Adherens Junction Proteins Research Articles

Euphorbium compositum SN improves the innate defenses of the airway mucosal barrier network during rhinovirus infection

Abstract Background Rhinoviruses (RV) are the major cause of common colds in healthy individuals and are associated with acute exacerbations in patients with chronic lung diseases. Yet, no vaccines or effective treatment against RV are available. This study investigated the effect of Euphorbium compositum SN (ECSN6), a multicomponent, multitarget medication made from natural ingredients, on the mucosal barrier network during RV infection. Methods Mucociliary-differentiated airway epithelial cell cultures were infected with RV or sham, and treated with 20% ECSN6 or placebo twice daily. Barrier integrity was assessed by measuring transepithelial resistance (TER), permeability to inulin, and expression and localization of intercellular junctions proteins (IJ). Ciliary beat frequency (CBF), expression of pro-inflammatory cytokines, antiviral interferons and mucins, and viral load were also measured. C57BL/6 mice were infected intranasally with RV or sham and treated with 40% ECSN6 or placebo twice daily. Inflammation of sinunasal mucosa, localization of E-cadherin, viral load and mucin gene expression were determined. Results ECSN6-treated, uninfected cell cultures showed small, but significant increase in TER over placebo, which was associated with enhanced localization of E-cadherin and ZO-1 to IJ. In RV-infected cultures, treatment with ECSN6, but not placebo prevented RV-induced (1) reduction in TER, (2) dissociation of E-cadherin and ZO-1 from the IJ, (3) mucin expression, and (4) CBF attenuation. ECSN6 also decreased RV-stimulated expression of pro-inflammatory cytokines and permeability to inulin. Although ECSN6 significantly increased the expression of some antiviral type I and type III interferons, it did not alter viral load. In vivo, ECSN6 reduced RV-A1-induced moderate inflammation of nasal mucosa, beneficially affected RV-A1-induced cytokine responses and Muc5ac mRNA expression and prevented RV-caused dissociation of E-cadherin from the IJ of nasal mucosa without an effect on viral clearance. Conclusions ECSN6 prevents RV-induced airway mucosal barrier dysfunction and improves the immunological and mucociliary barrier function. ECSN6 may maintain integrity of barrier function by promoting localization of tight and adherence junction proteins to the IJ. This in turn may lead to the observed decrease in RV-induced pro-inflammatory responses in vitro. By improving the innate defenses of the airway mucosal barrier network, ECSN6 may alleviate respiratory symptoms caused by RV infections.

EphA2 regulates vascular permeability and prostate cancer metastasis via modulation of cell junction protein phosphorylation.

Prostate cancer morbidity and mortality demonstrate a need for more effective targeted therapies. One potential target is EphA2, although paradoxically, pro- and anti-oncogenic effects have been shown to be mediated by EphA2. We demonstrate that unique activating and blocking EphA2-targeting monoclonal antibodies display opposing tumor-suppressive and oncogenic properties in vivo. To further explore this complexity, we performed detailed phosphoproteomic analysis following ligand-induced EphA2 activation. Our analysis identified altered phosphorylation of 73 downstream proteins related to the PI3K/AKT/mTOR and ERK/MAPK pathways, with the majority implicated in cell junction and cytoskeletal organization, cell motility, and tumor metastasis. We demonstrate that the adapter protein SHB is an essential component in mediating the inhibition of the ERK/MAPK pathway in response to EphA2 receptor activation. Furthermore, we identify the adherence junction protein afadin as an EphA2-regulated phosphoprotein which is involved in prostate cancer migration and invasion.

Airway epithelium damage in acute respiratory distress syndrome

BackgroundThe airway epithelium (AE) fulfils multiple functions to maintain pulmonary homeostasis, among which ensuring adequate barrier function, cell differentiation and polarization, and actively transporting immunoglobulin A (IgA), the predominant mucosal immunoglobulin in the airway lumen, via the polymeric immunoglobulin receptor (pIgR). Morphological changes of the airways have been reported in ARDS, while their detailed features, impact for mucosal immunity, and causative mechanisms remain unclear. Therefore, this study aimed to assess epithelial alterations in the distal airways of patients with ARDS.MethodsWe retrospectively analyzed lung tissue samples from ARDS patients and controls to investigate and quantify structural and functional changes in the small airways, using multiplex fluorescence immunostaining and computer-assisted quantification on whole tissue sections. Additionally, we measured markers of mucosal immunity, IgA and pIgR, alongside with other epithelial markers, in the serum and the broncho-alveolar lavage fluid (BALF) prospectively collected from ARDS patients and controls.ResultsCompared to controls, airways of ARDS were characterized by increased epithelial denudation (p = 0.0003) and diffuse epithelial infiltration by neutrophils (p = 0.0005). Quantitative evaluation of multiplex fluorescence immunostaining revealed a loss of ciliated cells (p = 0.0317) a trend towards decreased goblet cells (p = 0.056), and no change regarding cell progenitors (basal and club cells), indicating altered mucociliary differentiation. Increased epithelial permeability was also shown in ARDS with a significant decrease of tight (p < 0.0001) and adherens (p = 0.025) junctional proteins. Additionally, we observed a significant decrease of the expression of pIgR, (p < 0.0001), indicating impaired mucosal IgA immunity. Serum concentrations of secretory component (SC) and S-IgA were increased in ARDS (both p < 0.0001), along other lung-derived proteins (CC16, SP-D, sRAGE). However, their BALF concentrations remained unchanged, suggesting a spillover of airway and alveolar proteins through a damaged AE.ConclusionThe airway epithelium from patients with ARDS exhibits multifaceted alterations leading to altered mucociliary differentiation, compromised defense functions and increased permeability with pneumoproteinemia.

Butyrolactone-I from marine fungi alleviates intestinal barrier damage caused by DSS through regulating Lactobacillus johnsonii and its metabolites in the intestine of mice

Butyrolactone-I (BTL-1), a secondary metabolite from the marine fungus Aspergillus terreus, exhibits numerous biological activities. Previous research has indicated that Butyrolactone-I alleviates intestinal epithelial inflammation via the TLR4/NF-κB and MAPK pathways. However, the mechanisms underlying its protection against intestinal barrier damage remain unclear. This study aims to further elucidate these mechanisms. We observed that BTL-1 administration increased the abundance of Lactobacillus johnsonii (LJ) in both in vivo and in vitro experiments, prompting an investigation into the effects of LJ and its metabolites on DSS-induced inflammatory bowel disease (IBD). The results demonstrated that BTL-1 significantly upregulated tight junction (TJ) and adherens junction (AJ) proteins, maintained intestinal barrier integrity, and alleviated DSS-induced IBD in mice. These effects were associated with the proliferation of LJ and its metabolites, such as butyric and propionic acids, and the inhibition of the MAPK signaling pathway in the colon. Interestingly, administering LJ alone produced a protective effect against DSS-induced IBD similar to that observed with BTL-1. Furthermore, butyric acid, a metabolite of LJ, also upregulated TJ/AJ proteins in intestinal epithelial cells through the MAPK signaling pathway. Our findings suggest that BTL-1 regulates intestinal flora, promotes LJ proliferation, protects intestinal barrier integrity, increases the concentrations of butyric and propionic acids, and ultimately inhibits the activation of the MAPK signaling pathway in mice to alleviate IBD. Therefore, BTL-1 could potentially be used as a natural drug to prevent IBD and maintain intestinal flora balance. ImportanceWe explored how butyrolactone-I exerts a preventive effect on IBD through intestinal bacteria (Lactobacillus johnsonii).

Tailoring N, N, N‐trimethyl chitosan nanospheres as an efficient drug carrier to overcome endothelial barrier

AbstractEndothelial barriers, integral components of tissue barriers, are densely packed with adherens junction and tight junction proteins, hindering the passage of drugs to target sites. Herein, a novel nanosphere comprising N, N, N‐trimethylated chitosan (TMC), with the size of 51 ± 8 nm and zeta potential of 13.2 ± 5.3 mV, has been engineered as a drug carrier to facilitate the transport of hydrophobic drugs, that is, curcumin, across endothelial barriers efficiently. The effects of pH value and temperature on the release kinetics of curcumin from TMC nanospheres have been studied. In addition, the transportation of TMC nanospheres across an in vitro endothelial barrier was evaluated over a 72‐h period. The integrity of the in vitro endothelial barrier was assessed before and after the transport of TMC nanospheres by using transepithelial electrical resistance measurements and immunofluorescence staining of tight junction proteins. The results reveal that TMC nanospheres significantly enhance the transport of curcumin across the endothelial barrier, with approximately 30% of curcumin released as compared to less than 3.3% across the barrier without the assistance of TMC nanospheres. This investigation underscores the potential of small‐sized TMC nanospheres to enhance the transport of hydrophobic drugs across endothelial barriers.

ENPP1 induces blood-brain barrier dysfunction and promotes brain metastasis formation in HER2-positive breast cancer.

Brain metastasis (BrM) is a devastating end-stage neurological complication that occurs in up to 50% of HER2+ breast cancer patients. Understanding how disseminating tumor cells manage to cross the blood-brain barrier (BBB) is essential for developing effective preventive strategies. We identified the ecto-nucleotidase ENPP1 as specifically enriched in the secretome of HER2+ brain metastatic cells, prompting us to explore its impact on BBB dysfunction and BrM formation. We used in vitro BBB and in vivo premetastatic mouse models to evaluate the effect of tumor-secreted ENPP1 on brain vascular permeability. BBB integrity was analyzed by real-time fluorescence imaging of 20kDa Cy7.5-dextran extravasation and immunofluorescence staining of adherens and tight junction proteins. Pro-metastatic effects of ENPP1 were evaluated in an experimental brain metastatic model. Systemically secreted ENPP1 from primary breast tumors impaired the integrity of BBB with loss of tight and adherens junction proteins early before the onset of BrM. Mechanistically, ENPP1 induced endothelial cell dysfunction by impairing insulin signaling and its downstream AKT/GSK3β/β-catenin pathway. Genetic ablation of ENPP1 from HER2+ brain metastatic cells prevented endothelial cell dysfunction and reduced metastatic burden while prolonging the overall and metastasis-free survival of mice. Furthermore, plasmatic ENPP1 levels correlate with brain metastatic burden and inversely with overall survival. We demonstrated that metastatic breast cancer cells exploit the ENPP1 signaling for cell transmigration across the BBB and brain colonization. Our data implicate ENPP1 as a potential biomarker for poor prognosis and early detection of BrM in HER2+ breast cancer.

Molecular Crosstalk Between Adherens Junction Proteins, E-cadherin and Nectin-4

Cell-cell junctions formed by the association of cell adhesion molecules facilitate physiological events necessary for growth and development of multicellular organisms. Among them, cadherins and nectins organize and assemble to form adherens junction, which thereby mechanically couples interacting cells. A detailed understanding of the crosstalk involving these cell adhesion molecules is fundamental to the study of the various developmental processes. Although, cadherins and nectins can recruit each other in the adherens junction through an interplay of cytoplasmic adaptor molecules, here, we report a direct interaction between N-terminal extracellular domains of E-cadherin and nectin-4 as demonstrated by surface plasmon resonance (SPR) and Atomic Force Microscopy (AFM)-based single molecule force spectroscopy (SMFS). Kinetic studies using SPR demonstrate the binding between the ectodomains of E-cadherin and nectin-4 with a KD of 3.7 ± 0.7 µM and KD of 5.4 ± 0.2 µM (reciprocal experiment). AFM-based SMFS experiments also support interaction between the ectodomains of E-cadherin and nectin-4 with the koff value of 31.48 ± 1.53 s−1 and the lifetime of the complex of 0.036 ± 0.0026 s. We thus propose a cell adhesion mechanism mediated by E-cadherin and nectin-4, which can have functional significance in early embryogenesis as evident from the expression pattern of both the proteins during early development.

Bta-miR-149–3p suppresses inflammatory response in bovine Sertoli cells exposed to microcystin-leucine arginine (MC-LR) through TLR4/NF-kB signaling pathway

This study explored the regulatory role of bta-miR-149–3p in the inflammatory response induced by microcystin-leucine arginine (MC-LR) exposure in bovine Sertoli cells. The research endeavored to enhance the comprehension of the epigenetic mechanisms underlying MC-LR-induced cytotoxicity in Sertoli cells and establish a foundation for mitigating these effects in vitro. In this study, we elucidated the regulatory mechanism of bta-miR-149–3p in the MC-LR-induced inflammatory response by verifying the target gene of bta-miR-149–3p through luciferase assays and treating the cells with a bta-miR-149–3p inhibitor for 24 h. The results demonstrate that nuclear factor κB (NF-κB) acts as a downstream target gene of bta-miR-149–3p, which inhibits the MC-LR-induced inflammatory response in bovine Sertoli cells. This inhibition occurs by regulating the downregulation of tight junction constitutive proteins of the blood-testis barrier (BTB) through the suppression of the TLR-4/NF-κB signaling pathway (p < 0.05) and the up-regulation of the adhesion junction protein β-catenin (p < 0.05). Notably, MC-LR exposure resulted in the up-regulation (p < 0.05) of inflammatory cytokines (IL-6, IL-1β, and NLRP3) and the down-regulation (p < 0.05) of BTB tight junction constitutive proteins (ZO-1, Occludin) in Sertoli cells. Furthermore, the BTB constitutive protein ZO-1 exhibited significant down-regulation in Sertoli cells pretreated with the bta-miR-149–3p inhibitor compared to controls (p < 0.05), while Occludin showed no significant difference from CTNNB1 (p > 0.05). In summary, our findings suggest that bta-miR-149–3p suppresses the MC-LR-induced inflammatory response and alterations in the expression of BTB proteins in bovine Sertoli cells by inhibiting the TLR-4/NF-κB signaling pathway.

Spheroid Model of Mammary Tumor Cells: Epithelial-Mesenchymal Transition and Doxorubicin Response.

Breast cancer is the most prevalent cancer among women worldwide. Therapeutic strategies to control tumors and metastasis are still challenging. Three-dimensional (3D) spheroid-type systems more accurately replicate the features of tumors in vivo, working as a better platform for performing therapeutic response analysis. This work aimed to characterize the epithelial-mesenchymal transition and doxorubicin (dox) response in a mammary tumor spheroid (MTS) model. We evaluated the doxorubicin treatment effect on MCF-7 spheroid diameter, cell viability, death, migration and proteins involved in the epithelial-mesenchymal transition (EMT) process. Spheroids were also produced from tumors formed from 4T1 and 67NR cell lines. MTSs mimicked avascular tumor characteristics, exhibited adherens junction proteins and independently produced their own extracellular matrix. Our spheroid model supports the 3D culturing of cells isolated from mice mammary tumors. Through the migration assay, we verified a reduction in E-cadherin expression and an increase in vimentin expression as the cells became more distant from spheroids. Dox promoted cytotoxicity in MTSs and inhibited cell migration and the EMT process. These results suggest, for the first time, that this model reproduces aspects of the EMT process and describes the potential of dox in inhibiting the metastatic process, which can be further explored.

MiR-625-5p is a potential therapeutic target in sepsis by regulating CXCL16/CXCR6 axis and endothelial barrier

BackgroundMicroRNA plays an important role in the progression of sepsis. We found a significant increase of in miR-625-5p expression in the blood of patients with sepsis, and lipopolysaccharide (LPS)-stimulated EA.hy926 cells. To date, little is known about the specific biological function of miR-625-5p in sepsis. MethodsChanges in miR-625-5p expression were verified through quantitative real-time polymerase chain reaction in 45 patients with sepsis or septic shock and 30 healthy subjects. In vitro, EA.hy926 cells were treated with LPS. Transendothelial electrical resistance assay and FITC-dextran were used in evaluating endothelial barrier function. ResultsHerein, patients with sepsis or septic shock had significantly higher miR-625-5p expression levels, chemokine (C-X-C motif) ligand 16 (CXCL16) levels, and glycocalyx components than the healthy controls, and miR-625-5p level was positively correlated with disease. Kaplan–Meier analysis demonstrated a strong association between miR-625-5p level and 28-day mortality. Furthermore, the miR-625-5p inhibitor significantly alleviated LPS-induced endothelial barrier injury in vitro. Then, miR-625-5p positively regulated CXCL16 and down-regulated miR-625-5p attenuated CXCL16 transcription and expression in EA.hy926 cells. CXCL16 knockout significantly alleviated vascular barrier dysfunction in the LPS-induced EA.hy926 cells. sCXCL16 treatment in EA.hy926 cells significantly increased endothelial hyperpermeability by disrupting endothelial glycocalyx, tight junction proteins, and adherens junction proteins through the modulation of C-X-C chemokine receptor type 6 (CXCR6). ConclusionsIncrease in miR-625-5p level may be an effective biomarker for predicting 28-day mortality in patients with sepsis/septic shock. miR-625-5p is a critical pathogenic factor for endothelial barrier dysfunction in LPS-induced EA.hy926 cells because it activates the CXCL16/CXCR6 axis.

IL-17A Aggravated Blood-Brain Barrier Disruption via Activating Src Signaling in Epilepsy Mice.

Inflammation is an important pathogenic driving force in the genesis and development of epilepsy. The latest researches demonstrated that IL-17A mediated blood-brain barrier (BBB) dysfunction through disruption of tight junction protein expression. To investigate whether IL-17A is involved in BBB disruption after acute seizure attack, the pilocarpine model was established with C57BL/6J (wild type, WT) and IL-17R-deficient mice in vivo and with primary cultured rat brain microvascular endothelial cells in vitro. The mortality rate and brain water content were evaluated at 24h after status epilepticus, and IL-17A concentration, endothelial tight junction, adherens junction proteins, and albumin leakage were assessed at 0h, 4h, 12h, and 24h after status epilepticus (SE). IL-17R-deficient mice showed lessen severity of epilepsy than WT mice, accompanied by less albumin leakage, reduced brain water content, decreased IL-17A, and upregulated expression of target proteins (ZO-1, Occludin and VE-cadherin). IL-17R knockout abrogated abnormal upregulation of Src kinase and phosphorylated Src kinase in the setting of SE, and Src kinase inhibitor PP1 abrogated IL-17A-induced SE related endothelial injury in vitro. In conclusion, IL-17A inhibition might be a promising therapeutic option to attenuate endothelial cell injury and further BBB disruption by reducing Src kinase activation.

MARCH family E3 ubiquitin ligases selectively target and degrade cadherin family proteins.

Cadherin family proteins play a central role in epithelial and endothelial cell-cell adhesion. The dynamic regulation of cell adhesion is achieved in part through endocytic membrane trafficking pathways that modulate cadherin cell surface levels. Here, we define the role for various MARCH family ubiquitin ligases in the regulation of cadherin degradation. We find that MARCH2 selectively downregulates VE-cadherin, resulting in loss of adherens junction proteins at cell borders and a loss of endothelial barrier function. Interestingly, N-cadherin is refractory to MARCH ligase expression, demonstrating that different classical cadherin family proteins are differentially regulated by MARCH family ligases. Using chimeric cadherins, we find that the specificity of different MARCH family ligases for different cadherins is conferred by the cadherin transmembrane domain. Further, juxta-membrane lysine residues are required for cadherin degradation by MARCH proteins. These findings expand our understanding of cadherin regulation and highlight a new role for mammalian MARCH family ubiquitin ligases in differentially regulating cadherin turnover.

Loss of Intestinal SNX27 Promotes Barrier Dysfunction and Inflammatory Responses

SNX27 belongs to the sorting nexin (SNX) family of proteins that play a crucial role in the endocytic pathway. SNX27 carries a unique PDZ domain, mediates recycling of endocytosed transmembrane proteins, and is critical for neurodevelopmental processes. However, its role in the intestine has not been explored yet. Here we aim to determine the previously unknown roles of SNX27 in regulating intestinal tissue homeostasis, epithelial barrier integrity, and related disorders. We have generated a novel mouse model of SNX27 deletion form intestinal epithelial cells (SNX27ΔIEC). We challenged the mice with DSS-induced colitis and AOM/DSS-induced colorectal cancer (CRC). We performed in vitro SNX27 loss of function studies via siRNA knockdown (KD) in SKCO15 cells. We further analyzed SNX27 expression in IBD and CRC patients via online human datasets. SNX27ΔIEC mice had shorter colon, small intestines, and heavier spleens. We found increased intestinal permeability upon SNX27 deletion. SNX27ΔIEC mice had significantly upregulated cleaved Caspase-3 (pro-apoptotic) and downregulated Bcl-xL (anti-apoptotic) protein levels. TUNEL staining also showed greater apoptotic cells in SNX27ΔIEC mice. Tight-junction proteins Claudin10 and ZO-1, as well as adherens-junction proteins ꞵ-catenin and E-cadherin, were downregulated in SNX27ΔIEC mice. The histological inflammation score and mRNA levels of pro-inflammatory cytokines IL-6 and TNF-α were higher in SNX27ΔIEC mice. Protein expression level of phospho-p65 was increased in SNX27ΔIEC mice, indicating NFkB pathway activation. Upon challenging with intestinal disorders, SNX27ΔIEC mice were more sensitive towards DSS and AOM/DSS treatments as they lost more bodyweight, had higher intestinal permeability levels, and heavier spleens. SNX27 KD in vitro reduced protein levels of PCNA and delayed wound closure in a wound healing assay. SNX27-KD cells had higher nuclear translocation of phospho-p65. Additionally, we observed cytoplasmic localization rearrangement and downregulation of ꞵ-catenin and E-cadherin, key mediators of epithelial-mesenchymal transition. Lastly, human datasets GSE11223, 6731, 8671, 21510 revealed significant downregulation of SNX27 expression in IBD and CRC patients, suggesting towards clinical relevance of SNX27 in intestinal disorders. Overall, intestinal deletion of SNX27 promotes apoptosis, inflammation, and disrupts epithelial barrier that increases susceptibility towards colonic inflammation and tumorigenesis. Our results indicate a novel role of SNX27 in regulating intestinal tissue homeostasis. Understanding the mechanisms of lower SNX27 in IBD and CRC will provide insights into new prevention and targets against these chronic diseases. We would like to acknowledge the VA Merit Award 1 I01BX004824-01, DOD CDMRP BC191198, the NIDDK/National Institutes of Health grants R01 DK105118 and R01DK114126 to Jun Sun. The study sponsors performed no role in the study design, data collection, analysis, and interpretation of data. The contents do not represent the views of the United States Department of Veterans Affairs or the United States Government. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Actin cytoskeletal proteins in mice cortical microvessels decreased with aging

Background: The actin cytoskeleton regulates many important cellular processes in the brain, including cell division and proliferation, migration, and differentiation. The interactions of actin microfilaments with plaque proteins zonula occludens (ZO)-1, -2, -3 are required ensuring the formation of a polarized brain endothelium which ultimately drives development of blood-brain barrier (BBB). In this study, we carried out an extensive evaluation of proteins involved in the structure and function of mouse brain cortical microvessels (MVs) using a discovery-based quantitative proteomic approach. Methods: Cortical MVs were isolated from equal number of age-matched, male and female, young (4–6 months), middle-aged (12–14 months), and old (20–21 months) mice obtained from Jackson Laboratory [Tg(Thy1-EGFP)MJrs/J] and bred in a C57B16J background (n = 6/group). The presence of end-arterioles, capillaries, and venules in MVs was confirmed by light microscopy and by alkaline phosphate staining. Proteomics analysis was performed using liquid chromatography/mass spectrometry. Results: We quantified 4746, 4216, and 4579 differentially expressed proteins by proteomics in brain cortical MVs of young, middle-aged, and old mice, respectively. We found that several actin cytoskeletal-related alpha actinin-1, -2, -3 (ACTN1/2/4), actin-related protein -2, -3, -3B (ACTR2/3/3B), actin-related protein 2/3 complex subunit -1A, -2, -4, -5 (ARPC1A/2/4/5), and [F-actin]-monooxygenase MICAL3 proteins were significantly downregulated in mice cortical MVs with aging. Under physiological conditions, cerebral endothelial cells are connected by tight junctions (TJs) and adherens junctions (AJs). The TJ and AJ proteins are anchored to the actin cytoskeleton by multiple accessory proteins, ZO-1, ZO-2 and ZO-3. Interestingly, we observed that several TJ, AJ, and their signaling (PKC-α/γ/ε, RHOA, RAB3 and YES1) proteins were significantly downregulated in mice cortical MVs with aging. Moreover, we previously documented that oxidative stress during aging leads to adverse protein profile changes of brain cortical MVs that affect mRNA/protein stability and ATP synthesis capacity in mice. Conclusions: We suggest that increased oxidative stress during aging leads to protein expression profile changes of brain cortical MVs that affect ATP synthesis capacity, which leads to actin cytoskeletal dysregulation, and accelerating to endothelial and BBB dysfunction in the brain microvasculature with aging. Funding: AG075988, HL148836, AG063345, AG074489, NS114286, AG047296, HL141143, HL168568, and the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Paneth cell-specific TCF4 down-regulation potentiates alcohol-induced dysbiosis of gut microbiota and tissue injury at the gut-liver axis

Introduction: Alcohol misuse results in hepatitis, pancreatitis, and neurodegenerative disorders. Alcohol consumption alters gut microbiota and increases gut permeability, leading to endotoxemia and multiple organ damage. However, the cellular and molecular mechanisms of alcohol-induced microbiota dysbiosis and barrier dysfunction remain poorly defined. Antimicrobial peptides such as α-defensins, secreted by the Paneth cells, regulate gut microbiota homeostasis. We previously reported that ethanol (EtOH) feeding reduces α-defensin expression in mice. α-Defensin expression is regulated by Wnt signaling and T-cell factor 4 (TCF4). We investigated the role of Paneth cell TCF4 in alcohol-induced microbiota dysbiosis and tissue injury at the gut-liver axis. Materials and methods: TCF4fl/wt (WT) and TCF4fl/wt-Defa6Cre (KD; heterozygous Paneth cell-specific knockout of TCF4) mice were fed a Lieber-DiCarli liquid diet with or without EtOH (0% 2d, 1% 2d, 2% 2d, 4% 1wk, 5% 1wk, &amp; 6% 1wk) (EF). Control groups were pair-fed (PF) isocaloric EtOH-free diet. Intestinal permeability was measured by the vascular-to-luminal flux of FITC-inulin (6 kDa) in vivo, and tight junction (TJ) and adherens junction (AJ) integrity were assessed by confocal microscopy for occludin, ZO-1, E-cadherin, and β-catenin. Paneth cell function and mucosal inflammation were assessed by RT-PCR for defensins and cytokines. Gut microbiota was analyzed by 16S rRNA sequencing of colonic flushing. Plasma lipopolysaccharide (LPS) and cytokines were measured to evaluate endotoxemia and systemic inflammation. Liver damage was assessed by measuring plasma AST, liver triglyceride, and cytokine mRNA. Results and conclusion: EtOH reduced TCF4 expression in the ileum in both WT and KD mice; lowest TCF4 expression was observed in KD-EF mice. Similarly, the expression of DEFA5 and DEFA6 was reduced by EtOH in WT and KD mice, with the lowest expression in KD-EF mice. EtOH-induced mucosal permeability was associated with reduced staining for TJ and AJ proteins, indicating the mucosal barrier dysfunction. EtOH increased intestinal mRNA for IL-1β, IL-6, TNFα, and MCP-1, indicating the mucosal inflammatory response. EtOH-induced gut permeability, TJ/AJ disruption, and mucosal inflammation were significantly higher in KD-EF mice than in WT-EF mice. Shannon index analysis indicated that EtOH reduced α-diversity of microbiota in KD but not in WT mice. Firmicutes: Bacteriodetes ratio and the abundance of Verrucomicrobia were reduced by EtOH in WT but not in KD mice. The abundance of Bacillus was increased by EtOH both in WT and KD mice; however, this effect was more severe in KD mice. EtOH-induced plasma LPS and cytokine elevations were higher in KD mice than in WT mice. Elevation of plasma AST, liver triglyceride, and liver cytokine mRNA indicated EtOH-induced liver damage; the damage was more severe in KD mice. These data indicate that Paneth cell TCF4 downregulation exacerbates alcohol-induced microbiota dysbiosis, gut barrier dysfunction, endotoxemia, systemic inflammation, and liver damage; suggesting that Paneth cell Wnt signal downregulation plays a crucial role in alcohol-associated organ damage. NIH/NIAA - R01AA012307, AA029270; Veterans Administration - IO1BX003014. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neurotensin modulates ovarian vascular permeability via adherens junctions.

Neurotensin (NTS) is a 13-amino acid peptide which is highly expressed in the mammalian ovary in response to the luteinizing hormone surge. Antibody neutralization of NTS in the ovulatory follicle of the cynomolgus macaque impairs ovulation and induces follicular vascular dysregulation, with excessive pooling of red blood cells in the follicle antrum. We hypothesize that NTS is an essential intrafollicular regulator of vascular permeability. In the present study, follicle injection of the NTS receptor antagonist SR142948 also resulted in vascular dysregulation. To measure vascular permeability changes invitro, primary macaque ovarian microvascular endothelial cells (mOMECs) were enriched from follicle aspirates and studied invitro. When treated with NTS, permeability of mOMECs decreased. RNA sequencing (RNA-Seq) of mOMECs revealed high mRNA expression of the permeability-regulating adherens junction proteins N-cadherin (CDH2) and K-cadherin (CDH6). Immunofluorescent detection of CDH2 and CDH6 confirmed expression and localized these cadherins to the cell-cell boundaries, consistent with function as components of adherens junctions. mOMECs did not express detectable levels of the typical vascular endothelial cadherin, VE-cadherin (CDH5) as determined by RNA-Seq, qPCR, western blot, and immunofluorescence. Knockdown of CDH2 or CDH6 via siRNA abrogated the NTS effect on mOMEC permeability. Collectively, these data suggest that NTS plays an ovulation-critical role in vascular permeability maintenance, and that CDH2 and CDH6 are involved in the permeability modulating effect of NTS on the ovarian microvasculature. NTS can be added to a growing number of angiogenic regulators which are critical for successful ovulation.

Dietary copper improves intestinal structural integrity in juvenile grass carp (Ctenopharyngodon idella) probably related to its increased intestinal antioxidant capacity and apical junction complex

This research evaluated the effects of copper (Cu) on intestinal antioxidant capacity and apical junctional complex (AJC) in juvenile grass carp. A total of 1080 healthy juvenile grass carp (11.16 ± 0.01 g) were fed six diets including different dosages of Cu, namely 0, 2, 4, 6, 8 mg/kg (Cu citrate [CuCit] as Cu source) and 3 mg/kg (CuSO4·5H2O as Cu source). The trial lasted for 9 weeks. The findings revealed that dietary optimal Cu supplementation (2.2 to 4.1 mg/kg) promoted intestinal growth, including intestinal length, intestinal length index, intestinal weight, and intestinal somatic index (P < 0.05). Furthermore, optimal Cu boosted the intestinal mucosal barrier in juvenile grass carp. On the one hand, optimal Cu reduced diamine oxidase and D-lactate levels in serum (P < 0.05), reduced levels of the oxidative damage indicators malondialdehyde, reactive oxygen species (ROS), protein carbonyl, superoxide dismutase (P < 0.05), and catalase mRNA levels were elevated (P < 0.05), thus boosting intestinal antioxidant capacity, the binding protein Keap1a/1b/Nrf2 signaling pathway might be involved. Optimal Cu had no impact on glutathione peroxidase 1b (GPx1b) gene expression (P > 0.05). On the other hand, optimal Cu increased intestinal tight junction (TJ) proteins (except for claudin 15b) and adherens junction (AJ) proteins (E-cadherin, α-catenin, β-catenin, nectin and afadin) mRNA levels (P < 0.05), which could be connected to the signaling pathway formed by the Ras homolog gene family, member A (RhoA), Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK). Finally, based on serum indicator D-lactate and intestinal oxidative damage index (ROS), Cu requirement (CuCit as Cu source) for juvenile grass carp from initial weight to final weight (from 11 to 173 g) was determined to be 4.14 and 4.12 mg/kg diet, respectively. This work may provide a theoretical foundation for identifying putative Cu regulation pathways on fish intestinal health.

Adjudin protects blood–brain barrier integrity and attenuates neuroinflammation following intracerebral hemorrhage in mice

Secondary brain injury exacerbates neurological dysfunction and neural cell death following intracerebral hemorrhage (ICH), targeting the pathophysiological mechanism of the secondary brain injury holds promise for improving ICH outcomes. Adjudin, a potential male contraceptive, exhibits neuroprotective effects in brain injury disease models, yet its impact in the ICH model remains unknown. In this study, we investigated the effects of adjudin on brain injury in a mouse ICH model and explored its underlying mechanisms. ICH was induced in male C57BL/6 mice by injecting collagenase into the right striatum. Mice received adjudin treatment (50 mg/kg/day) for 3 days before euthanization and the perihematomal tissues were collected for further analysis. Adjudin significantly reduced hematoma volume and improved neurological function compared with the vehicle group. Western blot showed that Adjudin markedly decreased the expression of MMP-9 and increased the expression of tight junctions (TJs) proteins, Occludin and ZO-1, and adherens junctions (AJs) protein VE-cadherin. Adjudin also decreased the blood–brain barrier (BBB) permeability, as indicated by the reduced albumin and Evans Blue leakage, along with a decrease in brain water content. Immunofluorescence staining revealed that adjudin noticeably reduced the infiltration of neutrophil, activation of microglia/macrophages, and reactive astrogliosis, accompanied by an increase in CD206 positive microglia/macrophages which exhibit phagocytic characteristics. Adjudin concurrently decreased the generation of proinflammatory cytokines, such as TNF-α and IL-1β. Additionally, adjudin increased the expression of aquaporin 4 (AQP4). Furthermore, adjudin reduced brain cell apoptosis, as evidenced by increased expression of anti-apoptotic protein Bcl-2, and decreased expression of apoptosis related proteins Bax, cleaved caspase-3 and fewer TUNEL positive cells. Our data suggest that adjudin protects against ICH-induced secondary brain injury and may serve as a potential neuroprotective agent for ICH treatment.

Identification of host protein ENO1 (alpha-enolase) interacting with Cryptosporidium parvum sporozoite surface protein, Cpgp40

BackgroundCryptosporidium parvum is an apicomplexan zoonotic parasite causing the diarrheal illness cryptosporidiosis in humans and animals. To invade the host intestinal epithelial cells, parasitic proteins expressed on the surface of sporozoites interact with host cells to facilitate the formation of parasitophorous vacuole for the parasite to reside and develop. The gp40 of C. parvum, named Cpgp40 and located on the surface of sporozoites, was proven to participate in the process of host cell invasion.MethodsWe utilized the purified Cpgp40 as a bait to obtain host cell proteins interacting with Cpgp40 through the glutathione S-transferase (GST) pull-down method. In vitro analysis, through bimolecular fluorescence complementation assay (BiFC) and coimmunoprecipitation (Co-IP), confirmed the solid interaction between Cpgp40 and ENO1. In addition, by using protein mutation and parasite infection rate analysis, it was demonstrated that ENO1 plays an important role in the C. parvum invasion of HCT-8 cells.ResultsTo illustrate the functional activity of Cpgp40 interacting with host cells, we identified the alpha-enolase protein (ENO1) from HCT-8 cells, which showed direct interaction with Cpgp40. The mRNA level of ENO1 gene was significantly decreased at 3 and 24 h after C. parvum infection. Antibodies and siRNA specific to ENO1 showed the ability to neutralize C. parvum infection in vitro, which indicated the participation of ENO1 during the parasite invasion of HCT-8 cells. In addition, we further demonstrated that ENO1 protein was involved in the regulation of cytoplasmic matrix of HCT-8 cells during C. parvum invasion. Functional study of the protein mutation illustrated that ENO1 was also required for the endogenous development of C. parvum.ConclusionsIn this study, we utilized the purified Cpgp40 as a bait to obtain host cell proteins ENO1 interacting with Cpgp40. Functional studies illustrated that the host cell protein ENO1 was involved in the regulation of tight junction and adherent junction proteins during C. parvum invasion and was required for endogenous development of C. parvum.Graphical

ITF2357 regulates NF-κB signaling pathway to protect barrier integrity in retinal pigment epithelial cells.

The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 μM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1β, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.