Mustafa AKKİPRİK, Duygu ÇEVİK, Ayşe ÖZER, Kaya ERMERK

Homosisteinin insan göbek kordon ven endotel hücre kültüründe enos ve ddah gen ekspresyonları üzerine etkisi

Amaç: Artmış plazma homosistein seviyeleri, miyokard enfarktüsü, felç ve periferik vasküler hastalıklarda bağımsız bir risk faktördür. Bu çalışmada, homosisteinin nitrik oksit (NO) sentezini, biyoyararlanımını ve yıkımını nasıl etkilediğini ortaya koymak amacı ile endotel hücre kültüründe endotelyal nitrik oksit sentaz (eNOS) ve dimetilarjinin dimetilaminohidrolaz (DDAH) gen ekspresyonları ile NO düzeyleri araştırılmıştır. Yöntem: Çalışmada insan göbek kordon ven endotel hücre kültürü (HUVEC) yapılmış ve kültür hücreleri 10, 50, 100, 500 ve 1000 µM homosistein konsantrasyonları ile 24 saat inkübe edilmiştir. Daha sonra bu hücrelerden (~1x106 hücre) total RNA izolasyonu yapılarak ters transkriptaz (RT) reaksiyonu ile komplementer DNA (cDNA) eldesi sağlanmıştır. Elde edilen cDNA örnekleri eNOS ve DDAH transkriptlerine özgü primerler kullanılarak polimeraz zincir reaksiyonu (PZR) yöntemi ile çoğaltılmıştır. PZR ürünlerinin agaroz jel elektroforezi sonrasında dijital jel görüntüleme sistemi ile dansitometrik olarak ölçümleri yapılmıştır. Ayrıca hücre kültürü medyumlarından elektrokemilüminesans yöntemi ile NO analizleri yapılmıştır. Bulgular: Elde edilen veriler ışığında, homosisteinin endotel hücre kültüründe eNOS ve DDAH gen ekspresyonları üzerine istatistiksel anlamlılık yaratacak herhangi bir etkisinin olmadığı gösterilmiştir. Buna karşın homosisteinin NO düzeyine hafif bir baskılayıcı etkisi olduğu görülmüştür. Sonuç: Sonuç olarak, homosisteinin NO üzerine etkisinin eNOS ve DDAH gen ekspresyonları düzeyinde olmadığı bu nedenle özellikle sübstrat düzeyinde denetim mekanizmaları üzerine yoğunlaşılması gerektiği düşünülmektedir.

Effects of homocysteine on enos and ddah gene expression levels in primary human umbilical endothelial cell culture

Aim: Elevated plasma homocysteine levels is an independent risk factor for myocardial infarction, stroke and peripheral vascular disease. Aim of this study is to investigate effects of homocysteine on nitric oxide (NO) synthesis, bioavailability and degradation by analysis gene expression levels of endothelial nitric oxide synthase (eNOS) and dimethylarginine dimethylaminohydrolase (DDAH) and also NO levels in endothelial cell line. Material and Methods: Human umbilical vein endothelial cell culture (HUVEC) is done and incubated with various homocysteine concentrations (10µM, 50µM, 100µM, 500µM, 1000µM). After that, total RNA extracted from these cells and converted to cDNA. These cDNA samples are amplified with PCR for eNOS and DDAH genes. Then the products are run in agarose gel electrophoresis and analysed for densitometric measurement by gel imaging system. In addition, NOx is analysed in cell culture mediums with electrochemiluminesans method. Results: All these studies show that homocysteine has no statistically significant effect on eNOS and DDAH gene expressions in endothelial cell culture. On the other hand, homocysteine has a minor suppressive effect on NOx levels. Conclusion: These data suggest that the effect of homocysteine on NO levels is not through gene expression levels of eNOS and DDAH but it is at substrate level.

PDF

___

1) Finkelstein JD. Homocysteine: a history in progress. Nutrition Reviews 2000; 58: 193-204.
2) Hofmann MA, Lalla E, Lu Y, Gleason MR, Wolf BM, Tanji N, Ferran LJ Jr, Kohl B, Rao V, Kisiel W, Stern DM, Schmidt AM. Hyperhomocysteinemia enhances vascular inflammation and accelerates atherosclerosis in a murine model. J Clin Invest. 2001; 107: 675-683.
3) Stamler JS, Osborne JA, Jaraki O, Rabbani LE, Mullins M, Singel D, Loscalzo J. Adverse vascular effects of Homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen. J Clin Invest 1993; 91: 303-318.
4) Zhang XH. Effects of homocysteine on endothelial NO production. Am. J. Physiol.-Renal Physiol 2000; 279: F671-F678.
5) Tran CT, Leiper JM, Vallance P. The DDAH/ADMA/NOS pathway. Atheroscler Suppl 2003; 4: 33-40.
6) Vallance P, Leone A, Calver A, et al. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992; 339: 572-575.
7) MacAllister RJ, Parry H, Kimoto M, Ogawa T, Russell RJ, Hodson H, et al. Regulation of nitric oxide synthesis by dimethylaminohydrolase. Br J Pharmacol 1996; 119: 1533-1540.
8) Ito A, Tsao PS, Adimoolam S, Kimoto M, Ogawa T, and Cookie JP. Novel mechanism for endothelial dysfunction: Dysregulation of dimethylarginine dimethylaminohydrolase. Circulation 1999; 99: 3092-3095.
9) Jaffe EA.: Culture and identification of large vessel endothelial cells. In biology of Endothelial cells.Ed. EA Jaffe, Martinus Nijhoff Publishers, Boston, p 1-11, 1984.
10) Harker LA, Ross R, Slichter SJ, Scott CR. Homocystine-induced arteriosclerosis. The role of endothelial cell injury and platelet response in its genesis. J Clin Invest. 1976; 58: 731-741.
11) Wang G, O K. Homocysteine stimulates the expression of monocyte chemoattractant protein-1 receptor (CCR2) in human monocytes: possible involvement of oxygen free radicals. Biochem J. 2001; 357: 233-240.
12) Upchurch GR Jr, Welch GN, Fabian AJ, Pigazzi A, Keaney JF Jr, Loscalzo J. Stimulation of endothelial nitric oxide production by homocyst(e)ine. Atherosclerosis 1997; 132: 177-185.
13) Welch GN, Upchurch GR Jr, Farivar RS, Pigazzi A, Vu K, Brecher P, Keaney JF Jr, Loscalzo J. Homocysteine-induced nitric oxide production in vascular smooth-muscle cells by NF-kappa B-dependent transcriptional activation of Nos2. Proc Assoc Am Physicians 1998; 110: 22-31.
14) Ikeda U, Ikeda M, Minota S, Shimada K. Homocysteine increases nitric oxide synthesis in cytokine-stimulated vascular smooth muscle cells. Circulation 1999; 99: 1230-1235.
15) Wang S, Wright G, Harrah J, Touchon R, McCumbee W, Geng W, Fultz ME, Abdul-Jalil MN, Wright GL. Short-term exposure to homocysteine depresses rat aortic contractility by an endothelium-dependent mechanism. Can J Physiol Pharmacol 2000; 78: 500-506.
16) Chow K, Cheung F, Lao TT, O K. Effect of homocysteine on the production of nitric oxide in endothelial cells. Clin Exp Pharmacol Physiol 1999; 26: 817-818.
17) Starkebaum G, Harlan JM. Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest 1986; 77: 1370-1376.
18) Radi R, Beckman JS, Bush KM, Freeman BA. Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J Biol Chem 1991; 266: 4244-4250.
19) Jin L, Abou-Mohamed G, Caldwell RB, Caldwell RW. Endothelial cell dysfunction in a model of oxidative stress. Med Sci Monit 2001; 7: 585-591.
20) Tyagi SC, Smiley LM, Mujumdar VS. Homocyst(e)ine impairs endocardial endothelial function. Can J Physiol Pharmacol 1999; 77: 950-957.
21) Stuhlinger MC, Tsao PS, Her JH, Kimoto M, Balint RF, Cooke JP. Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine. Circulation 2001; 104: 2569-2575.