multiplexed quantitative proteomics identified 206 proteins that were significantly (FDR < 0.1) upregulated in GTM cells, 42 proteins that were downregulated, with 5270 non-candidates. Significant regulated pathways included extracellular matrix (DCN, COL4A1, CHI3L1), Wnt signaling (FZD1, FZD7, GSK3B), cytoskeletal regulation (ROCK2, MSN, TPM2, VIM, NF2), protein degradation (USP9X, LAMP1, SYNV1, UBE2L3), and nuclear proteins (LMNA, DFFA, CHMP3, RAD21). Western immunoblotting studies confirmed the TMTpro data. Immunofluorescence showed that the SNX7-stained nucleoli of GTM cells were significantly (p < 0.05) larger, and the DIAPH2 immunostaining was more distended into the cytosol than in NTM cells. This study identified many significantly regulated proteins in cultured GTM cells, and the results revealed several new avenues for developing clinical therapies for glaucoma patients.
Data availability
Data sets generated from the mass spectrometry proteomics analysis have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD06659888.
References
Jayaram, H., Kolko, M., Friedman, D. S. & Gazzard, G. Glaucoma: now and beyond. Lancet 402, 1788–1801 (2023).
Acott, T. S., Vranka, J. A., Keller, K. E., Raghunathan, V. & Kelley, M. J. Normal and glaucomatous outflow regulation. Prog. Retin Eye Res. 82, 100897 (2021).
Tamm, E. R., Braunger, B. M. & Fuchshofer, R. Intraocular pressure and the mechanisms involved in resistance of the aqueous humor flow in the trabecular meshwork outflow pathways. Prog. Mol. Biol. Transl. Sci. 134, 301–314 (2015).
Keller, K. E. & Peters, D. M. Pathogenesis of glaucoma: Extracellular matrix dysfunction in the trabecular meshwork-A review. Clin. Exp. Ophthalmol. 50, 63–182 (2022).
Tian, B., Gabelt, B. T., Geiger, B. & Kaufman, P. L. The role of the actomyosin system in regulating trabecular fluid outflow. Exp. Eye Res. 88, 713–717 (2009).
Alvarado, J., Murphy, C. & Juster, R. Trabecular meshwork cellularity in primary open-angle glaucoma and nonglaucomatous normals. Ophthalmology 91, 564–579 (1984).
Stamer, D. W., Roberts, B. C., Epstein, D. L. & Allingham, R. R. Isolation of primary open-angle glaucomatous trabecular meshwork cells from whole eye tissue. Curr. Eye Res. 20, 347–350 (2000).
Keller, K. E. et al. Consensus recommendations for trabecular meshwork cell isolation, characterization and culture. Exp. Eye Res. 171, 164–173 (2018).
Stamer, W. D. & Clark, A. F. The many faces of the trabecular meshwork cell. Exp. Eye Res. 158, 112–123 (2017).
Tovar-Vidales, T., Roque, R., Clark, A. F. & Wordinger, R. J. Tissue transglutaminase expression and activity in normal and glaucomatous human trabecular meshwork cells and tissues. Investig. Ophthalmol. Vis. Sci. 49, 622–628 (2008).
Balestrini, J. L., Chaudhry, S., Sarrazy, V., Koehler, A. & Hinz, B. The mechanical memory of lung myofibroblasts. Integr. Biol. (Camb). 4, 410–421 (2012).
Cambria, E. et al. Linking cell mechanical memory and cancer metastasis. Nat. Rev. Cancer. 24, 216–228 (2024).
Tamm, E. R., Siegner, A., Baur, A. & Lutjen-Drecoll, E. Transforming growth factor-beta 1 induces alpha-smooth muscle-actin expression in cultured human and monkey trabecular meshwork. Exp. Eye Res. 62, 389–397 (1996).
de Kater, A. W., Shahsafaei, A. & Epstein, D. L. Localization of smooth muscle and nonmuscle actin isoforms in the human aqueous outflow pathway. Investig. Ophthalmol. Vis. Sci. 33, 424–429 (1992).
Yang, Y. F. et al. Fibrosis-related gene and protein expression in normal and glaucomatous trabecular meshwork cells. Investig. Ophthalmol. Vis. Sci. 66, 48 (2025).
Sun, Y. Y., Bradley, J. M. & Keller, K. E. Phenotypic and functional alterations in tunneling nanotubes formed by glaucomatous trabecular meshwork cells. Investig. Ophthalmol. Vis. Sci. 60, 4583–4595 (2019).
Peters, J. C., Bhattacharya, S., Clark, A. F. & Zode, G. S. Increased endoplasmic reticulum stress in human glaucomatous trabecular meshwork cells and tissues. Investig. Ophthalmol. Vis. Sci. 56, 3860–3868 (2015).
Porter, K., Hirt, J., Stamer, W. D. & Liton, P. B. Autophagic dysregulation in glaucomatous trabecular meshwork cells. Biochim. Biophys. Acta. 1852, 379–385 (2015).
Bhattacharya, S. K. et al. Proteomics reveal Cochlin deposits associated with glaucomatous trabecular meshwork. J. Biol. Chem. 280, 6080–6084 (2005).
Elsobky, S., Crane, A. M., Margolis, M., Carreon, T. A. & Bhattacharya, S. K. Review of application of mass spectrometry for analyses of anterior eye proteome. World J. Biol. Chem. 5, 106–114 (2014).
Bollinger, K. E. et al. Quantitative proteomics: TGFbeta(2) signaling in trabecular meshwork cells. Investig. Ophthalmol. Vis. Sci. 52, 8287–8294 (2011).
Steely, H. T. et al. Protein expression in a transformed trabecular meshwork cell line: proteome analysis. Mol. Vis. 12, 372–383 (2006).
Clark, R. et al. Comparative genomic and proteomic analysis of cytoskeletal changes in dexamethasone-treated trabecular meshwork cells. Mol. Cell. Proteom. 12, 194–206 (2013).
Soundararajan, A. et al. Multiomics analysis reveals the mechanical stress-dependent changes in trabecular meshwork cytoskeletal-extracellular matrix interactions. Front. Cell. Dev. Biol. 10, 874828 (2022).
Chowdhury, U. R., Madden, B. J., Charlesworth, M. C. & Fautsch, M. P. Proteome analysis of human aqueous humor. Investig. Ophthalmol. Vis. Sci. 51, 4921–4931 (2010).
Hubens, W. H. G. et al. Aqueous humor proteome of primary open angle glaucoma: A combined dataset of mass spectrometry studies. Data Brief. 32, 106327 (2020).
Liu, X. et al. Proteome characterization of glaucoma aqueous humor. Mol. Cell. Proteom. 20, 100117 (2021).
McDonnell, F. S., Riddick, B. J., Roberts, H., Skiba, N. & Stamer, W. D. Comparison of the extracellular vesicle proteome between glaucoma and non-glaucoma trabecular meshwork cells. Front. Ophthalmol. 3, 1257737 (2023).
Stamer, W. D., Hoffman, E. A., Luther, J. M., Hachey, D. L. & Schey, K. L. Protein profile of exosomes from trabecular meshwork cells. J. Proteom. 74, 796–804 (2011).
Micera, A. et al. Differential protein expression profiles in glaucomatous trabecular meshwork: An evaluation study on a small primary open angle glaucoma population. Adv. Ther. 33, 252–267 (2016).
McAlister, G. C. et al. MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal. Chem. 86, 7150–7158 (2014).
Li, J. et al. TMTpro-18plex: The expanded and complete set of TMTpro reagents for sample multiplexing. J. Proteome Res. 20, 2964–2972 (2021).
Thompson, A. et al. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal. Chem. 75, 1895–1904 (2003).
Vranka, J. A. et al. Biomechanical rigidity and quantitative proteomics analysis of segmental regions of the trabecular meshwork at physiologic and elevated pressures. Investig. Ophthalmol. Vis. Sci. 59, 246–259 (2018).
Liang, X., Li, N., Rong, Y., Wang, J. & Zhang, H. Identification of proteomic changes for dexamethasone-induced ocular hypertension using a tandem mass tag (TMT) approach. Exp. Eye Res. 216, 108914 (2022).
Schneider, M. et al. A novel ocular function for decorin in the aqueous humor outflow. Matrix Biol. 97, 1–19 (2021).
Colorado, P. C. et al. Anti-angiogenic cues from vascular basement membrane collagen. Cancer Res. 60, 2520–2526 (2000).
Rao, P. V., Pattabiraman, P. P. & Kopczynski, C. Role of the Rho GTPase/Rho kinase signaling pathway in pathogenesis and treatment of glaucoma: Bench to bedside research. Exp. Eye Res. 158, 23–32 (2017).
Iontcheva, I., Amar, S., Zawawi, K. H., Kantarci, A. & Van Dyke, T. E. Role for moesin in lipopolysaccharide-stimulated signal transduction. Infect. Immun. 72, 2312–2320 (2004).
Stamenkovic, I. & Yu, Q. Merlin, a magic linker between extracellular cues and intracellular signaling pathways that regulate cell motility, proliferation, and survival. Curr. Protein Pept. Sci. 11, 471–484 (2010).
Lin, J. J., Eppinga, R. D., Warren, K. S. & McCrae, K. R. Human tropomyosin isoforms in the regulation of cytoskeleton functions. Adv. Exp. Med. Biol. 644, 201–222 (2008).
Yeoh, S., Pope, B., Mannherz, H. G. & Weeds, A. Determining the differences in actin binding by human ADF and cofilin. J. Mol. Biol. 315, 911–925 (2002).
Rao, V. P. & Epstein, D. L. Rho GTPase/Rho kinase inhibition as a novel target for the treatment of glaucoma. BioDrugs. 21, 167–177 (2007).
Keller, K. E. et al. Ankyrin repeat and suppressor of cytokine signaling box containing protein-10 is associated with ubiquitin-mediated degradation pathways in trabecular meshwork cells. Mol. Vis. 19, 1639–1655 (2013).
Liton, P. B. The autophagic lysosomal system in outflow pathway physiology and pathophysiology. Exp. Eye Res. 144, 29–37 (2016).
Scaffidi, P. & Misteli, T. Lamin A-dependent nuclear defects in human aging. Science 312, 1059–1063 (2006).
Liu, X., Zou, H., Slaughter, C. & Wang, X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89, 175–184 (1997).
Chen, J., Gao, G., Zhang, Y., Dai, P. & Huang, Y. Comprehensive analysis and validation of SNX7 as a novel biomarker for the diagnosis, prognosis, and prediction of chemotherapy and immunotherapy response in hepatocellular carcinoma. BMC Cancer. 23, 899 (2023).
Anton, Z. et al. A heterodimeric SNX4–SNX7 SNX-BAR autophagy complex coordinates ATG9A trafficking for efficient autophagosome assembly. J. Cell. Sci. 133, (2020).
van Zyl, T. et al. Cell atlas of the human ocular anterior segment: Tissue-specific and shared cell types. Proc. Natl. Acad. Sci. U S A. 119, e2200914119 (2022).
Gharahkhani, P. et al. Genome-wide meta-analysis identifies 127 open-angle glaucoma loci with consistent effect across ancestries. Nat. Commun. 12, 1258 (2021).
Khawaja, A. P. et al. Genome-wide analyses identify 68 new loci associated with intraocular pressure and improve risk prediction for primary open-angle glaucoma. Nat. Genet. 50, 778–782 (2018).
Ahmad, M. T., Zhang, P., Dufresne, C., Ferrucci, L. & Semba, R. D. The human eye proteome project: Updates on an emerging proteome. Proteomics. 18, e1700394 (2018).
Saccuzzo, E. G., Youngblood, H. A. & Lieberman, R. L. Myocilin misfolding and glaucoma: A 20-year update. Prog. Retin Eye Res. 95, 101188 (2023).
Howell, K. G., Vrabel, A. M., Chowdhury, U. R., Stamer, W. D. & Fautsch, M. P. Myocilin levels in primary open-angle glaucoma and pseudoexfoliation glaucoma human aqueous humor. J. Glaucoma. 19, 569–575 (2010).
Wang, W. H. et al. Increased expression of the WNT antagonist sFRP-1 in glaucoma elevates intraocular pressure. J. Clin. Investig. 118, 1056–1064 (2008).
Soundararajan, A., Wang, T., Ghag, S. A., Kang, M. H. & Pattabiraman, P. P. Novel insight into the role of clusterin on intraocular pressure regulation by modifying actin polymerization and extracellular matrix remodeling in the trabecular meshwork. J. Cell. Physiol. 237, 3012–3029 (2022).
Bachman, W. et al. Glucocorticoids preferentially influence expression of nucleoskeletal actin network and cell adhesive proteins in human trabecular meshwork cells. Front. Cell. Dev. Biol. 10, 886754 (2022).
Bermudez, J. Y., Montecchi-Palmer, M., Mao, W. & Clark, A. F. Cross-linked actin networks (CLANs) in glaucoma. Exp. Eye Res. 159, 16–22 (2017).
Keller, K. E. & Kopczynski, C. Effects of netarsudil on actin-driven cellular functions in normal and glaucomatous trabecular meshwork cells: A live imaging study. J. Clin. Med. 9, 3524 (2020).
Filla, M. S., Schwinn, M. K., Sheibani, N., Kaufman, P. L. & Peters, D. M. Regulation of cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells by convergence of distinct beta1 and beta3 integrin pathways. Investig. Ophthalmol. Vis. Sci. 50, 5723–5731 (2009).
Wang, S. K. & Chang, R. T. An emerging treatment option for glaucoma: Rho kinase inhibitors. Clin. Ophthalmol. 8, 883–890 (2014).
Gateva, G. et al. Tropomyosin isoforms specify functionally distinct actin filament populations in vitro. Curr. Biol. 27, 705–713 (2017).
Wallar, B. J. & Alberts, A. S. The formins: Active scaffolds that remodel the cytoskeleton. Trends Cell. Biol. 13, 435–446 (2003).
Zhang, X. et al. Mechanism and disease association with a ubiquitin conjugating E2 enzyme: UBE2L3. Front. Immunol. 13, 793610 (2022).
Heo, A. J. et al. The N-terminal cysteine is a dual sensor of oxygen and oxidative stress. Proc Natl. Acad. Sci. U S A 118, e2107993118 (2021).
McDowell, C. M., Tebow, H. E., Wordinger, R. J. & Clark, A. F. Smad3 is necessary for transforming growth factor-beta2 induced ocular hypertension in mice. Exp. Eye Res. 116, 419–423 (2013).
Pathak, R. U., Soujanya, M. & Mishra, R. K. Deterioration of nuclear morphology and architecture: A hallmark of senescence and aging. Ageing Res. Rev. 67, 101264 (2021).
Gauthier, B. R. & Comaills, V. Nuclear envelope integrity in health and disease: Consequences on genome instability and inflammation. Int. J. Mol. Sci. 22, 7281 (2021).
McDonald, O. G., Wu, H., Timp, W., Doi, A. & Feinberg, A. P. Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition. Nat. Struct. Mol. Biol. 18, 867–874 (2011).
Ghosh, R. et al. Altered nuclear dynamics in glaucomatous trabecular meshwork cell pathobiology. Investig. Ophthalmol. Visual Sci. 66, 3253 (2025).
Gutierrez, J. I. & Tyler, J. K. A mortality timer based on nucleolar size triggers nucleolar integrity loss and catastrophic genomic instability. Nat. Aging. 4, 1782–1793 (2024).
Liton, P. B. et al. Cellular senescence in the glaucomatous outflow pathway. Exp. Gerontol. 40, 745–748 (2005).
Shim, M. S., Nettesheim, A., Hirt, J. & Liton, P. B. The autophagic protein LC3 translocates to the nucleus and localizes in the nucleolus associated to NUFIP1 in response to cyclic mechanical stress. Autophagy 16, 1248–1261 (2020).
Chen, W. et al. ROCK2, but not ROCK1 interacts with phosphorylated STAT3 and co-occupies TH17/TFH gene promoters in TH17-activated human T cells. Sci. Rep. 8, 16636 (2018).
Batchelor, C. L., Woodward, A. M. & Crouch, D. H. Nuclear ERM (ezrin, radixin, moesin) proteins: regulation by cell density and nuclear import. Exp. Cell. Res. 296, 208–222 (2004).
Aebersold, R., Burlingame, A. L. & Bradshaw, R. A. Western blots versus selected reaction monitoring assays: time to turn the tables? Mol. Cell. Proteom. 12, 2381–2382 (2013).
Fingert, J. H., Stone, E. M., Sheffield, V. C. & Alward, W. L. Myocilin glaucoma. Surv. Ophthalmol. 47, 547–561 (2002).
Yang, Y. F., Sun, Y. Y., Peters, D. M. & Keller, K. E. The effects of mechanical stretch on integrins and filopodial-associated proteins in normal and glaucomatous trabecular meshwork cells. Front. Cell. Dev. Biol. 10, 886706 (2022).
Wilmarth, P. A., Riviere, M. A. & David, L. L. Techniques for accurate protein identification in shotgun proteomic studies of human, mouse, bovine, and chicken lenses. J. Ocul Biol. Dis. Infor. 2, 223–234 (2009).
Chambers, M. C. et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol. 30, 918–920 (2012).
Eng, J. K., Jahan, T. A. & Hoopmann, M. R. Comet: an open-source MS/MS sequence database search tool. Proteomics 13, 22–24 (2013).
Keller, A., Nesvizhskii, A. I., Kolker, E. & Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 74, 5383–5392 (2002).
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Robinson, M. D. & Oshlack, A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 11, R25 (2010).
Ge, S. X., Jung, D. & Yao, R. ShinyGO: A graphical gene-set enrichment tool for animals and plants. Bioinformatics. 36, 2628–2629 (2020).
Szklarczyk, D. et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 51, D638–D646 (2023).
Deutsch, E. W. et al. The ProteomeXchange consortium at 10 years: 2023 update. Nucleic Acids Res. 51, D1539–D1548 (2023).
Acknowledgements
We thank VisionGift, Portland, OR for helping to procure human donor eyes. Supported by NIH/NEI grants R01 EY032590 (KEK), R01 EY019643 (KEK), the Malcolm M. Marquis MD endowed fund for innovation, and an unrestricted grant from Research to Prevent Blindness (New York, NY) to the Casey Eye Institute, OHSU. Mass spectrometric analysis was performed by the OHSU Proteomics Shared Resource (RRID: SCR_009991) with partial support from NIH core grants P30EY010572, P30CA069533, and S10OD012246.
Funding
Supported by NIH/NEI grants R01 EY032590 (KEK), R01 EY019643 (KEK), the Malcolm M. Marquis MD endowed fund for innovation, and an unrestricted grant from Research to Prevent Blindness (New York, NY) to the Casey Eye Institute, OHSU. Mass spectrometric analysis was performed by the OHSU Proteomics Shared Resource (RRID: SCR_009991) with partial support from NIH core grants P30EY010572, P30CA069533, and S10OD012246.
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P.H., A.P.R., K.E.K. conceived and designed the study; P.H., Y.Y.S., K.Z. conducted experiments; P.A.W., A.P.R., K.E.K., P.H. analyzed data. K.E.K., P.A.W., P.H. wrote the manuscript. All authors reviewed the manuscript.
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Holden, P., Sun, Y.Y., Zientek, K. et al. Isobaric quantitative proteomics reveals altered extracellular matrix, cytoskeletal, and degradation pathways in glaucomatous trabecular meshwork cells. Sci Rep (2026). https://doi.org/10.1038/s41598-026-44561-x
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DOI: https://doi.org/10.1038/s41598-026-44561-x
