Cell Layer Integrity Research Articles

An Improved In Vitro Blood-Brain Barrier Model for the Evaluation of Drug Permeability Using Transwell with Shear Stress.

The development of drugs targeting the central nervous system (CNS) is challenging because of the presence of the Blood-Brain barrier (BBB). Developing physiologically relevant in vitro BBB models for evaluating drug permeability and predicting the activity of drug candidates is crucial. The transwell model is one of the most widely used in vitro BBB models. However, this model has limitations in mimicking in vivo conditions, particularly in the absence of shear stress. This study aimed to overcome the limitations of the transwell model using immortalized human endothelial cells (hCMEC/D3) by developing a novel dish design for an orbital shaker, providing shear stress. During optimization, we assessed cell layer integrity using trans-endothelial electrical resistance measurements and the % diffusion of lucifer yellow. The efflux transporter activity and mRNA expression of junctional proteins (claudin-5, occludin, and VE-cadherin) in the newly optimized model were verified. Additionally, the permeability of 14 compounds was evaluated and compared with published in vivo data. The cell-layer integrity was substantially increased using the newly designed annular shaking-dish model. The results demonstrate that our model provided robust conditions for evaluating the permeability of CNS drug candidates, potentially improving the reliability of in vitro BBB models in drug development.

Baicalin Attenuates Panton-Valentine Leukocidin (PVL)-Induced Cytoskeleton Rearrangement via Regulating the RhoA/ROCK/LIMK and PI3K/AKT/GSK-3β Pathways in Bovine Mammary Epithelial Cells.

Pore-forming toxins (PFTs) exert physiological effects by rearrangement of the host cell cytoskeleton. Staphylococcus aureus-secreted PFTs play an important role in bovine mastitis. In the study, we examined the effects of recombinant Panton-Valentine leukocidin (rPVL) on cytoskeleton rearrangement, and identified the signaling pathways involved in regulating the process in bovine mammary epithelial cells (BMECs) in vitro. Meanwhile, the underlying regulatory mechanism of baicalin for this process was investigated. The results showed that S. aureus induced cytoskeleton rearrangement in BMECs mainly through PVL. S. aureus and rPVL caused alterations in the cell morphology and layer integrity due to microfilament and microtubule rearrangement and focal contact inability. rPVL strongly induced the phosphorylation of cofilin at Ser3 mediating by the activation of the RhoA/ROCK/LIMK pathway, and resulted in the activation of loss of actin stress fibers, or the hyperphosphorylation of Tau at Ser396 inducing by the inhibition of the PI3K/AKT/GSK-3β pathways, and decreased the microtubule assembly. Baicalin significantly attenuated rPVL-stimulated cytoskeleton rearrangement in BMECs. Baicalin inhibited cofilin phosphorylation or Tau hyperphosphorylation via regulating the activation of RhoA/ROCK/LIMK and PI3K/AKT/GSK-3β signaling pathways. These findings provide new insights into the pathogenesis and potential treatment in S. aureus causing bovine mastitis.

MiRNAs as Predictors of Barrier Integrity.

The human body has several barriers that protect its integrity and shield it from mechanical, chemical, and microbial harm. The various barriers include the skin, intestinal and respiratory epithelia, blood-brain barrier (BBB), and immune system. In the present review, the focus is on the physical barriers that are formed by cell layers. The barrier function is influenced by the molecular microenvironment of the cells forming the barriers. The integrity of the barrier cell layers is maintained by the intricate balance of protein expression that is partly regulated by microRNAs (miRNAs) both in the intracellular space and the extracellular microenvironment. The detection of changes in miRNA patterns has become a major focus of diagnostic, prognostic, and disease progression, as well as therapy-response, markers using a great variety of detection systems in recent years. In the present review, we highlight the importance of liquid biopsies in assessing barrier integrity and challenges in differential miRNA detection.

Effects of lipid type and toxicological properties on the digestion of cellulose nanocrystals in simulated gastrointestinal tract

This study aimed to understand the impact of different oil types on the cellulose nanocrystals (CNCs) to modulate lipid digestion and the in vitro gastrointestinal toxicity of CNCs in food systems. We explored the ability of CNCs to modulate lipid digestion in a simulated gastrointestinal system and monitored the gastrointestinal fate of CNC-based emulsions with different oil types. Finally, a small intestine epithelial model was used to evaluate the influence of cytotoxicity. The results suggested that the addition of 0.6 wt% CNCs in the high-fat food model reduced the hydrolysis of free fatty acids (FFAs) from triglycerides by 37.8% after the small intestine phase. CNCs showed the best effect in reducing lipid digestion in emulsions with high unsaturation triglycerides. In addition, the toxicology results suggest that 0.6 wt% CNCs had only a slight effect on reactive oxygen species and cytotoxicity, and no significant change in cell-layer integrity.

Effect of Pre‐Conditioning with Copolymer‐Based Cell Membrane Stabilizer Poloxamer 188 on Adult Mouse Coronary Artery Endothelial Layer Integrity after Prolonged Hypoxia/Reoxygenation

Prolonged ischemia followed by reperfusion can cause significant tissue damage and cell death. This ischemia/reperfusion (IR) injury can be alleviated by administration of Copolymer‐based Cell Membrane Stabilizers (CCMS), like the tri‐block Poloxamer (P)188, thought to be able to seal gaps in cell membranes and/or tight junctions and, thus, repair leaks attributable to severe cellular stress. We have previously tested post‐conditioning, i.e. the administration of P188 upon reperfusion/reoxygenation, to attenuate IR injury in various models. Here, we hypothesized that pre‐conditioning, i.e. its administration before simulated ischemia (hypoxia), has a protective effect on endothelial cell layer integrity as assessed by Trans‐Endothelial Electrical Resistance (TEER).Confluent adult mouse coronary artery endothelial cell (ECs) layers were pre‐treated with 0, 30, 100, 300, 1,000, or 2,000 µM P188 during plating of cells before simulated IR with a 24‐hour hypoxic followed by a 2‐hour reoxygenation (HR) period. EC monolayer barrier integrity was assessed by TEER.In contrast to mere post‐conditioning, pre‐conditioning dose‐dependently attenuated HR injury compared to control/normoxia (Fig 1) even after prolonged HR for 24+2 hrs.These results suggest that CCMS may indeed elicit its cardioprotective effect, in part, through protection of the EC layer. However, more research is needed to determine optimal dosage and timing for CCMS administration.

Abstract 11809: Trans-Endothelial Electrical Resistance to Assess Cell Layer Integrity in Coronary Artery Endothelial Cells After Hypoxia/Reoxygenation Injury

Introduction: Cardiovascular disease remains one of the leading causes of complications and death worldwide. Therefore, accurate and reliable methods of mimicking ischemia/reperfusion (IR) injury in cardiac cells in vivo are crucial when testing drugs/substances for prevention and treatment. Coronary artery endothelial cells play a critical role in not only supplying blood to myocardial cells but protecting them from insult as well. However, the endothelial layer can be compromised by ischemic injury, heightening damage to the heart during reperfusion. Hypothesis: Varied ischemic insult of mouse coronary artery endothelial cells (MCAECs) affects cell layer integrity as measured by Trans-Endothelial Electrical Resistance (TEER). Methods: MCAECs were cultured on Grenier Bio-One ThinCert™-cell culture inserts for 72 hrs to allow for adequate confluency. Cells were then subjected to either continued normoxic conditions or hypoxia for 3, 6, 12, or 24 hrs, with a 2-hr reperfusion period immediately following. TEER was used to measure the integrity of the endothelial cell layer on the insert. Results: Our data showed a significant decrease in TEER between control and hypoxic groups after 6 hrs (p = 0.0400), 12 hrs (p = 0.0179) and 24 hrs (p = 0.0103), but not after 3 hrs (p = 0.4453) of hypoxia. Conclusion: This indicates that titrating the hypoxia time to a sufficient duration is necessary to achieve an adequate H/R injury which can then be used for potential cardioprotective agents and/or strategies to be tested. Furthermore, TEER is a reliable and reproducible method to assess the role of endothelial cell membrane integrity in cardioprotection.

Current-Driven Organic Electrochemical Transistors for Monitoring Cell Layer Integrity with Enhanced Sensitivity.

In this progress report an overview is given on the use of the organic electrochemical transistor (OECT) as a biosensor for impedance sensing of cell layers. The transient OECT current can be used to detect changes in the impedance of the cell layer, as shown by Jimison etal. To circumvent the application of a high gate bias and preventing electrolysis of the electrolyte, in case of small impedance variations, an alternative measuring technique based on an OECT in a current-driven configuration is developed. The ion-sensitivity is larger than 1200 mV V-1 dec-1 at low operating voltage. It can be even further enhanced using an OECT based complementary amplifier, which consists of a p-type and an n-type OECT connected in series, as known from digital electronics. The monitoring of cell layer integrity and irreversible disruption of barrier function with the current-driven OECT is demonstrated for an epithelial Caco-2 cell layer, showing the enhanced ion-sensitivity as compared to the standard OECT configuration. As a state-of-the-art application of the current-driven OECT, the in situ monitoring of reversible tight junction modulation under the effect of drug additives, like poly-l-lysine, is discussed. This shows its potential for in vitro and even in vivo toxicological and drug delivery studies.

Poloxamer 188 Dose‐Dependently Protects Adult Mouse Coronary Artery Endothelial Cell Layers from Reoxygenation Injury

Poloxamer (P)188 is a tri-block copolymer shown to provide a cardioprotective effect when administered at the beginning of reoxygenation in adult mouse cardiomyocyte hypoxia/reoxygenation models. It is thought that it seals cells damaged by hypoxia via membrane stabilization, thus, protecting the cells from additional injury during reoxygenation. To further explore the effects of P188 on maintaining endothelial cell layer integrity, we treated confluent adult mouse coronary artery endothelial cell layers with varying concentrations of P188 upon initiation of a 2-hr reoxygenation period immediately following hypoxic periods of 3, 12, and 48 hrs. Using trans-endothelial electrical resistance (TEER), we found that P188 dose-dependently protected cells from further damage after hypoxia, with some doses returning TEER measurements even back to control/normoxia levels. However, we also observed that similar concentrations of P188 varied in effects as hypoxia times increased. Our results indicate that P188 protects the endothelial layer in a dose-dependent manner as long as it is administered after an optimal ischemia time to effectively preserve membrane integrity in tissue barriers. More research will need to be conducted to confirm our findings and determine the optimal combination of dosage and timing of P188 administration.

Investigations of acute effects of polystyrene and polyvinyl chloride micro- and nanoplastics in an advanced in vitro triple culture model of the healthy and inflamed intestine

The continuous degradation of plastic waste in the environment leads to the generation of micro- and nanoplastic fragments and particles. Due to the ubiquitous presence of plastic particles in natural habitats as well as in food, beverages and tap water, oral exposure of the human population with plastic particles occurs worldwide. We investigated acute toxicological effects of polystyrene (PS) and polyvinyl chloride (PVC) micro- and nanoparticles in an advanced in vitro triple culture model (Caco-2/HT29-MTX-E12/THP-1) mimicking the healthy and inflamed human intestine to study the effect of inflammatory processes on plastic particle toxicity. We monitored barrier integrity, cytotoxicity, cell layer integrity, DNA damage, the release of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) and mucus distribution after 24 h of particle exposure. In addition, we investigated cytotoxicity, DNA damage and IL-1β release in monocultures of the three cell lines. Amine-modified polystyrene nanoparticles (PS–NH2) served as a positive control for particle-induced toxicity. No acute effects in the investigated endpoints were observed in the model of the healthy intestine after PS or PVC exposure. However, during active inflammatory processes, exposure to PVC particles was found to augment the release of IL-1β and to cause a loss of epithelial cells. Our results suggest that prevalent intestinal inflammation might be an important factor to consider when assessing the hazard of ingested micro- and nanoplastic particles.

Administration of all-trans retinoic acid after experimental traumatic brain injury is brain protective.

All-trans retinoic acid (ATRA) is a vitamin A metabolite, important in the developing and mature brain. Pre-injury ATRA administration ameliorates ischaemic brain insults in rodents. This study examined the effects of post-traumatic ATRA treatment in experimental traumatic brain injury (TBI). Male adult mice were subjected to the controlled cortical impact model of TBI or sham procedure and killed at 7 or 30 days post-injury (dpi). ATRA (10 mg kg-1, i.p.) was given immediately after the injury and 1, 2 and 3 dpi. Neurological function and sensorimotor coordination were evaluated. Brains were processed for (immuno-) histological, mRNA and protein analyses (qPCR and western blot). ATRA treatment reduced brain lesion size, reactive astrogliosis and axonal injury at 7 dpi, and hippocampal granule cell layer (GCL) integrity was protected at 7 and 30 dpi, independent of cell proliferation in neurogenic niches and blood-brain barrier damage. Neurological and motor deficits over time and the brain tissue loss at 30 dpi were not affected by ATRA treatment. ATRA decreased gene expression of markers for damage-associated molecular pattern (HMGB1), apoptosis (caspase-3 and Bax), activated microglia (TSPO), and reactive astrogliosis (GFAP, SerpinA3N) at 7 dpi and a subset of markers at 30 dpi (TSPO, GFAP). In experimental TBI, post-traumatic ATRA administration exerted brain protective effects, including long-term protection of GCL integrity, but did not affect neurological and motor deficits. Further investigations are required to optimize treatment regimens to enhance ATRA's brain protective effects and improve outcomes.

Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.

SummaryThe glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.

Effect of sevoflurane and propofol on tourniquet-induced endothelial damage: a pilot randomized controlled trial for knee-ligament surgery

BackgroundThe glycocalyx layer is a key structure in the endothelium. Tourniquet-induced ischemic periods are used during orthopedic surgery, and the reactive oxygen species generated after ischemia-reperfusion may mediate the shedding of the glycocalyx. Here, we describe the effects of tourniquet-induced ischemia-reperfusion and compare the effects of sevoflurane and propofol on the release of endothelial biomarkers after ischemia-reperfusion in knee-ligament surgery.MethodsThis pilot, single-center, blinded, randomized, controlled trial included 16 healthy patients. After spinal anesthesia, hypnosis was achieved with sevoflurane or propofol according to randomization. During the perioperative period, five venous blood samples were collected for quantification of syndecan-1, heparan sulfate, and thrombomodulin from blood serum by using ELISA assays kits. Sample size calculation was performed to detect a 25% change in the mean concentration of syndecan-1 with an alpha of 0.05 and power of 80%.ResultsFor our primary outcome, a two-way ANOVA with post-hoc Bonferroni correction analysis showed no differences in syndecan-1 concentrations between the sevoflurane and propofol groups at any time point. In the sevoflurane group, we noted an increase in syndecan-1 concentrations 90 min after tourniquet release in the sevoflurane group from 34.6 ± 24.4 ng/mL to 47.9 ± 29.8 ng/mL (Wilcoxon test, p < 0.01) that was not observed in patients randomized to the propofol group. The two-way ANOVA showed no intergroup differences in heparan sulfate and thrombomodulin levels.ConclusionsSuperficial endothelial damage without alterations in the cell layer integrity was observed after tourniquet knee-ligament surgery. There was no elevation in serum endothelial biomarkers in the propofol group patients. Sevoflurane did not show the protective effect observed in in vitro and in vivo studies.Trial registrationThe trial was registered in www.clinicaltrials.gov (ref: NCT03772054, Registered 11 December 2018).

Microcystin-LR Does Not Alter Cell Survival and Intracellular Signaling in Human Bronchial Epithelial Cells.

Changes in ecological and environmental factors lead to an increased occurrence of cyanobacterial water blooms, while secondary metabolites-producing cyanobacteria pose a threat to both environmental and human health. Apart from oral and dermal exposure, humans may be exposed via inhalation and/or swallowing of contaminated water and aerosols. Although many studies deal with liver toxicity, less information about the effects in the respiratory system is available. We investigated the effects of a prevalent cyanotoxin, microcystin-LR (MC-LR), using respiratory system-relevant human bronchial epithelial (HBE) cells. The expression of specific organic-anion-transporting polypeptides was evaluated, and the western blot analysis revealed the formation and accumulation of MC-LR protein adducts in exposed cells. However, MC-LR up to 20 μM neither caused significant cytotoxic effects according to multiple viability endpoints after 48-h exposure, nor reduced impedance (cell layer integrity) over 96 h. Time-dependent increase of putative MC-LR adducts with protein phosphatases was not associated with activation of mitogen-activated protein kinases ERK1/2 and p38 during 48-h exposure in HBE cells. Future studies addressing human health risks associated with inhalation of toxic cyanobacteria and cyanotoxins should focus on complex environmental samples of cyanobacterial blooms and alterations of additional non-cytotoxic endpoints while adopting more advanced in vitro models.

Evaluation of the cytotoxic and cellular proteome impacts of food-grade TiO2 (E171) using simulated gastrointestinal digestions and a tri-culture small intestinal epithelial model

Engineered nanomaterials (ENMs) are widely used in the food industry; however, regulations for ENMs in food are still in the early stages of development due to insufficient health data. This study investigated the cytotoxicity and changes to the proteomic profile in an in vitro small intestinal epithelium model after exposure to digested food models containing the ubiquitous engineered particulate food additive, TiO2 (E171) with an average size around 110 nm. TiO2 at 0.75% or 1.5% (w/w) concentrations in either a fasting food model (FFM) or a standardized food model (SFM) based on American diet were digested using an in vitro oral-gastric-small intestinal simulator, and the resulting digestas were applied to a small intestinal epithelium tri-culture cellular model. Effects on cell layer integrity, cytotoxicity, and oxidative stress were assessed. In order to explore the impact on cellular processes beyond basic cytotoxicity, mass spectrometry-based quantitative proteomic analyses of control and exposed tri-culture cells was performed. TiO2 in FFM, but not in SFM, produced significant, dose-dependent cytotoxicity (24%, p < 0.001), and at the higher dose caused significant oxidative stress (1.24-fold, p < 0.01), indicative of a food matrix effect. No significant perturbations of the cellular proteome were observed with TiO2 in either FFM or SFM food models. However, proteins involved in energy metabolism and protein synthesis were up-regulated by digestas from SFM compared to those from FFM, indicative of a food matrix effect on the cellular proteome. Interestingly, the differences in profiles between the two food models were more pronounced in the presence of TiO2. Together, these results indicate that TiO2 in a fasting diet may be slightly cytotoxic, and that ingested TiO2 does not significantly alter the epithelial proteome, whereas the food matrix alone can have a dramatic effect on the proteome.

HAX1 impact on collective cell migration, cell adhesion, and cell shape is linked to the regulation of actomyosin contractility.

HAX1 protein is involved in the regulation of apoptosis, cell motility and calcium homeostasis. Its overexpression was reported in several tumors, including breast cancer. This study demonstrates that HAX1 has an impact on collective, but not single-cell migration, thus indicating the importance of cell–cell contacts for the HAX1-mediated effect. Accordingly, it was shown that HAX1 knockdown affects cell–cell junctions, substrate adhesion, and epithelial cell layer integrity. As demonstrated here, these effects can be attributed to the modulation of actomyosin contractility through changes in RhoA and septin signaling. Additionally, it was shown that HAX1 does not influence invasive potential in the breast cancer cell line, suggesting that its role in breast cancer progression may be linked instead to collective invasion of the epithelial cells but not single-cell dissemination.

Short communication: Processed bovine colostrum milk protein concentrate increases epithelial barrier integrity of Caco-2 cell layers

Colostrum plays an important role in initiating the development of the intestinal barrier in newborn mammals. Given its bioactivity, there is much interest in the potential use of bovine colostrum to improve human gastrointestinal health throughout the life span. There is evidence that bovine colostrum is effective at improving small intestinal barrier integrity and some indication that it may alter colonic motility. However, for colostrum to be used as a product to improve intestinal health, it needs to be bioactive after processing. The aim of this study was to determine whether industrial processing of bovine colostrum affects its ability to improve small intestinal barrier integrity or alter distal colon motility. Three colostrum sample types were compared; raw whole colostrum powder (WCP), raw skim colostrum powder (SCP), and industrially produced colostrum milk protein concentrate (CMPC). To determine whether these colostrum powders had different effects on small intestinal barrier integrity, their effects on the transepithelial electrical resistance across an in vitro intestinal epithelial layer (Caco-2 cells) were measured, both with and without a challenge from the proinflammatory cytokine tumor necrosis factor-α. These results showed that CMPC enhanced transepithelial electrical resistance across unchallenged epithelial cell layers, whereas the raw colostrum samples, WCP and SCP, did not have an effect. The colostrum samples were also compared to determine how they affect contractility in the distal colon isolated from the rat. Skim colostrum powder was the only sample to act directly on colonic tissue to modulate motility, increasing the amplitude of contractions. The results show that bovine colostrum is able to improve small intestinal barrier integrity and alter colon motility, and they implicate different components. The barrier integrity enhancement was apparent only in the industrial CMPC, which may have been due to the increase in protein concentration or the release of small peptides as a result of processing. The ability to alter colon motility was present in SCP but absent in WCP, again implying that an increase in protein concentration is responsible for the effect. However, this effect was not apparent for the industrially processed CMPC, suggesting denaturation or degradation of the active component. The beneficial effect of colostrum on small intestinal barrier integrity was present after processing, confirming that it is feasible to industrially produce an active product for gut health.

Biocompatibility of Nanocellulose-Reinforced PVA Hydrogel with Human Corneal Epithelial Cells for Ophthalmic Applications.

Transparent composite hydrogel in the form of a contact lens made from poly(vinyl alcohol) (PVA) and cellulose nanocrystals (CNCs) was subjected to in vitro biocompatibility evaluation with human corneal epithelial cells (HCE-2 cells). The cell response to direct contact with the hydrogels was investigated by placing the samples on top of confluent cell layers and evaluating cell viability, morphology, and cell layer integrity subsequent to 24 h culture and removal of the hydrogels. To further characterize the lens–cell interactions, HCE-2 cells were seeded on the hydrogels, with and without simulated tear fluid (STF) pre-conditioning, and cell viability and morphology were evaluated. Furthermore, protein adsorption on the hydrogel surface was investigated by incubating the materials with STF, followed by protein elution and quantification. The hydrogel material was found to have affinity towards protein adsorption, most probably due to the interactions between the positively charged lysozyme and the negatively charged CNCs embedded in the PVA matrix. The direct contact experiment demonstrated that the physical presence of the lenses did not affect corneal epithelial cell monolayers in terms of integrity nor cell metabolic activity. Moreover, it was found that viable corneal cells adhered to the hydrogel, showing the typical morphology of epithelial cells and that such response was not influenced by the STF pre-conditioning of the hydrogel surface. The results of the study confirm that PVA-CNC hydrogel is a promising ophthalmic biomaterial, motivating future in vitro and in vivo biocompatibility studies.

Monitoring of Cell Layer Integrity with a Current-Driven Organic Electrochemical Transistor.

The integrity of CaCo-2 cell barriers is investigated by organic electrochemical transistors (OECTs) in a current-driven configuration. Ion transport through cellular barriers via the paracellular pathway is modulated by tight junctions between adjacent cells. Rupturing its integrity by H2 O2 is monitored by the change of the output voltage in the transfer characteristics. It is demonstrated that by operating the OECT in a current-driven configuration, the sensitive and temporal resolution for monitoring the cell barrier integrity is strongly enhanced as compared to the OECT transient response measurement. As a result, current-driven OECTs are useful tools to assess dynamic and critical changes in tight junctions, relevant for clinical applications as drug targeting and screening.

Effect of positive airway pressure oscillations on human epithelial cells

Lung supportive devices are widely used for respiratory ventilation and therapy to help providing breathing support for patients with various lung diseases including Obstructive Sleep Apnea. These devices deliver continuous air to the patient through a nasal or a facial mask. However, the use of these devices normally results in dryness in the upper airways. Various methods have been developed to improve the continuous positive pressure features using pressure oscillations, which can reduce the patient requirements such as reduction in the titration pressure and humidification requirements. Thus, the objective of this research is to investigate the differences between continuous positive pressure and positive pressure oscillation on the upper airway humidification. Human nasal (RPMI 2650) and bronchial (Calu-3) epithelial cells grown in air-liquid interface on permeable supports are used as a respiratory model. Trans-epithelial electrical resistance is measured before and after each experiment to evaluate the cell layer integrity and permeability.

Transcriptional Alterations by Ischaemic Postconditioning in a Pig Infarction Model: Impact on Microvascular Protection.

Although the application of cardioprotective ischaemia/reperfusion (I/R) stimuli after myocardial infarction (MI) is a promising concept for salvaging the myocardium, translation to a clinical scenario has not fulfilled expectations. We have previously shown that in pigs, ischaemic postconditioning (IPostC) reduces myocardial oedema and microvascular obstruction (MVO), without influencing myocardial infarct size. In the present study, we analyzed the mechanisms underlying the IPostC-induced microvascular protection by transcriptomic analysis, followed by pathway analysis. Closed-chest reperfused MI was induced by 90 min percutaneous balloon occlusion of the left anterior descending coronary artery, followed by balloon deflation in anaesthetised pigs. Animals were randomised to IPostC (n = 8), MI (non-conditioned, n = 8), or Control (sham-operated, n = 4) groups. After three hours or three days follow-up, myocardial tissue samples were harvested and subjected to RNA-seq analysis. Although the transcriptome analysis revealed similar expression between IPostC and MI in transcripts involved in cardioprotective pathways, we identified gene expression changes responding to IPostC at the three days follow-up. Focal adhesion signaling, downregulated genes participating in cardiomyopathy and activation of blood cells may have critical consequences for microvascular protection. Specific analyses of the gene subsets enriched in the endothelium of the infarcted area, revealed strong deregulation of transcriptional functional clusters, DNA processing, replication and repair, cell proliferation, and focal adhesion, suggesting sustentative function in the endothelial cell layer protection and integrity. The spatial and time-dependent transcriptome analysis of porcine myocardium supports a protective effect of IPostC on coronary microvasculature post-MI.