Vascular injury and endothelial dysfunction are recognised features of atherosclerosis, transplant
vasculopathy, SLE and rheumatoid. Heme oxygenase-1 (HO-1) and complement inhibitory proteins
(CIP) decay-accelerating factor (DAF) limit vascular injury. There is considerable interest in
therapeutic manipulation of cytoprotective genes and statins induce HO-1 and DAF expression,
while rapamycin (R) enhances HO-1 and protects against post-transplant vasculopathy. A
therapeutic regimen combining immunosuppression with vasculoprotection has the potential to
prevent accelerated atherosclerosis in systemic inflammatory diseases. Based on previous data
generated in the lab, my project explored the combination of atorvastatin and rapamycin, using
DAF and HO-1 as target genes.
A combination of AT 0.5μM and R 1μM led to a synergistic increase in EC DAF expression 24-72h
post-treatment (>200%, p<0.05). In contrast, no change in CD59 was seen. In vivo, AT+R enhanced
DAF expression in the murine aorta when compared to treatment with AT or R alone. The
functional relevance of this synergy was revealed by enhanced protection against complement-mediated
lysis, when compared to EC treated with R or AT (35% v 75% and 55% respectively,
p<0.05). The role of DAF was confirmed by loss of protection in the presence of a DAF inhibitory
mAb. Mechanistically, AT+R increased HO-1 expression and activity, while HO-1 inhibition with
ZnPP attenuated synergy. As a consequence of HO-1-activation, AT+R increased intracellular
ferritin, while Fe2+ chelation with DFO suggested depletion of intracellular Fe2+ is important for
synergy. AT+R enhanced PKCα phosphorylation, while a DN-PKCα adenovirus and a PKCα
inhibitory peptide abrogated DAF induction (AT + R 250% v DN-adv 95%, p<0.05). AT and R-induced
p38 MAPK phosphorylation and inhibition attenuated DAF upregulation (AT+R 270% v
120%, p<0.05). Transcriptionally, DAF induction by AT+R required activation and binding of CREB
to the DAF promoter. Synergistic upregulation of DAF expression was reproduced by simvastatin
and rapamycin, while combinations of AT and cyclosporine or mycophenolate were ineffective.
Treatment with rapamycin and atorvastatin synergistically enhances DAF expression and
protection against complement-mediated injury. Rapamycin increases HO-1 activity and
intracellular ferritin, and this combined with HMG-CoA reductase inhibition and activation of
PKCα, p38 MAPK and CREB, results in optimal DAF induction. Thus, rapamycin and statin
combination therapy may represent a means by which vascular endothelium can be
therapeutically conditioned against complement-mediated injury and is worthy of further study in
vascular diseases.