Abstract
Abstract: :
Purpose: To assess the ability of integrins to regulate CLAN formation in HTM cells. CLANs have been implicated as a structural change responsible for reduced outflow facility in steroid induced glaucoma. Methods: Immunofluorescence microscopy was used to quantitate CLAN formation in HTM cells plated and spread on coverslips coated with poly–L–lysine (PL), fibronectin (FN), type I collagen (Col I), type IV collagen (Col IV), vitronectin (VN), vascular cell adhesion molecule (VCAM), the III7–10, III12–14 or IIICS domains of FN , or activating antibodies to ß1, ß3 or α2ß1 integrins. In some instances, ß3 antibodies were used as soluble ligands. Cells were spread for 3hr, fixed and labeled with phalloidin. Results: Both adsorbed FN and ß1 antibodies induced CLAN formation in 13% and 11% of the cells respectively. In contrast, 4–5% of the cells formed CLANs on VN, Col IV or Col I. 13% of the cells formed CLANs on VCAM (an α4ß1 ligand) while 7% of the cells formed CLANS on the III7–10 FN fragment (an α5ß1 ligand). PL, the III12–14 and IIICS fragments of FN, adsorbed α2ß1 and adsorbed ß3 integrin antibodies all failed to induce CLAN formation in HTM cells. Soluble ß3 antibody also increased CLAN formation on HTM cells spread on FN, Col I or Col IV by 3–, 7– and 8–fold, respectively while there was no change in CLAN formation induced by VN. Soluble ß3 antibody also increased CLAN formation on adsorbed ß1 and α2ß1 antibodies by 2– and 10– fold respectively. Finally, soluble ß3 antibody increased CLAN formation by 3– and 4–fold on VCAM and the III7–10 FN fragment, respectively. Soluble ß3 antibody by itself induced CLANs in 4% of HTM cells. Conclusions: CLAN formation may be regulated by specific integrin signaling pathways involving ß1 and ß3 integrins. This suggests that ß1 and ß3 integrin co–signaling may contribute to the pathophysiology of steroid induced glaucoma as well as modulate outflow facility in general through its regulation of the HTM actin cytoskeleton.
Keywords: trabecular meshwork • cytoskeleton • signal transduction