Abstract
Abstract: :
Purpose: Hep II was recently shown to lower IOP in human eye organ cultures (Santas, et al., IOVS. 44:4796, 2003). To determine if Hep II lowers IOP by altering the cytoskeletal network, we studied its effect on stress fiber formation and cell spreading in HTM cells. Methods: HTM cells were serum starved for 24 hrs and plated on the III7–10 repeats of fibronectin in the absence or presence of Hep II for 3 hrs. In some experiments, either the Rho kinase inhibitor Y27632 or anti–α4 integrin antibodies were added in the presence of Hep II, or VCAM–1 (an α4ß1 ligand) was used in lieu of Hep II. Pretreatments with chlorate, heparinase or heparin were used to determine the role of heparan sulfate proteoglycans (HSPGs). Immunofluorescence microscopy studies used phalloidin and vinculin antibodies to visualize stress fiber and focal adhesion formation respectively. Results: In the absence of soluble Hep II, HTM cells spread on the III7–10 repeats had a bipolar morphology with few focal adhesions and stress fibers. Addition of soluble Hep II increased stress fiber formation (indicated by F–actin levels) by 51±2% (p<0.001) and enhanced cell spreading (cell length vs. width ratio) by 33±8% (p<0.001) compared to control cells. Both stress fiber formation and cell spreading were inhibited by Y27632 in the presence of Hep II. Neither treatment with chlorate, heparinase or soluble heparin prevented cell spreading in the presence of Hep II. In addition, a mutated Hep II/RK that no longer binds heparin was able to induce cell spreading. In contrast, soluble α4 integrin antibodies inhibited cell spreading mediated by both Hep II and Hep II/RK by 21 (p<0.005) and 24% (p<0.001) respectively. Similar to Hep II, soluble VCAM–1 also induced HTM cell spreading on the III7–10 repeats. Conclusions: Hep II mediates cell spreading and stress fiber formation through an α4ß1 integrin dependent and HSPG independent signaling pathway. This suggests that the Hep II domain may regulate IOP by altering the actin cytoskeleton via an α4ß1 integrin/Rho signaling pathway.
Keywords: cytoskeleton • extracellular matrix • trabecular meshwork