Changes in human umbilical vein endothelial cells induced by endothelial nitric oxide synthase traffic inducer

Abstract

This study investigated the changes in human umbilical vein endothelial cells (HUVECs) induced by overexpression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor VIII-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the proliferative ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS (eNOS) activity was assayed by using spectrophotometry, and NO₂⁻/NO₃⁻ levels were measured using nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma membrane and cytoplasm, and Western blot analysis confirmed that NOSTRIN levels were significantly higher in cells transfected with the NOSTRIN plasmid (P<0.01). The activity of eNOS and the levels of NO₂⁻/NO₃⁻ were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells (P<0.01 and P<0.01, respectively). Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOSTRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overexpression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.

Keywords

human umbilical vein endothelial cell / nitric oxide synthase traffic inducer / nitric oxide synthase / nitric oxide

References

[1] RudicRD, SheselyEG, MaedaN, et al.. Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. J Clin Invest, 1998, 101(4): 731-736

[2] GonzalezE, KouR, LinAJ, et al.. Subcellular targeting and agonist-induced site-specific phosphorylation of endothelial nitric-oxide synthase. J Biol Chem, 2002, 277(42): 39 554-39 560

[3] MichelJB, ForonO, SaseK, et al.. Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase. J Biol Chem, 1997, 272(41): 25 907-25 912

[4] García-CardeñaG, FanR, SternDF, et al.. Endothelial nitric oxide synthase is regulated by tyrosine phosphorylation and interacts with caveolin-1. J Biol Chem, 1996, 271(44): 27 237-27 240

[5] García-CardeñaG, FanR, ShahV, et al.. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature, 1998, 392(6678): 821-824

[6] ZimmermannK, OpitzN, DedioJ, et al.. NOSTRIN: a protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase. Proc Natl Acad Sci USA, 2002, 99(26): 17 167-17 172

[7] XiangW, ChenH, XuX, et al.. Expression of endothelial nitric oxide synthase traffic inducer in the placentas of women with pre-eclampsia. Int J Gynaecol Obstet, 2005, 89(2): 103-107

[8] XiangW, ChenH, HuL, et al.. Endothelial nitric oxide synthase traffic inducer in the umbilical vessels of the patients with pre-eclampsia. J Huazhong Univ Sci Technol Med Sci, 2009, 29(2): 243-245

[9] FeronO, SaldanaF, MichelJB, et al.. The endothelial nitric oxide synthase -caveolin regulatory cycle. J Biol Chem, 1998, 273(6): 3125-3128

[10] PrabhakarP, ThatteHS, GoetzRM, et al.. Receptor-regulated translocation of endothelial nitric-oxide synthase. J Biol Chem, 1998, 273(42): 27 383-27 388

[11] JuH, ZouR, VenemaVJ, et al.. Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J Biol Cell, 1997, 8(4): 595-605

[12] SchillingK, OpitzN, WiesenthalA, et al.. Translocation of endothelial nitricoxide synthase involves a ternary complex with caveolin-1 and NOSTRIN. Mol Biol Cell, 2006, 17(9): 3870-3880

[13] MoncadaS, PalmerRM, HiggsEA. Nitric oxide: Physiology, pathophysiology and pharmacology. Pharmacol Rev, 1991, 43(2): 109-142

[14] VallanceP, CollierJ, MoncadaS. Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet, 1989, 2(8670): 997-1000

[15] CannonRO3rd. Role of nitric oxide in cardiovascular disease: focus on the endothelium. Clin Chem, 1998, 44(8Pt2): 1809-1819