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Mark S. Filla, Harini Desikan, Donna M Peters; Activation of αvβ3 integrin in human trabecular meshwork cells enhances Rho-independent assembly of fibronectin fibrils.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5139.
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© ARVO (1962-2015); The Authors (2016-present)
The deposition of fibronectin (FN) within the extracellular matrix (ECM) is an integrin-mediated process and appears to be a factor in the pathogenesis of primary open angle glaucoma (POAG). In this study we examined the effects of αvβ3 integrin signaling on the assembly of FN fibrils into the ECM of trabecular meshwork (TM) cell cultures.
The SV40-immortalized TM-1 cell line was used to derive cells overexpressing either a wild type (WTβ3) or a constitutively active (CAβ3) β3 integrin subunit. Control cells expressing an empty vector (EV) were also generated. To distinguish between soluble FN on the cell surface and FN incorporated into DOC-insoluble fibrils, we used a differential extraction of monolayers with deoxycholic acid (DOC) together with immunoblotting, immunofluorescence microscopy and On-cell western (OCW) analysis. The FN-binding peptide FUD, which blocks FN fibril formation, the β1 integrin function-blocking antibody mAb 13 and the Rho GTPase inhibitor Y27632 were used to assess the FN fibril assembly process. Incorporation of EDA FN and EDB FN isoforms into DOC-insoluble fibrils was also assessed with the isoform-specific antibodies IST-9 and BC-1 respectively.
OCW analysis found that CAβ3 cells increased the assembly of DOC-insoluble FN fibrils 1.9-fold relative to EV cells. Increased FN fibril formation in CAβ3 cells was inhibited by FUD by 40% - 45% relative to untreated cells. In all 3 cell lines, mAb 13 decreased cell surface FN levels 32%-54% relative to untreated cells, but had no effect on DOC-insoluble FN fibril levels in CAβ3 cells. The level of DOC-insoluble FN fibrils was unaffected by Y27632. All 3 cell lines exhibited similar levels of EDA-FN. In contrast, EV cells showed higher levels of EDB-FN than WTβ3 or CAβ3 cells. CAβ3 cells, however, deposited 4-5-fold more EDA-FN and at least 17-fold more EDB-FN into DOC-insoluble fibrils compared to EV and WTβ3 cells.
The data suggest that activation of αvβ3 integrin signaling contributes to excessive deposition of multiple FN isoforms into the TM matrix using a novel mechanism that is independent of the canonical β1 integrin/Rho-mediated process. Together the data suggest that β3 integrin plays a significant role in POAG pathogenesis by increasing ECM assembly and deposition within the TM.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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