Prostaglandin E2 EP2 Receptor Deletion Attenuates Intracerebral Hemorrhage-Induced Brain Injury and Improves Functional Recovery

Intracerebral hemorrhage (ICH) is a devastating type of stroke characterized by bleeding into the brain parenchyma and secondary brain injury resulting from strong neuroinflammatory responses to blood components. Production of prostaglandin E₂ (PGE₂) is significantly upregulated following ICH and contributes to this inflammatory response in part through its E prostanoid receptor subtype 2 (EP2). Signaling through the EP2 receptor has been shown to affect outcomes of many acute and chronic neurological disorders; although, not yet explored in the context of ICH. Wildtype (WT) and EP2 receptor knockout (EP2^−/−) mice were subjected to ICH, and various anatomical and functional outcomes were assessed by histology and neurobehavioral testing, respectively. When compared with age-matched WT controls, EP2^−/− mice had 41.9 ± 4.7% smaller ICH-induced brain lesions and displayed significantly less ipsilateral hemispheric enlargement and incidence of intraventricular hemorrhage. Anatomical outcomes correlated with improved functional recovery as identified by neurological deficit scoring. Histological staining was performed to begin investigating the mechanisms involved in EP2-mediated neurotoxicity after ICH. EP2^−/− mice exhibited 45.5 ± 5.8% and 41.4 ± 8.1% less blood and ferric iron accumulation, respectively. Furthermore, significantly less striatal and cortical microgliosis, striatal and cortical astrogliosis, blood–brain barrier breakdown, and peripheral neutrophil infiltration were seen in EP2^−/− mice. This study is the first to suggest a deleterious role for the PGE₂-EP2 signaling axis in modulating brain injury, inflammation, and functional recovery following ICH. Targeting the EP2 G protein-coupled receptor may represent a new therapeutic avenue for the treatment of hemorrhagic stroke.     

Deletion of TRAAK Potassium Channel Affects Brain Metabolism and Protects against Ischemia

Cerebral stroke is a worldwide leading cause of disability. The two-pore domain K⁺ channels identified as background channels are involved in many functions in brain under physiological and pathological conditions. We addressed the hypothesis that TRAAK, a mechano-gated and lipid-sensitive two-pore domain K⁺ channel, is involved in the pathophysiology of brain ischemia. We studied the effects of TRAAK deletion on brain morphology and metabolism under physiological conditions, and during temporary focal cerebral ischemia in Traak^−/− mice using a combination of in vivo magnetic resonance imaging (MRI) techniques and multinuclear magnetic resonance spectroscopy (MRS) methods. We provide the first in vivo evidence establishing a link between TRAAK and neurometabolism. Under physiological conditions, Traak^−/− mice showed a particular metabolic phenotype characterized by higher levels of taurine and myo-inositol than Traak^+/+ mice. Upon ischemia, Traak^−/− mice had a smaller infarcted volume, with lower contribution of cellular edema than Traak^+/+ mice. Moreover, brain microcirculation was less damaged, and brain metabolism and pH were preserved. Our results show that expression of TRAAK strongly influences tissue levels of organic osmolytes. Traak^−/− mice resilience to cellular edema under ischemia appears related to their physiologically high levels of myo-inositol and of taurine, an aminoacid involved in the modulation of mitochondrial activity and cell death. The beneficial effects of TRAAK deletion designate this channel as a promising pharmacological target for the treatment against stroke.     

MAP Kinase Phosphatase-2 Plays a Key Role in the Control of Infection with Toxoplasma gondii by Modulating iNOS and Arginase-1 Activities in Mice

The dual specific phosphatase, MAP kinase phosphatase-2 (MKP-2) has recently been demonstrated to negatively regulate macrophage arginase-1 expression, while at the same time to positively regulate iNOS expression. Consequently, MKP-2 is likely to play a significant role in the host interplay with intracellular pathogens. Here we demonstrate that MKP-2^−/− mice on the C57BL/6 background have enhanced susceptibility compared with wild-type counterparts following infection with type-2 strains of Toxoplasma gondii as measured by increased parasite multiplication during acute infection, increased mortality from day 12 post-infection onwards and increased parasite burdens in the brain, day 30 post-infection. MKP-2^−/− mice did not, however, demonstrate defective type-1 responses compared with MKP-2^+/+ mice following infection although they did display significantly reduced serum nitrite levels and enhanced tissue arginase-1 expression. Early resistance to T. gondii in MKP-2^+/+, but not MKP-2^−/−, mice was nitric oxide (NO) dependent as infected MKP-2^+/+, but not MKP-2^−/− mice succumbed within 10 days post-infection with increased parasite burdens following treatment with the iNOS inhibitor L-NAME. Conversely, treatment of infected MKP-2^−/− but not MKP-2^+/+ mice with nor-NOHA increased parasite burdens indicating a protective role for arginase-1 in MKP-2^−/− mice. In vitro studies using tachyzoite-infected bone marrow derived macrophages and selective inhibition of arginase-1 and iNOS activities confirmed that both iNOS and arginase-1 contributed to inhibiting parasite replication. However, the effects of arginase-1 were transient and ultimately the role of iNOS was paramount in facilitating long-term inhibition of parasite multiplication within macrophages.     

Tenascin-R promotes assembly of the extracellular matrix of perineuronal nets via clustering of aggrecan

Perineuronal nets (PNs) in the brains of tenascin-R-deficient (tn-r^−/−) mice develop in temporal concordance with those of wild-type (tn-r^+/+) mice. However, the histological appearance of PNs is abnormal in adult tn-r^−/− mice. Here, we investigated whether similar defects are also seen in dissociated and organotypic cultures from hippocampus and forebrain of tn-r^−/− mice and whether the structure of PNs could be normalized. In tn-r^−/− cultures, accumulations of several extracellular matrix molecules were mostly associated with somata, whereas dendrites were sparsely covered, compared with tn-r^+/+ mice. Experiments to normalize the structure of PNs in tn-r^−/− organotypic slice cultures by depolarization of neurons, or by co-culturing tn-r^+/+ and tn-r^−/− brain slices failed to restore a normal PN phenotype. However, formation of dendritic PNs in cultures was improved by the application of tenascin-R protein and rescued by polyclonal antibodies to aggrecan and a bivalent, but not monovalent form of the lectin Wisteria floribunda agglutinin. These results show that tenascin-R and aggrecan are decisive contributors to formation and stabilization of PNs and that tenascin-R may implement these functions by clustering of aggrecan. Proposed approaches for restoration of normal PN structure are noteworthy in the context of PN abnormalities in neurological disorders, such as epilepsy, schizophrenia and addiction.     

Acid Sphingomyelinase Gene Knockout Ameliorates Hyperhomocysteinemic Glomerular Injury in Mice Lacking Cystathionine-β-Synthase

Acid sphingomyelinase (ASM) has been implicated in the development of hyperhomocysteinemia (hHcys)-induced glomerular oxidative stress and injury. However, it remains unknown whether genetically engineering of ASM gene produces beneficial or detrimental action on hHcys-induced glomerular injury. The present study generated and characterized the mice lacking cystathionine β-synthase (Cbs) and Asm mouse gene by cross breeding Cbs^+/− and Asm^+/− mice. Given that the homozygotes of Cbs^−/−/Asm^−/− mice could not survive for 3 weeks. Cbs^+/−/Asm^+/+, Cbs^+/−/Asm^+/− and Cbs^+/−/Asm^−/− as well as their Cbs wild type littermates were used to study the role of Asm^−/− under a background of Cbs^+/− with hHcys. HPLC analysis revealed that plasma Hcys level was significantly elevated in Cbs heterozygous (Cbs^+/−) mice with different copies of Asm gene compared to Cbs^+/+ mice with different Asm gene copies. Cbs^+/−/Asm^+/+ mice had significantly increased renal Asm activity, ceramide production and O₂.⁻ level compared to Cbs^+/+/Asm^+/+, while Cbs^+/−/Asm^−/− mice showed significantly reduced renal Asm activity, ceramide production and O₂.⁻ level due to increased plasma Hcys levels. Confocal microscopy demonstrated that colocalization of podocin with ceramide was much lower in Cbs^+/−/Asm^−/− mice compared to Cbs^+/−/Asm^+/+ mice, which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs^+/−/Asm^−/− mice. Immunofluorescent analyses of the podocin, nephrin and desmin expression also illustrated less podocyte damages in the glomeruli from Cbs^+/−/Asm^−/− mice compared to Cbs^+/−/Asm^+/+ mice. In in vitro studies of podocytes, hHcys-enhanced O₂.⁻ production, desmin expression, and ceramide production as well as decreases in VEGF level and podocin expression in podocytes were substantially attenuated by prior treatment with amitriptyline, an Asm inhibitor. In conclusion, Asm gene knockout or corresponding enzyme inhibition protects the podocytes and glomeruli from hHcys-induced oxidative stress and injury.     

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date^1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies³. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase⁴. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours⁵. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood⁶ as well as an ICH injection model of bacterial collagenase⁷, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 μl) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p<0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.     

Heterozygosity for Fibrinogen Results in Efficient Resolution of Kidney Ischemia Reperfusion Injury

Fibrinogen (Fg) has been recognized to play a central role in coagulation, inflammation and tissue regeneration. Several studies have used Fg deficient mice (Fg^−/−) in comparison with heterozygous mice (Fg^+/−) to point the proinflammatory role of Fg in diverse pathological conditions and disease states. Although Fg^+/− mice are considered ‘normal’, plasma Fg is reduced to ∼75% of the normal circulating levels present in wild type mice (Fg^+/+). We report that this reduction in Fg protein production in the Fg^+/− mice is enough to protect them from kidney ischemia reperfusion injury (IRI) as assessed by tubular injury, kidney dysfunction, necrosis, apoptosis and inflammatory immune cell infiltration. Mechanistically, we observed binding of Fg to ICAM-1 in kidney tissues of Fg^+/+ mice at 24 h following IRI as compared to a complete absence of binding observed in the Fg^+/− and Fg^−/− mice. Raf-1 and ERK were highly activated as evident by significantly higher phosphorylation in the Fg^+/+ kidneys at 24 h following IRI as compared to Fg^+/− and Fg^−/− mice kidneys. On the other hand Cyclin D1 and pRb, indicating higher cell proliferation, were significantly increased in the Fg^+/− and Fg^−/− as compared to Fg^+/+ kidneys. These data suggest that Fg heterozygosity allows maintenance of a critical balance of Fg that enables regression of initial injury and promotes faster resolution of kidney damage.     

Deletion of Nardilysin Prevents the Development of Steatohepatitis and Liver Fibrotic Changes

Nonalcoholic steatohepatitis (NASH) is an inflammatory form of nonalcoholic fatty liver disease that progresses to liver cirrhosis. It is still unknown how only limited patients with fatty liver develop NASH. Tumor necrosis factor (TNF)-α is one of the key molecules in initiating the vicious circle of inflammations. Nardilysin (N-arginine dibasic convertase; Nrd1), a zinc metalloendopeptidase of the M16 family, enhances ectodomain shedding of TNF-α, resulting in the activation of inflammatory responses. In this study, we aimed to examine the role of Nrd1 in the development of NASH. Nrd1^+/+ and Nrd1^−/− mice were fed a control choline-supplemented amino acid-defined (CSAA) diet or a choline-deficient amino acid-defined (CDAA) diet. Fatty deposits were accumulated in the livers of both Nrd1^+/+ and Nrd1^−/− mice by the administration of the CSAA or CDAA diets, although the amount of liver triglyceride in Nrd1^−/− mice was lower than that in Nrd1^+/+ mice. Serum alanine aminotransferase levels were increased in Nrd1^+/+ mice but not in Nrd1^−/− mice fed the CDAA diet. mRNA expression of inflammatory cytokines were decreased in Nrd1^−/− mice than in Nrd1^+/+ mice fed the CDAA diet. While TNF-α protein was detected in both Nrd1^+/+ and Nrd1^−/− mouse livers fed the CDAA diet, secretion of TNF-α in Nrd1^−/− mice was significantly less than that in Nrd1^+/+ mice, indicating the decreased TNF-α shedding in Nrd1^−/− mouse liver. Notably, fibrotic changes of the liver, accompanied by the increase of fibrogenic markers, were observed in Nrd1^+/+ mice but not in Nrd1^−/− mice fed the CDAA diet. Similar to the CDAA diet, fibrotic changes were not observed in Nrd1^−/− mice fed a high-fat diet. Thus, deletion of nardilysin prevents the development of diet-induced steatohepatitis and liver fibrogenesis. Nardilysin could be an attractive target for anti-inflammatory therapy against NASH.     

T Cell Hypo-Responsiveness against Leishmania major in MAP Kinase Phosphatase (MKP) 2 Deficient C57BL/6 Mice Does Not Alter the Healer Disease Phenotype

We have recently demonstrated that MAP kinase phosphatase 2 (MKP-2) deficient C57BL/6 mice, unlike their wild-type counterparts, are unable to control infection with the protozoan parasite Leishmania mexicana. Increased susceptibility was associated with elevated Arginase-1 levels and reduced iNOS activity in macrophages as well as a diminished T_H1 response. By contrast, in the present study footpad infection of MKP-2^−/− mice with L. major resulted in a healing response as measured by lesion size and parasite numbers similar to infected MKP-2^+/+ mice. Analysis of immune responses following infection demonstrated a reduced T_H1 response in MKP-2^−/− mice with lower parasite specific serum IgG2b levels, a lower frequency of IFN-γ and TNF-α producing CD4⁺ and CD8⁺ T cells and lower antigen stimulated spleen cell IFN-γ production than their wild-type counterparts. However, infected MKP-2^−/− mice also had similarly reduced levels of antigen induced spleen and lymph node cell IL-4 production compared with MKP-2^+/+ mice as well as reduced levels of parasite-specific IgG1 in the serum, indicating a general T cell hypo-responsiveness. Consequently the overall T_H1/T_H2 balance was unaltered in MKP-2^−/− compared with wild-type mice. Although non-stimulated MKP-2^−/− macrophages were more permissive to L. major growth than macrophages from MKP-2^+/+ mice, reflecting their reduced iNOS and increased Arginase-1 expression, LPS/IFN-γ activation was equally effective at controlling parasite growth in MKP-2^−/− and MKP-2^+/+ macrophages. Consequently, in the absence of any switch in the T_H1/T_H2 balance in MKP-2^−/− mice, no significant change in disease phenotype was observed.     

Calpain-dependent Cleavage of N-cadherin Is Involved in the Progression of Post-myocardial Infarction Remodeling

Enzymatic proteolysis by calpains, Ca²⁺-dependent intracellular cysteine proteases, has been implicated in pathological processes such as cellular degeneration or death. Here, we investigated the role of calpain activation in the hearts subjected to myocardial infarction. We produced myocardial infarction in Cast^−/− mice deficient for calpastatin, the specific endogenous inhibitory protein for calpains, and Cast^+/+ mice. The activity of cardiac calpains in Cast^+/+ mice was not elevated within 1 day but showed a gradual elevation after 7 days following myocardial infarction, which was further pronounced in Cast^−/− mice. Although the prevalence of cardiomyocyte death was indistinguishable between Cast^−/− and Cast^+/+ mice, Cast^−/− mice exhibited profound contractile dysfunction and chamber dilatation and showed a significant reduction in survival rate after myocardial infarction as compared with Cast^+/+ mice. Notably, immunofluorescence revealed that at 28 days after myocardial infarction, calpains were activated in cardiomyocytes exclusively at the border zone and that Cast^−/− mice showed higher intensity and a broader extent of calpain activation at the border zone than Cast^+/+ mice. In the border zone of Cast^−/− mice, pronounced activation of calpains was associated with a decrease in N-cadherin expression and up-regulation of molecular markers for cardiac hypertrophy and fibrosis. In cultured rat neonatal cardiomyocytes, calpain activation by treatment with ionomycin induced cleavage of N-cadherin and decreased expression levels of β-catenin and connexin 43, which was attenuated by calpain inhibitor. These results thus demonstrate that activation of calpains disassembles cell-cell adhesion at intercalated discs by degrading N-cadherin and thereby promotes left ventricular remodeling after myocardial infarction.     

Peroxisome Proliferator-activated Receptor-D (PPARD) Coordinates Mouse Spermatogenesis by Modulating Extracellular Signal-regulated Kinase (ERK)-dependent Signaling

Ppard^−/− mice exhibit smaller litter size compared with Ppard^+/+ mice. To determine whether peroxisome proliferator-activated receptor-D (PPARD) could possibly influence this phenotype, the role of PPARD in testicular biology was examined. Atrophic testes and testicular degeneration were observed in Ppard^−/− mice compared with Ppard^+/+ mice, indicating that PPARD modulates spermatogenesis. Higher expression of p27 and decreased expression of proliferating cellular nuclear antigen in Sertoli cells were observed in Ppard^+/+ mice as compared with Ppard^−/− mice, and these were associated with decreased Sertoli cell number in Ppard^+/+ mice. Cyclin D1 and cyclin D2 expression was lower in Ppard^+/+ as compared with Ppard^−/− mice. Ligand activation of PPARD inhibited proliferation of a mouse Sertoli cell line, TM4, and an inverse agonist of PPARD (DG172) rescued this effect. Temporal inhibition of extracellular signal-regulated kinase (ERK) activation by PPARD in the testis was observed in Ppard^+/+ mice and was associated with decreased serum follicle-stimulating hormone and higher claudin-11 expression along the blood-testis barrier. PPARD-dependent ERK activation also altered expression of claudin-11, p27, cyclin D1, and cyclin D2 in TM4 cells, causing inhibition of cell proliferation, maturation, and formation of tight junctions in Sertoli cells, thus confirming a requirement for PPARD in accurate Sertoli cell function. Combined, these results reveal for the first time that PPARD regulates spermatogenesis by modulating the function of Sertoli cells during early testis development.     

Three distinct roles of aquaporin-4 in brain function revealed by knockout mice

Aquaporin-4 (AQP4) is expressed in astrocytes throughout the central nervous system, particularly at the blood-brain and brain-cerebrospinal fluid barriers. Phenotype analysis of transgenic mice lacking AQP4 has provided compelling evidence for involvement of AQP4 in cerebral water balance, astrocyte migration, and neural signal transduction. AQP4-null mice have reduced brain swelling and improved neurological outcome in models of (cellular) cytotoxic cerebral edema including water intoxication, focal cerebral ischemia, and bacterial meningitis. However, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema including cortical freeze-injury, brain tumor, brain abscess and hydrocephalus, probably due to impaired AQP4-dependent brain water clearance. AQP4 deficiency or knock-down slows astrocyte migration in response to a chemotactic stimulus in vitro, and AQP4 deletion impairs glial scar progression following injury in vivo. AQP4-null mice also manifest reduced sound- and light-evoked potentials, and increased threshold and prolonged duration of induced seizures. Impaired K⁺ reuptake by astrocytes in AQP4 deficiency may account for the neural signal transduction phenotype. Based on these findings, we propose modulation of AQP4 expression or function as a novel therapeutic strategy for a variety of cerebral disorders including stroke, tumor, infection, hydrocephalus, epilepsy, and traumatic brain injury.     

Guanylate Cyclase C Deficiency Causes Severe Inflammation in a Murine Model of Spontaneous Colitis

Background

Guanylate Cyclase C (GC-C; Gucy2c) is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses.

Methods

We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C^+/+ and GC-C^−/− mice were bred with interleukin (IL)-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10^−/−, GC-C^+/+IL-10^−/− and GC-C^−/−IL-10^−/− mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line.

Results

Relative to GC-C^+/+ animals, intraperitoneal lipopolysaccharide injection into GC-C^−/− mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C^−/−IL-10^−/− animals was significantly more severe relative to GC-C^+/+IL-10^−/− mice. Unlike GC-C^+/+IL-10^−/− controls, colon pathology in GC-C^−/−IL-10^−/− animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C^−/−IL-10^−/− mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells.

Conclusions

The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by systemic cytokine burst or loss of mucosal immune cell immunosuppression. These data as well as the apparent intestinal inflammation in human GC-C mutant kindred underscore the importance of GC-C in regulating the response to injury and inflammation within the gut.     

The Soluble Interleukin-6 Receptor Is a Mediator of Hematopoietic and Skeletal Actions of Parathyroid Hormone

Both PTH and IL-6 signaling play pivotal roles in hematopoiesis and skeletal biology, but their interdependence is unclear. The purpose of this study was to evaluate the effect of IL-6 and soluble IL-6 receptor (sIL-6R) on hematopoietic and skeletal actions of PTH. In the bone microenvironment, PTH stimulated sIL-6R protein levels in primary osteoblast cultures in vitro and bone marrow in vivo in both IL-6^+/+ and IL-6^−/− mice. PTH-mediated hematopoietic cell expansion was attenuated in IL-6^−/− compared with IL-6^+/+ bone marrow, whereas sIL-6R treatment amplified PTH actions in IL-6^−/− earlier than IL-6^+/+ marrow cultures. Blocking sIL-6R signaling with sgp130 (soluble glycoprotein 130 receptor) inhibited PTH-dependent hematopoietic cell expansion in IL-6^−/− marrow. In the skeletal system, although intermittent PTH administration to IL-6^+/+ and IL-6^−/− mice resulted in similar anabolic actions, blocking sIL-6R significantly attenuated PTH anabolic actions. sIL-6R showed no direct effects on osteoblast proliferation or differentiation in vitro; however, it up-regulated myeloid cell expansion and production of the mesenchymal stem cell recruiting agent, TGF-β1 in the bone marrow microenvironment. Collectively, sIL-6R demonstrated orphan function and mediated PTH anabolic actions in bone in association with support of myeloid lineage cells in the hematopoietic system.     

Effect of ecto-5′-nucleotidase (eN) in astrocytes on adenosine and inosine formation

ATP is a gliotransmitter released from astrocytes. Extracellularly, ATP is metabolized by a series of enzymes, including ecto-5′-nucleotidase (eN; also known as CD73) which is encoded by the gene 5NTE and functions to form adenosine (ADO) from adenosine monophosphate (AMP). Under ischemic conditions, ADO levels in brain increase up to 100-fold. We used astrocytes cultured from 5NTE^+/+ or 5NTE^−/− mice to evaluate the role of eN expressed by astrocytes in the production of ADO and inosine (INO) in response to glucose deprivation (GD) or oxygen-glucose deprivation (OGD). We also used co-cultures of these astrocytes with wild-type neurons to evaluate the role of eN expressed by astrocytes in the production of ADO and INO in response to GD, OGD, or N-methyl-d-aspartate (NMDA) treatment. As expected, astrocytes from 5NTE^+/+ mice produced adenosine from AMP; the eN inhibitor α,β-methylene ADP (AOPCP) decreased ADO formation. In contrast, little ADO was formed by astrocytes from 5NTE^−/− mice and AOPCP had no significant effect. GD and OGD treatment of 5NTE^+/+ astrocytes and 5NTE^+/+ astrocyte-neuron co-cultures produced extracellular ADO levels that were inhibited by AOPCP. In contrast, these conditions did not evoke ADO production in cultures containing 5NTE^−/− astrocytes. NMDA treatment produced similar increases in ADO in both 5NTE^+/+ and 5NTE^−/− astrocyte-neuron co-cultures; dipyridamole (DPR) but not AOPCP inhibited ADO production. These results indicate that eN is prominent in the formation of ADO from astrocytes but in astrocyte-neuron co-cultures, other enzymes or pathways contribute to rising ADO levels in ischemia-like conditions.     

Potential Compensation among Group I PAK Members in Hindlimb Ischemia and Wound Healing

PAKs are serine/threonine kinases that regulate cytoskeletal dynamics and cell migration. PAK1 is activated by binding to the small EF hand protein, CIB1, or to the Rho GTPases Rac1 or Cdc42. The role of PAK1 in angiogenesis was established based only on in vitro studies and its role in angiogenesis in vivo has never been examined. Here we tested the hypothesis that PAK1 is an essential regulator of ischemic neovascularization (arteriogenesis and angiogenesis) and wound healing using a global PAK1 knockout mouse. Neovascularization was assessed using unilateral hindlimb ischemia. We found that plantar perfusion, limb use and appearance were not significantly different between 6–8 week old PAK1^−/− and PAK1^+/+ mice throughout the 21-day period following hindlimb ischemia; however a slightly delayed healing was observed in 16 week old PAK1^−/− mice. In addition, the wound healing rate, as assessed with an ear punch assay, was unchanged in PAK1^−/− mice. Surprisingly, however, we observed a notable increase in PAK2 expression and phosphorylation in ischemic gastrocnemius tissue from PAK1^−/− but not PAK1^+/+ mice. Furthermore, we observed higher levels of activated ERK2, but not AKT, in ischemic and non-ischemic muscle of PAK1^−/− mice upon hindlimb ischemic injury. A group I PAK inhibitor, IPA3, significantly inhibited endothelial cell sprouting from aortic rings in both PAK1^−/− and PAK1^+/+ mice, implying that PAK2 is a potential contributor to this process. Taken together, our data indicate that while PAK1 has the potential to contribute to neovascularization and wound healing, PAK2 may functionally compensate when PAK1 is deficient.     

Neutrophil-Derived Myeloperoxidase Aggravates Non-Alcoholic Steatohepatitis in Low-Density Lipoprotein Receptor-Deficient Mice

Background

Chronic inflammation and oxidative stress play fundamental roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). Previously, we reported that myeloperoxidase (MPO), an aggressive oxidant-generating neutrophil enzyme, is associated with NASH severity in man. We now investigated the hypothesis that MPO contributes to the development and progression of NASH.

Methodology

Low-density lipoprotein receptor-deficient mice with an MPO-deficient hematopoietic system (LDLR^−/−/MPO^−/−tp mice) were generated and compared with LDLR^−/−/MPO^+/+tp mice after induction of NASH by high-fat feeding.

Results

High-fat feeding caused a ∼4-fold induction of liver MPO in LDLR^−/−/MPO^+/+ mice which was associated with hepatic sequestration of MPO-positive neutrophils and high levels of nitrotyrosine, a marker of MPO activity. Importantly, LDLR^−/−/MPO^−/−tp mice displayed markedly reduced hepatic neutrophil and T-lymphocyte infiltration (p<0.05), and strong down regulation of pro-inflammatory genes such as TNF-α and IL-6 (p<0.05, p<0.01) in comparison with LDLR^−/−/MPO^+/+tp mice. Next to the generalized reduction of inflammation, liver cholesterol accumulation was significantly diminished in LDLR^−/−/MPO^−/−tp mice (p = 0.01). Moreover, MPO deficiency appeared to attenuate the development of hepatic fibrosis as evident from reduced hydroxyproline levels (p<0.01). Interestingly, visceral adipose tissue inflammation was markedly reduced in LDLR^−/−/MPO^−/−tp mice, with a complete lack of macrophage crown-like structures. In conclusion, MPO deficiency attenuates the development of NASH and diminishes adipose tissue inflammation in response to a high fat diet, supporting an important role for neutrophils in the pathogenesis of metabolic disease.     

Promyelocytic Leukemia (PML) Protein Plays Important Roles in Regulating Cell Adhesion,Morphology, Proliferation and Migration

PML protein plays important roles in regulating cellular homeostasis. It forms PML nuclear bodies (PML-NBs) that act like nuclear relay stations and participate in many cellular functions. In this study, we have examined the proteome of mouse embryonic fibroblasts (MEFs) derived from normal (PML^+/+) and PML knockout (PML^−/−) mice. The aim was to identify proteins that were differentially expressed when MEFs were incapable of producing PML. Using comparative proteomics, total protein were extracted from PML^−/− and PML^+/+ MEFs, resolved by two dimensional electrophoresis (2-DE) gels and the differentially expressed proteins identified by LC-ESI-MS/MS. Nine proteins (PML, NDRG1, CACYBP, CFL1, RSU1, TRIO, CTRO, ANXA4 and UBE2M) were determined to be down-regulated in PML^−/− MEFs. In contrast, ten proteins (CIAPIN1, FAM50A, SUMO2 HSPB1 NSFL1C, PCBP2, YWHAG, STMN1, TPD52L2 and PDAP1) were found up-regulated. Many of these differentially expressed proteins play crucial roles in cell adhesion, migration, morphology and cytokinesis. The protein profiles explain why PML^−/− and PML^+/+ MEFs were morphologically different. In addition, we demonstrated PML^−/− MEFs were less adhesive, proliferated more extensively and migrated significantly slower than PML^+/+ MEFs. NDRG1, a protein that was down-regulated in PML^−/− MEFs, was selected for further investigation. We determined that silencing NDRG1expression in PML^+/+ MEFs increased cell proliferation and inhibited PML expression. Since NDRG expression was suppressed in PML^−/− MEFs, this may explain why these cells proliferate more extensively than PML^+/+ MEFs. Furthermore, silencing NDRG1expression also impaired TGF-β1 signaling by inhibiting SMAD3 phosphorylation.     

Evidence That the EphA2 Receptor Exacerbates Ischemic Brain Injury

Ephrin (Eph) signaling within the central nervous system is known to modulate axon guidance, synaptic plasticity, and to promote long-term potentiation. We investigated the potential involvement of EphA2 receptors in ischemic stroke-induced brain inflammation in a mouse model of focal stroke. Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and EphA2-deficient (EphA2^−/−) mice by middle cerebral artery occlusion (MCAO; 60 min), followed by reperfusion (24 or 72 h). Brain infarction was measured using triphenyltetrazolium chloride staining. Neurological deficit scores and brain infarct volumes were significantly less in EphA2^−/− mice compared with WT controls. This protection by EphA2 deletion was associated with a comparative decrease in brain edema, blood-brain barrier damage, MMP-9 expression and leukocyte infiltration, and higher expression levels of the tight junction protein, zona occludens-1. Moreover, EphA2^−/− brains had significantly lower levels of the pro-apoptotic proteins, cleaved caspase-3 and BAX, and higher levels of the anti-apoptotic protein, Bcl-2 as compared to WT group. We confirmed that isolated WT cortical neurons express the EphA2 receptor and its ligands (ephrin-A1–A3). Furthermore, expression of all four proteins was increased in WT primary cortical neurons following 24 h of glucose deprivation, and in the brains of WT mice following stroke. Glucose deprivation induced less cell death in primary neurons from EphA2^−/− compared with WT mice. In conclusion, our data provide the first evidence that the EphA2 receptor directly contributes to blood-brain barrier damage and neuronal death following ischemic stroke.