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(Circulation. 2005;112:727-736.)
© 2005 American Heart Association, Inc.

Vascular Medicine

Upregulation of Nitric Oxide Production in Vascular Endothelial Cells by All-trans Retinoic Acid Through the Phosphoinositide 3-Kinase/Akt Pathway

Akira Uruno, MD, PhD; Akira Sugawara, MD, PhD; Hiroshi Kanatsuka, MD, PhD; Hiroyuki Kagechika, PhD; Akiko Saito, MD; Kazunori Sato, MD, PhD; Masataka Kudo, MD, PhD; Kazuhisa Takeuchi, MD, PhD; Sadayoshi Ito, MD, PhD

From the Division of Nephrology, Endocrinology, and Vascular Medicine, Department of Medicine (A.U., A.S., K.S., M.K., K.T., S.I.), Department of Pediatrics (A.S.), and Department of Comprehensive Medicine (A.S., H.K.), Tohoku University Graduate School of Medicine, Sendai, and School of Biomedical Science, Tokyo Medical and Dental University, Tokyo (H.K.), Japan.

Correspondence to Dr Akira Sugawara, Department of Medicine, Tohoku University Graduate School of Medicine, 1-1, Seiryo-cyo, Aoba-ku, Sendai, 980-8574 Japan. E-mail akiras2i{at}mail.tains.tohoku.ac.jp

Received May 1, 2003; de novo received August 27, 2004; revision received April 19, 2005; accepted April 22, 2005.

Background— A natural retinoid all-trans retinoic acid (ATRA) contains various beneficial effects on vasculature, including suppression of neointima formation after balloon injury. However, little is known about the effects of ATRA on vascular endothelial function. We therefore studied its role in nitric oxide (NO) production of vascular endothelial cells (ECs).

Methods and Results— Human dermal microvascular ECs, human umbilical vein ECs, and SV40-transformed rat lung vascular ECs were incubated with or without ATRA (1 µmol/L) for 48 hours. Their NO production was determined with the use of a fluorescent NO indicator, diaminofluorescein-2 diacetate. ATRA significantly increased their basal as well as acetylcholine-induced NO production. Treatment with N-nitro-L-arginine methyl ester or carboxy-PTIO suppressed their fluorescence. Increase of NO production was also observed by incubation with retinoic acid receptor (RAR) agonist Am580. ATRA-induced NO increase was abolished by coincubation with RAR antagonist LE540. Moreover, the NO increase was completely inhibited by the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and LY294002. ATRA as well as Am580 enhanced endothelial NO synthase (eNOS) phosphorylation at Ser-1177 as well as Akt phosphorylation at Ser-473 without changing their protein expression. Overexpression of dominant-negative Akt inhibited the eNOS phosphorylation. Moreover, ATRA increased PI3K activity as well as PI3K catalytic subunit p110ß protein expression, which was completely inhibited by LE540 treatment. Real-time polymerase chain reaction analyses demonstrated that ATRA increased PI3K catalytic subunit p110ß mRNA expression without affecting its stability. Finally, ATRA-induced NO increase was observed in COS-1 cells transfected with wild-type eNOS and RAR, but not with mutated eNOS whose Ser-1177 was substituted.

Conclusions— ATRA increases NO production by eNOS phosphorylation through RAR-mediated PI3K/Akt pathway activation in vascular ECs and possibly plays beneficial roles in vascular endothelium. Retinoids may therefore be candidates as novel therapeutic agents against vascular disorders with endothelial damage.

Key Words: endothelium • nitric oxide • receptors

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