Comparison of adipose‑ and bone marrow‑derived stem cells in protecting against ox‑LDL‑induced inflammation in M1‑macrophage‑derived foam cells

Abstract

Adipose‑derived stem cells (ADSCs) and bone marrow‑derived stem cells (BMSCs) are considered to be prospective sources of mesenchymal stromal cells (MSCs), that can be used in cell therapy for atherosclerosis. The present study investigated whether ADSCs co‑cultured with M1 foam macrophages via treatment with oxidized low‑density lipoprotein (ox‑LDL) would lead to similar or improved anti‑inflammatory effects compared with BMSCs. ADSCs, peripheral blood monocytes, BMSCs and ox‑LDL were isolated from ten coronary heart disease (CHD) patients. After three passages, the supernatants of the ADSCs and BMSCs were collected and systematically analysed by liquid chromatography‑quadrupole time‑of‑flight‑mass spectrometry (6530; Agilent Technologies, Inc., Santa Clara, CA, USA). Cis‑9, trans‑11 was deemed to be responsible for the potential differences in the metabolic characteristics of ADSCs and BMSCs. These peripheral blood monocytes were characterized using flow cytometry. Following peripheral blood monocytes differentiation into M1 macrophages, the formation of M1 foam macrophages was achieved through treatment with ox‑LDL. Overall, 2x106 ADSCs, BMSCs or BMSCs+cis‑9, trans‑11 were co‑cultured with M1 foam macrophages. Anti‑inflammatory capability, phagocytic activity, anti‑apoptotic capability and cell viability assays were compared among these groups. It was demonstrated that the accumulation of lipid droplets decreased following ADSCs, BMSCs or BMSCs+cis‑9, trans‑11 treatment in M1 macrophages derived from foam cells. Consistently, ADSCs exhibited great advantageous anti‑inflammatory capabilities, phagocytic activity, anti‑apoptotic capability activity and cell viability over BMSCs or BMSCs+cis‑9, trans‑11. Additionally, BMSCs+cis‑9, trans‑11 also demonstrated marked improvement in anti‑inflammatory capability, phagocytic activity, anti‑apoptotic capability activity and cell viability in comparison with BMSCs. The present results indicated that ADSCs would be more appropriate for transplantation to treat atherosclerosis than BMSCs alone or BMSCs+cis‑9, trans‑11. This may be an important mechanism to regulate macrophage immune function.

Figure 1.

Principal component analysis score plots for discriminating BMSCs and ADSCs in (A) ESI⁺ and (B) ESI⁻ modes. BMSCs, bone marrow-derived stem cells; ADSCs, adipose-derived stem cells; ESI, electrospray ionization; QC, quality control.

Figure 2.

PLS-DA score plots and validation plots for discriminating ADSCs and BMSCs in ESI⁺ and ESI⁻ modes. (A) PLS-DA plot in ESI⁺ mode. (B) Validation plot in ESI⁺ mode. (C) PLS-DA plot in ESI⁻ mode. (D) Validation plot in ESI⁻ mode. PLS-DA, partial least squares discriminant analysis; BMSCs, bone marrow-derived stem cells; ADSCs, adipose-derived stem cells; ESI, electrospray ionization; Q2, second quartile; R2, coefficient of determination.

Figure 3.

Metabolite profiles of potential biomarkers differing between epithelial BMSCs and ADSCs. The profiles are displayed as box & whiskers graphs (Min to Max). BMSCs, bone marrow-derived stem cells; ADSCs, adipose-derived stem cells.

Figure 4.

(A) Cells were treated with ox-LDL (100 mg/l) in the absence or presence of ADSCs, BMSCs or BMSCs+ cis-9, trans-11 for 24 h, and the intracellular lipid droplets were stained using Oil Red O. Representative lipid droplet staining images are shown. Scale bars, 25 µm. (B) The average IOD of lipid droplets stained with Oil Red O from differentiated macrophage foam cells was obtained by examining five fields in each condition (n=10). *P<0.05 and **P<0.01 vs. the ox-LDL group; ^##P<0.01 vs. the ADSCs group. ox-LDL, oxidized low-density lipoprotein; BMSCs, bone marrow-derived stem cells; ADSCs, adipose-derived stem cells; IOD, integrated optical density.

Figure 5.

ADSCs, BMSCs and BMSCs+cis-9, trans-11 decreased the secretion of inflammatory factors (TNF-α and IL-6) and increased the secretion of inflammatory factors (IL-10) in ox-LDL-stimulated macrophages. Cells were pre-incubated with ADSCs, BMSCs and BMSCs+cis-9, trans-11 following treatment with 100 g/ml ox-LDL for 24 h. The concentrations of (A) IL-6, (B) IL-8, (C) IL-10 and (D) TNF-α secretion from the medium of macrophages were measured using an ELISA kit. The results are expressed as a percentage of the results obtained with a blank. Data are presented as the mean ± standard deviation (n=10). *P<0.05, **P<0.01 and ***P<0.001 vs. the ox-LDL group; ^#P<0.05 vs. the ADSCs group; ^&P<0.05 vs. the BMSCs group. BMSCs, bone marrow-derived stem cells; ADSCs, adipose-derived stem cells; TNF-α, tumour necrosis factor-α; IL-, interleukin-; ox-LDL, oxidized low-density lipoprotein.

Figure 6.

Effects of ADSCs, BMSCs and BMSCs+cis-9, trans-11 on the vitality of Mrc-5 cells. M1 macrophage foam cells were seeded in 96-well plates and co-cultured with ADSCs, BMSCs or BMSCs+cis-9, trans-11, and the cell viability of M1 macrophage foam cells was assessed at 24 h using a Cell Counting kit-8 assay (n=10). *P<0.05 and **P<0.01 vs. the ox-LDL group; ^#P<0.05 vs. the ADSCs group. BMSCs, bone marrow-derived stem cells; ADSCs, adipose-derived stem cells; ox-LDL, oxidized low-density lipoprotein.

Figure 7.

ADSCs, BMSCs and BMSCs+cis-9, trans-11 attenuated ox-LDL-induced apoptosis of M1 macrophages. Cells were pre-incubated with cystathionine-γ-lyase (ADSCs, BMSCs and BMSCs+cis-9, trans-11), followed by treatment with 100 g/ml ox-LDL for 24 h. (A) The representative data of apoptotic cells stained with Annexin V-FITC/PI which was detected by flow cytometry analysis. (B) The percentage of apoptotic cells under different treatments was measured and expressed as mean ± standard deviation of at least three independent experiments (n=10). *P<0.05 and **P<0.01 vs. the ox-LDL group; ^#P<0.05 vs. the ADSCs group; ^&P<0.05 vs. the BMSCs group. BMSCs, bone marrow-derived stem cells; ADSCs, adipose-derived stem cells; ox-LDL, oxidized low-density lipoprotein; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 8.

Effects of the inhibition of proinflammatory factors on oxLDL-induced NFκBp65, TNF-α and IL-6 upregulation. Reverse transcription-quantitative polymerase chain reaction analysis of (A) IL-6, (B) NFκBp65 and (C) TNF-α mRNA level in macrophages co-cultured with ADSCs, BMSCs or BMSCs+cis-9, trans-11. Data are expressed as the mean ± standard deviation (n=10). ***P<0.001 vs. the ox-LDL group; ^#P<0.05 and ^###P<0.001 vs. the ADSCs group; ^&&&P<0.001 vs. the BMSCs group. NFκBp65, nuclear factor-κBp65; TNF-α, tumour necrosis factor-α; IL-, interleukin-; BMSCs, bone marrow-derived stem cells; ADSCs, adipose-derived stem cells; ox-LDL, oxidized low-density lipoprotein.

Figure 9.

ADSCs, BMSCs and BMSCs+cis-9, trans-11 suppressed the expression of nuclear protein NF-κBp65 protein and phosphorylated NF-κBp65. Western blot analyses of NF-κBp65 protein and phosphorylated NF-κBp65 expression in ox-LDL-induced of macrophages co-cultured with ADSCs, BMSCs and BMSCs+ cis-9, trans-11. Representative blots from 5 different experiments are shown (n=5). **P<0.01 vs. the ox-LDL group; ^#P<0.05 vs. the ADSCs group; ^&P<0.05 vs. the BMSCs group. BMSCs, bone marrow-derived stem cells; ADSCs, adipose-derived stem cells; NFκBp65, nuclear factor-κBp65; ox-LDL, oxidized low-density lipoprotein.