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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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Related in: MedlinePlus

Effect of PPARγ agonist rosiglitazone (ROSI, 10 μmol/L, 24 h) on expression of proangiogenic genes in PACs from wild type (WT) and diabetic (db/db) mice. PACs cultured without ROSI are used as a control. A: VEGF. B: KDR. C: Flt-1. D: SDF-1. E: CXCR4. F: CXCR7. G: KC. H: Angiotensinogen. I: PRG-4. Quantitative RT-PCR. EF2 serves as an internal control. Each bar represents mean + SEM. N = 8-10, *p < 0.05, **p < 0.01 versus WT, #p < 0.05, ##p < 0.01, ###p < 0.001 versus control.
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Fig3: Effect of PPARγ agonist rosiglitazone (ROSI, 10 μmol/L, 24 h) on expression of proangiogenic genes in PACs from wild type (WT) and diabetic (db/db) mice. PACs cultured without ROSI are used as a control. A: VEGF. B: KDR. C: Flt-1. D: SDF-1. E: CXCR4. F: CXCR7. G: KC. H: Angiotensinogen. I: PRG-4. Quantitative RT-PCR. EF2 serves as an internal control. Each bar represents mean + SEM. N = 8-10, *p < 0.05, **p < 0.01 versus WT, #p < 0.05, ##p < 0.01, ###p < 0.001 versus control.

Mentions: Next, we tested effects of rosiglitazone (10 μmol/L, 24 h) on expression of genes involved in angiogenesis, focusing on two major agents, VEGF and SDF-1. Expression of VEGF, similar in the cultured wild type and db/db PACs, was upregulated in response to rosiglitazone. This upregulation was slightly, but significantly weaker in cells isolated from diabetic mice (Figure 3A). However, we did not see changes on a protein level (data not shown). Expression of KDR (Flk-1, VEGFR-2), the major receptor mediating the proangiogenic VEGF action, was higher in db/db cells, and not modified by rosiglitazone (Figure 3B), while Flt-1 (VEGFR-1) level was affected neither by diabetes nor by rosiglitazone (Figure 3C). Like in the case of VEGF, expression of SDF-1 was similar in control wild type and db/db PACs. Treatment with rosiglitazone showed a tendency toward decrease in SDF-1 levels (Figure 3D), but this effect did not reach statistical significance (p = 0.06 in control PACs and p = 0.12 in db/db PACs). The same trend was observed for protein level (data not shown). Its major receptor, CXCR4 was, however, downregulated in db/db cells, the effect not modified by rosiglitazone (Figure 3E). Expression of CXCR-7 receptor was the same in all experimental groups (Figure 3F). Additionally, we found that both in wild type and db/db PACs rosiglitazone significantly increased production of KC, a potent proangiogenic chemokine (Figure 3G). Cells cultured with rosiglitazone showed also increased expression of two genes which may promote differentiation of proangiogenic precursors, namely angiotensinogen (Figure 3H) and proteolycan-4 (Figure 3I).Figure 3


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 PPARγ agonist rosiglitazone (ROSI, 10 μmol/L, 24 h) on expression of proangiogenic genes in PACs from wild type (WT) and diabetic (db/db) mice. PACs cultured without ROSI are used as a control. A: VEGF. B: KDR. C: Flt-1. D: SDF-1. E: CXCR4. F: CXCR7. G: KC. H: Angiotensinogen. I: PRG-4. Quantitative RT-PCR. EF2 serves as an internal control. Each bar represents mean + SEM. N = 8-10, *p < 0.05, **p < 0.01 versus WT, #p < 0.05, ##p < 0.01, ###p < 0.001 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4233236&req=5

Fig3: Effect of PPARγ agonist rosiglitazone (ROSI, 10 μmol/L, 24 h) on expression of proangiogenic genes in PACs from wild type (WT) and diabetic (db/db) mice. PACs cultured without ROSI are used as a control. A: VEGF. B: KDR. C: Flt-1. D: SDF-1. E: CXCR4. F: CXCR7. G: KC. H: Angiotensinogen. I: PRG-4. Quantitative RT-PCR. EF2 serves as an internal control. Each bar represents mean + SEM. N = 8-10, *p < 0.05, **p < 0.01 versus WT, #p < 0.05, ##p < 0.01, ###p < 0.001 versus control.
Mentions: Next, we tested effects of rosiglitazone (10 μmol/L, 24 h) on expression of genes involved in angiogenesis, focusing on two major agents, VEGF and SDF-1. Expression of VEGF, similar in the cultured wild type and db/db PACs, was upregulated in response to rosiglitazone. This upregulation was slightly, but significantly weaker in cells isolated from diabetic mice (Figure 3A). However, we did not see changes on a protein level (data not shown). Expression of KDR (Flk-1, VEGFR-2), the major receptor mediating the proangiogenic VEGF action, was higher in db/db cells, and not modified by rosiglitazone (Figure 3B), while Flt-1 (VEGFR-1) level was affected neither by diabetes nor by rosiglitazone (Figure 3C). Like in the case of VEGF, expression of SDF-1 was similar in control wild type and db/db PACs. Treatment with rosiglitazone showed a tendency toward decrease in SDF-1 levels (Figure 3D), but this effect did not reach statistical significance (p = 0.06 in control PACs and p = 0.12 in db/db PACs). The same trend was observed for protein level (data not shown). Its major receptor, CXCR4 was, however, downregulated in db/db cells, the effect not modified by rosiglitazone (Figure 3E). Expression of CXCR-7 receptor was the same in all experimental groups (Figure 3F). Additionally, we found that both in wild type and db/db PACs rosiglitazone significantly increased production of KC, a potent proangiogenic chemokine (Figure 3G). Cells cultured with rosiglitazone showed also increased expression of two genes which may promote differentiation of proangiogenic precursors, namely angiotensinogen (Figure 3H) and proteolycan-4 (Figure 3I).Figure 3

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