NADPH oxidase (NOX) 1 mediates cigarette smoke-induced superoxide generation in rat vascular smooth muscle cells.
Smoking is a well-established risk factor for cardiovascular diseases. Oxidative stress is one of the common etiological factors, and NADPH oxidase (NOX) has been suggested as a potential mediator of oxidative stress. In this study, cigarette smoke (CS)-induced superoxide production was characterized in vascular smooth muscle cells (VSMC). CS was prepared in forms of cigarette smoke extract (CSE) and total particulate matter (TPM). Several molecular probes for reactive oxygen species were trialed, and dihydroethidium (DHE) and WST-1 were chosen for superoxide detection considering the autofluorescence, light absorbance, and peroxidase inhibitory activity of CS. Both CSE and TPM generated superoxide in a VSMC culture system by stimulating cells to produce superoxide and by directly producing superoxide in the aqueous solution. NOX, specifically NOX1 was found to be an important cellular source of superoxide through experiments with the NOX inhibitors diphenyleneiodonium (DPI) and VAS2870 as well as isoform-specific NOX knockdown. NOX inhibitors and the superoxide dismutase mimetic TEMPOL reduced the cytotoxicity of CSE, thus suggesting the contribution of NOX1-derived superoxide to cytotoxicity. Since NOX1 is known to mediate diverse pathological processes in the vascular system, NOX1 may be a critical effector of cardiovascular toxicity caused by smoking.
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 10326, Republic of Korea.
Kyung-Hwa
KH
Chang
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 10326, Republic of Korea.
Jung-Min
JM
Park
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 10326, Republic of Korea.
Chang Hoon
CH
Lee
Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program), College of Veterinary Medicine, Chonbuk National University, Iksan, Jeollabuk-do 54596, Republic of Korea.
Bumseok
B
Kim
Laboratory of Biochemistry and Immunology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungcheongbuk-do 28644, Republic of Korea.
Kyung-Chul
KC
Choi
Inhalation Toxicology Research Center, Korea Institute of Toxicology, Jeongeup, Jeollabuk-do 56212, Republic of Korea.
Seong-Jin
SJ
Choi
Inhalation Toxicology Research Center, Korea Institute of Toxicology, Jeongeup, Jeollabuk-do 56212, Republic of Korea.
Kyuhong
K
Lee
College of Pharmacy, Dongguk University, Goyang, Gyeonggi-do 10326, Republic of Korea. Electronic address: mlee@dongguk.edu.
Moo-Yeol
MY
Lee
D000818:Animals; D001013:Aorta, Thoracic; D008297:Male; D009131:Muscle, Smooth, Vascular; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics; D052638:Particulate Matter / Q000633:toxicity*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D017207:Rats, Sprague-Dawley; D012906:Smoke / Q000009:adverse effects*; D013481:Superoxides / Q000378:metabolism*; D014026:Nicotiana
D052638:Particulate Matter; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012906:Smoke; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C000614712:NOX1 protein, rat; C498911:Nox4 protein, rat
Cigarette smoke; Cigarette smoke extract (CSE); NADPH oxidase (NOX); Superoxide; Total particulate matter (TPM); Vascular smooth muscle cells (VSMC)