Abstract
Abstract: :
Purpose: Alteration of several extracellular matrix molecules occurs during glaucoma pathogenesis and therefore an understanding of the molecular organization in the outflow pathway is essential. The present study was undertaken to elucidate the molecular distribution and supramolecular organization of several key trabecular meshwork components in the avian eye. Methods: Cryosections cut from adult or developing chicken anterior eyes were used in immunofluorescence assays to visualize the distribution of collagen types II and XII and of hyaluronan (HA). Collagen was detected using specific antibodies and hyaluronan was detected using a biotinylated HA–binding protein. Hyaluronan–collagen interactions were assayed in vitro using either a) direct binding of biotinylated hyaluronan to Western blots containing anterior eye extracts or b) an immunoprecipitation assay employing an anti–biotin antibody followed by protein A/G–agarose to pellet collagen–biotinylated hyaluronan complexes. Results: These studies show that collagen types II and XII and hyaluronan colocalize in both the developing and adult trabecular meshwork and that type II collagen is a specific marker for this region. Hyaluronan also is highly expressed in the trabecular meshwork and analysis of the type XII collagen sequence indicates that it contains numerous (13 per alpha chain, 39 total) BX7B hyaluronan–binding sites (B is a basic amino acid and X is any non–acidic amino acid preferably containing a basic residue). These sites are highly conserved in both location and sequence between chicken, mouse and human (98% sequence identity). In vitro assays demonstrate that type XII collagen and hyaluronan do, in fact, interact thus establishing this collagen as a hyaluronan–binding protein in the trabecular meshwork. Conclusions: The data suggest a novel architecture for collagen and hyaluronan interaction in the chicken trabecular meshwork and furthermore, that type XII collagen appears to be a major hyaladherin in the anterior eye. It is well established that hyaluronan is decreased in glaucoma and that alteration of the pericellular hyaluronan in a number of cell types leads to apoptosis, increased susceptibility to oxidative stress and to stimulation of inflammatory markers, all characteristics of the glaucomatous phenotype. It is hypothesized that type XII collagen binds to and stabilizes hyaluronan in the normal condition and that this stability may be compromised during glaucoma pathogenesis. A model is proposed in which pericellular hyaluronan is stabilized by type XII collagen that, in turn, is bound to type II collagen fibrils.
Keywords: extracellular matrix • trabecular meshwork • microscopy: light/fluorescence/immunohistochemistry