A novel role for P450 eicosanoids in the neurogenic control of cerebral blood flow in the rat

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1 Exp Physiol 92.4 pp Experimental Physiology Symposium Report A novel role for P450 eicosanoids in the neurogenic control of cerebral blood flow in the rat Jeffrey J. Iliff 1,2, Liesl N. Close 3, Nathan R. Selden 3 and Nabil J. Alkayed 1,2 Departments of 1 Anaesthesiology & Peri-Operative Medicine, 2 Physiology & Pharmacology and 3 Neurological Surgery, Oregon Health & Science University, Portland, OR, USA The P450 eicosanoids epoxyeicosatrienoic acids (EETs) are endogenous lipid mediators produced in the brain by P450 epoxygenases and metabolized through multiple pathways, including soluble epoxide hydrolase (seh). Epoxyeicosatrienoic acids play important functions in the brain, including regulation of cerebral blood flow and protection from ischaemic brain injury. We previously demonstrated that ischaemic preconditioning induces cytochrome P450 2C11 epoxygenase (CYP2C11) expression in the brain, and that pharmacological inhibition and genetic deletion of seh increases EETs and protects against stroke-induced brain damage. However, the expression profiles of CYP2C11 and seh in normal brain remain unknown. In agreement with previous reports in peripheral vessels, we here demonstrate by immunofluorescence double-labelling that within cerebral parenchymal microvessels, seh-immunoreactivity (IR) is localized to the vascular smooth muscle layer. Unexpectedly, however, analysis of large cerebral conduit arteries such as the middle cerebral artery revealed CYP2C11 and seh expression in extrinsic perivascular nerves. Double-labelling studies revealed that CYP2C11- and seh- IR predominantly colocalized with neuronal nitric oxide synthase-ir within perivascular nerve fibres. Significant colocalization for CYP2C11 and seh was also observed with the parasympatheticmarkersvasoactiveintestinalpeptideandcholineactetyltransferase,inaddition to the sensory fibre markers calcitonin gene-related peptide and substance P. No colocalization was observed for either CYP2C11 or seh with the sympathetic nerve markers dopamine β- hydroxylase or neuropeptide Y. The presence of enzymes involved in production and inactivation of EETs within extrinsic parasympathetic and sensory vasodilator fibres suggests a novel role for EETs in the neurogenic control of cerebral arteries. (Received 9 March 2007; accepted after revision 24 April 2007; first published online 27 April 2007) Corresponding author N. J. Alkayed: Department of Anesthesiology & Peri-Operative Medicine, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, UHS-2, Portland, OR , USA. alkayedn@ohsu.edu Cerebral blood flow (CBF) is intricately regulated through multiple mechanisms that operate at different hierarchical levels to maintain adequate tissue perfusion and prevent wide fluctuations in brain blood flow. Locally, cerebral blood vessels respond to changes in their physical (intraluminal pressure, longitudinal shear) and chemical (ph, partial pressures of O 2 and CO 2 ) environments, in addition to sensing and responding to changes in neuronal activity in order to fine-tune and dynamically regulate blood flow rates in accordance with local metabolic demands. This mechanism of blood flow regulation occurs within the so-called neurovascular unit, comprised of neurones, vascular smooth muscle (VSM), endothelium and intervening astrocytes. These different cell types work together to match blood flow with neuronal demands, a process termed neurovascular coupling which forms the basis of functional magnetic resonance imaging (fmri; Edvinsson et al. 1993; Girouard & Iadecola 2006). Neurovascular coupling is mediated in large part by astrocytes, whose processes ensheathe both neuronal synapses and parenchymal arterioles, and which are linked by gap junctions into an electrochemical syncytium (Koehler et al. 2006). According to this model, astrocytes sense neuronal activity through stimulation of metabotropic glutamate receptors (mglurs), which leads to the release of vasoactive substances, such as K + (Filosa DOI: /expphysiol.2006/036889

2 654 J. J. Iliff and others Exp Physiol 92.4 pp et al. 2006) cyclooxygenase (COX) and P450 eicosanoids (Alkayed et al. 1997; Niwa et al. 2000; Peng et al. 2002) to dilate adjacent arterioles and increase nutritive blood flow. In addition to this well-appreciated mode of blood flow metabolism coupling, large conduit arteries such as the middle cerebral artery (MCA) are subject to neurogenic regulation by extrinsic perivascular nerves. Three broad classes of nerve fibres innervate cerebral surface arteries: parasympathetic nitrergic vasodilator fibres, sympathetic adrenergic vasoconstrictor nerves and sensory vasodilator fibres (Hamel, 2006). These extrinsic perivascular nerves are believed to safeguard the brain against extreme fluctuations in CBF, such as occurs during transient hypoperfusion or acute hypertension. Impaired neurogenic control may also have pathophysiological consequences. For example, the recruitment of sensory and parasympathetic vasodilator fibres is believed to underlie the cortical hyperaemia associated with migraine (Edvinsson & Uddman, 2005). Figure 1. Synthesis and metabolism of EETs Epoxidation of arachidonic acid at 5 6, 8 9, or double bonds by cytochrome P450 (CYP450) epoxygenase enzymes results in the formation of four respective epoxyeicosatrienoic acid (EETs) regioisomers. Hydration of the epoxide by soluble epoxide hydrolase (seh) converts EETs to dihydroxyeicosatrieonic acids (DHETs). The schematic diagram depicts the formation and metabolism of 14,15-EET, the preferred substrate for seh. P450 Eicosanoids in CBF regulation Cytochrome P450 epoxygenases catalyse the formation of epoxyeicosatrienoic acids (EETs) from arachidonic acid (AA) via the epoxidation of one of four AA double bonds, resulting in four regioisomers of EETs (Fig. 1): 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET (Roman, 2002). In brain, EETs are vasodilators (Ellis et al. 1990) released from astrocytes following glutamate receptor activation (Alkayed et al. 1996, 1997). Inhibition of the synthesis of EETs blocks functional hyperaemia in brain (Alkayed et al. 1997; Peng et al. 2002, 2004), prompting the proposal that EETs are astrocyte-derived mediators of neurovascular coupling (Harder et al. 1998). Though principally known for their potent vasodilator action, EETs also exert anti-inflammatory, antipyretic, antithrombotic and pro-angiogenic effects (Larsen et al. 2006), in addition to conferring protection against ischaemic injury (Liu & Alkayed, 2005; Gross et al. 2007; Koerner et al. 2007). These varied protective properties have led to the targeting of EETs as potential neuroprotective agents against ischaemic injury in the brain (Liu & Alkayed, 2005; Koerner et al. 2007; Zhang et al. 2007). However, exogenous administration of EETs as therapeutic agents is hampered by their chemical instability and short halflife. As an alternative, we targeted the metabolic pathways of EETs as a means of increasing the bioavailability of endogenous brain EETs. The biological effects of EETs are terminated through multiple pathways, including hydration to dihydoxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (seh, Fig. 1). The inhibition of hydration of EETs by pharmacological blockade or gene deletion of seh is currently under investigation as a novel means of promoting EETs-mediated neuroprotection. Our studies suggest that both seh inhibition and gene deletion are protective against ischaemic brain damage (Zhang et al. 2006, 2007). In the case of seh gene deletion, we observed marked preservation of brain blood flow during the ischaemic period in seh knockout (sehko) mice compared with wild-type control animals (Zhang et al. 2006), an effect that we ascribe to an increase in bioavailable EETs and their vasodilator action within the cerebral circulation.

3 Exp Physiol 92.4 pp Signalling by epoxyeicosatrienoic acids in neurogenic vasodilatation 655 Expression of the EETs system within the neurovascular unit The observed cerebral haemodynamic preservation under ischaemic conditions in the sehko mouse prompted us to investigate the cerebrovascular expression of cytochrome P450 2C11 epoxygenase (CYP2C11) and seh. Using immunofluorescent double-labelling with cell-specific markers, we first confirmed the presence of CYP2C11 immunoreactivity (IR) within glial fibrillary acidic protein (GFAP)-positive astrocytes (Fig. 2A). We then determined seh expression in cerebral parenchymal microvessels. In these vessels, seh-ir colocalized with myosin heavy chain I (MHC-I), suggesting that seh-ir is specifically expressed within VSM cells (Fig. 2B). This finding is in broad agreement with results from peripheral vascular beds, where seh expression is primarily localized within the VSM, and where it is presumed to terminate the activity of endothelium-derived vasodilator EETs (Enayetallah et al. 2004, 2006). Analogously, we propose that astrocytic CYP2C11 (synthesizing EETs) and vascular smooth muscle seh (terminating the action of EETs) form the functional unit of EETs signalling within the neurovascular unit (Fig. 3). According to this model, the increased blood flow response to vascular occlusion observed in sehko mice is attributed to the loss of VSM seh, resulting in enhanced dilation by EETs within the neurovascular unit and preserved collateral blood flow during focal vascular occlusion (Zhang et al. 2006). Cytochrome P450 2C11 epoxygenase and seh within cerebral extrinsic perivascular nerves We then investigated the expression of seh and CYP2C11 in whole-mount large cerebral surface vessels, such as the MCA. In agreement with our findings in parenchymal vessels, we observed no CYP2C11 expression within the VSM or endothelium, whereas seh-ir was observed in vascular cells exhibiting the circumferential orientation characteristic of arterial VSM cells (data not shown). However, by closely examining the MCA main trunk, we unexpectedly observed CYP2C11- and seh-ir within perivascular nerves innervating the MCA (Fig. 2C and F). Innervation of the MCA by CYP2C11- and seh-positive fibres extended along the trunks of the conduit arteries and their most proximal branches, but terminated prior to distal surface branches and penetrating arterioles. As already mentioned, three distinct populations of nerve fibres are known to innervate the cerebral vasculature at this level: parasympathetic nitrergic vasodilator fibres originating in the sphenopalatine (SPG) and otic ganglia (OG), sympathetic adrenergic vasoconstrictor fibres originating in the superior cervical ganglia (SCG) and calcitonin gene-related peptide (CGRP)-releasing sensory fibres originating in the trigeminal ganglia (TG; Hamel, 2006). Given the vasodilator properties of EETs, we hypothesized that CYP2C11 and seh were expressed within extrinsic parasympathetic and sensory vasodilator fibres innervating the MCA. Figure 2. Expression of CYP2C11 and seh within the cerebral vasculature A, CYP2C11-IR is expressed in GFAP-positive cortical astrocytes. Using confocal microscopy ( 3000 magnification), CYP2C11-IR (green) is observed in the cell bodies corresponding to GFAP-positive (red) processes. B, seh-ir is expressed in cerebral parenchymal blood vessels. Confocal microscopy ( 3000), detected seh-ir (green) within the same cell layer as the vascular smooth muscle marker myosin heavy chain I (MHC-I, red). C, CYP2C11-IR (green) is observed in cerebral perivascular nerves innervating the MCA. Conventional fluorescence microscopy at 400 magnification is utilized to accentuate whole-vessel morphology. D and E, immunofluorescent double-labelling of CYP2C11 (green) and nnos (red) within perivascular fibres (confocal microscopy, 600 for D and 3000 for E). F, seh-ir is observed in cerebral perivascular nerves around the MCA ( 400 conventional fluorescence microscopy). G and H, immunofluorescent double-labelling of seh (green) and nnos (red) within perivascular fibres (confocal microscopy, 600 for G and 3000 for H).

4 656 J. J. Iliff and others Exp Physiol 92.4 pp Table 1. Co-localization of CYP2C11 and SEH with nerve population markers Parasympathetic Sensory Sympathetic CYP 2C11 seh ChAT VIP nnos CGRP SubP DBH NPY CYP2C seh CYP2C11, cytochrome P450 epoxygenase isoform 2C11; seh, soluble epoxide hydrolase; ChAT, choline acetyltransferase; VIP, vasoactive intestinal peptide; nnos, neuronal nitric oxide synthase; CGRP, calcitonin gene-related peptide; SubP, substance P; DBH, dopamine β-hydroxylase; and NPY, neuropeptide Y. Colocalization:, no colocalization; +, < 25%; ++, 25 50%; +++, 50 75%; ++++, > 75%. Using immunofluorescent double-labelling and confocal microscopy, we colocalized CYP2C11- and seh-ir with known markers of the parasympathetic, sympathetic and sensory nerve populations that Figure 3. Two proposed modes of CBF regulation by EETs Within the neurovascular unit (bottom), astrocytes respond to neuronal release of glutamate (GLUT) with production of EETs by CYP2C11 (P450) and their subsequent release from astrocytic end-feet encompassing parenchymal arterioles. The vasodilator effect of astrocyte-derived EETs is terminated by soluble epoxide hydrolase (seh) localized in the arteriolar vascular smooth muscle. At the level of the cerebral conduit arteries (top), EETs may be released from parasympathetic fibres or sensory fibres originating in the otic/sphenopalatine ganglia (SPG) or trigeminal ganglia (TG), respectively. These fibres are also known to release nitric oxide (NO, parasympathetic) and calcitonin gene-related peptide (CGRP, sensory) to mediate their vasodilator effects. Signalling by EETs is regulated and terminated by seh localized to the nerve fibres and the vascular smooth muscle. innervate large cerebral arteries. Table 1 summarizes the proportions of CYP2C11- or seh-ir fibres (rows) co-expressing CYP2C11, seh or nerve population markers (columns). In general, seh-positive fibres were more numerous around the MCA than CYP2C11-positive nerves. Double-labelling demonstrated that all (100%) CYP2C11-positive fibres were seh positive, whereas only a fraction (40%) of seh-positive fibres colabelled for CYP2C11 (n = 3 rats). Sample size (n) refers to the total number of rats examined for each double-labelling study. Two MCAs from each rat were labelled and analysed for colocalization by confocal microscopy. Colocalization was initially observed at 600 magnification in five to 20 fibres per artery. Specific colocalization within individual fibres was confirmed at higher magnification ( 3000) in two to five fibres per artery. Fig. 2D, E, G and H depicts representative fibres at these magnifications. Antibody specificity was confirmed by omitting the primary antibody, by using sehko mouse brain tissue and by performing antigen competition studies. In addition to nitric oxide (NO), parasympathetic fibres innervating the cerebral vessels release vasoactive intestinal peptide (VIP) and acetylcholine (ACh; Hamel, 2006). Therefore, we first colocalized CYP2C11-IR and seh- IR with these parasympathetic markers. We found that within perivascular fibres innervating the MCA, 100% of CYP2C11-positive fibres colabelled for neuronal nitric oxide synthase (nnos; n = 5, Fig. 2D and E). Fibres expressing VIP or choline acetyltransferae (ChAT) were less numerous than nnos-positive fibres, and represented a fraction (15%, n = 3 each) of CYP2C11-expressing fibres. Similarly, most seh-positive fibres (75%, n = 6) co-expressed nnos (Fig. 2G and H), whereas ChAT- and VIP-positive fibres represented a small portion (5 10%, n = 3 each) of the larger seh-positive fibre pool. Perivascular sensory fibres also express nnos, in addition to such peptidergic vasodilators as CGRP and substance P (SubP; Hamel, 2006). Both CYP2C11- and seh-ir were observed to colocalize with CGRP at a high frequency (60 and 50% of fibres, respectively; n = 3 each). Markedly fewer SubP- than CGRP-positive fibres were observed innervating the MCA, and these fibres represented a correspondingly small proportion of CYP2C11- and seh-expressing fibres (20 and 15%, respectively, n = 2 each).

5 Exp Physiol 92.4 pp Signalling by epoxyeicosatrienoic acids in neurogenic vasodilatation 657 Sympathetic innervation of the cerebral conduit vessels is primarily adrenergic, although many fibres also express neuropeptide Y (NPY; Hamel, 2006). Double-labelling studies demonstrated that CYP2C11 did not colocalize with the adrenergic marker dopamine β-hydroxylase (DBH) or NPY in these perivascular nerves. Similarly, seh expression was observed only in a small portion of DBHor NPY-positive fibres. However, this co-expression was infrequent and was observed only in those fibres exhibiting the weakest seh-ir. In summary, the results of these studies suggest that components of the EETs signalling system, EETs-synthetic CYP2C11 and EETs-metabolizing seh, are present within both parasympathetic and sensory vasodilator fibres innervating the MCA. Co-expression was observed to be greatest in nnos-positive fibres, which are likely to be comprised of nitrergic parasympathetic and sensory nerve populations. Conclusions Previous studies indicated that within the neurovascular unit, the primary cell type responsible for synthesis of EETs is astrocytes. Our present findings suggest that within the neurovascular unit, the site of metabolic termination for EETs is in the VSM. Furthermore, we uncovered in the present study a previously unknown potential mode of CBF regulation by EETs. The observations in our study that the biosynthetic and metabolic enzymes of EETs are expressed in perivascular nerve fibres suggest that EETs may serve as a nerve-derived vasodilator agent in large cerebral conduit arteries (Fig. 3). This novel role of EETs may contribute to the haemodynamic response to cerebral ischaemia observed in sehko mice (Zhang et al. 2006), and may play an important role in such disease states characterized by cerebrovascular dysregulation as migraine (Edvinsson & Uddman, 2005), cerebral ischaemia and vasospasm following subarachnoid haemorrhage. References Alkayed NJ, Birks EK, Narayanan J, Petrie KA, Kohler-Cabot AE & Harder DR (1997). Role of P-450 arachidonic acid epoxygenase in the response of cerebral blood flow to glutamate in rats. Stroke 28, Alkayed NJ, Narayanan J, Gebremedhin D, Medhora M, Roman RJ & Harder DR (1996). Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes. Stroke 27, Edvinsson L, MacKenzie ET & McCulloch J (1993). Cerebral Blood Flow and Metabolism. Raven Press, New York. Edvinsson L & Uddman R (2005). Neurobiology in primary headaches. Brain Res Brain Res Rev 48, Ellis EF, Police RJ, Yancey L, McKinney JS & Amruthesh SC (1990). 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6 658 J. J. Iliff and others Exp Physiol 92.4 pp Zhang W, Koop D & Alkayed NJ (2006). Soluble epoxide hydrolase gene deletion is associated with increased CBF and reduced stroke damage. Stroke 682 [Abstract P183]. Acknowledgements The authors acknowledge the generous contribution of seh antibody by Dr Bruce Hammock and graphical support from Andrew Rekito. Studies were supported by NINDS R01NS and PO1 NS to N.J.A. and the OHSU Brain Institute Neurobiology of Disease Award to J.J.I.