Reduction of monocyte chemoattractant protein-1 and interleukin-8 levels by ticlopidine in TNF-alpha stimulated human umbilical vein endothelial cells

Abstract

Atherosclerosis and its associated complications represent major causes of morbidity and mortality in the industrialized or Western countries. Monocyte chemoattractant protein-1 (MCP-1) is critical for the initiating and developing of atherosclerotic lesions. Interleukin-8 (IL-8), a CXC chemokine, stimulates neutrophil chemotaxis. Ticlopidine is one of the antiplatelet drugs used to prevent thrombus formation relevant to the pathophysiology of atherothrombosis. In this study, we found that ticlopidine dose-dependently decreased the mRNA and protein levels of TNF-alpha-stimulated MCP-1, IL-8, and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Ticlopidine declined U937 cells adhesion and chemotaxis as compared to TNF-alpha stimulated alone. Furthermore, the inhibitory effects were neither due to decreased HUVEC viability, nor through NF-kB inhibition. These results suggest that ticlopidine decreased TNF-alpha induced MCP-1, IL-8, and VCAM-1 levels in HUVECs, and monocyte adhesion. Therefore, the data provide additional therapeutic machinery of ticlopidine in treatment and prevention of atherosclerosis.

Figure 1

Reduction of MCP-1 mRNA and protein levels by ticlopidine in HUVECs. HUVECs were incubated with serum-free medium for 12 hours before different concentrations of ticlopidine were added, after 12 hours incubation cells were stimulated with/without TNF-α (10 ng/mL) for another 24 hours. (a) Cell growth was analyzed by MTT. Results are mean ± SEM (n = 3). (b) Total RNA was extracted and analyzed by RT-PCR. Lane 1, without TNF-α; lane 2, TNF-α (10 ng/mL) alone; lane 3, TNF-α + ticlopidine (0.1 μg/mL); lane 4 TNF-α + ticlopidine (30 μg/mL); M denotes molecular size marker. The lower panel is β-actin as internal control. Results are representative of one of three independent experiments. (c) Relative amount of MCP-1 mRNA level was determined by quantitative real-time RT-PCR. (d) Culture supernatants were analyzed by ELISA. Data are expressed as the mean ± SEM of duplicate wells and are representative of five individual experiments. Significantly different versus TNF-treated alone *P < .05, **P < .001.

Figure 2

Reduction of IL-8 mRNA and protein levels by ticlopidine in HUVECs. HUVECs were incubated with serum-free medium for 12 hours before different concentrations of ticlopidine were added, after 12 hours incubation cells were stimulated with/without TNF-α (10 ng/mL) for another 24 hours. (a) Total RNA was extracted and analyzed by RT-PCR. Lane 1, without TNF-α; lane 2, TNF-α (10 ng/mL) alone; lane 3, TNF-α + ticlopidine (0.1 μg/mL); lane 4 TNF-α + ticlopidine (30 μg/mL); M denotes molecular size marker. The lower panel is β-actin as the internal control. Results are representative one of three independent experiments. (b) Relative amount of IL-8 mRNA level was determined by quantitative real-time RT-PCR. (c) Culture supernatants were analyzed by ELISA. Data are expressed as the mean ± SEM of duplicate wells and are representative of six individual experiments. Significantly different versus TNF-treated alone*P < .05, **P < .001.

Figure 3

Reduction of VCAM-1 mRNA and protein levels by ticlopidine in HUVECs. HUVECs were incubated with serum-free medium for 12 hours before different concentrations of ticlopidine were added, after 12 hours incubation cells were stimulated with/without TNF-α (10 ng/mL) for another 24 hours. (a) Relative amount of VCAM-1 mRNA level was determined by quantitative real-time RT-PCR. (b) Culture supernatants were analyzed by ELISA. Data are expressed as the mean ± SEM of duplicate wells and are representative of three individual experiments. Significantly different versus TNF-treated alone *P < .05, **P < .001.

Figure 4

Reduction of monocyte (U937) adhesion by ticlopidine. HUVECs were incubated with serum-free medium for 12 hours before different concentrations of ticlopidine were added, after 12 hours incubation cells were stimulated with/without TNF-α (10 ng/mL) for another 24 hours. Adhesion of fluorescence-labeled U937 cells was determined by adhesion assay. Data are expressed as the mean ± SEM of five individual experiments. Significantly different versus TNF-treated alone *P < .05, **P < .001.

Figure 5

Reduction of U937 cells chemotaxis by ticlopidine. HUVECs were incubated at the upper wells of the transwell plates until confluency, then different concentrations of ticlopidine were added. After 30 minutes of incubation, cells were stimulated with/without TNF-α (10 ng/mL). U937 cells were added to the upper chamber wells and incubated for 1.5 hours. The cells chemotastic to the lower wells were counted. Data are expressed as the mean ± SEM of three individual experiments. Significantly different versus TNF-treated alone *P < .05, **P < .001.

Figure 6

Ticlopidine has no effect on NF-kB activation. HUVECs were incubated with different concentrations of ticlopidine, and 100 μg/mL of aspirin was added as the control. Cells were stimulated with/without TNF-α (10 ng/mL) for 30 minutes, nuclear and cytosolic proteins were collected. Briefly, the nuclear protein extracts were collected, and western blots were performed using anti-NFkB p65 and anti-B23 Ab (a). Cytosolic protein extracts were collected, and western blots were performed using anti-Ik-B-α and α-tubulin Ab (b). Lane 1, without TNF-α; lane 2, TNF-α (10 ng/mL) alone; lane 3, TNF-α + aspirin (100 μg/mL); lane 4, TNF-α + ticlopidine (1 μg/mL); lane 5, TNF-α + ticlopidine (10 μg/mL); lane 6, TNF-α + ticlopidine (30 μg/mL). Results are representative of one of three independent experiments.