Protein Permeability Research Articles

Hyperglycemia Increases the Production of Amyloid Beta-Peptide Leading to Decreased Endothelial Tight Junction.

Amyloid beta-peptide (Aβ), the main component of senile plaques in the Alzheimer's disease (AD) brains, is generated from sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretase. Hyperglycemia in diabetes may compromise barrier integrity in endothelial cells (ECs). However, the roles of endothelial APP in response to high glucose (HG) remain to be delineated. The aims of this study were to test whether HG may increase Aβ secretion, thereby leading to heightened paracellular permeability in ECs. We determined the effects of HG on production of Aβ, expression of full-length APP, intercellular permeability, and expression levels of specific junctional proteins in human umbilical vein endothelial cells (HUVECs). HG at 30mM significantly stimulated expression of full-length APP accompanied by heightened secretion of Aβ1-42, increased paracellular permeability, and attenuated expression of zona occluden-1 (ZO-1), claudin-5, occludin, and junctional adhesion molecule (JAM)-C in HUVECs; all of which were abolished by the γ-secretase inhibitor BMS299897. Exogenous application of Aβ1-42, but not the reverse peptide Aβ42-1, was sufficient to downregulate the expression of the same junction proteins. Hyperglycemia enhances APP expression with increased Aβ production, which downregulates junctional proteins causing increased intercellular permeability in ECs.

Determining Unitary Water Permeability of Membrane Proteins Reconstituted into Giant Unilamellar Vesicles

The high background water permeability of lipid bilayers may hamper determining unitary water permeability pf at low protein/lipid ratios. That is, reliable pf measurements require reconstitution of more than one water-channeling protein per large unilamellar vesicle (LUV)1. The copy number per LUV must be even higher for proteins that conduct water at slower rates than the most efficient aquaporins, which may prove impossible to achieve. Here we show that protein reconstitution into giant unilamellar vesicles (GUVs) provides a solution. Instead of assessing the volume change from the intensity of light scattering as performed with LUVs2, we implement the micropipette aspiration technique. It offers the possibility of measuring much smaller changes in relative volume. Additional advantages are that channel membrane abundancy and oligomeric state are directly determined by fluorescence correlation spectroscopy in the very same GUV from which the volume flow is extracted. In contrast, the channel number per LUV is extracted in retrospect: It is equal to the difference in particle number in the confocal volume of the correlation spectrometer before and subsequent to vesicle solubilization3. We test the new assay by reconstituting the purified and fluorescently-labeled glycerol facilitator from E.coli into GUVs. We found it mandatory to properly account for unstirred layer effects, i.e. for osmolyte and solute dilution adjacent to the external surface of the GUV and for solute concentration increases in the immediate vicinity of the internal leaflet. The pf value we determined with the novel assay agrees very well with recently published data2.1. Hoomann et al. (2013). PNAS 110, 10842-10847.2. Horner et al. (2015). Science Advances 1, e1400083.3. Knyazev et al. (2013). JBC 288, 17941-17946.

Alveolocapillary membrane permeability in experimental model of ventilator induced lung injury

Aim: to assess alveocapillary membrane permeability for the whole protein, middle molecular peptides and some lipoperoxidation markers depending on respiratory volume using in reproduction of ventilator induced lung injury model. Material and methods: Experiments were carried out on 15 laboratory rats- males (body mass 180–240 gr.) of “Vistar” line). The mechanical pulmonary ventilation in rats was carried out using tracheostomy cannula ALV Hamilton G 5 apparatus during 2 hours under the total anesthesia with sodium thiopental at a rate of 40 mg|kg of animal body mass. The initial parameters of ventilation were equal in all animals: Inspiratory time = 0,5 seconds; respiratory rate = 60 – 76/minute; pressure at the end of expiration (PEE) = 0 - 2 sm. of water column; inspiration-expiration ratio (I:E) = 1:1 or 1:2. Depending on the size of respiratory volume (RV) animal were divided into 3 groups (n=5). Animals with RV=7 ml/kg of body mass formed the first group (the control one). The second group included animals with RV = 20 ml/kg of body mass (the moderate volutrauma) and the third one included animals with RV = 40 ml/kg of body mass (the heavy volutrauma). The bronchoalveolar lavage was carried out on isolated lungs with the volume of filling at a rate 5 ml of 0,9 % sodium chloride solution for 1 g of pulmonary tissue and there was received nearly 2,5 + 0,5 ml of lavage liquid (sodium chloride solution + bronchoalveolar liquid). The alveolocapillary membrane permeability was assessed by detecting in the received liquid of bronchoalveolar lavage the concentration of whole protein on Lowry, the content of middle mass molecules on extinction at wave lengths 238, 254, 260, and 280 nm; the level of diene conjugates on V.B. Gavrilov and catalase activity on M.A. Koroliuk. The received data were processed using methods of nonparametric statistics. The revealed intergroup differences were assessed on Kruskall-Wallis «ANOVA» criterion. The differences at р < 0,05 were considered as reliable ones. Results: Alveolocapillary membrane permeability for the whole protein at the size of respiratory volume 20 ml/kg of body mass exceeds the values in control group in 12,5 times and at respiratory volume 40 ml/kg- in 20 times. Alveolocapillary membrane permeability for middle molecular peptides at the size of respiratory volume 20 ml/kg exceeds the values in the control group on extinction at 238 nm in 2 times; at 254 nm in 1,5 times; at 260 nm in 1,2 times and at 280 nm in 1,5 times. The double increase of respiratory volume at reproduction of ventilator induced lung injury model is attended with practically double increase of alveolocapillary barrier permeability for middle molecular peptides determined by detection at all wave lengths. The changes of alveolocapillary membrane permeability for diene conjugates in the conditions of ventilator induced lung injury model correspond to the one for protein and middle molecular peptides. The change of catalase activity as alveolocapillary membrane permeability marker is informative only in the model used at respiratory volume 40 ml/kg of animal body mass. Conclusions: The changes of alveolocapillary membrane permeability in ventilator induced lung injury model are proportional to the size of respiratory volume used for reproduction of the model

Inhibiting Integrin αvβ5 Reduces Ischemia-Reperfusion Injury in an Orthotopic Lung Transplant Model in Mice.

Primary graft dysfunction after lung transplantation is the leading cause of morbidity and mortality in the immediate posttransplant period and is characterized by endothelial and epithelial barrier disruption and the leakage of protein-rich edema fluid. Integrins are cell surface receptors that have an important role in maintenance of the cell barrier, and inhibition of integrins, such as αvβ5, can diminish alveolar flooding in lung injury models. We hypothesized that inhibition of αvβ5 during donor lung cold ischemia would reduce endothelial permeability during reperfusion. Using an orthotopic single lung transplantation model with and without cold ischemia, donor lungs were perfused with αvβ5-blocking antibody (ALULA) or control antibody at the time of collection, followed by transplantation, 8 h of reperfusion, and the measurement of lung injury parameters. Prolonged cold ischemia (18 h) produced increases in extravascular lung water, protein permeability, and neutrophilic alveolitis and decreased oxygenation compared with lungs without cold ischemia. Perfusion of lungs with αvβ5 antibody versus control antibody protected donor lungs from injury and significantly improved oxygenation. In summary, αvβ5 integrin blockade protects from the development of ischemia-reperfusion lung injury and is a promising approach to preventing primary graft dysfunction in human lung transplant procedures.

Deletion of P2X7 attenuates hyperoxia-induced acute lung injury via inflammasome suppression.

Increasing evidence shows that hyperoxia is a serious complication of oxygen therapy in acutely ill patients that causes excessive production of free radicals leading to hyperoxia-induced acute lung injury (HALI). Our previous studies have shown that P2X7 receptor activation is required for inflammasome activation during HALI. However, the role of P2X7 in HALI is unclear. The main aim of this study was to determine the effect of P2X7 receptor gene deletion on HALI. Wild-type (WT) and P2X7 knockout (P2X7 KO) mice were exposed to 100% O2 for 72 h. P2X7 KO mice treated with hyperoxia had enhanced survival in 100% O2 compared with the WT mice. Hyperoxia-induced recruitment of inflammatory cells and elevation of IL-1β, TNF-α, monocyte chemoattractant protein-1, and IL-6 levels were attenuated in P2X7 KO mice. P2X7 deletion decreased lung edema and alveolar protein content, which are associated with enhanced alveolar fluid clearance. In addition, activation of the inflammasome was suppressed in P2X7-deficient alveolar macrophages and was associated with suppression of IL-1β release. Furthermore, P2X7-deficient alveolar macrophage in type II alveolar epithelial cells (AECs) coculture model abolished protein permeability across mouse type II AEC monolayers. Deletion of P2X7 does not lead to a decrease in epithelial sodium channel expression in cocultures of alveolar macrophages and type II AECs. Taken together, these findings show that deletion of P2X7 is a protective factor and therapeutic target for the amelioration of hyperoxia-induced lung injury.

PROTEIN MARKERS OF THE HYPOXIC-ISCHEMIC INJURY OF THE CENTRAL NERVOUS SYSTEM IN THE PERINATAL PERIOD

Preeclampsia (gestosis) and eclampsia, as well as the acute intranatal hypoxy often act as trigger for the development of the perinatal hypoxic-ischemic brain injury. That is accompanied by such typical structural changes as periventricular leukomalacia (PVL) and intraventricular hemorrhages (IVH). The hypoxic-ischemic brain injury in the perinatal period can have such consequences as hydrocephaly, microcephaly, infantile cerebral paralysis (ICP), epilepsy and motormental retardation. It is not always possible to determine the disease gravity and forecast of the disease by routine methods of clinical, instrumental and laboratory examination. As it is proved, that the perinatal hypoxic-ischemic brain injury is always accompanied by the disruption of Blood-Brain-Barrier (BBB) permeability for neuron-specific proteins (NSP). NSP can be considered as markers of this pathologic process. Nowadays there are more or less details on 120 NSP, namely, non-enzyme neurospecific Са+ -binding proteins; non-enzyme NSP, responsible for adhesion and intercellular recognition processes; соntractile and cytoskeletal nervous tissue proteins; secreted regulatory and transport NSP; myeline proteins and glial NSP. To evaluate BBB status it is expedient to select from the variety of known NSP most studied proteins, being neurons’ and astrocytes’ markers. Such NSP are gliofibrillary acid protein, GFAP and neuronspecific enolase, NSE. In normal conditions the NSP are within BBB and can be almost undeterminable in the blood serum. In case of the BBB permeability disruption NSP penetrate to the peripheral blood flow and can be determined. The dynamic determination of the NSP in the blood serum can be expedient to evaluate the BBB resistance, to determine the degree of the CNS injury and the disease state forecast at children with perinatal hypoxic-ischemic brain injury.

Opposite Effects of Lysophosphatidylethanolamines on Conformation of OmpF-like Porin from Yersinia pseudotuberculosis.

Lysophosphatidyletnolamine (LPE) is one of enigmatic lipids of bacteria. It is generated from major membrane lipid - phosphatidylethanolamine at severe changes of the bacterial growth conditions. Accumulation of this phospholipid in cells of Gram-negative enterobacterium Yersinia pseudotuberculosis results in the enhanced thermostability of OmpF-like porin (YOmpF) from the same bacteria. The respective integral conformational rearrangements may disturb the channel permeability of protein under stress conditions. However, role of fatty acid composition of LPE in this effect remained unclear. Present work demonstrated that the level of unsaturated LPE is 3.5 times higher than saturated one in total LPE of bacterial cells exposed to stress (phenol treatment). Unsaturated 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (MOPE) and saturated LPE 1-palmitoyl-2- hydroxy-sn-glycero-3-phosphoethanolamine (MPPE) oppositely affect the conformation of YOmpF. MOPE increases the protein thermal stability due to more dense packing of monomers in porin and preserves its trimeric form at elevated temperature, while MPPE weakens the contact between monomers and promotes dissociation of the protein.

Optimisation of the extraction and purification of chondroitin sulphate from head by-products of Prionace glauca by environmental friendly processes

The goal of the present work was to optimise the different environmental friendly processes involved in the extraction and purification of chondroitin sulphate (CS) from Prionace glauca head wastes. The experimental development was based on second order rotatable designs and evaluated by response surface methodology combined with a previous kinetic approach. The sequential stages optimised were: (1) the enzymatic hydrolysis of head cartilage catalysed by alcalase (55.7°C/pH 8.2); (2) the chemical treatment of enzyme hydrolysates by means of alkaline-hydroalcoholic saline solutions (NaOH: 0.54M, EtOH: 1.17v, NaCl: 2.5%) to end the protein hydrolysis and to precipitate and selectively redissolve CS versus the peptidic material and (3) the selective purification and concentration of CS and the concomitant protein permeation of extracts which were obtained from previous treatment using ultrafiltration and diafiltration (UF–DF) technologies at two different cut-offs.

Proteins of the oral mucosa in experimental herpetic stomatitis

Aim. To conduct a comparative analysis of acute phase proteins levels and permeability proteins in the tissues of oral mucosa and blood plasma in case of experimental herpetic stomatitis.&#x0D; Methods. Levels of total protein, C-reactive protein, orosomucoid, α1-antitrypsin, prealbumin, albumin and microalbumin were determined in the homogenates of oral mucosa and blood plasma of 30 rats with experimental herpetic stomatitis.&#x0D; Results. Levels of C-reactive protein, orosomucoid and α1-antitrypsin in serum of rats with experimental herpetic stomatitis are increased. In the homogenates of the rat oral mucosa, levels of the studied proteins increased significantly, by 1.5-3.5 times compared to the control levels. Total protein, orosomucoid, prealbumin, and microalbumin levels increased not only in tissues affected by herpes, but also in the oral mucosa on the opposite side, indicating alterations of histochematic barrier transport of, most importantly, low molecular weight proteins, increased cell membrane permeability and changes in oral mucosa periodontal tissue structural integrity.&#x0D; Conclusion. Herpetic infection of the oral cavity is accompanied by increased levels of acute phase reactants in rat oral mucosa both on the side of oral mucosa infected by herpes and on healthy side. The level of not only acute phase proteins, but also low-molecular transport proteins allows to evaluation the alterations of oral mucosa membrane permeability in herpetic stomatitis.

Study on Changes of the Urea, Serum Creatinine and Glomerular Protein Permeability, after General Anesthesia with Sevoflurane

Abstract Introduction: The widespread use of sevoflurane as an induction and maintenance volatile agent of general anesthesia demostrates an increased safety profile. Sevoflurane contact with CO2 absorbents lead to the occurrence of toxic compounds such as Compund A and Compound B. Among the side efffects of Sevoflurane remember the renal toxic effect much discussed in the literature but still unresolved. In previous research we have demonstrated the glomerular protein changes as a result of exposure to Sevoflurane. In the current study we intend to monitor the changes in blood urea nitrogen and serum creatinine after exposure to Sevoflurane. Material and method: We included in our study 90 patients who were anesthetized in the Department of Anesthesiology of the County Mure Hospital during 01.10.2009-01.10.2014. They had normal values for blood urea nitrogen and serum creatinine and had no preoperative proteinuria. Serum and urine samples were taken preoperatively and at 24 and 72 hours postanesthetic and were analyzed in the laboratory. Proteinuria was determined by spectrophotometry. Results: After protein quantitative determination by spectrophotometry and statistical anaysis we obtained significant differences by comparing the average preoperative/24 hours total protein (p&lt;0.0001) and 24/72 hours (p&lt;0.0001). There are no significant statistical differences by comparing the blood urea nitrogen at the three intervals (p&lt;0.53) and no statistical changes for mean serum creatinine (p&lt;0.18). Conclusions: Changes in glomerular filtered proteins following exposure to Sevoflurane demonstrate its toxic effect on glomerular tubules. Lack of perioperative significant wich is why we recommend determining perioperative urinary protein as a marker of glomerular damage.

Permeation of water vapour, nitrogen, oxygen and carbon dioxide through whey protein isolate based films and coatings—Permselectivity and activation energy

The aim of this study was to evaluate the influence of relative humidity (RH) on the oxygen permeability and water vapour transmission rate (WVTR) of whey protein coated Polyethylene terephthalate (PET) or whey protein monolayer films. Furthermore, the activation energies for the permeability of oxygen, carbon dioxide and nitrogen as well as the permselectivities under different set of temperatures were measured. The results showed that the permeability values through the whey protein coating and whey protein film increased with increasing RH. The water vapour permeability measured at 50% RH of (9.3 ± 0.6)−10 cm3 (STP) cm cm−2 s−1 Pa−1 increased to (16.2 ± 0.9)−10 cm3 (STP) cm cm−2 s−1 Pa−1 at 85% RH. An increase in temperature showed an expected increase of the permeability in both uncoated PET and the whey protein monolayer. The permselectivity of whey protein film was determined and the calculated ratios of the permselectivity (P(N2)/P(O2)/P(CO2):1/(4–6/(34–35)) differed from the simplified ratios given in the literature (P(N2)/P(O2)/P(CO2):1/4/16). Furthermore the calculated activation energy values for whey (51 kJ mol−1) were in agreement with other studies.

Bioactive protein-based nanofibers interact with intestinal biological components resulting in transepithelial permeation of a therapeutic protein

Proteins originating from natural sources may constitute a novel type of material for use in drug delivery. However, thorough understanding of the behavior and effects of such a material when processed into a matrix together with a drug is crucial prior to further development into a drug product. In the present study the potential of using bioactive electrospun fish sarcoplasmic proteins (FSP) as a carrier matrix for small therapeutic proteins was demonstrated in relation to the interactions with biological components of the intestinal tract. The inherent structural and chemical properties of FSP as a biomaterial facilitated interactions with cells and enzymes found in the gastrointestinal tract and displayed excellent biocompatibility. More specifically, insulin was efficiently encapsulated into FSP fibers maintaining its conformation, and subsequent controlled release was obtained in simulated intestinal fluid. The encapsulation of insulin into FSP fibers provided protection against chymotrypsin degradation, and resulted in an increase in insulin transport to around 12% without compromising the cellular viability. This increased transport was driven by interactions upon contact between the nanofibers and the Caco-2 cell monolayer leading to the opening of the tight junction proteins. Overall, electrospun FSP may constitute a novel material for oral delivery of biopharmaceuticals.

Effect of Nutrient Formulations on Permeation of Proteins and Lipids through Porcine Intestine &lt;i&gt;In vitro&lt;/i&gt;

Purpose: To investigate the effect of nutrient formulations on the permeation of proteins and lipids through porcine intestine in vitro . Method: In vitro permeation studies of proteins and lipids of two peptide-based formulations, composed of various compounds and sources of hydrolyzed protein was carried out, and compared with a conservative polymeric formulation as control, The test was undertaken using Franz diffusion cell apparatus incorporating porcine intestine. Results: The peptide-based formulation demonstrated higher protein absorption than the conservative polymeric one. However, there were some differences in protein absorption rates between the peptide based formulations obtained from various sources. Formulation A with 1.0 and 1.5 kcal/mL exhibited significantly (p < 0.05) higher cumulative protein permeation (11.97 ± 0.23 and 12.54 ± 0.94 μg/cm 2 ) than formulations B (9.41 ± 0.36 and 9.67 ± 0.35 μg/cm 2 ) and C (8.34 ± 0.56 and 8.61 ± 0.71 μg/cm 2 ), respectively. Lipid permeation from formulations A and B (13.91 ± 0.26 and 12.94 ± 0.59 μg/cm 2 respectively for 1.0 kcal/mL formulation, and 13.31 ± 0.21 and 12.86 ± 0.16 for 1.5 kcal/mL formulation) which consist mainly of medium chain triglycerides (MCTs), were significantly (p <0.05) higher than those from formulation C (11.49 ± 0.43 and 12.62 ± 0.38 μg/cm 2 for 1.0 and 1.5 kcal/mL formulation, respectively) which mostly contained long chain triglycerides (LCTs). Conclusion: The results reveal that oligomeric formulations have higher absorption rate than polymeric formulations. However, the outcomes when administered to clinically ill patients need to be investigated. Keywords: Nutrient Formulations, Permeation, Proteins, Lipids, Porcine Intestine, Medium Chain Triglycerides, Long Chain Triglycerides

A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes.

In vitro blood-brain barrier (BBB) models based on primary brain endothelial cells (BECs) cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER) and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs) in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP) and breast cancer related protein (BCRP), and the transferrin receptor).

Genetic deficit of KCa 3.1 channels protects against pulmonary circulatory collapse induced by TRPV4 channel activation.

The intermediate conductance calcium/calmodulin-regulated K+ channel KCa 3.1 produces hyperpolarizing K+ currents that counteract depolarizing currents carried by transient receptor potential (TRP) channels, and provide the electrochemical driving force for Cl- and fluid movements. We investigated whether a deficiency in KCa 3.1 (KCa 3.1-/- ) protects against fatal pulmonary circulatory collapse in mice after pharmacological activation of the calcium-permeable TRP subfamily vanilloid type 4 (TRPV4) channels. An opener of TRPV4 channels, GSK1016790A, was infused in wild-type (wt) and KCa 3.1-/- mice; haemodynamic parameters, histology and pulmonary vascular reactivity were measured; and patch clamp was performed on pulmonary arterial endothelial cells (PAEC). In wt mice, GSK1016790A decreased right ventricular and systemic pressure leading to a fatal circulatory collapse that was accompanied by increased protein permeability, lung haemorrhage and fluid extravasation. In contrast, KCa 3.1-/- mice exhibited a significantly smaller drop in pressure to GSK1016790A infusion, no haemorrhage and fluid water extravasation, and the mice survived. Moreover, the GSK1016790A-induced relaxation of pulmonary arteries of KCa 3.1-/- mice was significantly less than that of wt mice. GSK1016790A induced TRPV4 currents in PAEC from wt and KCa 3.1-/- mice, which co-activated KCa 3.1 and disrupted membrane resistance in wt PAEC, but not in KCa 3.1-/- PAEC. Our findings show that a genetic deficiency of KCa 3.1 channels prevented fatal pulmonary circulatory collapse and reduced lung damage caused by pharmacological activation of calcium-permeable TRPV4 channels. Therefore, inhibition of KCa 3.1channels may have therapeutic potential in conditions characterized by abnormal high endothelial calcium signalling, barrier disruption, lung oedema and pulmonary circulatory collapse.

Intrinsic cell permeability of the GAGA zinc finger protein into HeLa cells

We examined the intrinsic cell permeability of a GAGA zinc finger obtained from the Drosophila melanogaster transcription factor and analyzed its mechanism of cellular uptake using confocal microscopy and flow cytometry. HeLa cells were treated with the Cy5-labeld GAGA peptides (containing a fluorescent chromophore) to detect fluorescence signals from the fluorescent labeling peptides by confocal microscopy. The results clearly indicated that GAGA peptides possess intrinsic cell permeability for HeLa cells. Based on the results of the flow cytometry analysis and the theoretical net positive charge of the GAGA peptides, the efficiency of cellular uptake of the GAGA peptides was predicted to depend on the net positive charge of the GAGA peptide as well as the cationic component ratio of Arg residues to Lys residues.

Permeation of Proteins, Oligonucleotide and Dextrans Across Ocular Tissues: Experimental Studies and a Literature Update

Proteins and oligonucleotides represent powerful tools for the treatment of several ocular diseases, affecting both anterior and posterior eye segments. Despite the potential of these compounds, their administration remains a challenge. The last years have seen a growing interest for the noninvasive administration of macromolecular drugs, but still there is only little information of their permeability across the different ocular barriers. The aim of this work was to evaluate the permeation of macromolecules of different size, shape and charge across porcine ocular tissues such as the isolated sclera, the choroid Bruch's membrane and the cornea, both intact and de-epitelialized. Permeants used were two proteins (albumin and cytochrome C), an oligonucleotide, two dextrans (4 and 40 kDa) and a monoclonal antibody (bevacizumab). Obtained data and its comparison with the literature highlight the difficulties in predicting the behavior of macromolecules based on their physicochemical properties, because the interplay between the charge, molecular radius and conformation prevent their analysis separately. However, the data can be of great help for a rough evaluation of the feasibility of a noninvasive administration and for building computational models to improve understanding of the interplay among static, dynamic and metabolic barriers in the delivery of macromolecules to the eye.

Therapeutic Effects of Human Mesenchymal Stem Cell-derived Microvesicles in Severe Pneumonia in Mice.

Microvesicles (MVs) are anuclear fragments of cells released from the endosomal compartment or shed from surface membranes. We and other investigators demonstrated that MVs released by mesenchymal stem cells (MSCs) were as effective as the cells themselves in inflammatory injuries, such as after endotoxin-induced acute lung injury. However, the therapeutic effects of MVs in an infectious model of acute lung injury remain unknown. We investigated the effects of human MSC MVs on lung inflammation, protein permeability, bacterial clearance, and survival after severe bacterial pneumonia. We tested the effects of MVs derived from human MSCs on Escherichia coli pneumonia in mice. We also studied the interactions between MVs and human monocytes and human alveolar epithelial type 2 cells. Administration of MVs derived from human MSCs improved survival in part through keratinocyte growth factor secretion and decreased the influx of inflammatory cells, cytokines, protein, and bacteria in mice injured with bacterial pneumonia. In primary cultures of human monocytes or alveolar type 2 cells, the uptake of MVs was mediated by CD44 receptors, which were essential for the therapeutic effects. MVs enhanced monocyte phagocytosis of bacteria while decreasing inflammatory cytokine secretion and increased intracellular ATP levels in injured alveolar epithelial type 2 cells. Prestimulation of MSCs with a toll-like receptor 3 agonist further enhanced the therapeutic effects of the released MVs. MVs derived from human MSCs were as effective as the parent stem cells in severe bacterial pneumonia.

Prevention of peritendinous adhesions with electrospun polyethylene glycol/polycaprolactone nanofibrous membranes

Postoperative adhesion formation is the major complication that could occur after acute tendon surgery. The application of an anti-adhesive membrane at the post-surgical site is deemed as a potential way to solve this problem by preventing adhesive fibrotic tissue development. In this study, we fabricated electrospun composite poly(ethylene glycol) (PEG)/poly(caprolactone) (PCL) nanofibrous membrane (NFM) to prevent peritendinous adhesions, which could act as a barrier between the tendon and surrounding tissues, without interrupting mass transfer and normal tendon gliding. PCL/PEG NFMs of 0% PEG (PCL), 25% PEG (25PECL), 50% PEG (50PECL) and 75% PEG (75PECL) were prepared and characterized for physico-chemical properties. The PCL NFM shows the lowest protein permeability while 25PECL NFM exhibited the largest fiber diameter, smallest pore size and the largest ultimate stress and strain. The 75PECL NFM had the lowest water contact angle and the highest Young's modulus. In vitro cell adhesion and migration experiments with fibroblasts indicate that all NFMs could prevent cell penetration, with 75PECL NFM having the least cell attachment. In vivo application of 75PECL NFM on the repaired site of rabbit flexor tendon rupture model demonstrated improved efficacy compared with the PCL NFM and a commercial anti-adhesion barrier (Seprafilm™), from gross observation, histological analysis and functional assays. We concluded that 75PECL NFM could function as an effective anti-adhesion membrane after tendon surgery in a clinical setting.

Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of adult mouse brain.

Fenestrated capillaries of the sensory circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis, the subfornical organ and the area postrema, lack completeness of the blood-brain barrier (BBB) to sense a variety of blood-derived molecules and to convey the information into other brain regions. We examine the vascular permeability of blood-derived molecules and the expression of tight-junction proteins in sensory CVOs. The present tracer assays revealed that blood-derived dextran 10k (Dex10k) having a molecular weight (MW) of 10,000 remained in the perivascular space between the inner and outer basement membranes, but fluorescein isothiocyanate (FITC; MW: 389) and Dex3k (MW: 3000) diffused into the parenchyma. The vascular permeability of FITC was higher at central subdivisions than at distal subdivisions. Neither FITC nor Dex3k diffused beyond the dense network of glial fibrillar acidic protein (GFAP)-positive astrocytes/tanycytes. The expression of tight-junction proteins such as occludin, claudin-5 and zonula occludens-1 (ZO-1) was undetectable at the central subdivisions of the sensory CVOs but some was expressed at the distal subdivisions. Electron microscopic observation showed that capillaries were surrounded with numerous layers of astrocyte processes and dendrites. The expression of occludin and ZO-1 was also observed as puncta on GFAP-positive astrocytes/tanycytes of the sensory CVOs. Our study thus demonstrates the heterogeneity of vascular permeability and expression of tight-junction proteins and indicates that the outer basement membrane and dense astrocyte/tanycyte connection are possible alternative mechanisms for a diffusion barrier of blood-derived molecules, instead of the BBB.