Nitric oxide synthase expression and activity in the tight-skin mouse model of fibrosis.
OBJECTIVE
Nitric oxide (*NO) is an important physiological signalling molecule and a potent vasodilator. We have previously demonstrated abnormal *NO metabolism in the plasma of patients with systemic sclerosis (SSc; scleroderma), a disease that features vascular dysfunction as well as collagen overproduction and fibrosis. The aim of the present study was to examine nitric oxide synthase (NOS) expression and activity and assess the potential role of antioxidants in the scleroderma-like syndrome of the tight-skin 1 (TSK-1/+) mouse, an experimental animal model for fibrosis.


METHODS
Skin, lung or plasma was taken from TSK-1/+ (n = 15) and wild-type (WT; n = 12) littermate mice. Type 1 collagen, endothelial NOS (eNOS), haemoxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein and gene expression were determined by western blot and reverse transcriptase-polymerase chain reaction. eNOS expression was further determined by immunohistochemistry. NOS activity was evaluated by conversion of [14C] L-arginine to [14C] L-citrulline. Levels of circulating plasma nitrite/nitrate (NO(x)) were also measured. Total antioxidant activity was evaluated by ABTS+ production (ABTS = 2,2'-azino-bis-[3-ethylbenz-thiazoline-6-sulphonic acid).


RESULTS
In the skin, eNOS was present in the epidermal layer, hair follicles and also in the endothelial cells lining the blood vessels. Expression of both the eNOS protein and gene was significantly reduced in TSK-1/+ skin tissue, while type 1 collagen protein was elevated compared with WT. Furthermore, there was decreased NOS activity in TSK-1/+ skin tissue; however, there was no measurable difference in plasma NO(x). Correspondingly, the protective antioxidant enzyme HO-1 and the associated transcription factor Nrf2 showed reduced protein and gene expression levels in TSK-1/+ skin, while there was also less total antioxidant activity. In TSK-1/+ lung tissue, however, we observed no difference in collagen protein expression, *NO metabolism or HO-1 expression and total antioxidant activity compared with WT.


CONCLUSIONS
The findings suggest that there is also abnormal *NO metabolism in the TSK-1/+ mouse model of fibrosis, particularly in the skin, while expression and activity of protective antioxidants are reduced. The TSK-1/+ mouse may also be useful for testing treatments that target vascular endothelial cell function in patients with SSc.
Dooley
A
A
Department of Biochemistry and Molecular Biology, Royal Free Campus, University College Medical School, London NW3 2PF, UK.
Low
S Y
SY
Holmes
A
A
Kidane
A G
AG
Abraham
D J
DJ
Black
C M
CM
Bruckdorfer
K R
KR
D000704:Analysis of Variance; D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D005355:Fibrosis / Q000201:enzymology* / Q000473:pathology*; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000378:metabolism*; D011336:Probability; D012313:RNA / Q000378:metabolism; D011897:Random Allocation; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012594:Scleroderma, Localized / Q000201:enzymology / Q000473:pathology; D012680:Sensitivity and Specificity
D000975:Antioxidants; D015415:Biomarkers; D012313:RNA; D019001:Nitric Oxide Synthase