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

Abstract

BackgroundPeroxisome 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).MethodsPACs 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.ResultsECs 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.Transcriptome analysis showed an upregulation of prooxidative and proinflammatory pathways, and downregulation of several proangiogenic genes in db/db PACs. Interestingly, db/db PACs had also a decreased level of PPARγ and changed expression of PPARγ-regulated genes. Using normoglycemic PPARγ+/− mice we demonstrated that reduced expression of PPARγ does not influence neovascularization either in wound healing or in hind limb ischemia models.ConclusionsIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-014-0150-7) contains supplementary material, which is available to authorized users.

PPAR Gamma and Angiogenesis: Endothelial Cells Perspective

“…Only the proliferation rate was not significantly affected [24]. Our data are in accordance with other studies showing decreased angiogenic potential in diabetic EPCs isolated from both humans and rodents [12, 79–82].…”

“…Our recent data also proved that conditioned media is more efficient than the injection of cells for restoring blood perfusion in a mouse ischemic limb model. It was also noteworthy that, in this model, approximately 70% of the injected cells were eluted from the site of injection within first 6 hours, and most remaining cells died during first three days after injection [23, 24]. …”

“…Interestingly, the treatment of pregnant wild-type mice with rosiglitazone also resulted in a disorganization of placental microvasculature [37]. However, our recently published data indicate that angiogenesis in wound healing and hind limb ischemia models is not affected by an ~50% decrease in the expression of PPAR γ in normoglycemic PPAR γ +/− mice [24]. …”

“…Finally, the use of different isolation protocols, different culture conditions, and even different markers for EPC characterization makes it difficult to compare results obtained by different research groups. For these reasons, in our studies, we use term EPCs only for cells analyzed by flow cytometry with strictly a defined phenotype (CD45−/Sca-1+/KDR+), whereas we refer to cells isolated and expanded in vitro as a bone marrow-derived proangiogenic cells (PACs) [24, 78]. …”

“…A paper published by Liang et al demonstrated that AGEs, which are present in diabetes, induce EPC apoptosis and impair SDF-1 and NO production [85]. Transcriptome analysis suggested that the impaired migration of PACs can be associated with upregulation of integrins and the concomitant downregulation of genes involved in filopodia formation, such as efexin or CCT2 [24, 91]. Furthermore, PACs isolated from db/db mice showed decreased paracrine potential, which could be related to decreased expression of VEGF-C, VEGF-D, FGF7, or angiogenin.…”