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W. J. O'Brien, T. Heimann, J. Joseph, A. Ahmed, B. Kalyanaraman; Corneal Stromal Cell Survival Modulated by Porphyrin Analogs. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4818.
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Porphyrins such as heme play critical roles in the survival of all mammalian cells. These studies investigate the influence of metalloporphyrins on corneal stromal cell survival and establish a mechanism by which they exert their effects.
Tetakis (4-benzoic acid) porphyrins (TBAPs) containing selected metal ions were synthesized, purified, and used to treat cultures of rabbit corneal stromal fibroblasts which had been induced to undergo apoptosis by serum deprivation. After 6hr of treatment the cells were harvested and caspase 3 activity assayed as a measure of the extent of apoptosis. Comparison of caspase activities among treated cultures by ANOVA was used to determine the effectiveness of treatments.
Serum deprivation induced apoptosis of rabbit corneal stromal fibroblasts by an extrinsic pathway as determined by TUNEL and annexin V staining, caspase 8 and 3 activation, and DNA laddering. Treatment of cultures with not only the iron, cobalt, and zinc derivatives of TBAP but also TBAP itself at concentrations as low as 5 to10 µM significantly reduced apoptosis (p < 0.001). Fe and Zn derivatives appeared more effective than the Co derivative. Treatment with the TBAPs significantly enhanced the anti-apoptotic activity of mixtures of the cytokines, IL-1β, TNF-α and rRaIFN-γ (p < 0.001). SnMPP did significantly inhibit the activity of FeTBAP, TBAP and the cytokine mixture but enhanced the anti-apoptotic activity of ZnTBAP (p < 0.001). TBAPs directly inhibited caspases 8 and 3 in assays of activities of pure enzymes. CoTBAP was the most potent inhibitor with Ki’s of 27 and 5 µM for caspases 8 and 3 respectively. TBAP itself also inhibited the caspases but the Ki’s were in the range of 200uM.
The metalloporphyrins elicit several biologically significant effects. The Fe and Mn but not Zn and Co derivatives efficiently scavenge superoxide. Co and Fe derivatives induce heme-oxygenase 1 (HO-1) more efficiently than the Zn derivative, while SnMPP is a very efficient inhibitor of HO-1. Our studies document that TBAPs possess potent anti-apoptotic activities. In vitro all derivatives except SnMPP enhanced the anti-apoptotic activities of the cytokine mixture which is known to induce HO-1. We document that the TBAPs are potent inhibitors of caspases 8 and 3 which likely contributed to the anti-apoptotic effects of the TBAPs independently of their effects on HO-1. The potent anti-apoptotic activities of the TBAPs make them potential therapeutic agents to treat corneal wound healing problems related to the apoptosis of corneal stromal fibroblasts.
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