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Vijaykrishna Raghunathan, Joshua Morgan, Yow-Ren Chang, Darren Michael Weber, Brett S Phinney, Christopher J Murphy, Paul Russell; Effect of TGFβ3 on ECM mechanics and composition. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3298.
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© ARVO (1962-2015); The Authors (2016-present)
Pseudoexfoliation syndrome is a systemic disorder of the extracellular matrix (ECM) with ocular manifestations in the form of glaucoma. Elevated levels of TGFβ3 in the aqueous humor of individuals with pseudoexfoliation glaucoma (PEX) have previously been reported. We wanted to determine how TGFβ3 influences the biochemical composition and biomechanics of ECM deposited by human trabecular meshwork (HTM) cells.
HTM cells from three eye bank donor eyes were each plated on functionalized glass substrates (25,000 cells/cm2) and cultured in the presence or absence of 1ng/ml TGFβ3 for four weeks with media changed twice every week. After incubation, samples were decellularized and verified by immunostaining. The mechanics of the remaining ECM that was deposited by the treated or the control cells were measured by atomic force microscopy (AFM). After force vs indentation curves were obtained, the ECM was rinsed, air dried and imaged by AFM. ECM derived from cells were also solubilized and the proteins present in the ECM were analyzed by shotgun proteomics.
Imaged by AFM, the surface features of the ECM from both sets of samples had a similar morphology; however, the ECM from the HTM cells treated with TGFβ3 was between 3 to 5 fold stiffer than that produced by the control HTM cells. Of the 4935 proteins identified by mass spectroscopy, more than 25 were increased greater than 1.5 fold between the treated and control cells across all donors. There were increases in Wnt related proteins such as secreted frizzled related protein 1 and 4, both greater than 2 fold. Gremlin expression was elevated 2.6 fold. Serpine 1 (plasminogen activator inhibitor 1) and serpine 2 (nexin) were increased in the ECM with TGFβ3 treatment as were TIMP 3, CTGF, periostin and extracellular sulfatase 1.
The presence of increased TGFβ3, observed with pseudoexfoliation syndrome, likely influences the ECM secretion by HTM cells. In vitro the matrix deposited by TGFβ3 cells was stiffer than the control group although there were no differences in surface morphology when visualized by AFM. Multiple proteins previously reported to be altered in association with glaucoma were changed in the ECM as a result of the presence of TGFβ3. These included inhibitors of the Wnt and BMP signaling pathways, certain protease inhibitors and connective tissue growth factor. The data suggest the increase in TGFβ3 may play a prominent role in pseudoexfoliation glaucoma.
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