Search Results (2,080)

Bluetongue (BT) is a Culicoides midge-borne hemorrhagic disease affecting cervids and ruminant livestock species, resulting in significant economic losses from animal production and trade restrictions. Experimental animal infections using the α/β interferon receptor knockout IFNAR mouse model and susceptible target species are critical for understanding viral pathogenesis, virulence, and evaluating vaccines. However, conducting experimental vector-borne transmission studies with the vector itself are logistically difficult and experimentally problematic. Therefore, experimental infections are induced by hypodermic injection with virus typically derived from baby hamster kidney (BHK) cells. Unfortunately, for many U.S. BTV serotypes, it is difficult to replicate the severity of the disease seen in natural, midge-transmitted infections by injecting BHK-derived virus into target host animals. Using the IFNAR BTV murine model, we compared the virulence of traditional BHK cell-derived BTV-17 with C. sonorensis midge (W8) cell-derived BTV-17 to determine whether using cells of the transmission vector would provide an in vitro virulence aspect of vector-transmitted virus. At both low and high doses, mice inoculated with W8-BTV-17 had an earlier onset of viremia, earlier onset and peak of clinical signs, and significantly higher mortality compared to mice inoculated with BHK-BTV-17. Our results suggest using a Culicoides W8 cell-derived inoculum may provide an in vitro vector-enhanced infection to more closely represent disease levels seen in natural midge-transmitted infections while avoiding the logistical and experimental complexity of working with live midges. Full article

In pancreatic cancer, the tumor microenvironment (TME) accounts for up to 90% of the tumor mass. Pancreatitis, characterized by the increased infiltration of macrophages into the pancreas, is a known risk factor for pancreatic cancer. The NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor regulates responses to oxidative stress and can promote cancer and chemoresistance. NRF2 also attenuates inflammation through the regulation of macrophage-specific genes. Heme oxygenase 1 (HO-1) is expressed by anti-inflammatory macrophages to degrade heme, and its expression is dependent on NRF2 translocation to the nucleus. In macrophages stimulated with conditioned media from pancreatic cancer cells, HO-1 protein levels increased, which correlated with higher NRF2 expression in the nuclear fraction. Significant differences in macrophage infiltration and HO-1 expression were detected in LSL-Kras^G12D/+; Pdx-1-Cre (KC) mice, Nrf2 whole-body knockout (KO) mice and wildtype mice with pancreatitis. Since epigenetic modulation is a mechanism used by tumors to regulate the TME, using small molecules as epigenetic modulators to activate immune recognition is therapeutically desirable. When the bromodomain inhibitor I-BET-762 was used to treat macrophages or mice with pancreatitis, high levels of HO-1 were reduced. This study shows that bromodomain inhibitors can be used to prevent physiological responses to inflammation that promote tumorigenesis. Full article

Plasminogen activation inhibitor-1 (PAI-1) plays a central role in thrombus formation leading to stroke; however, the contributions of PAI-1 to cellular damage in response to reactive oxygen species which are elevated during reperfusion are unknown. Given that PAI-1 can limit apoptosis, we hypothesized that PAI increases the resilience of cerebral arteries to H₂O₂ (200 µM). Cell death, mitochondrial membrane potential, and mitochondrial ROS production were evaluated in pressurized mouse posterior cerebral arteries from males and females. The effects of pharmacological and genetic inhibition of PAI-1 signaling were evaluated with the inhibitor PAI-039 (10 µM) and PAI-1 knockout mice, respectively. During exposure to H₂O₂, PCAs from male mice lacking PAI-1 had reduced mitochondrial depolarization and smooth muscle cell death, and PAI-039 increased EC death. In contrast, mitochondrial depolarization and cell death were augmented in female PCAs. With no effect of PAI-1 inhibition on resting mitochondrial ROS production, vessels from female PAI-1 knockout mice had increased mitochondrial ROS generation during H₂O₂ exposure. During acute exposure to oxidative stress, protein ablation of PAI-1 enhances cell death in posterior cerebral arteries from females while limiting cell death in males. These findings provide important considerations for blood flow restoration during stroke treatment. Full article

Atherosclerosis involves an inflammatory response due to plaque formation within the arteries, which can lead to ischemic stroke and heart disease. It is one of the leading causes of death worldwide, with various contributing factors such as hyperlipidemia, hypertension, obesity, diabetes, and smoking. Wall shear stress (WSS) is also known as a contributing factor of the formation of atherosclerotic plaques. Since the causes of atherosclerosis cannot be attributed to a single factor, clearly understanding the mechanisms and causes of its occurrence is crucial for preventing the disease and developing effective treatment strategies. To better understand atherosclerosis and define the correlation between various contributing factors, computational fluid dynamics (CFD) analysis is primarily used. CFD simulates WSS, the frictional force caused by blood flow on the vessel wall with various hemodynamic changes. Using apolipoprotein E knockout (ApoE-KO) mice subjected to partial ligation and a high-fat diet at 1-week, 2-week, and 4-week intervals as an atherosclerosis model, CFD analysis was conducted along with the reconstruction of carotid artery blood flow via magnetic resonance imaging (MRI) and compared to the inflammatory factors and pathological staining. In this experiment, a comparative analysis of the effects of high WSS and low WSS was conducted by comparing the standard deviation of time-averaged wall shear stress (TAWSS) at each point within the vessel wall. As a novel approach, the standard deviation of TAWSS within the vessel was analyzed with the staining results and pathological features. Since the onset of atherosclerosis cannot be explained by a single factor, the aim was to find the correlation between the thickness of atherosclerotic plaques and inflammatory factors through standard deviation analysis. As a result, the gap between low WSS and high WSS widened as the interval between weeks in the atherosclerosis mouse model increased. This finding not only linked the occurrence of atherosclerosis to WSS differences but also provided a connection to the causes of vulnerable plaques. Full article

Diabetic cardiomyopathy (DCM), one of the most serious long-term consequences of diabetes, is closely associated with myocardial fatty acid metabolism. Carnitine palmitoyltransferase-1β (CPT-1β) is the rate-limiting enzyme responsible for β-oxidation of long-chain fatty acids. Intermedin (IMD) is a pivotal bioactive small molecule peptide, participating in the protection of various cardiovascular diseases. However, the role and underlying mechanisms of IMD in DCM are still unclear. In this study, we investigated whether IMD alleviates DCM via regulating CPT-1β. A rat DCM model was established by having rats to drink fructose water for 12 weeks. A mouse DCM model was induced by feeding mice a high-fat diet for 16 weeks. We showed that IMD and its receptor complexes levels were significantly down-regulated in the cardiac tissues of DCM rats and mice. Reduced expression of IMD was also observed in neonatal rat cardiomyocytes treated with palmitic acid (PA, 300 μM) in vitro. Exogenous and endogenous IMD mitigated cardiac hypertrophy, fibrosis, dysfunction, and lipid accumulation in DCM rats and IMD-transgenic DCM mice, whereas knockout of IMD worsened these pathological processes in IMD-knockout DCM mice. In vitro, IMD alleviated PA-induced cardiomyocyte hypertrophy and cardiac fibroblast activation. We found that CPT-1β enzyme activity, mRNA and protein levels, and acetyl-CoA content were increased in T2DM patients, rats and mice. IMD up-regulated the CPT-1β levels and acetyl-CoA content in T2DM rats and mice. Knockdown of CPT-1β blocked the effects of IMD on increasing acetyl-CoA content and on inhibiting cardiomyocyte hypertrophy and cardiac fibroblast activation. IMD receptor antagonist IMD_17–47 and the phosphatidyl inositol 3 kinase (PI3K)/protein kinase B (Akt) inhibitor LY294002 reversed the effects of IMD on up-regulating CPT-1β and acetyl-CoA expression and on inhibiting cardiomyocyte hypertrophy and cardiac fibroblast activation. We revealed that IMD alleviates DCM by up-regulating CPT-1β via calcitonin receptor-like receptor/receptor activity-modifying protein (CRLR/RAMP) receptor complexes and PI3K/Akt signaling. IMD may serve as a potent therapeutic target for the treatment of DCM. Full article

Threonine phosphorylation promotes inflammatory functions of STAT1 while restricting its interferon (IFN) signaling in innate immune responses. However, it remains unclear whether the restriction of STAT1-mediated IFN signaling conferred by threonine phosphorylation is a ubiquitous mechanism or one that is context-dependent. To address this, we utilized pristane-induced lupus, a prototype IFN-driven systemic autoimmune disease model characterized by the production of high-titer autoantibodies against nucleic acid-associated antigens. Through genetic and biochemical assays, we demonstrate that Thr748 phosphorylation is dispensable for STAT1 functionality in pristane-induced lupus. Genetically engineered mice expressing the phospho-deficient threonine 748-to-alanine (T748A) mutant STAT1 exhibited similar survival rates, high titers of anti-dsDNA IgG, and nephritis compared to their wild-type littermates. In sharp contrast, STAT1 deficiency protected mice against pristane-induced lupus, as evidenced by increased survival, low titers of anti-dsDNA IgG, and less severe nephritis in the STAT1 knockout mice compared to their T748A littermates. Our study suggests a phosphorylation-dependent modularity that governs the spectrum of STAT1 functionality in inflammatory contexts: IFN phospho-tyrosine-dependent and inflammatory phospho-threonine-dependent, with Thr748 phosphorylation driving selective inflammatory activities, particularly those not driven by the canonical JAK pathway. From a broader perspective, our findings provide deeper insights into how distinct phosphorylation events shape the combinatorial logic of signaling cassettes, thereby regulating context-dependent responses. Full article

The vascular endothelium, a specialized monolayer of endothelial cells (ECs), is crucial for maintaining vascular homeostasis by controlling the passage of substances and cells. In the tumor microenvironment, Vascular Endothelial Growth Factor A (VEGF-A) drives tumor angiogenesis, leading to endothelial anergy and vascular immunosuppression—a state where ECs resist cytotoxic CD8⁺ T cell infiltration, hindering immune surveillance. Immunotherapies have shown clinical promise. However, their effectiveness is significantly reduced by tumor EC anergy. Anti-angiogenic treatments aim to normalize tumor vessels and improve immune cell infiltration. Despite their potential, these therapies often cause significant systemic toxicities, necessitating new treatments. The small GTPase Rap1B emerges as a critical regulator of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) signaling in ECs. Our studies using EC-specific Rap1B knockout mice show that the absence of Rap1B impairs tumor growth, alters vessel morphology, and increases CD8⁺ T cell infiltration and activation. This indicates that Rap1B mediates VEGF-A’s immunosuppressive effects, making it a promising target for overcoming vascular immunosuppression in cancer. Rap1B shares structural and functional similarities with RAS oncogenes. We propose that targeting Rap1B could enhance therapies’ efficacy while minimizing adverse effects by reversing endothelial anergy. We briefly discuss strategies successfully developed for targeting RAS as a model for developing anti-Rap1 therapies. Full article

GSK-3β plays a critical role in regulating the Wnt/β-catenin signaling pathway, and manipulating GSK-3β in dendritic cells (DCs) has been shown to improve the antitumor efficacy of DC vaccines. Since the inhibition of GSK-3β leads to the activation of β-catenin, we hypothesize that blocking GSK-3β in DCs negatively regulates DC-mediated CD8 T cell immunity and antitumor immunity. Using CD11c-GSK-3β^−/− conditional knockout mice in which GSK-3β is genetically deleted in CD11c-expressing DCs, we surprisingly found that the deletion of GSK-3β in DCs resulted in increased antitumor immunity, which contradicted our initial expectation of reduced antitumor immunity due to the presumed upregulation of β-catenin in DCs. Indeed, we found by both Western blot and flow cytometry that the deletion of GSK-3β in DCs did not lead to augmented expression of β-catenin protein, suggesting that GSK-3β exerts its function independent of β-catenin. Supporting this notion, our single-cell RNA sequencing (scRNA-seq) analysis revealed that GSK-3β-deficient DCs exhibited distinct gene expression patterns with minimally overlapping differentially expressed genes (DEGs) compared to DCs with activated β-catenin. This suggests that the deletion of GSK-3β in DCs is unlikely to lead to upregulation of β-catenin at the transcriptional level. Consistent with enhanced antitumor immunity, we also found that CD11c-GSK-3β^−/− mice exhibited significantly augmented cross-priming of antigen-specific CD8 T cells following DC-targeted vaccines. We further found that the deletion of GSK-3β in DCs completely abrogated memory CD8 T cell responses, suggesting that GSK-3β in DCs also plays a negative role in regulating the differentiation and/or maintenance of memory CD8 T cells. scRNA-seq analysis further revealed that although the deletion of GSK-3β in DCs positively regulated transcriptional programs for effector differentiation and function of primed antigen-specific CD8 T cells in CD11c-GSK-3β^−/− mice during the priming phase, it resulted in significantly reduced antigen-specific memory CD8 T cells, consistent with diminished memory responses. Taken together, our data demonstrate that GSK-3β in DCs has opposite functions in regulating cross-priming and memory CD8 T cell responses, and GSK-3β exerts its functions independent of its regulation of β-catenin. These novel insights suggest that targeting GSK-3β in cancer immunotherapies must consider its dual role in CD8 T cell responses. Full article

Cellular Communication Network Factor 2 (CCN2) is a matricellular protein implicated in cell communication and microenvironmental signaling. Overexpression of CCN2 has been documented in various cardiovascular pathologies, wherein it may exert either deleterious or protective effects depending on the pathological context, thereby suggesting that its role in the cardiovascular system is not yet fully elucidated. In this study, we aimed to investigate the effects of Ccn2 gene deletion on the progression of acute cardiac injury induced by doxorubicin (DOX), a widely utilized chemotherapeutic agent. To this end, we employed conditional knockout (KO) mice for the Ccn2 gene (CCN2-KO), which were administered DOX and compared to DOX-treated wild-type (WT) control mice. Our findings demonstrated that the ablation of CCN2 ameliorated DOX-induced cardiac dysfunction, as evidenced by improvements in ejection fraction (EF) and fractional shortening (FS) of the left ventricle. Furthermore, DOX-treated CCN2-KO mice exhibited a significant reduction in the gene expression and activation of oxidative stress markers (Hmox1 and Nfe2l2/NRF2) relative to DOX-treated WT controls. Additionally, the deletion of Ccn2 markedly attenuated DOX-induced cardiac fibrosis. Collectively, these results suggest that CCN2 plays a pivotal role in the pathogenesis of DOX-mediated cardiotoxicity by modulating oxidative stress and fibrotic pathways. These findings provide a novel avenue for future investigations to explore the therapeutic potential of targeting CCN2 in the prevention of DOX-induced cardiac dysfunction. Full article

Congenital proximal renal tubular acidosis (pRTA) is a rare systemic disease caused by mutations in the SLC4A4 gene that encodes the electrogenic sodium bicarbonate cotransporter, NBCe1. The major NBCe1 protein variants are designated NBCe1-A, NBCe1-B, and NBCe1-C. NBCe1-A expression is kidney-specific, NBCe1-B is broadly expressed and is the only NBCe1 variant expressed in the heart, and NBCe1-C is a splice variant of NBCe1-B that is expressed in the brain. No cardiac manifestations have been reported from patients with pRTA, but studies in adult rats with virally induced reduction in cardiac NBCe1-B expression indicate that NBCe1-B loss leads to cardiac hypertrophy and prolonged QT intervals in rodents. NBCe1-null mice die shortly after weaning, so the consequence of congenital, global NBCe1 loss on the heart is unknown. To circumvent this issue, we characterized the cardiac function of NBCe1-B/C-null (KO_b/c) mice that survive up to 2 months of age and which, due to the uninterrupted expression of NBCe1-A, do not exhibit the confounding acidemia of the globally null mice. In contrast to the viral knockdown model, cardiac hypertrophy was not present in KO_b/c mice as assessed by heart-weight-to-body-weight ratios and cardiomyocyte cross-sectional area. However, echocardiographic analysis revealed reduced left ventricular ejection fraction, and intraventricular pressure–volume measurements demonstrated reduced load-independent contractility. We also observed increased QT length variation in KO_b/c mice. Finally, using the calcium indicator Fura-2 AM, we observed a significant reduction in the amplitude of Ca²⁺ transients in paced KO_b/c cardiomyocytes. These data indicate that congenital, global absence of NBCe1-B/C leads to impaired cardiac contractility and increased QT length variation in juvenile mice. It remains to be determined whether the cardiac phenotype in KO_b/c mice is influenced by the absence of NBCe1-B/C from neuronal and endocrine tissues. Full article

Photoreceptors in the mammalian retina convert light signals into electrical and molecular signals through phototransduction and transfer the visual inputs to second-order neurons via specialized ribbon synapses. Two kinds of photoreceptors, rods and cones, possess distinct morphology and function. Currently, we have limited knowledge about rod versus (vs.) cone synapse development and the associated genes. The transcription factor neural retina leucine zipper (NRL) determines the rod vs. cone photoreceptor cell fate and is critical for rod differentiation. Nrl knockout mice fail to form rods, generating all cone or S-cone-like (SCL) photoreceptors in the retina, whereas ectopic expression of Nrl using a cone-rod homeobox (Crx) promoter (CrxpNrl) forms all rods. Here, we examined rod and cone pre-synapse development, including axonal elongation, terminal shaping, and synaptic lamination in the outer plexiform layer (OPL) in the presence or absence of Nrl. We show that NRL loss and knockdown result in delayed OPL maturation and plasticity with aberrant dendrites of bipolar neurons. The integrated analyses of the transcriptome in developing rods and SCLs with NRL CUT&RUN and synaptic gene ontology analyses identified G protein subunit beta (Gnb) 1 and p21 (RAC1) activated kinase 5 (Pak5 or Pak7) transcripts were upregulated in developing rods and down-regulated in developing SCLs. Notably, Gnb1 and Gnb5 are rod dominant, and Gnb3 is enriched in cones. NRL binds to the genes of Gnb1, Gnb3, and Gnb5. NRL also regulates pre-synapse ribbon genes, and their expression is altered in rods and SCLs. Our study of histological and gene analyses provides new insights into the morphogenesis of photoreceptor pre-synapse development and regulation of associated genes in the developing retina. Full article

Hypercholesterolemia forms the background of several cardiovascular pathologies. LDL receptor-knockout (LDLR-KO) mice kept on a high-fat diet (HFD) develop high cholesterol levels and atherosclerosis (AS). Cannabinoid type 1 receptors (CB₁Rs) induce vasodilation, although their role in cardiovascular pathologies is still controversial. We aimed to reveal the effects of CB₁Rs on vascular function and remodeling in hypercholesterolemic AS-prone LDLR-KO mice. Experiments were performed on a newly established LDLR and CB₁R double-knockout (KO) mouse model, in which KO and wild-type (WT) mice were kept on an HFD or a control diet (CD) for 5 months. The vascular functions of abdominal aorta rings were tested with wire myography. The vasorelaxation effects of acetylcholine (Ach, 1 nM–1 µM) were obtained after phenylephrine precontraction, which was repeated with inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX), Nω-nitro-L-arginine (LNA), and indomethacin (INDO), respectively. Blood pressure was measured with the tail-cuff method. Immunostaining of endothelial NOS (eNOS) was carried out. An HFD significantly elevated the cholesterol levels in the LDLR-KO mice more than in the corresponding WT mice (mean values: 1039 ± 162 mg/dL vs. 91 ± 18 mg/dL), and they were not influenced by the presence of the CB₁R gene. However, with the defect of the CB₁R gene, damage to the Ach relaxation ability was moderated. The blood pressure was higher in the LDLR-KO mice compared to their WT counterparts (systolic/diastolic values: 110/84 ± 5.8/6.8 vs. 102/80 ± 3.3/2.5 mmHg), which was significantly elevated with an HFD (118/96 ± 1.9/2 vs. 100/77 ± 3.4/3.1 mmHg, p < 0.05) but attenuated in the CB₁R-KO HFD mice. The expression of eNOS was depressed in the HFD WT mice compared to those on the CD, but it was augmented if CB₁R was knocked out. This newly established double-knockout mouse model provides a tool for studying the involvement of CB₁Rs in the development of hypercholesterolemia and atherosclerosis. Our results indicate that knocking out the CB₁R gene significantly attenuates vascular damage in hypercholesterolemic mice. Full article

Complement component 3 (C3) deficiency has recently been reported as one of the novel causes of constipation. To identify a unique gene specific to constipation caused by C3 deficiency, the total RNA extracted from the mid colon of C3 knockout (C3 KO) mice was hybridized to oligonucleotide microarrays, and the function of the candidate gene was verified in in vitro and in vivo models. C3 KO mice used for microarrays showed definite phenotypes of constipation. Overall, compared to the wild type (WT), 1237 genes were upregulated, and 1292 genes were downregulated in the C3 KO mice. Of these, the major genes included were lysine (K)-specific demethylase 5D (KDM5D), olfactory receptor 870 (Olfr870), pancreatic lipase (PNLIP), and alkaline phosphatase intestinal (ALPI). Specifically, the ALPI gene was selected as a novel gene candidate based on alterations during loperamide (Lop)-induced constipation and intestinal bowel disease (IBD). The upregulation of ALPI expression treated with acetate recovered the expression level of mucin-related genes in primary epithelial cells of C3 KO mice as well as most phenotypes of constipation in C3 KO mice. These results indicate that ALPI plays an important role as the novel gene associated with C3 deficiency-induced constipation. Full article

Angiotensin converting enzyme 2 (ACE2) presents pleiotropic actions. It hydrolyzes angiotensin I (AngI) and angiotensin II (AngII) into angiotensin-(1-9) (Ang-(1-9)) and angiotensin-(1-7) (Ang-(1-7)), respectively, as well as participates in tryptophan uptake in the gut and in COVID-19 infection. Our aim was to investigate the metabolic effect of ACE2 deletion in young adults and elderly mice under conditions of high calorie intake. Male C57Bl/6 (WT) and ACE2-deficient (ACE2^-/y) mice were analyzed at the age of 6 and 12 months under standard diet (StD) and high-fat diet (HFD). Under StD, ACE2^-/y showed lower body weight and fat depots, improved glucose tolerance, enhanced insulin sensitivity, higher adiponectin, and lower leptin levels compared to WT. This difference was even more pronounced after HFD in 6-month-old mice, but, interestingly, it was blunted at the age of 12 months. ACE2^-/y presented a decrease in adipocyte diameter and lipolysis, which reflected in the upregulation of lipid metabolism in white adipose tissue through the increased expression of genes involved in lipid regulation. Under HFD, both food intake and total energy expenditure were decreased in 6-month-old ACE2^-/y mice, accompanied by an increase in liquid intake, compared to WT mice, fed either StD or HFD. Thus, ACE2^-/y mice are less susceptible to HFD-induced obesity in an age-dependent manner, as well as represent an excellent animal model of human lipodystrophy and a tool to investigate new treatments. Full article

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is a critical enzyme that regulates nitric oxide (NO) signaling through the degradation of asymmetric dimethylarginine (ADMA). Previous studies have revealed a link between the beneficial effects of aerobic exercise and the upregulation of DDAH1 in bones and hearts. We previously reported that skeletal muscle DDAH1 plays a protective role in cardiotoxin (CTX)-induced skeletal muscle injury and regeneration. To determine the effects of aerobic exercise on CTX-induced skeletal muscle injury and the role of DDAH1 in this process, wild-type (WT) mice and skeletal muscle-specific Ddah1-knockout (Ddah1^MKO) mice were subjected to swimming training for 8 weeks and then injected with CTX. In WT mice, swimming training for 8 weeks significantly promoted skeletal muscle regeneration and attenuated inflammation, oxidative stress, and apoptosis in the gastrocnemius (GA) muscle after CTX injection. These phenomena were associated with increases in the protein expression of PAX7, myogenin, MEF2A, eNOS, SOD2, and peroxiredoxin 5 and decreases in iNOS expression in GA muscles. Swimming training also decreased serum ADMA levels and increased serum nitrate/nitrite (NOx) levels and skeletal muscle DDAH1 expression. Interestingly, swimming training in Ddah1^MKO mice had no obvious effect on CTX-induced skeletal muscle injury or regeneration and did not repress the CTX-induced inflammatory response, superoxide generation, or apoptosis. In summary, our data suggest that DDAH1 is important for the protective effect of aerobic exercise on skeletal muscle injury and regeneration. Full article