ABSTRACT
Intracellular renin-angiotensin system (RAS) can operate independently of the circulating RAS. Estrogens provide protective effects by modulating the RAS. Our aim was to investigate the effect of estradiol (E2) on angiotensin converting enzymes (ACE) 1 and ACE2 expression and activities in human endothelial cells (HUVEC), and the role of estrogen receptors (ER). The results confirmed the presence of active intracellular RAS in HUVEC. Physiological concentrations of E2 induced a concentration-dependent increase of ACE1 and ACE2 mRNA expression and ACE1, but not ACE2, protein levels. ACE1 and ACE2 enzymatic activities were also induced with E2. These effects were mediated through ERα activation, since ER antagonists ICI 182780 and MPP completely abolished the effect of E2. Moreover, the ERα agonist PPT mirrored the E2 effects on ACE1 and ACE2 protein expression and activity. Exposure of endothelial cells to E2 significantly increased Ang-(1-7) production. In conclusion, E2 increases Ang-(1-7) production, through ERα, involving increased ACE1 and ACE2 mRNA expression and activity and ACE1 protein levels.
Subject(s)
Angiotensin I/metabolism , Estradiol/pharmacology , Estrogen Receptor alpha/metabolism , Peptide Fragments/metabolism , Peptidyl-Dipeptidase A/genetics , Angiotensin-Converting Enzyme 2 , Dose-Response Relationship, Drug , Estradiol/analogs & derivatives , Estrogen Receptor Antagonists/pharmacology , Fulvestrant , Gene Expression Regulation/drug effects , Human Umbilical Vein Endothelial Cells , Humans , Peptidyl-Dipeptidase A/metabolism , Piperidines/pharmacology , Pyrazoles/pharmacologyABSTRACT
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase. ADMA accumulation, mainly due to a decreased dimethylarginine dimethylaminohydrolase (DDAH) activity, has been related to the development of cardiovascular diseases. We investigate whether estradiol prevents the changes induced by oxidized low density lipoprotein (oxLDL) on the DDAH/ADMA/NO pathway in human umbilical artery endothelial cells (HUAEC). HUAEC were exposed to estradiol, native LDL (nLDL), oxLDL and their combinations for 24 h. In some experiments, cells were also exposed to the unspecific estrogen receptor (ER) antagonist ICI 182780, the specific ERα antagonist MPP or specific agonists for ERα, ERß and GPER. ADMA concentration was measured by HPLC and concentration of NO by amperometry. Protein expression and DDAH activity were measured by immunoblotting and an enzymatic method, respectively. oxLDL, but not nLDL, increased ADMA concentration with a concomitant decrease on DDAH activity. oxLDL reduced eNOS protein and NO production. Estradiol alone had no effects on DDAH/ADMA/NO pathway, but increased the attenuated endothelial NO production induced by oxLDL by reduction in ADMA and preventing loss of eNOS protein levels. ICI 182780 and MPP completely abolished these effects of estradiol on oxLDL-exposed cells. ERα agonist, but not ERß and GPER agonists, mirrored estradiol effects on NO production. In conclusion, estradiol restores (1) DDAH activity, and therefore ADMA levels, and (2) NO production impaired by oxLDL in HUAEC acting through ERα.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Endothelium, Vascular/metabolism , Estradiol/metabolism , Estrogen Receptor alpha/metabolism , Lipoproteins, LDL/antagonists & inhibitors , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide/agonists , Amidohydrolases/chemistry , Amidohydrolases/metabolism , Arginine/adverse effects , Arginine/analogs & derivatives , Arginine/antagonists & inhibitors , Arginine/metabolism , Blotting, Western , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Estradiol/agonists , Estradiol/chemistry , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha/agonists , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor beta/agonists , Estrogen Receptor beta/antagonists & inhibitors , Estrogen Receptor beta/metabolism , Estrogens, Non-Steroidal/pharmacology , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/metabolism , Lipoproteins, LDL/adverse effects , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/antagonists & inhibitors , Nitric Oxide Synthase Type III/chemistry , Protein Stability , Protein-Arginine N-Methyltransferases/metabolism , Receptors, Estrogen/agonists , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/metabolism , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/metabolism , Repressor Proteins/metabolism , Umbilical Arteries/cytologyABSTRACT
OBJECTIVE: Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, is a novel cardiovascular risk factor produced by endothelial cells. ADMA levels are mainly regulated by the activity of dimethylarginine dimethylaminohydrolases (DDAH). Endothelial release of ADMA is increased in the presence of oxidized LDL cholesterol (oxLDL), whereas estrogens stimulate NO production by endothelial cells by increasing both expression and activity of NO synthase and by reducing ADMA levels. Thus, the aim of the present study was to evaluate the estradiol effects on the DDAH/ADMA/NO pathway in cultured human umbilical vein endothelial cells (HUVEC) exposed to LDL. METHODS: After 24 h of exposure to various treatments, culture medium was collected to measure NO production by using an amperometric sensor specific for NO, and to measure dimethylarginines by high-performance liquid chromatography (HPLC). DDAH-I and II mRNA expression and protein content were quantified by real-time PCR assay and immunoblotting, respectively. RESULTS: Exposure of HUVEC to 100 microg/mL oxLDL, but not to 100 microg/mL of native LDL (nLDL), reduced DDAH-II expression at both the mRNA as well as the protein levels, which in turn increased ADMA levels and reduced NO production. Estradiol (1 nM) alone increased DDAH-II mRNA and protein expression, which reduced ADMA levels and increased NO production. In cells exposed to estradiol in combination with either nLDL or oxLDL, levels of DDAH-II, ADMA, and NO were the same as those for estradiol alone. CONCLUSION: Estradiol completely reverses the effects induced by oxLDL on the DDAH/ADMA/NO pathway.
