Sıçan Koroner Mikrovasküler Endotel Hücre Kültüründe Östrojenin Oluşturduğu RhoA Up-regülasyonu Üzerine ICI182,780, Progesteron ve Testosteronun Etkisi

Abstract

Abstract The Effects of ICI182,780, Progesterone and Testosterone on Estrogen Induced Upregulation of RhoA Expressions in The Rat Coronary Microvascular Endothelium Cell Culture Method: Wistar rats were used in the study. Coronary microvascular endothelial cells were obtained by the mounted rat hearts on a constant-flow Langendorff system. The cell suspensions were plated and incubated at 37oC under 5% CO2. Confluent flasks were passaged. The third passage coronary microvascular endothelial cells were made quiscent 24 hours before experimentation. Next, it was incubated with either 17β-estradiol (10-8 -10-7M), or its combination with ICI182, 780 (10-5M), progesterone (10-8 - 10-7M), and testosterone (10-8 - 10-7M) for 24 hrs. Afterwards, the cells in flasks were homogenized and RhoA protein enzyme expression levels were measured by Western-Blotting technique. Results: 17-β estradiol causes RhoA up-regulation that can not be blocked by progesterone and testosterone in rat coronary microvascular endothelial cell culture. Conclusion: Activation of Rho/Rho-kinase pathway might be responsible for the development of estrogen associated cardiovascular complications.

Keywords

• endothelium; 17β-estradiol; progesterone; rhoA; testosterone

Sıçan Koroner Mikrovasküler Endotel Hücre Kültüründe Östrojenin Oluşturduğu RhoA Up-regülasyonu Üzerine ICI182,780, Progesteron ve Testosteronun Etkisi

Abstract

Türkçe Özet: Amaç: Bu çalışmada sıçan koroner mikrovasküler endotel hücrelerinde tek başına estradiol ve estradiolün progesteron, testosteron ve estrojen reseptör α ve β blokörü ile kombinasyonlarının RhoA ekspresyonu üzerine etkileri Western blot analizi ile değerlendirildi. Yöntem: Çalışmada Wistar türü sıçanlar kullanıldı. Kalpler Langendorff kalp perfüzyon sistemine asılarak koroner mikrovasküler endotel hücreleri elde edildi. Hücreler kültür flasklarına ekilerek 37ºC\'de %5 CO2\'li ortamda inkübe edildi. Konfluent olan flasklar pasajlanarak çoğaltıldı. 3. pasaj koroner mikrovasküler endotel hücreleri, deneylerden 24 saat önce bölünmeleri durdurulup 17β-estradiol (10-8 ve 10-7M) tek başına veya 17β-estradiol testosteron (10-8 ve 10-7M), progesteron (10-8 ve 10-7M) ve ICI 182, 780 (10-5M), kombinasyonu ilave edilerek 24 saat inkübe edildi. İnkübasyon sonrası flasklardaki hücreler homojenize edilerek Western-Blot yöntemi ile RhoA protein enzim ekspresyon düzeyleri ölçüldü. Bulgular: 17β-estradiol sıçan koroner mikrovasküler endotel hücre kültüründe progesteron ve testosteron ile bloke edilemeyen RhoA up-regülasyonu neden olmaktadır. Sonuç: Sonuç olarak östrojene bağlı olarak gözlemlenen kardiyovasküler komplikasyonlardan Rho/Rho-kinaz yolağının aktivasyonu sorumlu olabilir.

Keywords

• endotel; 17β-estradiol; progesteron; rhoA; testosteron

References

1. Koledova VV. Khalil RA. Sex hormone replacement therapy and modulation of vascular function in cardiovascular disease. Expert Rev Cardiovcmc Ther 2007:5(4):777-89.
2. Kuhlickiene K. Luksha L. Gender and the Endothcliutn. Pimrmacoı'ugiml Rep 2t)08'.60( 1 ):49-60.
3. Rosano GM. Sarrel PM. Poole—Wilson PA. Collins P. Beneficial effect oi" oestrogen on exercise-induced myocardial ischacmia in women with coronary artery disease. umre: 1993:342(8864):133—41.
4. Hulley S. Grady D. Bush T. Furberg C, Herrington D. Riggs B. Vittinghoff E. Randomized trial of estrogen plus progesıin for secondary prevention of coronary heart disease in postmenopausal women. Heart and Estrogen/progestin Replacement Study (HERS) Research Group. JAM/lı 1998:280(7]:605-l3.
5. Angerer P. Stork S. Kothny W. Schmitt P. von Schacky C. Effect of oral pestntenopausal hormone replacement on progression of atherosclerosis: a randomized. controlled trial. Arrerioscler Tltromb Vasc Bir) 200i :21(2):262-8.
6. Orshal .fM. Khalil RA. Gender. sex hormones. and vascular tone. Am J Pin-'sini Regal luregr Camp Pltysml 2004ı286(2):233—49.
7. Aydilck N. Aksakal M. Effects of testosterone on lipid pcroxidation. lipid profiles and some coagulation parameters in rabbits. J Vet Men A Physio] Pollin Clin Med 2()05:52(9):436—9.
8. Hclcr RF. Jacobs HS. Androgens estrogen and coronary heart disease. Br MedJ 1981;232(6262):43879.

9. Ross R. Atheroscleros is an inşammatory disease. N Engl J Merll9991340(2):115-26.
10. Rubio AR. Morales—Segura MA. Nitric Oxide. an Iceberg in Cardiovascular Physiology: Far Beyond Vessel Tone Control. Art'lıt'eı’cr (if/Medical Research 2004:35( l ): 1-1 1.
11. Wojciak-Stothard B. Ridley A]. Rho GTPases and the regulation ol' endothelial permeability. Vortal Plîarnrcrml 20021396): 187-99.
12. van Nieuw Amerongen GP. Beckers CM. Achekaı' LD. Zeeman S. Musters RJ. van Hinsbergh VW. Involvement of Rho kinase in endothelial barrier maintenance. Arreriorcler Tltroml) Vasc Biol 200727“ | l:2332-9.
13. Somlyo AP. Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 1994;372(6503}:23l— 6.
14. Somlyo AP, Somlyo AV. Signal transduction by G— Proteins. rhowkinase and protein phosphatase to smooth muscle and non-muscle miyosin 11. J Pltyst'oı' (loud) 2000ı522(2l: 177—85.
15. Kanım KE and Stull .lT. The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Anım Rei" Pharmm'rıl Toxic“ 1985125:593-620. lo.
16. Fu XD. Flamini M. Sanchez AM. Goglia L.. Giretti MS.
17. Mosca L. Manson JE. Sutherland SE. Langer RD. Manolio
18. Shimokawa H. Morisltige K. Miyata K. Kandabashi T. Eto lw...) ’Jı
19. Brouchet L. Krust A. Dupont S. Chambon P. Bayard F.
20. Arnal 1F. Estradiol accelerates reendothelialization in
21. mouse carotid artery through estrogen receptor—alpha but
22. not estrogen receptor-beta. Circulation 200l;103(3):423— 28.
23. Bath P. Gray LJ. Association between hormone replacement
24. therapy and subsuquent stroke: a meta analysis. BMJ 2005ı33(l(7487):34.
25. Peverill RE. Hormone therapy and venous
26. thı'ombocmholism. Bast Prue Res Clin Endor-rim) Metal: 2003:1711):149-64.