Upregulation of Nitric Oxide Production in Vascular Endothelial Cells by All-trans Retinoic Acid Through the Phosphoinositide 3-Kinase/Akt Pathway
Upregulation of Nitric Oxide Production in Vascular Endothelial Cells by All-trans Retinoic Acid Through the Phosphoinositide 3-Kinase/Akt Pathway
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DOI：
10.1161/circulationaha.104.500959
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发表时间：
2005-08
期刊：
影响因子：
37.8
通讯作者：
A. Uruno;A. Sugawara;H. Kanatsuka;H. Kagechika;A. Saito;Kazunori Sato;M. Kudo;K. Takeuchi;S. Ito
中科院分区：
文献类型：
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作者：
A. Uruno;A. Sugawara;H. Kanatsuka;H. Kagechika;A. Saito;Kazunori Sato;M. Kudo;K. Takeuchi;S. Ito
天然维甲酸全反式维甲酸（ATRA）对血管系统有多种有益作用，包括抑制球囊损伤后新生内膜的形成。然而，很少有人知道ATRA对血管内皮功能的影响。因此，我们研究了它在血管内皮细胞（EC）产生一氧化氮（NO）的作用。方法和结果：人真皮微血管内皮细胞、人脐静脉内皮细胞和SV 40转化的大鼠肺血管内皮细胞在ATRA（1 mol/L）存在或不存在的情况下孵育48小时。他们的NO生产测定与使用荧光NO指示剂，二氨基荧光素-2二乙酸酯。ATRA显着增加他们的基础以及乙酰胆碱诱导的NO产生。用N-硝基-L-精氨酸甲酯或羧基-PTIO处理抑制它们的荧光。通过与视黄酸受体（RAR）激动剂Am 580孵育也观察到NO产生的增加。与RAR拮抗剂LE 540共孵育可阻断ATRA诱导的NO升高。此外，NO的增加被磷酸肌醇3-激酶（PI 3 K）抑制剂wortmannin和LY 294002完全抑制。ATRA以及Am 580增强内皮NO合酶（eNOS）在Ser-1177的磷酸化以及Akt在Ser-473的磷酸化而不改变其蛋白表达。过表达显性失活Akt抑制eNOS磷酸化。ATRA还可增加PI 3 K活性和PI 3 K催化亚基p110β蛋白表达，而LE 540可完全抑制ATRA的作用。实时荧光定量聚合酶链反应（PCR）结果显示，ATRA可增加PI 3 K催化亚基p110β mRNA的表达，但不影响其稳定性。最后，在转染野生型eNOS和RARα的COS-1细胞中观察到ATRA诱导的NO增加，但未观察到Ser-1177被取代的突变型eNOS。结论：ATRA通过激活RAR介导的PI 3 K/Akt通路，使内皮细胞eNOS磷酸化，增加NO的产生，可能对血管内皮细胞具有保护作用。因此，类维生素A可能是候选人作为新的治疗药物对血管疾病的内皮损伤。
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 mol/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 p110β protein expression, which was completely inhibited by LE540 treatment. Real-time polymerase chain reaction analyses demonstrated that ATRA increased PI3K catalytic subunit p110β mRNA expression without affecting its stability. Finally, ATRA-induced NO increase was observed in COS-1 cells transfected with wild-type eNOS and RARα, 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.