Abstract
Purpose: :
To investigate a potential role of phagocytic function in outflow pathway tissue homeostasis.
Methods: :
PTM cells were challenged with different phagocytic ligands (FITC-labeled E.coli, latex beads, collagen-coated beads, pigment, and cell debris) for 2, 5, and 10 days. Levels of expression were quantified by qPCR and WB analysis. Cathepsin activities were evaluated using fluorogenic substrates (z-FR-AMC, z-RR-AMC, z-GPR-AMC, z-VVR-AMC, CTSD/E substrate). MMP-2 and MMP-9 activities were evaluated by gelatin and casein zymography. Gene expression profile analysis was performed using Affymetrix U133 plus 2.0 array and analyzed with Genespring GX and Metacore Softwares. Inhibition of NFΚB was achieved via transduction with a recombinant adenovirus containing a dominant negative of IkB (Ad-IkB-DN).
Results: :
PTM cells challenged to E. coli and collagen-coated beads showed a specific and sustained upregulation in the mRNA (9.6±3.3 fold, p2 fold), and activity of the lysosomal enzyme cathepsin B (CTSB, 65.45±19.66% and 50.3±9.3%, p<0.05). Transduction with Ad-Ik-DN further increased CTSB upregulation in response to phagocytic challenge (2.74±0.71 fold, p<0.05). TM cells demonstrated the secretion of CTSB into the culture media, which was significantly enhanced following phagocytosis. Furthermore, culture media from PTM cells exposed to E. coli and collagen-coated beads showed elevated MMP-2 and MMP-9 activities. Gene expression profile analysis identified the upregulation of MMP-3 (11.2 fold, p=0.004 and 2.09 fold, p=0.006) and MMP-1 (4.61 fold, p=0.003 and 1.98, p=0.006) in PTM cells phagocytically challenged to either E. coli or pigment.
Conclusions: :
Our data indicate a potential role of phagocytosis in outflow pathway tissue homeostasis through the upregulation and/or proteolytic activation of extracellular matrix remodeling genes. Furthermore, the constitutive upregulation of NF-kB described in glaucoma might negatively regulate such homeostatic mechanism.
Keywords: extracellular matrix • outflow: trabecular meshwork • phagocytosis and killing