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PPARγ activation but not PPARγ haplodeficiency affects proangiogenic potential of endothelial cells and bone marrow-derived progenitors.

Kotlinowski J, Grochot-Przeczek A, Taha H, Kozakowska M, Pilecki B, Skrzypek K, Bartelik A, Derlacz R, Horrevoets AJ, Pap A, Nagy L, Dulak J, Jozkowicz A - Cardiovasc Diabetol (2014)

Bottom Line: ECs and PACs isolated from diabetic db/db mice displayed a reduced angiogenic potential in ex vivo and in vitro assays, the effect partially rescued by incubation of cells with rosiglitazone (PPARγ activator).Correction of diabetes by administration of rosiglitazone in vivo did not improve angiogenic potential of isolated PACs or ECs.In summary, activation of PPARγ by rosiglitazone improves angiogenic potential of diabetic ECs and PACs, but decreased expression of PPARγ in diabetes does not impair angiogenesis.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Peroxisome proliferator-activated receptor-γ (PPARγ) agonists, which have been used as insulin sensitizers in diabetic patients, may improve functions of endothelial cells (ECs). We investigated the effect of PPARγ on angiogenic activities of murine ECs and bone marrow-derived proangiogenic cells (PACs).

Methods: PACs were isolated from bone marrow of 10-12 weeks old, wild type, db/db and PPARγ heterozygous animals. Cells were cultured on fibronectin and gelatin coated dishes in EGM-2MV medium. For in vitro stimulations, rosiglitazone (10 μmol/L) or GW9662 (10 μmol/L) were added to 80% confluent cell cultures for 24 hours. Angiogenic potential of PACs and ECs was tested in vitro and in vivo in wound healing assay and hind limb ischemia model.

Results: ECs and PACs isolated from diabetic db/db mice displayed a reduced angiogenic potential in ex vivo and in vitro assays, the effect partially rescued by incubation of cells with rosiglitazone (PPARγ activator). Correction of diabetes by administration of rosiglitazone in vivo did not improve angiogenic potential of isolated PACs or ECs. In a hind limb ischemia model we demonstrated that local injection of conditioned media harvested from wild type PACs improved the blood flow restoration in db/db mice, confirming the importance of paracrine action of the bone marrow-derived cells.

Conclusions: In summary, activation of PPARγ by rosiglitazone improves angiogenic potential of diabetic ECs and PACs, but decreased expression of PPARγ in diabetes does not impair angiogenesis.

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Effect of oral daily administration of PPARγ agonist rosiglitazone (ROSI, 10 mg/kg of body weight, 14 days) on endothelial progenitor cells and expression of proangiogenic genes in diabetic (db/db) mice. Control db/db and wild type (WT) mice were treated with vehicle. A: Percentage of CD45−KDR+Sca-1+ cells in bone marrow. B: Percentage of CD45−KDR+Sca-1+ cells in peripheral blood. C: Number of CD45−KDR+Sca-1+ cells in peripheral blood. Multicolor FACS phenotyping. D: Expression of VEGF mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. E: Concentration of VEGF protein in bone marrow. F: Concentration of VEGF protein in peripheral blood. ELISA. G: Expression of SDF-1 mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. H: Concentration of SDF-1 protein in bone marrow. I: Concentration of SDF-1 protein in peripheral blood. ELISA. J: Expression of PPARγ mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. Each bar represents mean + SEM. N = 8-10 (4A-C, F, I), N = 4-6 (4D, E, G, H, J), *p < 0.05, **p < 0.01, ***p < 0.001 versus WT, #p < 0.05 versus db/db.
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Fig4: Effect of oral daily administration of PPARγ agonist rosiglitazone (ROSI, 10 mg/kg of body weight, 14 days) on endothelial progenitor cells and expression of proangiogenic genes in diabetic (db/db) mice. Control db/db and wild type (WT) mice were treated with vehicle. A: Percentage of CD45−KDR+Sca-1+ cells in bone marrow. B: Percentage of CD45−KDR+Sca-1+ cells in peripheral blood. C: Number of CD45−KDR+Sca-1+ cells in peripheral blood. Multicolor FACS phenotyping. D: Expression of VEGF mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. E: Concentration of VEGF protein in bone marrow. F: Concentration of VEGF protein in peripheral blood. ELISA. G: Expression of SDF-1 mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. H: Concentration of SDF-1 protein in bone marrow. I: Concentration of SDF-1 protein in peripheral blood. ELISA. J: Expression of PPARγ mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. Each bar represents mean + SEM. N = 8-10 (4A-C, F, I), N = 4-6 (4D, E, G, H, J), *p < 0.05, **p < 0.01, ***p < 0.001 versus WT, #p < 0.05 versus db/db.

Mentions: Flow cytometric analysis showed that endothelial progenitors (EPCs), defined as CD45−KDR+Sca-1+ cells, were significantly less frequent in the bone marrow of diabetic mice. Treatment with rosiglitazone resulted in a partial restoration of bone marrow EPCs pool (Figure 4A, p = 0.06), which was complete after prolongation of treatment time to 28 days (data not shown). Decreased frequency of CD45−KDR+Sca-1+ cells was found also in the peripheral blood of diabetic animals, although this tendency did not reach statistical significance (Figure 4B). The number of circulating EPCs was, however, strongly reduced in db/db mice and not affected by rosiglitazone (Figure 4A-C).Figure 4


PPARγ activation but not PPARγ haplodeficiency affects proangiogenic potential of endothelial cells and bone marrow-derived progenitors.

Kotlinowski J, Grochot-Przeczek A, Taha H, Kozakowska M, Pilecki B, Skrzypek K, Bartelik A, Derlacz R, Horrevoets AJ, Pap A, Nagy L, Dulak J, Jozkowicz A - Cardiovasc Diabetol (2014)

Effect of oral daily administration of PPARγ agonist rosiglitazone (ROSI, 10 mg/kg of body weight, 14 days) on endothelial progenitor cells and expression of proangiogenic genes in diabetic (db/db) mice. Control db/db and wild type (WT) mice were treated with vehicle. A: Percentage of CD45−KDR+Sca-1+ cells in bone marrow. B: Percentage of CD45−KDR+Sca-1+ cells in peripheral blood. C: Number of CD45−KDR+Sca-1+ cells in peripheral blood. Multicolor FACS phenotyping. D: Expression of VEGF mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. E: Concentration of VEGF protein in bone marrow. F: Concentration of VEGF protein in peripheral blood. ELISA. G: Expression of SDF-1 mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. H: Concentration of SDF-1 protein in bone marrow. I: Concentration of SDF-1 protein in peripheral blood. ELISA. J: Expression of PPARγ mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. Each bar represents mean + SEM. N = 8-10 (4A-C, F, I), N = 4-6 (4D, E, G, H, J), *p < 0.05, **p < 0.01, ***p < 0.001 versus WT, #p < 0.05 versus db/db.
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Fig4: Effect of oral daily administration of PPARγ agonist rosiglitazone (ROSI, 10 mg/kg of body weight, 14 days) on endothelial progenitor cells and expression of proangiogenic genes in diabetic (db/db) mice. Control db/db and wild type (WT) mice were treated with vehicle. A: Percentage of CD45−KDR+Sca-1+ cells in bone marrow. B: Percentage of CD45−KDR+Sca-1+ cells in peripheral blood. C: Number of CD45−KDR+Sca-1+ cells in peripheral blood. Multicolor FACS phenotyping. D: Expression of VEGF mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. E: Concentration of VEGF protein in bone marrow. F: Concentration of VEGF protein in peripheral blood. ELISA. G: Expression of SDF-1 mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. H: Concentration of SDF-1 protein in bone marrow. I: Concentration of SDF-1 protein in peripheral blood. ELISA. J: Expression of PPARγ mRNA in bone marrow. Quantitative RT-PCR. EF2 serves as an internal control. Each bar represents mean + SEM. N = 8-10 (4A-C, F, I), N = 4-6 (4D, E, G, H, J), *p < 0.05, **p < 0.01, ***p < 0.001 versus WT, #p < 0.05 versus db/db.
Mentions: Flow cytometric analysis showed that endothelial progenitors (EPCs), defined as CD45−KDR+Sca-1+ cells, were significantly less frequent in the bone marrow of diabetic mice. Treatment with rosiglitazone resulted in a partial restoration of bone marrow EPCs pool (Figure 4A, p = 0.06), which was complete after prolongation of treatment time to 28 days (data not shown). Decreased frequency of CD45−KDR+Sca-1+ cells was found also in the peripheral blood of diabetic animals, although this tendency did not reach statistical significance (Figure 4B). The number of circulating EPCs was, however, strongly reduced in db/db mice and not affected by rosiglitazone (Figure 4A-C).Figure 4

Bottom Line: ECs and PACs isolated from diabetic db/db mice displayed a reduced angiogenic potential in ex vivo and in vitro assays, the effect partially rescued by incubation of cells with rosiglitazone (PPARγ activator).Correction of diabetes by administration of rosiglitazone in vivo did not improve angiogenic potential of isolated PACs or ECs.In summary, activation of PPARγ by rosiglitazone improves angiogenic potential of diabetic ECs and PACs, but decreased expression of PPARγ in diabetes does not impair angiogenesis.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Peroxisome proliferator-activated receptor-γ (PPARγ) agonists, which have been used as insulin sensitizers in diabetic patients, may improve functions of endothelial cells (ECs). We investigated the effect of PPARγ on angiogenic activities of murine ECs and bone marrow-derived proangiogenic cells (PACs).

Methods: PACs were isolated from bone marrow of 10-12 weeks old, wild type, db/db and PPARγ heterozygous animals. Cells were cultured on fibronectin and gelatin coated dishes in EGM-2MV medium. For in vitro stimulations, rosiglitazone (10 μmol/L) or GW9662 (10 μmol/L) were added to 80% confluent cell cultures for 24 hours. Angiogenic potential of PACs and ECs was tested in vitro and in vivo in wound healing assay and hind limb ischemia model.

Results: ECs and PACs isolated from diabetic db/db mice displayed a reduced angiogenic potential in ex vivo and in vitro assays, the effect partially rescued by incubation of cells with rosiglitazone (PPARγ activator). Correction of diabetes by administration of rosiglitazone in vivo did not improve angiogenic potential of isolated PACs or ECs. In a hind limb ischemia model we demonstrated that local injection of conditioned media harvested from wild type PACs improved the blood flow restoration in db/db mice, confirming the importance of paracrine action of the bone marrow-derived cells.

Conclusions: In summary, activation of PPARγ by rosiglitazone improves angiogenic potential of diabetic ECs and PACs, but decreased expression of PPARγ in diabetes does not impair angiogenesis.

Show MeSH
Related in: MedlinePlus