Tight Junction-associated Proteins Research Articles

Eucalyptol Inhibits Amyloid-β-Induced Barrier Dysfunction in Glucose-Exposed Retinal Pigment Epithelial Cells and Diabetic Eyes.

Hyperglycemia elicits tight junction disruption and blood-retinal barrier breakdown, resulting in diabetes-associated vison loss. Eucalyptol is a natural compound found in eucalyptus oil with diverse bioactivities. This study evaluated that eucalyptol ameliorated tight junctions and retinal barrier function in glucose/amyloid-β (Aβ)-exposed human retinal pigment epithelial (RPE) cells and in db/db mouse eyes. RPE cells were cultured in media containing 33 mM glucose or 5 μM Aβ for 4 days in the presence of 1–20 μM eucalyptol. The in vivo animal study employed db/db mice orally administrated with 10 mg/kg eucalyptol. Nontoxic eucalyptol inhibited the Aβ induction in glucose-loaded RPE cells and diabetic mouse eyes. Eucalyptol reversed the induction of tight junction-associated proteins of ZO-1, occludin-1 and matrix metalloproteinases in glucose- or Aβ-exposed RPE cells and in diabetic eyes, accompanying inhibition of RPE detachment from Bruch’s membrane. Adding eucalyptol to glucose- or Aβ-loaded RPE cells, and diabetic mouse eyes reciprocally reversed induction/activation of apoptosis-related bcl-2, bax, cytochrome C/Apaf-1 and caspases. Eucalyptol attenuated the generation of reactive oxygen species and the induction of receptor for advanced glycation end products in Aβ-exposed RPE cells and diabetic eyes. Eucalyptol may ameliorate RPE barrier dysfunction in diabetic eyes through counteracting Aβ-mediated oxidative stress-induced RPE cell apoptosis.

Improved binding of SARS-CoV-2 Envelope protein to tight junction-associated PALS1 could play a key role in COVID-19 pathogenesis

The Envelope (E) protein of SARS-CoV-2 is the most enigmatic protein among the four structural ones. Most of its current knowledge is based on the direct comparison to the SARS E protein, initially mistakenly undervalued and subsequently proved to be a key factor in the ER-Golgi localization and in tight junction disruption.We compared the genomic sequences of E protein of SARS-CoV-2, SARS-CoV and the closely related genomes of bats and pangolins obtained from the GISAID and GenBank databases. When compared to the known SARS E protein, we observed a significant difference in amino acid sequence in the C-terminal end of SARS-CoV-2 E protein.Subsequently, in silico modelling analyses of E proteins conformation and docking provide evidences of a strengthened binding of SARS-CoV-2 E protein with the tight junction-associated PALS1 protein. Based on our computational evidences and on data related to SARS-CoV, we believe that SARS-CoV-2 E protein interferes more stably with PALS1 leading to an enhanced epithelial barrier disruption, amplifying the inflammatory processes, and promoting tissue remodelling. These findings raise a warning on the underestimated role of the E protein in the pathogenic mechanism and open the route to detailed experimental investigations.

High expression of EphA2 led to secondary injury by destruction of BBB integrity though the ROCK pathway after diffuse axonal injury

Blood-brain barrier (BBB) disruption exacerbates diffuse axonal injury (DAI), but the underlying mechanisms are not fully understood. Inactivation or deletion of erythropoietin-producing hepatoma (EPH) receptor A2 (EphA2) attenuated BBB damage and promoted tight junction formation. In this study, we aimed to investigate the role of EphA2 in the protection of BBB integrity and the relevant mechanisms involved in a rat model of DAI. Blocking activation of the EphA receptor by EphA2-Fc ameliorated axonal injury, cell apoptosis, and glial activation, protected BBB integrity and increased expression of the tight junction-associated proteins ZO-1, claudin-5 and occludin-1. In vitro BBB models established by human brain microvascular endothelial cells (HBMECs) were subjected to oxygen deprivation (OGD). Treatment with EphrinA1, which activates EphA2, exacerbated the OGD-induced destruction of permeability and integrity of the BBB models by reducing the expression of tight junction-associated proteins. However, inhibition of Rho-associated coiled coil-containing protein kinases 1 and 2 (ROCK1 and 2) abrogated all of the effects of EphrinA1 on the BBB models in vitro. In conclusion, we provide evidence that EphA2 plays an important role in the destruction of BBB integrity by decreasing the expression of tight junction proteins through the ROCK pathway.

Mfsd2a Reverses Spatial Learning and Memory Impairment Caused by Chronic Cerebral Hypoperfusion via Protection of the Blood-Brain Barrier.

Disruption of the blood–brain barrier (BBB) can lead to cognitive impairment. Major facilitator superfamily domain-containing protein 2a (Mfsd2a) is a newly discovered protein that is essential for maintaining BBB integrity. However, the role of Mfsd2a in vascular cognitive impairment has not been explored yet. In this study, a rat model of chronic cerebral hypoperfusion (CCH) was established by producing permanent bilateral common carotid artery occlusion (2VO) in rats. We found that after the 2VO procedure, the rats exhibited cognitive impairment, showed increased BBB leakage within the hippocampus, and had reduced expression of the Mfsd2a protein. The overexpression of Mfsd2a in the rat hippocampus reversed these changes. Further investigations using transmission electron microscopy revealed a significantly increased rate of vesicular transcytosis in the BBB of the hippocampus of the CCH rats; the rate reduced after overexpression of Mfsd2a. Moreover, Mfsd2a overexpression did not cause changes in the expression of tight junction-associated proteins and in the ultrastructures of the tight junctions. In conclusion, Mfsd2a attenuated BBB damage and ameliorated cognitive impairment in CCH rats, and its protective effect on the BBB was achieved via inhibition of vesicular transcytosis.

Mechanism of Fisetin Inhibiting Oxidative Stress to Alleviate the Damage of Blood Brain Barrier, Around the Intracerebral Hemorrhag Lesions

Background: The paper determined the brain water content, blood-brain barrier permeability, oxidative stress-related indicators, expressions of tight junction-associated proteins ZO-1 and Occludin, as well as expressions of ERK, p38, and JNK proteins in cerebral hemorrhage rats. The paper explored whether Fisetin reduced the damage of blood-brain barrier caused by cerebral hemorrhage through inhibiting oxidative stress, and investigated whether MAPK pathway is the main pathway for BBB damage caused by oxidative stress. Material and Methods: Rat ICH model was established. After 72 hours, mNSS score was used to measure the behavior of rats. The water content of brain tissue was determined by dry-wet weight method. Evans blue method was applied to measure the blood-brain barrier permeability. Oxidative stress index was detected by spectrophotometer. The expressions of ZO-1, Occludin, ERK, p38 and JNK proteins in rat brain tissue were determined by Western blot. Results: Fisetin significantly reduced the mNSS score, brain water content, bloodbrain barrier permeability, oxidative stress index and the expressions of ERK, p38 and JNK in rat ICH model, and also significantly increased the antioxidant index of rat ICH model, as well as the expressions of ZO-1 and Occludin. The effect is becoming more significant as the therapeutic dose is increased. Conclusion: Fisetin can inhibit the oxidative stress and alleviate the damage of BBB around the lesions of rat ICH model, thus alleviating secondary brain injury. In the rat ICH model, the expressions of ERK, p38 and JNK in the brain tissue around the lesion are increased and oxidized. The stress response is enhanced, and Fisetin inhibits oxidative stress and the expressions of ERK, p38, and JNK. MAPK is imperative in the process of oxidative stress-induced BBB damage in rat ICH model.

Exploring blood-brain barrier hyperpermeability and potential biomarkers in traumatic brain injury

Blood-brain barrier breakdown and associated vascular hyperpermeability leads to vasogenic edema in traumatic brain injury (TBI). Tight junctions maintain blood-brain barrier integrity; their disruption in TBI holds significant promise for diagnosis and treatment. A controlled cortical impactor was used for TBI in mouse studies. Blood was collected 1 h after injury and sent for antibody microarray analysis. Twenty human subjects with radiographic evidence of TBI were enrolled and blood collected within 48 h of admission. Control subjects were individuals with nontrauma diagnoses. The subjects were matched by age and gender. Enzyme-linked immunosorbent assays were performed on each TBI and control sample for tight junction–associated proteins (TJPs), inflammatory markers, and S100β. Plasma was used to conduct in vitro monolayer permeability studies with human brain endothelial cells. S100β and the TJP occludin were significantly elevated in TBI plasma in both the murine and human studies. Monolayer permeability studies showed increased hyperpermeability in TBI groups. Plasma from TBI subjects increases microvascular hyperpermeability in vitro. TJPs in the blood may be a potential biomarker for TBI.

Protective effects of panax notoginseng saponin on dextran sulfate sodium-induced colitis in rats through phosphoinositide-3-kinase protein kinase B signaling pathway inhibition.

BACKGROUNDIntestinal inflammation is a common digestive tract disease, which is usually treated with hormone medicines. Hormone medicines are effective to some extent, but long-term use of them may bring about many complications.AIMTo explore the protective effects of panax notoginseng saponin (PNS) against dextran sulfate sodium (DSS)-induced intestinal inflammatory injury through phosphoinositide-3-kinase protein kinase B (PI3K/AKT) signaling pathway inhibition in rats.METHODSColitis rat models were generated via DSS induction, and rats were divided into control (no modeling), DSS, DSS + PNS 50 mg/k, and DSS + PNS 100 mg/kg groups. Then, the intestinal injury, oxidative stress parameters, inflammatory indices, tight junction proteins, apoptosis, macrophage polarization, and TLR4/AKT signaling pathway in colon tissues from rats in each of the groups were detected. The PI3K/AKT signaling pathway in the colon tissue of rats was blocked using the PI3K/AKT signaling pathway inhibitor, LY294002.RESULTSCompared with rats in the control group, rats in the DSS group showed significantly shortened colon lengths, and significantly increased disease activity indices, oxidative stress reactions and inflammatory indices, as well as significantly decreased expression of tight junction-associated proteins. In addition, the DSS group showed significantly increased apoptotic cell numbers, and showed significantly increased M1 macrophages in spleen and colon tissues. They also showed significantly decreased M2 macrophages in colon tissues, as well as activation of the PI3K/AKT signaling pathway (all P < 0.05). Compared with rats in the DSS group, rats in the DSS + PNS group showed significantly lengthened colon lengths, decreased disease activity indices, and significantly alleviated oxidative stress reactions and inflammatory responses. In addition, this group showed significantly increased expression of tight junction-associated proteins, significantly decreased apoptotic cell numbers, and significantly decreased M1 macrophages in spleen and colon tissues. This group further showed significantly increased M2 macrophages in colon tissues, and significantly suppressed activation of the PI3K/AKT signaling pathway, as well as a dose dependency (all P < 0.05). When the PI3K/AKT signaling pathway was inhibited, the apoptosis rate of colon tissue cells in the DSS + LY294002 group was significantly lower than that of the DSS group (P < 0.05).CONCLUSIONPNS can protect rats against DSS-induced intestinal inflammatory injury by inhibiting the PI3K/AKT signaling pathway, and therefore may be potentially used in the future as a drug for colitis.

The Integral Role of Tight Junction Proteins in the Repair of Injured Intestinal Epithelium.

The intestinal epithelial monolayer forms a transcellular and paracellular barrier that separates luminal contents from the interstitium. The paracellular barrier consists of a highly organized complex of intercellular junctions that is primarily regulated by apical tight junction proteins and tight junction-associated proteins. This homeostatic barrier can be lost through a multitude of injurious events that cause the disruption of the tight junction complex. Acute repair after injury leading to the reestablishment of the tight junction barrier is crucial for the return of both barrier function as well as other cellular functions, including water regulation and nutrient absorption. This review provides an overview of the tight junction complex components and how they link to other plasmalemmal proteins, such as ion channels and transporters, to induce tight junction closure during repair of acute injury. Understanding the components of interepithelial tight junctions and the mechanisms of tight junction regulation after injury is crucial for developing future therapeutic targets for patients experiencing dysregulated intestinal permeability.

Endothelial-specific Crif1 deletion induces BBB maturation and disruption via the alteration of actin dynamics by impaired mitochondrial respiration

Cerebral endothelial cells (ECs) require junctional proteins to maintain blood–brain barrier (BBB) integrity, restricting toxic substances and controlling peripheral immune cells with a higher concentration of mitochondria than ECs of peripheral capillaries. The mechanism underlying BBB disruption by defective mitochondrial oxidative phosphorylation (OxPhos) is unclear in a mitochondria-related gene-targeted animal model. To assess the role of EC mitochondrial OxPhos function in the maintenance of the BBB, we developed an EC-specific CR6-interactin factor1 (Crif1) deletion mouse. We clearly observed defects in motor behavior, uncompacted myelin and leukocyte infiltration caused by BBB maturation and disruption in this mice. Furthermore, we investigated the alteration in the actin cytoskeleton, which interacts with junctional proteins to support BBB integrity. Loss of Crif1 led to reorganization of the actin cytoskeleton and a decrease in tight junction-associated protein expression through an ATP production defect in vitro and in vivo. Based on these results, we suggest that mitochondrial OxPhos is important for the maturation and maintenance of BBB integrity by supplying ATP to cerebral ECs.

Effects of Brain-Derived Mitochondria on the Function of Neuron and Vascular Endothelial Cell After Traumatic Brain Injury

Mitochondrial dysfunction plays an essential role in secondary brain injury following traumatic brain injury (TBI). Interestingly, accumulating evidence has shown that therapeutic benefits of mitochondrial transplantation exist. Therefore, we hypothesized that the injection of exogenous mitochondria would contribute to the mitigation of cellular energy metabolism disorders and neurologic functions after TBI. We first extracted isolated mitochondria from fresh brain tissue using a kit and then identified their activity and purity. The role of exogenous mitochondria was assessed using the glucose oxygen deprivation-induced cellular damage model and controlled cortical impact-induced mice with TBI. The results showed that treatment with exogenous mitochondria improved the cellular respiratory control rate, the expression of tight junction-associated proteins, and synaptic plasticity-related proteins invitro. Moreover, the application of exogenous mitochondria significantly reduced cellular apoptosis, promoted angiogenesis and alleviated brain edema and blood-brain barrier leakage in mice subjected to TBI. Additionally, exogenous mitochondria significantly reduced excessive inhibition of long-term depression in the hippocampus 7 days after TBI. Taken together, the data suggested that exogenous mitochondrial intervention ameliorated glucose oxygen deprivation-induced cell damage and controlled cortical impact-induced TBI in a mouse model. The new discovery in the current study inspires us to suggest that mitochondrial transplantation might serve as a new therapeutic strategy for TBI.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of junctional adhesion molecule 1

Porphyromonas gingivalis is a major pathogen in severe and chronic manifestations of periodontal disease, which is one of the most common infections of humans. A central feature of P. gingivalis pathogenicity is dysregulation of innate immunity at the gingival epithelial interface; however, the molecular basis underlying P. gingivalis–dependent abrogation of epithelial barrier function remains unknown. Gingival epithelial cells express junctional adhesion molecule (JAM1), a tight junction–associated protein, and JAM1 homodimers regulate epithelial barrier function. Here we show that Arg-specific or Lys-specific cysteine proteases (gingipains) secreted by P. gingivalis can specifically degrade JAM1 at K134 and R234 in gingival epithelial cells, resulting in permeability of the gingival epithelium to 40 kDa dextran, lipopolysaccharide (LPS), and proteoglycan (PGN). A P. gingivalis strain lacking gingipains was impaired in degradation of JAM1. Knockdown of JAM1 in monolayer cells and a three-dimensional multilayered tissue model also increased permeability to LPS, PGN, and gingipains. Inversely, overexpression of JAM1 in epithelial cells prevented penetration by these agents following P. gingivalis infection. Our findings strongly suggest that P. gingivalis gingipains disrupt barrier function of stratified squamous epithelium via degradation of JAM1, allowing bacterial virulence factors to penetrate into subepithelial tissues.

β-Defensin 129 Attenuates Bacterial Endotoxin-Induced Inflammation and Intestinal Epithelial Cell Apoptosis.

Defensins have attracted considerable research interest worldwide because of their potential to serve as a substitute for antibiotics. In this study, we characterized a novel porcine β-defensin (pBD129) and explored its role in alleviating bacterial endotoxin-induced inflammation and intestinal epithelium atrophy. The pBD129 gene was cloned and expressed in Escherichia coli. A recombinant pBD129 protein was also purified. To explore its role in alleviating the endotoxin-induced inflammation, mice, with or without lipopolysaccharide (LPS) challenge were treated by pBD129 at different doses. The recombinant pBD129 showed significant antimicrobial activities against the E. coli and Streptococcus with a minimal inhibitory concentration (MICs) of 32 μg/mL. Hemolytic assays showed that the pBD129 had no detrimental impact on cell viabilities. Interestingly, we found that pBD129 attenuated LPS-induced inflammatory responses by decreasing serum concentrations of inflammatory cytokines, such as the IL-1β, IL-6, and TNF-α (P < 0.05). Moreover, pBD129 elevated the intestinal villus height (P < 0.05) and enhanced the expression and localization of the major tight junction-associated protein ZO-1 in LPS-challenged mice. Additionally, pDB129 at a high dose significantly decreased serum diamine oxidase (DAO) concentration (P < 0.05) and reduced intestinal epithelium cell apoptosis (P < 0.05) in LPS-challenged mice. Importantly, pBD129 elevated the expression level of Bcl-2-associated death promoter (Bcl-2), but down-regulated the expression levels of apoptosis-related genes such as the B-cell lymphoma-2-associated X protein (Bax), BH3-interacting domain death agonist (Bid), cysteinyl aspartate-specific proteinase-3 (Caspase-3), and caspase-9 in the intestinal mucosa (P < 0.05). These results suggested a novel function of the mammalian defensins, and the anti-bacterial and anti-inflammatory properties of pBD129 may allow it a potential substitute for conventionally used antibiotics or drugs.

Effects of oxygen-glucose deprivation (OGD) on barrier properties and mRNA transcript levels of selected marker proteins in brain endothelial cells/astrocyte co-cultures.

Ischemic stroke has been shown to induce breakdown of the blood-brain barrier, although these changes are not fully characterized. Oxygen-glucose deprivation (OGD) has been used to investigate the effects of ischemia in cultured brain capillary endothelial cells, however this involves a change of medium which in itself may affect the cells. The aim of the present study was to investigate the effect of OGD and simple medium exchange followed by 48 h of reperfusion on barrier properties of primary bovine endothelial cells co-cultured with rat astrocytes. Barrier properties were evaluated by transendothelial electrical resistance measurements, passive permeability of flux markers, RT-qPCR and immunocytochemistry. Both OGD and simple medium exchange caused an increase in endothelial monolayer permeability. This correlated with reduced transcript levels of a number of tight junction and tight junction-associated proteins (claudin-1, claudin-5, occludin, ZO-1, tricellulin, marveld3 and PECAM-1), as well as with altered transcript level of several transporters and receptors (GLUT-1, HB-EGF, InsR, TfR, two members of the low density lipoprotein receptor family, LDLR and LRP-1, and the efflux transporter BCRP). In contrast, effects induced specifically by OGD were transient de-localization of claudin-5 from the junction zone, increased InsR localization at the plasma membrane and transient downregulation of MRP-1 and P-gp transcript levels. In conclusion, OGD caused changes in claudin-5 and InsR localization, as well as in MRP-1 and P-gp transcript levels. Our results however also indicated that medium exchange alone caused changes in functional barrier properties and expression levels of wide range of proteins.

Molecular determinants of afferent sensitization in a rat model of cystitis with urothelial barrier dysfunction.

The internal surface of the urinary bladder is covered by the urothelium, a stratified epithelium that forms an impermeable barrier to urinary solutes. Increased urothelial permeability is thought to contribute to symptom generation in several forms of cystitis by sensitizing bladder afferents. In this report we investigate the physiological mechanisms that mediate bladder afferent hyperexcitability in a rat model of cystitis induced by overexpression in the urothelium of claudin-2 (Cldn2), a tight junction-associated protein upregulated in bladder biopsies from patients with interstitial cystitis/bladder pain syndrome. Patch-clamp studies showed that overexpression of Cldn2 in the urothelium sensitizes a population of isolectin GS-IB4-negative [IB4(-)] bladder sensory neurons with tetrodotoxin-sensitive (TTX-S) action potentials. Gene expression analysis revealed a significant increase in mRNA levels of the delayed-rectifier voltage-gated K+ channel (Kv)2.2 and the accessory subunit Kv9.1 in this population of bladder sensory neurons. Consistent with this finding, Kv2/Kv9.1 channel activity was greater in IB4(-) bladder sensory neurons from rats overexpressing Cldn2 in the urothelium than in control counterparts. Likewise, current density of TTX-S voltage-gated Na+ (Nav) channels was greater in sensitized neurons than in control counterparts. Significantly, guangxitoxin-1E (GxTX-1E), a selective blocker of Kv2 channels, blunted the repetitive firing of sensitized IB4(-) sensory neurons. In summary, our studies indicate that an increase in the activity of TTX-S Nav and Kv2/Kv9.1 channels mediates repetitive firing of sensitized bladder sensory neurons in rats with increased urothelial permeability.NEW & NOTEWORTHY Hyperexcitability of sensitized bladder sensory neurons in a rat model of interstitial cystitis/bladder pain syndrome (IC/BPS) results from increased activity of tetrodotoxin-sensitive voltage-gated Na+ and delayed-rectifier voltage-gated K+ (Kv)2/Kv9.1 channels. Of major significance, our studies indicate that Kv2/Kv9.1 channels play a major role in symptom generation in this model of IC/BPS by maintaining the sustained firing of the sensitized bladder sensory neurons.

MiR-24 Is Elevated in Ulcerative Colitis Patients and Regulates Intestinal Epithelial Barrier Function

Inflammatory bowel disease is characterized by high levels of inflammation and loss of barrier integrity in the colon. The intestinal barrier is a dynamic network of proteins that encircle intestinal epithelial cells. miRNAs regulate protein-coding genes. In this study, miR-24 was found to be elevated in colonic biopsies and blood samples from ulcerative colitis (UC) patients compared with healthy controls. In the colon of UC patients, miR-24 is localized to intestinal epithelial cells, which prompted an investigation of intestinal epithelial barrier function. Two intestinal epithelial cell lines were used to study the effect of miR-24 overexpression on barrier integrity. Overexpression of miR-24 in both cell lines led to diminished transepithelial electrical resistance and increased dextran flux, suggesting an effect on barrier integrity. Overexpression of miR-24 did not induce apoptosis or affect cell proliferation, suggesting that the effect of miR-24 on barrier function was due to an effect on cell-cell junctions. Although the tight junctions in cells overexpressing miR-24 appeared normal, miR-24 overexpression led to a decrease in the tight junction-associated protein cingulin. Loss of cingulin compromised barrier formation; cingulin levels negatively correlated with disease severity in UC patients. Together, these data suggest that miR-24 is a significant regulator of intestinal barrier that may be important in the pathogenesis of UC.

Blood-brain barrier function, cell viability, and gene expression of tight junction-associated proteins in the mouse are disrupted by crude oil, benzo[a]pyrene, and the dispersant COREXIT

Exposure to crude oil, its components, and oil dispersants during a major crude oil spill, such as the Deepwater Horizon Oil Spill, can elicit behavioral changes in animals and humans. However, the underlying mechanisms by which oil spill-related compounds alters behavior remains largely unknown. A major cause of behavioral changes generally is dysfunction of the blood-brain barrier (BBB). We investigated the impact of a crude oil high energy water accommodated fraction (HEWAF), benzo[a] pyrene (BaP; a major component of crude oil), and the oil dispersant COREXIT, on BBB function. BBB function was assessed by measuring transendothelial electrical resistance (TEER) of mouse brain microvascular endothelial cells (BMECs). Within 3 h after treatment, TEER was significantly reduced by exposure to high concentrations of all test compounds. TEER remained reduced in response to COREXIT after 48 h, but this effect waned in BMECs treated with HEWAF and BaP, with low-mid range concentrations inducing increased TEER compared to vehicle controls. At 48 h of treatment, BMEC viability was significantly reduced in response to 2% HEWAF, but was increased in response to BaP (25 and 50 μM). BMEC viability was increased with 80 ppm COREXIT, but was reduced with 160 ppm. Gene expression of tight junction-associated proteins (claudin-5 and tight junction protein-1), and cell adhesion receptor (vascular cell adhesion molecule-1) was reduced in response to HEWAF and COREXIT, but not BaP. Taken together, these data suggest that oil spill-related compounds markedly affect BBB function, and that these changes may underlie the observed behavioral changes due to crude oil exposure.

Bazedoxifene protects cerebral autoregulation after traumatic brain injury and attenuates impairments in blood-brain barrier damage: involvement of anti-inflammatory pathways by blocking MAPK signaling.

Traumatic brain injury (TBI) is a significant cause of death and long-term deficits in motor and cognitive functions for which there are currently no effective chemotherapeutic drugs. Bazedoxifene (BZA) is a third-generation selective estrogen receptor modulator (SERM) and has been investigated as a treatment for postmenopausal osteoporosis. It is generally safe and well tolerated, with favorable endometrial and breast safety profiles. Recent findings have shown that SERMs may have therapeutic benefits; however, the role of BZA in the treatment of TBI and its molecular and cellular mechanisms remain poorly understood. The aim of the present study was to examine the neuroprotective effects of BZA on early TBI in rats and to explore the underlying mechanisms of these effects. TBI was induced using a modified weight-drop method. Neurological deficits were evaluated according to the neurological severity score (NSS). Morris water maze and open-field behavioral tests were used to test cognitive functions. Brain edema was measured by brain water content, and impairments in the blood-brain barrier (BBB) were evaluated by expression analysis of tight junction-associated proteins, such as occludin and zonula occludens-1 (ZO-1). Neuronal injury was assessed by hematoxylin and eosin (H&E) staining. LC-MS/MS analysis was performed to determine the ability of BZA to cross the BBB. Our results indicated that BZA attenuated the impaired cognitive functions and the increased BBB permeability of rats subjected to TBI through activation of inflammatory cascades. In vivo experiments further revealed that BZA provided this neuroprotection by suppressing TBI-induced activation of the MAPK/NF-κB signaling pathway. Thus, mechanically, the anti-inflammatory effects of BZA in TBI may be partially mediated by blocking the MAPK signaling pathway. These findings suggest that BZA might attenuate neurological deficits and BBB damage to protect against TBI by blocking the MAPK/NF-κB signaling pathway.

Paraquat affects the differentiation of neural stem cells and impairs the function of vascular endothelial cells: a study of molecular mechanism

To investigate the effect of paraquat (PQ) exposure on gene expression in neural stem cells as well as structures and functions of vascular endothelial cells. RNA-Seq was used to explore the differentially expressed genes in human umbilical cord blood-neural stem cells (HUCB-NSCs) at different stages (eg, proliferation, early and late differentiation) in the presence of PQ. The effects of PQ on human umbilical vein endothelial cells (HUVECs), including cell proliferation, apoptosis, cytokines secretion, and expression of tight junction proteins, were assessed with CCK-8, flow cytometry, ELISA, and western blot analysis, individually. A total of 53 genes were up-regulated and 61 genes were down-regulated in PQ treated HUCB-NSCs, including seven genes associated with the differentiation of neural stem cells, for example, Gfap, S100B, Oct4, Gdf3, Sox1, Pax6, and Ngn1. PQ treatment significantly reduced the proliferation of HUVECs, inhibited cytokines secretion (VEGF, BFGF) and expressions of tight junction-associated protein (Claudin 1, Occludin, ZO-1), as well as induced significant apoptosis. Our study suggests that PQ impairs the development of nervous system by regulating the expression of genes associated with neural stem cell differentiation, as well as the structure and function of vascular endothelial cells, which together lead to abnormality in the nervous system.

Human Brain Endothelial CXCR2 is Inflammation-Inducible and Mediates CXCL5- and CXCL8-Triggered Paraendothelial Barrier Breakdown.

Chemokines (C-X-C) motif ligand (CXCL) 5 and 8 are overexpressed in patients with multiple sclerosis, where CXCL5 serum levels were shown to correlate with blood–brain barrier dysfunction as evidenced by gadolinium-enhanced magnetic resonance imaging. Here, we studied the potential role of CXCL5/CXCL8 receptor 2 (CXCR2) as a regulator of paraendothelial brain barrier function, using the well-characterized human cerebral microvascular endothelial cell line hCMEC/D3. Low basal CXCR2 mRNA and protein expression levels in hCMEC/D3 were found to strongly increase under inflammatory conditions. Correspondingly, immunohistochemistry of brain biopsies from two patients with active multiple sclerosis revealed upregulation of endothelial CXCR2 compared to healthy control tissue. Recombinant CXCL5 or CXCL8 rapidly and transiently activated Akt/protein kinase B in hCMEC/D3. This was followed by a redistribution of tight junction-associated protein zonula occludens-1 (ZO-1) and by the formation of actin stress fibers. Functionally, these morphological changes corresponded to a decrease of paracellular barrier function, as measured by a real-time electrical impedance-sensing system. Importantly, preincubation with the selective CXCR2 antagonist SB332235 partially prevented chemokine-induced disturbance of both tight junction morphology and function. We conclude that human brain endothelial CXCR2 may contribute to blood–brain barrier disturbance under inflammatory conditions with increased CXCL5 and CXCL8 expression, where CXCR2 may also represent a novel pharmacological target for blood–brain barrier stabilization.

Blood vessel epicardial substance (BVES) reduces LRP6 receptor and cytoplasmic -catenin levels to modulate Wnt signaling and intestinal homeostasis.

Blood vessel epicardial substance (BVES, otherwise known as POPDC1) is an integral membrane protein known to regulate tight junction formation and epithelial-mesenchymal transition. BVES is underexpressed in a number of malignancies, including colorectal cancer. BVES loss leads to activation of the Wnt pathway, suggesting that decreased BVES expression functionally contributes to tumorigenesis. However, the mechanism by which BVES modulates Wnt signaling are unknown. Here we confirm that BVES loss increases -catenin protein levels, leads to Wnt pathway activation in a ligand-independent fashion, and coordinates with Wnt ligand to further increase Wnt signaling. We show that BVES loss increases levels and activation of the Wnt co-receptor, LRP6, in cell lines, murine adenoma tumoroids, and human-derived colonoids. We also demonstrate that BVES interacts with LRP6. Finally, murine tumor modelling using a Wnt-driven genetic model and a chemically-induced model of colorectal carcinogenesis demonstrates that BVES loss increases tumor multiplicity and dysplasia. Together, these results implicate BVES as an inhibitor of Wnt signaling, provide one of the first examples of a tight junction-associated protein regulating Wnt receptor levels, and expand the number of putative molecular targets for therapeutic intervention in colorectal cancer.