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

Jerzy Kotlinowski,Magdalena Kozakowska,Bartosz Pilecki,Attila Pap,Aleksandra Bartelik,Laszlo Nagy,Anna Grochot-Przeczek,Klaudia Skrzypek,Alicja Jozkowicz,Jozef Dulak,Hevidar Taha,Anton J G Horrevoets,Rafal Derlacz

doi:10.1186/s12933-014-0150-7

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.

Highlights

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent transcription factor of nuclear receptor superfamily, involved in regulation of lipid metabolism, insulin sensitivity, and inflammatory response [1,2]
Anti-mouse antibodies against stem cell antigen-1 (Sca-1), chemokine receptor type-4 (CXCR4), CD45, and kinase insert domain receptor (KDR, VEGF receptor-2 (VEGFR-2)) used for immunophenotyping of proangiogenic cells (PACs), antibodies recognizing mouse CD31 used for immunohistochemistry and PharmLyse were obtained from BD Biosciences
Characterization of bone marrow-derived PACs Bone marrow-derived proangiogenic cells isolated from healthy C57BL6/J mice and cultured for 9–11 days under conditions promoting the endothelial differentiation, generated a heterogeneous population, with 80-90% of cells incorporating acLDL and binding BS1 lectin, capable of forming the capillary-like structures after seeding on Matrigel (Additional file 2: Figure S1A-D), which confirmed our earlier observation [12]

Summary

Introduction

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent transcription factor of nuclear receptor superfamily, involved in regulation of lipid metabolism, insulin sensitivity, and inflammatory response [1,2]. In further studies a broad spectrum of protocols for isolation or characterization of EPCs has been used, which makes the comparison of the results difficult It seems, that cells named EPCs are rather a heterogenous population containing mainly monocytes with angiogenic properties [9]. That cells named EPCs are rather a heterogenous population containing mainly monocytes with angiogenic properties [9] They can produce a range of growth factors including the vascular endothelial growth factor-A (VEGF-A), stromal cellderived factor-1 (SDF-1), or interleukin-8 (IL-8), and this paracrine activity is supposed to be the most important for compensatory vascularization in ischemic tissues [10,11,12]. 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

Results

Conclusion