Abstract
Purpose:
The expression of cellular (cFN) and plasma (pFN) fibronectin isoforms are induced by TGF-β2 in human trabecular meshwork (HTM) cultured cells. Expression of specific FN isoforms can alter ECM homeostasis, ECM-cell interactions, and gene expression. Our purpose is to determine cFN levels in HTM tissues and to explore the impact of FN isoforms on HTM cells by studying changes in adhesion, cytoskeletal organization and gene expression.
Methods:
Differences between cFN levels in normal (NTM) and glaucomatous (GTM) tissues were obtained by immunohistochemistry. NTM cell strains were cultured for 24-48 hrs on surfaces coated with cFN or pFN, and the responses were compared to PBS controls. Changes in formation and redistribution of F-actin fibers and adhesion proteins were analyzed by phalloidin staining, Western immunoblots, and immunocytochemistry. Gene expression changes were analyzed using PCR arrays.
Results:
GTM tissues exhibited significantly greater cFN levels (1.7-fold, p<0.05). NTM strains exposed to both FN isoforms showed increased F-actin formation and redistribution; however, the F-actin pattern and distribution was different between cFN and pFN. Similarly, adhesion molecules such as talin, vinculin, paxillin and integrin beta 1 were increased and redistributed. Both FN isoforms changed gene expression, including alpha-smooth muscle actin-2, metalloproteases and their inhibitors, inflammatory cytokines, and TGF-β related genes.
Conclusions:
Our results show that GTM tissues expressed more cFN and that NTM cells respond differently depending on the FN isoform. The relationship between TGF-β2 modulation of FN isoform expression and the effect of FN isoforms on NTM cells suggests that this type of ECM remodeling may contribute to the TM changes associated with glaucoma.
Keywords: 735 trabecular meshwork •
519 extracellular matrix •
493 cytoskeleton