Geranylgeranylaceton prevents hydrogen peroxide induced retardation of angiogenesis

Abstract

Background & Aim : Angiogenesis is one of the crucial events affecting ulcer healing. It has been reported that neutrophil-derived reactive oxygen species including hydrogen peroxide (HzOz) play an important role in endothelial cell damage leading to suppression of angiogenesis in the H.pylori-infected gastric mucosa. In this study, we aimed to investigate the protective effect of geranylgeranylaceton (GGA), a mucosal defensive agent, against endothelial cell injury caused by HzOz. Methods: HzOz (0.88mM) was added to human umbilical endothelial cells (HUVEC) cultured in DMEM F-12 / RPMIl640 medium containing 2 % FCS with or without 10M GGA that was added 12 h prior to the initiation of the experiments. Cell proliferation was assessed by BrdU staining and apoptotic cells were detected by the TUNEL method. The damage of actin was evaluated with rhodamine-phalloidin staining. To explore the mechanism(s) of the protective effect of GGA, production of prostaglandin E2 (PGE2) was measured by ELISA, expression of the transcription factor ETS-I was detected by RT-PCR, and the level of heat shock protein 70 (HSP70) was measured by Western blotting and immunohistochemistry. Results: Addition of HzOz reduced the growth of HUVEC by 87 %, from 42.5:t6.4 to 5.5:t 1.0 % BrdU staining index. Pre-treatment with GGA restored the cell growth by 60 % to 20.4:t2.3 %. Moreover, GGA prevented completely the HzOz-induced apoptosis (control 2.2+0.9, +HzOz 7.6:t1.7, GGA+HzOz 2.0:tl.l%). In the non-HzOz treated normal cells, polymerized actin was distributed abundantly beneath the plasma membrane and on the stressfibers. In marked contrast, HzOz-treatment reduced greatly polymerized actin. GGA diminished this HzOz-induced reduction of polymerized actin. Cytoplasmic HSP70 protein level was significantly higher in the GGA-treated group as compared to control whereas the production of PGE2 and expression of ETS-I were not altered by GGA pre-treatment. Conclusions: These results demonstrated that GGA prevents the HzOz-induced induction of apoptosis and retardation of proliferation in endothelial cells in vitro. It was postulated that this effect was due to upregulation of cellular HSP70. These results suggest that GGA might act as a muccosal protectant through preserving angiogenesis process during ulcer healing.