Elsevier

Brain Research

Volume 1708, 1 April 2019, Pages 160-170
Brain Research

Research report
Astrocytic cytochrome P450 4A/20-hydroxyeicosatetraenoic acid contributes to angiogenesis in the experimental ischemic stroke

https://doi.org/10.1016/j.brainres.2018.12.023Get rights and content

Highlights

  • OGD upregulates CYP4A expression and 20-HETE production in astrocytes.

  • Conditioned media from stimulated astrocytes promotes angiogenesis through 20-HETE.

  • HET0016 suppresses angiogenesis and worsens neurological outcomes 14 days poststroke.

  • 20-HETE mediates OGD activated VEGF and HIF-1α via JNK pathway in astrocytes.

  • HET0016 decreases VEGF and HIF-1α by inhibiting JNK pathway in ischemic brains.

Abstract

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 4A (CYP4A) metabolite of arachidonic acid, is one of the primary eicosanoids in most of microcirculatory beds. Studies have indicated that 20-HETE has important functions in the modulation of vascular tone, ion transport, inflammation reaction, and cellular proliferation. Both we and others have demonstrated that 20-HETE plays an important role in acute phase of ischemic stroke. However, little is known about the effect of 20-HETE on recovery phase of stroke. Crosstalk between the cells within the neurovascular unit is increasingly suspected of playing critical roles in stroke recovery. We found that CYP4A is upregulated in astrocytes exposed to oxygen-glucose deprivation (OGD), which increases the production of 20-HETE that promotes endothelial cell proliferation, tube formation and migration. siRNA suppression of CYP4A or 20-HETE inhibitor prevents this effect. In a mouse model of transient focal cerebral ischemia, inhibition of CYP4A reduces peri-infact angiogenesis and worsens neurological deficits 14 days after stroke. We further showed that ischemia injury increases VEGF and HIF-1α expression in cell cultures and ischemic brains, which is negated by a 20-HETE inhibitor-HET0016. Lastly, we showed that JNK signaling pathway is a component of 20-HETE regulated ischemic angiogenesis after stroke. Taken together, we demonstrated a positive influence of 20-HETE in angiogenesis in later stage of stroke. These molecular and in vivo findings also support a previously undescribed mechanism of crosstalk between reactive astrocytes and endothelial cells wherein 20-HETE promotes neurovascular remodeling and functional recovery after ischemic stroke.

Introduction

Arachidonic acid (AA) can be metabolized by enzymes of the cytochrome P450 4A (CYP4A) family to 20-hydroxyeicosatetraenoic acid (20-HETE) (Hoopes et al., 2015). 20-HETE is a potent mitogen in a variety of cell types and has also been reported to serve as a second messenger in the mitogenic actions of a number of growth factors (Roman and Fan, 2018). Amaral et al. first reported that 20-HETE plays a critical role in angiogenesis induced by chronic electrical stimulation of skeletal muscle (Amaral et al., 2003). Chen and colleagues demonstrated that 20-HETE induces angiogenic responses in rat cornea (Chen et al., 2005). They also found that 20-HETE contributes to ischemia-induced angiogenesis in a mouse hindlimb-ischemic angiogenesis model (Chen et al., 2016). However, little is known regarding the effects of CYP4A/20-HETE on angiogenesis in the recovery phase of ischemic stroke (IS).

Adaptive signaling within the neurovascular unit is critical for the balance between injury and repair after IS (Lo, 2008). Emerging data suggested that neurovascular repair might be induced by dynamic interactions among neuron, glia, and cerebral endothelial cell (EC) from days to weeks after stroke (Zhang and Chopp, 2009). Of these cells, astrocytes are the most numerous nonneuronal cell type in the mammalian brain (Iadecola, 2017). Traditionally, reactive astrocytes may form inhibitory glial scars that block neural remodeling (Fitch and Silver, 2008). However, it has been demonstrated that astrocytes may release angiogenic factors which could promote EC proliferation and angiogenesis during stroke recovery (Carmichael, 2010). Recent studies showed that CYP4A expresses in astrocytes and synthesized 20-HETE when exposed to AA or reactive oxygen species (ROS) (Gebremedhin et al., 2016, Han et al., 2018). Nevertheless, whether CYP4A/20-HETE involved in this astrocyte-EC interaction and promoted angiogenesis after IS remain poorly understood.

Furthermore, the cellular and molecular mechanism by which it may do so are completely unexplored. Since induction of HIF-1α/VEGF signaling is a fundamental component of ischemic angiogenesis induced by 20-HETE (Chen et al., 2012, Chen et al., 2016), we explored the possibility that CYP4A/20-HETE could mediate angiogenesis in response to cerebral ischemia through HIF-1α/VEGF signaling. In this study, we examined the hypothesis that CYP4A/20-HETE mediates the interaction between reactive astrocytes and ECs via HIF-1α/VEGF signaling pathway to promote angiogenesis following IS. In this work, we provide evidence that this is indeed the case by using an oxygen-glucose deprivation (OGD) cell model and a focal cerebral ischemia animal model.

Section snippets

OGD elevates the CYP4A expression and 20-HETE production of astrocytes

OGD significantly induced CYP4A protein activation after 24-hour exposure (Fig. 1A). CYP4A siRNA successfully suppressed CYP4A levels in OGD-stimulated astrocytes (Fig. 1B). Liquid chromatography-tandem mass spectrometry analysis demonstrated that 20-HETE synthesis was significantly increased under OGD stimulation (16.02 ± 3.21 versus 71.23 ± 5.13 ng/min/mg, p < 0.05). Furthermore, both 20-HETE synthesis inhibitor-HET0016 and CYP4A siRNA decreased the levels of 20-HETE in astrocyte-conditioned

Discussion

The study showed that ischemic injury activates the CYP4A in astrocytes and elevates the production of 20-HETE, which mediate the interaction between astrocytes and ECs. We also provided evidence that astrocytic CYP4A/20-HETE appears to track EC proliferation, tube formation and migration. Importantly, blockade of this signal interferes with angiogenesis and stroke recovery. The current study demonstrated that the ischemia-induced angiogenic response relies on a mechanism involving

Reagents and animals

20-HETE and HET0016 were purchased from Cayman Chemical Company (Ann Arbor, Michigan, USA). Antibodies against CYP4A, GFAP, VEGF, HIF-1α, JNK, and c-jun were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho JNK polyclonal antibody and anti-phospho c-jun polyclonal antibody were obtained from Cell Signaling Technology (Beverly, MA, USA).

Male C57BL/6J mice (10 weeks old) from Jackson Laboratories (Bar Harbor, Maine, USA) were used in the in vivo experiments. Adult male

Acknowledgments

This work was supported by National Key Research and Development Program of China (No. 2017YFA0205200), National Natural Science Foundation of China (No. 81571785, 81771957), Natural Science Foundation of Guangdong Province, China (No. 2016A030311055, 2016A030313770), and China Postdoctoral Science Foundation Grant (No. 2018M631038).

Declaration of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article.

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