Protective Effects of Resveratrol on TNF-α-Induced Endothelial Cytotoxicity in Baboon Femoral Arterial Endothelial Cells

Abstract

Endothelial injury induced by inflammatory factors plays a critical role in the pathogenesis of cardiovascular disease. Endothelial cell (EC) apoptosis, proliferation, migration, and cellular adhesion molecule (CAM) expression contribute to the development of atherosclerosis. We investigated the effects of resveratrol (0.1-100 μ M) on the proliferation, migration, and CAM expression of primary cultures of baboon arterial endothelial cells (BAECs). In addition, we tested its effects under normal conditions as well as under inflammatory conditions induced by tumour necrosis factor-α (TNF-α) administered either by cotreatment, pretreatment, or posttreatment. Immunocytochemistry, MTT, wound-healing, and flow cytometry assays were performed. The resveratrol treatment significantly enhanced BAEC proliferation and attenuated TNF-α-induced impairment of proliferation at the optimal doses of 1-50 µM. Resveratrol at a high dose (100 μ M) and TNF-α impaired BAEC migration, while low doses of resveratrol (1-50 μ M) attenuated TNF-α-induced impairment of BAEC migration. Moreover, resveratrol inhibited TNF-α-induced ICAM-1 and VCAM-1 expression. Taken together, our results suggest that the resveratrol protects BAECs after inflammatory stimulation as well as ameliorates inflammatory effects at low concentrations. Consequently, resveratrol should be considered as a candidate drug for the prevention and treatment of inflammatory vascular diseases.

Figure 1

Morphology of primary baboon ECs cultured at day 3 (a) and day 7 (b). Isolated baboon femoral artery ECs showed a typical cobble stone shape (magnification ×40). Cells were stained with specific endothelial cell surface markers for VE-cadherin (c) and vWF (d) (magnification ×100). Immunofluorescence staining of baboon ECs with antibody to VE-cadherin and von Willebrand factor was conducted using an FITC-labelled secondary antibody. DAPI was used to stain the nuclei. Images were taken with an Eclipse E800 microscope.

Figure 2

Effect of resveratrol on BAEC proliferation with or without TNF-α for 24 h. Resveratrol (0.1–100 μM) highly significantly enhanced EC proliferation after 24 h incubation in the optimal range of 10–50 μM (a). TNF-α (10 ng/mL) inhibited endothelial cell proliferation, while cotreatment with resveratrol (1–50 μM) reversed inhibition by TNF-α (b). BAECs were pretreated with resveratrol for 24 h then treated with TNF-α for 4 h; pretreatment with resveratrol ameliorated TNF-α-induced inhibition (c). BAECs were treated with TNF-α for 4 h then incubated with resveratrol for 24 h; posttreatment with resveratrol ameliorated TNF-α-induced inhibition (d). Data are expressed as a percentage of basal value (100%) and are the mean ± SEM from seven independent experiments, each conducted in quintuplicate. *P < 0.05, **P < 0.01 versus control and ^& P < 0.05; and ^&& P < 0.01 versus TNF-α alone.

Figure 3

Effect of resveratrol on baboon endothelial cell migration with or without TNF-α (10 ng/mL) for 15 h. 10 μM resveratrol significantly increased BAEC migration, and 100 μM resveratrol significantly decreased BAEC migration (a). TNF-α administration (b) decreased BAEC migration by comparison with controls. Resveratrol (1–50 μM) attenuated impairment of BAEC migration by TNF-α when during incubation for 15 h (b). Data are the mean ± SEM from five independent experiments; each conducted in triplicate. *P < 0.05; and **P < 0.01 versus control, ^& P < 0.05; and ^&& P < 0.01 versus TNF-α alone.

Figure 4

Resveratrol inhibited CAM expression induced by TNF-α (10 ng/mL). TNF-α significantly stimulated ICAM (CD54) and VCAM (CD106) expression after 4 H (a) and 24 h (b) treatment and significantly increased E-selectin (CD62e) expression after 4 h. Resveratrol (RSV) (10, 50 μM) inhibited expression of ICAM and VCAM in activated BAECs stimulated with TNF-α (10 ng/mL) during 24 h of cotreatment (b). Resveratrol (10, 50 μM) administered pretreatment (c) also significantly inhibited the expression of ICAM and VCAM activated by TNF-α, and significantly attenuated sustained ICAM and VCAM expression after TNF stimulation by posttreatment (d). Data are mean ± SEM from three independent experiments each performed in triplicate. *P < 0.05; and **P < 0.01 versus control and ^& P < 0.05; and ^&& P < 0.01 versus TNF-α alone.