In Vitro Anti-Inflammatory and Vasculoprotective Effects of Red Cell Extract from the Black Sea Urchin Arbacia lixula

Abstract

Sea urchins have emerged as an important source of bioactive compounds with anti-inflammatory and antioxidant properties relevant to human health. Since inflammation is a crucial pathogenic process in the development and progression of atherosclerosis, we here assessed the potential anti-inflammatory and vasculoprotective effects of coelomic red-cell methanolic extract of the black sea urchin Arbacia lixula in an in vitro model of endothelial cell dysfunction. Human microvascular endothelial cells (HMEC-1) were pretreated with A. lixula red-cell extract (10 and 100 μg/mL) before exposure to the pro-inflammatory cytokine tumor necrosis factor (TNF)-α. The extract was non-toxic after 24 h cell treatment and was characterized by antioxidant power and phenol content. The TNF-α-stimulated expression of adhesion molecules (VCAM-1, ICAM-1) and cytokines/chemokines (MCP-1, CCL-5, IL-6, IL-8, M-CSF) was significantly attenuated by A. lixula red-cell extract. This was functionally accompanied by a reduction in monocyte adhesion and chemotaxis towards activated endothelial cells. At the molecular level, the tested extract significantly counteracted the TNF-α-stimulated activation of the pro-inflammatory transcription factor NF-κB. These results provide evidence of potential anti-atherosclerotic properties of A. lixula red-cell extract, and open avenues in the discovery and development of dietary supplements and/or drugs for the prevention or treatment of cardiovascular diseases.

Keywords: NF-κB; adhesion molecule; atherosclerosis; chemokine; cytokine; endothelial dysfunction; gene expression; inflammation; monocyte adhesion; red cells; sea urchin.

Figure 1

Effect of A. lixula extract on endothelial cell viability. HMEC-1 were treated with A. lixula extract for 4 h at the concentrations indicated, and then either treated with 10 ng/mL TNF-α or left untreated for 18 h. (A) Cell viability was assessed by the MTT assay and data (means ± S.D., n = 3) expressed as percent of unstimulated control. In (B), representative phase-contrast images (10× magnification) of cells after treatments are shown. (a) control; (b) TNF-α 10 ng/mL; (c) A. lixula extract 10 µg/mL + TNF-α; (d) A. lixula extract 100 µg/mL + TNF-α.

Figure 2

Effect of A. lixula extract on TNF-α-induced endothelial cell–monocyte adhesion. HMEC-1 were treated with A. lixula extract for 4 h at the concentration indicated, and then either treated with 10 ng/mL TNF-α or left untreated for 18 h. THP-1 were added to the HMEC-1 monolayers. Images of HMEC-1 and adherent THP-1 cells were visualized and counted (A). Data (means ± S.D., n = 3) are expressed as number of adherent monocytes per field. In (B), images captured with a phase contrast microscope (10× magnification) are shown. (a) control; (b) TNF-α 10 ng/mL; (c) A. lixula extract 10 µg/mL + TNF-α; (d) A. lixula extract 100 µg/mL + TNF-α. *** p < 0.001 vs. basal (untreated) control; ## p < 0.01 vs. TNF-α alone.

Figure 3

Effect of A. lixula extract on TNF-α-induced expression of endothelial adhesion molecules. HMEC-1 were treated with A. lixula extract for 4 h at the concentration indicated, and then either treated with 10 ng/mL TNF-α or left untreated for 18 h. (A) mRNA levels of VCAM-1 and ICAM-1 were measured by qPCR. Data (means ± S.D., n = 3) are expressed as fold induction over basal (untreated) control. *** p < 0.001 vs. basal (untreated) control; # p < 0.05 vs. TNF-α alone; ## p < 0.01 vs. TNF-α alone. (B) Endothelial cell surface protein expression of VCAM-1 and ICAM-1 was assessed by EIA and expressed as percent of TNF-α. *** p < 0.001 vs. basal (untreated) control; ## p < 0.01 vs. TNF-α alone.

Figure 4

Effect of A. lixula extract on TNF-α-induced expression of inflammatory genes in human endothelial cells. HMEC-1 were treated with A. lixula extract for 4 h at the concentration indicated, and then either treated with 10 ng/mL TNF-α or left untreated for 18 h. mRNA levels of MCP-1, CCL-5, IL-8 (A), IL-6, and M-CSF (B) were measured by qPCR. Data (means ± S.D., n = 3) are expressed as fold induction over basal (untreated) control. *** p < 0.001 vs. basal (untreated) control; # p < 0.05 vs. TNF-α alone; ## p < 0.01 vs. TNF-α alone; ### p < 0.001 vs. TNF-α alone.

Figure 5

Effect of A. lixula extract on TNF-α-induced chemiotaxis of monocytes. HMEC-1 were treated with A. lixula extract for 4 h at the concentration indicated, and then either treated with 10 ng/mL TNF-α or left untreated for 18 h. Culture medium was collected and added to the lower chamber of a Boyden chamber. THP-1 were added to the upper chamber. Migrated THP-1 cells were then measured by the MTT assay. Data (means ± S.D., n = 3) are expressed as percent of untreated control. ** p < 0.01 vs. basal (untreated) control; ## p < 0.01 vs. TNF-α alone.

Figure 6

Effect of A. lixula extract on TNF-α-induced NF-κB activation. HMEC-1 were treated with A. lixula extract for 4 h at the concentration indicated, and then either treated with 10 ng/mL TNF-α or left untreated for 1 h. Nuclear proteins were analyzed for NF-κB p65 DNA-binding activity by ELISA. Data (means ± S.D., n = 3) are expressed as percent of TNF-α. *** p < 0.001 vs. basal (untreated) control; ## p < 0.01 vs. TNF-α alone.