Pioglitazone strengthen therapeutic effect of adipose-derived regenerative cells against ischemic cardiomyopathy through enhanced expression of adiponectin and modulation of macrophage phenotype

Fig. 1. Study protocol and scenery graft preparation. a Study protocol and groups of the in vivo experiment. UCG Ultrasonocardiography, PCR polymerase chain reaction, ELISA enzyme-linked immunosorbent assay, ADRCs adipose-derived regenerative cells, PGZ pioglitazone. b Formation of round-shaped grafts with cells suspended in fibrinogen and thrombin solution on culture dishes immediately after cell isolation and just before implantation. c Intraoperative photograph showing the grafts being placed onto the surfaces of the hearts

Fig. 2. Relationship between implanted graft and ischemic heart. The ADRCs and PGZ in graft was implanted on the surface of the heart. PGZ is thought to act on the cells in graft, pericardial adipocyte and residual cardiomyocytes, and enhance the APN production in these cells. Furthermore factors that affect the phenotypical change of macrophage and characteristics of these macrophage are shown

Fig. 3. In vitro analysis of ADRCs and its grafts. a Distribution patterns of the cells, plotted as FSC (cell size) versus SSC (granularity) with flow cytometric analysis. b, c Analysis of population 1 based on the stained makers CD11b, CD31, CD45, CD73, and CD90. The figure shows one representative result. d Macroscopic finding of a graft. e Cross-sectional view of hematoxylin and eosin-stained ADRC graft. f Magnified photograph of the rectangular area in b. Scale bar = 50 μm. g Oil red-O staining of ADRC grafts showing that mature adipocytes were more common in grafts containing PGZ after 72 h culture in vitro. Arrows indicate oil droplets in the adipocyte cytosol. Scale bar = 50 μm. h Whole section of graft showing scattered distribution of adipocytes. i ELISA analysis of ADRCs graft culture supernatant. Adiponectin (APN), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) secretion into the culture supernatant, measured by ELISA. j Inflammation-related cytokines from ADRCs measured by ELISA. k There was significant difference of cell sphericity index between before and after 72 h incubation of graft in AP group. Multiple comparisons made by one-way ANOVA. n…

Fig. 4. Engraftment of ADRC grafts on the cardiac surface and local APN production. a Macroscopically, the grafts covered the anterior walls of the heart. b Hematoxylin and eosin staining of the grafts showed connective tissue between the graft and the heart. Asterisk indicates implanted ADRCs. c ADRCs derived from GFP rats were implanted in the same manner. IHC showed that the connective tissue contained a vascular network in its stalk. c-2 Magnified photograph of the rectangular area in c-1. Arrow head shows GFP-positive cells in the graft that survived, indicating that the implanted ADRCs survived 14 days after transplantation. Scale bar = 50 μm. c-1 Magnified photograph of the rectangular area in c-2. Arrows show connective tissue between the graft and the heart. Scale bar = 100 μm. Green, GFP-positive cells; red, SMA; and blue, nuclei. d Representative pictures of APN positive cells in myocardium 3 weeks after implantation. Some cells in infarct areas and infarct border zones showed strong labelling for APN. The APN distribution was not only observed in the intravascular lumen, but also in suspected residual cardiomyocytes. Red, APN; Green, SMA; blue, nuclei. Scale bar = 50 μm

Fig. 5. Cardiac function and survival rate after transplantation. a ADRCs grafts improved left ventricular ejection fraction after implantation. b Left ventricular anterior wall thickness was preserved in A, AP and Si groups. c, d In A, AP and Si groups, the left ventricular end-systolic dimension was significantly smaller than that in the C group. However, the end-diastolic diameter was almost the same among the groups. e, f Representative M-mode pictures of echocardiography of control and ADRC groups. Arrows indicate anterior wall of the left ventricle. g The ADRC transplantation groups showed significantly better survival than did group C. There was no significant difference among the A, AP, and Si groups. Multiple comparisons made by one-way ANOVA. *P < 0.05 versus C group, **P < 0.05 versus A group, †P < 0.05 versus Si group, n = 8–10 per group

Fig. 6. Histological evaluation of left ventricular at 3 days and 8 weeks after implantation. a, b Representative whole microscopic images of Sirius red staining in each group. Quantification of percent fibrosis and fibrotic area. Fibrosis at areas remote to the infarct zone was significantly suppressed in the A, AP, and Si groups compared to that in the C group 8 weeks after transplantation. Scale bar = 50 μm. Evaluation of hypertrophy of cardiomyocytes. c, d Representative periodic acid-Schiff staining of tissue infarct border site. Quantification of cardiomyocyte diameter. Cardiomyocyte diameters in the borders of the infarct sites were significantly smaller in the A and AP groups than in the C group. Especially at 8 weeks diameters in AP group was significantly smaller than the other three groups. e, f Capillary density in the area bordering the infarct zone and areas remote to infarction. Capillaries stained by immunostaining with an anti-von Willebrand factor antibody were significantly denser in the A and AP groups than C and Si groups at 3 days. At 8 weeks the density in AP group was significantly larger than the other three groups. n = 8–10 per group; *P < 0.05 versus C group,…

Fig. 7. Evaluation of macrophage phenotype in infarct and border zone. a Representative immuno-histochemical staining of CD11c (M1 macrophage marker) and/or CD206 (M2 macrophage marker). b The ratio of the number of CD206- to CD11c-positive cells was significantly higher in the A and AP groups than in the C and Si groups at two points of observation. The highest ratio was maintained in the AP group. Green, CD11c-positive cell; red, CD206; and blue, nuclei. N = 8–10; *P < 0.05 versus C group, **P < 0.05 versus A group, †P < 0.05 versus P group. Scale bar = 50 μm

Fig. 8. Gene and protein expression in the heart. a Quantitative RT-PCR results for the expression levels of APN, CDH-13, Adipo-R1, Adipo-R2, SDF-1, VEGF, HGF, TNF-α, IL-6 and IL-10 genes in the infarct border area. APN was significantly higher in the ADRC groups than in the C group. ADRCs effect on VEGF and HGF expression after myocardial infarction during the early days of implantation. b Representative immunoblot of T-cadherin in total protein extracted from the heart 3 days and 8 weeks after implantation. c T-cadherin expression levels were higher in the A and AP groups than in the C and Si groups during the early days after implantation. At 8 weeks, T-cadherin levels were higher in the AP group than in the other groups. Multiple comparisons were made by one-way ANOVA. n = 8–10; *P < 0.05 versus C group, **P < 0.05 versus A group, †P < 0.05 versus Si group