Vascular injury and endothelial dysfunction is a recognized feature of SLE and RA. Haemoxygenase-1 (HO-1) and the complement regulatory proteins decay-accelerating factor (DAF) and CD59 are examples of endothelial cytoprotective genes which act to limit vascular injury. Amongst the lipid-independent effects of statins are induction of HO-1, DAF and CD59, while rapamycin, in addition to its potent immunosuppressant actions, also enhances HO-1 expression and protects against post-transplant

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... opathy. Herein, we have investigated the combined effects of rapamycin and statins in facilitating vascular endothelial cell (EC) protection against complement-mediated endothelial injury. Methods: Human umbilical vein EC were cultured with statin and/or rapamycin and complement regulatory protein expression and complement-mediated lysis assessed by flow cytometry. mRNA expression was quantified by competitive PCR. The role of HO-1 was investigated using bilirubin, desferrioxamine (DFO) and HO-1 antagonist zinc protoporphyrin (ZnPP). Results: Dose response studies demonstrated that treatment of EC with a combination of atorvastatin 0.5 M and rapamycin 1.25 M led to a synergistic increase in DAF expression 24 h post-treatment (>200%, P < 0.05), which was sustained at 72 h. Likewise a synergistic increase in DAF mRNA was apparent at 6-24 h. In contrast, no change in CD59 expression was seen. Atorvastatinþ rapamycin-induced DAF expression was dependent upon gene transcription and de novo protein synthesis. The functional relevance of the synergistic induction of DAF was revealed by enhanced protection against complement-mediated EC lysis, when compared with EC treated with rapamycin or atorvastatin alone (35% vs 75% and 55% lysis, respectively, P < 0.05). The role of DAF was confirmed by loss of cytoprotection in the presence of a DAF inhibitory mAb. Mevalonate reversed the synergistic response, reducing DAF expression to that seen with rapamycin alone, confirming a role for HMG-CoA reductase inhibition. Of note, HO-1 inhibition with ZnPP attenuated the synergistic response. Subsequent experiments using the iron chelator DFO suggested that increased intracellular ferritin, a consequence of HO-1-activation, was central to synergy. Finally, although the synergistic increase in DAF expression was reproduced by simvastatin and rapamycin, there was no evidence for synergy between cyclosporin and atorvastatin. Conclusions: Treatment of EC with rapamycin and atorvastatin results in synergistic enhancement of DAF expression and protection against complementmediated injury. Rapamycin increases HO-1 activity at the concentrations used and a subsequent increase in intracellular ferritin, combined with inhibition of HMG-CoA reductase, results in optimal DAF expression. Combination therapy with rapamycin and statins may represent a means by which vascular endothelium can be therapeutically conditioned against complement-mediated injury and is worthy of further exploration in rheumatic diseases.