Soluble Epoxide Hydrolase Inhibition Reverses Cognitive Dysfunction in a Mouse Model of Metabolic Syndrome by Modulating Inflammation.

Midlife metabolic syndrome (MetS) is associated with cognitive impairment in late life. The mechanism of delayed MetS-related cognitive dysfunction (MetSCD) is not clear, but it has been linked to systemic inflammation and chronic cerebral microangiopathy. Currently there is no treatment for late life MetSCD other than early risk factor modification. We investigated the effect of soluble epoxide hydrolase (sEH) inhibitor 4-[[trans-4-[[(tricyclo[3.3.1.13,7]dec-1-ylamino)carbonyl]amino]cyclohexyl]oxy]-benzoic acid (t-AUCB) on cognitive performance, cerebral blood flow (CBF), and central and peripheral inflammation in the high-fat diet (HFD) model of MetS in mice. At 6 weeks of age, male mice were randomly assigned to receive either HFD or standard chow (STD) for 6 months. Mice received either t-AUCB or vehicle for 4 weeks. Cognitive performance was evaluated, followed by CBF measurement using magnetic resonance imaging (MRI). At the end of the study, blood was collected for measurement of eicosanoids and inflammatory cytokines. The brains were then analyzed by immunohistochemistry for glial activation markers. The HFD caused a significant impairment in novel object recognition. Treatment with t-AUCB increased plasma levels of 14,15-EET, prevented this cognitive impairment and modified hippocampal glial activation and plasma cytokine levels, without affecting CBF in mice on HFD. In conclusion, sEH inhibition for four weeks prevents cognitive deficits in mice on chronic HFD by modulating inflammatory processes without affecting CBF.

Cytochrome P450-derived eicosanoids in brain: From basic discovery to clinical translation.

Cytochrome P450 metabolism of arachidonic acid produces epoxyeicosatrienoates (EETs) and hydroxyeicosatetraenoates (HETEs). Both classes of eicosanoids play important and opposing roles in brain function and disease. EETs promote vasodilation and exhibit antiinflammatory and cytoprotective properties; their biological action is blunted by metabolism to less active diols by the enzyme soluble epoxide hydrolase (sEH). EETs levels are dysregulated in disease states, primarily due to increased activity of sEH. Inhibition of sEH is a promising therapeutic approach for multiple brain disorders including stroke, dementia, subarachnoid hemorrhage and epilepsy. In this chapter, we summarize evidence implicating P450 eicosanoids and their synthetic and metabolizing enzymes in brain health and disease, and experimental and clinical studies targeting these pathways for brain disorders. We also discuss the diagnostic utility of quantifying P450 eicosanoids and their enzymes as disease biomarkers. Remarkable progress has been achieved in translating basic science discoveries in this field clinically.

Low-Intensity Ultrasound Reduces Brain Infarct Size by Upregulating Phosphorylated Endothelial Nitric Oxide in Mouse Model of Middle Cerebral Artery Occlusion.

OBJECTIVE: There have been attempts to use therapeutic ultrasound (US) for the treatment of both experimental and clinical stroke. We hypothesized that low-intensity US has direct beneficial effects on the brain independent of cerebral blood flow (CBF) during middle cerebral artery occlusion (MCAO). METHODS: Three groups of mice were studied. Group I included 84 mice with MCAO undergoing US treatment/no treatment at two US frequencies (0.25 and 1.05 MHz) with three different acoustic pressures at each frequency in which infarct size (IS) was measured 24 h later. Group II included 11 mice undergoing treatment based on best US results from group I animals in which the IS/risk area (RA) ratio was measured 24 h later. Group III included 38 normal mice undergoing US treatment/no treatment for assessment of CBF, tissue metabolite and protein expression and histopathology. DISCUSSION: Ultrasound at both frequencies and most acoustic pressures resulted in reduction in IS in group I animals, with the best results obtained with 0.25 MHz at 2.0 MPa: IS was reduced 4-fold in the cerebral cortex, 1.5-fold in the caudate putamen and 3.5-fold in the cerebral hemisphere compared with control. US application in group III animals elicited only a marginal increase in CBF despite a 2.6-fold increase in phosphorylated endothelial nitric oxide synthase (p-eNOS)-S1177 and a corresponding decrease in p-eNOS-T494. Histopathology revealed no evidence of hemorrhage, inflammation or necrosis. CONCLUSION: Low-intensity US at specific frequencies and acoustic pressures results in marked neuroprotection in a mouse model of stroke by modulation of p-eNOS independent of its effect on CBF.

Sex Difference in Capillary Reperfusion After Transient Middle Cerebral Artery Occlusion in Diabetic Mice.

BACKGROUND: Type 2 diabetes (DM2) exacerbates stroke injury, reduces efficacy of endovascular therapy, and worsens long-term functional outcome. Sex differences exist in stroke incidence, response to therapy, poststroke microvascular dysfunction, and functional recovery. In this study, we tested the hypotheses that poor outcome after stroke in the setting of DM2 is linked to impaired microvascular tissue reperfusion and that male and female DM2 mice exhibit different microvascular reperfusion response after transient middle cerebral artery occlusion (MCAO). METHODS: Transient MCAO was induced for 60 minutes using an intraluminal filament in young adult DM2 and nondiabetic control male and female mice. Capillary flux in deep cortical layers was assessed using optical coherence tomography-based optical microangiography (OMAG), and associated regional brain infarct size was evaluated by hematoxylin and eosin staining. RESULTS: Compared to baseline, MCAO reduced absolute capillary red blood cell flux by 84% at 24 hours post-MCAO in male DM2 (P<0.001) but not male control mice. When normalized to pre-MCAO baseline, red blood cell flux 24 hours after stroke was 64% lower in male DM2 mice than male nondiabetic controls (P<0.01). In females, MCAO decreased capillary flux by 48% at 24 hours post-MCAO compared with baseline in DM2 (P<0.05) but not in control mice. Red blood cell flux of female DM2 mice did not differ from that of nondiabetic controls either before or 24 hours after MCAO. Furthermore, normalized capillary flux 24 hours after MCAO failed to differ between female DM2 mice and nondiabetic controls. Concomitantly, male but not female DM2 mice experienced 25% larger infarct in caudate-putamen versus respective nondiabetic controls (P<0.05). CONCLUSIONS: DM2 impairs capillary perfusion and exacerbates ischemic deep brain injury in male but not female young adult mice. Premenopausal females appear to be protected against DM2-related capillary dysfunction and brain injury.

Role of Inflammatory Processes in Hemorrhagic Stroke.

Hemorrhagic stroke is the deadliest form of stroke and includes the subtypes of intracerebral hemorrhage and subarachnoid hemorrhage. A common cause of hemorrhagic stroke in older individuals is cerebral amyloid angiopathy. Intracerebral hemorrhage and subarachnoid hemorrhage both lead to the rapid collection of blood in the central nervous system and generate inflammatory immune responses that involve both brain resident and infiltrating immune cells. These responses are complex and can contribute to both tissue recovery and tissue injury. Despite the interconnectedness of these major subtypes of hemorrhagic stroke, few reviews have discussed them collectively. The present review provides an update on inflammatory processes that occur in response to intracerebral hemorrhage and subarachnoid hemorrhage, and the role of inflammation in the pathophysiology of cerebral amyloid angiopathy-related hemorrhage. The goal is to highlight inflammatory processes that underlie disease pathology and recovery. We aim to discuss recent advances in our understanding of these conditions and identify gaps in knowledge with the potential to develop effective therapeutic strategies.

Fluid dynamics in aging-related dementias.

Recent human and animal model experimental studies revealed novel pathways for fluid movement, immune cell trafficking and metabolic waste clearance in CNS. These studies raise the intriguing possibility that the newly discovered pathways, including the glymphatic system, lymphatic meningeal vessels and skull-brain communication channels, are impaired in aging and neurovascular and neurodegenerative diseases associated with dementia, including Alzheimer's disease (AD) and AD-related dementia. We provide an overview of the glymphatic and dural meningeal lymphatic systems, review current methods and approaches used to study glymphatic flow in humans and animals, and discuss current evidence and controversies related to its role in CNS flow homeostasis under physiological and pathophysiological conditions. Non-invasive imaging approaches are needed to fully understand the mechanisms and pathways driving fluid movement in CNS and their roles across lifespan including healthy aging and aging-related dementia.

GPR39 Knockout Worsens Microcirculatory Response to Experimental Stroke in a Sex-Dependent Manner.

No current treatments target microvascular reperfusion after stroke, which can contribute to poor outcomes even after successful clot retrieval. The G protein-coupled receptor GPR39 is expressed in brain peri-capillary pericytes, and has been implicated in microvascular regulation, but its role in stroke is unknown. We tested the hypothesis that GPR39 plays a protective role after stroke, in part due to preservation of microvascular perfusion. We generated GPR39 knockout (KO) mice and tested whether GPR39 gene deletion worsens capillary blood flow and exacerbates brain injury and functional deficit after focal cerebral ischemia. Stroke was induced in male and female GPR39 KO and WT littermates by 60-min middle cerebral artery occlusion (MCAO). Microvascular perfusion was assessed via capillary red blood cell (RBC) flux in deep cortical layers in vivo using optical microangiography (OMAG). Brain injury was assessed by measuring infarct size by 2,3,5-triphenyltetrazolium chloride staining at 24 h or brain atrophy at 3 weeks after ischemia. Pole and cylinder behavior tests were conducted to assess neurological function deficit at 1 and 3 weeks post-stroke. Male but not female GPR39 KO mice exhibited larger infarcts and lower capillary RBC flux than WT controls after stroke. Male GPR39 KO mice also exhibited worse neurologic deficit at 1 week post-stroke, though functional deficit disappeared in both groups by 3 weeks. GPR39 deletion worsens brain injury, microvascular perfusion, and neurological function after experimental stroke. Results indicate that GPR39 plays a sex-dependent role in re-establishing microvascular flow and limiting ischemic brain damage after stroke.

Role of Endothelial STAT3 in Cerebrovascular Function and Protection from Ischemic Brain Injury.

STAT3 plays a protective role against ischemic brain injury; however, it is not clear which brain cell type mediates this effect, and by which mechanism. We tested the hypothesis that endothelial STAT3 contributes to protection from cerebral ischemia, by preserving cerebrovascular endothelial function and blood-brain barrier (BBB) integrity. The objective of this study was to determine the role of STAT3 in cerebrovascular endothelial cell (EC) survival and function, and its role in tissue outcome after cerebral ischemia. We found that in primary mouse brain microvascular ECs, STAT3 was constitutively active, and its phosphorylation was reduced by oxygen-glucose deprivation (OGD), recovering after re-oxygenation. STAT3 inhibition, using two mechanistically different pharmacological inhibitors, increased EC injury after OGD. The sub-lethal inhibition of STAT3 caused endothelial dysfunction, demonstrated by reduced nitric oxide release in response to acetylcholine and reduced barrier function of the endothelial monolayer. Finally, mice with reduced endothelial STAT3 (Tie2-Cre; STAT3flox/wt) sustained larger brain infarcts after middle cerebral artery occlusion (MCAO) compared to wild-type (WT) littermates. We conclude that STAT3 is vital to maintaining cerebrovascular integrity, playing a role in EC survival and function, and protection against cerebral ischemia. Endothelial STAT3 may serve as a potential target in preventing endothelial dysfunction after stroke.

Age-dependent cognitive impairment, hydrocephalus and leukocyte infiltration in transgenic mice with endothelial expression of human EPHX2.

Soluble epoxide hydrolase (sEH) is upregulated in microvascular endothelium of human brain with vascular cognitive impairment (VCI). Transgenic endothelial expression of human sEH in mice (Tie2hsEH) induces endothelial dysfunction (ED), a pathogenetic mechanism of VCI. We sought to determine if endothelial upregulation of sEH is sufficient to cause cognitive impairment, and if cognitive impairment due to chronic hypoperfusion induced by unilateral common carotid artery occlusion (CCAO) is exacerbated in Tie2hsEH mice. Behavioral performance was assessed by the open field, rotarod, novel object, Morris water maze and fear conditioning tests. Cerebral blood flow and brain morphology were evaluated by MRI, and inflammatory changes investigated using immunohistochemistry and flow cytometry. We demonstrate that transgenic endothelial expression of sEH is sufficient to induce cognitive impairment, associated with leukocyte infiltration, brain atrophy and accelerated, age-dependent ventriculomegaly, identifying ED and sEH upregulation as potential underlying mechanisms and therapeutic targets for VCI.

GPR39 Deficiency Impairs Memory and Alters Oxylipins and Inflammatory Cytokines Without Affecting Cerebral Blood Flow in a High-Fat Diet Mouse Model of Cognitive Impairment.

Vascular cognitive impairment (VCI) is the second most common cause of dementia. There is no treatment for VCI, in part due to a lack of understanding of the underlying mechanisms. The G-protein coupled receptor 39 (GPR39) is regulated by arachidonic acid (AA)-derived oxylipins that have been implicated in VCI. Furthermore, GPR39 is increased in microglia of post mortem human brains with VCI. Carriers of homozygous GPR39 SNPs have a higher burden of white matter hyperintensity, an MRI marker of VCI. We tested the hypothesis that GPR39 plays a protective role against high-fat diet (HFD)-induced cognitive impairment, in part mediated via oxylipins actions on cerebral blood flow (CBF) and neuroinflammation. Homozygous (KO) and heterozygous (Het) GPR39 knockout mice and wild-type (WT) littermates with and without HFD for 8 months were tested for cognitive performance using the novel object recognition (NOR) and the Morris water maze (MWM) tests, followed by CBF measurements using MRI. Brain tissue and plasma oxylipins were quantified with high-performance liquid chromatography coupled to mass spectrometry. Cytokines and chemokines were measured using a multiplex assay. KO mice, regardless of diet, swam further away from platform location in the MWM compared to WT and Het mice. In the NOR test, there were no effects of genotype or diet. Brain and plasma AA-derived oxylipins formed by 11- and 15-lipoxygenase (LOX), cyclooxygenase (COX) and non-enzymatically were increased by HFD and GPR39 deletion. Interleukin-10 (IL-10) was lower in KO mice on HFD than standard diet (STD), whereas IL-4, interferon γ-induced protein-10 (IP-10) and monocyte chemotactic protein-3 (MCP-3) were altered by diet in both WT and KO, but were not affected by genotype. Resting CBF was reduced in WT and KO mice on HFD, with no change in vasoreactivity. The deletion of GPR39 did not change CBF compared to WT mice on either STD or HFD. We conclude that GPR39 plays a role in spatial memory retention and protects against HFD-induced cognitive impairment in part by modulating inflammation and AA-derived oxylipins. The results indicate that GPR39 and oxylipin pathways play a role and may serve as therapeutic targets in VCI.

Proteomic profiling of concurrently isolated primary microvascular endothelial cells, pericytes, and vascular smooth muscle cells from adult mouse heart.

The microcirculation serves crucial functions in adult heart, distinct from those carried out by epicardial vessels. Microvessels are governed by unique regulatory mechanisms, impairment of which leads to microvessel-specific pathology. There are few treatment options for patients with microvascular heart disease, primarily due to limited understanding of underlying pathology. High throughput mRNA sequencing and protein expression profiling in specific cells can improve our understanding of microvessel biology and disease at the molecular level. Understanding responses of individual microvascular cells to the same physiological or pathophysiological stimuli requires the ability to isolate the specific cell types that comprise the functional units of the microcirculation in the heart, preferably from the same heart, to ensure that different cells have been exposed to the same in-vivo conditions. We developed an integrated process for simultaneous isolation and culture of the main cell types comprising the microcirculation in adult mouse heart: endothelial cells, pericytes, and vascular smooth muscle cells. These cell types were characterized with isobaric labeling quantitative proteomics and mRNA sequencing. We defined microvascular cell proteomes, identified novel protein markers, and confirmed established cell-specific markers. Our results allow identification of unique markers and regulatory proteins that govern microvascular physiology and pathology.

Control of coronary vascular resistance by eicosanoids via a novel GPCR.

Arachidonic acid metabolites epoxyeicosatrienoates (EETs) and hydroxyeicosatetraenoates (HETEs) are important regulators of myocardial blood flow and coronary vascular resistance (CVR), but their mechanisms of action are not fully understood. We applied a chemoproteomics strategy using a clickable photoaffinity probe to identify G protein-coupled receptor 39 (GPR39) as a microvascular smooth muscle cell (mVSMC) receptor selective for two endogenous eicosanoids, 15-HETE and 14,15-EET, which act on the receptor to oppose each other's activity. The former increases mVSMC intracellular calcium via GPR39 and augments coronary microvascular resistance, and the latter inhibits these actions. Furthermore, we find that the efficacy of both ligands is potentiated by zinc acting as an allosteric modulator. Measurements of coronary perfusion pressure (CPP) in GPR39-null hearts using the Langendorff preparation indicate the receptor senses these eicosanoids to regulate microvascular tone. These results implicate GPR39 as an eicosanoid receptor and key regulator of myocardial tissue perfusion. Our findings will have a major impact on understanding the roles of eicosanoids in cardiovascular physiology and disease and provide an opportunity for the development of novel GPR39-targeting therapies for cardiovascular disease.

A Double-Blind, Randomized, Placebo-Controlled Trial of Soluble Epoxide Hydrolase Inhibition in Patients with Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: Epoxyeicosatrienoates (EETs) are endogenous regulators of neuroinflammation and cerebral blood flow. Their metabolism to dihydroxyeicosatrienoates (DHETs) is catalyzed by soluble epoxide hydrolase (sEH). After subarachnoid hemorrhage (SAH), EETs' pathway amplification may be a therapeutic target for the prevention of delayed cerebral ischemia (DCI). We conducted a double-blind, placebo-controlled, phase Ib randomized trial of GSK2256294, a pharmacologic inhibitor of sEH, to evaluate the safety profile and to assess biomarkers of neurovascular inflammation in patients with aneurysmal SAH. METHODS: Patients were randomly assigned to receive 10 mg of GSK2256294 or a placebo treatment once daily for 10 days, beginning within 72 hours after aneurysm rupture. The primary study end point was safety. Secondary end points included serum and cerebrospinal fluid (CSF) EETs-to-DHETs ratio, cytokine levels, and serum endothelial injury biomarkers, measured at day 7 and day 10 after SAH. Tertiary end points included neurologic status, disposition, length of stay, incidence of DCI, and mortality; these were assessed at hospital discharge and at 90 days. RESULTS: Ten patients received GSK2256294 and nine patients received a placebo. There were no adverse events related to the study drug. GSK2256294 administration resulted in a significant increase in the EET/DHET ratio at day 7 and day 10 in serum, but not in the CSF. There was a trend for decreased CSF inflammatory cytokines following GSK2256294 administration, but this did not reach statistical significance. CONCLUSIONS: GSK2256294 administration was safe and well tolerated in critically ill patients with SAH, producing an increase in serum EETs and the EET-to-DHET ratio. Our findings support future studies in a larger population to evaluate the role of sEH inhibition in the prevention of DCI after SAH and other forms of brain injury and inflammatory conditions. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT03318783.

A Novel Model of Tobacco Smoke-Mediated Aortic Injury.

OBJECTIVE: Tobacco smoke exposure is a major risk factor for aortic aneurysm development. However, the initial aortic response to tobacco smoke, preceding aneurysm formation, is not well understood. We sought to create a model to determine the effect of solubilized tobacco smoke (STS) on the thoracic and abdominal aorta of mice as well as on cultured human aortic smooth muscle cells (HASMCs). METHODS: Tobacco smoke was solubilized and delivered to mice via implanted osmotic minipumps. Twenty male C57BL/6 mice received STS or vehicle infusion. The descending thoracic, suprarenal abdominal, and infrarenal abdominal segments of the aorta were assessed for elastic lamellar damage, smooth muscle cell phenotype, and infiltration of inflammatory cells. Cultured HASMCs grown in media containing STS were compared to cells grown in standard media in order to verify our in vivo findings. RESULTS: Tobacco smoke solution caused significantly more breaks in the elastic lamellae of the thoracic and abdominal aorta compared to control solution (P< .0001) without inciting an inflammatory infiltrate. Elastin breaks occurred more frequently in the abdominal aorta than the thoracic aorta (P < .01). Exposure to STS-induced aortic microdissections and downregulation of α-smooth muscle actin (α-SMA) by vascular smooth muscle cells (VSMCs). Treatment of cultured HASMCs with STS confirmed the decrease in α-SMA expression. CONCLUSION: Delivery of STS via osmotic minipumps appears to be a promising model for investigating the early aortic response to tobacco smoke exposure. The initial effect of tobacco smoke exposure on the aorta is elastic lamellar damage and downregulation of (α-SMA) expression by VSMCs. Elastic lamellar damage occurs more frequently in the abdominal aorta than the thoracic aorta and does not seem to be mediated by the presence of macrophages or other inflammatory cells.

GPR39 localization in the aging human brain and correlation of expression and polymorphism with vascular cognitive impairment.

INTRODUCTION: The pathogenesis of vascular cognitive impairment (VCI) is not fully understood. GPR39, an orphan G-protein coupled receptor, is implicated in neurological disorders but its role in VCI is unknown. METHODS: We performed GPR39 immunohistochemical analysis in post mortem brain samples from mild cognitive impairment (MCI) and control subjects. DNA was analyzed for GPR39 single nucleotide polymorphisms (SNPs), and correlated with white matter hyperintensity (WMH) burden on pre mortem magnetic resonance imaging. RESULTS: GPR39 is expressed in aged human dorsolateral prefrontal cortex, localized to microglia and peri-capillary cells resembling pericytes. GPR39-capillary colocalization, and density of GPR39-expressing microglia was increased in aged brains compared to young. SNP distribution was equivalent between groups; however, homozygous SNP carriers were present only in the MCI group, and had higher WMH volume than wild-type or heterozygous SNP carriers. DISCUSSION: GPR39 may play a role in aging-related VCI, and may serve as a therapeutic target and biomarker for the risk of developing VCI.

(Phospho)Proteomic dataset of ischemia- and ultrasound- stimulated mouse cardiac endothelial cells in vitro.

Cardiac endothelial cells respond to both ischemia and therapeutic ultrasound; the proteomic changes underlying these responses are unknown. This data article provides raw and processed data resulting from our global, unbiased phosphoproteomics investigation conducted on primary mouse cardiac endothelial cells exposed to ischemia (2-hour oxygen glucose deprivation) and ultrasound (250 kHz, 1.2 MPa) in vitro [1]. Proteins were extracted from cell lysates and enriched phosphopeptides were analyzed with a high mass accuracy liquid chromatrography (LC) - tandem mass spectrometry (MS/MS) proteomic platform, yielding multiple alterations in both total protein levels and phosphorylation events in response to ischemic injury and ultrasound. This dataset can be used as a reference for future studies on the cardiac endothelial response to ischemia and the mechanistic underpinnings of the cellular response to ultrasound, with the potential to yield clinically relevant therapeutic targets.

Gene-Specific DNA Methylation Linked to Postoperative Cognitive Dysfunction in Apolipoprotein E3 and E4 Mice.

BACKGROUND: Geriatric surgical patients are at higher risk of developing postoperative neurocognitive disorders (NCD) than younger patients. The specific mechanisms underlying postoperative NCD remain unknown, but they have been linked to genetic risk factors, such as the presence of APOE4, compared to APOE3, and epigenetic modifications caused by exposure to anesthesia and surgery. OBJECTIVE: To test the hypothesis that compared to E3 mice, E4 mice exhibit a more pronounced postoperative cognitive impairment associated with differential DNA methylation in brain regions linked to learning and memory. METHODS: 16-month-old humanized apolipoprotein-E targeted replacement mice bearing E3 or E4 were subjected to surgery (laparotomy) under general isoflurane anesthesia or sham. Postoperative behavioral testing and genome-wide DNA methylation were performed. RESULTS: Exposure to surgery and anesthesia impaired cognition in aged E3, but not E4 mice, likely due to the already lower cognitive performance of E4 prior to surgery. Cognitive impairment in E3 mice was associated with hypermethylation of specific genes, including genes in the Ephrin pathway implicated in synaptic plasticity and learning in adults and has been linked to Alzheimer's disease. Other genes, such as the Scratch Family Transcriptional Repressor 2, were altered after surgery and anesthesia in both the E3 and E4 mice. CONCLUSION: Our findings suggest that the neurocognitive and behavioral effects of surgery and anesthesia depend on baseline neurocognitive status and are associated with APOE isoform-dependent epigenetic modifications of specific genes and pathways involved in memory and learning.