Tenascin-C produced by oxidized LDL-stimulated macrophages increases foam cell formation through Toll-like receptor-4

Abstract

Atherosclerosis is a chronic inflammatory disease in which both innate and adaptive immunity are involved. Although there have been major advances in the involvement of toll-like receptor 4 (TLR4) and CD36 in the initiation and development of this disease, detailed mechanisms remain unknown. Here, we show that tenascin-C (TN-C) can stimulate foam cell formation and this can be inhibited by a TLR4-blocking antibody or CD36 gene silencing. Our results identify TN-C-TLR4 activation as a common molecular mechanism in oxLDL-stimulated foam cell formation and atherosclerosis. In addition, CD36 is the major scavenger receptor responsible for the TN-C-mediated foam cell formation. Taken together, we have identified that TNC produced by oxLDL-stimulated macrophages increases foam cell formation through TLR4 and scavenger receptor CD36.

Fig. 1

OxLDL induced the expression of TN-C in macrophages in a time- and dose-dependent manner. (A) Macrophages were stimulated with different concentrations of oxLDL (0, 1, 5, 10, 20 or 50 μg/ml); (B) Macrophages were stimulated with 5 μg/ml oxLDL for different time; (C): The expression of TN-C protein in macrophages induced with 5 μg/ml oxLDL for different times were measured by ELISA. ^*(P < 0.05) represents significant difference to control (0 μg/ml or 0 h).

Fig. 2

Blocking antibodies against TNF-α reduced the expression of TN-C in oxLDL-stimulated macrophages. Different concentration of blocking antibodies against TNF-α were added to macrophages 0.5 h before or 12 h after oxLDL incubation, and TN-C expression was measured by ELISA kit. ^*(P < 0.05), significant difference to group with oxLDLD treatment only.

Fig. 3

Exogenous TN-C could increase the differentiation of macrophages into foam cells in a time- and dose-dependent manner. (A) Macrophages were induced with 5 μg/ml oxLDL and different concentration of exogenous TN-C; (B) Macrophages were stimulated with 5 μg/ml oxLDL and 1 μM TN-C for the indicated time. CE/TC contents were measured by HPLC. ^*(P < 0.05) and ^*(P < 0.01), significant difference to control (0 μM or 0 h).

Fig. 4

Blocking of TLR4 significantly reduced TN-C-induced foam cell formation. Different concentrations of antibody against TLR4 or control antibody were added to macrophages 1 h prior to oxLDL and TN-C stimulation, and foam cell formation was detected by measuring CE/TC contents. ^*(P < 0.05), significant difference to group with oxLDL and TN-C treatment only.

Fig. 5

Exogenous TN-C stimulation significantly increased CD36 expression by macrophages (P < 0.05). (B) Macrophages without TN-C treatment were analyzed for CD36 expression using flow cytometry. (C) Macrophages were treated with 1 μM TN-C for 24 h, and CD36 expression was determined by flow cytometry. Significant differences were observed between resting cells and TN-C-treated cells (P < 0.05).

Fig. 6

CD36-specific siRNA transfection reduced CE/TC production in macrophages. Macrophages were transfected with scramble siRNA sequence or CD36 specific siRNA 1 h before cells were stimulated with 5 μg/ml oxLDL and 1 μM TN-C. (A, B) Cells lysates were prepared from these cells, equal amount of proteins were resolved by SDS-PAGE and CD36 expression were detected by Western blotting. Beta-actin was used as a loading control. (C) CE/TC contents were measured by HPLC 48 h after stimulation. ^*(P < 0.05), significant difference to group with oxLDl treatment only or cells transfected with scramble sequence.