Abstract 1231
Neonatal Disease Oriented Research: Molecular Events and Brain Injury Poster Symposium, Tuesday, 5/4
Retinal vasoconstriction and vasoobliteration which results from capillary degeneration, are relatively early events which precede abnormal neovascularization in ROP. Peroxides, which are involved in ROP, cause retinal vasoconstriction and endothelial cell (EC) death. 8-Iso-PGF2α and 8-iso-PGE2 are abundant peroxidation products which cause marked vasoconstriction. We postulated that isoprostanes may reproduce essential features of ROP including oxidant stress-induced EC death and vasoobliteration. Using MTT assay and propidium iodide (PI) incorporation we found that 8-iso-PGF2α (EC50=0.1 nM), but not 8-iso-PGE2, caused (∼35%) death of cultured fetal (0.9 gestation) porcine retinal EC but not of smooth muscle cells, starting 24 h after 8-iso-PGF2α exposure; similar effect was observed with H2O2 (0.1 mM). Apoptosis was minimally evident as nuclear condensation and DNA fragmentation (TUNEL) were hardly detected at all times after 8-iso-PGF2α exposure; inhibition of major apoptosis-involved poly-ADP-ribose polymerase (using nicotinamide) also did not prevent EC death. In contrast, indicators of necrosis, PI incorporation and lactate dehydrogenase release, were increased by 8-iso-PGF2α. Based on our previous work, the mechanism of 8-iso-PGF2α-induced EC death appeared to involve TXA2. 8-Iso-PGF2α evoked a sharp increase in EC TXA2 formation which remained high for the first 24 h. TXA2 synthase inhibitors (CGS12970 and U63557A) prevented EC death by 8-iso-PGF2α and (0.1 mM) H2O2; TXA2 mimetics U46619 and IBOP evoked EC death to the same degree as 8-iso-PGF2α (35%). In in vivo model of retinal vasoobliteration, alternate day intravitreal injection of 8-iso-PGF2α (1 µM final) to rats from postnatal days 8-12 caused a reduction (∼35%) of peripheral vascular density at 15 days old; no adverse effects were seen on saline-injected contralateral eye. This microvascular degeneration by 8-iso-PGF2α as well as that induced by 80% O2 (first 10 postnatal days) which evokes an oxidant stress in the rat pup, was prevented by TXA2 synthase inhibitor CGS12970 (50 mg/kg/day ip). Thus, peroxidation product 8-iso-PGF2α via TXA2 causes not only potent vasoconstriction but also participates in retinal EC death leading to vasoobliteration. Our findings provide a so far unknown mechanism for critically important oxidant stress-induced vasoobliteration of ROP; TXA2 synthase/receptor blockers may help in prevention of ROP and its sequelae.
