TNF-α induces endothelial dysfunction via PKC-ζ-dependent NADPH oxidase activation

Abstract

Endothelial dysfunction is implicated in a variety of cardiovascular diseases although the detailed mechanisms are not yet completely understood. A relationship has been suggested to exist between inflammation and endothelial dysfunction. TNF-α serves as one of the most important pro-inflammatory cytokines. The main objectives of the present study were to explore the effect of PKC-ζ on TNF-α-impaired endothelial function as well as the underlying mechanisms. Acetylcholine-induced endothelium-dependent vasodilation of mouse thoracic aorta stimulated by TNF-α was initially determined. PKC-ζ deficient mice and the specific inhibitor of NADPH oxidase were respectively applied to elucidate their roles in TNF-α-induced endothelial dysfunction. In vitro superoxide generation in HAECs was detected by DHE staining after administration of TNF-α. Meanwhile, the regulatory p47^phox subunit of NADPH oxidase was evaluated by Western blotting and RT-PCR. The results showed that TNF-α conspicuously impaired endothelium-dependent vasodilation and the impairment was attenuated by either depleting PKC-ζ or inhibiting NADPH oxidase. In vitro TNF-α increased superoxide production and p47^phox expression in HAECs, and such increases could be ameliorated by the specific PKC-ζ inhibitor. Our findings suggest that superoxide over-production triggered by PKC-ζ-dependent NADPH oxidase activation contributes to TNF-α-induced endothelial dysfunction.

Keywords

endothelial dysfunction / TNF-α / PKC-ζ / NADPH oxidase

References

[1] FurchgottR.F., ZawadzkiJ.V.. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 1980, 288(5789): 373-376

[2] BaracA., CampiaU., PanzaJ.A.. Methods for evaluating endothelial function in humans. Hypertension, 2007, 49(4): 748-60

[3] FeletouM., VanhoutteP.M.. Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture). Am J Physiol Heart Circ Physiol, 2006, 291(3): H985-H1002

[4] StenvinkelP.. Endothelial dysfunction and inflammation-is there a link?. Nephrol Dial Transplant, 2001, 16(10): 1968-1971

[5] PoberJ.S., MinW.. Endothelial cell dysfunction, injury and death. Handb Exp Pharmacol, 2006, 176(Pt2): 135-156

[6] NapoliC.F., de Nigris, PalinskiW.. Multiple role of reactive oxygen species in the arterial wall. J Cell Biochem, 2001, 82(4): 674-682

[7] CaiH., HarrisonD.G.. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res, 2000, 87(10): 840-844

[8] TouyzR.M., SchiffrinE.L.. Reactive oxygen species in vascular biology: implications in hypertension. Histochem Cell Biol, 2004, 122(4): 339-352

[9] CaveA.C., BrewerA.C., NarayanapanickerA., et al.. NADPH oxidases in cardiovascular health and disease. Antioxid Redox Signal, 2006, 8(5–6): 691-728

[10] LiJ.M., ShahA.M.. Endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology. Am J Physiol Regul Integr Comp Physiol, 2004, 287(5): R1014-R1030

[11] FreyR.S., GaoX., JavaidK., et al.. Phosphatidylinositol 3-kinase gamma signaling through protein kinase Czeta induces NADPH oxidase-mediated oxidant generation and NF-kappaB activation in endothelial cells. J Biol Chem, 2006, 281(23): 16128-16138

[12] Thallas-BonkeV., ThorpeS.R., CoughlanM.T., et al.. Inhibition of NADPH oxidase prevents advanced glycation end product-mediated damage in diabetic nephropathy through a protein kinase C-alpha-dependent pathway. Diabetes, 2008, 57(2): 460-469

[13] FathiR., HaluskaB., IsbelN., et al.. The relative importance of vascular structure and function in predicting cardiovascular events. J Am Coll Cardiol, 2004, 43(4): 616-623

[14] CaballeroA.E.. Endothelial dysfunction, inflammation, and insulin resistance: a focus on subjects at risk for type 2 diabetes. Curr Diab Rep, 2004, 4(4): 237-246

[15] LusterM.I., SimeonovaP.P., GallucciR., et al.. Tumor necrosis factor alpha and toxicology. Crit Rev Toxicol, 1999, 29(5): 491-511

[16] GaoX., BelmadaniS., PicchiA., et al.. Tumor necrosis factor-alpha induces endothelial dysfunction in Lepr(db) mice. Circulation, 2007, 115(2): 245-254

[17] PicchiA., GaoX., BelmadaniS., et al.. Tumor necrosis factor-alpha induces endothelial dysfunction in the prediabetic metabolic syndrome. Circ Res, 2006, 99(1): 69-77

[18] De KeulenaerG.W., AlexanderR.W., Ushio-FukaiM., et al.. Tumour necrosis factor alpha activates a p22phox-based NADH oxidase in vascular smooth muscle. Biochem J, 1998, 329(Pt3): 653-657

[19] MuzaffarS., ShuklaN., AngeliniG., et al.. Nitroaspirins and morpholinosydnonimine but not aspirin inhibit the formation of superoxide and the expression of gp91phox induced by endotoxin and cytokines in pig pulmonary artery vascular smooth muscle cells and endothelial cells. Circulation, 2004, 10(9): 1140-1147

[20] FreyR.S., RahmanA., KeferJ.C., et al.. PKCzeta regulates TNF-alpha-induced activation of NADPH oxidase in endothelial cells. Circ Res, 2002, 90(9): 1012-1019

[21] DangP.M., FontayneA., HakimJ., et al.. Protein kinase C zeta phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst. J Immunol, 2001, 166(2): 1206-1213

[22] LiJ.M., MullenA.M., YunS., et al.. Essential role of the NADPH oxidase subunit p47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor-alpha. Circ Res, 2002, 90(2): 143-150

[23] NigroP., AbeJ., WooC.H., et al.. PKCzeta decreases eNOS protein stability via inhibitory phosphorylation of ERK5. Blood, 2010, 116(11): 1971-1979