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Abstract:
Background. Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de novo synthesis of endothelial cell-derived inflammatory mediators, including the leukocyte-recruiting adhesion molecule E-selectin. Here we investigate the role and mechanism of complement in human serum-induced porcine endothelial cell activation.
Methods. An in vitro xenotransplantation method was designed using porcine aortic endothelial cells stimulated with human serum in micro-culture wells. E-selectin expression was measured by cell-enzyme immunoassay. Complement inhibitors acting at different levels in the cascade were investigated for their effect on E-selectin expression.
Results. E-selectin was strongly induced by normal human serum, but not by heat-inactivated serum. Compstatin, a synthetic C3 inhibitor, markedly reduced human serum-induced E-selectin expression. Purified C1-inhibitor suppressed E-selectin induction completely, indicating activation through the classical or lectin pathway. Furthermore, a mAb that inhibits cleavage of C5 or another mAb that blocks the function of C7, completely inhibited the expression of serum-induced E-selectin, consistent with the terminal C5b-9 complement complex being the mediator of the endothelial cell activation. Inhibition of the alternative pathway using a novel anti-factor D mAb did not reduce E-selectin expression.
Conclusion. Human serum-induced expression of porcine E-selectin is totally complement dependent, induced by a C1-inhibitor regulated pathway, and mediated through the terminal complement complex. The data may have implications for therapeutic strategies, particularly of C1-inhibitor and anti-C5 mAb, to protect against endothelial cell activation and subsequent AVR of porcine xenografts.
Abstract:
Background. Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de novo synthesis of endothelial cell-derived inflammatory mediators, including the leukocyte-recruiting adhesion molecule E-selectin. Here we investigate the role and mechanism of complement in human serum-induced porcine endothelial cell activation.
Methods. An in vitro xenotransplantation method was designed using porcine aortic endothelial cells stimulated with human serum in micro-culture wells. E-selectin expression was measured by cell-enzyme immunoassay. Complement inhibitors acting at different levels in the cascade were investigated for their effect on E-selectin expression.
Results. E-selectin was strongly induced by normal human serum, but not by heat-inactivated serum. Compstatin, a synthetic C3 inhibitor, markedly reduced human serum-induced E-selectin expression. Purified C1-inhibitor suppressed E-selectin induction completely, indicating activation through the classical or lectin pathway. Furthermore, a mAb that inhibits cleavage of C5 or another mAb that blocks the function of C7, completely inhibited the expression of serum-induced E-selectin, consistent with the terminal C5b-9 complement complex being the mediator of the endothelial cell activation. Inhibition of the alternative pathway using a novel anti-factor D mAb did not reduce E-selectin expression.
Conclusion. Human serum-induced expression of porcine E-selectin is totally complement dependent, induced by a C1-inhibitor regulated pathway, and mediated through the terminal complement complex. The data may have implications for therapeutic strategies, particularly of C1-inhibitor and anti-C5 mAb, to protect against endothelial cell activation and subsequent AVR of porcine xenografts.
Abstract:
Background. Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de n...