Crif1 deficiency in dopamine neurons triggers early-onset parkinsonism.

Mitochondrial dysfunction has been implicated in Parkinson's Disease (PD) progression; however, the mitochondrial factors underlying the development of PD symptoms remain unclear. One candidate is CR6-interacting factor1 (CRIF1), which controls translation and membrane insertion of 13 mitochondrial proteins involved in oxidative phosphorylation. Here, we found that CRIF1 mRNA and protein expression were significantly reduced in postmortem brains of elderly PD patients compared to normal controls. To evaluate the effect of Crif1 deficiency, we produced mice lacking the Crif1 gene in dopaminergic neurons (DAT-CRIF1-KO mice). From 5 weeks of age, DAT-CRIF1-KO mice began to show decreased dopamine production with progressive neuronal degeneration in the nigral area. At ~10 weeks of age, they developed PD-like behavioral deficits, including gait abnormalities, rigidity, and resting tremor. L-DOPA, a medication used to treat PD, ameliorated these defects at an early stage, although it was ineffective in older mice. Taken together, the observation that CRIF1 expression is reduced in human PD brains and deletion of CRIF1 in dopaminergic neurons leads to early-onset PD with stepwise PD progression support the conclusion that CRIF1-mediated mitochondrial function is important for the survival of dopaminergic neurons.

Characterization of acute pain-induced behavioral passivity in mice: Insights from statistical modeling.

Affective-motivational disturbances are highly inconsistent in animal pain models. The reproducibility of the open-field test in assessing anxiety, malaise or disability remains controversial despite its popularity. While traumatic, persistent or multiregional pain models are commonly considered more effective in inducing negative affect or functional impairment, the early psychobehavioral changes before pain chronification are often underexplored. Here, we aimed to clarify the fundamental relationship between hypernociception and passive distress-like behavior using a model of transient inflammatory pain. To minimize latent confounders and increase data consistency, male C57BL/6N mice were habituated to the open-field arena 6 times before receiving the unilateral intraplantar injection of prostaglandin E2 (PGE2) or vehicle. Open-field (40-min exploration) and nociceptive behavior were evaluated repeatedly along the course of hypernociception in both wild-type and transgenic mice with a known pronociceptive phenotype. To reduce subjectivity, multivariate open-field behavioral outcomes were analyzed by statistical modeling based on exploratory factor analyses, which yielded a 2-factor solution. Within 3 hr after PGE2 injection, mice developed significantly reduced center exploration (factor 1) and a marginally significant increase in their habituation tendency (factor 2), which were not apparent in vehicle-injected mice. The behavioral passivity generally improved as hypernociception subsided. Therefore, transient inflammatory irritation is sufficient to suppress mouse open-field exploratory activity. The apparent absence of late affective-motivational changes in some rodents with prolonged hypernociception may not imply a lack of preceding or underlying neuropsychological alterations. Procedural pain after invasive animal experiments, however small, should be assessed and adequately controlled as a potential research confounder.

PICK1 inhibits the E3 ubiquitin ligase activity of Parkin and reduces its neuronal protective effect.

Parkin functions as a multipurpose E3 ubiquitin ligase, and Parkin loss of function is associated with both sporadic and familial Parkinson's disease (PD). We report that the Bin/Amphiphysin/Rvs (BAR) domain of protein interacting with PRKCA1 (PICK1) bound to the really interesting new gene 1 (RING1) domain of Parkin and potently inhibited the E3 ligase activity of Parkin by disrupting its interaction with UbcH7. Parkin translocated to damaged mitochondria and led to their degradation in neurons, whereas PICK1 robustly inhibited this process. PICK1 also impaired the protective function of Parkin against stresses in SH-SY5Y cells and neurons. The protein levels of several Parkin substrates were reduced in young PICK1-knockout mice, and these mice were resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated toxicity. Taken together, the results indicate that PICK1 is a potent inhibitor of Parkin, and the reduction of PICK1 enhances the protective effect of Parkin.

Adipocyte fatty acid-binding protein exacerbates cerebral ischaemia injury by disrupting the blood-brain barrier.

AIMS: Adipocyte fatty acid-binding protein (A-FABP) is an adipokine implicating in various metabolic diseases. Elevated circulating levels of A-FABP correlate positively with poor prognosis in ischaemic stroke (IS) patients. No information is available concerning the role of A-FABP in the pathogenesis of IS. Experiments were designed to determine whether or not A-FABP mediates blood-brain barrier (BBB) disruption, and if so, to explore the molecular mechanisms underlying this deleterious effects. METHODS AND RESULTS: Circulating A-FABP and its cerebral expression were increased in mice after middle cerebral artery occlusion. Genetic deletion and pharmacological inhibition of A-FABP alleviated cerebral ischaemia injury with reduced infarction volume, cerebral oedema, neurological deficits, and neuronal apoptosis; BBB disruption was attenuated and accompanied by reduced degradation of tight junction proteins and induction of matrix metalloproteinases-9 (MMP-9). In patients with acute IS, elevated circulating A-FABP levels positively correlated with those of MMP-9 and cerebral infarct volume. Mechanistically, ischaemia-induced elevation of A-FABP selectively in peripheral blood monocyte-derived macrophages and cerebral resident microglia promoted MMP-9 transactivation by potentiating JNK/c-Jun signalling, enhancing degradation of tight junction proteins and BBB leakage. The detrimental effects of A-FABP were prevented by pharmacological inhibition of MMP-9. CONCLUSION: A-FABP is a key mediator of cerebral ischaemia injury promoting MMP-9-mediated BBB disruption. Inhibition of A-FABP is a potential strategy to improve IS outcome.

Aldose Reductase and the Polyol Pathway in Schwann Cells: Old and New Problems.

Aldose reductase (AR) is a member of the reduced nicotinamide adenosine dinucleotide phosphate (NADPH)-dependent aldo-keto reductase superfamily. It is also the rate-limiting enzyme of the polyol pathway, catalyzing the conversion of glucose to sorbitol, which is subsequently converted to fructose by sorbitol dehydrogenase. AR is highly expressed by Schwann cells in the peripheral nervous system (PNS). The excess glucose flux through AR of the polyol pathway under hyperglycemic conditions has been suggested to play a critical role in the development and progression of diabetic peripheral neuropathy (DPN). Despite the intensive basic and clinical studies over the past four decades, the significance of AR over-activation as the pathogenic mechanism of DPN remains to be elucidated. Moreover, the expected efficacy of some AR inhibitors in patients with DPN has been unsatisfactory, which prompted us to further investigate and review the understanding of the physiological and pathological roles of AR in the PNS. Particularly, the investigation of AR and the polyol pathway using immortalized Schwann cells established from normal and AR-deficient mice could shed light on the causal relationship between the metabolic abnormalities of Schwann cells and discordance of axon-Schwann cell interplay in DPN, and led to the development of better therapeutic strategies against DPN.

Exchange protein directly activated by cAMP (Epac) 1 plays an essential role in stress-induced exercise capacity by regulating PGC-1α and fatty acid metabolism in skeletal muscle.

Exchange protein directly activated by cAMP (Epac) mediates cAMP-mediated cell signal independent of protein kinase A (PKA). Mice lacking Epac1 displayed metabolic defect suggesting possible functional involvement of skeletal muscle and exercise capacity. Epac1 was highly expressed, but not Epac 2, in the extensor digitorum longus (EDL) and soleus muscles. The exercise significantly increased protein expression of Epac 1 in EDL and soleus muscle of wild-type (WT) mice. A global proteomics and pathway analyses revealed that Epac 1 deficiency mainly affected "the energy production and utilization" process in the skeletal muscle. We have tested their forced treadmill exercise tolerance. Epac1-/- mice exhibited significantly reduced exercise capacity in the forced treadmill exercise and lower number of type 1 fibers than WT mice. The basal protein level of proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) was reduced in the Epac1-/- mice. Furthermore, increasing expression of PGC-1α by exercise was also significantly attenuated in the skeletal muscle of Epac1-/- mice. The expressions of downstream target genes of PGC-1α, which involved in uptake and oxidation of fatty acids, ERRα and PPARδ, and fatty acid content were lower in muscles of Epac1-/-, suggesting a role of Epac1 in forced treadmill exercise capacity by regulating PGC-1α pathway and lipid metabolism in skeletal muscle. Taken together, Epac1 plays an important role in exercise capacity by regulating PGC-1α and fatty acid metabolism in the skeletal muscle.

Astrocytic endothelin-1 overexpression promotes neural progenitor cells proliferation and differentiation into astrocytes via the Jak2/Stat3 pathway after stroke.

BACKGROUND: Endothelin-1 (ET-1) is synthesized and upregulated in astrocytes under stroke. We previously demonstrated that transgenic mice over-expressing astrocytic ET-1 (GET-1) displayed more severe neurological deficits characterized by a larger infarct after transient middle cerebral artery occlusion (tMCAO). ET-1 is a known vasoconstrictor, mitogenic, and a survival factor. However, it is unclear whether the observed severe brain damage in GET-1 mice post stroke is due to ET-1 dysregulation of neurogenesis by altering the stem cell niche. METHODS: Non-transgenic (Ntg) and GET-1 mice were subjected to tMCAO with 1 h occlusion followed by long-term reperfusion (from day 1 to day 28). Neurological function was assessed using a four-point scale method. Infarct area and volume were determined by 2,3,5-triphenyltetra-zolium chloride staining. Neural stem cell (NSC) proliferation and migration in subventricular zone (SVZ) were evaluated by immunofluorescence double labeling of bromodeoxyuridine (BrdU), Ki67 and Sox2, Nestin, and Doublecortin (DCX). NSC differentiation in SVZ was evaluated using the following immunofluorescence double immunostaining: BrdU and neuron-specific nuclear protein (NeuN), BrdU and glial fibrillary acidic protein (GFAP). Phospho-Stat3 (p-Stat3) expression detected by Western-blot and immunofluorescence staining. RESULTS: GET-1 mice displayed a more severe neurological deficit and larger infarct area after tMCAO injury. There was a significant increase of BrdU-labeled progenitor cell proliferation, which co-expressed with GFAP, at SVZ in the ipsilateral side of the GET-1 brain at 28 days after tMCAO. p-Stat3 expression was increased in both Ntg and GET-1 mice in the ischemia brain at 7 days after tMCAO. p-Stat3 expression was significantly upregulated in the ipsilateral side in the GET-1 brain than that in the Ntg brain at 7 days after tMCAO. Furthermore, GET-1 mice treated with AG490 (a JAK2/Stat3 inhibitor) sh owed a significant reduction in neurological deficit along with reduced infarct area and dwarfed astrocytic differentiation in the ipsilateral brain after tMCAO. CONCLUSIONS: The data indicate that astrocytic endothelin-1 overexpression promotes progenitor stem cell proliferation and astr ocytic differentiation via the Jak2/Stat3 pathway.

A spontaneously immortalized Schwann cell line from aldose reductase-deficient mice as a useful tool for studying polyol pathway and aldehyde metabolism.

The increased glucose flux into the polyol pathway via aldose reductase (AR) is recognized as a major contributing factor for the pathogenesis of diabetic neuropathy, whereas little is known about the functional significance of AR in the peripheral nervous system. Spontaneously immortalized Schwann cell lines established from long-term cultures of AR-deficient and normal C57BL/6 mouse dorsal root ganglia and peripheral nerves can be useful tools for studying the physiological and pathological roles of AR. These cell lines, designated as immortalized knockout AR Schwann cells 1 (IKARS1) and 1970C3, respectively, demonstrated distinctive Schwann cell phenotypes, such as spindle-shaped morphology and immunoreactivity to S100, p75 neurotrophin receptor, and vimentin, and extracellular release of neurotrophic factors. Conditioned media obtained from these cells promoted neuronal survival and neurite outgrowth of cultured adult mouse dorsal root ganglia neurons. Microarray and real-time RT-PCR analyses revealed significantly down-regulated mRNA expression of polyol pathway-related enzymes, sorbitol dehydrogenase and ketohexokinase, in IKARS1 cells compared with those in 1970C3 cells. In contrast, significantly up-regulated mRNA expression of aldo-keto reductases (AKR1B7 and AKR1B8) and aldehyde dehydrogenases (ALDH1L2, ALDH5A1, and ALDH7A1) was detected in IKARS1 cells compared with 1970C3 cells. Exposure to reactive aldehydes (3-deoxyglucosone, methylglyoxal, and 4-hydroxynonenal) significantly up-regulated the mRNA expression of AKR1B7 and AKR1B8 in IKARS1 cells, but not in 1970C3 cells. Because no significant differences in viability between these two cell lines after exposure to these aldehydes were observed, it can be assumed that the aldehyde detoxification is taken over by AKR1B7 and AKR1B8 in the absence of AR.

Role of nuclear factor of activated T-cells 5 in regulating hypertonic-mediated secretin receptor expression in kidney collecting duct cells.

A growing body of evidence suggests that secretin (SCT) is an important element in the osmoregulatory pathway. It is interesting to note that both SCT and its receptor (SCTR) gene are activated upon hyperosmolality in the kidney. However, the precise molecular mechanisms underlying the induction of the SCTR gene expression in response to changes in osmolality have yet to be clarified. Detailed DNA sequence analysis of the promoter regions of the SCTR gene reveals the presence of multiple osmotic response elements (ORE). The ORE is the binding site of a key osmosensitive transactivator, namely, the nuclear factor of activated T-cells 5 (NFAT5). SCTR and NFAT5 are co-expressed in the kidney cortex and medulla collecting duct cells. We therefore hypothesize that NFAT5 is responsible for modulating SCTR expression in hypertonic environments. In this study, we found hypertonicity stimulates the promoter activities and endogenous gene expression of SCTR in mouse kidney cortex collecting duct cells (M1) and inner medulla collecting duct cells (mIMCD3). The overexpression and silencing of NFAT5 further confirmed it to be responsible for the up-regulation of the SCTR gene under hypertonic conditions. A significant increase in the interaction between NFAT5 and the SCTR promoter was also observed following chromatin immunoprecipitation assay. In vivo, osmotic stress up-regulates the SCTR gene in the kidney cortex and medulla of wild-type mice, but does not do so in NFAT5(+/-) animals. Hence, this study provides comprehensive information on how NFAT5 regulates SCTR expression in different osmotic environments.

Epac Activation Regulates Human Mesenchymal Stem Cells Migration and Adhesion.

How to enhance the homing of human mesenchymal stem cells (hMSCs) to the target tissues remains a clinical challenge nowadays. To overcome this barrier, the mechanism responsible for the hMSCs migration and engraftment has to be defined. Currently, the exact mechanism involved in migration and adhesion of hMSCs remains unknown. Exchange protein directly activated by cAMP (Epac), a novel protein discovered in cAMP signaling pathway, may have a potential role in regulating cells adhesion and migration by triggering the downstream Rap family signaling cascades. However, the exact role of Epac in cells homing is elusive. Our study evaluated the role of Epac in the homing of hMSCs. We confirmed that hMSCs expressed functional Epac and its activation enhanced the migration and adhesion of hMSCs significantly. The Epac activation was further found to be contributed directly to the chemotactic responses induced by stromal cell derived factor-1 (SDF-1) which is a known chemokine in regulating hMSCs homing. These findings suggested Epac is connected to the SDF-1 signaling cascades. In conclusion, our study revealed that Epac plays a role in hMSCs homing by promoting adhesion and migration. Appropriate manipulation of Epac may enhance the homing of hMSCs and facilitate their future clinical applications.

Endothelial overexpression of endothelin-1 modulates aortic, carotid, iliac and renal arterial responses in obese mice.

Endothelin-1 (ET-1) is essential for mammalian development and life, but it has also been implicated in increased cardiovascular risk under pathophysiological conditions. The aim of this study was to determine the impact of endothelial overexpression of the prepro-endothelin-1 gene on endothelium-dependent and endothelium-independent responses in the conduit and renal arteries of lean and obese mice. Obesity was induced by high-fat-diet (HFD) consumption in mice with Tie-1 promoter-driven, endothelium-specific overexpression of the prepro-endothelin-1 gene (TEThet) and in wild-type (WT) littermates on a C57BL/6N background. Isometric tension was measured in rings (with endothelium) of the aorta (A), carotid (CA) and iliac (IA) arteries as well as the main (MRA) and segmental renal (SRA) arteries; all experiments were conducted in the absence or presence of L-NAME and/or the COX inhibitor meclofenamate. The release of prostacyclin and thromboxane A2 was measured by ELISA. In the MRA, TEThet per se increased contractions to endothelin-1, but the response was decreased in SRA in response to serotonin; there were also improved relaxations to acetylcholine but not insulin in the SRA in the presence of L-NAME. HFD per se augmented the contractions to endothelin-1 (MRA) and to the thromboxane prostanoid (TP) receptor agonist U46619 (CA, MRA) as well as facilitated relaxations to isoproterenol (A). The combination of HFD and TEThet overexpression increased the contractions of MRA and SRA to vasoconstrictors but not in the presence of meclofenamate; this combination also augmented further relaxations to isoproterenol in the A. Contractions to endothelin-1 in the IA were prevented by endothelin-A receptor antagonist BQ-123 but only attenuated in obese mice by BQ-788. The COX-1 inhibitor FR122047 abolished the contractions of CA to acetylcholine. The release of prostacyclin during the latter condition was augmented in samples from obese TEThet mice and abolished by FR122047. These findings suggest that endothelial TEThet overexpression in lean animals has minimal effects on vascular responsiveness. However, if comorbid with obesity, endothelin-1-modulated, prostanoid-mediated renal arterial dysfunction becomes apparent.

Crosstalk between astrocytic CXCL12 and microglial CXCR4 contributes to the development of neuropathic pain.

BACKGROUND: Chemokine axis chemokine C-X-C motif ligand 12/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) is an emerging pain modulator, but mechanisms for its involvement in neuropathic pain remain unclear. Here, we aimed to study whether CXCL12/CXCR4 axis modulated the development of neuropathic pain via glial mechanisms. In this study, two mouse models of neuropathic pain, namely partial sciatic nerve ligation (pSNL) model and chronic post-ischemia pain (CPIP) model, were used. RESULTS: In the dorsal horn of L3-L5 segment of spinal cord, CXCL12 and CXCR4 were expressed in both astrocyte and microglia in normal mice. In the pSNL or CPIP model, the expression level of CXCL12 in the ipsilateral L3-L5 segment of mice spinal cord was increased in an astrocyte-dependent manner on post-operative day (POD) 3. Intrathecal administration of CXCL12 with AMD3100 (CXCR4 antagonist) or minocycline (microglia activation inhibitor), but not fluorocitrate (astrocyte activation inhibitor), reversed CXCL12-indued mechanical allodynia in naïve mice. In these models, AMD3100 and AMD3465 (CXCR4 antagonist), administered daily from 1 h before surgery and up to POD 3, attenuated the development of mechanical allodynia. Moreover, AMD3100 administered daily from 1 h before surgery and up to POD 3 downregulated mRNA levels of tumor necrosis factor alpha, interleukin 1ß, and interleukin 6 in the ipsilateral L3-L5 segment of spinal cord in the pSNL and CPIP models on POD 3. CONCLUSION: This study demonstrates the crosstalk between astrocytic CXCL12 and microglial CXCR4 in the pathogenesis of neuropathic pain using pSNL and CPIP models. Our results offer insights for the future research on CXCL12/CXCR4 axis and neuropathic pain therapy.

Polarized Macrophages Have Distinct Roles in the Differentiation and Migration of Embryonic Spinal-cord-derived Neural Stem Cells After Grafting to Injured Sites of Spinal Cord.

Spinal cord injury (SCI) frequently provokes serious detrimental outcomes because neuronal regeneration is limited in the central nervous system (CNS). Thus, the creation of a permissive environment for transplantation therapy with neural stem/progenitor cells (NS/PCs) is a promising strategy to replace lost neuronal cells, promote repair, and stimulate functional plasticity after SCI. Macrophages are important SCI-associated inflammatory cells and a major source of secreted factors that modify the lesion milieu. Here, we used conditional medium (CM) from bone marrow-derived M1 or M2 polarized macrophages to culture murine NS/PCs. The NS/PCs showed enhanced astrocytic versus neuronal/oligodendrocytic differentiation in the presence of M1- versus M2-CM. Similarly, cotransplantation of NS/PCs with M1 and M2 macrophages into intact or injured murine spinal cord increased the number of engrafted NS/PC-derived astrocytes and neurons/oligodendrocytes, respectively. Furthermore, when cotransplantated with M2 macrophages, the NS/PC-derived neurons integrated into the local circuitry and enhanced locomotor recovery following SCI. Interesting, engrafted M1 macrophages promoted long-distance rostral migration of NS/PC-derived cells in a chemokine (C-X-C motif) receptor 4 (CXCR4)-dependent manner, while engrafted M2 macrophages resulted in limited cell migration of NS/PC-derived cells. Altogether, these findings suggest that the cotransplantation of NS/PCs together with polarized macrophages could constitute a promising therapeutic approach for SCI repair.

Deficiency of aldose reductase attenuates inner retinal neuronal changes in a mouse model of retinopathy of prematurity.

Retinopathy of prematurity (ROP) is a leading cause of childhood blindness where vascular abnormality and retinal dysfunction are reported. We showed earlier that genetic deletion of aldose reductase (AR), the rate-limiting enzyme in the polyol pathway, reduced the neovascularization through attenuating oxidative stress induction in the mouse oxygen-induced retinopathy (OIR) modeling ROP. In this study, we further investigated the effects of AR deficiency on retinal neurons in the mouse OIR. Seven-day-old wild-type and AR-deficient mice were exposed to 75 % oxygen for 5 days and then returned to room air. Electroretinography was used to assess the neuronal function at postnatal day (P) 30. On P17 and P30, retinal cytoarchitecture was examined by morphometric analysis and immunohistochemistry for calbindin, protein kinase C alpha, calretinin, Tuj1, and glial fibrillary acidic protein. In OIR, attenuated amplitudes and delayed implicit time of a-wave, b-wave, and oscillatory potentials were observed in wild-type mice, but they were not significantly changed in AR-deficient mice. The morphological changes of horizontal, rod bipolar, and amacrine cells were shown in wild-type mice and these changes were partly preserved with AR deficiency. AR deficiency attenuated the Müller cell gliosis induced in OIR. Our observations demonstrated AR deficiency preserved retinal functions in OIR and AR deficiency could partly reduce the extent of retinal neuronal histopathology. These findings suggested a therapeutic potential of AR inhibition in ROP treatment with beneficial effects on the retinal neurons.

Over-expression of astrocytic ET-1 attenuates neuropathic pain by inhibition of ERK1/2 and Akt(s) via activation of ETA receptor.

A differential role of endothelin-1 (ET-1) in pain processing has recently been suggested. However, the function of central ET-1 in neuropathic pain (NP) has not been fully elucidated to date. We report here the action of endogenous central ET-1 in sciatic nerve ligation-induced NP (SNL-NP) in a transgenic animal model that over-expresses ET-1 in the astrocytes (GET-1 mice). We hypothesized that the over-expression of astrocytic ET-1 would exert anti-allodynic and anti-hyperalgesic effects in NP, as demonstrated by mechanical threshold and plantar withdrawal latency using the von Frey filament and heat stimuli. In our animal model, GET-1 mice showed an increase in the withdrawal threshold and latency in response to the mechanical and thermal stimuli, respectively, in pain behavior tests after SNL. ET-1 and endothelin type A receptor (ETA-R) levels were increased significantly in L4-L6 segments of the spinal cord (ipsilateral to SNL) of GET-1 mice at 7 and 21days after surgery. Moreover, intrathecal administration of a specific ETA-R antagonist, BQ-123, attenuated the anti-allodynic and anti-hyperalgesic phenotype in GET-1 mice. The effects of BQ-123 on the mRNA expression of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and protein kinase B/serine protein kinase (Akt(s)) were assessed in the ipsilateral L4-L6 segments harvested 30min after BQ-123 administration on day 7 after surgery. Phosphorylation of ERK1/2 and Akt(s) in the ipsilateral spinal cord of GET-1 mice was reduced following SNL, whereas no reduction was observed after intrathecal injection of BQ-123. In conclusion, our results showed that the xover-expression of astrocytic ET-1 reduced SNL-induced allodynia and hyperalgesia by inhibiting the activation of ERK1/2 and Akt(s) via the ETA-R-mediated pathway.

The inhibition of aldose reductase attenuates hepatic ischemia-reperfusion injury through reducing inflammatory response.

OBJECTIVE: We aim to investigate the role of aldose reductase (AR) in hepatic ischemia-reperfusion injury (IRI) of normal and fatty livers and to explore the underlying mechanisms. BACKGROUND: Hepatic IRI is a typical inflammatory response during liver surgery. It contributes to liver graft failure or nonfunction after transplantation. Increasing evidence implicates that AR plays a key role in a number of inflammatory diseases. However, the role of AR in hepatic IRI is still unknown. METHODS: Intragraft AR expression profile and the association with liver graft injury were investigated in both human and rat liver transplantation using normal or fatty graft. The direct role of AR in hepatic IRI was studied in the AR knockout mice IRI model with or without fatty liver. They were further validated by the simulated IRI in vitro model using fatty LO2 cells with or without AR inhibitor zopolrestat and primary peritoneal macrophages isolated from AR knockout and wild-type mice. Gene expression of inflammatory cytokines/chemokines, the infiltration of macrophages/neutrophils, and NF-κB pathway activation were compared among different groups. RESULTS: AR was overexpressed in liver graft after human and rat liver transplantation and correlated with consequent liver injuries. The knockout of AR significantly attenuated hepatic sinusoidal damage and apoptosis in both normal and fatty livers after IRI. The expression of proinflammatory cytokines/chemokines and neutrophil chemoattractants, infiltration of macrophage and neutrophil, and activation of inflammation-associated NF-κB and JNK pathway were downregulated in AR knockout mice. Furthermore, the inhibition of AR effectively suppressed macrophage migration and decreased lipopolysaccharide (LPS)-induced production of proinflammatory cytokines/chemokines in isolated macrophages. CONCLUSIONS: The deficiency of AR attenuated hepatic IRI in both normal and fatty livers by reducing liver inflammatory responses.

Exchange protein activated by cAMP 1 (Epac1)-deficient mice develop ß-cell dysfunction and metabolic syndrome.

Previously, exchange protein directly activated by cAMP 2 (Epac2) and PKA were known to play a role in glucose-stimulated insulin secretion (GSIS) by pancreatic ß cells. The present study shows that Epac1 mRNA is also expressed by ß cells. Therefore, we generated mice and embryonic stem (ES) cells with deletion of the Epac1 gene to define its role in ß-cell biology and metabolism. The homozygous Epac1-knockout (Epac1(-/-)) mice developed impaired glucose tolerance and GSIS with deranged islet cytoarchitecture, which was confirmed by isolated islets from adult Epac1(-/-) mice. Moreover, Epac1(-/-) mice developed more severe hyperglycemia with increased ß-cell apoptosis and insulitis after multiple low-dose streptozotocin (MLDS; 40 mg/kg) treatment than Epac1(+/+) mice. Interestingly, Epac1(-/-) mice also showed metabolic defects, including increased respiratory exchange ratio (RER) and plasma triglyceride (TG), and more severe diet-induced obesity with insulin resistance, which may contributed to ß-cell dysfunction. However, islets differentiated from Epac1(-/-) ES cells showed insulin secretion defect, reduced Glut2 and PDX-1 expression, and abolished GLP-1-stimulated PCNA induction, suggesting a role of Epac1 in ß-cell function. The current study provides in vitro and in vivo evidence that Epac1 has an important role in GSIS of ß cells and phenotype resembling metabolic syndrome.

Targeted over-expression of endothelin-1 in astrocytes leads to more severe brain damage and vasospasm after subarachnoid hemorrhage.

BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictor, and astrocytic ET-1 is reported to play a role in the pathogenesis of cerebral ischemic injury and cytotoxic edema. However, it is still unknown whether astrocytic ET-1 also contributes to vasogenic edema and vasospasm during subarachnoid hemorrhage (SAH). In the present study, transgenic mice with astrocytic endothelin-1 over-expression (GET-1 mice) were used to investigate the pathophysiological role of ET-1 in SAH pathogenesis. RESULTS: The GET-1 mice experienced a higher mortality rate and significantly more severe neurological deficits, blood-brain barrier breakdown and vasogenic edema compared to the non-transgenic (Ntg) mice following SAH. Oral administration of vasopressin V1a receptor antagonist, SR 49059, significantly reduced the cerebral water content in the GET-1 mice. Furthermore, the GET-1 mice showed significantly more pronounced middle cerebral arterial (MCA) constriction after SAH. Immunocytochemical analysis showed that the calcium-activated potassium channels and the phospho-eNOS were significantly downregulated, whereas PKC-α expression was significantly upregulated in the MCA of the GET-1 mice when compared to Ntg mice after SAH. Administration of ABT-627 (ETA receptor antagonist) significantly down-regulated PKC-α expression in the MCA of the GET-1 mice following SAH. CONCLUSIONS: The present study suggests that astrocytic ET-1 involves in SAH-induced cerebral injury, edema and vasospasm, through ETA receptor and PKC-mediated potassium channel dysfunction. Administration of ABT-627 (ETA receptor antagonist) and SR 49059 (vasopressin V1a receptor antagonist) resulted in amelioration of edema and vasospasm in mice following SAH. These data provide a strong rationale to investigate SR 49059 and ABT-627 as therapeutic drugs for the treatment of SAH patients.

Transgenic mice over-expressing endothelial endothelin-1 show cognitive deficit with blood-brain barrier breakdown after transient ischemia with long-term reperfusion.

Increased level of endothelin-1 (ET-1), a potent vasoconstrictor, has been found in the cerebral spinal fluid (CSF) of patients with multi-infarction dementia, suggesting a possible role of ET-1 in cognitive deficit associated with stroke. Previously, we have reported that synthesis of ET-1 is induced in endothelial cells in hypoxic/ischemic conditions. Transgenic mice over-expressing endothelin-1 in endothelial cells (TET-1) developed systemic hypertension and showed more severe brain damage after transient ischemia. To further understand the significance of endothelial ET-1 in cognitive deficit, we subjected adult TET-1 mice to 30 min middle cerebral artery occlusion (MCAO) with 7 days reperfusion. At baseline, TET-1 mice showed similar locomotor activity, emotion and cognitive function compared to non-transgenic (NTg) mice. However, after 30 min MCAO and 7 days reperfusion, although the sensorimotor function measured by neurological scores was recovered in both genotypes, TET-1 mice showed increased anxiety-like behavior in the open field test and impaired spatial learning and reference memory in the Morris water maze. Parallel with these behavioral changes, TET-1 mice showed more severe brain damage with blood-brain-barrier breakdown (BBB), reactive astrogliosis, increased caspase-3, and increased peroxiredoxin 6 (Prx6) expressions around blood vessels in the ipsilateral hippocampus, compared to that of NTg mice, suggesting that ET-1 over-expression in the endothelial cells leads to more severe BBB breakdown and increased oxidative stress which may resulted in neuronal apoptosis and glial reactivity, which might contribute to the emotional changes and cognitive deficits after short-term ischemia with long-term reperfusion.