Abstract
Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the hypoxic induction of >300 genes required for survival and adaptation under oxygen deprivation. Inhibition of HIF-P4H-2 has been shown to be protective in focal cerebral ischemia rodent models, while that of HIF-P4H-1 has no effects and inactivation of HIF-P4H-3 has adverse effects. A transmembrane prolyl 4-hydroxylase (P4H-TM) is highly expressed in the brain and contributes to the regulation of HIF, but the outcome of its inhibition on stroke is yet unknown. To study this, we subjected WT and P4htm−/− mice to permanent middle cerebral artery occlusion (pMCAO). Lack of P4H-TM had no effect on lesion size following pMCAO, but increased inflammatory microgliosis and neutrophil infiltration was observed in the P4htm−/− cortex. Furthermore, both the permeability of blood brain barrier and ultrastructure of cerebral tight junctions were compromised in P4htm−/− mice. At the molecular level, P4H-TM deficiency led to increased expression of proinflammatory genes and robust activation of protein kinases in the cortex, while expression of tight junction proteins and the neuroprotective growth factors erythropoietin and vascular endothelial growth factor was reduced. Our data provide the first evidence that P4H-TM inactivation has no protective effect on infarct size and increases inflammatory microgliosis and neutrophil infiltration in the cortex at early stage after pMCAO. When considering HIF-P4H inhibitors as potential therapeutics in stroke, the current data support that isoenzyme-selective inhibitors that do not target P4H-TM or HIF-P4H-3 would be preferred.
Highlights
Chemical inhibition of HIF-P4Hs has been shown to have protective effects in rodent models of permanent or transient focal cerebral ischemia (1523)
The earliest pathological hallmarks comprise the breakdown of transcellular ion gradients due to reduced oxygen and energy supply, cytotoxic edema, production of toxic free radicals, a progressive thrombus formation in the cerebral microvasculature and a strong inflammatory response leading to increased reduced apoptosis and blood brain barrier (BBB) disruption [19,24,25]
As HIFP4Hs are currently regarded as potential therapeutic targets in ischemic conditions [33,34,35], we used P4htm-/- mice to evaluate the role of this enzyme in the early response to cerebral ischemia
Summary
To study the effect of P4H-TM inactivation on acute outcome of cerebral ischemia, we subjected P4htm-/- mice to permanent middle cerebral artery occlusion (MCAO) for 24 h, after which the infarct volume and location were quantified by magnetic resonance imaging (MRI) analysis (Figure 1A-D). QRT-PCR analysis revealed significant downregulation of the mRNA qRT-PCR analysis of the gene expression for the expression of occludin and a trend for decreased microglia activation markers CD11b and CD16 expression of claudin-5 in the injured cortex of the [50] in cortical tissue revealed slight upregulation WT mice 24 h after the onset of the MCAO, while no difference was seen in the expression of ZO-1 these two conditions are not equivalent as, for (Figure 9A-C). Our data show that inactivation of P4H-TM results in a variety of molecular level effects, including alterations in HIF stabilization, MAPK signaling, complement C1q amount and BBB function, which jointly can contribute to the observed increased inflammatory response following MCAO.
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