MiRNA-224-5p regulates the defective permeability barrier in sensitive skin by targeting claudin-5.

BACKGROUND: Sensitive skin is hypersensitive to various external stimuli and a defective epidermal permeability barrier is an important clinical feature of sensitive skin. Claudin-5 (CLDN5) expression levels decrease in sensitive skin. This study aimed to explore the impact of CLDN5 deficiency on the permeability barrier in sensitive skin and the regulatory role of miRNAs in CLDN5 expression. MATERIALS AND METHODS: A total of 26 patients were retrospectively enrolled, and the CLDN5 expression and permeability barrier dysfunction in vitro were assessed. Then miRNA-224-5p expression was also assessed in sensitive skin. RESULTS: Immunofluorescence and electron microscopy revealed reduced CLDN5 expression, increased miR-224-5p expression, and disrupted intercellular junctions in sensitive skin. CLDN5 knockdown was associated with lower transepithelial electrical resistance (TEER) and Lucifer yellow penetration in keratinocytes and organotypic skin models. The RNA-seq and qRT-PCR results indicated elevated miR-224-5p expression in sensitive skin; MiR-224-5p directly interacted with the 3`UTR of CLDN5, resulting in CLDN5 deficiency in the luciferase reporter assay. Finally, miR-224-5p reduced TEER in keratinocyte cultures. CONCLUSION: These results suggest that the miR-224-5p-induced reduction in CLDN5 expression leads to impaired permeability barrier function, and that miR-224-5p could be a potential therapeutic target for sensitive skin.

Synergistic induction of blood-brain barrier properties.

Blood-brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug development for the human brain. Yet providing stem cell-derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vascular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/ß-catenin signaling and inhibition of the TGF-ß pathway in endothelial cells robustly induce BBB properties in vitro. To target this interaction, we present a small-molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/ß-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell-derived endothelial cells toward the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis, and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility, and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories.

Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4-NOTCH-CLDN5 pathway and is vulnerable to neonatal hyperoxia.

The mechanisms governing brain vascularization during development remain poorly understood. A key regulator of developmental vascularization is delta like 4 (DLL4), a Notch ligand prominently expressed in endothelial cells (EC). Exposure to hyperoxia in premature infants can disrupt the development and functions of cerebral blood vessels and lead to long-term cognitive impairment. However, its role in cerebral vascular development and the impact of postnatal hyperoxia on DLL4 expression in mouse brain EC have not been explored. We determined the DLL4 expression pattern and its downstream signalling gene expression in brain EC using Dll4+/+ and Dll4+/LacZ mice. We also performed in vitro studies using human brain microvascular endothelial cells. Finally, we determined Dll4 and Cldn5 expression in mouse brain EC exposed to postnatal hyperoxia. DLL4 is expressed in various cell types, with EC being the predominant one in immature brains. Moreover, DLL4 deficiency leads to persistent abnormalities in brain microvasculature and increased vascular permeability both in vivo and in vitro. We have identified that DLL4 insufficiency compromises endothelial integrity through the NOTCH-NICD-RBPJ-CLDN5 pathway, resulting in the downregulation of the tight junction protein claudin 5 (CLDN5). Finally, exposure to neonatal hyperoxia reduces DLL4 and CLDN5 expression in developing mouse brain EC. We reveal that DLL4 is indispensable for brain vascular development and maintaining the blood-brain barrier's function and is repressed by neonatal hyperoxia. We speculate that reduced DLL4 signalling in brain EC may contribute to the impaired brain development observed in neonates exposed to hyperoxia. KEY POINTS: The role of delta like 4 (DLL4), a Notch ligand in vascular endothelial cells, in brain vascular development and functions remains unknown. We demonstrate that DLL4 is expressed at a high level during postnatal brain development in immature brains and DLL4 insufficiency leads to abnormal cerebral vasculature and increases vascular permeability both in vivo and in vitro. We identify that DLL4  regulates endothelial integrity through NOTCH-NICD-RBPJ-CLDN5 signalling. Dll4 and Cldn5 expression are decreased in mouse brain endothelial cells exposed to postnatal hyperoxia.

Mosaic deletion of claudin-5 reveals rapid non-cell-autonomous consequences of blood-brain barrier leakage.

Claudin-5 (CLDN5) is an endothelial tight junction protein essential for blood-brain barrier (BBB) formation. Abnormal CLDN5 expression is common in brain disease, and knockdown of Cldn5 at the BBB has been proposed to facilitate drug delivery to the brain. To study the consequences of CLDN5 loss in the mature brain, we induced mosaic endothelial-specific Cldn5 gene ablation in adult mice (Cldn5iECKO). These mice displayed increased BBB permeability to tracers up to 10 kDa in size from 6 days post induction (dpi) and ensuing lethality from 10 dpi. Single-cell RNA sequencing at 11 dpi revealed profound transcriptomic differences in brain endothelial cells regardless of their Cldn5 status in mosaic mice, suggesting major non-cell-autonomous responses. Reactive microglia and astrocytes suggested rapid cellular responses to BBB leakage. Our study demonstrates a critical role for CLDN5 in the adult BBB and provides molecular insight into the consequences and risks associated with CLDN5 inhibition.

Potential Therapeutic Strategies and Drugs That Target Vascular Permeability in Severe Infectious Diseases.

Severe infection pathogenicity is induced by processes such as pathogen exposure, immune cell activation, inflammatory cytokine production, and vascular hyperpermeability. Highly effective drugs, such as antipathogenic agents, steroids, and antibodies that suppress cytokine function, have been developed to treat the first three processes. However, these drugs cannot completely suppress severe infectious diseases, such as coronavirus disease 2019 (COVID-19). Therefore, developing novel drugs that inhibit vascular hyperpermeability is crucial. This review summarizes the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced vascular hyperpermeability and identifies inhibitors that increase endothelial cell (EC) junction-related proteins and determines their efficacy in COVID-19 and endotoxemia models. Analyzing the effects of SARS-CoV-2 on vascular permeability revealed that SARS-CoV-2 suppresses Claudin-5 (CLDN5) expression, which is responsible for adhesion between ECs, thereby increasing vascular permeability. Inhibiting CLDN5 function in mice induced vascular hyperpermeability and pulmonary edema. In contrast, Enhancing CLDN5 expression suppressed SARS-CoV-2-induced endothelial hyperpermeability, suggesting that SARS-CoV-2-induced vascular hyperpermeability contributes to pathological progression, which can be suppressed by upregulating EC junction proteins. Based on these results, we focused on Roundabout4 (Robo4), another EC-specific protein that stabilizes EC junctions. EC-specific Robo4 overexpression suppressed vascular hyperpermeability and mortality in lipopolysaccharide-treated mice. An ALK1 inhibitor (a molecule that increases Robo4 expression), suppressed vascular hyperpermeability and mortality in lipopolysaccharide- and SARS-CoV-2-treated mice. These results indicate that Robo4 expression-increasing drugs suppress vascular permeability and pathological phenotype in COVID-19 and endotoxemia models.

Early Increase in Blood-Brain Barrier Permeability in a Murine Model Exposed to Fifteen Days of Intermittent Hypoxia.

Obstructive sleep apnea (OSA) is characterized by intermittent repeated episodes of hypoxia-reoxygenation. OSA is associated with cerebrovascular consequences. An enhanced blood-brain barrier (BBB) permeability has been proposed as a marker of those disorders. We studied in mice the effects of 1 day and 15 days intermittent hypoxia (IH) exposure on BBB function. We focused on the dorsal part of the hippocampus and attempted to identify the molecular mechanisms by combining in vivo BBB permeability (Evans blue tests) and mRNA expression of several junction proteins (zona occludens (ZO-1,2,3), VE-cadherin, claudins (1,5,12), cingulin) and of aquaporins (1,4,9) on hippocampal brain tissues. After 15 days of IH exposure we observed an increase in BBB permeability, associated with increased mRNA expressions of claudins 1 and 12, aquaporins 1 and 9. IH seemed to increase early for claudin-1 mRNA expression as it doubled with 1 day of exposure and returned near to its base level after 15 days. Claudin-1 overexpression may represent an immediate response to IH exposure. Then, after 15 days of exposure, an increase in functional BBB permeability was associated with enhanced expression of aquaporin. These BBB alterations are possibly associated with a vasogenic oedema that may affect brain functions and accelerate neurodegenerative processes.

Xuanfu Daizhe Tang alleviates reflux esophagitis in rats by inhibiting the STAT1/TREM-1 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Reflux esophagitis (RE) is a common chronic inflammatory disease of the esophageal mucosa with a high prevalence and recurrence rate, for which a satisfactory therapeutic strategy is still lacking. Chinese medicine has its characteristics and advantages in treating RE, and the clinical application of Xuanfu Daizhe Tang (XDT) in treating RE has achieved sound therapeutic effects. However, there needs to be more research on its mechanism of action. AIM OF THE STUDY: The present work aimed to investigate the mechanism of XDT action in RE through the Signal Transducer and Activator of Transcription 1 (STAT1)/Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) pathway. MATERIALS AND METHODS: The main active components of XDT were analyzed by ultra-performance liquid chromatography-mass spectrometer (UPLC-MS). The effect of XDT on RE was evaluated in a rat model of RE induced by "Cardioplasty + pyloric ligation + Roux-en-Y esophagojejunostomy". Each administration group was treated by gavage. The degree of damage to the esophageal mucosa was evaluated by visual observation, and the Potential of Hydrogen (PH) method and Hematoxylin-eosin staining (HE) staining were performed. Serum levels of Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6), Tumor Necrosis Factor alpha (TNF-α), and Inducible Nitric Oxide Synthase (iNOS) were measured by ELISA. Quantitative Real-time PCR (qPCR), Western Blot (WB), and Immunofluorescence (IF) methods were used to detect Claudin-4, Claudin-5, TREM-1, and p-STAT1 in esophageal tissues for studying the mechanism of action and signaling pathway of XDT. Immunohistochemistry (IHC) analysis was used to detect the expression of TREM-1 and CD68 in esophageal tissues. Flow Cytometry (FC) was used to detect the polarization of macrophages in the blood. After conducting preliminary experiments to verify our hypothesis, we performed molecular docking between the active component of XDT and STAT1 derived from rats and parallel experiments with STAT1 inhibitor. The selective increaser of STAT1 transcription (2-NP) group was used to validate the mechanism by which XDT acts. RESULTS: XDT alleviated esophageal injury and attenuated histopathological changes in RE rats. XDT also inhibited the inflammatory response and decreased serum IL-1ß, IL-6, TNF-α, and iNOS levels in RE rats. qPCR and WB results revealed that XDT inhibited the expression of Claudin-4, Claudin-5, TREM-1, and STAT1 in the esophageal mucosa of RE rats. IHC and FC results showed that XDT reduced TREM-1 levels in esophageal tissues and polarized macrophages toward M2. The molecular docking results showed that rat-derived STAT1 can strongly bind to Isochronogenic acid A in XDT. The parallel experimental results of STAT1 inhibitor showed that XDT has anti-inflammatory effects similar to STAT1 inhibitors. The 2-NP group confirmed that XDT exerts its therapeutic effect on reflux esophagitis through the STAT1/TREM-1 pathway, with STAT1 as the upstream protein. CONCLUSIONS: This study suggests that XDT may treat reflux esophagitis by modulating the STAT1/TREM-1 pathway.

ZNF787 and HDAC1 Mediate Blood-Brain Barrier Permeability in an In Vitro Model of Alzheimer's Disease Microenvironment.

The permeability of the blood-brain barrier (BBB) is increased in Alzheimer's disease (AD). This plays a key role in the instigation and maintenance of chronic inflammation during AD. Experiments using AD models showed that the increased permeability of the BBB was mainly caused by the decreased expression of tight junction-related proteins occludin and claudin-5. In this study, we found that ZNF787 and HDAC1 were upregulated in ß-amyloid (Aß)1-42-incubated endothelial cells, resulting in increased BBB permeability. Conversely, the silencing of ZNF787 and HDAC1 by RNAi led to reduced BBB permeability. The silencing of ZNF787 and HDAC1 enhanced the expression of occludin and claudin-5. Mechanistically, ZNF787 binds to promoter regions for occludin and claudin-5 and functions as a transcriptional regulator. Furthermore, we demonstrate that ZNF787 interacts with HDAC1, and this resulted in the downregulation of the expression of genes encoding tight junction-related proteins to increase in BBB permeability. Taken together, our study identifies critical roles for the interaction between ZNF787 and HDAC1 in regulating BBB permeability and the pathogenesis of AD.

Association of Plasma Claudin-5 with Age and Alzheimer Disease.

The blood-brain barrier (BBB) plays pivotal roles in synaptic and neuronal functioning by sealing the space between adjacent microvascular endothelial cells. BBB breakdown is present in patients with mild cognitive impairment (MCI) or Alzheimer disease (AD). Claudin-5 (CLDN-5) is a tetra-spanning protein essential for sealing the intercellular space between adjacent endothelial cells in the BBB. In this study, we developed a blood-based assay for CLDN-5 and investigated its diagnostic utility using 100 cognitively normal (control) subjects, 100 patients with MCI, and 100 patients with AD. Plasma CLDN-5 levels were increased in patients with AD (3.08 ng/mL) compared with controls (2.77 ng/mL). Plasma levels of phosphorylated tau (pTau181), a biomarker of pathological tau, were elevated in patients with MCI or AD (2.86 and 4.20 pg/mL, respectively) compared with control subjects (1.81 pg/mL). In patients with MCI or AD, plasma levels of CLDN-5-but not pTau181-decreased with age, suggesting some age-dependent BBB changes in MCI and AD. These findings suggest that plasma CLDN-5 may a potential biochemical marker for the diagnosis of AD.

[Mechanobiological Mechanisms Involved in the Regualation of the Blood-Brain Barrier by Fluid Shear Force]. / æµä½“å‰ªåˆ‡åŠ›è°ƒæŽ§è¡€è„‘å±éšœçš„åŠ›å­¦ç”Ÿç‰©å­¦æœºåˆ¶ç ”ç©¶.

Objective: To explore the mechanobiological mechanism of fluid shear force (FSF) on the protection, injury, and destruction of the structure and function of the blood-brain barrier (BBB) under normal physiological conditions, ischemic hypoperfusion, and postoperative hyperperfusion conditions. BBB is mainly composed of brain microvascular endothelial cells. Rat brain microvascular endothelial cells (rBMECs) were used as model cells to conduct the investigation. Methods: rBMECs were seeded at a density of 1×105 cells/cm2 and incubated for 48 h. FSF was applied to the rBMECs at 0.5, 2, and 20 dyn/cm2, respectively, simulating the stress BBB incurs under low perfusion, normal physiological conditions, and high FSF after bypass grafting when there is cerebral vascular stenosis. In addition, a rBMECs static culture group was set up as the control (no force was applied). Light microscope, scanning electron microscope (SEM), and laser confocal microscope (LSCM) were used to observe the changes in cell morphology and cytoskeleton. Transmission electron microscope (TEM) was used to observe the tight junctions. Immunofluorescence assay was performed to determine changes in the distribution of tight junction-associated proteins claudin-5, occludin, and ZO-1 and adherens junction-associated proteins VE-cadherin and PECAM-1. Western blot was performed to determine the expression levels of tight junction-associated proteins claudin-5, ZO-1, and JAM4, adherens junction-associated protein VE-cadherin, and key proteins in Rho GTPases signaling (Rac1, Cdc42, and RhoA) under FSF at different intensities. Results: Microscopic observation showed that the cytoskeleton exhibited disorderly arrangement and irregular orientation under static culture and low shear force (0.5 dyn/cm2). Under normal physiological shear force (2 dyn/cm2), the cytoskeleton was rearranged in the orientation of the FSF and an effective tight junction structure was observed between cells. Under high shear force (20 dyn/cm2), the intercellular space was enlarged and no effective tight junction structure was observed. Immunofluorescence results showed that, under low shear force, the gap between the cells decreased, but there was also decreased distribution of tight junction-associated proteins and adherens junction-associated proteins at the intercellular junctions. Under normal physiological conditions, the cells were tightly connected and most of the tight junction-associated proteins were concentrated at the intercellular junctions. Under high shear force, the gap between the cells increased significantly and the tight junction and adherens junction structures were disrupted. According to the Western blot results, under low shear force, the expression levels of claudin-5, ZO-1, and VE-cadherin were significantly up-regulated compared with those of the control group (P<0.05). Under normal physiological shear force, claudin-5, ZO-1, JAM4, and VE-cadherin were highly expressed compared with those of the control group (P<0.05). Under high shear force, the expressions of claudin-5, ZO-1, JAM4, and VE-cadherin were significantly down-regulated compared with those of the normal physiological shear force group (P<0.05). Under normal physiological shear force, intercellular expressions of Rho GTPases proteins (Rac1, Cdc42, and RhoA) were up-regulated and were higher than those of the other experimental groups (P<0.05). The expressions of Rho GTPases under low and high shear forces were down-regulated compared with that of the normal physiological shear force group (P<0.05). Conclusion: Under normal physiological conditions, FSF helps maintain the integrity of the BBB structure, while low or high shear force can damage or destroy the BBB structure. The regulation of BBB by FSF is closely related to the expression and distribution of tight junction-associated proteins and adherens junction-associated proteins.

SH2 domain protein E and ABL signaling regulate blood vessel size.

Blood vessels in different vascular beds vary in size, which is essential for their function and fluid flow along the vascular network. Molecular mechanisms involved in the formation of a vascular lumen of appropriate size, or tubulogenesis, are still only partially understood. Src homology 2 domain containing E (She) protein was previously identified in a screen for proteins that interact with Abelson (Abl)-kinase. However, its biological role has remained unknown. Here we demonstrate that She and Abl signaling regulate vessel size in zebrafish embryos and human endothelial cell culture. Zebrafish she mutants displayed increased endothelial cell number and enlarged lumen size of the dorsal aorta (DA) and defects in blood flow, eventually leading to the DA collapse. Vascular endothelial specific overexpression of she resulted in a reduced diameter of the DA, which correlated with the reduced arterial cell number and lower endothelial cell proliferation. Chemical inhibition of Abl signaling in zebrafish embryos caused a similar reduction in the DA diameter and alleviated the she mutant phenotype, suggesting that She acts as a negative regulator of Abl signaling. Enlargement of the DA size in she mutants correlated with an increased endothelial expression of claudin 5a (cldn5a), which encodes a protein enriched in tight junctions. Inhibition of cldn5a expression partially rescued the enlarged DA in she mutants, suggesting that She regulates DA size, in part, by promoting cldn5a expression. SHE knockdown in human endothelial umbilical vein cells resulted in a similar increase in the diameter of vascular tubes, and also increased phosphorylation of a known ABL downstream effector CRKL. These results argue that SHE functions as an evolutionarily conserved inhibitor of ABL signaling and regulates vessel and lumen size during vascular tubulogenesis.

Irisin links Claudin-5 preservation and Mfn2-mediated mitochondrial dynamics to resist doxorubicin-induced cardiac endothelial damage.

Irisin is protective in the cardiac microenvironment and can resist doxorubicin-induced cardiotoxicity. The purpose of this study was to investigate the connection between Irisin, endothelial cell integrity, and mitochondrial dynamics. Primary cardiac microvascular endothelial cells (CMECs) were used to explore the regulatory effects of Irisin on tight junction proteins, mitochondrial dynamics, ß-catenin expression, and transcriptional activity. Results showed that Irisin can suppress doxorubicin-induced upregulation of MMP2 and MMP9, thereby reducing the degradation of tight junction proteins (ZO-1 and Claudin-5) and VE-cadherin. The preservation of Claudin-5 contributes to maintaining Mfn2 expression, which in turn supports mitochondrial fusion. Although Irisin restores doxorubicin-induced downregulation of ß-catenin, it concurrently limits ß-catenin transcriptional activity via Mfn2-mediated sulfenylation. Therefore, this study revealed a novel mechanism linking the protective effects of Irisin on the tight junction proteins and mitochondrial dynamics upon doxorubicin exposure.

CLDN5: From structure and regulation to roles in tumors and other diseases beyond CNS disorders.

Claudin-5 (CLDN5) is an essential component of tight junctions (TJs) and is critical for the integrity of the blood-brain barrier (BBB), ensuring homeostasis and protection from damage to the central nervous system (CNS). Currently, many researchers have summarized the role and mechanisms of CLDN5 in CNS diseases. However, it is noteworthy that CLDN5 also plays a significant role in tumor growth and metastasis. In addition, abnormal CLDN5 expression is involved in the development of respiratory diseases, intestinal diseases, cardiac diseases, and diabetic ocular complications. This paper aims to review the structure, expression, and regulation of CLDN5, focusing on its role in tumors, including its expression and regulation, effects on malignant phenotypes, and clinical significance. Furthermore, this paper will provide an overview of the role and mechanisms of CLDN5 in respiratory diseases, intestinal diseases, cardiac diseases, and diabetic ocular complications.

Reduction of claudin-5 and aquaporin-4 in the rat hippocampal CA-1 and CA-3 regions of a learned helplessness model of depression.

BACKGROUND: Although findings from both animal and clinical research indicate that the blood-brain barrier (BBB) contributes to the pathogenesis of various psychiatric disorders (including depression), the underlying mechanisms are unknown. We investigated the levels of the tight-junction proteins claudin-5 and aquaporin-4 (AQP-4) in astrocytes of learned helplessness (LH) rats (an animal model of depression) and non-LH rats (a model of resilience). METHODS: We administered inescapable mild electric shock to rats and then identified the LH and non-LH rats by a post-shock test. The expressions of claudin-5 and AQP-4 in several brain regions of the LH and non-LH rats were then evaluated by a western blot analysis. RESULTS: The levels of both claudin-5 and AQP-4 in the CA-1 and CA-3 hippocampal areas of the LH group were significantly lower than those of the control group, whereas those of the non-LH rats were not significantly different from those of the control and LH rats. CONCLUSIONS: These results suggest that LH rats but not non-LH rats experienced down-regulations of claudin-5 and AQP-4 in the CA-1 and CA-3. It is possible that a region-specific modulation of claudin-5 and AQP-4 is involved in the mechanisms of vulnerability but not resilience in depression.

Hypoxia-Induced miR-101 Impairs Endothelial Barrier Integrity Through Altering VE-Cadherin and Claudin-5.

Stroke is a life-threatening medical condition across the world that adversely affects the integrity of the blood-brain barrier (BBB). The brain microvascular endothelial cells are the important constituent of the BBB. These cells line the blood vessels and form a semipermeable barrier. Disruptions in adherens junction and tight junction proteins of brain microvascular endothelial cells compromise the integrity of BBB. The Vascular Endothelial (VE)-cadherin is an integral adherens junction protein required for the establishment and maintenance of the endothelial barrier integrity. This study aims to investigate the role of miRNA in hypoxia-induced endothelial barrier disruption. In this study, brain endothelial cells were exposed to hypoxic conditions for different time points. Western blotting, overexpression and knockdown of miRNA, real-time PCR, TEER, and sodium fluorescein assay were used to examine the effect of hypoxic conditions on brain endothelial cells. Hypoxic exposure was validated using HIF-1α protein. Exposure to hypoxic conditions resulted to a significant decrease in endothelial barrier resistance and an increase in sodium fluorescein migration across the endothelial barrier. Reduction in endothelial barrier resistance demonstrated compromised barrier integrity, whereas the increase in migration of sodium fluorescein across the barrier indicated the increase in barrier permeability. The present study revealed microRNA-101 decreases the expression of VE-cadherin and claudin-5 in brain endothelial cells exposed to the hypoxic conditions.

Heterogeneity of endothelial VE-PTP downstream polarization, Tie2 activation, junctional claudin-5, and permeability in the aorta and vena cava.

Endothelial cells of mammalian blood vessels have multiple levels of heterogeneity along the vascular tree and among different organs. Further heterogeneity results from blood flow turbulence and variations in shear stress. In the aorta, vascular endothelial protein tyrosine phosphatase (VE-PTP), which dephosphorylates tyrosine kinase receptor Tie2 in the plasma membrane, undergoes downstream polarization and endocytosis in endothelial cells exposed to laminar flow and high shear stress. VE-PTP sequestration promotes Tie2 phosphorylation at tyrosine992 and endothelial barrier tightening. The present study characterized the heterogeneity of VE-PTP polarization, Tie2-pY992 and total Tie2, and claudin-5 in anatomically defined regions of endothelial cells in the mouse descending thoracic aorta, where laminar flow is variable and IgG extravasation is patchy. We discovered that VE-PTP and Tie2-pY992 had mosaic patterns, unlike the uniform distribution of total Tie2. Claudin-5 at tight junctions also had a mosaic pattern, whereas VE-cadherin at adherens junctions bordered all endothelial cells. Importantly, the amounts of Tie2-pY992 and claudin-5 in aortic endothelial cells correlated with downstream polarization of VE-PTP. VE-PTP and Tie2-pY992 also had mosaic patterns in the vena cava, but claudin-5 was nearly absent and extravasated IgG was ubiquitous. Correlation of Tie2-pY992 and claudin-5 with VE-PTP polarization supports their collective interaction in the regulation of endothelial barrier function in the aorta, yet differences between the aorta and vena cava indicate additional flow-related determinants of permeability. Together, the results highlight new levels of endothelial cell functional mosaicism in the aorta and vena cava, where blood flow dynamics are well known to be heterogeneous.

Metabolomics study reveals increased deoxycholic acid contributes to deoxynivalenol-mediated intestinal barrier injury.

AIMS: Deoxynivalenol (DON), namely vomitoxin, is one of the most prevalent fungal toxins in cereal crops worldwide. However, the underlying toxic mechanisms of DON remain largely unknown. MAIN METHODS: DON exposure-caused changes in the murine plasma metabolome and gut microbiome were investigated by an LC-MS/MS-based nontargeted metabolomics approach and sequencing of 16S rRNA in fecal samples, respectively. Cellular models were then used to validate the findings from the metabolomics study. KEY FINDINGS: DON exposure increased intestinal barrier permeability evidenced by its-mediated decrease in colonic Claudin 5 and E-cadherin, as well as increases in colonic Ifn-γ, Cxcl9, Cxcl10, and Cxcr3. Furthermore, DON exposure resulted in a significant increase in murine plasma levels of deoxycholic acid (DCA). Also, DON exposure led to gut microbiota dysbiosis, which was associated with DON exposure-caused increase in plasma DCA. In addition, we found not only DON but also DCA dose-dependently caused a significant increase in the levels of IFN-γ, CXCL9, CXCL10, and/or CXCR3, as well as a significant decrease in the expression levels of Claudin 5 and/or E-cadherin in the human colonic epithelial cells (NCM460). SIGNIFICANCE: DON-mediated increase in DCA contributes to DON-caused intestinal injury. DCA may be a potential therapeutic target for DON enterotoxicity.

Teriflunomide Promotes Blood-Brain Barrier Integrity by Upregulating Claudin-1 via the Wnt/ß-catenin Signaling Pathway in Multiple Sclerosis.

The blood-brain barrier (BBB) and tight junction (TJ) proteins maintain the homeostasis of the central nervous system (CNS). The dysfunction of BBB allows peripheral T cells infiltration into CNS and contributes to the pathophysiology of multiple sclerosis (MS). Teriflunomide is an approved drug for the treatment of MS by suppressing lymphocytes proliferation. However, whether teriflunomide has a protective effect on BBB in MS is not understood. We found that teriflunomide restored the injured BBB in the EAE model. Furthermore, teriflunomide treatment over 6 months improved BBB permeability and reduced peripheral leakage of CNS proteins in MS patients. Teriflunomide increased human brain microvascular endothelial cell (HBMEC) viability and promoted BBB integrity in an in vitro cell model. The TJ protein claudin-1 was upregulated by teriflunomide and responsible for the protective effect on BBB. Furthermore, RNA sequencing revealed that the Wnt signaling pathway was affected by teriflunomide. The activation of Wnt signaling pathway increased claudin-1 expression and reduced BBB damage in cell model and EAE rats. Our study demonstrated that teriflunomide upregulated the expression of the tight junction protein claudin-1 in endothelial cells and promoted the integrity of BBB through Wnt signaling pathway.

Protective Effects of Danmu Extract Syrup on Acute Lung Injury Induced by Lipopolysaccharide in Mice through Endothelial Barrier Repair.

OBJECTIVE: To investigate the effects of Danmu Extract Syrup (DMS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism. METHODS: Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table (n=12), including control (normal saline), LPS (5 mg/kg), LPS+DMS 2.5 mL/kg, LPS+DMS 5 mL/kg, LPS+DMS 10 mL/kg, and LPS+Dexamethasone (DXM, 5 mg/kg) groups. After pretreatment with DMS and DXM, the ALI mice model was induced by LPS, and the bronchoalveolar lavage fluid (BALF) were collected to determine protein concentration, cell counts and inflammatory cytokines. The lung tissues of mice were stained with hematoxylin-eosin, and the wet/dry weight ratio (W/D) of lung tissue was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1 ß in BALF of mice were detected by enzyme linked immunosorbent assay. The expression levels of Claudin-5, vascular endothelial (VE)-cadherin, vascular endothelial growth factor (VEGF), phospho-protein kinase B (p-Akt) and Akt were detected by Western blot analysis. RESULTS: DMS pre-treatment significantly ameliorated lung histopathological changes. Compared with the LPS group, the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment (P<0.05 or P<0.01). The number of cells in BALF and myeloperoxidase (MPO) activity decreased significantly after DMS pretreatment (P<0.05 or P<0.01). DMS pre-treatment decreased the levels of TNF-α, IL-6 and IL-1 ß (P<0.01). Meanwhile, DMS activated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway and reversed the expressions of Claudin-5, VE-cadherin and VEGF (P<0.01). CONCLUSIONS: DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier. It might be a potential therapeutic drug for LPS-induced lung injury.