PTX3 Controls Activation of Matrix Metalloproteinase 1 and Apoptosis in Conjunctivochalasis Fibroblasts

PURPOSE. Conjunctivochalasis (CCh) is an age-related inflammatory ocular surface disease manifesting redundant, loose conjunctiva folds. The pathogenic role of Pentraxin 3 (PTX3) in controlling upregulation of matrix metalloproteinase 1 (MMP-1) and MMP-3 in CCh remains undefined. METHODS. Cytolocation of PTX3 and apoptosis were compared by immunostaining and terminal deoxyribonucleotidyl transferase- mediated FITC-linked dUTP nick-end DNA labeling (TUNEL) assay between normal and CCh specimens containing the conjunctiva and the Tenon. Second to third cultures of normal and CCh fibroblasts were treated with or without Aprotinin, Batimastat, or N-isobutyl-N-(4-methoxyphenylsulfonyl)- glycylhydroxamic acid (NNGH), followed by transfection with or without PTX3 siRNA, and TNF-alpha or IL-1 beta. Cell lysates and culture media were collected to assess apoptosis measured by the Cell Death Detection ELISA and expression of PTX3, MMP-1, and MMP-3 transcripts and proteins by quantitative RTPCR and Western blot, respectively. RESULTS. PTX3 immunostaining was negative in normal specimens, but strongly positive in the subconjunctival stroma of CCh specimens. More apoptotic cells were found in CCh samples than in normal specimens. Expression of PTX3 transcripts and protein was not constitutive in resting normal fibroblasts but was in resting CCh fibroblasts and was upregulated by IL-1 beta in both cell lysates and culture media of both fibroblasts. PTX3 siRNA further upregulated MMP-1 and MMP-3 transcripts in resting normal fibroblasts, but synergistically with IL-1 beta upregulated the expression of MMP-1 and MMP-3 transcripts only in CCh fibroblasts, with activation of MMP-1 more so than MMP-3. PTX3 siRNA knockdown also promoted cell death characterized by apoptosis and necrosis, and such cell death could be rescued by inhibitors against serine proteinase, MMP1, or MMP3. CONCLUSIONS. Perturbation of PTX3 expression might partake in apoptosis and pathogenesis of CCh by upregulating expression of MMP-1 and MMP-3, and activation of MMP-1 and MMP-3. (Invest Ophthalmol Vis Sci. 2012;53:3414-3423) DOI: 10.1167/iovs.11-9103