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 Tc99m-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.Key messages 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. Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-013-1115-0) contains supplementary material, which is available to authorized users.
Highlights
Peripheral artery disease (PAD) is a common disorder due to atherosclerosis of the leg arteries [1]
We have recently described a model by which mitochondria-derived signals [including mitochondria-derived reactive oxygen species and α-ketoglutarate, a diffusible Krebs' cycle product] can directly regulate hypoxiainducible factor 1α (HIF-1α) in cancer cells, leading to decreased paracrine angiogenic signaling in the tumor [12]
Our findings are clinically relevant, in that impaired angiogenesis might account for an unexpected finding of the PROactive trial, which found that pioglitazone was associated with increased rates of lower extremity revascularization in the 1,274 subjects with concomitant PAD [9]
Summary
Peripheral artery disease (PAD) is a common disorder due to atherosclerosis of the leg arteries [1]. The PROactive trial, a randomized trial of pioglitazone versus placebo in 5238 patients with diabetes [8], unexpectedly showed that pioglitazone was associated with increased hazard for surgical or percutaneous lower extremity revascularization (hazard ratio 1.69, 95 % CI 1.153, 2.484) [9]. Those with PAD did not benefit from pioglitazone with respect to other endpoints like cardiovascular death or acute coronary syndrome
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