Permeability of the blood–tumor barrier is enhanced by combining vascular endothelial growth factor with papaverine

This study aims to determine the effects of vascular endothelial growth factor (VEGF), papaverine (PA), and the combination of VEGF and PA on the permeability of the blood–tumor barrier (BTB) and to determine possible molecular mechanisms contributing to the effects. In the rat C₆ glioma model, the extravasation of Evans blue (EB) through the BTB was increased significantly by VEGF and PA. VEGF‐induced and PA‐induced increase of EB extravasation was further increased after combining VEGF with PA infusion. Transmission electron microscopy (TEM) showed that the combination of VEGF and PA not only opened tight junctions (TJ) dramatically but increased the presence of pinocytotic vesicles of brain microvascular endothelial cells (BMECs) significantly. Meanwhile, the downregulation of the TJ‐associated proteins occludin and claudin‐5 and the upregulation of the caveolae structure proteins caveolin‐1 and caveolin‐2 caused by the combination of VEGF and PA were observed by Western blot and immunohistochemistry, which were more remarkable than those by the two strategies separately. In addition, after VEGF and PA infusion, the results of radioimmunoassay, Western blot, and enzyme‐linked immunosorbent assay (ELISA) revealed a significant increase in expression levels of cGMP and protein kinase G‐1 (PKG‐1) and the activation of nuclear factor‐κB (NF‐κB) p65. This study demonstrates that combination of VEGF and PA can increase the permeability of the BTB by a paracellular pathway (downregulation of occludin and claudin‐5) and a transcellular pathway (upregulation of caveolin‐1 and caveolin‐2) and that the cGMP/PKG/NF‐κB signal pathway might be involved in the modulation process. © 2014 Wiley Periodicals, Inc.     

PPARγ Agonist Pioglitazone Reverses Memory Impairment and Biochemical Changes in a Mouse Model of Type 2 Diabetes Mellitus

Aims: Pioglitazone, known as a peroxisome proliferator‐activated receptor γ (PPARγ) agonist, is used to treat type 2 diabetes mellitus (T2DM). T2DM has been associated with reduced performance on numerous domains of cognitive function. Here, we investigated the effects of pioglitazone on memory impairment in a mouse model with defects in insulin sensitivity and secretion, namely high‐fat diet (HFD) streptozotocin (STZ)‐induced diabetic mice. Methods: ICR mice were fed with HFD for 4 weeks and then injected with a single low dose of STZ followed by continued HFD feeding for an additional 4 weeks. Pioglitazone (18 mg/kg, 9 mg/kg body weight) was orally administered for 6 weeks once daily. Y‐maze test and Morris water maze test (MWM) were employed for testing learning and memory. Serum glucose, serum insulin, serum triglyceride, brain β‐amyloid peptide (Aβ), brain β‐site amyloid precursor protein cleaving enzyme (BACE1), brain nuclear factor κB (NF‐κB), and brain receptor for advanced glycation end products (RAGE) were also tested. Results: The STZ/HFD diabetic mice, characterized by hyperglycemia, hyperlipemia and hypoinsulinemia, performed poorly on Y‐maze and MWM hence reflecting impairment of learning and memory behavior with increases of Aβ40/Aβ42, BACE1, NF‐κB, and RAGE in brain. Treatment of PPARγ agonist, pioglitazone (18 or 9 mg/kg body weight), significantly reversed diabetes‐induced impairment of learning and memory behavior, which is involved in decreases of Aβ40/Aβ42 via inhibition of NF‐κB, BACE1 and RAGE in brain as well as attenuation of hyperglycemia, hyperlipemia, and hypoinsulinemia. Conclusions: It is concluded that PPARγ agonist pioglitazone may be considered as potential pharmacological agents for the management of cognitive dysfunction in T2DM.     

Upregulation of RAGE at the blood‐brain barrier in streptozotocin‐induced diabetic mice

Deposition of amyloid‐β peptide (Aβ) in the brain of diabetes is poorly understood. The receptor for advanced glycation end products (RAGE) at the blood‐brain barrier (BBB) is critical for regulation of Aβ homeostasis in the brain. In this studies, we used streptozotocin‐induced diabetic mice to observe the expression of RAGE at the BBB by Western blot and immunocytochemical analysis, and the in vivo blood‐to‐brain influx transport of ¹²⁵I‐Aβ_1–40 using the permeability surface area product (PS) and brain capillary uptake. In the diabetic mice with hyperglycemia (>16.0 mmol/L) at 6 weeks, RAGE expression at the BBB was significantly upregulated, no significant changes of RAGE levels were found at 1 and 3 weeks after diabetes induction. The data of PS and brain capillary uptake for Aβ showed significant RAGE‐dependent transport of Aβ across the BBB and substantial RAGE‐dependent brain capillary uptake at 6 weeks after diabetes induction. We conclude that the upregulation of RAGE at the BBB contributes to cerebral Aβ deposition in the diabetes. Synapse 63:636–642, 2009. © 2009 Wiley‐Liss, Inc.     

Degeneration of noradrenergic fibres from the locus coeruleus causes tight-junction disorganisation in the rat brain

Although functional studies demonstrate that noradrenaline controls the permeability of the blood-brain barrier, it has never been determined whether this neurotransmitter regulates the tight junction (TJ) assembly that confers the barrier property to brain microvessels. We thus tested in rats the effect of pharmacological depletion of noradrenaline with the noradrenergic toxin DSP4 (5 mg/kg) on the expression of the TJ proteins zonula occludens-1 (ZO1) and occludin. The effectiveness of the lesion was confirmed by tyrosine hydroxylase immunoreactivity, which showed noradrenergic fibre reduction accompanied by debris and swollen fibres in DSP4-treated brains. Noradrenergic fibre degeneration caused: (i) gliosis; (ii) disappearance of TJ proteins in vascular cell-to-cell contacts (49.9 and 38.3% reductions for occludin and ZO1, respectively); (iii) a 49.2% decrease in total ZO1 protein, measured by Western blot analysis, parallel to a 39.5% decrease in ZO1 mRNA, measured by real-time PCR; and (iv) a relative increase in the beta occludin isoform (62.9%), with no change in total occludin protein or mRNA. The expression of endothelial brain antigen, a marker of a functionally competent brain endothelium, was also reduced. We conclude that damage to the ascending fibres from the locus coeruleus caused TJ disruption and gliosis, a sign of inflammation. These results imply that the locus coeruleus degeneration reported in Alzheimer's and Parkinson's diseases may contribute to these disorders by causing blood-brain barrier dysfunction. Whether the vascular damage is the result of impaired noradrenergic transmission or secondary to the inflammatory reaction remains to be determined.     

Specific Role of Tight Junction Proteins Claudin-5, Occludin,and ZO-1 of the Blood–Brain Barrier in a Focal Cerebral Ischemic Insult

Blood–brain barrier (BBB) leakage plays a key role in cerebral ischemia–reperfusion injury. It is quite necessary to further explore the characteristic and mechanism of BBB leakage during stroke. We induced a focal cerebral ischemia model by transient middle cerebral artery occlusion in male rats for defining the time course of BBB permeability within 120 h following reperfusion and evaluate the specific role of tight junction (TJ) associated proteins claudin-5, occludin, and ZO-1 as well as protein kinase C delta (PKCδ) pathway in BBB leakage induced by reperfusion injury. We verified a bimodal increase in the permeability of the BBB following focal ischemia by Evans blue assay. Two peaks of BBB permeability appeared at 3 h and 72 h of reperfusion after 2 h focal ischemia, respectively. The leak at the endothelial cell was represented at the level of transmission electron microscopy. TTC staining results showed increased infarct size with time after cerebral ischemia reperfusion. The mRNA and protein expression levels of these three TJ associated proteins were significantly decreased compared with the sham-operated group within 120 h of reperfusion, corresponding to the time-dependent change of the biphasic pattern in BBB leakage. The redistribution of claudin-5, occludin, and ZO-1 in ischemia brain microvascular endothelial cells was observed at the same time points. In addition, Western blot assay revealed PKCδ level was also significantly increased in a similar biphasic pattern to above results within 120 h after cerebral ischemia–reperfusion. This study demonstrates the timing of TJ associated proteins claudin-5, occludin, and ZO-1 in light of BBB permeability associated with cerebral ischemia reperfusion, and suggests PKCδ pathway may participate in TJ barrier open and BBB leakage during reperfusion injury in a time-dependent manner.     

RAGE-NF-κB-PPARγ Signaling is Involved in AGEs-Induced Upregulation of Amyloid-β Influx Transport in an In Vitro BBB Model

The receptor for advanced glycation end products (RAGE) at the blood-brain barrier (BBB) is critical for regulation of amyloid-β (Aβ) homeostasis in the diabetic brain. In this study, we used an in vitro BBB model consisting of mouse brain capillary endothelial cells (MBCECs) to investigate whether advanced glycation end products (AGEs) increase Aβ influx transport across the BBB and the underlying mechanisms. We found that AGEs induced Aβ influx transport across the BBB in concentration- and time-dependent manner, accompanied by increased RAGE expression and nuclear factor-kappa B p65 (NF-κB p65), and decreased nuclear peroxisome proliferator-activated receptor γ (PPARγ). Blockade of RAGE with its antibody and inhibition of NF-κB signaling with PDTC as well as activation of PPARγ with rosiglitazone significantly decreased Aβ transport across the BBB from the periphery to the brain. These treatments also pronouncedly suppressed AGEs-induced increases in RAGE expression and nuclear NF-κB p65 and reversed the decrease in nuclear PPARγ. These results suggest that RAGE-NF-κB-PPARγ signaling is involved in regulation of AGEs-induced influx transport of Aβ across the BBB and targeting the signaling pathway could serve as a novel strategy to modify such Aβ transport.     

Oxidative stress increases blood�Cbrain barrier permeability and induces alterations in occludin during hypoxia�Creoxygenation

The blood–brain barrier (BBB) has a critical role in central nervous system homeostasis. Intercellular tight junction (TJ) protein complexes of the brain microvasculature limit paracellular diffusion of substances from the blood into the brain. Hypoxia and reoxygenation (HR) is a central component to numerous disease states and pathologic conditions. We have previously shown that HR can influence the permeability of the BBB as well as the critical TJ protein occludin. During HR, free radicals are produced, which may lead to oxidative stress. Using the free radical scavenger tempol (200 mg/kg, intraperitoneal), we show that oxidative stress produced during HR (6% O₂ for 1 h, followed by room air for 20 min) mediates an increase in BBB permeability in vivo using in situ brain perfusion. We also show that these changes are associated with alterations in the structure and localization of occludin. Our data indicate that oxidative stress is associated with movement of occludin away from the TJ. Furthermore, subcellular fractionation of cerebral microvessels reveals alterations in occludin oligomeric assemblies in TJ associated with plasma membrane lipid rafts. Our data suggest that pharmacological inhibition of disease states with an HR component may help preserve BBB functional integrity.     

Chinese medicine Tongxinluo capsule protects against blood-brain barrier disruption after ischemic stroke by inhibiting the low-density lipoprotein receptor-related protein 1 pathway in mice

BackgroundChinese medicine Tongxinluo capsule (TXL) has been extensively used to treat ischemic stroke in China, and one of its mechanisms is to protect against blood brain barrier (BBB) disruption after stroke. However, the underlying protective mechanisms are not fully illuminated. It is reported that the low-density lipoprotein receptor-related protein 1 (LRP-1) is involved in BBB disruption after brain ischemia. In this study, we explored whether TXL could downregulate LRP-1 expression and subsequently protect against BBB disruption after stroke using permanent middle cerebral artery occlusion (pMCAO) in mice.MethodsThe animal model of ischemic stroke was induced by pMCAO in male adult C57BL/6J mice. The mice were orally administered TXL (3.0 g/kg) at 1, 3 and 21 h after pMCAO. Meanwhile, the LRP-1 antagonist receptor associated protein (RAP) was intracerebroventricularly injected at 1 and 21 h after stroke. We measured the following parameters at 6 and 24 h: LRP-1 protein level, BBB leakage, and the expression of tight junction (TJ) proteins including occludin, claudin‐5 and zonula occludens‐1 (ZO‐1).ResultsOur results showed that TXL downregulated LRP-1 level, upregulated these TJ proteins level, and reduced BBB leakage in peri-infarct regions after pMCAO. Further study found that the inhibitor RAP played the same role as did TXL in upregulating these TJ proteins level and reducing BBB leakage after stroke.ConclusionOur study demonstrates that TXL protects against BBB disruption after stroke via inhibiting the LRP-1 pathway.     

Aβ????-RAGE interaction disrupts tight junctions of the blood-brain barrier via Ca2?-calcineurin signaling

The blood-brain barrier (BBB), which is formed by adherens and tight junctions (TJs) of endothelial cells, maintains homeostasis of the brain. Disrupted intracellular Ca2? homeostasis and breakdown of the BBB have been implicated in the pathogenesis of Alzheimer's disease (AD). The receptor for advanced glycation end products (RAGE) is known to interact with amyloid β-peptide (Aβ) and mediate Aβ transport across the BBB, contributing to the deposition of Aβ in the brain. However, molecular mechanisms underlying Aβ-RAGE interaction-induced alterations in the BBB have not been identified. We found that Aβ???? induces enhanced permeability, disruption of zonula occludin-1 (ZO-1) expression in the plasma membrane, and increased intracellular calcium and matrix metalloproteinase (MMP) secretion in cultured endothelial cells. Neutralizing antibodies against RAGE and inhibitors of calcineurin and MMPs prevented Aβ????-induced changes in ZO-1, suggesting that Aβ-RAGE interactions alter TJ proteins through the Ca2?-calcineurin pathway. Consistent with these in vitro findings, we found disrupted microvessels near Aβ plaque-deposited areas, elevated RAGE expression, and enhanced MMP secretion in microvessels of the brains of 5XFAD mice, an animal model for AD. We have identified a potential molecular pathway underlying Aβ-RAGE interaction-induced breakage of BBB integrity. This pathway might play an important role in the pathogenesis of AD.     

Redox regulation of NF‐κB p50 and M1 polarization in microglia

Redox‐signaling is implicated in deleterious microglial activation underlying CNS disease, but how ROS program aberrant microglial function is unknown. Here, the oxidation of NF‐κB p50 to a free radical intermediate is identified as a marker of dysfunctional M1 (pro‐inflammatory) polarization in microglia. Microglia exposed to steady fluxes of H₂O₂ showed altered NF‐κB p50 protein–protein interactions, decreased NF‐κB p50 DNA binding, and augmented late‐stage TNFα expression, indicating that H₂O₂ impairs NF‐κB p50 function and prolongs amplified M1 activation. NF‐κB p50^?/? mice and cultures exhibited a disrupted M2 (alternative) response and impaired resolution of the M1 response. Persistent neuroinflammation continued 1 week after LPS (1 mg/kg, IP) administration in the NF‐κB p50^?/? mice. However, peripheral inflammation had already resolved in both strains of mice. Treatment with the spin‐trap DMPO mildly reduced LPS‐induced 22 h TNFα in the brain in NF‐κB p50^+/+ mice. Interestingly, DMPO failed to reduce and strongly augmented brain TNFα production in NF‐κB p50^?/? mice, implicating a fundamental role for NF‐κB p50 in the regulation of chronic neuroinflammation by free radicals. These data identify NF‐κB p50 as a key redox‐signaling mechanism regulating the M1/M2 balance in microglia, where loss of function leads to a CNS‐specific vulnerability to chronic inflammation. GLIA 2015;63:423–440     

Tight junctions in the blood–brain barrier promote edema formation and infarct size in stroke – Ambivalent effects of sealing proteins

The outcome of stroke is greatly influenced by the state of the blood–brain barrier (BBB). The BBB endothelium is sealed paracellularly by tight junction (TJ) proteins, i.e., claudins (Cldns) and the redox regulator occludin. Functions of Cldn3 and occludin at the BBB are largely unknown, particularly after stroke. We address the effects of Cldn3 deficiency and stress factors on the BBB and its TJs. Cldn3 tightened the BBB for small molecules and ions, limited endothelial endocytosis, strengthened the TJ structure and controlled Cldn1 expression. After middle cerebral artery occlusion (MCAO) and 3-h reperfusion or hypoxia of isolated brain capillaries, Cldn1, Cldn3 and occludin were downregulated. In Cldn3 knockout mice (C3KO), the reduction in Cldn1 was even greater and TJ ultrastructure was impaired; 48 h after MCAO of wt mice, infarct volumes were enlarged and edema developed, but endothelial TJs were preserved. In contrast, junctional localization of Cldn5 and occludin, TJ density, swelling and infarction size were reduced in affected brain areas of C3KO. Taken together, Cldn3 and occludin protect TJs in stroke, and this keeps the BBB intact. However, functional Cldn3, Cldn3-regulated TJ proteins and occludin promote edema and infarction, which suggests that TJ modulation could improve the outcome of stroke.     

Insulin-induced generation of reactive oxygen species and uncoupling of nitric oxide synthase underlie the cerebrovascular insulin resistance in obese rats

Hyperinsulinemia accompanying insulin resistance (IR) is an independent risk factor for stroke. The objective is to examine the cerebrovascular actions of insulin in Zucker obese (ZO) rats with IR and Zucker lean (ZL) control rats. Diameter measurements of cerebral arteries showed diminished insulin-induced vasodilation in ZO compared with ZL. Endothelial denudation revealed vasoconstriction to insulin that was greater in ZO compared with ZL. Nonspecific inhibition of nitric oxide synthase (NOS) paradoxically improved vasodilation in ZO. Scavenging of reactive oxygen species (ROS), supplementation of tetrahydrobiopterin (BH(4)) precursor, and inhibition of neuronal NOS or NADPH oxidase or cyclooxygenase (COX) improved insulin-induced vasodilation in ZO. Immunoblot experiments revealed that insulin-induced phosphorylation of Akt, endothelial NOS, and expression of GTP cyclohydrolase-I (GTP-CH) were diminished, but phosphorylation of PKC and ERK was enhanced in ZO arteries. Fluorescence studies showed increased ROS in ZO arteries in response to insulin that was sensitive to NOS inhibition and BH(4) supplementation. Thus, a vicious cycle of abnormal insulin-induced ROS generation instigating NOS uncoupling leading to further ROS production underlies the cerebrovascular IR in ZO rats. In addition, decreased bioavailability and impaired synthesis of BH(4) by GTP-CH induced by insulin promoted NOS uncoupling.     

Hemoglobin-Induced Nitric Oxide Synthase Overexpression and Nitric Oxide Production Contribute to Blood–Brain Barrier Disruption in the Rat

Hemoglobin (Hb) released from extravasated erythrocytes may have a critical role in the process of blood–brain barrier (BBB) disruption and subsequent edema formation after intracerebral hemorrhage (ICH). Excessive nitric oxide (NO) production synthesized by nitric oxide synthase (NOS) has been well documented to contribute to BBB disruption. However, considerably less attention has been focused on the role of NO in Hb-induced BBB disruption. This study was designed to examine the hypothesis that Hb-induced NOS overexpression and excessive NO production may contribute to the changes of tight junction (TJ) proteins and subsequent BBB dysfunction. Hemoglobin was infused with stereotactic guidance into the right caudate nucleus of male Sprague Dawley rats. Then, we investigated the effect of Hb on the BBB permeability, changes of TJ proteins (claudin-5, occludin, zonula occludens-1 (ZO-1), and junctional adhesion molecule-1 (JAM-1)), iron deposition, expression of inducible NOS (iNOS) and endothelial NOS (eNOS), as well as NO production. Hb injection caused a significant increase in BBB permeability. Significant reduction of claudin-5, ZO-1, and JAM-1 was observed after Hb injection as evidenced by PCR and immunofluorescence. After a decrease at early stage, occludin showed a fivefold increase in mRNA level at 7 days. Significant iron deposition was detectable from 48 h to 7 days in a time-dependent manner. The iNOS and eNOS levels dramatically increased after Hb injection concomitantly with large quantities of NO released. Furthermore, enhanced iNOS or eNOS immunoreactivity was co-localized with diffused or diminished claudin-5 staining. We concluded that overexpressed NOS and excessive NO production induced by Hb may contribute to BBB disruption, which may provide an important potential therapeutic target in the treatment of ICH.     

Nuclear factor‐κB/p65 responds to changes in the Notch signaling pathway in murine BV‐2 cells and in amoeboid microglia in postnatal rats treated with the γ‐secretase complex blocker DAPT

Microglial cells constitutively express Notch‐1 and nuclear factor‐κB/p65 (NF‐κB/p65), and both pathways modulate production of inflammatory mediators. This study sought to determine whether a functional relationship exists between them and, if so, to investigate whether they synergistically regulate common microglial cell functions. By immunofluorescence labeling, real‐time polymerase chain reaction (RT‐PCR), flow cytometry, and Western blot, BV‐2 cells exhibited Notch‐1 and NF‐κB/p65 expression, which was significantly up‐regulated in cells challenged with lipopolysaccharide (LPS). This was coupled with an increase in expression of Hes‐1, tumor necrosis factor‐α (TNF‐α), and interleukin‐1β (IL‐1β). In BV‐2 cells pretreated with N‐[N‐(3,5‐difluorophenacetyl)‐1‐alany1]‐S‐phenyglycine t‐butyl ester (DAPT), a γ‐secretase inhibitor, followed by LPS stimulation, Notch‐1 expression level was enhanced but that of all other markers was suppressed. Additionally, Hes‐1 expression and NF‐κB nuclear translocation decreased as shown by flow cytometry. Notch‐1's modulation of NF‐κB/p65 was also evidenced in amoeboid microglial cells (AMC) in vivo. In 5‐day‐old rats given intraperitoneal injections of LPS, Notch‐1, NF‐κB/p65, TNF‐α, and IL‐1β immunofluorescence in AMC was markedly enhanced. However, in rats given an intraperitoneal injection of DAPT prior to LPS, Notch‐1 labeling was augmented, but that of TNF‐α and IL‐1β was reduced. The results suggest that blocking of Notch‐1 activation with DAPT would reduce the level of its downstream end product Hes‐1 along with suppression of NF‐κB/p65 translocation, resulting in suppressed production of proinflammatory cytokines. It is concluded that Notch‐1 signaling can trans‐activate NF‐κB/p65 by amplifying NF‐κB/p65‐dependent proinflammatory functions in activated microglia. © 2010 Wiley‐Liss, Inc.     

Biphasic cytoarchitecture and functional changes in the BBB induced by chronic inflammatory pain

The blood-brain barrier (BBB) is a dynamic system which maintains brain homeostasis and limits CNS penetration via interactions of transmembrane and intracellular proteins. Inflammatory pain (IP) is a condition underlying several diseases with known BBB perturbations, including stroke, Parkinson's, multiple sclerosis and Alzheimer's. Exploring the underlying pathology of chronic IP, we demonstrated alterations in BBB paracellular permeability with correlating changes in tight junction (TJ) proteins: occludin and claudin-5. The present study examines the IP-induced molecular changes leading to a loss in functional BBB integrity. IP was induced by injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the right hindpaw of female Sprague-Dawley rats. Inflammation and hyperalgesia were confirmed, and BBB paracellular permeability was assessed by in situ brain perfusion of [14C]sucrose (paracellular diffusion marker). The permeability of the BBB was significantly increased at 24 and 72 h post-CFA. Analysis of the TJ proteins, which control the paracellular pathway, demonstrated decreased claudin-5 expression at 24 h, and an increase at 48 and 72 h post-injection. Occludin expression was significantly decreased 72 h post-CFA. Expression of junction adhesion molecule-1 (JAM-1) increased 48 h and decreased by 72 h post-CFA. Confocal microscopy demonstrated continuous expression of both occludin and JAM-1, each co-localizing with ZO-1. The increased claudin-5 expression was not limited to the junction. These results provide evidence that chronic IP causes dramatic alterations in specific cytoarchitectural proteins and demonstrate alterations in molecular properties during CFA, resulting in significant changes in BBB paracellular permeability.     

Assessments of tight junction proteins occludin, claudin 5 and scaffold proteins ZO1 and ZO2 in endothelial cells of the rat blood-brain barrier: cellular responses to neurotoxicants malathion and lead acetate

The blood-brain barrier (BBB) is essential for central nervous system (CNS) normal function. It is formed by endothelial cells with special characteristics, which confer the BBB with low permeability and high transendothelial electrical resistance (TEER). We previously demonstrated that malathion and lead, two neurotoxicants widely present in the environment, decrease TEER and increase permeability in in vitro models of the BBB. In this study we assessed tight junction disruption at the protein and gene expression levels using a rat brain microvascular endothelial cell line (RBE4) exposed to lead acetate at 10(-5)M and 10(-6)M, malathion at 10(-5)M, malaoxon at 10(-6)M, and their combinations. Cells were incubated with treatments for 2h, 4h, 8h, 16h, and 24h periods. Immunoblotting assessments demonstrated that protein levels of tight junction proteins occludin and claudin 5, and scaffold proteins ZO1 and ZO2 were decreased after treatments. Gene expression determinations did not correlate with the decreases in protein, indicating that the effects on these proteins were post-translational.