Publication: İnsan göbek kordon ven endotel hücre kültüründe homosisteinin enzim ekspresyonu üzerine etkisi: eNOS ve DDAH
Abstract
Homosistein, metiyonin metaboliti olarak bulunan, sülfür taşıyan bir amino asittir. Plazmada birçok formda bulunur: % 70-80 proteine bağlı, % 20-30 disülfid ve çok az bir miktarıda serbest ve indirgenmiş halde bulunur. Homosistein ya transsülfürasyon yolu ile sistatiniyonine sonra da sisteine katabolize olur ya da metiyonin haline yeniden metillenir. Homosisteininin metillenmesi, folat ve kobalamin bağımlı bir enzim olan metiyonin sentaz tarafından yapılır. Artmış plazma homosistein seviyeleri miyokard enfarktüsü, felç, demans ve periferik vasküler hastalıklarda bağımsız bir risk faktörü olarak belirtilmiştir. Folik asit, vitamin B6 ve vitamin B12 gibi kofaktörlerin ve sistatiniyon - sentaz ve 5,10- metilentetrahidrofolat redüktaz gibi metabolizmasında yer alan değişik enzimlerin eksiklikleri plazma homosistein düzeyinin artmasına yol açmaktadır. Homosistein vasküler fonksiyonu bozarak, aterosklerozda komplikasyon riskini artırır. Hiperhomosisteineminin ateroskleroza yol açma mekanizmalarından bir tanesi de NO (Nitrik Oksit) üzerine etkileridir. Endotelyal NO vazodilatatördür ve anti-trombosit etkileri vardır. Hiperhomosisteinemide endotel bağımlı relaksasyonun bozulmasının NO'nun bioyararlanımının azalması sonucunda olduğu söylenmektedir. Biyoyararlanımın azalmasının nedeninin üretimin azalması, degradasyonun artması ya da nitrozotiyol türevlerinin oluşması olduğu düşünülmektedir. Homosisteinin NO üretimini etkilediği bilinen bir konu olmasına rağmen mekanizması tam olarak anlaşılamamıştır. Bu çalışmada homosisteinin NO sentezini nasıl etkilediği araştırılmıştır. İnsan göbek kordon ven endotel hücre kültürü (HUVEC) yapılmış ve kültür hücreleri değişik homosistein konsantrasyonları (10µM, 50µM, 100µM, 500µM ve1000µM) ile inkübe edilmiştir. Daha sonra bu hücrelerden elde edilen RNA'lar cDNA'ya dönüştürülmüştür. Bu cDNA örnekleri eNOS ve dimetilarjinin dimetilaminohidrolaza (DDAH) özgü primerler kullanılarak polimeraz zincir reaksiyonu (PZR) yöntemi ile çoğaltılmıştır. PZR ürünleri daha sonra agaroz jel elektroforezi ve Kodak jel görüntüleme sistemi ile incelenmiştir. Ayrıca hücre kültürü medyumlarından elektrokemilüminesans yöntemi ile NO analizleri yapılmıştır. Yapılan incelemeler homosisteinin eNOS ve DDAH'nin gen ekspresyonları üzerine herhangi bir etkisi olmadığını göstermiştir. NO düzeylerine ise hafif bir baskılayıcı etkisi olduğu görülmüştür. Elde edilen bu veriler homosisteinin NO üzerine etkisinin gen ekspresyonu düzeyinde olmadığını göstermektedir. II.
Homocysteine (HCy) is a naturally occuring, sulphur-containing amino acid , that appears in the blood as a metabolite of methionine. HCy is formed in the liver and other tissues as a response to the intake of methionine, which in itself is derived from protein-rich foods. In the plasma: 70-80% of HCy is protein bound, 20-30% is disulfide, and small amount is free i.e. reduced. Homocysteine may either be catabolised in the transsulfuration pathway via cystathionine to cysteine or remetylated back to methionine mainly by the folate and cobalamine dependent enzyme methionine synthase. Elevated plasma homocysteine levels are independent risk factor for myocardial infarction, stroke, alzeimer and peripheral vascular disease. In deficiency of folic acid, vitamin B6, vitamin B12, cystatinione - synthase and 5,10-methylenetetrahydrofolatreductase the plasma homocysteine levels are elevated. Hyperhomocysteinemia may alter vascular function in a way that increases risk for complications of atherosclerosis. Hyperhomocysteinemia have effects on the NO modulated pathways. Endothelial NO is a vasodilator and has antitrombolytic effects. Impairment of endothelium-dependent relaxation in hyperhomocysteinemia appears to be mediated in part by decreased bioavailability of endothelium-derived NO, which may be caused either by decreased production or increased degradation of NO or the formation of nitrosothiol derivatives. Expression of eNOS does not appear to be decreased during hyperhomocysteinemia, but production of NO by eNOS may be limited by endogenous inhibitors. It is known that homocysteine effects the NO production but the mechanism is not clear yet . In this study the effect of homocysteine on NO synthesis is analyzed. Primary human umbilical vein endothelial cells are incubated with various homocysteine concentrations (10µM, 50µM, 100µM, 500µM, ve1000µM). For 24 hours, mRNA extracted from these is converted to cDNA. Using primers specific for DNA sequences of eNOS and DDAH .These cDNA samples are amplified with polymerase chain reaction. Then the PCR products were run in agarose gel electrophoresis and analyzed with Kodak gel imaging system. In addition to these NO is assayed in the cell culture mediums using electrochemiluminesans method. The results show that homocysteine has no effect on eNOS and DDAH gene exspressions but has a minor depressive effect on NO levels.
Homocysteine (HCy) is a naturally occuring, sulphur-containing amino acid , that appears in the blood as a metabolite of methionine. HCy is formed in the liver and other tissues as a response to the intake of methionine, which in itself is derived from protein-rich foods. In the plasma: 70-80% of HCy is protein bound, 20-30% is disulfide, and small amount is free i.e. reduced. Homocysteine may either be catabolised in the transsulfuration pathway via cystathionine to cysteine or remetylated back to methionine mainly by the folate and cobalamine dependent enzyme methionine synthase. Elevated plasma homocysteine levels are independent risk factor for myocardial infarction, stroke, alzeimer and peripheral vascular disease. In deficiency of folic acid, vitamin B6, vitamin B12, cystatinione - synthase and 5,10-methylenetetrahydrofolatreductase the plasma homocysteine levels are elevated. Hyperhomocysteinemia may alter vascular function in a way that increases risk for complications of atherosclerosis. Hyperhomocysteinemia have effects on the NO modulated pathways. Endothelial NO is a vasodilator and has antitrombolytic effects. Impairment of endothelium-dependent relaxation in hyperhomocysteinemia appears to be mediated in part by decreased bioavailability of endothelium-derived NO, which may be caused either by decreased production or increased degradation of NO or the formation of nitrosothiol derivatives. Expression of eNOS does not appear to be decreased during hyperhomocysteinemia, but production of NO by eNOS may be limited by endogenous inhibitors. It is known that homocysteine effects the NO production but the mechanism is not clear yet . In this study the effect of homocysteine on NO synthesis is analyzed. Primary human umbilical vein endothelial cells are incubated with various homocysteine concentrations (10µM, 50µM, 100µM, 500µM, ve1000µM). For 24 hours, mRNA extracted from these is converted to cDNA. Using primers specific for DNA sequences of eNOS and DDAH .These cDNA samples are amplified with polymerase chain reaction. Then the PCR products were run in agarose gel electrophoresis and analyzed with Kodak gel imaging system. In addition to these NO is assayed in the cell culture mediums using electrochemiluminesans method. The results show that homocysteine has no effect on eNOS and DDAH gene exspressions but has a minor depressive effect on NO levels.