Mesenchymal stem cells promote type 2 macrophage polarization to ameliorate the myocardial injury caused by diabetic cardiomyopathy

Abstract

Background: Diabetic cardiomyopathy (DCM) is a common complication of diabetes and is characterized by chronic myocardial inflammation. Mesenchymal stem cell (MSC) infusions have recently been suggested to alleviate myocardial injury and ameliorate cardiac function. However, few studies have focused on the effects of MSCs in DCM. Therefore, we explored the effects of MSC-regulated macrophage polarization on myocardial repair in DCM.

Methods: A DCM rat model was induced by a high-fat diet and streptozotocin (STZ) administration and infused 4 times with MSCs. Rat blood and heart tissue were analyzed for blood glucose levels, lipid levels, echocardiography, histopathology, macrophage phenotype ratios and inflammatory cytokines, respectively. We mimicked chronic inflammation in vitro by inducing peritoneal macrophages with high glucose and LPS, then cocultured these macrophages with MSCs to explore the specific mechanism of MSCs on macrophage polarization.

Results: DCM rats exhibited abnormal blood glucose levels and lipid metabolism, cardiac inflammation and dysfunction. MSC infusion ameliorated metabolic abnormalities and preserved cardiac structure and function in DCM rats. Moreover, MSC infusion significantly increased the M2 phenotype macrophages and alleviated cardiac inflammation. Interestingly, this in vitro study revealed that the MSCs pretreated with a COX-2 inhibitor had little effect on M2 macrophage polarization, but this phenomenon could be reversed by adding prostaglandin E2 (PGE2).

Conclusions: Our results suggested that MSC infusions can protect against cardiac injury in DCM rats. The underlying mechanisms may include MSC-enhanced M2 macrophage polarization via the COX-2-PGE2 pathway.

Keywords: Diabetic cardiomyopathy; Macrophage polarization; Mesenchymal stem cell.

Fig. 1

Identification of adipose-derived MSCs and peritoneal macrophages. Cultivated Passage 3 MSCs stained positive for CD29-APC and CD90-APC and negative for CD34-FITC, CD45-FITC and CD11b-FITC via flow cytometry. Negative control was also presented. The target cells we chose were in the FSC-Height and SSC-Height quadrant to separate cell debris (a). MSC morphology was observed via light microscopy, and MSCs were stained by Oil Red O or Alizarin Red to indicate differentiation into adipocytes or osteoblasts (b). Macrophage morphology was observed via light microscopy, and more than 99% of the cultured macrophages stained positive for CD11b and CD68 via flow cytometry (c). Scale bar: 100 μm

Fig. 2

Characterization of a diabetic cardiomyopathy rat model. Rats were fed either a normal or HFD for 8 weeks and DCM rats were injected intraperitoneally with STZ (25 mg/kg). After that, all rats fed a normal chow diet (NCD). Echocardiography was performed on the rats 4 weeks after STZ injection (a). Body weight (b), fasting blood glucose (c), and fasting serum insulin (d) were assessed at baseline, 8 weeks and 12 weeks, and the insulin sensitivity index (e) was calculated by 1/FBG*FINS. Blood total cholesterol (f) and blood triglycerides (g) were also analyzed to further characterize the lipid metabolism. n = 3 per group in the echocardiogram detection, n = 5–10 per group in the body weight and blood detection, * P < 0.05, ** P < 0.01 vs. normal group

Fig. 3

MSCs improved cardiac function, blood glucose and lipid metabolism in DCM rats. MSCs were infused 4 times, and cardiac function was measured by echocardiography (a) at 20 weeks. Fasting blood glucose (b) was assessed throughout the MSC infusion process. Intraperitoneal glucose tolerance testing (c), fasting serum insulin (d) and the insulin sensitivity index (e) were used to evaluate glucose metabolism. Blood total cholesterol (f) and blood triglycerides (g) were also analyzed to assess lipid metabolism at 19 weeks. n = 3–6 per group for echocardiographic assessment, n = 5–8 per group for hematological assessment, ** P < 0.01 vs. normal group, ## P < 0.01 vs. DCM group

Fig. 4

MSC infusion ameliorated abnormal metabolism, cardiac structure, inflammation and fibrosis in DCM rats. MSCs were infused 4 times, then rats were sacrificed, and their hearts were removed and weighed immediately (a), the ratio of heart weight to body weight (HW/BW) was calculated (b). Heart samples were fixed with paraformaldehyde, and cross sections of the ventricular wall and chamber were stained with H&E at the papillary muscle level (c). H&E staining (d, scale bar = 20 μm) was performed to reflect cardiomyocyte morphology and cardiac inflammation, and cardiomyocyte cross-sectional area (CSA) was also measured, analyzed and presented (e). Masson’s trichrome staining was performed to observe cardiac fibrosis (f, scale bar = 50 μm) and the quantification of myocardial fibrosis was also analyzed (g). n = 5–8 per group, **P < 0.01 vs. normal group, #P < 0.05, ##P < 0.01 vs. DCM group

Fig. 5

MSCs promoted M2 macrophage polarization in DCM hearts and relieved chronic inflammation in DCM rats. Heart tissues from the different groups were digested and stained with the cell surface markers CD68 (total macrophages), CD11c (M1), and CD163 (M2) and were analyzed by flow cytometry at 20 weeks. Ratios of total macrophages (a), M1 (b) and M2 (c) in total cells were analyzed. The percentage of M1 macrophages and M2 macrophages among CD68-positive total macrophages of normal (d, g), DCM (e, h) and DCM + MSCs (f, i) groups was presented and analyzed. IL-6, TNF-α and IL-10 concentrations in the heart homogenates (j, k, l) and blood serum (m, n, o) were detected by ELISA. n = 3–5 per group, *P < 0.05, **P < 0.01 vs. normal group, #P < 0.05, ##P < 0.01 vs. DCM group

Fig. 6

MSCs promoted M2 macrophage polarization and anti-inflammatory effects in high glucose conditions with LPS stimulation. Rat peritoneal macrophages were separated and divided into the normal, HG + LPS and HG + LPS + MSCs groups. After treatment, macrophages from the different groups were collected, and the M1 ratios, M2 ratios and the ratios of M1 to M2 phenotypes were detected by flow cytometry (a). The data were analyzed using Cellquest software (b). The IL-6, TNF-α and IL-10 concentrations in the supernatants were determined by ELISA (c). n = 4 per group. *P < 0.05, **P < 0.01 vs. normal group, ##P < 0.01 vs. HG + LPS group

Fig. 7

MSCs promoted M2 macrophage polarization and anti-inflammatory effects in an HG + LPS environment via the COX-2-PGE2 pathway. MSCs were incubated in normal control, HG + LPS or HG + LPS + COX-2-inhibitor media, and the PGE2 concentration in the supernatant was detected by ELISA (a). n = 4–5, **P < 0.01. The HG + LPS-induced macrophages were cocultured with control MSCs or MSCs pretreated with COX-2 inhibitor in the presence or absence of exogenous PGE2. After treatment, macrophages were collected, and the M1 and M2 phenotypes were detected by flow cytometry (b, c). The IL-6, TNF-α and IL-10 concentrations in the supernatants were detected by ELISA (d). n = 3–4 per group, *P < 0.05, **P < 0.01