Research reportAstrocytic cytochrome P450 4A/20-hydroxyeicosatetraenoic acid contributes to angiogenesis in the experimental 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.
References (36)
- et al.
Repeated administration of exendin-4 reduces focal cerebral ischemia-induced infarction in rats
Brain Res.
(2012) - et al.
20-HETE in neovascularization
Prostaglandins Other Lipid Mediat.
(2012) - et al.
20-HETE contributes to ischemia-induced angiogenesis
Vasc. Pharmacol.
(2016) - et al.
Inhibitors of cytochrome P450 4A suppress angiogenic responses
Am. J. Pathol.
(2005) - et al.
CNS injury, glial scars, and inflammation: inhibitory extracellular matrices and regeneration failure
Exp. Neurol.
(2008) - et al.
Expression of CYP 4A omega-hydroxylase and formation of 20-hydroxyeicosatetreanoic acid (20-HETE) in cultured rat brain astrocytes
Prostaglandins Other Lipid Mediat.
(2016) - et al.
NOX2, NOX4, and mitochondrial-derived reactive oxygen species contribute to angiopoietin-1 signaling and angiogenic responses in endothelial cells
Vasc. Pharmacol.
(2017) - et al.
Vascular actions of 20-HETE
Prostaglandins Other Lipid Mediat.
(2015) The neurovascular unit coming of age: a journey through neurovascular coupling in health and disease
Neuron
(2017)- et al.
The protective effect of nordihydroguaiaretic acid on cerebral ischemia/reperfusion injury is mediated by the JNK pathway
Brain Res.
(2012)
The protective effect of HET0016 on brain edema and blood-brain barrier dysfunction after cerebral ischemia/reperfusion
Brain Res.
12/15-lipoxygenase inhibition or knockout reduces warfarin-associated hemorrhagic transformation after experimental stroke
Stroke
Continuous inhibition of 20-HETE synthesis by TS-011 improves neurological and functional outcomes after transient focal cerebral ischemia in rats
Neurosci. Res.
Optimization of behavioural tests for the prediction of outcomes in mouse models of focal middle cerebral artery occlusion
Brain Res.
Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic
Lancet Neurol.
CYP4A metabolites of arachidonic acid and VEGF are mediators of skeletal muscle angiogenesis
Am. J. Physiol. Heart. Circ. Physiol.
The adhesive removal test: a sensitive method to assess sensorimotor deficits in mice
Nat. Protoc.
Targets for neural repair therapies after stroke
Stroke
Cited by (19)
Cross-talk between bioactive lipid mediators and the unfolded protein response in ischemic stroke
2023, Prostaglandins and Other Lipid MediatorsUrinary 20-HETE: A prospective Non-Invasive prognostic and diagnostic marker for diabetic kidney disease: Urinary 20-HETE: Prognostic and Diagnostic Marker in Diabetic Kidney Disease
2023, Journal of Advanced ResearchCitation Excerpt :Our findings indicate that urinary 20-HETE levels increase with hypertension, coronary artery disease, stroke, dyslipidemia, and metabolic syndrome (Table 1). These findings can be explained by the predominant effect of 20-HETE on the vascular system, where it stimulates vasoconstriction, modulates endothelial function, and regulates angiogenesis and vascular remodeling [24]. A recent study has well described a positive correlation between the polymorphisms of CYP enzymes producing 20-HETE and systemic hypertension in humans [16].
Dexamethasone impairs neurofunctional recovery in rats following traumatic brain injury by reducing circulating endothelial progenitor cells and angiogenesis
2019, Brain ResearchCitation Excerpt :The strong correlations between these results indicate that the decreased circulating EPC levels and angiogenesis around the lesion may contribute to DEX-induced neurofunctional impairment after TBI. A majority of studies have revealed that angiogenesis is indispensable for improving blood flow and tissue repair after TBI and stroke and is directly associated with improved neurological outcomes (Liu, et al., 2019; Pang et al., 2017; Kaufer et al., 2010; Salehi et al., 2017; Wang et al., 2015). The mobilization of EPCs, a population of bone marrow-derived cells with angiovasculogenic capabilities, is reported after TBI (Li et al., 2012; Liu et al., 2007).
Astrocyte Endfeet in Brain Function and Pathology: Open Questions
2023, Annual Review of NeuroscienceRecent advances in deorphanization of G-protein-coupled receptor 75 and its functions in related diseases
2022, Chinese Journal of Pathophysiology