Reduced Barrier Function Research Articles

SLC37A4, gene responsible for glycogen storage disease type 1b, regulates gingival epithelial barrier function via JAM1 expression

Solute carrier family 37 member 4 (SLC37A4) is known to regulate glucose-6-phosphate transport from cytoplasm to the lumen of the endoplasmic reticulum, which serves to maintain glucose homeostasis. Glycogen storage disease type 1b (GSD1b) is caused by a mutation of SLC37A4, leading to a glycogenolysis defect. Although GSD1b cases are known to be complicated by periodontitis, the etiological molecular basis remains unclear. The present study investigated the effects of SLC37A4 on gingival barrier function. Examinations of immortalized human gingival epithelial (IHGE) cells showed SLC37A4 localized in the endoplasmic reticulum. SLC37A4 knockout decreased expression of JAM1, a tight junction-related protein, in IHGE cells. Using in silico analysis to investigate potential transcription factor binding sites, H6 family homeobox 3 (HMX3) was shown to be related to JAM1 expression. In HMX3-knockdown IHGE cells, JAM1 expression was markedly suppressed. Furthermore, HMX3 was scarcely detected in SLC37A4-knockout cells, while HMX3 overexpression restored JAM1 expression in those cells. Finally, using a three-dimensional multilayered gingival epithelial tissue model, knockout of SLC37A4 was also found to increase permeability to lipopolysaccharide and peptidoglycan, which was dependent on JAM1 expression. Specific downregulation of HMX3 by SLC37A4 and the consequent decrease in JAM1 expression provides findings indicating a molecular basis for the reduction in barrier function of gingival epithelial tissues in GSD1b cases.

Lesional Psoriasis is Associated With Alterations in the Stratum Corneum Ceramide Profile and Concomitant Decreases in Barrier Function.

Psoriasis is an inflammatory skin disease associated with an impaired skin barrier. The skin barrier function is dependent on the extracellular lipid matrix which surrounds the corneocytes in the stratum corneum. Ceramides comprise essential components of this matrix. Alterations in the stratum corneum ceramide profile have been directly linked to barrier dysfunction and might be an underlying factor of the barrier impairment in psoriasis. In this study, we investigated the ceramide profile and barrier function in psoriasis. Lesional and non-lesional skin of 26 patients and 10 healthy controls were analysed using in-depth ceramide lipidomics by liquid chromatography-mass spectrometry. Barrier function was assessed by measuring transepidermal water loss. Lesional skin showed a significant decrease in the abundance of total ceramides with significant alterations in the ceramide subclass composition compared to control and non-lesional skin. Additionally, the percentage of monounsaturated ceramides was significantly increased, and the average ceramide chain length significantly decreased in lesional skin. Altogether, this resulted in a markedly different profile compared to controls for lesional skin, but not for non-lesional skin. Importantly, the reduced barrier function in lesional psoriasis correlated to alterations in the ceramide profile, highlighting their interdependence. By assessing the parameters 2 weeks apart, we are able to highlight the reproducibility of these findings, which further affirms this connection. To conclude, we show that changes in the ceramide profile and barrier impairment are observed in, and limited to, lesional psoriatic skin. Their direct correlation provides a further mechanistic basis for the concomitantly observed impairment of barrier dysfunction.

Monocyte-driven inflamm-aging reduces intestinal barrier function in females.

The intestinal barrier encompasses physical and immunological components that act to compartmentalize luminal contents, such as bacteria and endotoxins, from the host. It has been proposed that an age-related decline of intestinal barrier function may allow for the passage of luminal contents into the bloodstream, triggering a low-grade systemic inflammation termed inflamm-aging. Although there is mounting evidence to support this hypothesis in model species, it is unclear if this phenomenon occurs in humans. In addition, despite being well-established that biological sex impacts aging physiology, its influence on intestinal barrier function and inflamm-aging has not been explored. In this study, we observed sex differences in markers of intestinal barrier integrity, where females had increased epithelial permeability throughout life as compared to males. With age, females had an age-associated increase in circulating bacterial products and metabolites such as LPS and kynurenine, suggesting reduced barrier function. Females also had age-associated increases in established markers of inflamm-aging, including peripheral blood monocytes as well as TNF and CRP. To determine if impaired barrier function was driving inflamm-aging, we performed a mediation analysis. The results show that the loss of intestinal barrier integrity was not the mediator of inflamm-aging in humans. Instead, persistent, low-grade inflammation with age preceded the increase in circulating bacterial products, which we confirmed using animal models. We found, as in humans, that sex modified age-associated increases in circulating monocytes in mice, and that inflammation mediates the loss of intestinal barrier function. Taken together, our results suggest that higher basal intestinal permeability in combination with age-associated inflammation, increases circulating LPS in females. Thus, targeting barrier permeability in females may slow the progression of inflamm-aging, but is unlikely to prevent it.

CLDN1 knock out keratinocytes as a model to investigate multiple skin disorders.

The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitisand various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis.

Do high-protein diets have the potential to reduce gut barrier function in a sex-dependent manner?

PurposeImpaired gut barrier function is associated with systemic inflammation and many chronic diseases. Undigested dietary proteins are fermented in the colon by the gut microbiota which produces nitrogenous metabolites shown to reduce barrier function in vitro. With growing evidence of sex-based differences in gut microbiotas, we determined whether there were sex by dietary protein interactions which could differentially impact barrier function via microbiota modification.MethodsFermentation systems were inoculated with faeces from healthy males (n = 5) and females (n = 5) and supplemented with 0.9 g of non-hydrolysed proteins sourced from whey, fish, milk, soya, egg, pea, or mycoprotein. Microbial populations were quantified using fluorescence in situ hybridisation with flow cytometry. Metabolite concentrations were analysed using gas chromatography, solid phase microextraction coupled with gas chromatography-mass spectrometry and ELISA.ResultsIncreased protein availability resulted in increased proteolytic Bacteroides spp (p < 0.01) and Clostridium coccoides (p < 0.01), along with increased phenol (p < 0.01), p-cresol (p < 0.01), indole (p = 0.018) and ammonia (p < 0.01), varying by protein type. Counts of Clostridium cluster IX (p = 0.03) and concentration of p-cresol (p = 0.025) increased in males, while females produced more ammonia (p = 0.02), irrespective of protein type. Further, we observed significant sex-protein interactions affecting bacterial populations and metabolites (p < 0.005).ConclusionsOur findings suggest that protein fermentation by the gut microbiota in vitro is influenced by both protein source and the donor’s sex. Should these results be confirmed through human studies, they could have major implications for developing dietary recommendations tailored by sex to prevent chronic illnesses.

Preeclampsia impedes foetal kidney development by delivering placenta-derived exosomes to glomerular endothelial cells

BackgroundFoetal renal dysplasia is still the main cause of adult renal disease. Placenta-derived exosomes are an important communication tool, and they may play an important role in placental (both foetal and maternal) function. We hypothesize that in women with preeclampsia, foetal renal dysplasia is impeded by delivering placenta-derived exosomes to glomerular endothelial cells.MethodsIn the present study, we established a PE trophoblast oxidative stress model to isolate exosomes from supernatants by ultracentrifugation (NO-exo and H/R-exo) and collected normal and PE umbilical cord blood plasma to isolate exosomes by ultracentrifugation combined with sucrose density gradient centrifugation (N-exo and PE-exo), then we investigated their effects on foetal kidney development by in vitro, ex vivo and in vivo models.ResultsThe PE trophoblast oxidative stress model was established successfully. After that, in in vitro studies, we found that H/R-exo and PE-exo could adversely affect glomerular endothelial cell proliferation, tubular formation, migration, and barrier functions. In ex vivo studies, H/R-exo and PE-exo both inhibited the growth and branch formation of kidney explants, along with the decrease of VE-cadherin and Occludin. In in vivo studies, we also found that H/R-exo and PE-exo could result in renal dysplasia, reduced glomerular number, and reduced barrier function in foetal mice.ConclusionsIn conclusion, we demonstrated that PE placenta-derived exosomes could lead to foetal renal dysplasia by delivering placenta-derived exosomes to foetal glomerular endothelial cells, which provides a novel understanding of the pathogenesis of foetal renal dysplasia.-vEjVMVHpL9r-BDAXuozRHVideo

Human organoids are superior to cell culture models for intestinal barrier research.

Loss of intestinal epithelial barrier function is a hallmark in digestive tract inflammation. The detailed mechanisms remain unclear due to the lack of suitable cell-based models in barrier research. Here we performed a detailed functional characterization of human intestinal organoid cultures under different conditions with the aim to suggest an optimized ex-vivo model to further analyse inflammation-induced intestinal epithelial barrier dysfunction. Differentiated Caco2 cells as a traditional model for intestinal epithelial barrier research displayed mature barrier functions which were reduced after challenge with cytomix (TNFα, IFN-γ, IL-1ß) to mimic inflammatory conditions. Human intestinal organoids grown in culture medium were highly proliferative, displayed high levels of LGR5 with overall low rates of intercellular adhesion and immature barrier function resembling conditions usually found in intestinal crypts. WNT-depletion resulted in the differentiation of intestinal organoids with reduced LGR5 levels and upregulation of markers representing the presence of all cell types present along the crypt-villus axis. This was paralleled by barrier maturation with junctional proteins regularly distributed at the cell borders. Application of cytomix in immature human intestinal organoid cultures resulted in reduced barrier function that was accompanied with cell fragmentation, cell death and overall loss of junctional proteins, demonstrating a high susceptibility of the organoid culture to inflammatory stimuli. In differentiated organoid cultures, cytomix induced a hierarchical sequence of changes beginning with loss of cell adhesion, redistribution of junctional proteins from the cell border, protein degradation which was accompanied by loss of epithelial barrier function. Cell viability was observed to decrease with time but was preserved when initial barrier changes were evident. In summary, differentiated intestinal organoid cultures represent an optimized human ex-vivo model which allows a comprehensive reflection to the situation observed in patients with intestinal inflammation. Our data suggest a hierarchical sequence of inflammation-induced intestinal barrier dysfunction starting with loss of intercellular adhesion, followed by redistribution and loss of junctional proteins resulting in reduced barrier function with consecutive epithelial death.

Патогенетические особенности формирования несостоятельности тазового дна у женщин репродуктивного возраста

Background. Today, the issue of genital prolapse remains in the spotlight all over the world. This condition accounts for 30–40% of all cases of gynecological diseases. It should be noted that a third of women are women of child-bearing age. The problem of understanding true prevalence of pelvic organ prolapse (РОР) in various age groups is that there are no severe clinical symptoms during early stages of the disease, which ham-pers the diagnosis and, consequently, assessment of the prevalence of genital prolapse. That is why the prevalence of genital prolapse in women of child-bearing age has been little studied in the world literature. The other feature of the РОР diagnosis in young women is that it is difficult to verify pelvic floor dysfunction (PFD) before POP manifestation. Patients often do not seek medical care, since the complaints they present with are not typical for the concept of PFD. The patients’ complaints most often turn out to be related to abnormal vaginal biocenosis or even inflam-matory diseases of the pelvis, sexual dysfunctions. No treatment of such conditions results in abnormal anatomic and topographic interaction be-tween vaginal walls, reduced barrier function of introitus vaginae and altered vaginal microbiota composition, therefore, the woman suffers from recurrent pain that significantly reduces her quality of life. Aim. To study the perineal tissue state during the development of PFD. Methods. The prospective randomized comparative cohort study involved 155 women, who contacted the Department of Gynecology for elective surgical treatment. All patients underwent standard clinical and instrumental assessment for gynecological patients: history taking, physical ex-amination, advanced gynecological assessment, pelvic ultrasound. The assessment program was expanded to complex perineological examination including pelvic floor evaluation according to the method by L.R. Toktar, perineal manometry using the iEase XFT-0010 perineometer (according to the unit user manual), transperineal ultrasound. In addition, evaluation of vaginal biotope and morphological assessment of the levator com-plex were performed. Results. According to the data obtained, abnormal vaginal biotope is the major atypical PFD symptom. This condition is recurrent. The vaginal wall loss of elasticity and abnormal collagenization result in the more profound perineal tissue alterations. Morphological assessment of perineal tissues has revealed levator ani inflammation in 20% of cases and fibrosis in 100% of cases. Conclusions. Recurrent vaginal biocenosis abnormality is a clinical symptom of PFD. Vaginal dysbiosis results in morphological alterations in the vaginal wall structure and probably in the entire levator complex.

M2a macrophages facilitate resolution of chemically-induced colitis in TLR4-SNP mice.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, impacts millions of individuals worldwide and severely impairs the quality of life for patients. Dysregulation of innate immune signaling pathways reduces barrier function and exacerbates disease progression. Macrophage (Mφ) signaling pathways are potential targets for IBD therapies. While multiple treatments are available for IBD, (i) not all patients respond, (ii) responses may diminish over time, and (iii) treatments often have undesirable side effects. Genetic studies have shown that the inheritance of two co-segregating SNPs expressed in the innate immune receptor, TLR4, is associated with human IBD. Mice expressing homologous SNPs ("TLR4-SNP" mice) exhibited more severe colitis than WT mice in a DSS-induced colonic inflammation/repair model. We identified a critical role for M2a "tissue repair" Mφ in the resolution of colitis. Our findings provide insight into potential development of novel therapies targeting Mφ signaling pathways that aim to alleviate the debilitating symptoms experienced by individuals with IBD.

Fiber deprivation and microbiome-borne curli shift gut bacterial populations and accelerate disease in a mouse model of Parkinson’s disease

Parkinson's disease (PD) is a neurological disorder characterized by motor dysfunction, dopaminergic neuron loss, and alpha-synuclein (αSyn) inclusions. Many PD risk factors are known, but those affecting disease progression are not. Lifestyle and microbial dysbiosis are candidates in this context. Diet-driven gut dysbiosis and reduced barrier function may increase exposure of enteric neurons to toxins. Here, we study whether fiber deprivation and exposure to bacterial curli, a protein cross-seeding with αSyn, individually or together, exacerbate disease in the enteric and central nervous systems of a transgenic PD mouse model. We analyze the gut microbiome, motor behavior, and gastrointestinal and brain pathologies. We find that diet and bacterial curli alter the microbiome and exacerbate motor performance, as well as intestinal and brain pathologies, but to different extents. Our results shed important insights on how diet and microbiome-borne insults modulate PD progression via the gut-brain axis and have implications for lifestyle management of PD.

Brain microvascular endothelial cells and blood-brain barrier dysfunction in psychotic disorders.

Although there is convergent evidence for blood-brain barrier (BBB) dysfunction and peripheral inflammation in schizophrenia (SZ) and bipolar disorder (BD), it is unknown whether BBB deficits are intrinsic to brain microvascular endothelial cells (BMECs) or arise via effects of peripheral inflammatory cytokines. We examined BMEC function using stem cell-based models to identify cellular and molecular deficits associated with BBB dysfunction in SZ and BD. Induced pluripotent stem cells (iPSCs) from 4 SZ, 4 psychotic BD and 4 healthy control (HC) subjects were differentiated into BMEC-"like" cells. Gene expression and protein levels of tight junction proteins were assessed. Transendothelial electrical resistance (TEER) and permeability were assayed to evaluate BBB function. Cytokine levels were measured from conditioned media. BMECs derived from human iPSCs in SZ and BD did not show differences in BBB integrity or permeability compared to HC BMECs. Outlier analysis using TEER revealed a BBB-deficit (n = 3) and non-deficit (n = 5) group in SZ and BD lines. Stratification based on BBB function in SZ and BD patients identified a BBB-deficit subtype with reduced barrier function, tendency for increased permeability to smaller molecules, and decreased claudin-5 (CLDN5) levels. BMECs from the BBB-deficit group show increased matrix metallopeptidase 1 (MMP1) activity, which correlated with reduced CLDN5 and worse BBB function, and was improved by tumor necrosis factor α (TNFα) and MMP1 inhibition. These results show potential deficits in BMEC-like cells in psychotic disorders that result in BBB disruption and further identify TNFα and MMP1 as promising targets for ameliorating BBB deficits.

Altered structure indicating reduced barrier function of lesional compared to non-lesional psoriatic skin-A non-invasive in vivo study of the human stratum corneum with confocal Raman micro-spectroscopy.

Psoriasis, one of the most common skin diseases affecting roughly 2%-3% of the world population, is associated with a reduced skin barrier function (SBF) that might play an important role in its pathophysiology. The SBF is provided primarily by the stratum corneum (SC) of the skin. Previous studies have revealed a higher trans-epidermal water loss, lower hydration, abnormal concentration and composition of intercellular lipids, as well as alterations in secondary keratin structure in the psoriatic SC. We compared on molecular level lesional psoriatic skin (LPS) with non-lesional psoriatic skin (nLPS) from 19 patients non-invasively in vivo, using confocal Raman micro-spectroscopy. By analysing the corresponding Raman spectra, we determined SBF-defining parameters of the SC depth-dependently. Our results revealed a lower total lipid concentration, a shift of lamellar lipid organisation towards more gauche-conformers and an increase of the less dense hexagonal lateral packing of the intercellular lipids in LPS. Furthermore, we observed lower natural moisturising factor concentration, lower total water as well as a strong tendency towards less strongly bound and more weakly bound water molecules in LPS. Finally, we detected a less stable secondary keratin structure with increased β-sheets, in contrast to the tertiary structure, showing a higher degree of folded keratin in LPS. These findings clearly suggest structural differences indicating a reduced SBF in LPS, and are discussed in juxtaposition to preceding outcomes for psoriatic and healthy skin. Understanding the alterations of the psoriatic SC provides insights into the exact pathophysiology of psoriasis and paves the way for optimal future treatments.

Healthy and diseased placental barrier on-a-chip models suitable for standardized studies.

Pathologies associated with uteroplacental hypoxia, such as preeclampsia are among the leading causes of maternal and perinatal morbidity in the world. Its fundamental mechanisms are yet poorly understood due to a lack of good experimental models. Here we report an in vitro model of the placental barrier, based on co-culture of trophoblasts and endothelial cells against a collagen extracellular matrix in a microfluidic platform. The model yields a functional syncytium with barrier properties, polarization, secretion of relevant extracellular membrane components, thinning of the materno-fetal space, hormone secretion, and transporter function. The model is exposed to low oxygen conditions and perfusion flow is modulated to induce a pathological environment. This results in reduced barrier function, hormone secretion, and microvilli as well as an increased nuclei count, characteristics of preeclamptic placentas. The model is implemented in a titer plate-based microfluidic platform fully amenable to high-throughput screening. We thus believe this model could aid mechanistic understanding of preeclampsia and other placental pathologies associated with hypoxia/ischemia, as well as support future development of effective therapies through target and compound screening campaigns. STATEMENT OF SIGNIFICANCE: : The human placenta is a unique organ sustaining fetus growth but is also the source of severe pathologies, such as Preeclampsia. Though leading cause of perinatal mortality in the world, preeclampsia remains untreatable due to a lack of relevant in vitro placenta models. To better understand the pathology, we have developed 3D placental barrier models in a microfluidic device. The platform allows parallel culture of 40 perfused physiological miniaturized placental barriers, comprising a differentiated syncytium and endothelium that have been validated for transporter functions. Exposure to a hypoxic and ischemic environment enabled the mimicking of preeclamptic characteristics in high-throughput, which we believe could lead to a better understanding of the pathology as well as support future effective therapies development.

Shiga Toxin (Stx) Type 1a and Stx2a Translocate through a Three-Layer Intestinal Model.

Shiga toxins (Stxs) produced by ingested E. coli can induce hemolytic uremic syndrome after crossing the intact intestinal barrier, entering the bloodstream, and targeting endothelial cells in the kidney. The method(s) by which the toxins reach the bloodstream are not fully defined. Here, we used two polarized cell models to evaluate Stx translocation: (i) a single-layer primary colonic epithelial cell model and (ii) a three-cell-layer model with colonic epithelial cells, myofibroblasts, and colonic endothelial cells. We traced the movement of Stx types 1a and 2a across the barrier models by measuring the toxicity of apical and basolateral media on Vero cells. We found that Stx1a and Stx2a crossed both models in either direction. However, approximately 10-fold more Stx translocated in the three-layer model as compared to the single-layer model. Overall, the percentage of toxin that translocated was about 0.01% in the epithelial-cell-only model but up to 0.09% in the three-cell-layer model. In both models, approximately 3- to 4-fold more Stx2a translocated than Stx1a. Infection of the three-cell-layer model with Stx-producing Escherichia coli (STEC) strains showed that serotype O157:H7 STEC reduced barrier function in the model and that the damage was not dependent on the presence of the eae gene. Infection of the three-layer model with O26:H11 STEC strain TW08571 (Stx1a+ and Stx2a+), however, allowed translocation of modest amounts of Stx without reducing barrier function. Deletion of stx2a from TW08571 or the use of anti-Stx1 antibody prevented translocation of toxin. Our results suggest that single-cell models may underestimate the amount of Stx translocation and that the more biomimetic three-layer model is suited for Stx translocation inhibitor studies.

Transcriptomic analysis of a 3D blood–brain barrier model exposed to disturbed fluid flow

Cerebral aneurysms are more likely to form at bifurcations in the vasculature, where disturbed fluid is prevalent due to flow separation at sufficiently high Reynolds numbers. While previous studies have demonstrated that altered shear stress exerted by disturbed flow disrupts endothelial tight junctions, less is known about how these flow regimes alter gene expression in endothelial cells lining the blood–brain barrier. Specifically, the effect of disturbed flow on expression of genes associated with cell–cell and cell–matrix interaction, which likely mediate aneurysm formation, remains unclear. RNA sequencing of immortalized cerebral endothelial cells isolated from the lumen of a 3D blood–brain barrier model reveals distinct transcriptional changes in vessels exposed to fully developed and disturbed flow profiles applied by both steady and physiological waveforms. Differential gene expression, validated by qRT-PCR and western blotting, reveals that lumican, a small leucine-rich proteoglycan, is the most significantly downregulated gene in endothelial cells exposed to steady, disturbed flow. Knocking down lumican expression reduces barrier function in the presence of steady, fully developed flow. Moreover, adding purified lumican into the hydrogel of the 3D blood–brain barrier model recovers barrier function in the region exposed to fully developed flow. Overall, these findings emphasize the importance of flow regimes exhibiting spatial and temporal heterogeneous shear stress profiles on cell–matrix interaction in endothelial cells lining the blood–brain barrier, while also identifying lumican as a contributor to the formation and maintenance of an intact barrier.

EpCAM proteolysis and release of complexed claudin-7 repair and maintain the tight junction barrier.

TJs maintain the epithelial barrier by regulating paracellular permeability. Since TJs are under dynamically fluctuating intercellular tension, cells must continuously survey and repair any damage. However, the underlying mechanisms allowing cells to sense TJ damage and repair the barrier are not yet fully understood. Here, we showed that proteinases play an important role in the maintenance of the epithelial barrier. At TJ break sites, EpCAM-claudin-7 complexes on the basolateral membrane become accessible to apical membrane-anchored serine proteinases (MASPs) and the MASPs cleave EpCAM. Biochemical data and imaging analysis suggest that claudin-7 released from EpCAM contributes to the rapid repair of damaged TJs. Knockout (KO) of MASPs drastically reduced barrier function and live-imaging of TJ permeability showed that MASPs-KO cells exhibited increased size, duration, and frequency of leaks. Together, our results reveal a novel mechanism of TJ maintenance through the localized proteolysis of EpCAM at TJ leaks, and provide a better understanding of the dynamic regulation of epithelial permeability.

Role of Endothelial STAT3 in Cerebrovascular Function and Protection from Ischemic Brain Injury.

STAT3 plays a protective role against ischemic brain injury; however, it is not clear which brain cell type mediates this effect, and by which mechanism. We tested the hypothesis that endothelial STAT3 contributes to protection from cerebral ischemia, by preserving cerebrovascular endothelial function and blood–brain barrier (BBB) integrity. The objective of this study was to determine the role of STAT3 in cerebrovascular endothelial cell (EC) survival and function, and its role in tissue outcome after cerebral ischemia. We found that in primary mouse brain microvascular ECs, STAT3 was constitutively active, and its phosphorylation was reduced by oxygen-glucose deprivation (OGD), recovering after re-oxygenation. STAT3 inhibition, using two mechanistically different pharmacological inhibitors, increased EC injury after OGD. The sub-lethal inhibition of STAT3 caused endothelial dysfunction, demonstrated by reduced nitric oxide release in response to acetylcholine and reduced barrier function of the endothelial monolayer. Finally, mice with reduced endothelial STAT3 (Tie2-Cre; STAT3flox/wt) sustained larger brain infarcts after middle cerebral artery occlusion (MCAO) compared to wild-type (WT) littermates. We conclude that STAT3 is vital to maintaining cerebrovascular integrity, playing a role in EC survival and function, and protection against cerebral ischemia. Endothelial STAT3 may serve as a potential target in preventing endothelial dysfunction after stroke.

Interferon gamma upregulates the cytokine receptors IFNGR1 and TNFRSF1A in HT-29-MTX E12 cells.

The intestinal mucosa protects the body from physical damage, pathogens, and antigens. However, inflammatory bowel diseases (IBDs) patients suffer from poor mucosal tissue function, including the lack of an effective cellular and/or mucus barrier. We investigated the mucus producing human colonic epithelial cell line HT29-MTX E12 to study its suitability as an in vitro model of cell/mucus barrier adaption during IBD. It was found that the proinflammatory cytokine interferon-gamma (IFN-γ), but not tumor necrosis factor-alpha (TNF-α), reduced cell viability. IFN-γ and TNF-α were found to synergize to decrease barrier function, as measured by trans-epithelial electric resistance (TER) and molecular flux assays. Cells cultured under an air-liquid interface produced an adherent mucus layer, and under these conditions reduced barrier function was found after cytokine exposure. Furthermore, IFN-γ, but not TNF-α treatment, upregulated the IFN-γ receptor 1 (IFNGR1) and TNF-α receptor super family 1A (TNFRSF1A) subunit mRNA in vitro. Co-stimulation resulted in increased mRNA expression of CLDN 2 and 5, two gene known to play a role in epithelial barrier integrity. Analysis of IBD patient samples revealed IFNGR1 and TNFRSF mRNA increased coincidently with guanylate binding protein 1 (GBP1) expression, an indicator of NFkB activity. Lastly, CLDN2 was found at higher levels in IBD patients while HNF4a was suppressed with disease. In conclusion, IFN-γ and TNF-α degrade epithelial/mucus barriers coincident with changes in CLDN gene and cytokine receptor subunit mRNA expression in HT29-MTX E12 cells. These changes largely reflect those observed in IBD patient samples.

Bioresonance According to Paul Schmidt (BaPS) and its Beneficial Effects on the Integrity of the Intestinal Barrier in vitro

The intestinal epithelium, which is only one cell layer thick, has two essential tasks. The first is to create a physical barrier between the contents of the intestinal lumen and the rest of our body. The second is to ensure an efficient absorption of essential nutrients from the gut lumen and to produce mucus, anti-microbial peptides and cytokines with both protective and immune-regulatory properties. Thus, a reduced barrier function may have consequences, not only for intestinal, but also for systemic health. In this study we investigated whether the use of a Mini-Rayonex device, which has been developed to emit a bioresonance frequency spectrum according to Paul Schmidt (BaPS), might be able to promote and maintain the integrity of the intestinal barrier. Intestinal epithelial cells (IPEC-J2) were cultivated on microporous transwell plates to build up an intestinal barrier after exposure to a Mini-Rayonex device with the dipole antenna system (= verum). Corresponding control cultures were treated in the same manner but were exposed to a non-working Mini-Rayonex device without the dipole antenna system (= placebo). The transepithelial electrical resistance was measured as an indicator of intestinal barrier integrity. Thereafter, the achieved intestinal barrier from both Mini-Rayonex devices was exposed to 500 and 1,000 µM H2 O2 for 30 hours and the transepithelial electrical resistance was measured again. Moreover, the effect of both Mini-Rayonex devices (verum and placebo) on the regeneration process of intestinal epithelial cells at conditions of oxidative stress was investigated via examination of the closure of a cell-free area. Exposure of the cells to the actively emitting Mini-Rayonex device strengthened the intestinal barrier more than 30 % in comparison to the non-emitting Mini-Rayonex device. Moreover, the strengthened intestinal barrier was significantly more resistant by 20 % towards 500 µM H2 O2 and about 30 % towards 1,000 µM H2 O2 after exposure to the actively emitting Mini-Rayonex device than the “normal” intestinal barrier after exposure to the non-emitting device. In accordance with this observation was the result that the regeneration process after traumatization or injury of intestinal epithelial cells was increased by the actively emitting Mini-Rayonex device by more than 12 % in direct comparison with the non-emitting device. We conclude that the use of the actively emitting Mini-Rayonex device in vivo might also result in an improved intestinal epithelial barrier integrity, function and regeneration, which might improve and maintain well-being and systemic health.

Electric Cell-Substrate Impedance Sensing (ECIS) as a Platform for Evaluating Barrier-Function Susceptibility and Damage from Pulmonary Atelectrauma.

Biophysical insults that either reduce barrier function (COVID-19, smoke inhalation, aspiration, and inflammation) or increase mechanical stress (surfactant dysfunction) make the lung more susceptible to atelectrauma. We investigate the susceptibility and time-dependent disruption of barrier function associated with pulmonary atelectrauma of epithelial cells that occurs in acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). This in vitro study was performed using Electric Cell-substrate Impedance Sensing (ECIS) as a noninvasive evaluating technique for repetitive stress stimulus/response on monolayers of the human lung epithelial cell line NCI-H441. Atelectrauma was mimicked through recruitment/derecruitment (RD) of a semi-infinite air bubble to the fluid-occluded micro-channel. We show that a confluent monolayer with a high level of barrier function is nearly impervious to atelectrauma for hundreds of RD events. Nevertheless, barrier function is eventually diminished, and after a critical number of RD insults, the monolayer disintegrates exponentially. Confluent layers with lower initial barrier function are less resilient. These results indicate that the first line of defense from atelectrauma resides with intercellular binding. After disruption, the epithelial layer community protection is diminished and atelectrauma ensues. ECIS may provide a platform for identifying damaging stimuli, ventilation scenarios, or pharmaceuticals that can reduce susceptibility or enhance barrier-function recovery.