NADPH oxidase mediates angiotensin II-induced endothelin-1 expression in vascular adventitial fibroblasts.
OBJECTIVE
We have recently reported that adventitial fibroblasts are able to express endothelin-1 (ET-1) in response to angiotensin II (Ang II) stimulation. However, the mechanism by which this occurs in the adventitia remains unclear. As Ang II has been reported to increase oxidant production by NADPH oxidase, we examined the role of this complex in Ang II stimulated ET-1 expression in vascular adventitial fibroblasts.


RESULTS
Adventitial fibroblasts were isolated and cultured from mouse aorta. Cells were treated with Ang II (100 nmol/L) in the presence or absence of NADPH oxidase inhibitors, apocynin (100 micromol/L) and diphenyleneiodonium (10 micromol/L), superoxide scavengers, SOD (350 units/mL), tempol (100 micromol/L), tiron (100 micromol/L), and ET-receptor antagonists (10 microM), BQ123 (for ET(A)-) and BQ788 (for ET(B)-). PreproET-1 mRNA and ET-1 level were determined by relative RT-PCR and ELISA, respectively. Type I procollagen-alpha-I (collagen) level was detected by Western blot. Superoxide anion (superoxide) production was determined by coelenterazine or lucigenin chemiluminescence. Ang II-induced collagen expression was inhibited by BQ123, suggesting that adventitial ET-1 plays a significant role in regulating the extracellular matrix. NADPH oxidase inhibitors and superoxide scavengers significantly decreased Ang II-induced ET-1 mRNA and peptide expression, superoxide production as well as collagen expression. Furthermore, deletion of gp91(phox) (a key subunit of NADPH oxidase) and overexpression of SOD1 attenuated Ang II-induced responses.


CONCLUSIONS
Ang II-evoked expression of ET-1 in adventitial fibroblasts appears to be mediated, at least in part, by NADPH oxidase. Functionally, this mechanism stimulates collagen expression thereby implicating the adventitia as a potential contributor to the vascular pathophysiology associated with oxidative stress and vascular remodeling.
An
Sheng Jun
SJ
Department of Community Health Sciences, Brock University, St. Catharines, ON, Canada.
Boyd
Ryan
R
Zhu
Min
M
Chapman
Alexander
A
Pimentel
David R
DR
Wang
Hui Di
HD
D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000098:Acetophenones / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D000804:Angiotensin II / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001011:Aorta; D015415:Biomarkers / Q000032:analysis; D002395:Catecholamines / Q000494:pharmacology; D002478:Cells, Cultured; D024042:Collagen Type I / Q000032:analysis / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D003238:Connective Tissue / Q000201:enzymology*; D003497:Cyclic N-Oxides / Q000494:pharmacology; D018491:Dopamine Agonists / Q000494:pharmacology; D065130:Endothelin A Receptor Antagonists; D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D007091:Image Processing, Computer-Assisted; D048288:Imidazolines / Q000494:pharmacology; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011498:Protein Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods
D000098:Acetophenones; D000975:Antioxidants; D015415:Biomarkers; D002395:Catecholamines; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D003497:Cyclic N-Oxides; D018491:Dopamine Agonists; D065130:Endothelin A Receptor Antagonists; D065131:Endothelin B Receptor Antagonists; D019332:Endothelin-1; D048288:Imidazolines; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D010456:Peptides, Cyclic; D010880:Piperidines; D011498:Protein Precursors; D012333:RNA, Messenger; D013113:Spin Labels; D000804:Angiotensin II; C086539:BQ 788; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; C056165:acetovanillone; C011775:(3,4-dihydroxyphenylamino)-2-imidazoline; D013482:Superoxide Dismutase; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C072247:cyclo(Trp-Asp-Pro-Val-Leu); C001803:tempol