[The role RNA interference of alpha1, 3GT plays in resistance to complement mediated cytotoxicity of porcine endothelial cells].

OBJECTIVE: To evaluate whether RNA interference can protect porcine endothelial cells from complement mediated cytotoxicity. METHODS: Immortalized porcine aortic endothelial cells of the line PED were cultured and transfected with alpha1,3-galactosyltransferase (alpha1, 3-GT) specific siRNAs. Cells transfected with mismatch SiRNA was used as negative controls. Forty-eight hours later the cells were collected. The expression of alpha1, 3-GT mRNA was examined by RT-PCR. The expression of alpha-Gal was examined by flow cytometry. PED cells ere labeled with (51)Cr and mixed with normal human serum (NHS). The release of (51)Cr was measured by gamma-ray counter. Heat inactivated NHS (HINHS) was used as control. RESULTS: Two isoforms (isoform 1 and isoform 2) were amplified from the PED cells. The expression of alpha1, 3-GT in the PED cells transfected with SiRNA-1 was lower by 70% in comparison with the mock group (69% for the isoform 1 and 72% for the isoform 2, both P < 0.05). However, the expression of alpha1, 3-GT in the PED cells transfected with SiRNA21 was not different from those in the mock group and mismatch group (both P > 0.05). Flow cytometry showed that the average fluorescence intensity of the PED cells transfected with SiRNA-1 was 52, 9, significantly lower than that of the mismatch group and mock group (493.9 and 5-5.7 respectively, both P < 0.0). Fluorescence microscopy observed the "silence effect" of alphaGal after SiRNA-1 transfection. Added with 20% and 40% NHS, the cell dissolution rate of the SiRNA transfection group was lower than that of the mock group by 70% and 60% respectively. CONCLUSIONS: alpha1, 3GT gene silencing actually occurs following transfection of SiRNA-1. Porcine endothelial cells can be the targets of RNAi.

Inhibition of xenogeneic response in porcine endothelium using RNA interference.

BACKGROUND: Rejection mediated by antibody recognition of the alpha-Gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) is a major barrier in porcine-to-human xenotransplantation. Because the synthesis of alpha-Gal is dependent on alpha1,3 galactosyltransferase (alpha1,3GT), methods of blocking this enzyme are needed. RNA interference induced by small interfering RNA (siRNA) is a powerful technique for allowing the silencing of mammalian genes with great specificity and potency. In this study, we use siRNA for silencing of alpha1,3GT with the purpose of reducing expression of the alpha-Gal epitope and subsequently decreasing immunogenicity of porcine endothelial cells. METHODS: alpha1,3GT-specific and control siRNAs were transfected into the porcine aortic endothelial cell line, PED. alpha-Gal expression was assessed by Western blotting, flow cytometry, and immunofluorescence. Protection from human-complement and natural killer (NK)-cell-mediated cytotoxicity was evaluated by Cr-release assays after incubation of PED with normal human serum (NHS) and NK92 cell, respectively. RESULTS: RNA interference was successfully achieved in PED as witnessed by the specific knock-down of alpha1,3GT mRNA levels. Flow cytometric analysis using the Griffonia simplicifolia isolectin B4 lectin confirmed the suppression of alpha1,3GT activity as evidenced by decreased alpha-Gal. Functional relevance of the knock-down phenotype was illustrated by the finding that silenced PED were protected from cytotoxicity of NHS. Protection from NK-mediated cytotoxicity was not observed. CONCLUSIONS: Our data are the first to demonstrate that RNA interference is a potent tool to down modulate alpha-Gal expression and to protect endothelial cells from complement-mediated cytotoxicity. Gene silencing by siRNA may represent a new approach for overcoming hyperacute and acute vascular rejection.