Polarized HLA Class I Expression on Renal Tubules Hinders the Detection of Donor-Specific Urinary Extracellular Vesicles.

Purpose: Kidney transplantation is the optimal treatment for patients with end-stage kidney disease. Donor-specific urinary extracellular vesicles (uEVs) hold potential as biomarkers for assessing allograft status. We aimed to develop a method for identifying donor-specific uEVs based on human leukocyte antigen (HLA) mismatching with the kidney transplant recipients (KTRs). Patients and Methods: Urine and plasma were obtained from HLA-A2+ donors and HLA-A2- KTRs pre-transplant. CD9 (tetraspanin, EV marker) and HLA-A2 double-positive (CD9+ HLA-A2+) EVs were quantified using isolation-free imaging flow cytometry (IFCM). Healthy individuals' urine was used to investigate CD9+ HLA-class-I+ uEV quantification using IFCM, time-resolved fluoroimmunoassay (TR-FIA), and immunogold staining cryo-electron microscopy (cryo-EM). Culture-derived CD9+ HLA-class-I+ EVs were spiked into the urine to investigate urine matrix effects on uEV HLA detection. Deceased donor kidneys and peritumoral kidney tissue were used for HLA class I detection with histochemistry. Results: The concentrations of CD9+ HLA-A2+ EVs in both donor and recipient urine approached the negative (detergent-treated) control levels for IFCM and were significantly lower than those observed in donor plasma. In parallel, universal HLA class I+ uEVs were similarly undetectable in the urine and uEV isolates compared with plasma, as verified by IFCM, TR-FIA, and cryogenic electron microscopy. Culture supernatant containing HLA class I+ vesicles from B, T, and human proximal tubule cells were spiked into the urine, and these EVs remained stable at 37°C for 8 hours. Immunohistochemistry revealed that HLA class I was predominantly expressed on the basolateral side of renal tubules, with limited expression on their urine/apical side. Conclusion: The detection of donor-specific uEVs is hindered by the limited release of HLA class I+ EVs from the kidney into the urine, primarily due to the polarized HLA class I expression on renal tubules. Identifying donor-specific uEVs requires further advancements in recognizing transplant-specific uEVs and urine-associated markers.

An in vitro model system based on calcium- and phosphate ion-induced hMSC spheroid mineralization.

A challenge in regenerative medicine is creating the three-dimensional organic and inorganic in vitro microenvironment of bone, which would allow the study of musculoskeletal disorders and the generation of building blocks for bone regeneration. This study presents a microwell-based platform for creating spheroids of human mesenchymal stromal cells, which are then mineralized using ionic calcium and phosphate supplementation. The resulting mineralized spheroids promote an osteogenic gene expression profile through the influence of the spheroids' biophysical environment and inorganic signaling and require less calcium or phosphate to achieve mineralization compared to a monolayer culture. We found that mineralized spheroids represent an in vitro model for studying small molecule perturbations and extracellular mediated calcification. Furthermore, we demonstrate that understanding pathway signaling elicited by the spheroid environment allows mimicking these pathways in traditional monolayer culture, enabling similar rapid mineralization events. In sum, this study demonstrates the rapid generation and employment of a mineralized cell model system for regenerative medicine applications.

Charging of Vitreous Samples in Cryogenic Electron Microscopy Mitigated by Graphene.

Cryogenic electron microscopy can provide high-resolution reconstructions of macromolecules embedded in a thin layer of ice from which atomic models can be built de novo. However, the interaction between the ionizing electron beam and the sample results in beam-induced motion and image distortion, which limit the attainable resolutions. Sample charging is one contributing factor of beam-induced motions and image distortions, which is normally alleviated by including part of the supporting conducting film within the beam-exposed region. However, routine data collection schemes avoid strategies whereby the beam is not in contact with the supporting film, whose rationale is not fully understood. Here we characterize electrostatic charging of vitreous samples, both in imaging and in diffraction mode. We mitigate sample charging by depositing a single layer of conductive graphene on top of regular EM grids. We obtained high-resolution single-particle analysis (SPA) reconstructions at 2 Å when the electron beam only irradiates the middle of the hole on graphene-coated grids, using data collection schemes that previously failed to produce sub 3 Å reconstructions without the graphene layer. We also observe that the SPA data obtained with the graphene-coated grids exhibit a higher b factor and reduced particle movement compared to data obtained without the graphene layer. This mitigation of charging could have broad implications for various EM techniques, including SPA and cryotomography, and for the study of radiation damage and the development of future sample carriers. Furthermore, it may facilitate the exploration of more dose-efficient, scanning transmission EM based SPA techniques.

In Situ Covalent Reinforcement of a Benzene-1,3,5-Tricarboxamide Supramolecular Polymer Enables Biomimetic, Tough, and Fibrous Hydrogels and Bioinks.

Synthetic hydrogels often lack the load-bearing capacity and mechanical properties of native biopolymers found in tissue, such as cartilage. In natural tissues, toughness is often imparted via the combination of fibrous noncovalent self-assembly with key covalent bond formation. This controlled combination of supramolecular and covalent interactions remains difficult to engineer, yet can provide a clear strategy for advanced biomaterials. Here, a synthetic supramolecular/covalent strategy is investigated for creating a tough hydrogel that embodies the hierarchical fibrous architecture of the extracellular matrix (ECM). A benzene-1,3,5-tricarboxamide (BTA) hydrogelator is developed with synthetically addressable norbornene handles that self-assembles to form a and viscoelastic hydrogel. Inspired by collagen's covalent cross-linking of fibrils, the mechanical properties are reinforced by covalent intra- and interfiber cross-links. At over 90% water, the hydrogels withstand up to 550% tensile strain, 90% compressive strain, and dissipated energy with recoverable hysteresis. The hydrogels are shear-thinning, can be 3D bioprinted with good shape fidelity, and can be toughened via covalent cross-linking. These materials enable the bioprinting of human mesenchymal stromal cell (hMSC) spheroids and subsequent differentiation into chondrogenic tissue. Collectively, these findings highlight the power of covalent reinforcement of supramolecular fibers, offering a strategy for the bottom-up design of dynamic, yet tough, hydrogels and bioinks.

Molecular Tuning of a Benzene-1,3,5-Tricarboxamide Supramolecular Fibrous Hydrogel Enables Control over Viscoelasticity and Creates Tunable ECM-Mimetic Hydrogels and Bioinks.

Traditional synthetic covalent hydrogels lack the native tissue dynamics and hierarchical fibrous structure found in the extracellular matrix (ECM). These dynamics and fibrous nanostructures are imperative in obtaining the correct cell/material interactions. Consequently, the challenge to engineer functional dynamics in a fibrous hydrogel and recapitulate native ECM properties remains a bottle-neck to biomimetic hydrogel environments. Here, the molecular tuning of a supramolecular benzene-1,3,5-tricarboxamide (BTA) hydrogelator via simple modulation of hydrophobic substituents is reported. This tuning results in fibrous hydrogels with accessible viscoelasticity over 5 orders of magnitude, while maintaining a constant equilibrium storage modulus. BTA hydrogelators are created with systematic variations in the number of hydrophobic carbon atoms, and this is observed to control the viscoelasticity and stress-relaxation timescales in a logarithmic fashion. Some of these BTA hydrogels are shear-thinning, self-healing, extrudable, and injectable, and can be 3D printed into multiple layers. These hydrogels show high cell viability for chondrocytes and human mesenchymal stem cells, establishing their use in tissue engineering applications. This simple molecular tuning by changing hydrophobicity (with just a few carbon atoms) provides precise control over the viscoelasticity and 3D printability in fibrillar hydrogels and can be ported onto other 1D self-assembling structures. The molecular control and design of hydrogel network dynamics can push the field of supramolecular chemistry toward the design of new ECM-mimicking hydrogelators for numerous cell-culture and tissue-engineering applications and give access toward highly biomimetic bioinks for bioprinting.

Extracellular histone release by renal cells after warm and cold ischemic kidney injury: Studies in an ex-vivo porcine kidney perfusion model.

Extracellular histones are cytotoxic molecules involved in experimental acute kidney injury. In patients receiving a renal transplant from donors after circulatory death, who suffer from additional warm ischemia, worse graft outcome is associated with higher machine perfusate extracellular histone H3 concentrations. We now investigated temperature-dependent extracellular histone release in an ex vivo porcine renal perfusion model, and subsequently studied histone release in the absence and presence of non-anticoagulant heparin. Seven pairs of ischemically damaged porcine kidneys were machine perfused at 4°C (cold ischemia) or 28°C (warm ischemia). Perfusate histone H3 concentration was higher after warm as compared to cold ischemia (median (IQR) = 0.48 (0.20-0.83) µg/mL vs. 0.02 (0.00-0.06) µg/mL; p = .045, respectively). Employing immune-electron microscopy (EM), histone containing cytoplasmic protrusions of tubular and endothelial cells were found after warm ischemic injury. Furthermore, abundant histone localization was detected in debris surrounding severely damaged glomerular cells, in a "buck shot" pattern. In vitro, histones were cytotoxic to endothelial and kidney epithelial cells in a temperature-dependent manner. In a separate ex vivo experiment, addition of heparin did not change the total histone H3 levels observed in the perfusate but revealed a continuous increase in the level of a lower molecular weight histone H3 variant. Our findings show that ischemically damaged kidneys release more extracellular histones in warm ischemia, which by EM was due to histone release by renal cells. Blocking of histone-mediated damage during transplantation may be beneficial in prevention of renal injury.

Desymmetrization via Activated Esters Enables Rapid Synthesis of Multifunctional Benzene-1,3,5-tricarboxamides and Creation of Supramolecular Hydrogelators.

Supramolecular materials based on the self-assembly of benzene-1,3,5-tricarboxamide (BTA) offer an approach to mimic fibrous self-assembled proteins found in numerous natural systems. Yet, synthetic methods to rapidly build complexity, scalability, and multifunctionality into BTA-based materials are needed. The diversity of BTA structures is often hampered by the limited flexibility of existing desymmetrization routes and the purification of multifunctional BTAs. To alleviate this bottleneck, we have developed a desymmetrization method based on activated ester coupling of a symmetric synthon. We created a small library of activated ester synthons and found that a pentafluorophenol benzene triester (BTE) enabled effective desymmetrization and creation of multifunctional BTAs in good yield with high reaction fidelity. This new methodology enabled the rapid synthesis of a small library of BTA monomers with hydrophobic and/or orthogonal reactive handles and could be extended to create polymeric BTA hydrogelators. These BTA hydrogelators self-assembled in water to create fiber and fibrous sheet-like structures as observed by cryo-TEM, and the identity of the BTA conjugated can tune the mechanical properties of the hydrogel. These hydrogelators display high cytocompatibility for chondrocytes, indicating potential for the use of these systems in 3D cell culture and tissue engineering applications. This newly developed synthetic strategy facilitates the simple and rapid creation of chemically diverse BTA supramolecular polymers, and the newly developed and scalable hydrogels can unlock exploration of BTA based materials in a wider variety of tissue engineering applications.

Secretion of pro-angiogenic extracellular vesicles during hypoxia is dependent on the autophagy-related protein GABARAPL1.

Tumour hypoxia is a hallmark of solid tumours and contributes to tumour progression, metastasis development and therapy resistance. In response to hypoxia, tumour cells secrete pro-angiogenic factors to induce blood vessel formation and restore oxygen supply to hypoxic regions. Extracellular vesicles (EVs) are emerging as mediators of intercellular communication in the tumour microenvironment. Here we demonstrate that increased expression of the LC3/GABARAP protein family member GABARAPL1, is required for endosomal maturation, sorting of cargo to endosomes and the secretion of EVs. Silencing GABARAPL1 results in a block in the early endosomal pathway and impaired secretion of EVs with pro-angiogenic properties. Tumour xenografts of doxycycline inducible GABARAPL1 knockdown cells display impaired vascularisation that results in decreased tumour growth, elevated tumour necrosis and increased therapy efficacy. Moreover, our data show that GABARAPL1 is expressed on the EV surface and targeting GABARAPL1+ EVs with GABARAPL1 targeting antibodies results in blockade of pro-angiogenic effects in vitro. In summary, we reveal that GABARAPL1 is required for EV cargo loading and secretion. GABARAPL1+ EVs are detectable and targetable and are therefore interesting to pursue as a therapeutic target.

Deletion of mTOR in liver epithelial cells enhances hepatic metastasis of colon cancer.

Activation of the mechanistic target of rapamycin (mTOR) pathway is frequently found in cancer, but mTOR inhibitors have thus far failed to demonstrate significant antiproliferative efficacy in the majority of cancer types. Besides cancer cell-intrinsic resistance mechanisms, it is conceivable that mTOR inhibitors impact on non-malignant host cells in a manner that ultimately supports resistance of cancer cells. Against this background, we sought to analyze the functional consequences of mTOR inhibition in hepatocytes for the growth of metastatic colon cancer. To this end, we established liver epithelial cell (LEC)-specific knockout (KO) of mTOR (mTORLEC ) mice. We used these mice to characterize the growth of colorectal liver metastases with or without partial hepatectomy to model different clinical settings. Although the LEC-specific loss of mTOR remained without effect on metastasis growth in intact liver, partial liver resection resulted in the formation of larger metastases in mTORLEC mice compared with wildtype controls. This was accompanied by significantly enhanced inflammatory activity in LEC-specific mTOR KO livers after partial liver resection. Analysis of NF-ĸB target gene expression and immunohistochemistry of p65 displayed a significant activation of NF-ĸB in mTORLEC mice, suggesting a functional importance of this pathway for the observed inflammatory phenotype. Taken together, we show an unexpected acceleration of liver metastases upon deletion of mTOR in LECs. Our results support the notion that non-malignant host cells can contribute to resistance against mTOR inhibitors and encourage testing whether anti-inflammatory drugs are able to improve the efficacy of mTOR inhibitors for cancer therapy. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interface culture system which was characterized by confocal and electron microscopy and single-cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing fetal lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive alveolar type II-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.

Electron microscopic observations in perfusion-fixed human non-alcoholic fatty liver disease biopsies.

Non-alcoholic fatty liver disease (NAFLD) is a widespread liver disease in Western society, but its multifactorial pathogenesis is not yet fully understood. Ultrastructural analysis of liver sinusoidal endothelial cells (LSECs) in animal models and in vitro studies shows defenestration early in the course of NAFLD, promoting steatosis. LSECs and fenestrae are important in the transport of lipids across the sinusoids. However, human ultrastructural data, especially on LSECs and fenestrae, are scarce. This study aimed to explore the ultrastructural changes in perfusion type fixed liver biopsies of NAFLD patients with and without non-alcoholic steatohepatitis (NASH), with a special focus on LSECs and their fenestration. Liver biopsies from patients with NAFLD were fixed using two perfusion techniques, jet and injection fixation, for needle and wedge biopsies, respectively. Ultrastructural changes were studied using transmission electron microscopy. NASH was diagnosed by bright-field microscopy using the SAF score (steatosis, activity, fibrosis). Thirty-seven patients were included, of which 12 (32.4%) had NASH. Significantly less defenestration was found in NASH compared to noNASH samples (p=0.002). Other features, i.e., giant mitochondria and fenestrae size did not differ between groups. Furthermore, we described new structures, i.e., single cell steatonecrosis and inflammatory fat follicles (IFF) that were observed in both groups. Concluding, defenestration was more common in noNASH compared to NASH in human liver samples. Defenestration was not related to the degree of steatosis or fibrosis. We speculate that defenestration can be a protective mechanism in simple steatosis which is lacking in NASH.

SARS-CoV-2 productively infects human gut enterocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause coronavirus disease 2019 (COVID-19), an influenza-like disease that is primarily thought to infect the lungs with transmission through the respiratory route. However, clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids (hSIOs), enterocytes were readily infected by SARS-CoV and SARS-CoV-2, as demonstrated by confocal and electron microscopy. Enterocytes produced infectious viral particles, whereas messenger RNA expression analysis of hSIOs revealed induction of a generic viral response program. Therefore, the intestinal epithelium supports SARS-CoV-2 replication, and hSIOs serve as an experimental model for coronavirus infection and biology.

Super-resolution microscopy localizes perilipin 5 at lipid droplet-mitochondria interaction sites and at lipid droplets juxtaposing to perilipin 2.

OBJECTIVE: Intramyocellular lipid droplets (LD) and their coat proteins PLIN2 and PLIN5 are involved in lipolysis, with a putative role for PLIN5 in mitochondrial tethering. Reportedly, these proteins co-localize and cover the surface of the LD. To provide the spatial basis for understanding how these proteins possess their distinct roles, we examined the precise location of PLIN2 and PLIN5 and explored PLIN5 presence at LD-mitochondria contact sites using Stimulated emission depletion (STED) microscopy and correlative light-electron microscopy (CLEM) in human skeletal muscle sections. METHODS: LDs were stained by MDH together with combinations of mitochondrial proteins and PLINs. Subcellular distribution and co-localization of PLIN proteins and mitochondria was imaged by STED microscopy (Leica TCS SP8) and quantified using Pearson's correlation coefficients and intensity profile plots. CLEM was employed to examine the presence of PLIN5 on mitochondria-LD contact sites. RESULTS: Both PLIN2 and PLIN5 localized to the LD in a dot-like, juxtaposed fashion rather than colocalizing and covering the entire LD. Both STED and CLEM revealed a high fraction of PLIN5 at the LD-mitochondria interface, but not at mitochondrial cristae, as suggested previously. CONCLUSION: Using two super-resolution imaging approaches, this is the first study to show that in sections of human skeletal muscle PLIN2 and PLIN5 localize to the LD at distinct sites, with abundance of PLIN5 at LD-mitochondria tethering sites. This novel spatial information uncovers that PLIN proteins do not serve as lipolytic barriers but rather are docking sites for proteins facilitating selective lipase access under a variety of lipolytic conditions.

Folic acid reduces doxorubicin-induced cardiomyopathy by modulating endothelial nitric oxide synthase.

The use of doxorubicin (DOXO) as a chemotherapeutic drug has been hampered by cardiotoxicity leading to cardiomyopathy and heart failure. Folic acid (FA) is a modulator of endothelial nitric oxide (NO) synthase (eNOS), which in turn is an important player in diseases associated with NO insufficiency or NOS dysregulation, such as pressure overload and myocardial infarction. However, the role of FA in DOXO-induced cardiomyopathy is poorly understood. The aim of this study was to test the hypothesis that FA prevents DOXO-induced cardiomyopathy by modulating eNOS and mitochondrial structure and function. Male C57BL/6 mice were randomized to a single dose of DOXO (20 mg/kg intraperitoneal) or sham. FA supplementation (10 mg/day per oral) was started 7 days before DOXO injection and continued thereafter. DOXO resulted in 70% mortality after 10 days, with the surviving mice demonstrating a 30% reduction in stroke volume compared with sham groups. Pre-treatment with FA reduced mortality to 45% and improved stroke volume (both P < 0.05 versus DOXO). These effects of FA were underlain by blunting of DOXO-induced cardiomyocyte atrophy, apoptosis, interstitial fibrosis and impairment of mitochondrial function. Mechanistically, pre-treatment with FA prevented DOXO-induced increases in superoxide anion production by reducing the eNOS monomer:dimer ratio and eNOS S-glutathionylation, and attenuated DOXO-induced decreases in superoxide dismutase, eNOS phosphorylation and NO production. Enhancing eNOS function by restoring its coupling and subsequently reducing oxidative stress with FA may be a novel therapeutic approach to attenuate DOXO-induced cardiomyopathy.

Silencing of Dok-7 in Adult Rat Muscle Increases Susceptibility to Passive Transfer Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies that target proteins at the neuromuscular junction, primarily the acetylcholine receptor (AChR) and the muscle-specific kinase. Because downstream of kinase 7 (Dok-7) is essential for the full activation of muscle-specific kinase and consequently for dense clustering of AChRs, we hypothesized that reduced levels of Dok-7 increase the susceptibility to passive transfer MG. To test this hypothesis, Dok-7 expression was reduced by transfecting shRNA-coding plasmids into the tibialis anterior muscle of adult rats by in vivo electroporation. Subclinical MG was subsequently induced with a low dose of anti-AChR monoclonal antibody 35. Neuromuscular transmission was significantly impaired in Dok-7-siRNA-electroporated legs compared with the contralateral control legs, which correlated with a reduction of AChR protein levels at the neuromuscular junction (approximately 25%) in Dok-7-siRNA-electroporated muscles, compared with contralateral control muscles. These results suggest that a reduced expression of Dok-7 may play a role in the susceptibility to passive transfer MG, by rendering AChR clusters less resistant to the autoantibody attack.

Can the Hydroxyapatite-Coated Skin-Penetrating Abutment for Bone Conduction Hearing Implants Integrate with the Surrounding Skin?

INTRODUCTION: Percutaneous implants, such as bone conduction hearing implants, suffer from complications that include inflammation of the surrounding skin. A sealed skin-abutment interface can prevent the ingress of bacteria, which should reduce the occurrence of peri-abutment dermatitis. It was hypothesized that a hydroxyapatite (HA)-coated abutment in conjunction with soft tissue preservation surgery should enable integration with the adjacent skin. Previous research has confirmed that integration is never achieved with as-machined titanium abutments. Here, we investigate, in vivo, if skin integration is achievable in patients using a HA-coated abutment. MATERIALS AND METHODS: One titanium abutment (control) and one HA-coated abutment (case) together with the surrounding skin were surgically retrieved from two patients who had a medical indication for this procedure. Histological sections of the skin were investigated using light microscopy. The abutment was qualitatively analyzed using scanning electron microscopy. RESULTS: The titanium abutment only had a partial and thin layer of attached amorphous biological material. The HA-coated abutment was almost fully covered by a pronounced thick layer of organized skin, composed of different interconnected structural layers. CONCLUSION: Proof-of-principle evidence that the HA-coated abutment can achieve integration with the surrounding skin was presented for the first time.

Proteasome inhibition with bortezomib depletes plasma cells and specific autoantibody production in primary thymic cell cultures from early-onset myasthenia gravis patients.

Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from nine early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 h and were no longer detectable at 24 h. Bortezomib therefore appears promising for treating MG and possibly other Ab-mediated autoimmune or allergic disorders, especially when given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.

Alveolocapillary model system to study alveolar re-epithelialization.

In the present study an in vitro bilayer model system of the pulmonary alveolocapillary barrier was established to investigate the role of the microvascular endothelium on re-epithelialization. The model system, confluent monolayer cultures on opposing sides of a porous membrane, consisted of a human microvascular endothelial cell line (HPMEC-ST1.6R) and an alveolar type II like cell line (A549), stably expressing EGFP and mCherry, respectively. These fluorescent proteins allowed the real time assessment of the integrity of the monolayers and the automated analysis of the wound healing process after a scratch injury. The HPMECs significantly attenuated the speed of re-epithelialization, which was associated with the proximity to the A549 layer. Examination of cross-sectional transmission electron micrographs of the model system revealed protrusions through the membrane pores and close contact between the A549 cells and the HPMECs. Immunohistochemical analysis showed that these close contacts consisted of heterocellular gap-, tight- and adherens-junctions. Additional analysis, using a fluorescent probe to assess gap-junctional communication, revealed that the HPMECs and A549 cells were able to exchange the fluorophore, which could be abrogated by disrupting the gap junctions using connexin mimetic peptides. These data suggest that the pulmonary microvascular endothelium may impact the re-epithelialization process.

Effects of ethanol and acetaldehyde on tight junction integrity: in vitro study in a three dimensional intestinal epithelial cell culture model.

BACKGROUND: Intestinal barrier dysfunction and translocation of endotoxins are involved in the pathogenesis of alcoholic liver disease. Exposure to ethanol and its metabolite, acetaldehyde at relatively high concentrations have been shown to disrupt intestinal epithelial tight junctions in the conventional two dimensional cell culture models. The present study investigated quantitatively and qualitatively the effects of ethanol at concentrations detected in the blood after moderate ethanol consumption, of its metabolite acetaldehyde and of the combination of both compounds on intestinal barrier function in a three-dimensional cell culture model. METHODS AND FINDINGS: Caco-2 cells were grown in a basement membrane matrix (Matrigel™) to induce spheroid formation and were then exposed to the compounds at the basolateral side. Morphological differentiation of the spheroids was assessed by immunocytochemistry and transmission electron microscopy. The barrier function was assessed by the flux of FITC-labeled dextran from the basal side into the spheroids' luminal compartment using confocal microscopy. Caco-2 cells grown on Matrigel assembled into fully differentiated and polarized spheroids with a central lumen, closely resembling enterocytes in vivo and provide an excellent model to study epithelial barrier functionality. Exposure to ethanol (10-40 mM) or acetaldehyde (25-200 µM) for 3 h, dose-dependently and additively increased the paracellular permeability and induced redistribution of ZO-1 and occludin without affecting cell viability or tight junction-encoding gene expression. Furthermore, ethanol and acetaldehyde induced lysine residue and microtubules hyperacetylation. CONCLUSIONS: These results indicate that ethanol at concentrations found in the blood after moderate drinking and acetaldehyde, alone and in combination, can increase the intestinal epithelial permeability. The data also point to the involvement of protein hyperacetylation in ethanol- and acetaldehyde-induced loss of tight junctions integrity.