Inhibition of endogenous HIF inactivation induces angiogenesis in ischaemic skeletal muscles of mice

Abstract

Hypoxia-inducible factor (HIF) modulates transcriptional control of several genes involved in vascular growth and cellular metabolism. HIF activity can be enhanced by suppression of prolyl and asparaginyl hydroxylase activity by dimethyloxalylglycine (DMOG). We have compared the effects of DMOG treatment and femoral artery ligation individually or in combination on HIF-1alpha protein level, HIF-dependent gene expression and capillary-to-fibre ratio (C: F) in extensor digitorum longus and tibialis anterior muscles of mice. Immunohistochemical examination revealed that HIF-1alpha is present in non-ischaemic mouse skeletal muscles, but its amount increased profoundly in response to the combination of DMOG treatment and ischaemia. Combined treatment resulted in 39% increase in C: F in ischaemic muscles (P < 0.0001 versus controls) whereas individual treatments produced little effect under our conditions. Combined treatment led to a significant increase in endogenous HIF-1alpha protein (6.14 +/- 1.1 versus 1.17 +/- 0.2 in controls; P < 0.05) that was not apparent in mice treated with DMOG or femoral artery ligation alone. Ischaemia increased vascular endothelial growth factor (VEGF) protein production by 2.5-fold (P < 0.05 versus controls), irrespective of DMOG treatment. However, production of the VEGF receptor Flk-1 was more enhanced in ischaemic + DMOG-treated muscles (P < 0.001 and P < 0.05 compared with controls and untreated ischaemic muscles, respectively), which may explain the intensive growth of capillaries in those muscles. The findings indicate that treatment with DMOG has a potential therapeutic use in promoting angiogenesis in ischaemic diseases, and perhaps for improving muscle recovery after injury, exercise or training.

Figure 1. Immunohistochemistry of HIF-1α protein expression in mouse skeletal muscle

Photomicrographs of controls (A) and DMOG-treated (B), ischaemic + DMOG-treated (C) TA muscles, and high power micrographs of ischaemic + DMOG-treated TA muscles (D). Arrows indicate HIF-1α-positive nuclei visualised with DAB (brown stain). Sections were counterstained with haematoxylin (blue stain). Scale bars = 20 μm.

Figure 2. Effect of DMOG treatment on protein expression by Western analysis in ischaemic and non-ischaemic mouse EDL (A), quantification of HIF-1α expression (B), and levels of VEGF and Flk-1 expression with treatment (C)

Each bar represents mean ± s.e.m. (n = 4 control, 4 DMOG, 6 ischaemic, 11 ischaemic + DMOG). *P < 0.05 vs. control and DMOG, ^#P < 0.05 vs. ischaemic.