Effects of estrogen on nitric oxide synthase expression in rat aorta allograft and smooth muscle cells.
BACKGROUND
We find that chronic estradiol treatment inhibits the development of transplant arteriosclerosis (TA). The mechanism of this inhibition remains unclear. The objective of this study is to investigate in a non-cyclosporin-requiring TA model whether estradiol-17beta treatment modulates the expression of both endothelial nitric oxide synthase (ecNOS) and inducible nitric oxide synthase (iNOS) in the early phase following transplantation.


METHODS
Orthotopic abdominal aorta allograft transplantation was performed in male rats using Brown-Norway rats as donors and Lewis rats as recipients. The recipients (n = 50) were treated with estradiol 20 microg/kg/day or placebo by osmotic minipump from 2 days prior to surgery until sacrifice on post-operative days 1, 3, 7, 14, and 21. The allografts were harvested and cross-sections of the vascular tissues were used for immunohistochemical staining of ecNOS and iNOS. The effects of estradiol on cytokine-induced (tumor necrosis factor-alpha and interleukin-1 beta iNOS protein and messenger RNA (mRNA) expression were also evaluated on rat aorta smooth muscle cells by Western blotting and RT-PCR in vitro, respectively.


RESULTS
The expression of ecNOS and iNOS was graded semiquantitatively from 0 to +3. Estrogen elevates ecNOS expression in the intima in the early phase following transplantation, 0.85 +/- 0.14 (day 7) and 1.08 +/- 0.11 (day 14) vs 1.53 +/- 0.25 (day 7) and 1.60 +/- 0.17 (day 14) for placebo and estradiol treated groups respectively, p < 0.01. Estrogen suppresses iNOS expression in neointima (0.67 +/- 0.17 vs 0.24 +/- 0.04, p < 0.01, day 14), media (1.03 +/- 0.15 vs 0.4 +/- 0.09, p < 0.01, day 7), and adventitia (1.55 +/- 0.12 vs 1.02 +/- 0.10, p < 0.05, day 14) in the same phase. Estradiol treatment inhibits cytokine-induced iNOS mRNA expression in cultured smooth muscle cells.


CONCLUSIONS
Chronic estrogen treatment modulates both ecNOS and iNOS expression in the early phase following transplantation. This is associated with the estrogen-protective effects on TA.
Saito
S
S
Department of Surgery, Georgetown University Medical Center, Washington, DC 20007, USA.
Aras
R S
RS
Lou
H
H
Ramwell
P W
PW
Foegh
M L
ML
D000818:Animals; D001012:Aorta, Abdominal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000637:transplantation*; D001161:Arteriosclerosis / Q000139:chemically induced / Q000201:enzymology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004958:Estradiol / Q000494:pharmacology*; D007150:Immunohistochemistry; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000187:drug effects*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D011897:Random Allocation; D051381:Rats; D011917:Rats, Inbred Lew; D013997:Time Factors; D014184:Transplantation, Homologous
D004958:Estradiol; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496318:Nos2 protein, rat; C496316:Nos3 protein, rat