Effect of human CRPs and Bcl-xl combined transgenes on protection of porcine endothelial cells
Introduction In this study we investigated, respectively, the effect of expression of double human complement regulatory proteins (CRPs) and double human decay accelerating factor (hDAF) and Bcl-xl in porcine endothelial cells (PEC) on prevention of PEC activation by human complement.
Materials and methods Using PEC in vitro culture model, human membrane cofactor protein (MCP), CD59 and Bcl-xl expression plasmids were transfected into hDAF-PEC, respectively. The death rates of PEC caused by human complement, and the levels of E-selectin expression stimulated with recombinant human TNF-α were analyzed.
Results In different concentration of human serum 1:1, 1:2 and 1:4, the death rates of hDAF-PEC transfected with hMCP were 18 ± 4%, 10 ± 3%, 4 ± 1%, respectively, and that of hDAF-PEC transfected with hCD59 were 15 ± 3%, 8 + 2% and 4 ± 2%. Both were significantly lower compared with that of simple hDAF-PEC (30 ± 5%, 18 ± 4%, 10 ± 3%) (P < 0.05). But flow cytometry displayed similar expression level of E-selection. The death rates of hDAF-PEC transfected with Bcl-xl expression plasmid were 25 ± 6% (1:1), 15 ± 4% (1:2) and 10 ± 2% (1:4), and similar to that of simple hDAF-PECs (P > 0.05). The levels of E-selectin and hTNF-α expression in hDAF-PEC tansfected with Bcl-x1 group were significantly lower compared with that of simple hDAF-PEC (P < 0.05) and unmodified PEC (P < 0.01). Immunohistochemistry examination disclosed that transgene of human DAF could reduce apoptosis of EC, which could be improved by further transfection of Bcl-xl plasmid.
Conclusions 1. The results indicated that further transfection of hMCP or hCD59 could provide stronger protection to hDAF-PEC, but none of hDAF, hMCP or hCD59 gene could be of benefit to the prevention of EC activation. 2. Bcl-xl was not useful to prevent complement activation, but was helpful to reduced E-selectin expression. 3. Human DAF may not resist the expression of E-selection, but may reduce complement activation. 4. Moreover, the presence of hDAF and Bcl-xl expressed in double transgenic PEC could benefit to reduce EC apoptosis and inhibit complement activation which to prevent xenograft rejection.