Abstract
Purpose: :
Intraocular pressure (IOP) elevation, common in primary open-angle glaucoma, results from increased resistance to aqueous humor (AH) outflow. Type VI collagen, an extracellular matrix (ECM) component of the juxtacanalicular region (JCT) and Schlemm’s canal (SC), may be important to the aqueous outflow resistance, and therefore, to IOP. Synergistic interactions between the cells and the ECM of the JCT and SC inner wall, as well as cell-cell interactions may regulate the outflow resistance. To explore aspects of this JCT/SC interface, we studied trabecular type VI collagen and its integrin and non-integrin receptors, as well as cell-cell interactions involving zonula occludens -1 (ZO-1) protein.
Methods: :
Western immunoblotting, immunohistochemistry (IHC) with confocal microscopy, as well as qRT-PCR were used to study molecular changes in human (HTM) and porcine trabecular meshwork (PTM). Type VI collagen and its primary integrin (α3β1) and non-integrin (NG2) receptors on TM were evaluated after being mechanically stretched or subjected to increases in pressure in anterior segment perfusion culture. In addition to the cell-matrix interactions, ZO-1, which is known to downregulate expression with elevated IOP, was examined for co-localization with other proteins, as well as at specialized TM cell complexes, podosome- and invadopodia-like structures (PILS).
Results: :
Type VI collagen and integrin β1 showed increases at 12 and 24 hrs in stretched PTM cell culture respectively, while integrin α3 was elevated at 24 hrs. NG2 was augmented with stretch in PTM cells and in HTM tissue, as assessed with western immunoblots and IHC. TGF-β2 addition at 24 hrs to PTM also amplified NG2. Double immunofluorescence labeling showed that type VI collagen co-localized with integrin α3. With co-immunoprecipitation, however, type VI collagen precipitated with NG2 in stretched PTM cells, but not with integrin α3 or integrin β1. When normal and glaucomatous HTM tissues were compared in anterior segment perfusion culture, the glaucomatous tissue showed increased degradation of NG2. Further immunofluorescence established that ZO-1 partially co-localized with integrin α5β1, c-jun, PKC epsilon, and also with cortactin, a marker component of PILS in PTM. Upon treatment of PTM cells with the phorbol diester PDBu, cortactin and ZO-1 co-localization increased in PILS.
Conclusions: :
The structural integrity of type VI collagen and its integrin and non-integrin receptors may contribute to the proper maintenance of the AH outflow resistance. ZO-1 and similar proteins also may be involved in intercellular actions at the junction of the JCT and the inner wall of SC, and also may modulate PILS formation and function in TM cells.
Keywords: trabecular meshwork • extracellular matrix • cell adhesions/cell junctions