title: Pioglitazone inhibits HIF-1α-dependent angiogenesis in rats by paracrine and direct effects on endothelial cells.
abstract: Pioglitazone was associated with increased hazard for surgical or percutaneous lower extremity revascularization in patients with diabetes in a large clinical trial, but this clinical finding has not been adequately explored in animal models. We hypothesized that pioglitazone would decrease hypoxia-inducible factor 1α (HIF-1α)-dependent angiogenesis in rat ischemic hindlimb models by altering mitochondrial-derived signals supporting HIF-1α activation. We tested oral pioglitazone (10 mg/kg/day) versus placebo in two cohorts of rats with hindlimb ischemia (normal Sprague-Dawley rats and insulin-resistant JCR:La-cp rats), and evaluated direct and paracrine effects of pioglitazone on angiogenesis in vitro using human skeletal muscle and endothelial cells. Pioglitazone treatment was associated with reductions in limb perfusion at 2 weeks measured by contrast-enhanced ultrasound and Tc(99m)-Sestamibi SPECT-CT. Ischemic muscle capillary density was also reduced by pioglitazone. HIF-1α and vascular endothelial growth factor (VEGF) expression in ischemic muscle were also reduced by pioglitazone. In vitro, pioglitazone's effects on both skeletal muscle cells and microvascular endothelial cells were associated with a decrease in autocrine and paracrine angiogenesis measured by matrigel assay, decreased HIF-1α expression and activation, as well as increases in both mitochondrial reactive oxygen species and α-ketoglutarate, both mitochondria-derived signals which promote HIF-1α degradation. We conclude that pioglitazone is associated with decreased ischemic limb perfusion and capillary density in relevant rat models of hindlimb ischemia, and these effects are mediated by mitochondria-dependent reductions in HIF-1α-dependent angiogenesis.
  
  
  CONCLUSIONS
  Pioglitazone inhibits angiogenesis in rats with and without insulin resistance. Pioglitazone inhibits HIF-1α by inhibiting mitochondrial stabilization of HIF-1. Pioglitazone inhibits both autocrine and paracrine angiogenesis. Inhibition of angiogenesis may explain unexpected results of a pioglitazone human clinical trial.
authors.affiliation: Department of Medicine, University of Alberta, 8440 112th Street, Edmonton, AB, T6G 2B7, AB, Canada.
authors.forename: Peter
authors.initials: P
authors.lastname: Dromparis
authors.forename: Gopinath
authors.initials: G
authors.lastname: Sutendra
authors.forename: Roxane
authors.initials: R
authors.lastname: Paulin
authors.forename: Spencer
authors.initials: S
authors.lastname: Proctor
authors.forename: Evangelos D
authors.initials: ED
authors.lastname: Michelakis
authors.forename: M Sean
authors.initials: MS
authors.lastname: McMurtry
mesh_terms: D020533:Angiogenesis Inhibitors / Q000627:therapeutic use*; D000818:Animals; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D006614:Hindlimb / Q000098:blood supply* / Q000187:drug effects; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007511:Ischemia / Q000188:drug therapy* / Q000378:metabolism; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000098:blood supply / Q000187:drug effects / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D019899:Paracrine Communication / Q000187:drug effects; D000077205:Pioglitazone; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D045162:Thiazolidinediones / Q000627:therapeutic use*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D020533:Angiogenesis Inhibitors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D045162:Thiazolidinediones; D042461:Vascular Endothelial Growth Factor A; D000077205:Pioglitazone