CNS border-associated macrophages in the homeostatic and ischaemic brain.

CNS border-associated macrophages (BAMs) are a small population of specialised macrophages localised in the choroid plexus, meningeal and perivascular spaces. Until recently, the function of this elusive cell type was poorly understood and largely overlooked, especially in comparison to microglia, the primary brain resident immune cell. However, the recent single cell immunophenotyping or transcriptomic analysis of BAM subsets in the homeostatic brain, coupled with the rapid emergence of new studies exploring BAM functions in various cerebral pathologies, including Alzheimer's disease, hypertension-induced neurovascular and cognitive dysfunction, and ischaemic stroke, has unveiled previously unrecognised heterogeneity and spatial-temporal complexity in BAM populations as well as their contributions to brain homeostasis and disease. In this review, we discuss the implications of this new-found knowledge on our current understanding of BAM function in ischaemic stroke. We first provide a comprehensive overview and discussion of the cell-surface expression profiles, transcriptional signatures and potential functional phenotypes of homeostatic BAM subsets described in recent studies. Evidence for their putative physiological roles is examined, including their involvement in immunological surveillance, waste clearance, and vascular permeability. We discuss the evidence supporting the accumulation and genetic transformation of BAMs in response to ischaemia and appraise the experimental evidence that BAM function might be deleterious in the acute phase of stroke, while considering the mechanisms by which BAMs may influence stroke outcomes in the longer term. Finally, we review the therapeutic potential of immunomodulatory strategies as an approach to stroke management, highlighting current challenges in the field and key issues relating to BAMs, and how BAMs could be harnessed experimentally to support future translational research.

UK consensus on pre-clinical vascular cognitive impairment functional outcomes assessment: Questionnaire and workshop proceedings.

Assessment of outcome in preclinical studies of vascular cognitive impairment (VCI) is heterogenous. Through an ARUK Scottish Network supported questionnaire and workshop (mostly UK-based researchers), we aimed to determine underlying variability and what could be implemented to overcome identified challenges. Twelve UK VCI research centres were identified and invited to complete a questionnaire and attend a one-day workshop. Questionnaire responses demonstrated agreement that outcome assessments in VCI preclinical research vary by group and even those common across groups, may be performed differently. From the workshop, six themes were discussed: issues with preclinical models, reasons for choosing functional assessments, issues in interpretation of functional assessments, describing and reporting functional outcome assessments, sharing resources and expertise, and standardization of outcomes. Eight consensus points emerged demonstrating broadly that the chosen assessment should reflect the deficit being measured, and therefore that one assessment does not suit all models; guidance/standardisation on recording VCI outcome reporting is needed and that uniformity would be aided by a platform to share expertise, material, protocols and procedures thus reducing heterogeneity and so increasing potential for collaboration, comparison and replication. As a result of the workshop, UK wide consensus statements were agreed and future priorities for preclinical research identified.

Microglial regulation of satiety and cognition.

Microglia have been known for decades as key immune cells that shape the central nervous system (CNS) during development and respond to brain pathogens and injury in adult life. Recent findings now suggest that these cells also play a highly complex role in several other functions of the CNS. In this review, we provide a brief overview of the established microglial functions in development and disease. We also discuss emerging research suggesting that microglia are important for both cognitive function and the regulation of food intake. With respect to cognitive function, current data suggest microglia are not indispensable for neurogenesis, synaptogenesis or cognition in the healthy young adult, although they crucially modulate and support these functions. In doing so, they are likely important in supporting the balance between apoptosis and survival of newborn neurones and in orchestrating appropriate synaptic remodelling in response to a learning stimulus. We also explore the possibility of a role for microglia in feeding and satiety. Microglia have been implicated in both appetite suppression with sickness and obesity and in promoting feeding under some conditions and we discuss these findings here, highlighting the contribution of these cells to healthy brain function.

Cigarette smoke extract exacerbates hyperpermeability of cerebral endothelial cells after oxygen glucose deprivation and reoxygenation.

Cigarette smoking is a risk factor for stroke and is linked to stroke severity. Previous studies have shown that cigarette smoke extract (CSE) triggers endothelial dysfunction in vitro by initiating oxidative stress and/or an inflammatory response. In addition, cerebral endothelial dysfunction (particularly at the level of the blood-brain barrier [BBB]) contributes to stroke pathogenesis. Therefore, we hypothesized that cigarette smoking may influence stroke, at least in part, by exacerbating ischaemia-induced BBB disruption. To test this, we examined the effect of CSE on the permeability of cerebral endothelial cells exposed to oxygen glucose deprivation and reoxygenation (OGD + RO). We found that the loss of BBB integrity following ischaemic/reperfusion-like conditions was significantly worsened by CSE. Despite this being associated with increased mRNA expression of Nox catalytic subunits, reactive oxygen species (ROS) levels were however markedly lower. Furthermore, this occurred in association with elevated expression of antioxidant enzymes (SOD1, SOD2, and Gpx-1), suggesting an antioxidant defence response. Lastly, we found that CSE significantly upregulated mRNA expression of cytokines (IL-6 and TGF-ß). Collectively, these results show that acute exposure to CSE worsens BBB disruption caused by OGD + RO, however, this is not linked to elevated ROS levels but may involve inflammatory mechanisms.

Ischaemic stroke in mice induces lung inflammation but not acute lung injury.

Stroke is a major cause of death worldwide and ischemic stroke is the most common subtype accounting for approximately 80% of all cases. Pulmonary complications occur in the first few days to weeks following ischemic stroke and are a major contributor to morbidity and mortality. Acute lung injury (ALI) occurs in up to 30% of patients with subarachnoid haemorrhage but the incidence of ALI after ischemic stroke is unclear. As ischemic stroke is the most common subtype of stroke, it is important to understand the development of ALI following the initial ischemic injury to the brain. Therefore, this study investigated whether focal ischemic stroke causes lung inflammation and ALI in mice. Ischemic stroke caused a significant increase in bronchoalveolar lavage fluid (BALF) macrophages and neutrophils and whole lung tissue proinflammatory IL-1ß mRNA expression but this did not translate into histologically evident ALI. Thus, it appears that lung inflammation, but not ALI, occurs after experimental ischemic stroke in mice. This has significant implications for organ donors as the lungs from patient's dying of ischemic stroke are not severely damaged and could thus be used for transplantation in people awaiting this life-saving therapy.

The inositol-requiring enzyme 1 (IRE1α) RNAse inhibitor, 4µ8C, is also a potent cellular antioxidant.

Inositol-requiring enzyme 1 alpha (IRE1α) is an endoplasmic reticulum (ER)-transmembrane endonuclease that is activated in response to ER stress as part of the unfolded protein response (UPR). Chronic activation of the UPR has been implicated in the pathogenesis of many common diseases including diabetes, cancer, and neurological pathologies such as Huntington's and Alzheimer's disease. 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (4µ8C) is widely used as a specific inhibitor of IRE1α ribonuclease activity (IC50 of 6.89 µM in cultured cells). However, in this paper, we demonstrate that 4µ8C acts as a potent reactive oxygen species (ROS) scavenger, both in a cell-free assay and in cultured cells, at concentrations lower than that widely used to inhibit IRE1α activity. In vitro we show that, 4µ8C effectively decreases xanthine/xanthine oxidase catalysed superoxide production with an IC50 of 0.2 µM whereas in cultured endothelial and clonal pancreatic ß-cells, 4µ8C inhibits angiotensin II-induced ROS production with IC50 values of 1.92 and 0.29 µM, respectively. In light of this discovery, conclusions reached using 4µ8C as an inhibitor of IRE1α should be carefully evaluated. However, this unexpected off-target effect of 4µ8C may prove therapeutically advantageous for the treatment of pathologies that are thought to be caused by, or exacerbated by, both oxidative and ER stress such as endothelial dysfunction and/or diabetes.

Angiotensin II Causes ß-Cell Dysfunction Through an ER Stress-Induced Proinflammatory Response.

The metabolic syndrome is associated with an increase in the activation of the renin angiotensin system, whose inhibition reduces the incidence of new-onset diabetes. Importantly, angiotensin II (AngII), independently of its vasoconstrictor action, causes ß-cell inflammation and dysfunction, which may be an early step in the development of type 2 diabetes. The aim of this study was to determine how AngII causes ß-cell dysfunction. Islets of Langerhans were isolated from C57BL/6J mice that had been infused with AngII in the presence or absence of taurine-conjugated ursodeoxycholic acid (TUDCA) and effects on endoplasmic reticulum (ER) stress, inflammation, and ß-cell function determined. The mechanism of action of AngII was further investigated using isolated murine islets and clonal ß cells. We show that AngII triggers ER stress, an increase in the messenger RNA expression of proinflammatory cytokines, and promotes ß-cell dysfunction in murine islets of Langerhans both in vivo and ex vivo. These effects were significantly attenuated by TUDCA, an inhibitor of ER stress. We also show that AngII-induced ER stress is required for the increased expression of proinflammatory cytokines and is caused by reactive oxygen species and IP3 receptor activation. These data reveal that the induction of ER stress is critical for AngII-induced ß-cell dysfunction and indicates how therapies that promote ER homeostasis may be beneficial in the prevention of type 2 diabetes.

Chronic NaHS treatment decreases oxidative stress and improves endothelial function in diabetic mice.

Hydrogen sulphide (H2S) is endogenously produced in vascular tissue and has anti-oxidant and vasoprotective properties. This study investigates whether chronic treatment using the fast H2S donor NaHS could elicit a vasoprotective effect in diabetes. Diabetes was induced in male C57BL6/J mice with streptozotocin (60 mg/kg daily, ip for 2 weeks) and confirmed by elevated blood glucose and glycated haemoglobin levels. Diabetic mice were then treated with NaHS (100 µmol/kg/day) for 4 weeks, and aortae collected for functional and biochemical analyses. In the diabetic group, both endothelium-dependent vasorelaxation and basal nitric oxide (NO•) bioactivity were significantly reduced ( p < 0.05), and maximal vasorelaxation to the NO• donor sodium nitroprusside was impaired ( p < 0.05) in aorta compared to control mice. Vascular superoxide generation via nicotine adenine dinucleotide phosphate (NADPH) oxidase ( p < 0.05) was elevated in aorta from diabetic mice which was associated with increased expression of NOX2 ( p < 0.05). NaHS treatment of diabetic mice restored endothelial function and exogenous NO• efficacy back to control levels. NaHS treatment also reduced the diabetes-induced increase in NADPH oxidase activity, but did not affect NOX2 protein expression. These data show that chronic NaHS treatment reverses diabetes-induced vascular dysfunction by restoring NO• efficacy and reducing superoxide production in the mouse aorta.

Protective actions of des-acylated ghrelin on brain injury and blood-brain barrier disruption after stroke in mice.

The major ghrelin forms, acylated ghrelin and des-acylated ghrelin, are novel gastrointestinal hormones. Moreover, emerging evidence indicates that these peptides may have other functions including neuro- and vaso-protection. Here, we investigated whether post-stroke treatment with acylated ghrelin or des-acylated ghrelin could improve functional and histological endpoints of stroke outcome in mice after transient middle cerebral artery occlusion (tMCAo). We found that des-acylated ghrelin (1 mg/kg) improved neurological and functional performance, reduced infarct and swelling, and decreased apoptosis. In addition, it reduced blood-brain barrier (BBB) disruption in vivo and attenuated the hyper-permeability of mouse cerebral microvascular endothelial cells after oxygen glucose deprivation and reoxygenation (OGD + RO). By contrast, acylated ghrelin (1 mg/kg or 5 mg/kg) had no significant effect on these endpoints of stroke outcome. Next we found that des-acylated ghrelin's vasoprotective actions were associated with increased expression of tight junction proteins (occludin and claudin-5), and decreased cell death. Moreover, it attenuated superoxide production, Nox activity and expression of 3-nitrotyrosine. Collectively, these results demonstrate that post-stroke treatment with des-acylated ghrelin, but not acylated ghrelin, protects against ischaemia/reperfusion-induced brain injury and swelling, and BBB disruption, by reducing oxidative and/or nitrosative damage.

COPD and stroke: are systemic inflammation and oxidative stress the missing links?

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and loss of lung function, and is currently the third largest cause of death in the world. It is now well established that cardiovascular-related comorbidities such as stroke contribute to morbidity and mortality in COPD. The mechanisms linking COPD and stroke remain to be fully defined but are likely to be interconnected. The association between COPD and stroke may be largely dependent on shared risk factors such as aging and smoking, or the association of COPD with traditional stroke risk factors. In addition, we propose that COPD-related systemic inflammation and oxidative stress may play important roles by promoting cerebral vascular dysfunction and platelet hyperactivity. In this review, we briefly discuss the pathogenesis of COPD, acute exacerbations of COPD (AECOPD) and cardiovascular comorbidities associated with COPD, in particular stroke. We also highlight and discuss the potential mechanisms underpinning the link between COPD and stroke, with a particular focus on the roles of systemic inflammation and oxidative stress.

Early life overfeeding impairs spatial memory performance by reducing microglial sensitivity to learning.

BACKGROUND: Obesity can lead to cognitive dysfunction including poor performance in memory tasks. However, poor memory is not seen in all obese humans and takes several months to develop in animal models, indicating the adult brain is relatively resistant to obesity's cognitive effects. We have seen that, in the rat, overfeeding for as little as 3 weeks in early life leads to lasting obesity and microglial priming in the hypothalamus. Here we hypothesized that microglial hyper-sensitivity in the neonatally overfed rats extends beyond the hypothalamus into memory-associated brain regions, resulting in cognitive deficits. METHODS: We tested this idea by manipulating Wistar rat litter sizes to suckle pups in litters of 4 (overfed) or 12 (control). RESULTS: Neonatally overfed rats had microgliosis in the hippocampus after only 14 days overfeeding, and this persisted into adulthood. These changes were coupled with poor performance in radial arm maze and novel object recognition tests relative to controls. In controls, the experience of the radial arm maze reduced cell proliferation in the dentate gyrus and neuron numbers in the CA3. The learning task also suppressed microglial number and density in hippocampus and retrosplenial cortex. Neonatally overfed brains had impaired sensitivity to learning, with no neuronal or cell proliferative effects and less effective microglial suppression. CONCLUSIONS: Thus, early life overfeeding contributes to a long-term impairment in learning and memory with a likely role for microglia. These data may partially explain why some obese individuals display cognitive dysfunction and some do not, i.e. the early life dietary environment is likely to have a vital long-term contribution.

Characterisation of a mouse cerebral microvascular endothelial cell line (bEnd.3) after oxygen glucose deprivation and reoxygenation.

Studies have utilised immortalised mouse cerebral endothelial cells (bEnd.3) exposed to oxygen glucose deprivation (OGD) to study blood-brain barrier (BBB) disruption after ischaemia. However, there is a paucity of literature describing the duration of OGD (and reoxygenation [RO]) required to best simulate BBB disruption in vivo. In this study we assessed BBB disruption in bEnd.3 cells after exposure to a range of OGD periods, and also after OGD + RO. Exposure of bEnd.3 monolayers to 4, 6, 16, or 24 hours of OGD resulted in a significant increase in permeability. The hyperpermeability after 16 or 24 hours was associated with decreased expression of tight junction proteins (occludin and claudin-5). Furthermore, there was a decrease in cell viability and increased expression of the pro-apoptotic protein, cleaved caspase-3. Exposure of bEnd.3 monolayers to 1 hour OGD+ 23 hours RO exacerbated hyperpermeability relative to 1 hour OGD, which was associated with decreased expression levels of occludin and ZO-1, but no change in cell viability or caspase-3. 4 hours OGD + 23 hours RO exacerbated hyperpermeability, decreased expression levels of tight junction proteins, decreased cell viability, and increased caspase-3 expression. Thus, bEnd.3 cells exhibit hyperpermeability, a loss of tight junction proteins, and undergo cell death, after exposure to prolonged periods of OGD. Moreover, they exhibit exacerbated hyperpermeability, a loss of tight junction proteins, and increased expression of caspase-3 after OGD + RO. These findings will facilitate the use of this cell line in studies of BBB disruption and for the testing of therapeutics.

Cerebral Small Vessel Disease: Targeting Oxidative Stress as a Novel Therapeutic Strategy?

Cerebral small vessel disease (SVD) is a major contributor to stroke, and a leading cause of cognitive impairment and dementia. Despite the devastating effects of cerebral SVD, the pathogenesis of cerebral SVD is still not completely understood. Moreover, there are no specific pharmacological strategies for its prevention or treatment. Cerebral SVD is characterized by marked functional and structural abnormalities of the cerebral microcirculation. The clinical manifestations of these pathological changes include lacunar infarcts, white matter hyperintensities, and cerebral microbleeds. The main purpose of this review is to discuss evidence implicating oxidative stress in the arteriopathy of both non-amyloid and amyloid (cerebral amyloid angiopathy) forms of cerebral SVD and its most important risk factors (hypertension and aging), as well as its contribution to cerebral SVD-related brain injury and cognitive impairment. We also highlight current evidence of the involvement of the NADPH oxidases in the development of oxidative stress, enzymes that are a major source of reactive oxygen species in the cerebral vasculature. Lastly, we discuss potential pharmacological strategies for oxidative stress in cerebral SVD, including some of the historical and emerging NADPH oxidase inhibitors.

Ghrelin-related peptides do not modulate vasodilator nitric oxide production or superoxide levels in mouse systemic arteries.

The ghrelin gene is expressed in the stomach where it ultimately encodes up to three peptides, namely, acylated ghrelin, des-acylated ghrelin and obestatin, which all have neuroendocrine roles. Recently, the authors' reported that these peptides have important physiological roles in positively regulating vasodilator nitric oxide (NO) production in the cerebral circulation, and may normally suppress superoxide production by the pro-oxidant enzyme, Nox2-NADPH oxidase. To date, the majority of studies using exogenous peptides infer that they may have similar roles in the systemic circulation. Therefore, this study examined whether exogenous and endogenous ghrelin-related peptides modulate NO production and superoxide levels in mouse mesenteric arteries and/or thoracic aorta. Using wire myography, it was found that application of exogenous acylated ghrelin, des-acylated ghrelin or obestatin to mouse thoracic aorta or mesenteric arteries failed to elicit a vasorelaxation response, whereas all three peptides elicited vasorelaxation responses of rat thoracic aorta. Also, none of the peptides modulated mouse aortic superoxide levels as measured by L-012-enhanced chemiluminescence. Next, it was found that NO bioactivity and superoxide levels were unaffected in the thoracic aorta from ghrelin-deficient mice when compared with wild-type mice. Lastly, using novel GHSR-eGFP reporter mice in combination with double-labelled immunofluorescence, no evidence was found for the growth hormone secretagogue receptor (GHSR1a) in the throracic aorta, which is the only functional ghrelin receptor identified to date. Collectively these findings demonstrate that, in contrast to systemic vessels of other species (e.g. rat and human) and mouse cerebral vessels, ghrelin-related peptides do not modulate vasodilator NO production or superoxide levels in mouse systemic arteries.

Ghrelin-related peptides exert protective effects in the cerebral circulation of male mice through a nonclassical ghrelin receptor(s).

The ghrelin-related peptides, acylated ghrelin, des-acylated ghrelin, and obestatin, are novel gastrointestinal hormones. We firstly investigated whether the ghrelin gene, ghrelin O-acyltransferase, and the ghrelin receptor (GH secretagogue receptor 1a [GHSR1a]) are expressed in mouse cerebral arteries. Secondly, we assessed the cerebrovascular actions of ghrelin-related peptides by examining their effects on vasodilator nitric oxide (NO) and superoxide production. Using RT-PCR, we found the ghrelin gene and ghrelin O-acyltransferase to be expressed at negligible levels in cerebral arteries from male wild-type mice. mRNA expression of GHSR1a was also found to be low in cerebral arteries, and GHSR protein was undetectable in GHSR-enhanced green fluorescent protein mice. We next found that exogenous acylated ghrelin had no effect on the tone of perfused cerebral arteries or superoxide production. By contrast, exogenous des-acylated ghrelin or obestatin elicited powerful vasodilator responses (EC50 < 10 pmol/L) that were abolished by the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester. Furthermore, exogenous des-acylated ghrelin suppressed superoxide production in cerebral arteries. Consistent with our GHSR expression data, vasodilator effects of des-acylated ghrelin or obestatin were sustained in the presence of YIL-781 (GHSR1a antagonist) and in arteries from Ghsr-deficient mice. Using ghrelin-deficient (Ghrl(-/-)) mice, we also found that endogenous production of ghrelin-related peptides regulates NO bioactivity and superoxide levels in the cerebral circulation. Specifically, we show that NO bioactivity was markedly reduced in Ghrl(-/-) vs wild-type mice, and superoxide levels were elevated. These findings reveal protective actions of exogenous and endogenous ghrelin-related peptides in the cerebral circulation and show the existence of a novel ghrelin receptor(s) in the cerebral endothelium.

Obesity and neuroinflammation: a pathway to cognitive impairment.

Obesity is a growing problem worldwide and is associated with a range of comorbidities, including cognitive dysfunction. In this review we will address the evidence that obesity and high fat feeding can lead to cognitive dysfunction. We will also examine the idea that obesity-associated systemic inflammation leads to inflammation within the brain, particularly the hypothalamus, and that this is partially responsible for these negative cognitive outcomes. Thus, obesity, and high fat feeding, lead to systemic inflammation and excess circulating free fatty acids. Circulating cytokines, free fatty acids and immune cells reach the brain at the level of the hypothalamus and initiate local inflammation, including microglial proliferation. This local inflammation likely causes synaptic remodeling and neurodegeneration within the hypothalamus, altering internal hypothalamic circuitry and hypothalamic outputs to other brain regions. The result is disruption to cognitive function mediated by regions such as hippocampus, amygdala, and reward-processing centers. Central inflammation is also likely to affect these regions directly. Thus, central inflammation in obesity leads not just to disruption of hypothalamic satiety signals and perpetuation of overeating, but also to negative outcomes on cognition.

Direct angiotensin AT2 receptor stimulation using a novel AT2 receptor agonist, compound 21, evokes neuroprotection in conscious hypertensive rats.

BACKGROUND: In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents. METHODS AND RESULTS: Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury. CONCLUSION: These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development.

Endothelial cell mineralocorticoid receptors regulate deoxycorticosterone/salt-mediated cardiac remodeling and vascular reactivity but not blood pressure.

Recent studies have identified novel pathological roles for mineralocorticoid receptors (MR) in specific cell types in cardiovascular disease. The mechanisms by which MR promotes inflammation and fibrosis involve multiple cell-specific events. To identify the role of MR in endothelial cells (EC-MR), the current study explored the vascular responses to aldosterone in wild-type (WT) and EC-null mice (EC-MRKO). Nitric oxide function was impaired in the thoracic aorta and mesenteric arteries of aldosterone-treated WT mice. Although endothelial nitric oxide function was equivalently impaired in the mesenteric arteries of aldosterone-treated EC-MRKO mice, endothelial function was unaffected in the aorta, suggesting a differential role for EC-MR depending on the vascular bed. Second, the contribution of EC-MR to cardiovascular inflammation, fibrosis, and hypertension was determined in WT and EC-MRKO treated with deoxycorticosterone/salt for 8 days or 8 weeks. At 8 days, loss of EC-MR prevented macrophage infiltration and the expression of proinflammatory genes in the myocardium. Increased cardiac fibrosis was not detected in either genotype at this time, mRNA levels of profibrotic genes were significantly lower in EC-MRKO mice versus WT. At 8 weeks, deoxycorticosterone/salt treatment increased macrophage recruitment and proinflammatory gene expression in WT but not in EC-MRKO. Collagen deposition and connective tissue growth factor expression were significantly reduced in EC-MRKO versus WT. Interestingly, systolic blood pressure was equivalently elevated in deoxycorticosterone/salt treated WT and EC-MRKO. Our data demonstrate that (1) EC-MR signaling contributes to vascular nitric oxide function in large conduit arteries but not in resistance vessels and (2) an independent role for EC-MR in the inflammatory and profibrotic response to deoxycorticosterone/salt.

Sex-dependent effects of G protein-coupled estrogen receptor activity on outcome after ischemic stroke.

BACKGROUND AND PURPOSE: Experimental studies indicate that estrogen typically, but not universally, has a neuroprotective effect in stroke. Ischemic stroke increases membrane-bound G protein-coupled estrogen receptor (GPER) distribution and expression in the brain of male but not female mice. We hypothesized that GPER activation may have a greater neuroprotective effect in males than in females after stroke. METHODS: Vehicle (dimethyl sulfoxide), a GPER agonist (G-1, 30 µg/kg), or a GPER antagonist (G-15, 300 µg/kg) were administered alone or in combination to young or aged male mice, or young intact or ovariectomized female mice, 1 hour before or 3 hours after cerebral ischemia-reperfusion. Some mice were treated with a combination of G-1 and the pan-caspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh), 1 hour before stroke. We evaluated functional and histological end points of stroke outcome up to 72 hours after ischemia-reperfusion. In addition, apoptosis was examined using cleaved caspase-3 immunohistochemistry. RESULTS: Surprisingly, G-1 worsened functional outcomes and increased infarct volume in males poststroke, in association with an increased expression of cleaved caspase-3 in peri-infarct neurons. These effects were blocked by G-15 or Q-VD-OPh. Conversely, G-15 improved functional outcomes and reduced infarct volume after stroke in males, whether given before or after stroke. In contrast to findings in males, G-1 reduced neurological deficit, apoptosis, and infarct volume in ovariectomized females, but had no significant effect in intact females. CONCLUSIONS: Future therapies for acute stroke could exploit the modulation of GPER activity in a sex-specific manner.

Hydrogen sulfide protects endothelial nitric oxide function under conditions of acute oxidative stress in vitro.

The aim of this study was to examine the ability of H2S, released from NaHS to protect vascular endothelial function under conditions of acute oxidative stress by scavenging superoxide anions (O2(-)) and suppressing vascular superoxide anion production. O2(-) was generated in Krebs' solution by reacting hypoxanthine with xanthine oxidase (Hx-XO) or with the O2(-) generator pyrogallol to model acute oxidative stress in vitro. O2(-) generation was measured by lucigenin-enhanced chemiluminescence. Functional responses in mouse aortic rings were assessed using a small vessel myograph. NaHS scavenged O2(-) in a concentration-dependent manner. Isolated aortic rings exposed to either Hx-XO or pyrogallol displayed significantly attenuated maximum vasorelaxation responses to the endothelium-dependent vasodilator acetylcholine, and significantly reduced NO bioavailability, which was completely reversed if vessels were pre-incubated with NaHS (100 µM). NADPH-stimulated aortic O2(-) production was significantly attenuated by the NADPH oxidase inhibitor diphenyl iodonium. Prior treatment of vessels with NaHS (100 nM-100 µM; 30 min) inhibited NADPH-stimulated aortic O2(-) production in a concentration-dependent manner. This effect persisted when NaHS was washed out prior to measuring NADPH-stimulated O2(-) production. These data show for the first time that NaHS directly scavenges O2(-) and suppresses vascular NADPH oxidase-derived O2(-) production in vitro. Furthermore, these properties protect endothelial function and NO bioavailability in an in vitro model of acute oxidative stress. These results suggest that H2S can elicit vasoprotection by both scavenging O2(-) and by reducing vascular NADPH oxidase-derived O2(-) production.