ß-Sitosterol Enhances Lung Epithelial Cell Permeability by Suppressing the NF-κB Signaling Pathway.

BACKGROUND: The dysregulation between pro-inflammatory and anti-inflammatory responses during sepsis is a crucial factor in driving sepsis progression. Acute lung injury (ALI) resulting from excessive production and accumulation of inflammatory mediators in the lungs contributes to impaired lung barrier function. The activation of the NF-κB signaling pathway during inflammation leads to the transcriptional activation of multiple inflammatory genes. Given the plausible impact of NF-κB signaling suppression in mitigating lung injury, substantive evidence demonstrates beta-sitosterol (BS)'s proficient ability to block NF-κB activation. Therefore, the aim of the present investigation was to delve into the impacts of BS in the context of sepsis-induced acute lung injury, employing both a mouse model and a model involving lung epithelial cells. METHODS: Sepsis-induced lung injury was simulated in mice through cecum ligation and puncture (CLP). To emulate injury in murine lung epithelial (MLE-12) cells, an experiment involving lipopolysaccharide (LPS) was administered. Evaluation of alterations in lung tissue permeability encompassed techniques such as lung wet/dry (W/D) mass ratio, Evans blue staining, and quantification of total protein concentration in bronchoalveolar lavage fluid (BALF). Lung tissue histopathological shifts were ascertained via hematoxylin and eosin (HE) staining. Additionally, the concentrations of inflammatory cytokines IL-6 and TNF-α were quantified in every lung tissue and cell group by implementing enzyme-linked immunosorbent assay (ELISA). Protein quantification for signal biomarkers was carried out using Western blotting and immunofluorescence methodologies. In tandem, the assessment of MLE-12 cell permeability was conducted by evaluating fluorescein isothiocyanate (FITC)-dextran extravasation. RESULTS: BS mitigated lung tissue pathologies, reduced inflammatory factors, and lowered tissue and cell permeability. BS inhibited NF-κB signaling and increased claudin-4 and claudin-5 expression, enhancing septic lung epithelial cell permeability. CONCLUSIONS: Through suppressing the NF-κB signaling cascade, BS effectively curtails the levels of inflammatory mediators. Simultaneously, it orchestrates the modulation of claudin-4 and claudin-5 expression, culminating in the augmentation of lung epithelial cell barrier competence, thus improving sepsis-induced lung injury.

Paritaprevir ameliorates experimental acute lung injury in vitro and in vivo.

Paritaprevir is a potent inhibitor of the NS3/4A protease used to treat chronic hepatitis C virus infection. However, its therapeutic effect on acute lung injury (ALI) remains to be elucidated. In this study, we investigated the effect of paritaprevir on a lipopolysaccharide (LPS)-induced two-hit rat ALI model. The anti-ALI mechanism of paritaprevir was also studied in human pulmonary microvascular endothelial (HM) cells following LPS-induced injury in vitro. Administration of 30 mg/kg paritaprevir for 3 days protected rats from LPS-induced ALI, as reflected by the changes in the lung coefficient (from 0.75 to 0.64) and lung pathology scores (from 5.17 to 5.20). Furthermore, the levels of the protective adhesion protein VE-cadherin and tight junction protein claudin-5 increased, and the cytoplasmic p-FOX-O1 and nuclear ß-catenin and FOX-O1 levels decreased. Similar effects were observed in vitro with LPS-treated HM cells, including decreased nuclear ß-catenin and FOX-O1 levels and higher VE-cadherin and claudin-5 levels. Moreover, ß-catenin inhibition resulted in higher p-FOX-O1 levels in the cytoplasm. These results suggested that paritaprevir could alleviate experimental ALI via the ß-catenin/p-Akt/ FOX-O1 signaling pathway.

D-beta-hydroxybutyrate reduced the enhanced cardiac microvascular endothelial FoxO1 to play protective roles in diabetic rats and high glucose-stimulated human cardiac microvascular endothelial cells.

The O subfamily of forkhead (FoxO) 1 may participate in the pathogenesis of diabetic microvascular endothelial injury. However, it is unknown whether D-beta-hydroxybutyrate (BHB) regulates cardiac microvascular endothelial FoxO1 to play protective roles in diabetes. In the study, limb microvascular morphological changes, endothelial distribution of the tight junction protein Claudin-5 and FoxO1, and FoxO1 content in limb tissue from clinical patients were evaluated. Then the effects of BHB on cardiac microvascular morphological changes, cardiac FoxO1 generation and its microvascular distribution in diabetic rats were measured. And the effects of BHB on FoxO1 generation in high glucose (HG)-stimulated human cardiac microvascular endothelial cells (HCMECs) were further analyzed. The results firstly confirmed the enhanced limb microvascular FoxO1 distribution, with reduced Claudin-5 and endothelial injury in clinical patients. Then the elevated FoxO1 generation and its enhanced cardiac microvascular distribution were verified in diabetic rats and HG-stimulated HCMECs. However, BHB inhibited the enhanced cardiac FoxO1 generation and its microvascular distribution with attenuation of endothelial injury in diabetic rats. Furthermore, BHB reduced the HG-stimulated mRNA expression and protein content of FoxO1 in HCMECs. In conclusion, BHB reduced the enhanced cardiac microvascular endothelial FoxO1 to play protective roles in diabetic rats and HG-stimulated HCMECs.

Lipopolysaccharide downregulates the expression of ZO-1 protein through the Akt pathway.

BACKGROUND: Neonatal bacterial meningitis is a common neonatal disease with high morbidity, and can cause serious sequelae when left untreated. Escherichia coli is the common pathogen, and its endotoxin, lipopolysaccharide (LPS) can damage the endothelial cells, increasing the permeability of the blood-brain barrier (BBB), leading to intracranial inflammation. However, the specific mechanism of bacterial meningitis induced by LPS damaging BBB remains unclear. In this study, the mouse brain microvascular endothelial (bEND.3) cells were used as a research object to investigate whether LPS damage BBB through the PI3K/Akt pathway. METHODS: The bEND.3 cells were stimulated with different concentrations of LPS for 12 h, and the expression of tight junction proteins (ZO-1, claudin-5, occludin) was detected using western blotting. The cells were challenged with the same concentration of LPS (1ug/ml) across different timepoints (0, 2 h, 4 h, 6 h, 12 h, 24 h). Expression of TJ proteins and signal pathway molecules (PI3K, p-PI3K, Akt, p-Akt) were detected. The distribution of ZO-1 in bEND.3 cells were detected by immunofluorescence staining. RESULTS: A negative correlation is observed between ZO-1 and LPS concentration. Moreover, a reduced expression of ZO-1 was most significant under 1 ug/ml of LPS, and the difference was statistically significant (P < 0.05). Additionally, there is a negative correlation between ZO-1 and LPS stimulation time. Meanwhile, the expression of claudin-5 and occludin did not change significantly with the stimulation of LPS concentration and time. The immunofluorescence assay showed that the amount of ZO-1 on the surface of bEND.3 cells stimulated with LPS was significantly lower than that of the control group. After LPS stimulation, p-Akt protein increased at 2 h and peaked at 4 h. The titer of p-PI3K did not change significantly with time. CONCLUSION: LPS can downregulate the expression of ZO-1; however, its effect on claudin-5 and occludin is minimal. Akt signal pathway may be involved in the regulation of ZO-1 expression induced by LPS in bEND.3 cells.

The Protective Effects of Acetazolamide Against Homocysteine-Induced Blood-Brain-Barrier Disruption by Regulating the Activation of the Wnt/ß-Catenin Signaling Pathway.

Acetazolamide (AZA) is a carbonic anhydrase inhibitor (CAI) with neuroprotective effects. Hyperhomocysteinemia is associated with blood-brain-barrier (BBB) disruption in brain disorders. A previous study indicated that AZA might have a new role in brain disorders. However, its function in hyperhomocysteinemia-related BBB disruption has not been reported. Here, we aim to clarify the role of AZA in homocysteine (Hcy)-mediated BBB dysfunction using both in vivo and in vitro assays. We found that AZA improved memory and cognitive function, and reduced brain edema in Hcy-stimulated hyperhomocysteinemia model rats. This protective effect of AZA on hyperhomocysteinemia rats was accompanied by improved BBB permeability and increased expression levels of the tight junction proteins, occludin, and claudin-5. The in vitro assay results show that AZA prevented Hcy-induced cell injury and attenuated the increased permeability in Hcy-treated bEnd.3 brain endothelial cells. The Hcy-induced decrease in occludin and claudin-5, and increase in MMP-2 and MMP-9 expression levels were attenuated by AZA in bEnd.3 cells. Moreover, the Hcy-induced downregulation of the Wnt/ß-catenin signaling pathway in bEnd.3 cells was abolished by AZA. Inhibition of Wnt/ß-catenin by ICG-001 reversed the protective effects of AZA in Hcy-treated bEnd.3 cells. We also prove that this process is mediated by WTAP. These findings suggest that acetazolamide mitigated the Hcy-induced compromised brain vascular endothelial integrity by regulating the activation of the Wnt/ß-catenin signaling pathway.

Effects of alkaline mineral complex water supplementation on growth performance, inflammatory response, and intestinal barrier function in weaned piglets.

The purpose of the present study was to investigate the effects of drinking water alkaline mineral complex (AMC) supplementation on growth performance, intestinal morphology, inflammatory response, immunity, antioxidant defense system, and barrier functions in weaned piglets. In a 15-d trial, 240 weaned piglets (9.35 ± 0.86 kg) at 28 d of age (large white × landrace × Duroc) were randomly divided into two groups: the control (Con) group and the AMC group. Drinking water AMC supplementation improved (P < 0.01) final body weight (BW) and average daily gain (ADG) in weaned piglets compared to the Con group. Importantly, AMC reduced (P < 0.01) the feed-to-gain (F:G) ratio. AMC water improved the physical health conditions of piglets under weaning stress, as reflected by the decreased (P < 0.05) hair score and conjunctival score. Moreover, there was no significant (P > 0.05) difference in relatively small intestinal length, organ (liver, spleen, and kidney) indices, or gastrointestinal pH value in weaned piglets between the two groups. Of note, AMC significantly promoted the microvilli numbers in the small intestine and effectively ameliorated the gut morphology damage induced by weaning stress, as evidenced by the increased (P < 0.05) villous height (VH) and ratio of VH to crypt depth. Additionally, AMC lessened the levels of lipopolysaccharide (LPS, P < 0.01) and the contents of IL1ß (P<0.05), and TNF-α (P<0.05) in the weaned piglet small intestine. Conversely, the gut immune barrier marker, secretory immunoglobulin A (sIgA) levels in serum and small intestine mucosa were elevated after AMC water treatment (P < 0.01). Furthermore, AMC elevated the antioxidant mRNA levels of (P < 0.05) SOD 1-2, (P < 0.01) CAT, and (P < 0.01) GPX 1-2 in the small intestine. Likewise, the mRNA levels of the small intestine tight junction factors Occludin (P < 0.01), ZO-1 (P < 0.05), Claudin 2 (P < 0.01), and Claudin 5 (P<0.01) in the AMC treatment group were notably higher than those in the Con group. In conclusion, drinking water AMC supplementation has an accelerative effect on growth performance by elevating gut health by improving intestinal morphology, the inflammatory response, the antioxidant defense system, and barrier function in weaned piglets.

The piglet suffers vital physiological, environmental, and social challenges when it is weaned from the sow that can predispose the piglet to subsequent diseases and other production losses, and these challenges are responsible for serious economic losses to the swine industry. Weaning stress induces intestinal injury, decreased immunity, and digestive system dysfunction, which then reduces feed intake and inhibits the growth performance of piglets. It is well known that alternatives to antibiotics for preventing weaning stress in weaned farm animals are sorely needed. The biologically beneficial effects of alkaline mineral water are widely reported. Alkaline mineral complex (AMC), as an immunomodulator, is considered to have antistress effects in the swine industry. In addition, treatment through drinking water is considered to be an efficient and low-cost feasible disease control strategy. Drinking water AMC supplementation is expected to exert health benefits in pigs; however, the responses of weaned piglets to water supplemented with AMC have not been fully explored. Thus, this study explored the effects of drinking water AMC supplementation on growth performance and gut health in weaned piglets. Our results showed that AMC water supplementation conspicuously enhanced the growth performance by improving the gut health.

Defibrotide suppresses brain metastasis by activating the adenosine A2A receptors.

Brain metastasis is a devastating clinical condition globally as one of the most common central nervous system malignancies. The current study aimed to assess the effect of defibrotide, an Food and Drug Administration-approved drug, against brain metastasis and the underlying molecular mechanisms. Two tumor cell lines with high brain metastasis potential, PC-9 and 231-BR, were subjected to defibrotide treatment of increasing dosage. The metastasis capacity of the tumor cells was evaluated by cell invasion and migration assays. Western blotting was employed to determine the levels of tight junction proteins in the blood-brain barrier (BBB) including Occludin, Zo-1, and Claudin-5, as well as metastasis-related proteins including CXCR4, MMP-2, and MMP-9. The in-vitro observations were further verified in nude mice, by monitoring the growth of xenograft tumors, mouse survival and brain metastasis foci following defibrotide treatment. Defibrotide inhibited proliferation, migration, invasion, and promotes lactate dehydrogenase release of brain metastatic tumor cells, elevated the levels of BBB tight junction proteins and metastasis-related proteins. Such beneficial role of defibrotide was mediated by its inhibitory action on the SDF-1/CXCR4 signaling axis both in vitro and in vivo , as CXCR4 agonist SDF1α negated the anti-tumoral effect of defibrotide on mouse xenograft tumor growth, mouse survival and brain metastasis. Defibrotide inhibits brain metastasis through activating the adenosine A2A receptors, which in turn inhibits the SDF-1/CXCR4 signaling axis. Our study hereby proposes defibrotide as a new and promising candidate drug against brain metastasis of multiple organ origins.

Sinomenine alleviates glomerular endothelial permeability by activating the C/EBP-α/claudin-5 signaling pathway.

Diabetic nephropathy (DN) is one of the main complications of diabetes. It is closely associated with the dysfunction of glomerular endothelial cells (GECs) under hyperglycemia. Severe inflammation is an important inducer for the development of GECs dysfunction, and it contributes to the disruption of tight junctions in GECs and the increased endothelial permeability. Sinomenine, an alkaloid monomer extracted from the rhizome of Sinomenium acutum, is recognized for its multiple pharmacological functions, including an anti-DN property. The present study aimed to explore the potential functional mechanism of Sinomenine against DN. Animals were randomly divided into Sham, DN, DN + Sinomenine (20 mg/kg), and DN + Sinomenine (40 mg/kg) groups. The Sinomenine or vehicle was administered every day for 6 weeks, followed by collecting renal tissues for further detection. Increased body weights, elevated blood glucose levels and UAE values, aggravated renal tissue pathology, higher concentrations of IL-18 and IL-1ß in renal tissues, and reduced claudin-5 expression were observed in DN rats. However, the administration of Sinomenine significantly alleviated all these DN-related changes. Furthermore, human renal glomerular endothelial cells (HrGECs) were treated with high glucose (HG, 30 mM) with or without Sinomenine (50, 100 µM) for 24 h. We found that Sinomenine treatment ameliorated the elevated production of IL-18 and IL-1ß, increased fluorescence intensity of FITC-dextran, declined trans-endothelial electrical resistance (TEER) value, and reduction of claudin-5 and C/EBP-α in HG-treated HrGECs. Moreover, the regulatory effect of Sinomenine on endothelial monolayer permeability in HG-treated HrGECs was abolished by the knockdown of C/EBP-α, indicating C/EBP-α is required for the effect of Sinomenine. We concluded that Sinomenine alleviated diabetic nephropathy-induced renal glomerular endothelial dysfunction via activating the C/EBP-α/claudin-5 axis.

Lowering of brain endothelial cell barrier function by exposure to 4'-iodo-α-pyrrolidinononanophenone.

Overuse of pyrrolidinophenones (PPs) is known to cause damage to vascular and central nervous systems, but little is known about its effect on brain endothelial barrier function. In this study, we found that exposure to 4'-iodo-α-pyrrolidinononanophenone (I-α-PNP), one of the most potently cytotoxic PPs, at sublethal concentrations decreases trans-endothelial electrical resistance and increases paracellular permeability across a monolayer of human brain microvascular endothelial cells. Treatment with I-α-PNP also elevated the production of superoxide anion. Furthermore, the treatment reduced the expression and plasma membrane localization of a tight junction protein claudin-5 (CLDN5), which was almost restored by pretreatment with an antioxidant N-acetyl-l-cysteine. These results indicate that I-α-PNP treatment may down-regulate the plasma membrane-localized CLDN5 by elevating the production of reactive oxygen species (ROS). The treatment with I-α-PNP increased the nuclear translocation of Forkhead box protein O1 (FoxO1), an oxidative stress-responsive transcription factor, and pretreating with a FoxO1 inhibitor ameliorated the decrease in CLDN5 mRNA. In addition, I-α-PNP treatment up-regulated the expression and secretion of matrix metalloproteinase-2 (MMP2) and MMP9, and the addition of an MMP inhibitor reversed the degradation of CLDN5 by I-α-PNP. Moreover, I-α-PNP treatment facilitated the activation of 26S proteasome-based proteolytic activity and pretreatment with an inhibitor of 26S proteasome, but not autophagy, suppressed the CLDN5 degradation by I-α-PNP. Accordingly, it is suggested that the down-regulation of CLDN5 by exposure to I-α-PNP is ascribable to suppression of the gene transcription due to FoxO1 nuclear translocation through ROS production and to acceleration both of the MMPs (MMP2 and MMP9)- and 26S proteasome-based proteolysis.

Verdiperstat attenuates acute lung injury by modulating MPO/µ-calpain/ß-catenin signaling.

Verdiperstat, a myeloperoxidase (MPO) inhibitor, is a well-known drug used for the treatment of multisystem atrophy. However, its therapeutic effect on acute lung injury (ALI) remains to be elucidated. In this study, the effect of verdiperstat on lipopolysaccharide (LPS)-induced two-hit rat ALI model was studied in vivo. Subsequently, to explore the anti-ALI mechanism of verdiperstat, an LPS-induced injury in human pulmonary microvascular endothelial cells (HMs) was studied in vitro. The continuous administration of verdiperstat at 120 mg/kg for 3 days exerted a protective effect on the LPS-induced two-hit rat ALI model, as reflected by the change in the lung coefficient and lung pathology scores from 0.72 to 0.61 and 6.08 to 4.37, respectively. Furthermore, the values of protective adhesion protein VE-cadherin and tight junction protein claudin 5 changed from 0.42 to 0.97 and 0.25 to 0.72, but MPO, the ratio of N-µ-calpain to µ-calpain, and the distribution of ß-catenin in the nucleus changed from 3.04 to 2.17, 0.62 to 0.38 and 2.25 to 0.76, respectively. LPS-induced HMs in vitro also showed similar results, including lower MPO and the distribution of ß-catenin in the nucleus, but higher VE-cadherin claudin 5 and N-µ-calpain. Moreover, MPO inhibition resulted in lower µ-calpain activation and lower ß-catenin in the nucleus. Our cumulative results suggest that verdiperstat alleviates ALI by strengthening VE-cadherin and claudin 5 through the inhibition of MPO/µ-calpain/ß-catenin activation.

The protective effects of butorphanol tartrate against homocysteine-induced blood-brain barrier dysfunction.

A high concentration of homocysteine (Hcy) has been recently reported to be closely associated with the development of stroke, which is related to the Hcy-induced blood-brain barrier (BBB) dysfunction. Butorphanol tartrate is a promising analgesic agent that targets the opiate receptor and shows promising protective effects on ischemia/reperfusion injury. The present research proposes to investigate the protective effect of butorphanol tartrate on Hcy-induced BBB disruption to explore the potential application of butorphanol tartrate in treating Hcy-induced stroke. Hcy was utilized to establish both an in vivo animal model and in vitro human brain vascular endothelial cells (HBVECs) injury model. We found that the increased diffusion of sodium fluorescein and Evan's blue, declined expression of Claudin-5, and increased production of interleukin- 6 (IL-6) and tumor necrosis factor-α (TNF-α) were observed in Hcy-treated mice, which were all significantly reversed by butorphanol tartrate. In Hcy-stimulated HBVECs, increased endothelial permeability and reduced expression levels of Claudin-5 and Krüppel-like factor 5 (KLF5) were observed, all of which were dramatically rescued by 2 and 5 µM butorphanol tartrate. Lastly, the protective function of butorphanol tartrate in Hcy-stimulated HBVECs was dramatically abolished by the knockdown of KLF5. Collectively, butorphanol tartrate showed protective effects on Hcy-induced BBB disruption by upregulating the KLF5/Claudin-5 axis.

Claudin-5 binder enhances focused ultrasound-mediated opening in an in vitro blood-brain barrier model.

Rationale: The blood-brain barrier (BBB) while functioning as a gatekeeper of the brain, impedes cerebral drug delivery. An emerging technology to overcome this limitation is focused ultrasound (FUS). When FUS interacts with intravenously injected microbubbles (FUS+MB), the BBB opens, transiently allowing the access of therapeutic agents into the brain. However, the ultrasound parameters need to be tightly tuned: when the acoustic pressure is too low there is no opening, and when it is too high, tissue damage can occur. We therefore asked whether barrier permeability can be increased by combining FUS+MB with a second modality such that in a clinical setting lower acoustic pressures could be used. Methods: Given that FUS+MB achieves BBB opening in part by disruption of tight junction (TJ) proteins such as claudin-5 of brain endothelial cells, we generated a stable MDCK (Madin-Darby Canine Kidney) II cell line (eGFP-hCldn5-MDCK II) that expresses fluorescently tagged human claudin-5. Two claudin-5 binders, the peptide mC5C2 and cCPEm (truncated form of an enterotoxin), reported previously to weaken the barrier, were synthesized and assessed for their abilities to enhance the permeability of cellular monolayers. We then performed a comparative analysis of single and combination treatments, measuring transendothelial electrical resistance (TEER) and cargo leakage, combined with confocal image analysis. Results: We successfully generated a novel cell line that formed functional monolayers as validated by an increased TEER reading and a low (< 0.2%) permeability to sodium fluorescein (376 Da). We found that the binders exerted a time- and concentration-dependent effect on barrier opening when incubated over an extended period, whereas FUS+MB caused a rapid opening followed by recovery after 12 hours within the tested pressure range. Importantly, preincubation with cCPEm prior to FUS+MB treatment resulted in greater barrier opening compared to either FUS+MB or cCPEm alone as measured by reduced TEER values and an increased permeability to fluorescently labelled 40 kDa dextran (FD40). Conclusion: The data suggest that pre incubation with clinically suitable binders to TJ proteins may be a general strategy to facilitate safer and more effective ultrasound-mediated BBB opening in cellular and animal systems and potentially also for the treatment of human diseases of the brain.

Response of immature rats to a low dose of nanoparticulate silver: Alterations in behavior, cerebral vasculature-related transcriptome and permeability.

The increasing production and use of silver nanoparticles (AgNPs) as antimicrobial agents in medicinal and commercial products creates a substantial risk of exposure, especially for infants and children. Our current knowledge concerning the impact of AgNPs on developing brain is insufficient. Therefore we investigated the temporal profile of transcriptional changes in cellular components of the neurovascular unit in immature rats exposed to a low dose of AgNPs. The behavior of animals under these conditions was also monitored. Significant deposition of AgNPs in brain of exposed rats was identified and found to persist over the post-exposure time. Substantial changes were noted in the transcriptional profile of tight junction proteins such as occludin and claudin-5, and pericyte-related molecules such as angiopoietin-1. Moreover, downregulation of platelet-derived growth factor (PDGFß) and its receptor (PDGFßR) which constitute the main signaling pathway between endothelial cells and pericytes was observed. These were long-lasting effects, accompanied by overexpression of astroglial-specific GFAP mRNA and endothelial cell adhesion molecule, ICAM-1, involved in the pathomechanism of neuroinflammation. The profile of changes indicates that even low doses of AgNPs administered during the early stage of life induce dysregulation of neurovascular unit constituents which may lead to disintegration of the blood-brain barrier. This was confirmed by ultrastructural analysis that revealed enhanced permeability of cerebral microvessels resulting in perivascular edema. Changes in the behavior of exposed rats indicating pro-depressive and anti-anxiety impacts were also identified. The results show a high risk of using AgNPs in medical and consumer products dedicated for infants and children.

Claudin peptidomimetics modulate tissue barriers for enhanced drug delivery.

The blood-brain barrier (BBB) formed by the microvascular endothelium limits cerebral drug delivery. The paraendothelial cleft is sealed by tight junctions (TJs) with a major contribution from claudin-5, which we selected as a target to modulate BBB permeability. For this purpose, drug-enhancer peptides were designed based on the first extracellular loop (ECL) of claudin-5 to allow transient BBB permeabilization. Peptidomimetics (C5C2 and derivatives, nanomolar affinity to claudin-5) size-selectively (≤40 kDa) and reversibly (12-48 h) increased the permeability of brain endothelial and claudin-5-transfected epithelial cell monolayers. Upon peptide uptake, the number of TJ strand particles diminished, claudin-5 was downregulated and redistributed from cell-cell contacts to the cytosol, and the cell shape was altered. Cellular permeability of doxorubicin (cytostatic drug, 580 Da) was enhanced after peptide administration. Mouse studies (3.5 µmol/kg i.v.) confirmed that, for both C5C2 and a d-amino acid derivative, brain uptake of Gd-diethylene-triamine penta-acetic acid (547 Da) was enhanced within 4 h of treatment. On the basis of our functional data, circular dichroism measurements, molecular modeling, and docking experiments, we suggest an association model between ß-sheets flanked by α-helices, formed by claudin-5 ECLs, and the peptides. In conclusion, we identified claudin-5 peptidomimetics that improve drug delivery through endothelial and epithelial barriers expressing claudin-5.

Acute effects of short-chain alkylglycerols on blood-brain barrier properties of cultured brain endothelial cells.

BACKGROUND AND PURPOSE: The blood-brain barrier (BBB) restricts drug penetration to the brain preventing effective treatment of patients suffering from brain tumours. Intra-arterial injection of short-chain alkylglycerols (AGs) opens the BBB and increases delivery of molecules to rodent brain parenchyma in vivo. The mechanism underlying AG-mediated modification of BBB permeability is still unknown. Here, we have tested the effects of AGs on barrier properties of cultured brain microvascular endothelial cells. EXPERIMENTAL APPROACH: The effects of two AGs, 1-O-pentylglycerol and 2-O-hexyldiglycerol were examined using an in vitro BBB model consisting of primary cultures of rat brain endothelial cells, co-cultured with rat cerebral glial cells. Integrity of the paracellular, tight junction-based, permeation route was analysed by functional assays, immunostaining for junctional proteins, freeze-fracture electron microscopy, and analysis of claudin-claudin trans-interactions. KEY RESULTS: AG treatment (5 min) reversibly reduced transendothelial electrical resistance and increased BBB permeability for fluorescein accompanied by changes in cell morphology and immunostaining for claudin-5 and ß-catenin. These short-term changes were not accompanied by alterations of inter-endothelial tight junction strand complexity or the trans-interaction of claudin-5. CONCLUSION AND IMPLICATIONS: AG-mediated increase in brain endothelial paracellular permeability was short, reversible and did not affect tight junction strand complexity. Redistribution of junctional proteins and alterations in the cell shape indicate the involvement of the cytoskeleton in the action of AGs. These data confirm the results from in vivo studies in rodents characterizing AGs as adjuvants that transiently open the BBB.