Hepatocyte growth factor enhances the ability of dental pulp stem cells to ameliorate atherosclerosis in apolipoprotein E-knockout mice

Figure 1 Ad-Null and Ad-hepatocyte growth factor modified dental pulp stem cells treatments alleviated atherosclerotic lesions in apolipoprotein E-knockout mice. A: Representative images of oil Red O-stained aortas; B: Atherosclerotic plaque areas are expressed as the ratio of oil Red O-stained areas to total aortic areas (n = 3); C: Representative images of hematoxylin and eosin (HE) stained sections showing the plaque size in the aortic lumen (scale bar = 100 μm); D: Representative images of aortic arch sections stained with an anti-mCD80 antibody (scale bar = 100 μm); E: The CD80-positive areas in the aortic tissues are expressed as the ratios of positively stained cells to total cells (n = 3); F: Representative images of intercellular cell adhesion molecule-1 (ICAM-1) expression in atherosclerotic lesions of the aortic arch (scale bar = 50 μm); G: The ICAM-1 expression levels are shown as the ratios of positively stained cells to total endothelial cells (n = 3). All results are presented as the means ± SEMs; ^bP < 0.01, ^cP < 0.001 vs. the normal fat diet group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs. the high-fat diet group; ^gP < 0.05 vs. the Ad-Null modified dental pulp stem cells group. DPSC-HGF: Ad-hepatocyte growth factor modified dental pulp stem cells; HFD: High-fat diet; NFD: Normal formula diet; DPSC: Dental pulp stem cell.

Figure 2 Neither Ad-Null modified dental pulp stem cells nor Ad-hepatocyte growth factor modified dental pulp stem cells altered the biochemical parameters of apolipoprotein E-knockout mice fed a high-fat diet. A: Serum concentrations of triglycerides, total cholesterol, low-density lipoprotein, and high-density lipoprotein in apolipoprotein E-knockout (ApoE^-/-) mice at weeks 6 and 12 (n = 4); B: Serum concentrations of glucose (GLU) and glycosylated serum protein (GSP) in ApoE^-/- mice at weeks 6 and 12 (n = 4); C: Body weight (n = 9). All results are presented as the means ± SEMs; ^bP < 0.01, ^cP < 0.001 vs. the normal fat diet group. ^eP < 0.01 vs. high-fat diet group. DPSC-Null: Ad-Null modified dental pulp stem cells; DPSC-HGF: Ad-hepatocyte growth factor modified dental pulp stem cells; HFD: High-fat diet; NFD: Normal formula diet; TG: Triglycerides; CHO: Cholesterol; LDL: Low-density lipoprotein; HDL: High-density lipoprotein; GLU: Glucose; GSP: Glycosylated serum protein.

Figure 3 Ad-Null and Ad-hepatocyte growth factor modified dental pulp stem cells treatments reduced the percentages of monocytes, neutrophils and inflammatory macrophages in apolipoprotein E-knockout mice. Peripheral blood cells were stained with anti-mouse CD11b, anti-mouse Ly6G, and anti-mouse Ly6C at weeks 6 and 12 to determine the percentages of CD11b⁺Ly6G⁺ and CD11b⁺Ly6C⁺ cells (n = 3). A: Representative flow cytometry images of peripheral blood; B: Statistical charts of peripheral blood are shown; C: At the end of the experiment, peripheral blood cells were stained with anti-mouse CD86 and anti-mouse CD206, and the mean fluorescence intensity (MFI) of CD86 and CD206 were analyzed (n = 3). Splenocytes were stained with anti-mCD11b, anti-mLy6G, and anti-mLy6C to determine the percentages of CD11b⁺Ly6G⁺ and CD11b⁺Ly6C⁺ cells (n = 3); D: Representative flow cytometry images of the spleen; E: Statistical graphs of the spleen are shown; F: Splenocytes were stained with anti-mCD86 and anti-mCD206 antibodies, and the MFI of CD86 and CD206 were analyzed (n = 3). All results are represented as the mean ± SEM; ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs. normal fat diet group; ^dP < 0.05, ^eP < 0.01 vs. high-fat diet group. DPSC-Null: Ad-Null modified dental pulp stem cells; DPSC-HGF: Ad-hepatocyte growth factor modified dental pulp stem cells; HFD: High-fat diet; NFD: Normal formula diet.

Figure 4 Ad-hepatocyte growth factor modified dental pulp stem cells treatment reduces the expression of inflammatory cytokines in aortic tissues and serum. The expression levels of matrix metallopeptidase-2 (MMP-2), MMP-9, interferon-γ, interleukin-1β (IL-1β), monocyte chemotactic protein-1 and IL-10 in aortic tissue were detected by real-time reverse transcription polymerase chain reaction (n = 3). A: The serum inflammatory factor expression levels of interleukin-1α (IL-1α), IL-1β and tumor necrosis factor-α were detected by enzyme-linked immunosorbent assay (n = 3); B: All results are represented as the mean ± SEM; ^cP < 0.001 vs. normal fat diet group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs. high fat diet group; ^gP < 0.05, ^hP < 0.01, ⁱP < 0.001 vs. the Ad-Null modified dental pulp stem cells group. DPSC-Null: Ad-Null modified dental pulp stem cells; DPSC-HGF: Ad-hepatocyte growth factor modified dental pulp stem cells; HFD: High-fat diet; NFD: Normal formula diet; MMP: Matrix metallopeptidase; IFN: Interferon; IL: Interleukin; MCP: Monocyte chemotactic protein; TNF: Tumor necrosis factor.

Figure 5 Effects of the supernatant of Ad-Null and Ad-hepatocyte growth factor modified dental pulp stem cells on the polarization and inflammatory cytokine expression of RAW264. 7 mouse macrophages under inflammatory stimulation. RAW264.7 cells were indirectly co-cultured with the supernatant of Ad-Null modified dental pulp stem cells (DPSCs) and Ad-hepatocyte growth factor modified DPSCs for 24 h under lipopolysaccharide (150 ng/mL) stimulation. The mean fluorescence intensity of the anti-CD86 antibody was detected by flow cytometry. A: Representative image; B: A statistical plot of the mean fluorescence intensity (MFI) of CD86 was generated (n = 3); C: The mRNA expression levels of the M1 macrophage markers tumor necrosis factor-α (TNF-α) and arginase 2 (Arg2) and the M2 macrophage marker Arg1 were detected by real-time reverse transcription polymerase chain reaction (RT-PCR) under inflammatory stimulation (n = 3); D: The mRNA expression levels of the inflammatory factors interleukin-1β (IL-1β), IL-6, and cyclooxygenase-2 were detected by RT-PCR under inflammatory stimulation (n = 3); E: The expression of phosphorylated p65 and p65 in RAW264.7 cells under inflammatory stimulation conditions was investigated using western blotting; F: The gray values were analyzed; G: The expression levels of phosphorylated p65 and p65 in RAW264.7 cells under nuclear factor-κB (NF-κB) activator treatment were detected by western blotting; H: Analyzed for gray values; I: After NF-κB activator treatment, the CD86 MFI was detected by flow cytometry; J: A statistical plot of the MFI of CD86 was generated (n = 3). All results are representative of the mean ± SEM; ^bP < 0.01, ^cP < 0.001 vs. control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs. lipopolysaccharide group; ^gP < 0.05, ⁱP < 0.001 vs. supernatant of Ad-Null modified dental pulp stem cells group. LPS: Lipopolysaccharide; DPSC-Null-CM: Supernatant of Ad-Null modified dental pulp stem cells; DPSC-HGF-CM: Supernatant of Ad-hepatocyte growth factor modified dental pulp stem cells; IL: Interleukin; TNF: Tumor necrosis factor; Arg2: Arginase 2; COX-2: Cyclooxygenase-2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 6 Effects of the supernatant of Ad-Null and Ad-hepatocyte growth factor modified dental pulp stem cells on the expression of adhesion molecules by human aortic endothelial cells under inflammatory stimulation. A and B: Human aortic endothelial cells (HAOECs) were indirectly co-cultured with the supernatant of Ad-Null modified dental pulp stem cells (DPSC-Null-CM) or Ad-hepatocyte growth factor modified DPSCs (DPSC-HGF-CM) for 2 h or 8 h under tumor necrosis factor-α (TNF-α) (100 ng/mL) stimulation. The expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1, E-selectin, and monocyte chemotactic protein-1 in HAOECs under inflammatory stimulation was detected by real-time reverse transcription polymerase chain reaction (RT-PCR) at 2 h (A) and 8 h (B) (n = 3). HAOECs were pretreated with DPSC-Null-CM or DPSC-HGF-CM for 4 h, and TNF-α (100 ng/mL) was then added for 5 min; C: The expression of phosphorylated p65 and p65 in inflammation-stimulated HAOECs was investigated using western blotting; D: Gray values analysis of Figure C; E: The expression of phosphorylated p65 and p65 in HAOECs under nuclear factor-κB (NF-κB) activator treatment was investigated using western blotting; F: Gray values analysis of Figure E; G and H: VCAM-1 was detected in HAOECs treated with the NF-κB activator by RT-PCR at 2 h (G) and 8 h (H) (n = 3). All results are represented as the mean ± SEM; ^bP < 0.01, ^cP < 0.001 vs. control group; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001 vs. tumor necrosis factor-α group; ^gP < 0.05, ^hP < 0.01, ⁱP < 0.001 vs. supernatant of Ad-Null modified dental pulp stem cells group. DPSC-Null-CM: Supernatant of Ad-Null modified dental pulp stem cells; DPSC-HGF-CM: Supernatant of Ad-hepatocyte growth factor modified dental pulp stem cells; TNF: Tumor necrosis factor; VCAM-1: Vascular cell adhesion molecule-1; ICAM-1: Intercellular adhesion molecule-1; MCP-1: Monocyte chemotactic protein-1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.