Soluble epoxide inhibition is protective against cerebral ischemia via vascular and neural protection.
ABSTRACT: Inhibition of soluble epoxide hydrolase (SEH), the enzyme responsible for degradation of vasoactive epoxides, protects against cerebral ischemia in rats. However, the molecular and biological mechanisms that confer protection in normotension and hypertension remain unclear. Here we show that 6 weeks of SEH inhibition via 2 mg/day of 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) in spontaneously hypertensive stroke-prone (SHRSP) rats protects against cerebral ischemia induced by middle cerebral artery occlusion, reducing percent hemispheric infarct and neurodeficit score without decreasing blood pressure. This level of cerebral protection was similar to that of the angiotensin-converting enzyme inhibitor, enalapril, which significantly lowered blood pressure. SEH inhibition is also protective in normotensive Wistar-Kyoto (WKY) rats, reducing both hemispheric infarct and neurodeficit score. In SHRSP rats, SEH inhibition reduced wall-to-lumen ratio and collagen deposition and increased cerebral microvessel density, although AUDA did not alter middle cerebral artery structure or microvessel density in WKY rats. An apoptosis mRNA expression microarray of brain tissues from AUDA-treated rats revealed that AUDA modulates gene expression of mediators involved in the regulation of apoptosis in neural tissues of both WKY and SHRSP rats. Hence, we conclude that chronic SEH inhibition protects against cerebral ischemia via vascular protection in SHRSP rats and neural protection in both the SHRSP and WKY rats, indicating that SEH inhibition has broad pharmacological potential for treating ischemic stroke.
Project description:We tested the hypothesis that apneas during the sleep cycle exacerbate hypertension and accelerate changes that occur with cerebral small vessel disease. Obstructive sleep apnea was modeled by intermittent inflations of a chronically implanted tracheal balloon to occlude the airway during the sleep cycle (termed OSA) in spontaneously hypertensive stroke-prone (SHRSP) rats, a model of cerebral small vessel disease. SHRSP rats and their parent strain, Wistar Kyoto (WKY) rats, were exposed to OSA for 2 weeks (from 9 to 11 or from 18 to 20 weeks). At 9 weeks, hypertension was developing in the SHRSP rats and was firmly established by 18 weeks. OSA exposure increased systolic blood pressure in SHRSP rats by ?30 mm Hg in both age groups compared with shams that were surgically prepared but not exposed to OSA (P<0.05). OSA exposure also increased systolic blood pressure in WKY rats by 20 and 37 mm Hg at 11 and 20 weeks, respectively (P<0.05). OSA exposure in SHRSP rats compromised blood-brain barrier integrity in white matter at both 11 and 20 weeks of age when compared with SHRSP sham rats (P<0.05). Microglia were activated in SHRSP rats exposed to OSA but not in sham rats at 11 weeks (P<0.05). At 20 weeks, microglia were activated in sham SHRSP rats (P<0.05) compared with WKY sham rats and were not further activated by OSA. Neither was blood-brain barrier integrity altered nor microglia activated in any of the WKY groups. We conclude that OSA accelerates the onset of the cerebral pathologies associated with cerebral small vessel disease in SHRSP, but not WKY, rats.
Project description:The P450 eicosanoids epoxyeicosatrienoic acids (EETs) are produced in brain and perform important biological functions, including protection from ischemic injury. The beneficial effect of EETs, however, is limited by their metabolism via soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH inhibition is protective against ischemic brain damage in vivo by a mechanism linked to enhanced cerebral blood flow (CBF). We determined expression and distribution of sEH immunoreactivity (IR) in brain, and examined the effect of sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE) on CBF and infarct size after experimental stroke in mice. Mice were administered a single intraperitoneal injection of AUDA-BE (10 mg/kg) or vehicle at 30 mins before 2-h middle cerebral artery occlusion (MCAO) or at reperfusion, in the presence and absence of P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH). Immunoreactivity for sEH was detected in vascular and non-vascular brain compartments, with predominant expression in neuronal cell bodies and processes. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester was detected in plasma and brain for up to 24 h after intraperitoneal injection, which was associated with inhibition of sEH activity in brain tissue. Finally, AUDA-BE significantly reduced infarct size at 24 h after MCAO, which was prevented by MS-PPOH. However, regional CBF rates measured by iodoantipyrine (IAP) autoradiography at end ischemia revealed no differences between AUDA-BE- and vehicle-treated mice. The findings suggest that sEH inhibition is protective against ischemic injury by non-vascular mechanisms, and that sEH may serve as a therapeutic target in stroke.
Project description:We hypothesized that perinatal inhibition of soluble epoxide hydrolase (SEH), which metabolizes epoxyeicosatrienoic acids in the arachidonic acid (AA) cascade, with an orally active SEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), would persistently reduce blood pressure (BP) in adult SHR despite discontinuation of AUDA at 4 wk of age. Renal cytoplasmic epoxide hydrolase-2 (Ephx2) gene expression was enhanced in SHR vs. WKY from 2 days to 24 wk. Effects of perinatal treatment with AUDA, supplied to SHR dams until 4 wk after birth, on BP in female and male offspring and renal oxylipin metabolome in female offspring were observed and contrasted to female SHR for direct effects of AUDA (8-12 wk). Briefly, inhibition of SEH was effective in persistently reducing BP in female SHR when applied during the perinatal phase. This was accompanied by marked increases in major renal AA epoxides and decreases in renal lipoxygenase products of AA. Early inhibition of SEH induced a delayed increase in renal 5-HETE at 24 wk, in contrast to a decrease at 2 wk. Inhibition of SEH in female SHR from 8 to 12 wk did not reduce BP but caused profound decreases in renal 15(S)-HETrE, LTB4, TBX2, 5-HETE, and 20-HETE and increases in TriHOMEs. In male SHR, BP reduction after perinatal AUDA was transient. Thus, Ephx2 transcription and SEH activity in early life may initiate mechanisms that eventually contribute to high BP in adult female SHR. However, programmed BP-lowering effects of perinatal SEH inhibition in female SHR cannot be simply explained by persistent reduction in renal SEH activity but rather by more complex and temporally dynamic interactions between the renal SEH, lipoxygenase, and cyclooxygenase pathways.
Project description:Hypertension is associated with cerebral small vessel disease (SVD) and with diffuse white matter hyperintensities (WMH) on T2-weighted magnetic resonance imaging (MRI). We tested whether stroke-prone spontaneously hypertensive rats (SHRSP), a model of chronic hypertension, exhibit WMH. Male SHRSP (age 10 months) without stroke symptoms were compared with age-matched male WKY rats. Stroke-prone spontaneously hypertensive rats exhibited no WMH on MRI scans (T2, T2*, diffusion tensor imaging) and no neuropathological lesions. While leptomeningeal arteries exhibited fibrohyaline wall thickening, with decreased smooth muscle actin relative to WKY, deep penetrating arterioles within the caudate nuclei had no vasculopathy. We conclude that WMH are not an obligate feature of stroke-free SHRSP aged up to 10 months.
Project description:Inhibition and deletion of soluble epoxide hydrolase (sEH) has been suggested to ameliorate infarction in experimental ischemic stroke possibly via vasoactive epoxyeicosatrienoic acids. However, it is unknown whether the neuroprotective mechanisms involve alteration of post-ischemic neuronal transmission and neurotrophic signaling. We used a permanent middle cerebral artery occlusion (MCAO) model in adult wild-type mice with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA) post-treatment and in sEH knockout (sEH KO) mice. We found that sensorimotor recovery was significantly enhanced after MCAO in both AUDA-treated and sEH KO mice, with decreased sEH activity and brain infarction. Decreased post-ischemic long-term potentiation (iLTP) was observed in an ex vivo hippocampal oxygen-glucose deprivation model. Tropomyosin receptor kinase B (TrkB) activation, rather than glutamate receptor alteration, was consistently found after the different manipulations. Immunohistochemistry further revealed peri-infarct neuronal TrkB activation and microvasculature augmentation in AUDA-treated and sEH KO mice, suggesting parallel neurovascular enhancement. Mechanistically, pretreatment with a selective TrkB antagonist ANA12 countered the effect of iLTP attenuation induced by sEH deletion ex vivo and abolished the infarct reduction in vivo. Together, the neuroprotective effects of sEH inhibition and gene deletion can both be mediated partially via enhancement of TrkB signaling which attenuated post-ischemic neuroexcitation and neurological deficits.
Project description:Metabolic syndrome is associated with an increased risk of colorectal cancer. This study investigated the impact of hypertension, a component of metabolic syndrome, on azoxymethane (AOM)-induced colorectal carcinogenesis using SHRSP/Izm (SHRSP) non-diabetic/hypertensive rats and SHRSP.Z-Leprfa/IzmDmcr (SHRSP-ZF) diabetic/hypertensive rats. Male 6-week-old SHRSP, SHRSP-ZF, and control non-diabetic/normotensive Wister Kyoto/Izm (WKY) rats were given 2 weekly intraperitoneal injections of AOM (20 mg/kg body weight). Two weeks after the last injection of AOM, the SHRSP and SHRSP-ZF rats became hypertensive compared to the control WKY rats. Serum levels of angiotensin-II, the active product of the renin-angiotensin system, were elevated in both SHRSP and SHRSP-ZF rats, but only the SHRSP-ZF rats developed insulin resistance, dyslipidemia, and hyperleptinemia and exhibited an increase in adipose tissue. The development of AOM-induced colonic preneoplastic lesions and aberrant crypts foci, was significantly accelerated in both SHRSP and SHRSP-ZF hypertensive rats, compared to WKY normotensive rats. Furthermore, induction of oxidative stress and exacerbation of inflammation were observed in the colonic mucosa and systemically in SHRSP and SHRSP-ZF rats. Our findings suggest that hypertension plays a role in the early stage of colorectal carcinogenesis by inducing oxidative stress and chronic inflammation, which might be associated with activation of the renin-angiotensin system.
Project description:OBJECTIVE:Hypertension is one of the most prevalent diseases in humans who live a modern lifestyle. Alongside more effective care, clarification of the genetic background of hypertension is urgently required. Gene expression in mesenteric resistance arteries of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and two types of renal hypertensive Wistar Kyoto rats (WKY), two kidneys and one clip renal hypertensive rat (2K1C) and one kidney and one clip renal hypertensive rat (1K1C), was compared using DNA microarrays. METHODS:We used a simultaneous equation and comparative selection method to identify genes associated with hypertension using the Reactome analysis tool and GenBank database. RESULTS:The expression of 298 genes was altered between SHR and WKY (44 upregulated and 254 downregulated), while the expression of 290 genes was altered between SHRSP and WKY (83 upregulated and 207 downregulated). For SHRSP versus SHR, the expression of 60 genes was altered (36 upregulated and 24 downregulated). Several genes expressed in SHR and SHRSP were also expressed in the renovascular hypertensive 2K1C and 1K1C rats, indicative of the existence of hyper-renin and/or hypervolemic pathophysiological changes in SHR and SHRSP. CONCLUSION:The overexpression of Kcnq1, Crlf1, Alb and Xirp1 and the inhibition of Galr2, Kcnh1, Ache, Chrm2 and Slc5a7 expression may indicate that a relationship exists between these genes and the cause and/or worsening of hypertension in SHR and SHRSP.
Project description:Inflammatory responses significantly contribute to neuronal damage and poor functional outcomes following intracerebral hemorrhage (ICH). Soluble epoxide hydrolase (sEH) is known to induce neuroinflammatory responses via degradation of anti-inflammatory epoxyeicosatrienoic acids (EET), and sEH is upregulated in response to brain injury. The present study investigated the involvement of sEH in ICH-induced neuroinflammation, brain damage, and functional deficits using a mouse ICH model and microglial cultures.ICH was induced by injecting collagenase in both wild-type (WT) C57BL/6 mice and sEH knockout (KO) mice. WT mice were injected intracerebroventricularly with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a selective sEH inhibitor, 30 min before ICH. Expression of sEH in the hemorrhagic hemisphere was examined by immunofluorescence and Western blot analysis. The effects of genetic deletion or pharmacological inhibition of sEH by AUDA on neuroinflammatory responses, EET degradation, blood-brain barrier (BBB) permeability, histological damage, and functional deficits were evaluated. The anti-inflammatory mechanism of sEH inactivation was investigated in thrombin- or hemin-stimulated cultured microglia.ICH induced an increase in sEH protein levels in the hemorrhagic hemisphere from 3 h to 4 days. sEH was expressed in microglia/macrophages, astrocytes, neurons, and endothelial cells in the perihematomal region. Genetic deletion of sEH significantly attenuated microglia/macrophage activation and expression of inflammatory mediators and reduced EET degradation at 1 and 4 days post-ICH. Deletion of sEH also reduced BBB permeability, matrix metalloproteinase (MMP)-9 activity, neutrophil infiltration, and neuronal damage at 1 and 4 days. Likewise, administration of AUDA attenuated proinflammatory microglia/macrophage activation and EET degradation at 1 day post-ICH. These findings were associated with a reduction in functional deficits and brain damage for up to 28 days. AUDA also ameliorated neuronal death, BBB disruption, MMP-9 activity, and neutrophil infiltration at 1 day. However, neither gene deletion nor pharmacological inhibition of sEH altered the hemorrhage volume following ICH. In primary microglial cultures, genetic deletion or pharmacological inhibition of sEH by AUDA reduced thrombin- and hemin-induced microglial activation. Furthermore, AUDA reduced thrombin- and hemin-induced P38 MAPK and NF-?B activation in BV2 microglia cultures. Ultimately, AUDA attenuated N2A neuronal death that was induced by BV2 microglial conditioned media.Our results suggest that inhibition of sEH may provide a potential therapy for ICH by suppressing microglia/macrophage-mediated neuroinflammation.
Project description:Soluble epoxide hydrolase (sEH) in endothelial cells determines the plasma concentrations of epoxyeicosatrienoic acids (EETs), which may act as vasoactive agents to control vascular tone. We hypothesized that the regulation of sEH activity may have a therapeutic value in preventing acute kidney injury by controlling the concentration of EETs. In this study, we therefore induced ischemia-reperfusion injury (IRI) in C57BL/6 mice and controlled sEH activity by intraperitoneal administration of the sEH inhibitor 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA). The deterioration of kidney function induced by IRI was partially moderated and prevented by AUDA treatment. In addition, AUDA treatment significantly attenuated tubular necrosis induced by IRI. Ischemic injury induced the down-regulation of sEH, and AUDA administration had no effect on the expression pattern of sEH induced by IRI. In vivo sEH activity was assessed by measuring the substrate epoxyoctadecenoic acid (EpOME) and its metabolite dihydroxyoctadec-12-enoic acid (DHOME). Ischemic injury had no effects on the plasma concentrations of EpOME and DHOME, but inhibition of sEH by AUDA significantly increased plasma EpOME and the EpOME/DHOME ratio. The protective effect of the sEH inhibitor was achieved by suppression of proinflammatory cytokines and up-regulation of regulatory cytokines. AUDA treatment prevented the intrarenal infiltration of inflammatory cells, but promoted endothelial cell migration and neovascularization. The results of this study suggest that treatment with sEH inhibitors can reduce acute kidney injury.
Project description:Traumatic brain injury (TBI) induces a series of inflammatory processes that contribute to neuronal damage. The present study investigated the involvement of soluble epoxide hydrolase (sEH) in neuroinflammation and brain damage in mouse TBI and in microglial cultures. The effects of genetic deletion of sEH and treatment with an sEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), on brain damage and inflammatory responses were evaluated in mice subjected to controlled cortical impact. The anti-inflammatory mechanism of sEH inhibition/deletion was investigated in vitro. TBI-induced an increase in sEH protein level in the injured cortex from 1 h to 4 days and sEH was expressed in microglia. Genetic deletion of sEH significantly attenuated functional deficits and brain damage up to 28 days post-TBI. Deletion of sEH also reduced neuronal death, apoptosis, brain edema, and BBB permeability at 1 and 4 day(s). These changes were associated with markedly reduced microglial/macrophage activation, neutrophil infiltration, matrix metalloproteinase-9 activity, inflammatory mediator expression at 1 and 4 day(s), and epoxyeicosatrienoic acid (EET) degradation at 1 and 4 day(s). Administration of AUDA attenuated brain edema, apoptosis, inflammatory mediator upregulation and EET degradation at 4 days. In primary microglial cultures, AUDA attenuated both LPS- or IFN-?-stimulated nitric oxide (NO) production and reduced LPS- or IFN-?-induced p38 MAPK and NF-?B signaling. Deletion of sEH also reduced IFN-?-induced NO production. Moreover, AUDA attenuated N2A neuronal death induced by BV2 microglial-conditioned media. Our results suggest that inhibition of sEH may be a potential therapy for TBI by modulating the cytotoxic functions of microglia.