Protein Permeability Research Articles

Effect of temperature and relative humidity on the water vapour permeability and mechanical properties of cassava starch and soy protein concentrate based edible films.

The effect of temperature and relative humidity on the water vapour permeability (WVP) and mechanical properties of cassava starch and soy protein concentrate (SPC) based edible films containing 20% glycerol level were studied. Tensile strength and elastic modulus of edible films increased with increase in temperature and decreased with increase in relative humidity, while elongation at break decreased. Water vapour permeability of the films increased (2.6-4.3g.mm/m(2).day.kPa) with increase in temperature and relative humidity. The temperature dependence of water vapour permeation of cassava starch-soy protein concentrate films followed Arrhenius relationship. Activation energy (Ea) of water vapour permeation of cassava starch-soy protein concentrate edible films ranged from 1.9 to 5.3kJ/mol (R (2) ≥ 0.93) and increased with increase in SPC addition. The Ea values were lower for the bio-films than for polyvinylidene chloride, polypropylene and polyethylene which are an indication of low water vapour permeability of the developed biofilms compared to those synthetic films.

Exposure to vehicle emissions results in altered blood brain barrier permeability and expression of matrix metalloproteinases and tight junction proteins in mice

BackgroundTraffic-generated air pollution-exposure is associated with adverse effects in the central nervous system (CNS) in both human exposures and animal models, including neuroinflammation and neurodegeneration. While alterations in the blood brain barrier (BBB) have been implicated as a potential mechanism of air pollution-induced CNS pathologies, pathways involved have not been elucidated.ObjectivesTo determine whether inhalation exposure to mixed vehicle exhaust (MVE) mediates alterations in BBB permeability, activation of matrix metalloproteinases (MMP) -2 and −9, and altered tight junction (TJ) protein expression.MethodsApolipoprotein (Apo) E−/− and C57Bl6 mice were exposed to either MVE (100 μg/m3 PM) or filtered air (FA) for 6 hr/day for 30 days and resulting BBB permeability, expression of ROS, TJ proteins, markers of neuroinflammation, and MMP activity were assessed. Serum from study mice was applied to an in vitro BBB co-culture model and resulting alterations in transport and permeability were quantified.ResultsMVE-exposed Apo E−/− mice showed increased BBB permeability, elevated ROS and increased MMP-2 and −9 activity, compared to FA controls. Additionally, cerebral vessels from MVE-exposed mice expressed decreased levels of TJ proteins, occludin and claudin-5, and increased levels of inducible nitric oxide synthase (iNOS) and interleukin (IL)-1β in the parenchyma. Serum from MVE-exposed animals also resulted in increased in vitro BBB permeability and altered P-glycoprotein transport activity.ConclusionsThese data indicate that inhalation exposure to traffic-generated air pollutants promotes increased MMP activity and degradation of TJ proteins in the cerebral vasculature, resulting in altered BBB permeability and expression of neuroinflammatory markers.

Interferon-γ-Induced Intestinal Epithelial Barrier Dysfunction by NF-κB/HIF-1α Pathway

Interferon-γ (IFN-γ) plays an important role in intestinal barrier dysfunction. However, the mechanisms are not fully understood. As hypoxia-inducible factor-1 (HIF-1) is a critical determinant response to hypoxia and inflammation, which has been shown to be deleterious to intestinal barrier function, we hypothesized that IFN-γ induces loss of barrier function through the regulation of HIF-1α activation and function. In this study, we detected the expressions of HIF-1α and tight junction proteins in IFN-γ-treated T84 intestinal epithelial cell line. IFN-γ led to an increase of HIF-1α expression in time- and dose-dependent manners but did not change the expression of HIF-1β. The IFN-γ-induced increase in HIF-1α was associated with an activation of NF-κB. Treatment with the NF-κB inhibitor, pyrolidinedithiocarbamate (PDTC), significantly suppressed the activation of NF-κB and the expression of HIF-1α. In addition, IFN-γ also increased intestinal epithelial permeability and depletion of tight junction proteins; inhibition of NF-κB or HIF-1α prevented the increase in intestinal permeability and alteration in tight junction protein expressions. Interestingly, we demonstrated that a significant portion of IFN-γ activation NF-kB and modulation tight junction expression is mediated through HIF-1α. Taken together, this study suggested that IFN-γ induced the loss of epithelial barrier function and disruption of tight junction proteins, by upregulation of HIF-1α expression through NF-κB pathway.

S98 Vitamin D deficiency increases bacterial load in a murine model of sepsis-induced lung injury

IntroductionWe have previously shown that patients with Acute Lung Injury (ALI) are severely vitamin D deficient. Several studies have reported a high prevalence of vitamin D deficiency in critically ill...

Structure design and performance study on 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 PVC concentration and poly(ethylene glycol) (PEG) molecular weight in polymer solutions on structure and performance of HR membranes were investigated. The results showed that the HR PVC membranes had a dense and smooth outer surface compared with matrix membrane. The maximum pure water flux and protein permeation flux of HR PVC membranes were obtained as the PVC concentration reached to 10wt.% and PEG molecular weight was 2000. The increase of PVC concentration and PEG molecular weight brought about the increase of protein rejection rate and the decrease of membrane porosity. The high flux recovery rate (>85.00%) indicated that the HR membranes had excellent anti-fouling property. Moreover, the HR PVC membranes had a favorable interfacial bonding force between the coating layer and matrix membrane. The tensile strength of HR membranes decreased slightly compared with matrix membrane and was nearly 19MPa, but the elongation at break was opposite which was nearly 102%.

PSS151 - Restoration of NAD Redox Balance Ameliorates Pressure Overload-induced Cardiac Hypertrophy and Dysfunction Via Regulation of Mitochondrial Protein Acetylation and Permeability Transition

PSS151 - Restoration of NAD Redox Balance Ameliorates Pressure Overload-induced Cardiac Hypertrophy and Dysfunction Via Regulation of Mitochondrial Protein Acetylation and Permeability Transition

The protective effect of VSL#3 on intestinal permeability in a rat model of alcoholic intestinal injury

BackgroundThis study aimed to investigate the mechanism of the probiotic VSL#3 in acute alcoholic intestinal injury, and evaluate the effect of VSL#3, glutamine,VSL#3+glutamine and heat-killed VSL#3 therapy in a rat model.MethodsSix- to eight-week-old male wild-type rats were divided into seven groups. To establish the acute alcohol liver disease model, rats received three doses of corn starch dissolved in PBS/40% alcohol administered intra-gastrically every 12 hours. Treatment groups received an intra-gastric dose of VSL#3, Glutamine, heat-killed VSL#3, or VSL#3+Glutamine 30 minutes prior to alcohol administration. The placebo group was treated with PBS prior to alcohol administration. TNFα and endotoxin in plasma was measured by ELISA and Tachypleus Ameboctye Lysate assays, and electron microscopy, Western blotting, and reverse transcription polymerase chain reaction were used to identify the mechanisms of VSL#3 in the regulation of epithelial permeability.ResultsFirst, compared with control group, endotoxin and TNFα in alcohol group was obviously high. At the same time, in VSL#3 group,the expression of endotoxin and TNFα obviously lower than the alcohol group. And the trends of the expression of tight junction proteins in these groups were reversed with the change of endotoxin and TNFα. Second, compared the groups of VSL#3 with glutamine,VSL#3+glutamine and heat-killed VSL#3,we found that both VSL#3 and heat-killed VSL#3, glutamine were as effective as VSL#3+glutamine in the treatment of acute alcohol liver disease, the expression of endotoxin and TNFα were lower than the alcohol group, and tight junction proteins were higher than the alcohol group whereas the expression of tight junction proteins were higher in VSL#3 + glutamine group than either agent alone, but have no significant difference.ConclusionWe conclude that VSL#3 treatment can regulate the ecological balance of the gut microflora, preventing passage of endotoxin and other bacterial products from the gut lumen into the portal circulation and down-regulating the expression of TNFα, which could otherwise down-regulate the expression of tight junction proteins and increase epithelial permeability.

Combination of homogenization and cross-flow microfiltration to remove microorganisms from industrial buttermilks with an efficient permeation of proteins and lipids

Abstract Buttermilk is a source of interesting nutritional and functional components, e.g. polar lipids and proteins. However, it is still considered as a low-value by-product of the dairy industry with high variations in biochemical composition and bacterial contaminations. The objective of this study was to develop a process based on microfiltration, permitting the removal of microorganisms to ensure the safety of buttermilk components for human nutrition. Industrial buttermilks and the products collected during microfiltration were characterized using particle size measurements, biochemical and microbiological analysis. The combination of homogenization at 80 MPa and cross-flow microfiltration successfully removed bacteria from skimmed buttermilk: bacterial reduction > 4.8 log 10 with 0 cfu/ml in the permeate using the 0.8 μm pore size membrane and 1 cfu/ml with 1.4 μm membrane. Chemical analysis revealed the efficient permeation of proteins, total lipids and polar lipids. Polar lipid classes permeated equally the membrane. This work will contribute in improving the safety of buttermilk-based ingredients. Industrial relevance This work describes the development of an innovative process combining homogenization and cross-flow microfiltration for the selective removal of bacteria from industrial buttermilks. This process is an alternative to heat treatments that alter the nutritional and organoleptic properties of food products. The safety of buttermilk-based ingredients containing milk polar lipids of interest will contribute in their economic valorization for human nutrition.

Fluid of choroidal origin

AbstractThe choroidal circulation is crucial in supplying nutrients as well as oxygen to the outer retina. The capillaries of the choriocapillaris are fenestrated and have a high protein permeability that allows the establishment of a high oncotic pressure, presumably contributing to movement of fluid out of the retina through the stroma to the sclera. The pores of the choriocapillaris are facing the RPE. Bruchs membrane acts as a sieve depending on the amount and chemical properties of the deposited waste products. The accumulation of fluid under the RPE or within the retina implies a tight Bruchs membrane. Lipids as result of the the outer segment turn over accumulate and decrease the permeability of Bruchs membrane precluding the evacuation of the waste products and initiate a vicious circle. Active transport by the RPE is impaired. The tight junctions of the RPE are ruptured, the fluid irruption into the retina results in the disorganisation of the outer layers of the retina. A progressive breakdown of all defense mechanisms especially the reabsorbtion pump tips over the balance. The mechanismsm of the restauration are partially known. All choroidal diseases can induce these featuree and clinicians may analyze the resulting steps

In Vivo Analysis of Aquaporin 0 Function in Zebrafish: Permeability Regulation Is Required for Lens Transparency

The zebrafish lens is well suited for studies of physiology and development due to its rapid formation in the embryo and genetic accessibility. Aquaporin 0 (AQP0), a lens-specific membrane protein, is required for lens clarity. Zebrafish have two copies of AQP0 (Aqp0a and b), whereas mammals have a single, multifunctional protein. Here we demonstrate a reliable knockdown/rescue system in zebrafish and use it to provide evidence for subfunctionalization of Aqp0a and b, as well as to show that calcium-mediated regulation of Aqp0a in zebrafish lenses is necessary for transparency. Coinjection of antisense oligonucleotides and DNA rescue constructs into zebrafish embryos, followed by evaluation of the developing fish for cataracts, was used to analyze the functions of Aqp0a and b. The water permeability and regulation characteristics of each rescue protein were tested in a Xenopus oocyte swelling assay. Both copies of AQP0 are necessary for lens clarity in the zebrafish, and neither is sufficient. Water permeability is necessary but also insufficient. Phosphorylation and regulation of Aqp0a are required for its function. In the zebrafish lens, the two closely related AQP0s have acquired distinct functions that are both necessary for lens development and clarity. Regulation of AQP0 water permeability, a well-studied phenomenon in vitro, may be physiologically relevant in the living lens.

Adult stem cells for acute lung injury: Remaining questions and concerns

Acute lung injury (ALI) or acute respiratory distress syndrome remains a major cause of morbidity and mortality in hospitalized patients. The pathophysiology of ALI involves complex interactions between the inciting event, such as pneumonia, sepsis or aspiration, and the host immune response resulting in lung protein permeability, impaired resolution of pulmonary oedema, an intense inflammatory response in the injured alveolus and hypoxemia. In multiple preclinical studies, adult stem cells have been shown to be therapeutic due to both the ability to mitigate injury and inflammation through paracrine mechanisms and perhaps to regenerate tissue by virtue of their multi-potency. These characteristics have stimulated intensive research efforts to explore the possibility of using stem or progenitor cells for the treatment of lung injury. A variety of stem or progenitor cells have been isolated, characterized and tested experimentally in preclinical animal models of ALI. However, questions remain concerning the optimal dose, route and the adult stem or progenitor cell to use. Here, the current mechanisms underlying the therapeutic effect of stem cells in ALI as well as the questions that will arise as clinical trials for ALI are planned are reviewed.

Inhibition of GSK3α/β promotes increased pulmonary endothelial permeability to albumin by reactive oxygen/nitrogen species

Glycogen synthase kinase 3α/β (GSK3α/β) is a serine/threonine kinase that participates in numerous processes in many cell types. Importantly, the role of GSK3α/β in homeostatic maintenance of the pulmonary endothelial cell barrier to protein is not known. We tested the hypothesis that GSK3α/β regulates endothelial barrier function by measuring the permeability to albumin of a rat pulmonary microvessel endothelial cell monolayer (PMECM) treated with and without the selective GSK3α/β inhibitor SB 216763 (1.0, 5.0 and 10 uM) for 1 h. The treatment with the inhibitor SB 216763 caused a dose dependent decrease in phospho-β-catenin-Ser33/37 levels indicating effective suppression of GSK3α/β. SB216763 caused an increase in both permeability to albumin and DCFDA (6-Carboxy-2′,7′-Dichlorodihydrofluorescein Diacetate, Di(Acetoxymethyl Ester)) oxidation that were prevented by co-treatment with the anti-oxidant tiron or the nitric oxide synthase inhibitor L-NAME (Nω-nitro-l-arginine-methyl ester). In separate studies PMECMs were treated with the Akt inhibitor triciribine (12.5 uM) for 1 h to unmask Akt dependent constitutive suppression of GSK3α/β. Triciribine decreased phospho-GSK3α/β-Ser21/9 (i.e., the product of Akt) which was associated with an increase in phospho-β-catenin-Ser33/37 (i.e., the product of GSK3α/β) indicating constitutive activity of Akt for GSK3α/β-Ser21/9. The data indicates GSK3α/β inhibition causes increased endothelial monolayer protein permeability which is mediated by reactive oxygen/nitrogen species.

Novel mechanism of cytokine-induced disruption of epithelial barriers

The ductal epithelium plays a key role in physiological secretion of pancreatic enzymes into the digestive system. Loss of barrier properties of the pancreatic duct may contribute to the development of pancreatitis and metastatic dissemination of pancreatic tumors. Proinflammatory cytokines are essential mediators of pancreatic inflammation and tumor progression; however, their effects on the integrity and barrier properties of the ductal epithelium have not been previously addressed. In the present study, we investigate mechanisms of cytokine-induced disassembly of tight junctions (TJs) and adherens junctions (AJs) in a model pancreatic epithelium. Exposure of HPAF-II human pancreatic epithelial cell monolayers to interferon (IFN)γ disrupted integrity and function of apical junctions as manifested by increased epithelial permeability and cytosolic translocation of AJ and TJ proteins. Tumor necrosis factor (TNF)α potentiated the effects of IFNγ on pancreatic epithelial junctions. The cytokine-induced increase in epithelial permeability and AJ/TJ disassembly was attenuated by pharmacological inhibition of Janus kinase (JAK) and protein kinase D (PKD). Loss of apical junctions in IFNγ/TNFα-treated HPAF-II cells was accompanied by JAK and PKD dependent decrease in expression of AJ (E-cadherin, p120 catenin) and TJ (occludin, ZO-1) proteins. Depletion of E-cadherin or p120 catenin recapitulated the effects of cytokines on HPAF-II cell permeability and junctions. Our data suggests that proinflammatory cytokines disrupt pancreatic epithelial barrier via expressional downregulation of key structural components of AJs and TJs. This mechanism is likely to be important for pancreatic inflammatory injury and tumorigenesis.

The Caco-2 cell culture model enables sensitive detection of enhanced protein permeability in the presence of N-decyl-β-d-maltopyranoside

Appropriate assessment of transepithelial permeability in vitro is needed to estimate and model trans-mucosal bioavailability to achieve oral delivery of protein biopharmaceuticals. The Caco-2 cell-based intestinal epithelium model is widely used for this purpose for low molecular mass drugs. The aim of this study was to test the suitability of the Caco-2 model for assessing enhanced transepithelial permeability to proteins. Four unrelated proteins were chosen to test the permeability of Caco-2 monolayers. It was found that proteins could cross the epithelium model, in spite of their size. All tested proteins had similar very low apparent permeability coefficients (Papp) of around 4×10(-10)cm/s. Protein stability over three-hour exposure to Caco-2 was also confirmed. Their crossing rate in a cell-free setup was also measured, to determine the upper limit of permeability to proteins. An epithelium permeability enhancer N-decyl-β-d-maltopyranoside (MP C10) was used to demonstrate accelerated permeability conditions. Papp values could be increased dose dependently up to about 1×10(-7)cm/s, close to the level in the cell-free setup, indicating distinctive potential of the model. This along with enhancing effect of known specific route permeators suggests involvement of the paracellular route in protein transport. Our results thus indicate that the Caco-2 model is a suitable tool for in vitro assessment of enhanced permeability to proteins.

SOX8 Regulates Permeability of the Blood-Testes Barrier That Affects Adult Male Fertility in the Mouse1

Sertoli cells provide nutritional and physical support to germ cells during spermatogenesis. Sox8 encodes a member of the high mobility group of transcription factors closely related to Sox9 and Sox10. Sertoli cells express SOX8 protein, and its elimination results in an age-dependent dysregulation of spermatogenesis, causing adult male infertility. Among the claudin genes with altered expression in the Sox8(-/-) testes, was claudin-3, which is required for the regulation and maintenance of the blood-testes barrier (BTB). Because the BTB is critical in restricting small molecules in the luminal compartment of the seminiferous tubules, the aim of this study was to analyze the level of tight junction proteins (claudin-3, claudin-11, and occludin) and BTB permeability in Sox8(-/-) adult testes. The acetylation level of alpha-tubulin and microtubule organization was also evaluated because microtubules are critical in maintaining the microenvironment of the seminiferous epithelium. Western blot analysis shows that claudin-3 protein is decreased in Sox8(-/-) testes. Chromatin immunoprecipitation confirmed that SOX8 binds at the promoter region of claudin-3. Claudin-3 was localized to the Sertoli cell tight junctions of wild-type testes and significantly decreased in the Sox8(-/-) testes. The use of biotin tracers showed increased BTB permeability in the Sox8(-/-) adult testes. Electron microscopy analysis showed that microtubule structures were destabilized in the Sox8(-/-) testes. These results suggest that Sox8 is essential in Sertoli cells for germ cell differentiation, partly by controlling the microenvironment of the seminiferous epithelium.

Predicting Susceptibility to Alveolar Flooding in Terms of Critical Cardiovascular Parameters

Modest increases in pulmonary interstitial fluid pressure (Pi) can lead to alveolar flooding and respiratory failure. Arising from the complex interaction of microvascular filtration, lymphatic drainage, as well as cardiac, renal and venous function, Pi is typically predicted by mathematical models that are notoriously complex, difficult to interpret, and solvable only by those with advanced programming skills. Therefore, we developed a simple model that predicts Pi as a function of critical cardiovascular parameters. Microvascular filtration into the pulmonary interstitium is characterized by the Starling‐Landis equation, with parameters for protein and water permeability. Lymph flow out of the interstitium is characterized by a lymphatic model with parameters for sensitivity to Pi and central venous pressure. Finally, capillary and central venous pressures are characterized by a standard minimal closed‐loop model with parameters for ventricular compliances and contractilities, arterial and venous compliances, and arterial and venous resistances. By linearization and judicious use of simplifying assumptions, we developed algebraic solutions predicting Pi. This simple model is comprehensive enough to predict the susceptibility of different patient populations to alveolar flooding with such diverse conditions as sepsis, hemodilution, and fluid volume retention with acute heart failure.

Glomerular Endothelial Surface Layer Acts as a Barrier against Albumin Filtration

Glomerular endothelium is highly fenestrated, and its contribution to glomerular barrier function is the subject of debate. In recent years, a polysaccharide-rich endothelial surface layer (ESL) has been postulated to act as a filtration barrier for large molecules, such as albumin. To test this hypothesis, we disturbed the ESL in C57Bl/6 mice using long-term hyaluronidase infusion for 4 weeks and monitored albumin passage using immunolabeling and correlative light-electron microscopy that allows for complete and integral assessment of glomerular albumin passage. ESL ultrastructure was visualized by transmission electron microscopy using cupromeronic blue and by localization of ESL binding lectins using confocal microscopy. We demonstrate that glomerular fenestrae are filled with dense negatively charged polysaccharide structures that are largely removed in the presence of circulating hyaluronidase, leaving the polysaccharide surfaces of other glomerular cells intact. Both retention of native ferritin [corrected] in the glomerular basement membrane and systemic blood pressure were unaltered. Enzyme treatment, however, induced albumin passage across the endothelium in 90% of glomeruli, whereas this could not be observed in controls. Yet, there was no net albuminuria due to binding and uptake of filtered albumin by the podocytes and parietal epithelium. ESL structure and function completely recovered within 4 weeks on cessation of hyaluronidase infusion. Thus, the polyanionic ESL component, hyaluronan, is a key component of the glomerular endothelial protein permeability barrier.

Human chorionic gonadotropin controls luteal vascular permeability via vascular endothelial growth factor by down-regulation of a cascade of adhesion proteins

To study the functional interactions of junctional proteins acting as regulators of vascular permeability in the human corpus luteum. We investigated the role of vascular endothelial (VE)-cadherin, nectin 2, and claudin 5 as controllers of vascular endothelial cell permeability. Performing immunohistochemical dual staining, we colocalized the above-mentioned proteins in the human corpus luteum. Not applicable. Not applicable. Not applicable. Using a granulosa-endothelial coculture system, we revealed that hCG-treatment down-regulates VE-cadherin, nectin 2, and claudin 5 in endothelial cells via vascular endothelial growth factor (VEGFA). Furthermore, the interaction of VE-cadherin, nectin 2, and claudin 5 was investigated by silencing these proteins that perform siRNA knockdown. Interestingly, knockdown of VE-cadherin and claudin 5 induced a decrease of the respective other protein. This down-regulation was associated with changed rates of vascular permeability: hCG induced a VEGFA-dependent down-regulation of VE-cadherin, nectin 2, and claudin 5, which increased the endothelial permeability in the coculture system. Furthermore, knockdown of VE-cadherin, nectin-2, and claudin 5 also resulted in a consecutive increase of endothelial permeability for each different protein. These results demonstrate for the first time that VE-cadherin, nectin 2, and claudin 5 are involved in the regulation of vascular permeability in a mutually interacting manner, which indicates their prominent role for the functionality of the human corpus luteum.

Erectile Dysfunction Precedes Other Systemic Vascular Diseases Due to Incompetent Cavernous Endothelial Cell-Cell Junctions

Erectile dysfunction is often a harbinger of cardiovascular disease. We sought to gain mechanistic insight at the cellular and molecular levels into why erectile dysfunction precedes the clinical consequences of cardiovascular disease. Diabetes was induced by intraperitoneal streptozotocin injection in 8-week-old C57BL/6J mice. At 8 weeks after diabetes induction, we determined the expression of endothelial cell-cell junction proteins and vascular endothelial permeability in the penis, heart and hind limb by systemic injection of various vascular space markers (350 Da to 2,000 kDa) or by immunohistochemical staining with antibody to oxidized low density lipoprotein. We also investigated the effect of recombinant Ang1 protein on cavernous endothelial permeability. Alterations in the integrity of the endothelial cell-cell junction, including a decrease in endothelial cell-cell junction proteins and an increase in vascular permeability to fluorescent tracers or oxidized low density lipoprotein, were prominent in the cavernous tissue of diabetic mice. In contrast, no significant changes in endothelial cell-cell junction proteins or vascular permeability were noted in heart or hind limb tissue according to the diabetic condition. Intracavernous injection of Ang1 protein, an anti-permeability factor, significantly decreased cavernous endothelial permeability to oxidized low density lipoprotein by restoring endothelial cell-cell junction proteins in diabetic mice. The incompetent cavernous endothelial cell-cell junction in the diabetic condition provides an important clue to why erectile dysfunction is highly prevalent and often precedes other systemic vascular diseases.

Peptide Derived from HIV-1 TAT Protein Destabilizes a Monolayer of Endothelial Cells in an in Vitro Model of the Blood-Brain Barrier and Allows Permeation of High Molecular Weight Proteins

Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3-0.6 μmol/ml of this C-TAT peptide, for a period of 1-2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.