Abstract
Ischemic stroke results in disruption of the blood-brain barrier (BBB), edema formation and neuronal cell loss. Some neuroprotective factors such as vascular endothelial growth factor (VEGF) favor edema formation, while others such as erythropoietin (Epo) can mitigate it. Both factors are controlled by hypoxia inducible transcription factors (HIF) and the activity of prolyl hydroxylase domain proteins (PHD). We hypothesize that activation of the adaptive hypoxic response by inhibition of PHD results in neuroprotection and prevention of vascular leakage. Mice, subjected to cerebral ischemia, were pre- or post-treated with the novel PHD inhibitor FG-4497. Inhibition of PHD activity resulted in HIF-1α stabilization, increased expression of VEGF and Epo, improved outcome from ischemic stroke and reduced edema formation by maintaining BBB integrity. Additional in vitro studies using brain endothelial cells and primary astrocytes confirmed that FG-4497 induces the HIF signaling pathway, leading to increased VEGF and Epo expression. In an in vitro ischemia model, using combined oxygen and glucose deprivation, FG-4497 promoted the survival of neurons. Furthermore, FG-4497 prevented the ischemia-induced rearrangement and gap formation of the tight junction proteins zonula occludens 1 and occludin, both in cultured endothelial cells and in infarcted brain tissue in vivo. These results indicate that FG-4497 has the potential to prevent cerebral ischemic damage by neuroprotection and prevention of vascular leakage.
Highlights
Stroke is one of the most relevant causes of death in the western world and responsible for enormous economic costs caused by illness
To hypoxia inducible transcription factors (HIF)-1a, FG-4497 and DMOG significantly increased the protein abundance of HIF-2a in endothelial cells and astrocytes (Fig. 1). In both cell types expression of the HIF target gene PHD3 on protein level was induced upon treatment with DMOG or FG-4497 (Fig. 1)
Endothelial transcription of the HIF target genes vascular endothelial growth factor (VEGF) and PHD2 was significantly increased upon treatment with either FG-4497 or DMOG (Fig. 2A)
Summary
Stroke is one of the most relevant causes of death in the western world and responsible for enormous economic costs caused by illness. Treatment with recombinant human Epo was shown to preserve the BBB integrity in mice undergoing ischemic stroke [11,12] The majority of these oxygen-dependent regulated genes including VEGF and Epo are target genes of hypoxia-inducible transcription factors (HIFs), heterodimeric proteins consisting of an a (1a, 2a or 3a) and a b subunit [13]. The a subunit is hydroxylated on conserved proline residues, which results in recruitment of the von Hippel-Lindau protein E3 ubiquitin ligase and immediate proteasomal degradation of HIF-a [13] This prolyl hydroxylation is mediated through a family of prolyl-4-hydroxylase domain (PHD) proteins, whose activity is dependent on molecular oxygen, ferrous iron and 2oxoglutarate. Genetic knockout studies in mice showed that PHD2 deficiency leads to embryonic lethality, while PHD1 and PHD3 null mice were viable and apparently normal, substantiating the key role of PHD2 as the master regulator of the hypoxic response [14]
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