Genotype-dependent expression of endothelial nitric oxide synthase (eNOS) and its regulatory proteins in cultured endothelial cells.
DNA polymorphisms in endothelial nitric oxide synthase (eNOS) gene have been shown to be associated with constitutive eNOS expression and coronary artery disease (CAD). In the present study we explored the hypothesis whether genotype-dependent effects can be maintained in vitro during replication, or the effect is conditional on in vivo biological environments. Human umbilical vein endothelial cells (HUVEC) were collected and cultured from 89 normal deliveries of Mexican Americans. The cells were treated with or without cigarette smoking extracts (CSE) and genotypes of eNOS polymorphisms were determined by PCR. We measured the levels of eNOS by ELISA and its binding proteins including heat-shock protein 90 (Hsp-90) and caveolin-1 by Western blotting. The rare C allele for the promoter T786C polymorphism (0.2), and the rare 4 x 27-bp repeat allele in the intron 4 (0.30) were different from those reported in other populations. Yet, the rare T allele in the exon 7 (G894T polymorphism) was similar as others. After four passages in vitro, both the intron 4 and promoter polymorphisms maintained significant effects on eNOS mRNA levels in HUVECs (P < 0.05). However, the effects on eNOS protein and enzyme activity were less consistent. Although primary smokers had significantly lower eNOS protein levels (P < 0.05), the in vitro CSE treatment on cultured HUVECs only resulted in a significant reduction in NO levels as measured by the stable metabolites of nitrite/nitrate (P < 0.001). Neither Hsp-90 nor caveolin-1--important eNOS regulators--appears to mediate the genotypesmoking effects on eNOS expression although HUVECs did produce more Hsp-90 when exposed to CSE. Our study demonstrates that endothelial cells maintain genotype-dependent expression even after the deprivation of in vivo environment. However, the cigarette smoking-genotype interaction may require such in vivo conditions to be manifested.
Senthil
Duraisamy
D
Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas 77030, USA.
Raveendran
Muthuswamy
M
Shen
Ying H
YH
Utama
Budi
B
Dudley
Donald
D
Wang
Jian
J
Wang
Xing Li
XL
D000704:Analysis of Variance; D015153:Blotting, Western; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002478:Cells, Cultured; D017931:DNA Primers; D042783:Endothelial Cells / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics*; D005838:Genotype; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D017058:Mexican Americans; D009154:Mutation / Q000235:genetics; D009566:Nitrates / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D009573:Nitrites / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000378:metabolism*; D013638:Tars / Q000633:toxicity; D014471:Umbilical Veins / Q000166:cytology*
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D017931:DNA Primers; D018841:HSP90 Heat-Shock Proteins; D009566:Nitrates; D009573:Nitrites; D012333:RNA, Messenger; D013638:Tars; C024746:tobacco tar; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III