Protein Permeability Research Articles

Relation between the tissue structure and protein permeability of decellularized porcine aorta

The aim of this study was to assess the suitability of decellularized porcine aorta as a vascular graft material by measuring its permeability to protein. Aorta samples were decellularized by treatment with either high hydrostatic pressurization (HHP) or sodium dodecyl sulfate (SDS). Histological evaluation showed that the structure of an HHP-treated sample was similar to that of an untreated sample, while the structure of an SDS-treated sample was surfactant-damaged. A two-chamber diffusion system was used to measure permeability to lysozyme and bovine serum albumin. The lysozyme and bovine serum albumin mass transfer coefficients calculated for an SDS-treated sample were significantly larger than those calculated for an untreated sample, while the mass transfer coefficients for an HHP-treated sample were similar to those for an untreated sample. The mass transfer coefficients showed very good agreement with the tissue structure characterization, which means that differences in permeability reflected differences in tissue structure.

Tuning the architecture of three-dimensional collagen hydrogels by physiological macromolecular crowding

Macromolecular crowding is an optimal physiological feature in intracellular and extracellular spaces, and results from a variety of macromolecules occupying space and contributing to a fractional volume occupancy. Here, we show that soft collagen hydrogels assembled in nature-inspired crowded conditions feature enhanced biophysical properties. We demonstrate that crowding tunes the rate of collagen nucleation and fiber growth, affecting fiber diameter and organization. Adjustments of crowding levels during collagen assembly tune the gel pore size, protein permeability, transparency and resistance to enzymatic degradation. Furthermore, gels assembled in crowded conditions are twice as resistant to mechanical stress as the controls, inducing a 70% boost of proliferation of stem cells cultured on tuned hydrogels. Emulating the crowdedness of interstitial fluids therefore represents a way to optimize the properties of soft collagen gels, with promising applications in soft biomaterials design.

Effect of Ethanol Composition in Water Coagulation Bath on Structure and Performance of Homogeneous-Reinforced Polyvinyl Chloride Hollow Fiber Membranes

Homogeneous-reinforced (HR) polyvinyl chloride (PVC) hollow fiber membranes consisting of coating layer and matrix layer were fabricated via coating process. The mixtures of polymer solutions were uniformly coated on the homogeneous PVC matrix membrane which was prepared by melt-spinning method. The influences of ethanol composition in water coagulation bath on structure and performance of HR membranes were investigated. The results showed that the pure water flux of the HR PVC hollow fiber membranes increased with the increase of ethanol weight fraction in coagulation bath, while the porosity changed slightly. The increase of ethanol weight fraction in coagulation bath brought about the decrease of the protein permeation flux and rejection rate. The tensile strength of HR PVC hollow fiber membranes was about 9.5 MPa, while the elongation at break was higher than 97 %.

In vitro permeability of a model protein across ocular tissues and effect of iontophoresis on the transscleral delivery

The aim of this work was to study the penetration of cytochrome c, a positively charged model protein (MW 12.4kDa, charge at pH 8.2: +9), across different ocular tissues, and to evaluate the potential of iontophoresis to enhance and control the transscleral transport.The passive transport of cytochrome c across the sclera and across the bilayer choroid-Bruch’s membrane was evaluated using Franz diffusion cells and porcine tissues. The affinity of cytochrome c for melanin was measured by means of in vitro binding experiments. The iontophoretic (anodal) permeation was studied as a function of donor concentration (from 5 to 70mg/ml) and current intensity (from 0.9 to 3.5mA; density from 1.5 to 5.8mA/cm2), and the contribution of electroosmosis on cytochrome c transport was evaluated by using a high molecular weight fluorescent dextran (FD-150, 149kDa) as neutral marker. Finally, the possibility of tuning cytochrome c permeation rate was investigated on a 70mg/ml cytochrome c solution, by alternating passive permeation and iontophoresis at different intensities.Cytochrome c permeated the sclera with a passive permeability coefficient of about 2.5×10−6cm/s, comparable to molecules of similar molecular radius. The choroid-Bruch’s layer was an important barrier to penetration, since its presence reduced 5–7 times the amount permeated after 5h, also because of the presence of melanin that binds cytochrome. Iontophoresis (2.9mA/cm2) enhanced cytochrome c penetration across the sclera at all the concentrations tested, increasing about ten times the amount permeated after 2h. The effect was proportional to current density: the enhancement factor (measured on a 10mg/ml solution), resulted 6.0±4.3 (i=0.9mA; density=1.5mA/cm2), 10.6±4.1 (i=1.75mA; density=2.9mA/cm2), 33.2±8.3 (i=1.75mA; density=5.8mA/cm2). Iontophoretic (density=2.9mA/cm2) experiments performed with FD-150, an electroosmotic flow (EO) marker, demonstrated that cytochrome c, at concentration higher that 1mg/ml, dramatically reduced the EO flow and that, despite the high MW, the main mechanism for cytochrome c iontophoretic permeation is electrorepulsion. Finally, by alternating in the same experiment passive permeation and iontophoresis at different current intensities, a precise modulation of cytochrome c release was obtained, thus indicating the possibility of tuning the release as a function of specific therapeutic needs.

Porin involvement in cephalosporin and carbapenem resistance of Burkholderia pseudomallei.

Background Burkholderia pseudomallei (Bps) is a Gram-negative bacterium that causes frequently lethal melioidosis, with a particularly high prevalence in the north and northeast of Thailand. Bps is highly resistant to many antimicrobial agents and this resistance may result from the low drug permeability of outer membrane proteins, known as porins.Principal FindingsMicrobiological assays showed that the clinical Bps strain was resistant to most antimicrobial agents and sensitive only to ceftazidime and meropenem. An E. coli strain defective in most porins, but expressing BpsOmp38, exhibited considerably lower antimicrobial susceptibility than the control strain. In addition, mutation of Tyr119, the most prominent pore-lining residue in BpsOmp38, markedly altered membrane permeability, substitution with Ala (mutant BpsOmp38Y119A) enhanced uptake of the antimicrobial agents, while substitution with Phe (mutant BpsOmp38Y119F) inhibited uptake. Channel recordings of BpsOmp38 reconstituted in a planar black lipid membrane (BLM) suggested that the higher permeability of BpsOmp38Y119A was caused by widening of the pore interior through removal of the bulky side chain. In contrast, the lower permeability of BpsOmp38Y119F was caused by introduction of the hydrophobic side chain (Phe), increasing the ‘greasiness’ of the pore lumen. Significantly, liposome swelling assays showed no permeation through the BpsOmp38 channel by antimicrobial agents to which Bps is resistant (cefoxitin, cefepime, and doripenem). In contrast, high permeability to ceftazidime and meropenem was observed, these being agents to which Bps is sensitive.Conclusion/SignificanceOur results, from both in vivo and in vitro studies, demonstrate that membrane permeability associated with BpsOmp38 expression correlates well with the antimicrobial susceptibility of the virulent bacterium B. pseudomallei, especially to carbapenems and cephalosporins. In addition, substitution of the residue Tyr119 affects the permeability of the BpsOmp38 channel to neutral sugars and antimicrobial agents.

Effects of alcohol on intestinal epithelial barrier permeability and expression of tight junction-associated proteins

The present study aimed to investigate the effects of alcohol on intestinal epithelial barrier permeability and expression of the tight junction-associated proteins, zonula occludens-1 (ZO-1) and claudin-1. An alcohol-treated Caco-2 intestinal epithelial cell monolayer in vitro model was used to investigate whether alcohol is able to induce intestinal barrier dysfunction and decrease expression of ZO-1 and claudin-1. MTT assay results revealed unaltered cell viability at alcohol concentrations of <5%. Caco-2 cells in the 5% alcohol-treated groups exhibited a significant time-dependent decrease in transepithelial electrical resistance (TEER) and an increase in fluorescent yellow flux rate compared with the control cells. ZO‑l expression exhibited a progressive decline following 20 min of incubation, reached its minimum levels at 60 min and then showed an increasing trend following 60 min of incubation. In addition, claudin-1 expression exhibited a progressive increase following 60 min of incubation, reached its maximum levels at 60 min and then showed an increasing trend following 60 min of incubation. Alterations in the expression of the ZO-l and claudin-1 proteins revealed trends consistent with changes in the TEER value and the fluorescent yellow transmittance rate in the Caco-2 cells. The results of this study indicate that alcohol is able to increase the intestinal epithelial barrier permeability in a dose- and time-dependent manner. Alcohol induces a change in the expression of the tight junction-associated proteins, ZO-1 and claudin-1, which are two major sites of alcohol action, thus increasing intestinal epithelial barrier permeability.

Tobacco smoke induces epithelial barrier dysfunction via receptor EphA2 signaling

Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors are the largest family of receptor tyrosine kinases (RTKs) that mediate various cellular and developmental processes. The degrees of expression of these key molecules control the cell-cell interactions. Although the role of Eph receptors and their ligand Ephrins is well studied in developmental processes, their function in tobacco smoke (TS)-induced epithelial barrier dysfunction is unknown. We hypothesized that TS may induce permeability in bronchial airway epithelial cell (BAEpC) monolayer by modulating receptor EphA2 expression, actin cytoskeleton, adherens junction, and focal adhesion proteins. Here we report that in BAEpCs, acute TS exposure significantly upregulated EphA2 and EphrinA1 expression, disrupted the actin filaments, decreased E-cadherin expression, and increased protein permeability, whereas the focal adhesion protein paxillin was unaffected. Silencing the receptor EphA2 expression with silencing interference RNA (siRNA) significantly attenuated TS-induced hyperpermeability in BAEpCs. In addition, when BAEpC monolayer was transfected with EphA2-expressing plasmid and treated with recombinant EphrinA1, the transepithelial electrical resistance decreased significantly. Furthermore, TS downregulated E-cadherin expression and induced hyperpermeability across BAEpC monolayer in a Erk1/Erk2, p38, and JNK MAPK-dependent manner. TS induced hyperpermeability in BAEpC monolayer by targeting cell-cell adhesions, and interestingly cell-matrix adhesions were unaffected. The present data suggest that TS causes significant damage to the BAEpCs via induction of EphA2 and downregulation of E-cadherin. Induction of EphA2 in the BAEpCs exposed to TS may be an important signaling event in the pathogenesis of TS-induced epithelial injury.

Conditioned media from mesenchymal stromal cells restore sodium transport and preserve epithelial permeability in an in vitro model of acute alveolar injury

Mesenchymal stromal cells (MSCs) or their media (MSC-M) were reported to reverse acute lung injury (ALI)-induced decrease of alveolar fluid clearance. To determine the mechanisms by which MSC-M exert their beneficial effects, an in vitro model of alveolar epithelial injury was created by exposing primary rat alveolar epithelial cells (AECs) to hypoxia (3% O2) plus cytomix, a combination of IL-1β, TNF-α, and IFN-γ. MSC-M were collected from human MSCs exposed for 12 h to either normoxia (MSC-M) or to hypoxia plus cytomix (HCYT-MSC-M). This latter condition was used to model the effect of alveolar inflammation and hypoxia on paracrine secretion of MSCs in the injured lung. Comparison of paracrine soluble factors in MSC media showed that the IL-1 receptor antagonist and prostaglandin E2 were markedly increased while keratinocyte growth factor (KGF) was twofold lower in HCYT-MSC-M compared with MSC-M. In AECs, hypoxia plus cytomix increased protein permeability, reduced amiloride-sensitive short-circuit current (AS-Isc), and also decreased the number of α-epithelial sodium channel (α-ENaC) subunits in the apical membrane. To test the effects of MSC media, MSC-M and HCYT-MSC-M were added for an additional 12 h to AECs exposed to hypoxia plus cytomix. MSC-M and HCYT-MSC-M completely restored epithelial permeability to normal. MSC-M, but not HCYT-MSC-M, significantly prevented the hypoxia plus cytomix-induced decrease of ENaC activity and restored apical α-ENaC channels. Interestingly, KGF-deprived MSC-M were unable to restore amiloride-sensitive sodium transport, indicating a possible role for KGF in the beneficial effect of MSC-M. These results indicate that MSC-M may be a preferable therapeutic option for ALI.

Intracellular Ca2+ Release Mediates Cationic but Not Anionic Poly(amidoamine) (PAMAM) Dendrimer-Induced Tight Junction Modulation

Poly(amidoamine) (PAMAM) dendrimers show great promise for utilization as oral drug delivery vehicles. These polymers are capable of traversing epithelial barriers, and have been shown to translocate by both transcellular and paracellular routes. While many proof-of-concept studies have shown that PAMAM dendrimers improve intestinal transport, little information exists on the mechanisms of paracellular transport, specifically dendrimer-induced tight junction modulation. Using anionic G3.5 and cationic G4 PAMAM dendrimers with known absorption enhancers, we investigated tight junction modulation in Caco-2 monolayers by visualization and mannitol permeability and compared dendrimer-mediated tight junction modulation to that of established permeation enhancers. [(14)C]-Mannitol permeability in the presence and absence of phospholipase C-dependent signaling pathway inhibitors was also examined and indicated that this pathway may mediate dendrimer-induced changes in permeability. Differences between G3.5 and G4 in tight junction protein staining and permeability with inhibitors were evident, suggesting divergent mechanisms were responsible for tight junction modulation. These dissimilarities are further intimated by the intracellular calcium release caused by G4 but not G3.5. Based on our results, it is apparent that the underlying mechanisms of dendrimer permeability are complex, and the complexities are likely a result of the density and sign of the surface charges of PAMAM dendrimers. The results of this study will have implications on the future use of PAMAM dendrimers for oral drug delivery.

A Mechanism Enhancing Macromolecule Transport Through Paracellular Spaces Induced by Poly-L-Arginine: Poly-L-Arginine Induces the Internalization of Tight Junction Proteins via Clathrin-Mediated Endocytosis

Poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 cell monolayer to hydrophilic macromolecules by disappearance of tight junction (TJ) proteins from cell-cell junctions. However, the mechanism of the disappearance of TJ proteins in response to PLA has been unclear. In this study, we investigated the mechanism of disappearance of TJ proteins from cell-cell junctions after the application of PLA to Caco-2 cell monolayers. The membrane conductance (Gt), FITC-dextran (FD-4) permeability, and localization of TJ proteins were examined after the treatment of Caco-2 cell monolayers with PLA in the presence of various endocytosis inhibitors. In addition, the localization of endosome marker proteins was also observed. Clathrin-mediated endocytosis inhibitors suppressed the increase in Gt and Papp of FD-4 induced by PLA, and also significantly suppressed the disappearance of TJ proteins induced by PLA. Furthermore, occludin, one of the TJ proteins, colocalized with early endosome and recycling endosomes after the internalization of occludin induced by PLA, and then was recycled to the cell-cell junctions. PLA induced the transient internalization of TJ proteins in cell-cell junctions via clathrin-mediated endocytosis, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.

Establishment and genetic characteristics analysis of in vitro culture a fibroblast cell line derived from Wuzhishan miniature pig

Establishment of fibroblast cell lines of endangered pig breeds and research on the gene functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. The Wuzhishan miniature pig ear marginal tissue fibroblast cell line (WPF22) from 22 samples, stocking 87 cryogenically-preserved vials, was successfully established by using primary explants technique and cell cryopreservation techniques. WPF22 cells were adherent, with a population doubling time of 30.2h. Chromosome karyotyping and G-banding analysis showed that >90.2% of cells were diploid (2n=38) prior to the 4th generation. Neither microbial contamination nor cross-contamination was detected by isoenzyme analyses. Cell viability was 97.8% before cryopreservation and 94.9% after recovery. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, six fluorescent protein genes were transferred into fibroblasts by lipofectamine-mediated method. The transfection efficiency of six fluorescent protein genes fluctuated between 8.1% and 42.6%. ECFP and DsRed were mostly shown in cytoplasmic in dots around the nucleus, and EYFP and EGFP had a slightly stronger expression in the nucleus than in the cytoplasm, but without expression in some vacuoles. Every index of the WPF22 cell line meets all the standard quality controls of American type Culture Collection (ATCC). This research thus does not only preserve important genetic resources of Wuzhishan miniature pig at the cell level, but also serve as a valuable resource for genome, postgenome and somacloning research.

Vitamin D deficiency and bacterial load in a murine model of sepsis-induced lung injury

BackgroundWe have previously shown that patients with acute lung injury are severely vitamin D deficient. Several studies have reported a high prevalence of vitamin D deficiency in critically ill patients with sepsis, associated with increased morbidity and mortality, but whether this association is cause or effect is unknown. Bacteraemic sepsis is more common in the winter months when serum vitamin D concentrations are lowest. The purpose of this study was to investigate the local and systemic effects of vitamin D deficiency in a murine model of sepsis-induced lung injury where we can predictably time the initiating insult. MethodsWe fed eight wild-type C57BL/6 mice a diet completely devoid of vitamin D for 6 weeks to induce severe vitamin D deficiency (serum concentration 9 nmol/L) and compared them with seven mice fed a vitamin D sufficient diet (42 nmol/L). We used caecal ligation and puncture (CLP) to establish sepsis. Animals were culled 16 h after CLP, and blood, peritoneal lavage fluid (PLF), and bronchoalveloar lavage fluid (BALF) were collected. Cell infiltrates were assessed by flow cytometry. Fluid protein concentrations were measured, and tissue protein permeability index was calculated as the ratio between fluid and serum protein. Bacterial load was evaluated as colony-forming units (CFU) per ml after 24 h incubation on appropriate media. Statistical analysis was done with Minitab 16 statistical software. FindingsVitamin D deficient mice had significantly increased bacterial load compared with dietary sufficient mice in BALF, blood, and PLF (BALF median 2·51 × 103 CFU per mL [IQR 0·65 × 103–1097 ×103] vs 0·51 ×103 [0·0–1·15 × 103], p=0·038; blood 66·1 × 103 [1·93 × 103–175 × 103] vs 0·56 × 103 [0·0–5·56 × 103], p=0·042; and PLF 336 × 103 [52 × 103–115 × 105] vs 8·22 × 103 [1·54 ×103–20·6 × 103], p=0·005). BALF protein permeability index was higher in deficient mice than in sufficient mice (median 3·3 [IQR 2·61–4·64] vs 1·8 [1·22–2·09], p=0·0003) but there was no difference in cell numbers recruited to the lung. PLF protein permeability index was also higher in the deficient group than in sufficient mice (46·8 [28·1–59·3] vs 27·2 [14·2–37·3], p=0·05), with an associated increase in neutrophils recruited to the peritoneum (p=0·04). InterpretationVitamin D deficiency significantly increased the bacterial load systemically, locally, and within the lung in a murine model of peritonitis. This increase was associated with a rise in tissue permeability locally and within the lung. These data support pre-existing vitamin D deficiency as a determinant of the severity of bacteraemic sepsis and might account for some of the seasonal variations observed in the incidence of sepsis. FundingUK Medical Research Council.

CO2 Transport by PIP2 Aquaporins of Barley

CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2;1, HvPIP2;2, HvPIP2;3 and HvPIP2;5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2;4 that is highly homologous to HvPIP2;3 did not. The isoleucine residue at position 254 of HvPIP2;3 was conserved in PIP2 aquaporins of barley, except HvPIP2;4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2;3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity.

Improving Stability and Biocompatibility of Alginate/Chitosan Microcapsule by Fabricating Bi‐Functional Membrane

Cell encapsulation technology holds promise for the cell-based therapy. But poor mechanical strength and biocompatibility of microcapsule membrane are still obstacles for the clinical applications. A novel strategy is presented to prepare AC₁ C₂ A microcapsules with bi-functional membrane (that is, both desirable biocompatibility and membrane stability) by sequentially complexing chitosans with higher deacetylation degree (C₁) and lower deacetylation degree (C₂) on alginate (A) gel beads. Both in vitro and in vivo evaluation of AC₁C₂ A microcapsules demonstrate higher membrane stability and less cell adhesion, because the introduction of C₂ increases membrane strength and decreases surface roughness. Moreover, diffusion test of AC₁C₂ A microcapsules displays no inward permeation of IgG protein suggesting good immunoisolation function. The results demonstrate that AC₁C₂ A microcapsules with bi-functional membrane could be a promising candidate for microencapsulated cell implantation with cost effective usage of naturally biocompatible polysaccharides.

HYDROGELS FROM POLYACRYLIC ACID FOR REDUCTION OF BIOADHESION ON SILICONE CONTACT LENSES

Contact lenses suffer from two limitations: low oxygen permeability and deposition of protein and lipids. In order to prevent bioadhesion, surface must be completely inert to all biological reactions. To achieve this, surface properties must be tailored. Also, to improve comfort, surface must be highly wettable and lubricous. In this paper the surface of silicone contact lenses was modified by plasma induced copolymerization of acrylic acid. A wettable surface was generated and in addition carboxyl groups that were created on the surface provided an ideal reactive platform for subsequent grafting of polyethylene glycol. Each surface modification step was analysed by XPS and contact angle measurements. Lysozyme adsorption on modified silicone contact lenses was analysed by surface-MALDI-ToF-MS and XPS. After incubation with lysozyme, surface-MALDI-TOF-MS and XPS analysis showed a reduction of adsorbed lysozyme on hydrogel modified contact lenses. Surface modification of silicone with PEG is a method for reduction of protein adsorption on contact lenses.

Sucrose laurate-enhanced transcutaneous immunization with a solid-in-oil nanodispersion

A novel transcutaneous immunization system was developed using a solid-in-oil (S/O) nanodispersion, consisting of a nano-sized particle of a protein–surfactant complex dispersed in an oil vehicle. Permeability of the model antigen protein, ovalbumin, was enhanced by coating with sucrose laurate, which is a hydrophobic surfactant. The nanodispersion prepared with sucrose laurate induced a 5-fold increase in mouse antigen-specific antibody production compared with that observed using sucrose erucate, indicating that stability of the S/O nanodispersion in the epidermis plays a key role in enabling effective immunization. In spite of the coating with the surfactants, only fluorescence-labeled ovalbumin permeated the deep epidermis beneath the stratum corneum and was observed by laser scanning confocal microscopy. These findings will lead the way to an improved transcutaneous immunization system based on S/O nanodispersion.

液体の統計力学理論によるチャネルタンパクの透過現象解析

液体の統計力学理論によるチャネルタンパクの透過現象解析

Human Mesenchymal Stem Cell Microvesicles for Treatment of Escherichia coli Endotoxin-Induced Acute Lung Injury in Mice

We previously found that human mesenchymal stem cells (MSC) or its conditioned medium restored lung protein permeability and reduced alveolar inflammation following Escherichia coli endotoxin-induced acute lung injury (ALI) in an ex vivo perfused human lung in part through the secretion of soluble factors such as keratinocyte growth factor (KGF). Recently, MSC were found to release microvesicles (MVs) that were biologically active because of the presence of mRNA or miRNA with reparative properties. MVs are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the surface membranes. These studies were designed to determine if MVs released by human bone marrow derived MSCs would be effective in restoring lung protein permeability and reducing inflammation in E. coli endotoxin-induced ALI in C57BL/6 mice. The intratracheal instillation of MVs improved several indices of ALI at 48 hours. Compared to endotoxin-injured mice, MVs reduced extravascular lung water by 43% and reduced total protein levels in the bronchoalveolar lavage (BAL) fluid by 35%, demonstrating a reduction in pulmonary edema and lung protein permeability. MVs also reduced the influx of neutrophils and macrophage inflammatory protein-2 levels in the BAL fluid by 73% and 49%, respectively, demonstrating a reduction in inflammation. KGF siRNA-pretreatment of MSC partially eliminated the therapeutic effects of MVs released by MSCs, suggesting that KGF protein expression was important for the underlying mechanism. In summary, human MSC-derived MVs were therapeutically effective following E. coli endotoxin-induced ALI in mice in part through the expression of KGF mRNA in the injured alveolus.

Hypoxia/Aglycemia-Induced Endothelial Barrier Dysfunction and Tight Junction Protein Downregulation Can Be Ameliorated by Citicoline

This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs.

Circulating Angiotensin II Gains Access to the Hypothalamus and Brain Stem During Hypertension via Breakdown of the Blood–Brain Barrier

Angiotensin II-mediated vascular brain inflammation emerged as a novel pathophysiological mechanism in neurogenic hypertension. However, the precise underlying mechanisms and functional consequences in relation to blood-brain barrier (BBB) integrity and central angiotensin II actions mediating neurohumoral activation in hypertension are poorly understood. Here, we aimed to determine whether BBB permeability within critical hypothalamic and brain stem regions involved in neurohumoral regulation was altered during hypertension. Using digital imaging quantification after intravascularly injected fluorescent dyes and immunohistochemistry, we found increased BBB permeability, along with altered key BBB protein constituents, in spontaneously hypertensive rats within the hypothalamic paraventricular nucleus, the nucleus of the solitary tract, and the rostral ventrolateral medulla, all critical brain regions known to contribute to neurohumoral activation during hypertension. BBB disruption, including increased permeability and downregulation of constituent proteins, was prevented in spontaneously hypertensive rats treated with the AT1 receptor antagonist losartan, but not with hydralazine, a direct vasodilator. Importantly, we found circulating angiotensin II to extravasate into these brain regions, colocalizing with neurons and microglial cells. Taken together, our studies reveal a novel angiotensin II-mediated feed-forward mechanism during hypertension, by which circulating angiotensin II evokes increased BBB permeability, facilitating in turn its access to critical brain regions known to participate in blood pressure regulation.