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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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Angiogenic potential of PACs and endothelial cells from wild type (WT) and diabetic (db/db) mice. A: Proliferation of PACs. Immunohistochemical detection of PCNA-positive cells. B: Migration of PACs in modified Boyden chambers. C: Formation of sprouts from PACs spheroids embedded in collagen gel, shown as a total length of sprouts per one spheroid. D: Formation of cords by PACs seeded on Matrigel shown as a number of cord junctions per microscopic field. E: Formation of capillaries by endothelial cells from aortic rings isolated from WT or db/db mice and plated on Matrigel. F: Formation of cords by HUVECs seeded on Matrigel and stimulated with empty medium (NC, negative control) or conditioned media from WT PACs and db/db PACs, shown as a number of cord junctions per microscopic field. Each bar represents mean + SEM. N = 4-5 (1A-E), N = 8-9 (1F), *p < 0.05, **p < 0.01 versus WT, ###p < 0.001 versus NC.
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Fig1: Angiogenic potential of PACs and endothelial cells from wild type (WT) and diabetic (db/db) mice. A: Proliferation of PACs. Immunohistochemical detection of PCNA-positive cells. B: Migration of PACs in modified Boyden chambers. C: Formation of sprouts from PACs spheroids embedded in collagen gel, shown as a total length of sprouts per one spheroid. D: Formation of cords by PACs seeded on Matrigel shown as a number of cord junctions per microscopic field. E: Formation of capillaries by endothelial cells from aortic rings isolated from WT or db/db mice and plated on Matrigel. F: Formation of cords by HUVECs seeded on Matrigel and stimulated with empty medium (NC, negative control) or conditioned media from WT PACs and db/db PACs, shown as a number of cord junctions per microscopic field. Each bar represents mean + SEM. N = 4-5 (1A-E), N = 8-9 (1F), *p < 0.05, **p < 0.01 versus WT, ###p < 0.001 versus NC.

Mentions: In a set of in vitro experiments we compared angiogenic potential of wild type and db/db PACs. Spontaneous proliferation, measured using immunohistochemical detection of PCNA, was similar in cells isolated from healthy and diabetic animals (Figure 1A). All other activities measured, namely migration (Figure 1B), sprouting of capillaries from PACs spheroids embedded in collagen (Figure 1C), and formation of cord-like structures on Matrigel (Figure 1D) were attenuated in db/db cells, indicating their impaired angiogenic potential. Similarly, angiogenic activity of mature endothelial cells derived from aorta of db/db mice tended to be weaker, as illustrated by the results of semiquantitative ring assay (Figure 1E).Figure 1


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)

Angiogenic potential of PACs and endothelial cells from wild type (WT) and diabetic (db/db) mice. A: Proliferation of PACs. Immunohistochemical detection of PCNA-positive cells. B: Migration of PACs in modified Boyden chambers. C: Formation of sprouts from PACs spheroids embedded in collagen gel, shown as a total length of sprouts per one spheroid. D: Formation of cords by PACs seeded on Matrigel shown as a number of cord junctions per microscopic field. E: Formation of capillaries by endothelial cells from aortic rings isolated from WT or db/db mice and plated on Matrigel. F: Formation of cords by HUVECs seeded on Matrigel and stimulated with empty medium (NC, negative control) or conditioned media from WT PACs and db/db PACs, shown as a number of cord junctions per microscopic field. Each bar represents mean + SEM. N = 4-5 (1A-E), N = 8-9 (1F), *p < 0.05, **p < 0.01 versus WT, ###p < 0.001 versus NC.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4233236&req=5

Fig1: Angiogenic potential of PACs and endothelial cells from wild type (WT) and diabetic (db/db) mice. A: Proliferation of PACs. Immunohistochemical detection of PCNA-positive cells. B: Migration of PACs in modified Boyden chambers. C: Formation of sprouts from PACs spheroids embedded in collagen gel, shown as a total length of sprouts per one spheroid. D: Formation of cords by PACs seeded on Matrigel shown as a number of cord junctions per microscopic field. E: Formation of capillaries by endothelial cells from aortic rings isolated from WT or db/db mice and plated on Matrigel. F: Formation of cords by HUVECs seeded on Matrigel and stimulated with empty medium (NC, negative control) or conditioned media from WT PACs and db/db PACs, shown as a number of cord junctions per microscopic field. Each bar represents mean + SEM. N = 4-5 (1A-E), N = 8-9 (1F), *p < 0.05, **p < 0.01 versus WT, ###p < 0.001 versus NC.
Mentions: In a set of in vitro experiments we compared angiogenic potential of wild type and db/db PACs. Spontaneous proliferation, measured using immunohistochemical detection of PCNA, was similar in cells isolated from healthy and diabetic animals (Figure 1A). All other activities measured, namely migration (Figure 1B), sprouting of capillaries from PACs spheroids embedded in collagen (Figure 1C), and formation of cord-like structures on Matrigel (Figure 1D) were attenuated in db/db cells, indicating their impaired angiogenic potential. Similarly, angiogenic activity of mature endothelial cells derived from aorta of db/db mice tended to be weaker, as illustrated by the results of semiquantitative ring assay (Figure 1E).Figure 1

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