Fibronectin fibrils are a major component of the extracellular matrix (ECM) of the trabecular meshwork (TM). They are a key mediator of the formation of the ECM which controls aqueous humor outflow and contributes to the pathogenesis of glaucoma. The purpose of this work was to determine if a fibronectin-binding peptide called FUD, derived from the Streptococcus pyogenes Functional Upstream Domain of the F1 adhesin protein, could be used to control fibronectin fibrillogenesis and hence ECM formation under conditions where its expression was induced by treatment with the glucocorticoid dexamethasone. FUD was very effective at preventing fibronectin fibrillogenesis in the presence or absence of steroid treatment as well as the removal of existing fibronectin fibrils. Disruption of fibronectin fibrillogenesis by FUD also disrupted the incorporation of type IV collagen, laminin and fibrillin into the ECM. The effect of FUD on these other protein matrices, however, was found to be dependent upon the maturity of the ECM when FUD was added. FUD effectively disrupted the incorporation of these other proteins into matrices when added to newly confluent cells that were forming a nascent ECM. In contrast, FUD had no effect on these other protein matrices if the cell cultures already possessed a preformed, mature ECM. Our studies indicate that FUD can be used to control fibronectin fibrillogenesis and that these fibrils play a role in regulating the assembly of other ECM protein into matrices involving type IV collagen, laminin, and fibrillin within the TM. This suggests that under in vivo conditions, FUD would selectively disrupt fibronectin fibrils and de novo assembly of other proteins into the ECM. Finally, our studies suggest that targeting fibronectin fibril assembly may be a viable treatment for POAG as well as other glaucomas involving excessive or abnormal matrix deposition of the ECM.

We previously demonstrated that an αvβ5 integrin/FAK- mediated pathway regulated the phagocytic properties of human trabecular meshwork (HTM) cells. Here we demonstrate that this process is mediated by Rac-1 and a previously unreported signaling pathway that utilizes the Tiam1 as well as a novel ILK/RhoG/ELMO2 signaling pathway. Phagocytosis in both a TM-1 cell line and normal HTM cells was mediated by Rac1 and could be significantly decreased by >75% using the Rac1 inhibitor EHop-016. Knockdown of Rac1 in TM-1 cells also inhibited phagocytosis by 40% whereas overexpression of a constitutively active Rac1 or stimulation with PDGF increased phagocytosis by 83% and 32% respectively. Tiam1 was involved in regulating phagocytosis. Knockdown of Tiam1 inhibited phagocytosis by 72% while overexpression of Tiam1 C1199 increased phagocytosis by 75%. Other upstream effectors of Rac1 found to be involved included ELMO2, RhoG, and ILK. Knockdowns of ELMO2, ILK, and RhoG caused a reduction in phagocytosis by 51%, 55% and 46% respectively. In contrast, knockdown of Vav2 and Dock1 or overexpression of Vav2 Y159/172F did not cause a significant change in phagocytosis. These data suggest a novel link between Tiam1 and RhoG/ILK/ELMO2 pathway as upstream effectors of the Rac1-mediated phagocytic process in TM cells.

Glucocorticoids such as dexamethasone can cause an increase in intraocular pressure (IOP) in some of the population, but not all. In this paper we used a mouse model of glucocorticoid induced ocular hypertension to examine the changes in the anterior segment of the eye in mice that failed to respond to glucocorticoid treatment with a sustained increase in IOP. C57BL/6J mice were treated with either 0.1% dexamethasone sodium phosphate ophthalmic solution or sterile PBS 3 times daily for up to 5 weeks. IOP was measured weekly at approximately the same time of the day. After 3–5 weeks of treatment, eyes were enucleated and evaluated for changes associated with steroid induced glaucoma. These studies showed that IOP was significantly elevated in dexamethasone (DEX) treated mice compared to PBS treated mice after 3 weeks of treatment, but IOP in DEX treated mice returned to baseline levels after 5 weeks of treatment. All the mice demonstrated a response to the glucocorticoid treatments and showed an elevation in FKBP5 expression after both 3 and 5 weeks of DEX treatment (primary glucocorticoid response protein) and a weight loss. Western blot analysis of anterior segments from treated mice, however, did not show an increase in secondary glucocorticoid response proteins such as β3 integrin or myocilin. Fibronectin levels were also not statistically different. The data suggest that in mice, which do not exhibit a prolonged increase in IOP in response to the DEX treatment, there is a compensatory mechanism that can prevent or turn off the secondary glucocorticoid response.