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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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Proinflammatory phenotype of PPARγ haplodeficient mice. A-B: Expression of PPARγ in wild type (WT) and PPARγ heterozygotic (PPARγ+/−) mice. A: bone marrow. B: PACs. Quantitative RT-PCR. EF2 serves as an internal control. C-F: Concentration of inflammatory mediators in the blood plasma of WT and PPARγ-HT mice. C: sVCAM-1. D: sICAM-1. E: IL-6. F: IL-1β. Milliplex®MAP platform. Each bar represents mean + SEM. N = 4-6, *p < 0.05, **p < 0.01 versus WT.
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Fig7: Proinflammatory phenotype of PPARγ haplodeficient mice. A-B: Expression of PPARγ in wild type (WT) and PPARγ heterozygotic (PPARγ+/−) mice. A: bone marrow. B: PACs. Quantitative RT-PCR. EF2 serves as an internal control. C-F: Concentration of inflammatory mediators in the blood plasma of WT and PPARγ-HT mice. C: sVCAM-1. D: sICAM-1. E: IL-6. F: IL-1β. Milliplex®MAP platform. Each bar represents mean + SEM. N = 4-6, *p < 0.05, **p < 0.01 versus WT.

Mentions: To verify this supposition, in the last set of experiments we investigated angiogenesis in normoglycemic mice carrying one dysfunctional allele of PPARγ (PPARγ+/−). Such animals had a decreased PPARγ mRNA expression both in the bone marrow and cultured PACs (Figure 7A,B), to a similar extent as in db/db mice. They also had a more proinflammatory phenotype, as illustrated by higher concentrations of sVCAM-1, soluble intercellular adhesion mulecule-1 (sICAM-1), IL-6, and IL-1β in the blood (Figure 7C-F). No differences were found, however, between expressions of genes regulating angiogenic activity (VEGF, KDR, SDF-1, and CXCR4) in PACs isolated form PPARγ+/+ and PPARγ+/− animals (Additional file 4: Figure S3A-D). Also frequency of CD45−KDR+Sca-1+ endothelial precursors were similar in both groups (Additional file 4: Figure S3E-F). Based on transcriptome analysis of PACs isolated from WT and db/db mice we selected a candidate genes to verify effects of PPARγ haplodeficiency and hypoxia on their expression. Cells isolated from PPARγ+/− animals did not display different expression levels of pro-oxidative genes (Nox4, p22phox), nor lower levels of cytoprotective genes (GST-3, Table 2). Despite lower expression of PPARγ in such cells, than in WT ones they were characterized by the same mRNA amount of proinflammatory genes (TNFR-1, P-selectin ligand) and matrix metalloproteinases MMP-2, MMP-9 (Table 2). Similarly, genes associated with proangiogenic properties of PACs (VEGF, SDF-1 and their receptors) were expressed at the same level in PPARγ+/+ and PPARγ+/− cells. However, CAV1 was upregulated in PPARγ+/− PAC (Table 2).Figure 7


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)

Proinflammatory phenotype of PPARγ haplodeficient mice. A-B: Expression of PPARγ in wild type (WT) and PPARγ heterozygotic (PPARγ+/−) mice. A: bone marrow. B: PACs. Quantitative RT-PCR. EF2 serves as an internal control. C-F: Concentration of inflammatory mediators in the blood plasma of WT and PPARγ-HT mice. C: sVCAM-1. D: sICAM-1. E: IL-6. F: IL-1β. Milliplex®MAP platform. Each bar represents mean + SEM. N = 4-6, *p < 0.05, **p < 0.01 versus WT.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4233236&req=5

Fig7: Proinflammatory phenotype of PPARγ haplodeficient mice. A-B: Expression of PPARγ in wild type (WT) and PPARγ heterozygotic (PPARγ+/−) mice. A: bone marrow. B: PACs. Quantitative RT-PCR. EF2 serves as an internal control. C-F: Concentration of inflammatory mediators in the blood plasma of WT and PPARγ-HT mice. C: sVCAM-1. D: sICAM-1. E: IL-6. F: IL-1β. Milliplex®MAP platform. Each bar represents mean + SEM. N = 4-6, *p < 0.05, **p < 0.01 versus WT.
Mentions: To verify this supposition, in the last set of experiments we investigated angiogenesis in normoglycemic mice carrying one dysfunctional allele of PPARγ (PPARγ+/−). Such animals had a decreased PPARγ mRNA expression both in the bone marrow and cultured PACs (Figure 7A,B), to a similar extent as in db/db mice. They also had a more proinflammatory phenotype, as illustrated by higher concentrations of sVCAM-1, soluble intercellular adhesion mulecule-1 (sICAM-1), IL-6, and IL-1β in the blood (Figure 7C-F). No differences were found, however, between expressions of genes regulating angiogenic activity (VEGF, KDR, SDF-1, and CXCR4) in PACs isolated form PPARγ+/+ and PPARγ+/− animals (Additional file 4: Figure S3A-D). Also frequency of CD45−KDR+Sca-1+ endothelial precursors were similar in both groups (Additional file 4: Figure S3E-F). Based on transcriptome analysis of PACs isolated from WT and db/db mice we selected a candidate genes to verify effects of PPARγ haplodeficiency and hypoxia on their expression. Cells isolated from PPARγ+/− animals did not display different expression levels of pro-oxidative genes (Nox4, p22phox), nor lower levels of cytoprotective genes (GST-3, Table 2). Despite lower expression of PPARγ in such cells, than in WT ones they were characterized by the same mRNA amount of proinflammatory genes (TNFR-1, P-selectin ligand) and matrix metalloproteinases MMP-2, MMP-9 (Table 2). Similarly, genes associated with proangiogenic properties of PACs (VEGF, SDF-1 and their receptors) were expressed at the same level in PPARγ+/+ and PPARγ+/− cells. However, CAV1 was upregulated in PPARγ+/− PAC (Table 2).Figure 7

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