| Objective The number of patients requiring organ or tissue transplantation far outweighs the availability of suitable human donor organs. Thus, the past several years have witnessed a resurgence of interest in interspecies transplantation, i.e. xenotransplantation. Transplantation between species has been considered to be a potential solution to the very urgent shortage of human donor organs and may allow the expanded application of transplantation. The pig is viewed as the xenogeneic donor species of choice .The first major obstacle in discordant pig-to-human xenotransplantation is hyperacute rejection (HAR). In view of the recent successful development of transgenic pigs to overcome HAR, delayed xenograft rejection (DXR) now represents the next immunological hurdle for pig-to-human xenotransplantation. In this setting, xenografts elicit severe and acute rejection linked to endothelial-cell activation. DXR is also characterized by recognition, infiltration and damage to grafts by various leukocyte subsets, including natural killer (NK) cells. We try to investigate the effect and mechanism of inducted expression of heme oxygenase-1 (HO-1), a protective gene, to protect endothelial cell from xenogenic impair in vitro and in vivo. Methods Cultured porcine aorta endothelial cells (PAEC) exposed to 20% human serum (HS) was used as in vitro model of xenotransplantation from pig to human. HO-1 was induced by pretreating PAEC with 100 μ M CoPP for 12h. Protein expression and enzyme activity of HO-1 were assessed by Western blot and bilirubin production, respectively. To identify the activation of PAEC and the effect of HO-1, immunofluorescence confocal microscopy and ELISA were respectively used to detect changes in nuclear factor kappa B (NF- k B) and E-selectin. The apoptosis of PAEC induced by HS and anti-apoptosis effect of HO-1 were also analyzed. TUNEL and FACS with double stains by Annexin V-FITC/PI were used to detect the apoptosis of PAEC. In addition, two anti-apoptosis genes A20 and Bcl-2 were investigated by Western blot to reveal whether they anticipated in the effect of HO-1. Because NK cell may play an important role in xenotransplantation, the cytotoxic effect of humanNK cell (NK92) to PAEC was measured by Cr51 release assay and the activity of NK92 was assessed by detecting the level of IFN-? in supernatant by ELISA analysis. We further investigated the effect of HO-1 on rejection of xenotransplantation in vivo. A liver xenotransplantation model from guinea-pig to rat was established with some improvements, the rates of successful operation and reperfusion of grafts were compared with that in conventional operation. Recipient rats were administered with 50 u g/kg Cobra venom factor (CVF) to exhaust complement and avoid HAR, and donors were injected with 5mg/kg CoPP to induce HO-1 overexpression in grafts 24h before transplantation, respectively. The effects of HO-1 on rejection were reflected by histological examination of xenografts and survival time of recipients. Results CoPP induced PAEC to overexpress HO-1 and then significantly inhibited the activation of NF-?B and expression of E-selectin of PAEC, suggesting HO-1 protected PAEC from stimulation by HS. Incubating PAEC with 20% HS for 24h resulted about 40% of PAEC to undergo apoptosis, however, overexpression of HO-1 markedly prevented this apoptosis (about 20%, PO.01). The protein expression of A20 had no change in this process, in contrast, that of Bcl-2 was overtly upregulated and the latter effect could be abrogated by preconditioning with HO-1 inhibitor ZnPP, suggesting that HO-1 may perform anti-apoptosis effect by Bcl-2. NK92 killed PAEC by a dose-effect and time-effect manner. When the ratio of effecter to target was 20, 46.7% of PAEC were lysed by NK92 within 12h, but HO-1 protected PAEC from cytotoxic effect of NK92 ( 24.6%, P<0.05). IFN-? secreted by NK92 was also inhibited by HO-1 overexpression (3. 28±0.47 vs 7. 55±0.52, P<0.01). Exogenous carbon monoxide (CO) could mimic the effects, indicating HO-I/CO directly inhibited NK cell activity and CO might be the major effecter. The protective effect of HO-1 was also observed in vivo. CoPP induced HO-1 significantly inhibited E-selectin expression (0.112+0.039 vs 0.211 ±0.030, PO.01) in the grafts after liver xenotransplantation, this correlated with amelioration of histological features, improved liver function, and depressed infiltration by leukocytes. Moreover, recipient rat bearing HO-1 overexpression liver graft exhibited a significantly prolonged...
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