Ox-LDL promotes migration and adhesion of bone marrow-derived mesenchymal stem cells via regulation of MCP-1 expression

Abstract

Bone marrow-derived mesenchymal stem cells (bmMSCs) are the most important cell source for stem cell transplant therapy. The migration capacity of MSCs is one of the determinants of the efficiency of MSC-based transplant therapy. Our recent study has shown that low concentrations of oxidized low-density lipoprotein (ox-LDL) can stimulate proliferation of bmMSCs. In this study, we investigated the effects of ox-LDL on bmMSC migration and adhesion, as well as the related mechanisms. Our results show that transmigration rates of bmMSCs and cell-cell adhesion between bmMSCs and monocytes are significantly increased by treatments with ox-LDL in a dose- and time-dependent manner. Expressions of ICAM-1, PECAM-1, and VCAM-1 as well as the levels of intracellular Ca(2+) are also markedly increased by ox-LDL in a dose-dependent manner. Cytoskeleton analysis shows that ox-LDL treatment benefits to spreading of bmMSCs and organization of F-actin fibers after being plated for 6 hours. More interestingly, treatments with ox-LDL also markedly increase expressions of LOX-1, MCP-1, and TGF- β ; however, LOX-1 antibody and MCP-1 shRNA markedly inhibit ox-LDL-induced migration and adhesion of bmMSCs, which suggests that ox-LDL-induced bmMSC migration and adhesion are dependent on LOX-1 activation and MCP-1 expression.

Figure 1

Uptake of Dil-ox-LDL and LOX-1 expression in bmMSCs. (a) Morphology of primary bmMSCs; (b) Dil-ox-LDL uptake in primary bmMSCs; (c) Morphology of the 3rd-passage bmMSCs; (d) Dil-ox-LDL uptake in the 3rd passage bmMSCs; (e) RT-PCR and Western-blotting assays show LOX-1 expression in the primary and the 3rd-passage bmMSCs.

Figure 2

Ox-LDL promotes transmigration of bmMSCs and enhances cell adhesion between bmMSCs (grey color) and monocytes (red color). (a) Transmigration rates of bmMSCs after exposure to 0, 5, 10, and 20 μg/mL ox-LDL for 6 hours; (b) transmigration rates of bmMSCs after exposure to 10 μg/mL for 0, 3, 6, and 12 hours; (c) the merged phase contrast and fluorescence images show adhesion between bmMSCs and monocytes after treatment with 0, 5, 10, and 20 μg/mL ox-LDL for 6 hours; (d) the merged phase contrast and fluorescence images show adhesion between bmMSCs and monocytes after treatment with 10 μg/mL for 0, 3, 6, and 12 hours; (e) the relative adhesive rate of monocytes onto bmMSCs after treatment with 0, 5, 10 and 20 μg/mL ox-LDL for 6 hours; (f) the relative adhesive rate of monocytes onto bmMSCs after treatment with 10 μg/mL ox-LDL for 0, 3, 6, and 12 hours. Bar graphs represent mean ± SD (n = 4 per group). *P < 0.01 versus control.

Figure 3

Ox-LDL increases expression of ICAM-1, PECAM-1, and VCAM-1 in a dose-dependent manner in bmMSCs. (a) Immunofluorescence assay shows expression of ICAM-1, PECAM-1 and VACM-1 in bmMSCs exposed to 0, 5, 10 and 20 μg/mL ox-LDL for 6 hour; (b)–(d) Relative fluorescence density of ICAM-1, PECAM-1, and VCAM-1; (e)–(g) Western-blotting assay shows expression of ICAM-1, PECAM-1, and VCAM-1 in bmMSCs exposed to 0, 5, 10, and 20 μg/mL ox-LDL for 6 hours. Bar graphs represent mean ± SD (n = 4 per group). *P < 0.01 versus Control.

Figure 4

Flowcytometry assay shows the levels of intracellular Ca²⁺ of bmMSCs exposed to 0, 5, 10, and 20 μg/mL ox-LDL for 6 hours. Bar graphs represent mean ± SD (n = 4 per group). *P < 0.01 versus control.

Figure 5

Cytoskeleton (F-actin fibers) organization in bmMSCs after exposure to 0, 5, 10, and 20 μg/mL ox-LDL for 6 hours.

Figure 6

Role of LOX-1 and MCP-1 in ox-LDL-mediated migration and adhesion of bmMSCs. (a)–(c) Western-blotting assay shows LOX-1, MCP-1 and TGF-β expression in bmMSCs after exposure to 0, 5, 10, and 20 μg/mL ox-LDL for 6 hours. (d) Western-blotting assay shows that LOX-1 antibody inhibits ox-LDL-induced MCP-1 expression. (e) Transwell assay shows that LOX-1 antibody inhibits ox-LDL-induced transmigration of bmMSCs. (f) LOX-1 antibody inhibits ox-LDL-induced cell adhesion between bmMSCs and monocytes. (g) Western-blotting assay shows that LOX-1 antibody decreases ICAM-1 expression. (h) Western-blotting assay shows MCP-1 expression after transfection of noneffective shRNA and MCP-1 shRNA. (i) MCP-1 knockdown inhibits ox-LDL-induced migration of bmMSCs. (j) MCP-1 knockdown inhibits ox-LDL-induced cell-cell adhesion between monocytes and bmMSCs. (k) MCP-1 knockdown decreases ox-LDL-induced ICAM-1 expression. Bar graphs represent mean ± SD (n = 4 per group). *P < 0.01 versus control, ox-LDL treatment, or negative control transfection.