Targeting monocytic Occludin impairs transendothelial migration and HIV neuroinvasion.

Transmigration of circulating monocytes from the bloodstream to tissues represents an early hallmark of inflammation. This process plays a pivotal role during viral neuroinvasion, encephalitis, and HIV-associated neurocognitive disorders. How monocytes locally unzip endothelial tight junction-associated proteins (TJAPs), without perturbing impermeability, to reach the central nervous system remains poorly understood. Here, we show that human circulating monocytes express the TJAP Occludin (OCLN) to promote transmigration through endothelial cells. We found that human monocytic OCLN (hmOCLN) clusters at monocyte-endothelium interface, while modulation of hmOCLN expression significantly impacts monocyte transmigration. Furthermore, we designed OCLN-derived peptides targeting its extracellular loops (EL) and show that transmigration of treated monocytes is inhibited in vitro and in zebrafish embryos, while preserving vascular integrity. Monocyte transmigration toward the brain is an important process for HIV neuroinvasion and we found that the OCLN-derived peptides significantly inhibit HIV dissemination to cerebral organoids. In conclusion, our study identifies an important role for monocytic OCLN during transmigration and provides a proof-of-concept for the development of mitigation strategies to prevent monocyte infiltration and viral neuroinvasion.

Tight junction protein occludin is an internalization factor for SARS-CoV-2 infection and mediates virus cell-to-cell transmission.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads efficiently by spike-mediated, direct cell-to-cell transmission. However, the underlying mechanism is poorly understood. Herein, we demonstrate that the tight junction protein occludin (OCLN) is critical to this process. SARS-CoV-2 infection alters OCLN distribution and expression and causes syncytium formation that leads to viral spread. OCLN knockdown fails to alter SARS-CoV-2 binding but significantly lowers internalization, syncytium formation, and transmission. OCLN overexpression also has no effect on virus binding but enhances virus internalization, cell-to-cell transmission, and replication. OCLN directly interacts with the SARS-CoV-2 spike, and the endosomal entry pathway is involved in OCLN-mediated cell-to-cell fusion rather than in the cell surface entry pathway. All SARS-CoV-2 strains tested (prototypic, alpha, beta, gamma, delta, kappa, and omicron) are dependent on OCLN for cell-to-cell transmission, although the extent of syncytium formation differs between strains. We conclude that SARS-CoV-2 utilizes OCLN as an internalization factor for cell-to-cell transmission.

Roles of epidermal growth factor receptor, claudin-1 and occludin in multi-step entry of hepatitis C virus into polarized hepatoma spheroids.

The multi-step process of hepatitis C virus (HCV) entry is facilitated by various host factors, including epidermal growth factor receptor (EGFR) and the tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), which are thought to function at later stages of the HCV entry process. Using single particle imaging of HCV infection of polarized hepatoma spheroids, we observed that EGFR performs multiple functions in HCV entry, both phosphorylation-dependent and -independent. We previously observed, and in this study confirmed, that EGFR is not required for HCV migration to the tight junction. EGFR is required for the recruitment of clathrin to HCV in a phosphorylation-independent manner. EGFR phosphorylation is required for virion internalization at a stage following the recruitment of clathrin. HCV entry activates the RAF-MEK-ERK signaling pathway downstream of EGFR phosphorylation. This signaling pathway regulates the sorting and maturation of internalized HCV into APPL1- and EEA1-associated early endosomes, which form the site of virion uncoating. The tight junction proteins, CLDN1 and OCLN, function at two distinct stages of HCV entry. Despite its appreciated function as a "late receptor" in HCV entry, CLDN1 is required for efficient HCV virion accumulation at the tight junction. Huh-7.5 cells lacking CLDN1 accumulate HCV virions primarily at the initial basolateral surface. OCLN is required for the late stages of virion internalization. This study produced further insight into the unusually complex HCV endocytic process.

Occludin is a target of Src kinase and promotes lipid secretion by binding to BTN1a1 and XOR.

Lipid droplets (LDs) have increasingly been recognized as an essential organelle for eukaryotes. Although the biochemistry of lipid synthesis and degradation is well characterized, the regulation of LD dynamics, including its formation, maintenance, and secretion, is poorly understood. Here, we report that mice lacking Occludin (Ocln) show defective lipid metabolism. We show that LDs were larger than normal along its biogenesis and secretion pathway in Ocln null mammary cells. This defect in LD size control did not result from abnormal lipid synthesis or degradation; rather, it was because of secretion failure during the lactation stage. We found that OCLN was located on the LD membrane and was bound to essential regulators of lipid secretion, including BTN1a1 and XOR, in a C-terminus-dependent manner. Finally, OCLN was a phosphorylation target of Src kinase, whose loss causes lactation failure. Together, we demonstrate that Ocln is a downstream target of Src kinase and promotes LD secretion by binding to BTN1a1 and XOR.

Small noncoding vault RNA2-1 disrupts gut epithelial barrier function via interaction with HuR.

Vault RNAs (vtRNAs) are small noncoding RNAs and highly expressed in many eukaryotes. Here, we identified vtRNA2-1 as a novel regulator of the intestinal barrier via interaction with RNA-binding protein HuR. Intestinal mucosal tissues from patients with inflammatory bowel diseases and from mice with colitis or sepsis express increased levels of vtRNAs relative to controls. Ectopically expressed vtRNA2-1 decreases the levels of intercellular junction (IJ) proteins claudin 1, occludin, and E-cadherin and causes intestinal epithelial barrier dysfunction in vitro, whereas vtRNA2-1 silencing promotes barrier function. Increased vtRNA2-1 also decreases IJs in intestinal organoid, inhibits epithelial renewal, and causes Paneth cell defects ex vivo. Elevating the levels of tissue vtRNA2-1 in the intestinal mucosa increases the vulnerability of the gut barrier to septic stress in mice. vtRNA2-1 interacts with HuR and prevents HuR binding to claudin 1 and occludin mRNAs, thus decreasing their translation. These results indicate that vtRNA2-1 impairs intestinal barrier function by repressing HuR-facilitated translation of claudin 1 and occludin.

Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle.

Occludin is a tetramembrane-spanning tight junction protein. The long C-terminal cytoplasmic domain, which represents nearly half of occludin sequence, includes a distal bundle of three α-helices that mediates interactions with other tight junction components. A short unstructured region just proximal to the α-helical bundle is a phosphorylation hotspot within which S408 phosphorylation acts as molecular switch that modifies tight junction protein interactions and barrier function. Here, we used NMR to define the effects of S408 phosphorylation on intramolecular interactions between the unstructured region and the α-helical bundle. S408 pseudophosphorylation affected conformation at hinge sites between the three α-helices. Further studies using paramagnetic relaxation enhancement and microscale thermophoresis indicated that the unstructured region interacts with the α-helical bundle. These interactions between the unstructured domain are enhanced by S408 phosphorylation and allow the unstructured region to obstruct the binding site, thereby reducing affinity of the occludin tail for zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation attenuates intramolecular interactions, exposes the binding site, and increases the affinity of occludin binding to ZO-1. Consistent with an increase in binding to ZO-1, intravital imaging and fluorescence recovery after photobleaching (FRAP) analyses of transgenic mice demonstrated increased tight junction anchoring of enhanced green fluorescent protein (EGFP)-tagged nonphosphorylatable occludin relative to wild-type EGFP-occludin. Overall, these data define the mechanisms by which S408 phosphorylation modifies occludin tail conformation to regulate tight junction protein interactions and paracellular permeability.

Hypoxia impairs urothelial barrier function by inhibiting the expression of tight junction proteins in SV-HUC-1 cells.

Hypoxia plays an important role in the pathological process of bladder outlet obstruction. Previous research has mostly focused on the dysfunction of bladder smooth muscle cells, which are directly related to bladder contraction. This study delves into the barrier function changes of the urothelial cells under exposure to hypoxia. Results indicated that after a 5-day culture, SV-HUC-1 formed a monolayer and/or bilayer of cell sheets, with tight junction formation, but no asymmetrical unit membrane was observed. qPCR and western blotting revealed the expression of TJ-associated proteins (occludin, claudin1 and ZO-1) was significantly decreased in the hypoxia group in a time-dependent manner. No expression changes were observed in uroplakins. When compared to normoxic groups, immunofluorescent staining revealed a reduction in the expression of TJ-associated proteins in the hypoxia group. Transepithelial electrical resistance (TEER) revealed a statistically significant decrease in resistance in the hypoxia group. Fluorescein isothiocyanate-conjugated dextran assay was inversely proportional to the results of TEER. Taken together, hypoxia down-regulates the expression of TJ-associated proteins and breaks tight junctions, thus impairing the barrier function in human urothelial cells.

Gingipain-carrying outer membrane vesicles from Porphyromonas gingivalis cause barrier dysfunction of Caco-2 cells by releasing gingipain into the cytosol.

Ingestion of Porphyromonas gingivalis, a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis, affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases.

Hepatitis C Virus Disrupts Annexin 5-Mediated Occludin Integrity through Downregulation of Protein Kinase Cα (PKCα) and PKCη Expression, Thereby Promoting Viral Propagation.

Annexins (ANXs) comprise a family of calcium- and phospholipid-binding proteins and are implicated in the hepatitis C virus (HCV) life cycle. Here, we demonstrate a novel role of ANX5 in the HCV life cycle. Comparative analysis by quantitative PCR in human hepatoma cells revealed that ANX2, ANX4, and ANX5 were highly expressed among the ANX family proteins. Knockdown of ANX5 mRNA resulted in marked enhancement of HCV RNA replication but had no effect on either HCV translation or assembly. Using the HCV pseudoparticle (HCVpp) system, we observed enhancement of HCVpp infectivity in ANX5 knockdown Huh-7OK1 cells, suggesting that ANX5 is involved in suppression of HCV entry. Additionally, we observed that subcellular localizations of tight-junction proteins, such as claudin 1 (CLDN1) and occludin (OCLN), were disrupted in the ANX5 knockdown cells. It was reported that HCV infection was facilitated by disruption of OCLN distribution and that proper distribution of OCLN was regulated by its phosphorylation. Knockdown of ANX5 resulted in a decrease of OCLN phosphorylation, thereby disrupting OCLN distribution and HCV infection. Further analysis revealed that protein kinase C (PKC) isoforms, including PKCα and PKCη, play important roles in the regulation of ANX5-mediated phosphorylation and distribution of OCLN and in the restriction of HCV infection. HCV infection reduced OCLN phosphorylation through the downregulation of PKCα and PKCη expression. Taken together, these results suggest that ANX5, PKCα, and PKCη contribute to restriction of HCV infection by regulating OCLN integrity. We propose a model that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting HCV propagation. IMPORTANCE Host cells have evolved host defense machinery to restrict viral infection. However, viruses have evolved counteracting strategies to achieve their infection. In the present study, we obtained results suggesting that ANX5 and PKC isoforms, including PKCα and PKCη, contribute to suppression of HCV infection by regulating the integrity of OCLN. The disruption of OCLN integrity increased HCV infection. We also found that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting viral infection. We propose that HCV disrupts ANX5-mediated OCLN integrity to establish a persistent infection. The disruption of tight-junction assembly may play important roles in the progression of HCV-related liver diseases.

Glaesserella parasuis serotype 5 breaches the porcine respiratory epithelial barrier by inducing autophagy and blocking the cell membrane Claudin-1 replenishment.

Glaesserella parasuis (G. parasuis), the primary pathogen of Glässer's disease, colonizes the upper respiratory tract and can break through the epithelial barrier of the respiratory tract, leading to lung infection. However, the underlying mechanisms for this adverse effect remain unclear. The G. parasuis serotype 5 SQ strain (HPS5-SQ) infection decreased the integrity of piglets' lung Occludin and Claudin-1. Autophagy regulates the function of the epithelial barrier and tight junction proteins (TJs) expression. We tested the hypothesis that HPS5-SQ breaking through the porcine respiratory epithelial barrier was linked to autophagy and Claudin-1 degradation. When HPS5-SQ infected swine tracheal epithelial cells (STEC), autophagosomes encapsulated, and autolysosomes degraded oxidatively stressed mitochondria covered with Claudin-1. Furthermore, we found that autophagosomes encapsulating mitochondria resulted in cell membrane Claudin-1 being unable to be replenished after degradation and damaged the respiratory tract epithelial barrier. In conclusion, G. parasuis serotype 5 breaks through the porcine respiratory epithelial barrier by inducing autophagy and interrupting cell membrane Claudin-1 replenishment, clarifying the mechanism of the G. parasuis infection and providing a new potential target for drug design and vaccine development.

Attenuated replication and damaging effects of SARS-CoV-2 Omicron variants in an intestinal epithelial barrier model.

Many COVID-19 patients suffer from gastrointestinal symptoms and impaired intestinal barrier function is thought to play a key role in Long COVID. Despite its importance, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on intestinal epithelia is poorly understood. To address this, we established an intestinal barrier model integrating epithelial Caco-2 cells, mucus-secreting HT29 cells and Raji cells. This gut epithelial model allows efficient differentiation of Caco-2 cells into microfold-like cells, faithfully mimics intestinal barrier function, and is highly permissive to SARS-CoV-2 infection. Early strains of SARS-CoV-2 and the Delta variant replicated with high efficiency, severely disrupted barrier function, and depleted tight junction proteins, such as claudin-1, occludin, and ZO-1. In comparison, Omicron subvariants also depleted ZO-1 from tight junctions but had fewer damaging effects on mucosal integrity and barrier function. Remdesivir, the fusion inhibitor EK1 and the transmembrane serine protease 2 inhibitor Camostat inhibited SARS-CoV-2 replication and thus epithelial barrier damage, while the Cathepsin inhibitor E64d was ineffective. Our results support that SARS-CoV-2 disrupts intestinal barrier function but further suggest that circulating Omicron variants are less damaging than earlier viral strains.

Thrombolytic tPA-induced hemorrhagic transformation of ischemic stroke is mediated by PKCß phosphorylation of occludin.

The current standard of care for moderate to severe ischemic stroke is thrombolytic therapy with tissue plasminogen activator (tPA). Treatment with tPA can significantly improve neurologic outcomes; however, thrombolytic therapy is associated with an increased risk of intracerebral hemorrhage (ICH). The risk of hemorrhage significantly limits the use of thrombolytic therapy, and identifying pathways induced by tPA that increase this risk could provide new therapeutic options to extend thrombolytic therapy to a wider patient population. Here, we investigate the role of protein kinase Cß (PKCß) phosphorylation of the tight junction protein occludin during ischemic stroke and its role in cerebrovascular permeability. We show that activation of this pathway by tPA is associated with an increased risk of ICH. Middle cerebral artery occlusion (MCAO) increased phosphorylation of occludin serine 490 (S490) in the ischemic penumbra in a tPA-dependent manner, as tPA-/- mice were significantly protected from MCAO-induced occludin phosphorylation. Intraventricular injection of tPA in the absence of ischemia was sufficient to induce occludin phosphorylation and vascular permeability in a PKCß-dependent manner. Blocking occludin phosphorylation, either by targeted expression of a non-phosphorylatable form of occludin (S490A) or by pharmacologic inhibition of PKCß, reduced MCAO-induced permeability and improved functional outcome. Furthermore, inhibiting PKCß after MCAO prevented ICH associated with delayed thrombolysis. These results show that PKCß phosphorylation of occludin is a downstream mediator of tPA-induced cerebrovascular permeability and suggest that PKCß inhibitors could improve stroke outcome and prevent ICH associated with delayed thrombolysis, potentially extending the window for thrombolytic therapy in stroke.

Astragalus Polysaccharide improves immunogenicity of influenza vaccine as well as modulate gut microbiota in BALB/c mice.

BACKGROUND: Vaccination is the best way to prevent influenza virus infection, and insufficient antibodies make it difficult to resist influenza virus invasion. Astragalus Polysaccharide (APS) has a boosting effect on immunity, so we evaluate the effect of APS as an immune adjuvant for H1N1 influenza vaccines in this study. METHODS: The mice were immunized twice with influenza A (H1N1) vaccine and APS. Subsequently, the serum antibody levels were assessed using enzyme-linked immunosorbent assay (ELISA). The frequency of peripheral immune T cells was determined by flow cytometry. Following this, the immunized mice were exposed to a lethal dose of the virus, and changes in body weight and survival rates were recorded. Hematoxylin-eosin staining was employed to observe pathological alterations in lung and intestinal tissues. Western blot analysis was conducted to detect the expression of intestinal barrier function proteins (Occludin and Claudin-1). ELISA was utilized to measure the expression level of serum inflammatory cytokine TNF-α. Fresh mouse feces were collected after the initial immunization as well as after viral infection for 16S rRNA analysis aimed at detecting alterations in gut microbiota. RESULTS: Compared to the Hemagglutinin (HA) group, the APS group demonstrated higher levels of immunoglobulin G (IgG), IgG1, and IgG3, as well as neutralizing antibody levels. Additionally, it increased the frequency of CD8+ cells to enhance resistance against lethal infection. On day 14 post-infection, the high-dose APS group exhibited a higher survival rate (71.40 %) compared to the HA group (14.28 %), along with faster weight recovery. Furthermore, APS was found to ameliorate alveolar damage in lung tissue and rectify intestinal structural disorder. It also upregulated the expression levels of tight junction proteins Occludin and Claudin-1 in intestinal tissue while reducing serum TNF-α expression levels. In addition, populations of Colidextribacter, Peptococcaceae, and Ruminococcaceae were the dominant gut microbiota in the APS group after viral infection. CONCLUSION: APS has an immune-enhancing effect and is expected to be a novel adjuvant in the H1N1 influenza vaccine.

Contribution of platelets to disruption of the blood-brain barrier during arterial baroreflex dysfunction.

BACKGROUND: Arterial baroreflex dysfunction, like many other central nervous system disorders, involves disruption of the blood-brain barrier, but what causes such disruption in ABR dysfunction is unclear. Here we explored the potential role of platelets in this disruption. METHODS: ABR dysfunction was induced in rats using sinoaortic denervation, and the effects on integrity of the blood-brain barrier were explored based on leakage of Evans blue or FITC-dextran, while the effects on expression of CD40L in platelets and of key proteins in microvascular endothelial cells were explored using immunohistochemistry, western blotting and enzyme-linked immunosorbent assay. Similar experiments were carried out in rat brain microvascular endothelial cell line, which we exposed to platelets taken from rats with ABR dysfunction. RESULTS: Sinoaortic denervation permeabilized the blood-brain barrier and downregulated zonula occludens-1 and occludin in rat brain, while upregulating expression of CD40L on the surface of platelets and stimulating platelet aggregation. Similar effects of permeabilization and downregulation were observed in healthy rats that received platelets from animals with ABR dysfunction, and in rat brain microvascular endothelial cells, but only in the presence of lipopolysaccharide. These effects were associated with activation of NF-κB signaling and upregulation of matrix metalloprotease-9. These effects of platelets from animals with ABR dysfunction were partially blocked by neutralizing antibody against CD40L or the platelet inhibitor clopidogrel. CONCLUSION: During ABR dysfunction, platelets may disrupt the blood-brain barrier when CD40L on their surface activates NF-kB signaling within cerebral microvascular endothelial cells, leading to upregulation of matrix metalloprotease-9. Our findings imply that targeting CD40L may be effective against cerebral diseases involving ABR dysfunction.

TRPA1 protects mice from pathogenic Citrobacter rodentium infection via maintaining the colonic epithelial barrier function.

Transient receptor potential ankyrin 1 (TRPA1) is expressed in gastrointestinal tract and plays important roles in intestinal motility and visceral hypersensitivity. However, the potential role of TRPA1 in host defense, particularly against intestinal pathogens, is unknown. Here, we show that Trpa1 knockout mice exhibited increased susceptibility to Citrobacter rodentium infection, associated with the increased severity of diarrhea and intestinal permeability associated with the disrupted tight junctions (TJs) in colonic epithelia. We further demonstrated the expression of TRPA1 in murine colonic epithelial cells (CECs) and human epithelial Caco-2 cells both at protein level and transcription level. Using calcium imaging, TRPA1 agonists allyl isothiocyanates (AITC) and hydrogen peroxide were observed to induce a transient Ca2+ response in Caco-2 cells, respectively. Moreover, TRPA1 knockdown in Caco-2 cells resulted in the decreased expression of TJ proteins, ZO-1 and Occludin, and in the increased paracellular permeabilities and the reduced TEER values of Caco-2 monolayers in vitro. Furthermore, inhibition of TRPA1 by HC-030031 in the confluent Caco-2 cells caused the altered distribution and expression of TJ proteins, ZO-1, Occludin, and Claudin-3, and exacerbated the bacterial endotoxin lipopolysaccharide (LPS)-induced damage to these TJ proteins and actin cytoskeleton. By contrast, AITC pretreatment restored the distribution and expression of these TJ proteins in the confluent Caco-2 cells upon LPS challenge. Our results identify an unrecognized protective role of TRPA1 in host defense against an enteric bacterial pathogen by maintaining colonic epithelium barrier function, at least in part, via preserving the distribution and expression of TJ proteins in CECs.

Interleukin-23-Independent IL-17 Production Regulates Intestinal Epithelial Permeability.

Whether interleukin-17A (IL-17A) has pathogenic and/or protective roles in the gut mucosa is controversial and few studies have analyzed specific cell populations for protective functions within the inflamed colonic tissue. Here we have provided evidence for IL-17A-dependent regulation of the tight junction protein occludin during epithelial injury that limits excessive permeability and maintains barrier integrity. Analysis of epithelial cells showed that in the absence of signaling via the IL-17 receptor adaptor protein Act-1, the protective effect of IL-17A was abrogated and inflammation was enhanced. We have demonstrated that after acute intestinal injury, IL-23R(+) γδ T cells in the colonic lamina propria were the primary producers of early, gut-protective IL-17A, and this production of IL-17A was IL-23 independent, leaving protective IL-17 intact in the absence of IL-23. These results suggest that IL-17-producing γδ T cells are important for the maintenance and protection of epithelial barriers in the intestinal mucosa.

Endothelial BACE1 Impairs Cerebral Small Vessels via Tight Junctions and eNOS.

BACKGROUND: Cerebral small vessel injury, including loss of endothelial tight junctions, endothelial dysfunction, and blood-brain barrier breakdown, is an early and typical pathology for Alzheimer's disease, cerebral amyloid angiopathy, and hypertension-related cerebral small vessel disease. Whether there is a common mechanism contributing to these cerebrovascular alterations remains unclear. Studies have shown an elevation of BACE1 (ß-site amyloid precursor protein cleaving enzyme 1) in cerebral vessels from cerebral amyloid angiopathy or Alzheimer's disease patients, suggesting that vascular BACE1 may involve in cerebral small vessel injury. METHODS: To understand the contribution of vascular BACE1 to cerebrovascular impairments, we combined cellular and molecular techniques, mass spectrometry, immunostaining approaches, and functional testing to elucidate the potential pathological mechanisms. RESULTS: We observe a 3.71-fold increase in BACE1 expression in the cerebral microvessels from patients with hypertension. Importantly, we discover that an endothelial tight junction protein, occludin, is a completely new substrate for endothelial BACE1. BACE1 cleaves occludin with full-length occludin reductions and occludin fragment productions. An excessive cleavage by elevated BACE1 induces membranal accumulation of caveolin-1 and subsequent caveolin-1-mediated endocytosis, resulting in lysosomal degradation of other tight junction proteins. Meanwhile, membranal caveolin-1 increases the binding to eNOS (endothelial nitric oxide synthase), together with raised circulating Aß (ß-amyloid peptides) produced by elevated BACE1, leading to an attenuation of eNOS activity and resultant endothelial dysfunction. Furthermore, the initial endothelial damage provokes chronic reduction of cerebral blood flow, blood-brain barrier leakage, microbleeds, tau hyperphosphorylation, synaptic loss, and cognitive impairment in endothelial-specific BACE1 transgenic mice. Conversely, inhibition of aberrant BACE1 activity ameliorates tight junction loss, endothelial dysfunction, and memory deficits. CONCLUSIONS: Our findings establish a novel and direct relationship between endothelial BACE1 and cerebral small vessel damage, indicating that abnormal elevation of endothelial BACE1 is a new mechanism for cerebral small vessel disease pathogenesis.

Occludin and collagen IV degradation mediated by the T9SS effector SspA contributes to blood-brain barrier damage in ducks during Riemerella anatipestifer infection.

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.

Docosahexaenoic acid (DHA) alleviates inflammation and damage induced by experimental colitis.

PURPOSE: Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are chronic gastrointestinal disorders associated with significant morbidity and complications. This study investigates the therapeutic potential of docosahexaenoic acid (DHA) in a trinitrobenzene sulfonic acid (TNBS) induced colitis model, focusing on inflammation, oxidative stress, and intestinal membrane permeability. METHODS: Wistar albino rats were divided into Control, Colitis, and Colitis + DHA groups (n = 8-10/group). The Colitis and Colitis + DHA groups received TNBS intrarectally, while the Control group received saline. DHA (600 mg/kg/day) or saline was administered via gavage for six weeks. Macroscopic and microscopic evaluations of colon tissues were conducted. Parameters including occludin and ZO-1 expressions, myeloperoxidase (MPO) activity, malondialdehyde (MDA), glutathione (GSH), total antioxidant status (TAS), total oxidant status (TOS), Interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) levels were measured in colon tissues. RESULTS: Colitis induction led to significantly higher macroscopic and microscopic damage scores, elevated TOS levels, reduced occludin and ZO-1 intensity, decreased mucosal thickness, and TAS levels compared to the Control group (p < 0.001). DHA administration significantly ameliorated these parameters (p < 0.001). MPO, MDA, TNF-α, and IL-6 levels were elevated in the Colitis group but significantly reduced in the DHA-treated group (p < 0.001 for MPO, MDA; p < 0.05 for TNF-α and IL-6). CONCLUSION: DHA demonstrated antioxidant and anti-inflammatory effects by reducing reactive oxygen species production, enhancing TAS capacity, preserving GSH content, decreasing proinflammatory cytokine levels, preventing neutrophil infiltration, reducing shedding in colon epithelium, and improving gland structure and mucosal membrane integrity. DHA also upregulated the expressions of occludin and ZO-1, critical for barrier function. Thus, DHA administration may offer a therapeutic strategy or supplement to mitigate colitis-induced adverse effects.

Netrin-1 protects blood-brain barrier (BBB) integrity after cerebral ischemia-reperfusion by activating the Kruppel-like factor 2 (KLF2)/occludin pathway.

Ischemia/reperfusion (I/R)-induced neural damage and neuroinflammation have been associated with pathological progression during stroke. Netrin-1 is an important member of the family of laminin-related secreted proteins, which plays an important role in governing axon elongation. However, it is unknown whether Netrin-1 possesses a beneficial role in stroke. Here, we employed the middle cerebral artery occlusion (MCAO) model to study the function of Netrin-1 in alleviating brain injuries. Our results demonstrate that Netrin-1 rescued poststroke neurological deficits and inhibited production of the inflammatory cytokines such as interleukin 6 (IL-6) and endothelial chemokine (C-X-C motif) ligand 1 (Cxcl1). Importantly, Netrin-1 protected against MCAO-induced dysfunction of the blood-brain barrier (BBB) in mice and a reduction in the expression of the tight junction (TJ) protein occludin. Additionally, we report that Netrin-1 could ameliorate oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury and prevent aggravation in endothelial monolayer permeability in bEnd.3 human brain microvascular endothelial cells (HBMVECs). Mechanistically, Netrin-1 ameliorated OGD/R-induced decrease in occludin and Kruppel-like factor 2 (KLF2) in HBMVECs. Notably, silencing of KLF2 abolished the beneficial effects of Netrin-1 in protecting endothelial permeability and occludin expression, suggesting that these effects are mediated by KLF2. In conclusion, our findings suggest that Netrin-1 could constitute a novel therapeutic strategy for ischemic stroke.