Effect of Targeted Deletion of the Heme Oxygenase-2 Gene on Hemoglobin Toxicity in the Striatum

The heme oxygenase (HO) enzymes catalyze the rate-limiting step in the breakdown of heme to iron, carbon monoxide, and biliverdin. A prior cell culture study demonstrated that deletion of HO-2, the isoform constitutively expressed in neurons, attenuated hemoglobin (Hb) neurotoxicity. The present study tested the hypothesis that HO-2 gene deletion is cytoprotective in a model of Hb toxicity in vivo. Stereotactic injection of 6 μL stroma-free Hb (SFHb) into the striatum significantly increased protein oxidation in wild-type mice at 24 to 72 h, as detected by an assay for carbonyl groups. At 72 h, carbonylation was increased 2.5-fold compared with that in the contralateral striatum. In HO-2 knockout mice, protein oxidation was not increased at 24 h, and was increased by only 1.7-fold at 72 h. Similarly, striatal lipid peroxidation, as detected by the malondialdehyde assay, was significantly greater in the SFHb-injected striata of wild-type mice than in knockout mice. Striatal cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, was 45.0%±6.3% of that in contralateral striata in wild-type mice at 72 h; it was increased to 85%±8% in knockouts. Heme oxygenase-2 gene deletion did not alter weight loss or mortality after SFHb injection. Baseline striatal HO-1 expression was similar in knockout and wild-type mice; induction after SFHb injection occurred more rapidly in the latter. These results suggest that HO-2 gene deletion protects striatal cells from the oxidative toxicity of Hb in vivo. Pharmacologic or genetic strategies that target HO-2 may be beneficial after central nervous system hemorrhage, and warrant further investigation.

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Preoperative administration of the 5-HT4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons

Gut ◽

10.1136/gutjnl-2018-317263 ◽

2018 ◽

Vol 68 (8) ◽

pp. 1406-1416 ◽

Cited By ~ 15

Author(s):

Nathalie Stakenborg ◽

Evelien Labeeuw ◽

Pedro J Gomez-Pinilla ◽

Sebastiaan De Schepper ◽

Raymond Aerts ◽

...

Keyword(s):

Knockout Mice ◽

Postoperative Ileus ◽

Intestinal Inflammation ◽

Electrical Field Stimulation ◽

Enteric Neurons ◽

Postoperative Treatment ◽

Wild Type ◽

Clinical Recovery ◽

Preoperative Administration

ObjectivesVagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human.DesignUsing Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1–5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI.ResultsEFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery.ConclusionEnteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI.Trial registration numberNCT02425774.

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Direct evidence of iNOS-mediated in vivo free radical production and protein oxidation in acetone-induced ketosis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00015.2008 ◽

2008 ◽

Vol 295 (2) ◽

pp. E456-E462 ◽

Cited By ~ 28

Author(s):

Krisztian Stadler ◽

Marcelo G. Bonini ◽

Shannon Dallas ◽

Danielle Duma ◽

Ronald P. Mason ◽

...

Keyword(s):

Lipid Peroxidation ◽

Free Radical ◽

Nadph Oxidase ◽

Knockout Mice ◽

Protein Oxidation ◽

Ketone Bodies ◽

Free Radical Production ◽

Radical Production ◽

Inos Knockout Mice

Diabetic patients frequently encounter ketosis that is characterized by the breakdown of lipids with the consequent accumulation of ketone bodies. Several studies have demonstrated that reactive species are likely to induce tissue damage in diabetes, but the role of the ketone bodies in the process has not been fully investigated. In this study, electron paramagnetic resonance (EPR) spectroscopy combined with novel spin-trapping and immunological techniques has been used to investigate in vivo free radical formation in a murine model of acetone-induced ketosis. A six-line EPR spectrum consistent with the α-(4-pyridyl-1-oxide)- N-t-butylnitrone radical adduct of a carbon-centered lipid-derived radical was detected in the liver extracts. To investigate the possible enzymatic source of these radicals, inducible nitric oxide synthase (iNOS) and NADPH oxidase knockout mice were used. Free radical production was unchanged in the NADPH oxidase knockout but much decreased in the iNOS knockout mice, suggesting a role for iNOS in free radical production. Longer-term exposure to acetone revealed iNOS overexpression in the liver together with protein radical formation, which was detected by confocal microscopy and a novel immunospin-trapping method. Immunohistochemical analysis revealed enhanced lipid peroxidation and protein oxidation as a consequence of persistent free radical generation after 21 days of acetone treatment in control and NADPH oxidase knockout but not in iNOS knockout mice. Taken together, our data demonstrate that acetone administration, a model of ketosis, can lead to protein oxidation and lipid peroxidation through a free radical-dependent mechanism driven mainly by iNOS overexpression.

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Soluble epoxide hydrolase inhibition and gene deletion are protective against myocardial ischemia-reperfusion injury in vivo

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00428.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. H2128-H2134 ◽

Cited By ~ 73

Author(s):

Atsuko Motoki ◽

Matthias J. Merkel ◽

William H. Packwood ◽

Zhiping Cao ◽

Lijuan Liu ◽

...

Keyword(s):

Myocardial Ischemia ◽

Reperfusion Injury ◽

Epoxide Hydrolase ◽

Gene Deletion ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Wild Type ◽

Myocardial Ischemia Reperfusion Injury ◽

Myocardial Ischemia Reperfusion

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ischemia-reperfusion injury in vivo. Male C57BL\6J wild-type or sEH knockout mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. Wild-type mice were injected intraperitoneally with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min before LCA occlusion or during ischemia 10 min before reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min before reperfusion. The EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE) was given intravenously 15 min before reperfusion. Area at risk (AAR) and I were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and I was expressed as I/AAR. I was significantly reduced in animals treated with AUDA-BE or 14,15-EET, independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. Immunohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia-reperfusion injury.

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Villin and actin in the mouse kidney brush-border membrane bind to and are degraded by meprins, an interaction that contributes to injury in ischemia-reperfusion

AJP Renal Physiology ◽

10.1152/ajprenal.00703.2010 ◽

2011 ◽

Vol 301 (4) ◽

pp. F871-F882 ◽

Cited By ~ 21

Author(s):

Elimelda Moige Ongeri ◽

Odinaka Anyanwu ◽

W. Brian Reeves ◽

Judith S. Bond

Keyword(s):

Brush Border ◽

Knockout Mice ◽

Brush Border Membrane ◽

Ischemia Reperfusion ◽

Kidney Injury ◽

Mouse Kidney ◽

Cytoskeletal Proteins ◽

Kidney Tubules ◽

Wild Type

Meprins, metalloproteinases abundantly expressed in the brush-border membranes (BBMs) of rodent proximal kidney tubules, have been implicated in the pathology of renal injury induced by ischemia-reperfusion (IR). Disruption of the meprin β gene and actinonin, a meprin inhibitor, both decrease kidney injury resulting from IR. To date, the in vivo kidney substrates for meprins are unknown. The studies herein implicate villin and actin as meprin substrates. Villin and actin bind to the cytoplasmic tail of meprin β, and both meprin A and B are capable of degrading villin and actin present in kidney proteins as well as purified recombinant forms of these proteins. The products resulting from degradation of villin and actin were unique to each meprin isoform. The meprin B cleavage site in villin was Glu744-Val745. Recombinant forms of rat meprin B and homomeric mouse meprin A had Km values for villin and actin of ∼1 μM (0.6–1.2 μM). The kcat values varied substantially (0.6–128 s−1), resulting in different efficiencies for cleavage, with meprin B having the highest kcat/ Km values (128 M−1·s−1 × 106). Following IR, meprins and villin redistributed from the BBM to the cytosol. A 37-kDa actin fragment was detected in protein fractions from wild-type, but not in comparable preparations from meprin knockout mice. The levels of the 37-kDa actin fragment were significantly higher in kidneys subjected to IR. The data establish that meprins interact with and cleave villin and actin, and these cytoskeletal proteins are substrates for meprins.

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ChREBP-Knockout Mice Show Sucrose Intolerance and Fructose Malabsorption

10.20944/preprints201802.0005.v1 ◽

2018 ◽

Author(s):

Takehiro Kato ◽

Katsumi Iizuka ◽

Ken Takao ◽

Yukio Horikawa ◽

Tadahiro Kitamura ◽

...

Keyword(s):

Plasma Glucose ◽

Knockout Mice ◽

Gene Deletion ◽

Body Weight Loss ◽

Mrna Levels ◽

Wild Type ◽

Glucose Levels ◽

Fructose Malabsorption ◽

Sucrose Diet ◽

Alpha Glucosidase

We have previously reported that 60% sucrose diet-fed ChREBP knockout mice (KO) showed body weight loss resulting in lethality. We aimed to elucidate whether sucrose and fructose metabolism are impaired in KO. Wild type mice (WT) and KO were fed a diet containing 30% sucrose with/without 0.08% miglitol, an α-glucosidase inhibitor, and these effects on phenotypes were tested. Furthermore, we compared metabolic changes of oral and peritoneal fructose injection. Thirty percent sucrose diet feeding did not affect phenotypes in KO. However, miglitol induced lethality in 30% sucrose-fed KO. Thirty percent sucrose plus miglitol diet-fed KO showed increased cecal contents, increased fecal lactate contents, increased growth of lactobacillales and Bifidobacterium and decreased growth of clostridium cluster XIVa. ChREBP gene deletion suppressed the mRNA levels of sucrose and fructose related genes. Next, oral fructose injection did not affect plasma glucose levels and liver fructose contents; however, intestinal sucrose and fructose related mRNA levels were increased only in WT. In contrast, peritoneal fructose injection increased plasma glucose levels in both mice; however, the hepatic fructose content in KO was much higher owing to decreased hepatic Khk mRNA expression. Taken together, KO showed sucrose intolerance and fructose malabsorption owing to decreased gene expression.

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IL-17 Activates the IL-6/STAT3 Signal Pathway in the Proliferation of Hepatitis B Virus-Related Hepatocellular Carcinoma

Cellular Physiology and Biochemistry ◽

10.1159/000484390 ◽

2017 ◽

Vol 43 (6) ◽

pp. 2379-2390 ◽

Cited By ~ 14

Author(s):

Zongqiang Hu ◽

Ding Luo ◽

Dongdong Wang ◽

Linjie Ma ◽

Yingpeng Zhao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Knockout Mice ◽

Hepg2 Cells ◽

Wild Type ◽

B Virus ◽

And Migration ◽

Proliferation And Migration

Background/Aims: We performed this study to determine the role of IL-17 in the immune microenvironment of hepatitis B virus- (HBV-) related hepatocellular carcinoma (HCC). Methods: HepG2 cells were treated with IL-17, STAT3 inhibitor S31-201 or IL-6 neutralizing monoclonal antibody (IL-6 mAb). Cell proliferation and migration were compared using the Cell Counting kit-8 (CCK-8) and Transwell assays, respectively. Real-time quantitative PCR (RT-qPCR), Western Blot, ELISA, immunofluorescence and histological staining were used for determining the expression levels of IL-17, IL-6, MCP-1, CCL5, VEGF, STAT3 and p-STAT3. HCC xenograft models were constructed in wild type and IL-17 knockout mice to clarify the effects of IL-17 on HCC in vivo. Results: Exogenous IL-17 enhanced the proliferation and migration of HepG2 cells, and it activated the phosphorylation of STAT3. RT-qPCR and ELISA showed that IL-17 promoted the expression of IL-6. The CCK-8 and Transwell assays showed that S31-201 or IL-6 mAb remarkably reversed the promotion effects of proliferation and migration by exogenous IL-17 in HepG2 cells. Additionally, IL-6 could promote the phosphorylation of STAT3, while IL-6 mAb acted as an inhibitor, and exogenous IL-17 could neutralize the inhibitory effects of IL-6 mAb. In vivo, compared to the wild type mice, the tumor volume, weight, density and size were decreased in IL-17 knockout mice. Additionally, the expression levels of p-STAT3, IL-6, MCP-1, CCL5 and VEGF decreased in IL-17 knockout mice. Conclusions: IL-17 can enhance the proliferation of HepG2 cells in vitro and in vivo via activating the IL-6/STAT3 pathway. Therefore, the IL-17/IL-6/STAT3 signaling pathway is a potential therapeutic target for HBV-related HCC.

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Kindlin-2 suppresses transcription factor GATA4 through interaction with SUV39H1 to attenuate hypertrophy

Cell Death and Disease ◽

10.1038/s41419-019-2121-0 ◽

2019 ◽

Vol 10 (12) ◽

Cited By ~ 2

Author(s):

Lihua Qi ◽

Xiaochun Chi ◽

Xi Zhang ◽

Xueqian Feng ◽

Wenhui Chu ◽

...

Keyword(s):

Transcription Factor ◽

Pathological Process ◽

Structure And Function ◽

Wild Type ◽

Cardiac Tissues ◽

Targeted Deletion ◽

Progressive Heart Failure ◽

Pathological Hypertrophy ◽

And Function

AbstractKindlin-2 plays an important role in the regulation of cardiac structure and function. Depletion of Kindlin-2 contributes to cardiac hypertrophy and progressive heart failure, however, the precise mechanisms involved in this process remain unclear. GATA4 is a critical transcription factor in regulating cardiogenesis. We found that Kindlin-2 suppresses the expression of GATA4 through binding to its promoter and prevents cardiomyocytes from hypertrophy induced by isoproterenol (ISO) treatment. Mechanistically, Kindlin-2 interacts with histone methyltransferase SUV39H1 and recruits it to GATA4 promoter leading to the occupancy of histone H3K9 di- and tri-methylation. Furthermore, to confirm the function of Kindlin-2 in vivo, we generated mice with targeted deletion of cardiac Kindlin-2. We found that 6-month-old Kindlin-2 cKO mice have developed hypertrophic cardiomyopathy and that this pathological process can be accelerated by ISO-treatment. GATA4 expression was markedly activated in cardiac tissues of Kindlin-2 cKO mice compared to wild-type animals. Collectively, our data revealed that Kindlin-2 suppresses GATA4 expression by triggering histone H3K9 methylation in part and protects heart from pathological hypertrophy.

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Ficolins Promote Fungal Clearance in vivo and Modulate the Inflammatory Cytokine Response in Host Defense against Aspergillus fumigatus

Journal of Innate Immunity ◽

10.1159/000447714 ◽

2016 ◽

Vol 8 (6) ◽

pp. 579-588 ◽

Cited By ~ 17

Author(s):

Ninette Genster ◽

Elisabeth Præstekjær Cramer ◽

Anne Rosbjerg ◽

Katrine Pilely ◽

Jack Bernard Cowland ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Host Defense ◽

Knockout Mice ◽

Fungal Infections ◽

Inflammatory Cells ◽

Lectin Pathway ◽

Wild Type ◽

Opportunistic Fungal Pathogen

Aspergillus fumigatus is an opportunistic fungal pathogen that causes severe invasive infections in immunocompromised patients. Innate immunity plays a major role in protection against A. fumigatus. The ficolins are a family of soluble pattern recognition receptors that are capable of activating the lectin pathway of complement. Previous in vitro studies reported that ficolins bind to A. fumigatus, but their part in host defense against fungal infections in vivo is unknown. In this study, we used ficolin-deficient mice to investigate the role of ficolins during lung infection with A. fumigatus. Ficolin knockout mice showed significantly higher fungal loads in the lungs 24 h postinfection compared to wild-type mice. The delayed clearance of A. fumigatus in ficolin knockout mice could not be attributed to a compromised recruitment of inflammatory cells. However, it was revealed that ficolin knockout mice exhibited a decreased production of proinflammatory cytokines in the lungs compared to wild-type mice following A. fumigatus infection. The impaired clearance and cytokine production in ficolin knockout mice was independent of complement, as shown by equivalent levels of A. fumigatus-mediated complement activation in ficolin knockout mice and wild-type mice. In conclusion, this study demonstrates that ficolins are important in initial innate host defense against A. fumigatus infections in vivo.

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