Estradiol counteracts oxidized LDL-induced asymmetric dimethylarginine production by cultured human endothelial cells.
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.


CONCLUSIONS
Estradiol completely reverses the effects induced by oxLDL on the DDAH/ADMA/NO pathway.
Monsalve
Elena
E
Department of Physiology, University of Valencia, Spain.
Oviedo
Pilar J
PJ
García-Pérez
Miguel Angel
MA
Tarín
Juan J
JJ
Cano
Antonio
A
Hermenegildo
Carlos
C
D000328:Adult; D000581:Amidohydrolases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000704:Analysis of Variance; D001120:Arginine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular; D004958:Estradiol / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008077:Lipoproteins, LDL / Q000378:metabolism / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D008077:Lipoproteins, LDL; D012333:RNA, Messenger; C487735:dimethylarginine; C099252:oxidized low density lipoprotein; D009569:Nitric Oxide; D004958:Estradiol; D001120:Arginine; D019001:Nitric Oxide Synthase; D000581:Amidohydrolases; C059999:dimethylargininase