Modulation of vasoactive and cytotrophic factors in developing mouse brain by pharmacological stabilization of hypoxia-inducible transcription factors (HIFs)

Aims: Hypoxia-inducible transcription factors (HIFs) are characterized as the most important mediators of adaptive mechanisms to hypoxia. During brain development, specific HIF target genes are involved in vasculogenesis, metabolic processes, cell migration and differentiation. The aim of our ongoing study is to investigate in vivo effects of HIF stabilization on vasoactive and neurotrophic HIF-regulated factors in developing mouse brain using a prolyl hydroxylase inhibitor (PHI).

Methods: Adult (n=5) and neonatal (P7, n=15) C57/BL6 wild-type mice were treated with PHI FG-4497 (30–100mg/kg, i.p.). Upon an incubation period of 6h, brains were analyzed in comparison to vehicle-treated (VT) and non-treated controls (NT). Gene expression (VEGF, vascular endothelial growth factor; ADM, adrenomedullin; EPO, erythropoietin; chemokine receptor CXCR4) was determined by TaqMan RT PCR. Protein levels were quantified by ELISA.

Results: Dose-response studies revealed systemic effects of PHI at a dose of 60mg/kg, exemplified by a 2-fold increase of VEGF serum levels and cerebral VEGF mRNA up-regulation in adult mice compared to controls (mean±SEM). By optimizing the dose of PHI, we observed most prominent effects in neonatal mouse brains (P7) at a dose of 100mg/kg. Compared to controls, PHI significantly increased mRNA levels of VEGF (PHI, 1.25±0.14, n=4; vs. VT, 0.48±0.02, n=5; NT, 0.52±0.05, n=5; p<0.05) and ADM (PHI, 0.69±0.21, n=4; vs. VT, 0.20±0.03, n=5; NT, 0.13±0.02, n=5; p<0.05). In addition, PHI treatment significantly up-regulated mRNA levels of cytotrophic factor EPO (PHI, 0.62±0.27; vs. VT, 0.07±0.03; NT, 0.05±0.01; p<0.05) and chemokine receptor CXCR4 (PHI, 1.05±0.24; vs. VT, 0.27±0.05; NT, 0.39±0.07; p<0.05) compared to controls.

Conclusions: PHI penetrates blood-brain-barrier at early stage of mouse brain maturation (P7). Suggested by significant up-regulation of specific HIF-regulated target genes, PHI might modulate vasoactive and neurotrophic regulatory processes known to be crucially involved in early brain maturation and hypoxia-induced brain pathology.