Wide-type Mice Research Articles

Role of Receptor for Advanced Glycation End Products in Regulating Lung Fluid Balance in Lipopolysaccharide-induced Acute Lung Injury and Infection-Related Acute Respiratory Distress Syndrome.

Receptor for advanced glycation end products (RAGE) is implicated in inflammatory responses in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), but its role in pulmonary edema formation remains unclear, especially in infection-related ARDS mainly caused by pneumonia or sepsis. In this study, we investigated the role of RAGE in alveolar fluid regulation by using RAGE gene knockout (RAGE) mice in a murine ALI model induced by lipopolysaccharide (LPS), and by comparing soluble RAGE (sRAGE) levels in serum and bronchial alveolar lavage fluid between ARDS patients and control subjects. We found that RAGE knockout significantly improved alveolar fluid clearance and reduced pulmonary vascular albumin leakage upon LPS challenge. Furthermore, LPS-induced substantial decrease in lung expression of sodium-potassium ATPase (Na,K-ATPase), epithelial sodium channel, and zonula occluden-1 (ZO-1) were fully or partially restored by the deletion of RAGE. In addition to this, LPS-induced lung leukocyte infiltration and inflammatory cytokine and chemokine release were all attenuated in RAGE mice as compared to wide-type mice. In infection-related ARDS patients, both serum and bronchial alveolar lavage fluid levels of the sRAGE were much higher than those in control subjects, and they were positively correlated with pulmonary vascular permeability and levels of interleukin (IL)-6, IL-8, and macrophage inflammatory protein (MIP)-2. Taken together, we provided the first direct evidence for the essential role of RAGE in regulating lung fluid balance in infection-related ARDS/ALI. The underlying mechanisms may involve the downregulation of both ion-channel and tight junction proteins mediated by RAGE signaling in bacterial endotoxin-induced lung injury.

Modulation of hepatic lipidome by rhodioloside in high-fat diet fed apolipoprotein E knockout mice

BackgroundRhodioloside is a glucoside of tyrosol isolated from Rhodiola rosea. However, its regulating effect on hepatic dyslipidemia of atherogenic mice has rarely been studied. PurposeThe specific aims of current study included to clarify lipidomic perturbation in liver tissues of apolipoprotein E deficient (apoE−/−) mice fed with high-fat diet, and to examine the effects of rhodioloside against atherosclerosis and dyslipidemia. Study DesignThe comparisons of hepatic lipidome were executed between wide type (WT) mice fed with normal diet (NDC) and apoE−/− mice fed with high-fat diet (Model), WT mice fed with high-fat diet (HFDC) versus the model mice, as well as the model mice versus rhodioloside-treated atherosclerotic mice. MethodsUltra high performance liquid chromatography coupled with a Q exactive hybrid quadrupole-orbitrap mass spectrometry (UPLC-MS/MS) was employed to provide an unbiased and simultaneous measurement of individual lipid species in liver tissues. ResultsMultivariate statistical analysis derived from LC-MS spectra revealed that high-fat diet and apoE deficiency caused a series of disturbances on glyerolipid metabolism, glycerophospholipid metabolism and sphingolipid metabolism. Rhodioloside administration showed atheroprotective effects on the apoE−/− mice with regulating the levels of 1 phosphatidylcholine, 2 phosphatidylserines, 5 alkyldiacylglycerols and 3 alkenyldiacylglycerols back to normal. In particular, PC (4:0/15:0) was positively associated with high-density lipoprotein cholesterol in blood, both of which could be ameliorated by rhodioloside. ConclusionOur results identified the abnormal hepatic lipids in atherosclerosis progression that could efficiently improved by rhodioloside. These lipids contributed to biological understanding of atherogenic dyslipidemia in liver and could also served as sensitive indicators for drug target screening.

MiR-188-3p upregulation results in the inhibition of macrophage proinflammatory activities and atherosclerosis in ApoE-deficient mice

BackgroundAtherosclerosis occurs as a result of a chronic inflammatory response in the arterial wall associated with an increased uptake of low-density lipoprotein by macrophages and the subsequent transformation of this lipoprotein into foam cells. It has been found that miR-188-3p can suppress autophagy and myocardial infarction. Therefore, we conducted the present study with determining the suppressive role played by miR-188-3p in atherosclerosis. MethodsThe atherosclerosis model was established using ApoE knockout mice. The healthy C57BL/6J wide-type mice were used as control, while miR-188-3p mimics or inhibitors were applied for the elevation or the depletion of the miR-188-3p expression in mice. The macrophage content was observed in atherosclerotic plaque. Once the miR-188-3p expression was determined, the effects of the over-expression of miR-188-3p on the lipid accumulation and macrophage inflammatory response were accessed. The plasma levels of pro-inflammatory factors and serum RANTES level, as well as OLR1, iNOS, ABCA1 and KLF2 expression were determined in order to evaluate the potential anti-inflammatory and antioxidative activities of miR-188-3p. ResultsApoE knockout mice with atherosclerosis presented with increased lipid accumulation and macrophage content. MiR-188-3p was found to reduce intravascular lipid accumulation in atherosclerotic mice. In addition to the alleviation of macrophage inflammatory response, the upregulation of miR-188-3p also leads to the suppression of oxidation with reduced macrophage accumulation, plasma expression of pro-inflammatory factors and serum RANTES level, OLR1 and iNOS, while it increases ABCA1 and KLF2. ConclusionsIn conclusion, the findings from our study found a new potential therapy for atherosclerosis by investigating the inhibitory effects of miR-188-3p on macrophage inflammatory response and oxidation.

Cerebral cavernous malformation 3 gene deficiency promotes early changes in Alzheimer disease-like lesions induced by low lead exposure

Objective: To investigate the effects of cerebral cavernous malformation 3 (CCM3) gene knockout on the lead exposure-induced blood-brain barrier malfunction in mice brain, and the relationship between CCM3 knockout and the Alzheimer's disease (AD). Methods: Wide type (WT) mice and CCM3(+)/- mice were divided into 4 groups, control group and lead exposed group in WT as well as CCM3(+/-) mice. Lead exposed groups were treated with 0.05% lead acetate in drinking water for 12 weeks, while control group drink deionized water freely. Blood lead and brain lead levels in each group were detected by graphite furnace atomic absorption spectrometry. The brain tissue of each group was made into paraffin sections, whose morphology were observed by HE staining. The expression of Aβ(1-42) in brain tissue was detected by immunohistochemistry and the brain capillaries were labeled by VRGFR2. The protein expression of Claudin-5, ZO-1, and p-Tau was detected by Western blot. The brain tissue RNA was extracted and the relative expression of LRP-1 mRNA was detected by qRT-PCR. Results: The levels of blood lead WT (216.07±84.16) and CCM3(+/-) (189.64±101.86) μg/L in lead exposed group were higher than those in control group WT (19.52±11.46) and CCM3(+/-) (11.79±8.20) μg/L, the difference was statistically significant (t=4.18, P=0.006; t=3.79, P=0.016). The levels of brain lead WT (1.78±0.69) and CCM3(+/-) (1.74±0.66) μg/L were higher than those in control group WT (1.06±0.87) and CCM3(+/-) (0.97±0.64) μg/L, the difference was statistically significant (t=3.67, P=0.018; t=3.88, P=0.015). The HE staining showed no obvious lesions in the brain of each group of mice. The results of immunohistochemistry showed that there was no Aβ(1)-42 deposition in the brain of mice in each group. The numbers of microvessels in the brain of CCM3(+/-) mice in the lead exposed group were decreased. Compared with the relative expression levels of Claudin-5 (WT: 1.30±0.03, CCM3(+/-): 1.07±0.08) in control group mice brain, the relative expression of Claudin-5 (WT: 0.96±0.04, CCM3(+/-): 0.59±0.01) was decreased with statistical significance (F=199.27, P<0.001). The relative expression level of LRP-1 gene mRNA in brain of lead exposed group (WT: 0.32±0.10, CCM3(+/-): 0.06±0.01) was higher than that of unexposed group (WT:1.00±0.06, CCM3(+/-):2.12±0.18), the difference was statistically significant (F=288.29, P<0.001). The relative expression level of LRP-1 gene mRNA in brain of CCM3(+)/- mice exposed to lead was lower than that of WT mice ((0.06±0.01)vs(0.32±0.10), t=26.90, P<0.001). Conclusion: The mice did not show significant AD-like lesions under low-does lead exposure, but resulted in early damage of brain blood-brain barrier and early changes of AD-like lesions in mice, with CCM3(+/-) mice being sensitive to lead exposure stronger than that of WT mice, suggesting that deletion of CCM3 gene may be one of the potential risk factors for accelerating the development of AD in mice exposed to lead.

A Novel Murine Chronic Obstructive Pulmonary Disease Model and the Pathogenic Role of MicroRNA-21

Chronic obstructive pulmonary disease (COPD) is a multi-pathogenesis chronic lung disease. The mechanisms underlying COPD have not been adequately illustrated. Many reseachers argue that microRNAs (miRs) could play a crucial role in COPD. The classic animal model of COPD is both time consuming and costly. This study proposes a novel mice COPD model and explores the role of miR-21 in COPD. A total of 50 wide-type (WT) C57BL/6 mice were separated into five euqlly-sized groups—(1) control group (CG), (2) the novel combined method group (NCM, cigarette smoke (CS) exposure for 28 days combined with cigarette smoke extract (CSE) intraperitoneal injection), (3) the short-term CS exposure group (SCSE, CS exposure for 28 days), (4) the CSE intraperitoneal injection group (CSEII, 28 days CSE intraperitoneal injection), and (5) the long-term CS exposure group (LCSE, CS exposure).The body weight gain of mice were recorded and lung function tested once the modeling was done. The pathological changes and the inflammation level by hematoxylin eosin (H&E) staining and immunohistochemical staining (IHS) on the lung tissue sections were also evaluated. The level of miR-21 in the mice lungs of the mice across all groups was detected by RT-qPCR and the effects of miR-21 knock-down in modeled mice were observed. The mice in LCSE and NCM exhibited the most severe inflammation levels and pathological and pathophysiological changes; while the changes for the mice in SCSE and CSEII were less, they remained more severe than the mice in the CG. The level of miR-21 was found to be negatively correlated with lung functions. Moreover, knocking miR-21 down from the modeled mice, ameliorated all those tested COPD-related changes. Our novel modeling method detected virtually the same changes as those detected in the classic method in WT mice, but in less time and cost. Further, it was determined that the level of miR-21 in the lungs could be an indicator of COPD severity and blocking functions of miR-21 could be a potential treatment for early stage COPD.

IL-17 deficiency attenuates acetaminophen-induced hepatotoxicity in mice

Acetaminophen (APAP) overdose results in the production of reactive oxygen species (ROS), hepatocyte necrosis, and cell death, and leads to acute liver failure. Interleukin-17 (IL-17), a pro-inflammatory cytokine, plays a key role in the recruitment of neutrophils into sites of inflammation and subsequent damage after liver ischemia-reperfusion injury. In this study, we employed IL-17 knockout (KO) mice to investigate the role of IL-17 in APAP-induced hepatotoxicity. IL-17 wide type (WT) and IL-17 KO mice received an intraperitoneal injection of APAP (300 mg/kg). After 16 h of treatment, the hepatic injury, inflammatory mediators, immune cell infiltration, and western blotting were examined and analyzed. The serum alanine transferase (ALT) enzyme levels and hepatic myeloperoxidase (MPO) activity were significantly elevated 16 h after APAP treatment in the WT mice. IL-17 deficiency significantly attenuates APAP-induced liver injury, MPO activity, pro-inflammatory cytokines (tumor necrosis factor-α, IL-6 and interferon-γ) levels and inflammatory cell (neutrophils, macrophage) infiltration in the liver. Moreover, phosphorylated extracellular signal-regulated kinase (ERK) was significantly decreased at 16 h after APAP treatment in the IL-17 KO mice compared with the IL-17 WT mice. Our data suggests that IL-17 plays a pivotal role in APAP-induced hepatotoxicity through modulation of inflammatory response, and perhaps in part through the ERK signaling pathway. Blockade of IL-17 could be a potential therapeutic target for APAP-induced hepatotoxicity.

Abnormal circadian locomotor rhythms and Per gene expression in six-month-old triple transgenic mice model of Alzheimer’s disease

Circadian rhythm disturbance (CRD) is one of the iconic manifestations in Alzheimer’s disease (AD), a disease tightly associated with age, but the characteristics and gender difference of CRD occurred in AD have not been well demonstrated. Using 6-month-old triple transgenic AD mouse model (3xTg-AD) without obvious brain pathological changes, we demonstrated the gender difference of CRD at this age. We further showed abnormal Per gene expression in the central clock suprachiasmatic nucleus (SCN) of the 3xTg-AD mice. Specifically, compared with the wide type (WT) mice, the 3xTg-AD mice showed disrupted circadian locomotor rhythms both at LD (light-dark 12 h:12 h) and DD (constant dark) conditions, such as increased activities in the resting phase, decreased and scattered activities in the active phase, decreased overall activity intensities, amplitude, robustness, and increased intradaily variability. We further observed that 3xTg-AD female mice showed obviously less CRD compared with the 3xTg-AD male mice, and female mice of both WT and 3xTg-AD were more active in locomotor activity. Accordingly, 3xTg-AD mice showed a phase delay in the expression of Per1 and Per2 mRNA in the SCN, with the levels of Per1 and Per2 mRNA were significantly lower than that of WT mice at specific time points. We conclude that 3xTg-AD mice exhibit behavioral CRD at the age of six months with male gender preference, and these phenomena are at least partly associated with the alteration of Per1 and Per2 transcription patterns in the SCN.

A Disintegrin and Metalloproteinase 15‐Mediated Glycocalyx Disruption Contributes to Vascular Leakage During Septic Injury

Disruption of vascular endothelial barrier results in excessive accumulation of plasma proteins and fluid within the interstitial space, exacerbating tissue injury caused by infection or sepsis. There is an urgent need to explore effective strategies to restore vascular barrier integrity in patients with sepsis. Glycocalyx, an endothelial surface coating matrix composed of glycoproteins and proteinglycans attached to glycoaminoglycan chains, has been shown to be protective of vascular barrier function. Degradation of glycocalyx has been implicated in the progression of septic injury, evidenced by increased serum levels of glycocalyx degradation products, including soluble CD44 and hyaluronic acid in sepsis patients, correlating with severity of injury. Currently, the molecular mechanisms underlying glycocalyx degradation are poorly understood. Here, we investigated whether and how this process involves a disintegrin and metalloproteinase 15 (ADAM15), a transmembrane sheddase capable of cleaving the ectodomains of membrane‐bounded proteins. Based on intravital microscopic measurements of dextran (150 kD) exclusion, we comparatively assessed glycocalyx thickness in wild‐type and ADAM15 knockout mice subjected to septic insult via lipopolysaccharides (LPS) injection. Our results indicated that LPS treatment induced a remarkable reduction in glycocalyx thickness in wide type mice, a response significantly attenuated in ADAM15−/− mice, suggesting the critical role of ADAM15 in LPS‐induced glycocalyx injury. Moreover, glycocalyx in mouse pulmonary microvasculature was observed by electron microscopy. Consistently, ADAM15 deficiency prevented the loss of glycocalyx coverage on the endothelial surface in pulmonary vessels following LPS challenge. To elucidate the underlying molecular details, we performed an in vitro cleavage assay, which confirmed the ability of recombinant ADAM15 to cleave CD44, an important glycoprotein expressed in glycocalyx, in a dose‐dependent manner. Further proteomic analysis revealed that the cleavage site of CD44 fell within the membrane‐proximal region of its ectodomain. Functionally, incubating endothelial cells with CD44 ectodomain resulted in barrier dysfunction and adherens junction disorganization. In line with these in vitro findings, mice with impaired glycocalyx exhibited vascular hyperpermeability after LPS challenge, as indicated by increased Evans Blue‐bound albumin extravasation in the lungs as well as enhanced albumin leakage from mesenteric vessels. In contrast, microvessels from ADAM15−/− mice with well‐preserved glycocalyx structure displayed an attenuated permeability response. Taken together, our studies suggest a novel function of ADAM15 in mediating glycocalyx injury during septic challenge.Support or Funding InformationThis work was supported by the National Institutes of Health Grants GM097270 and HL070752.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract TP257: Fasl Enhances Cytotoxicity of CD8 + T Cells After Ischemic Stroke

Introduction: After the onset of stroke, infiltrated CD8 + T cells aggravate brain injury through FasL or cytotoxic granules. We previously reported that FasL mutation could attenuate brain inflammation in experimental stroke, however, the exact mechanisms were undefined. Thus, this study tested the hypothesis whether FasL could modulate the cytotoxicity of CD8 + T cells or not. Methods: Middle cerebral artery occlusion was induced in both wide-type (WT) and FasL-mutant ( gld ) mice. Magnetic activated cell sorting was used to isolate CD8 + T cells from the spleen. In vitro , CD8 + T cells were cocultured with neurons or microglia for 24 hours. In some experiments, FasL neutralizing antibody were used to block FasL on CD8 + T cells. Flow cytometry and q-PCR were performed to measure the cytotoxicity of CD8 + T cells and polarization of microglia. Cytometric Bead Array was used to evaluate the inflammatory cytokines in the coculture supernatant. The cell viability of oxygen glucose deprivation (OGD) neurons was measured by lactate dehydrogenase assay and propidium iodide and calcein AM staining. Results: CD8 + T cells were activated and expressed more cytotoxic cytokines when cocultured with microglia. Nevertheless, this induction was attenuated when CD8 + T cells were pretreated with FasL neutralizing antibody. Meanwhile, FasL mutation or functional block abrogated CD8 + T cell-mediated killing of neurons and M1 microglia polarization. In addition, the CD8 + T cell-microglia coculture supernatant contained less pro-inflammatory cytokines and resulted in enhanced cell viability of post-OGD neuron when FasL on CD8 + T cells was mutant or blocked. Conclusions: FasL enhances cytotoxicity of CD8 + T cells after ischemic stroke.

SNRK (Sucrose Nonfermenting 1-Related Kinase) Promotes Angiogenesis In Vivo.

SNRK (sucrose nonfermenting 1-related kinase) is a novel member of the AMPK (adenosine monophosphate-activated protein kinase)-related superfamily that is activated in the process of angiogenesis. Currently, little is known about the function of SNRK in angiogenesis in the physiological and pathological conditions. In this study, in Snrk global heterozygous knockout mice, retina angiogenesis and neovessel formation after hindlimb ischemia were suppressed. Consistently, mice with endothelial cell (EC)-specific Snrk deletion exhibited impaired retina angiogenesis, and delayed perfusion recovery and exacerbated muscle apoptosis in ischemic hindlimbs, compared with those of littermate wide-type mice. Endothelial SNRK expression was increased in the extremity vessel samples from nonischemic human. In ECs cultured in hypoxic conditions, HIF1α (hypoxia inducible factor 1α) bound to the SNRK promoter to upregulate SNRK expression. In the nuclei of hypoxic ECs, SNRK complexed with SP1 (specificity protein 1), and together, they bound to an SP1-binding motif in the ITGB1 (β1 integrin) promoter, resulting in enhanced ITGB1 expression and promoted EC migration. Furthermore, SNRK or SP1 deficiency in ECs ameliorated hypoxia-induced ITGB1 expression and, consequently, inhibited EC migration and angiogenesis. Taken together, our data have revealed that SNRK/SP1-ITGB1 signaling axis promotes angiogenesis in vivo.

MiR-17-92 ameliorates renal ischemia reperfusion injury

There is limited information on the role of miR-17-92 in renal tubular pathophysiology. Therefore, the present study was performed to determine whether miR-17-92 plays a role in ischemia-reperfusion injury (IRI)-induced acute kidney injury. We originally demonstrated that miR-17-92 is up-regulated following IRI in vivo. To explore the roles of miR-17-92 in the IRI process, we first generated a renal proximal tubule-specific miR-17-92 deletion (PT-miR-17-92−/−) knockout mouse model with Cre driven by the Kap promoter. We found that PT-deficient miR-17-92 mice had more severe renal dysfunction and renal structures than their littermates. Compared with sham-operated mice, both wide-type (WT) mice and PT-miR-17-92−/− mice showed increased serum levels of creatinine and urea. However, the levels of serum urea and creatinine in PT-miR-17-92−/− mice after the IRI operation were significantly higher than the levels in WT mice. In addition, PT-miR-17-92−/− mice showed higher levels of serum potassium and phosphonium after the IRI operation. Histological analysis revealed that PT-miR-17-92−/− mice had substantial histopathologic changes, such as tubular dilation and tubular necrosis. Overexpression of miR-17-92 could partially reverse the side-effects of IRI on the proximal tubules in vivo. Furthermore, we employed a quantitative proteomic strategy and identified 16 proteins as potential targets of miR-17-92. Taken together, our findings suggested that miR-17-92 may ameliorates IRI-induced acute kidney injury. Our results indicate that pharmacologic modulation of these miRNAs may have therapeutic potential for acute kidney injury.

MiR-551b-5p Contributes to Pathogenesis of Vein Graft Failure via Upregulating Early Growth Response-1 Expression.

Background:Vein graft failure (VGF) is a serious complication of coronary artery bypass graft, although the mechanism remains unclear. The study aimed to investigate the effects of microRNAs (miRNAs) on the endothelial dysfunction involved in VGF.Methods:Human umbilical vein endothelial cells (HUVECs) were subjected to mechanical stretch stimulation to induce endothelial dysfunction. Genome-wide transcriptome profiling was performed using the Human miRNA OneArray® V4 (PhalanxBio Inc., San Diego, USA). The miRNA-messenger RNA (mRNA) network was investigated using gene ontology and Kyoto Encyclopedia of Genes and Genomes. The miR-551b-5p mimic and inhibitor were applied to regulate miR-551b-5p expression in the HUVECs. The 5-ethynyl-2’-deoxyuridine assay, polymerase chain reaction (PCR), and Western blotting (WB) were used to assess HUVECs proliferation, mRNA expression, and protein expression, respectively. The vein graft model was established in early growth response (Egr)-1 knockout (KO) mice and wide-type (WT) C57BL/6J mice for pathological and immunohistochemical analysis. Endothelial cells isolated from the veins of WT and Egr-1 KO mice were subjected to mechanical stretch stimulation; PCR and WB were conducted to confirm the regulatory effect of Egr-1 on Intercellular adhesion molecule (Icam-1). One-way analysis of variance and independent t-test were performed for data analysis.Results:Thirty-eight miRNAs were differentially expressed in HUVECs after mechanical stretch stimulation. The bioinformatics analysis revealed that Egr-1 might be involved in VGF and was a potential target gene of miR-551b-5p. The mechanical stretch stimulation increased miR-551b-5p expression by 2.93 ± 0.08 fold (t = 3.07, P < 0.05), compared with the normal HUVECs. Transfection with the miR-551b-5p mimic or inhibitor increased expression of miR-551b-5p by 793.1 ± 171.6 fold (t = 13.84, P < 0.001) or decreased by 26.3% ± 2.4% (t = 26.39, P < 0.05) in the HUVECs, respectively. HUVECs proliferation and EGR-1 mRNA expression were significantly suppressed by inhibiting miR-551b-5p expression (P < 0.05). The lumens of the vein grafts in the Egr-1 KO mice were wider than that in the WT mice. Icam-1 expression was suppressed significantly in the Egr-1 KO vein grafts (P < 0.05).Conclusions:Increased miR-551b-5p expression leads to endothelial dysfunction by upregulating Egr-1 expression. EGR-1 KO can improve the function of a grafted vein through suppressing Icam-1.

Leptin differentially regulates chondrogenesis in mouse vertebral and tibial growth plates

BackgroundLeptin plays an important role in mediating chondrogenesis of limb growth plate. Previous studies suggest that bone structures and development of spine and limb are different. The expression of Ob-Rb, the gene that encodes leptin receptors, is vertebral and appendicular region-specific, suggesting the regulation of leptin on VGP and TGP chondrogenesis may be very different. The aim of the present study was to investigate the differential regulation of leptin on the chondrogenesis of vertebral growth plate (VGP) and tibial growth plate (TGP).MethodsWe compared the VGP and TGP from wild type (C57BL/6) and leptin-deficient (ob/ob) mice. We then generated primary cultures of TGP and VGP chondrocytes. By treating the primary cells with different concentrations of leptin in vitro, we analyzed proliferation and apoptosis of the primary chondrocytes from TGP and VGP. We further measured expression of chondrogenic-related genes in these cells that had been incubated with different doses of leptin.ResultsLeptin-deficient mice of 8-week-old had shorter tibial and longer vertebral lengths than the wide type mice. Disturbed columnar structure was observed for TGP but not for VGP. In primary chondrocyte cultures, leptin inhibited VGP chondrocyte proliferation but promoted their apoptosis. Collagen IIA and aggrecan mRNA, and the protein levels of proliferation- and chondrogenesis-related markers, including PCNA, Sox9, and Smad4, were downregulated by leptin in a dose-dependent manner. In contrast, leptin stimulated the proliferation and chondrogenic differentiation of TGP chondrocytes at physiological levels (i.e., 10 and 50 ng/mL) but not at high levels (i.e., 100 and 1000 ng/mL).ConclusionLeptin exerts a stimulatory effect on the proliferation and chondrogenic differentiation of the long bone growth plate but an inhibitory effect on the spine growth plate. The ongoing study will shed light on the regulatory mechanisms of leptin in bone development and metabolism.

Abstract 377: Mast Cell Serotonin Controls Subcutaneous Adipose Tissue Browning and Systemic Energy Expenditure in Mice

Adipose tissue browning and systemic energy expenditure provide a defense mechanism against obesity and associated metabolic diseases. In western diet-fed mice, inactivation of mast cells (MCs) ameliorates obesity and insulin resistance along with improved metabolic rate. Yet, a direct role of MCs in adipose tissue thermogenesis and browning remains unclear. Here we report that, in the context of mice on a chew diet, norepinephrine (NE)-stimulated metabolic rate is increased in MC-deficient Kit w-sh/w-sh mice and MC-stabilized wide-type (WT) mice. Such functional inactivation of MCs enhances thermogenesis and browning in subcutaneous adipose tissues (SAT), but not in brown (BAT) and epididymal adipose tissues (EAT). MC reconstitution to SAT blocks the aforesaid changes in Kit w-sh/w-sh mice. Mechanistic studies demonstrate that functional inactivation of MCs not only elevates the numbers of PDGFRα + bipotential adipocyte precursors but also accelerates beige adipocyte differentiation in SAT. Using tryptophan hydroxylase 1 inhibitor, we show that MC-derived serotonin inhibits SAT beige adipocyte biogenesis and systemic energy expenditure. Together, functional inactivation of MCs or inhibition of MC serotonin synthesis in SAT promotes adipocyte browning and systemic energy metabolism in mice.

DSS‐induced intestinal damage exacerbates liver injury by acute alcohol exposure in mice: role of intestinal hypoxia‐inducible factor 1α

Inflammatory bowel diseases (IBD) are characterized by repeated injury of the mucosa and loss of the intestinal epithelial barrier function, which has also been considered one of the major contributors to the development of alcoholic liver disease (ALD). Hypoxia induced factor 1α (HIF1α) plays a critical role in the intestinal barrier integrity by regulating several mucus and tight junction protecting genes. In this study, we hypothesize that intestinal HIF1α deletion increases intestinal permeability resulting in an elevation of endotoxemia in the dextran sulfate sodium (DSS)‐induced colitis murine model, and this exacerbates acute alcohol‐induced liver injury. To test this hypothesis, Wide type (WT) and intestinal epithelial‐specific HIF1α knockout (IEhif‐1α−/−) mice were fed 3% dextran sodium sulfate (DSS) in drinking water for 7 days and a bolus of alcohol at 6g/kg was gavaged 6 hours before sacrificing. DSS decreased body weight of the WT mice, and this decrease was more significant in the IEhif‐1α−/− mice. Intestinal inflammation and mucosal abnormality were increased in the IEhif‐1α−/− mice than that in the WT mice, along with an elevated serum LPS concentration, indicating an increase in intestinal barrier permeability. Binge alcohol, in addition to DSS, further damaged barrier function and increased serum LPS concentrations. Further analyses showed that DSS+alcohol induced a decrease in the expression of caludin‐1 and intestinal trefoil factor, which play an important role in intestinal barrier integrity. Intestine epithelial HIF1α deletion aggravated this effect. Unlike alcohol exposure, DSS treatment alone did not affect liver function in either WT or IEhif‐1α−/− mice, as determined by serum ALT and AST analysis. However, ALT and AST levels were significantly elevated in DSS + alcohol treatment groups, and these elevations were more pronounced in IEhif‐1α−/− mice. HIF‐1α deletion also enhanced alcohol‐induced hepatic triglyceride concentration in DSS‐treated mice. Hepatic histology analysis showed that IEhif‐1α−/− mice had an increased fat accumulation and inflammatory cell infiltration, confirming the hepatic steatosis and liver injury. In conclusion, our results demonstrated that DSS‐induced colitis increases intestinal permeability and endotoxemia, resulting in an worsened liver steatosis and injury due to binge alcohol exposure. Deleting intestinal HIF1α further exacerbates these deleterious effects. Targeting intestinal HIF1α might be an effective strategy for the treatment of colitis‐associated ALD.Support or Funding InformationSupported by NIH and Veterans Administration.

Deficiency of TREK-1 potassium channel exacerbates secondary injury following spinal cord injury in mice.

Spinal cord injury (SCI) involves complex pathological process which can be complicated by secondary injury. TREK-1 is a member of the two-pore domain potassium (K2P) channel family, which can be modulated by a number of physiological and pathological stimuli. Recent studies suggest that TREK-1 plays an active role in depression, pain and neuroprotection. However, its role in the pathological process after SCI remains unclear. In this study, we tested the expression and function of TREK-1 in spinal cord of mice after traumatic SCI. TREK-1 was widely expressed in mice spinal cord, including astrocytes and neurons. Deficiency of TREK-1 significantly exacerbated focal inflammatory responses as indicated by the increased accumulation of microglia/macrophage as well as pro-inflammatory factor interleukin-1 beta (IL-1β) and tumor necrosis factor alpha expression. Meanwhile, TREK-1 knockout mice showed enhanced reactive astrogliosis, chondroitin sulphate proteoglycans (CSPGs) production and decreased glutamate transporter-1 expression compared to the wide-type mice after SCI. Furthermore, TREK-1 deficiency promoted neurons and oligodendrocytes apoptosis, aggravated demyelination, cavity formation and retarded motor recovery. In summary, our findings provide the first invivo evidence suggesting that TREK-1 may thereby constitute a promising therapeutic target to treat acute SCI.

Co-housing reverses memory decline by epigenetic regulation of brain-derived neurotrophic factor expression in an animal model of Alzheimer’s disease

Co-housing with a company exerts profound effects on memory decline in animal model of Alzheimer's disease (AD). Recently, we found that APP/PS1 mice of 9-month-old improved their memories after co-housing with wide-type mice for 3months by increasing hippocampal brain-derived neurotrophic factor (BDNF) expression. However, the mechanism of how co-housing could induce BDNF expression remains elusive. Here we examined epigenetic changes in the mouse hippocampus that accompanied the co-housing-induced memory improvement. We found that the level of histone deacetylase 2 (HDAC2), but not that of HDAC1, was significantly lower in the memory improved mice than in the control and memory un-improved APP/PS1 mice after co-housing. Knockdown of Hdac2 resulted in a higher freezing response after co-housing. Conversely, over-expression of HDAC2 blocked co-housing-induced memory improvement. The level of Bdnf exon IV mRNA increased significantly after knockdown of Hdac2. ChIP assay revealed a decreased occupancy of HDAC2 in the promoter region of Bdnf exon IV of memory improved mice but not memory un-improved and control APP/PS1 mice. Consistently, the acetylation of histone 3 on Lys 9 (H3K9) and histone 4 on Lys12 (H4K12) increased significantly in the promoter region of Bdnf exon IV. These results suggest HDAC2 expression is reduced after co-housing resulting in a decreased occupancy of HDAC2 and increased histone H3K9 and H4K12 acetylation in the promoter region of Bdnf exon IV, leading to increased BDNF expression in the hippocampus that improves memory.

A Novel Testis-Specific Gene, Ccdc136, Is Required for Acrosome Formation and Fertilization in Mice.

Testis-specific genes are essential for the spermatogenesis in mammalian male reproduction. In this study, we have identified a novel testis-specific gene, Ccdc136 (coiled-coil domain containing 136), from the results of high-throughput gene expression profiling in the developmental stage of mouse testes. Ccdc136 was conserved across species in evolution. Quantitative real-time polymerase chain reaction and Western blot analyses showed that Ccdc136 messenger RNA and protein were extraordinarily expressed in mouse testes, which was first presented at postnatal 3 week and increased in an age-dependent manner before adulthood. Immunofluorescence staining revealed that CCDC136 protein was most abundantly located in the acrosome of round spermatids and elongating spermatids within seminiferous tubules of the adult mouse testes. To investigate the function of Ccdc136 in mouse testes, we generated the Ccdc136-knockout mice using Cas9/RNA-mediated gene targeting technology. Interestingly, we found Ccdc136(-/-) males were infertile, due to severe defect of disrupting acrosome formation. The expression levels of proteins (SPACA1 and PICK1) involved in acrosome formation were significantly downregulated in the testes of Ccdc136(-/-) mice than wide-type mice. Moreover, in vitro fertilization assay revealed that anti-CCDC136 antibody could remarkably inhibit fertilization, suggesting CCDC136 also plays an important role in fertilization. All of these demonstrated the essential role of CCDC136-mediated acrosome formation in spermatogenesis and fertilization, which might also provide new insight into the genetic causes of human infertility.

Progressive hearing loss and degeneration of hair cell stereocilia in taperin gene knockout mice

The TPRN gene encodes taperin, which is prominently present at the taper region of hair cell stereocilia. Mutations in TPRN have been reported to cause autosomal recessive nonsyndromic deafness 79(DFNB 79). To investigate the role of taperin in pathogenesis of hearing loss, we generated TPRN knockout mice using TALEN technique. Sanger sequencing confirmed an 11 bp deletion at nucleotide 177–187 in exon 1 of TPRN, which results in a truncated form of taperin protein. Heterozygous TPRN+/− mice showed apparently normal auditory phenotypes to their wide-type (WT) littermates. Homozygous TPRN−/− mice exhibited progressive sensorineural hearing loss as reflected by auditory brainstem response to both click and tone burst stimuli at postnatal days 15 (P15), 30 (P30), and 60 (P60). Alex Fluor-594 phalloidin labeling showed no obvious difference in hair cell numbers in the cochlea between TPRN−/− mice and WT mice under light microscope. However, scanning electronic microscopy revealed progressive degeneration of inner hair cell stereocilia, from apparently normal at postnatal days 3 (P3) to scattered absence at P15 and further to substantial loss at P30. The outer hair cell stereocilia also showed progressive degeneration, though much less severe, Collectively, we conclude that taperin plays an important role in maintenance of hair cell stereocilia. Establishment of TPRN knockout mice enables further investigation into the function of this gene.

Sema4D is required in both the adaptive and innate immune responses of contact hypersensitivity.

Originally recognized as a regulator of axon guidance in the nervous system, Semaphorin 4D (Sema4D, CD100) also participates in various immune responses and many immune-related diseases. However, whether Sema4D is involved in the pathogenesis of contact hypersensitivity (CHS) remains unclear. In this study, we explored the role of Sema4D in oxazolone-induced CHS using Sema4D knockout (KO) mice. We found that Sema4D KO mice developed attenuated CHS responses, as indicated by milder ear-swelling, lower expression of IL-1β, IL-6, CXCL2 and CXCL5, and decreased recruitment of neutrophils, CD8+ T cells and CD4+ T cells. CHS was impaired in the wide type (WT) mice reconstituted with bone marrow from Sema4D KO mice, indicating that deletion of Sema4D gene in hematopoietic cells played a key role in the alleviated CHS in Sema4D KO mice. CHS was also attenuated in the WT mice transferred with draining lymph nodes (dLNs) cells from oxazolone-sensitized Sema4D KO mice, and the activation and differentiation of hapten-specific CD8+ T cells were impaired in Sema4D KO mice. Furthermore, Sema4D KO mice expressed less IL-1β and CXCL2 than WT mice after oxazolone sensitization, and after transferred with dLNs cells from oxazolone-sensitized WT mice, naïve Sema4D KO mice showed attenuated CHS responses upon oxazolone challenge, indicating that the innate immune response of CHS in Sema4D KO mice was also abrogated. Taken together, our findings revealed for the first time that Sema4D positively regulated both the adaptive and innate immune responses in CHS.