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B. Chaqour, H. Liu, R. Yang, A. Choudhry; Expression of Cysteine-Rich Protein 61 (Cyr61) and Connective Tissue Growth Factor (CTGF) in Retinal Pericytes Leads to Matrix Metalloproteinase (MMP)-2-Dependent Apoptosis. Invest. Ophthalmol. Vis. Sci. 2007;48(13):559.
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The cysteine-rich protein 61 (Cyr61) and connective tissue growth factor (CTGF) are structurally-related, multifaceted matricellular proteins with a function in cell adhesion and survival. Studies of tissues from human clinical specimens and animal models reported the accumulation of these matricellular proteins in the retinal vasculature under diabetic conditions. The present study used an in vitro model of retinal pericytes in culture to examine the expression and function of Cyr61 and CTGF in retinal pericytes.
Expression of Cyr61 and CTGF was examined in primary cultures of rat retinal pericytes exposed to either advanced glycosylation end (AGE)-bovine serum albumin (BSA) [100 µg/ml] or high glucose concentration (30 mM). The proper effects of Cyr61 and CTGF were examined by adenovirus-mediated expression of Cyr61 and/or CTGF. The levels of mRNA and protein were measured by Real-time PCR and Western blotting techniques respectively. Detection of apoptotic cells was performed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and subcellular localization of cytochrome c. Collagenolytic activity in cell medium was determined by gel zymography.
AGE-BSA or high glucose levels induced a biphasic increase at 4 h and 5 days of Cyr61 and CTGF mRNA and protein levels. The stronger increase of Cyr61 and CTGF levels at 5 days coincided with an increased number of TUNEL positive cells. Adenovirus-mediated gene transfer experiments showed that 50% and 31% of cells underwent apoptosis upon expression of Cyr61 and CTGF respectively. Overexpression of both Cyr61 and CTGF in pericytes had an additive effects and amplified the death signal. Inhibition of either all caspases or caspase-3 significantly reduced the number of apoptotic pericytes overexpressing Cyr61 or CTGF while inhibition of caspase-8 had no significant effect. Furthermore, overexpression of Cyr61 and CTGF increased matrix metalloproteinase-2 (MMP-2) expression and activity. Incubation of the cells with SB3CT, a specific inhibitor of MMP-2, significantly reduced the number of apoptotic cells overexpressing either Cyr61 or CTGF.
Cyr61 and CTGF activate the intrinsic apoptotic pathway in retinal pericytes. Cyr61- and CTGF-induced MMP-2 activity is, at least in part, responsible for retinal pericyte death. Cyr61 and CTGF may contribute to premature death of retinal pericytes which is a pathophysiological hallmark of background diabetic retinopathy.
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