Overexpression of miR‐18a negatively regulates myocyte enhancer factor 2D to increase the permeability of the blood–tumor barrier via Krüppel‐like factor 4‐mediated downregulation of zonula occluden‐1, claudin‐5, and occludin

miR‐18a represses angiogenesis and tumor evasion by weakening vascular endothelial growth factor and transforming growth factor‐β signaling to prolong the survival of glioma patients, although it is thought to be an oncogene. This study investigates the potential effects of miR‐18a on the permeability of the blood–tumor barrier (BTB) and its possible molecular mechanisms. An in vitro BTB model was successfully established. The endogenous expression of miR‐18a in glioma vascular endothelial cells (GECs) was significantly lower than that in normal vascular ECs, and the overexpression of miR‐18a significantly increased the permeability of the BTB as well as downregulating the mRNA and protein expressions of tight junction‐related proteins zonula occluden‐1 (ZO‐1), claudin‐5, and occludin in GECs. Dual luciferase reporter assays revealed that miR‐18a bound to the 3′‐untranslated region (3′UTR) of myocyte enhancer factor 2D (MEF2D). The overexpression of both miR‐18a and MEF2D with the 3′UTR significantly weakened the effect caused by miR‐18a of decreasing the mRNA and protein expressions of ZO‐1, claudin‐5 and occludin and of increasing the permeability of the BTB. Chromatin immunoprecipitation showed that MEF2D could directly bind to KLF4 promoter. This study shows that miR‐18a targets and negatively regulates MEF2D, which further regulates tight junction‐related proteins ZO‐1, claudin‐5, and occludin through transactivation of KLF4 and, finally, changes the permeability of the BTB. MiR‐18a should garner growing attention because it might serve as a potential target in opening the BTB and providing a new strategy for the treatment of gliomas. © 2015 Wiley Periodicals, Inc.     

Increasing of blood-tumor barrier permeability through paracellular pathway by low-frequency ultrasound irradiation in vitro

The research was conducted to study the increase of blood-tumor barrier (BTB) permeability through paracellular pathway by low-frequency ultrasound (LFU) irradiation in vitro. LFU (frequency = 1.0 MHz) was performed to irradiate BTB model from the co-culture of rat C6 glioma cells and rat brain microvascular endothelial cells (RBMECs). The permeability of BTB was measured by transendothelial electrical resistance (TEER) and flux of horseradish peroxidase (HRP) assays after LFU irradiation. Western-blotting, immunohistochemistry, and immunofluorescence assays were used to investigate the changes of expressions and distributions of tight junction (TJ)-associated proteins ZO-1, occludin, and claudin-5. The TEER value began to decrease, and the minimum value appeared at 2 h, then gradually returned to the original level at 24 h after LFU irradiation. With time, flux of HRP gradually increased and reached the peak 2 h after LFU irradiation. The expressions of ZO-1, occludin, and claudin-5 in RBMECs decreased, and decreased most significantly at 2 h, then gradually restored to the original level at 24 h. Meanwhile, they were discontinuously distributed in the cellular boundaries after LFU irradiation. In summary, the expresstion of TJ-associated proteins was down-regulated, TJ was opened, and the permeability of BTB was increased through paracellular pathway by LFU irradiation.     

Bradykinin increases blood-tumor barrier permeability by down-regulating the expression levels of ZO-1, occludin, and claudin-5 and rearranging actin cytoskeleton

Bradykinin (BK) has been shown to open blood-tumor barrier (BTB) selectively and to increase permeability of the BTB transiently, but the mechanism is unclear. This study was performed to determine whether BK opens the BTB by affecting the tight junction (TJ)-associated proteins zonula occluden-1 (ZO-1), occludin, and caludin-5 and cytoskeleton protein filamentous actin (F-actin). In rat brain glioma model and BTB model in vitro, we find that the protein expression levels of ZO-1, occludin, and claudin-5 are attenuated by BK induction. Immunohistochemistry and immunofluorescence assays show that the attenuated expression of ZO-1, occludin, and claudin-5 and F-actin is most obvious in the smaller tumor capillaries (<20 microm) after BK infusion, and there is no change in the larger tumor capillaries (>20 microm). The redistribution of ZO-1, occludin, and claudin-5 and rearrangement of F-actin in brain microvascular endothelial cells are observed at the same time. Meanwhile, Evans blue assay shows that the permeability of BTB increases after BK infusion. Transmission electron microscopy indicates that TJ is opened and that pinocytotic vesicular density is increased. Transendothelial electrical resistance (TEER) and horseradish peroxidase flux assays also reveal that TJ is opened by BK induction. In addition, radioimmunity and Western blot assay reveal a significant decrease in expression levels of cAMP and catalytic subunit of protien kinase A (PKAcs) of tumor tissue. This study demonstrates that the increase of BK-mediated BTB permeability is associated with the down-regulation of ZO-1, occludin, and claudin-5 and the rearrangement of F-actin and that cAMP/PKA signal transduction system might be involved in the modulating process.     

Effects of insulin combined with idebenone on blood–brain barrier permeability in diabetic rats

This study investigates the effect of insulin combined with idebenone on blood–brain barrier (BBB) permeability in experimental streptozotocin‐induced diabetic rats as well as the underlying mechanisms. With a diabetic rat model, we show that insulin and idebenone normalize body weight and water intake and restore BBB permeability and that their combination displays a synergistic effect. The results from transmission electron microscopy show that the combination of insulin and idebenone significantly closed the tight junction (TJ) in diabetic rats. The results from Western blotting in diabetic rats show that the upregulation of TJ‐associated proteins occludin, and zonula occludens (ZO)‐1 caused by the combination of insulin and idebenone is more remarkable than that with either agent alone. In addition, the activations of reactive oxygen species (ROS) and advanced glycation end products (AGEs) and the expression levels of receptors for advanced glycation end‐products (RAGE) and nuclear factor‐κB (NF‐κB) were significantly decreased after treatment with insulin and idebenone in diabetic rats. These results suggest that the combination of insulin and idebenone could decrease the BBB permeability in diabetic rats by upregulating the expression of occludin, claudin‐5, and ZO‐1 and that the ROS/AGE/RAGE/NF‐κB signal pathway might be involved in the process. © 2014 Wiley Periodicals, Inc.     

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.     

Mechanism of Low-Frequency Ultrasound in Opening Blood–Tumor Barrier by Tight Junction

The clinical chemotherapy of brain tumors has been limited by the blood–tumor barrier (BTB). Low-frequency ultrasound (LFU) in combination with microbubbles might be a useful method for local drug delivery. However, the underlying mechanism remains unclear. In this study, we asked whether LFU changed the permeability of BTB by regulating the tight junction-related proteins. The permeability of BTB was evaluated by Evans blue dye, and the protein and mRNA expression levels of tight junction-related proteins claudin-5, occludin, and ZO-1 were determined by immunohistochemical staining, RT-PCR, and western blot assays. We found that the permeability of BTB increased significantly after LFU exposure in the presence of Optison. The mRNA and protein expression levels of claudin-5, occludin, and ZO-1 decreased significantly at 3 h, restored gradually and nearly recovered after 12 h. The correlation between the increase of BTB permeability and the reduction of tight junction-related proteins suggests that LFU combined with microbubbles may be involved in the opening of the BTB by the tight junction-related proteins.     

RhoA-Mediated Potential Regulation of Blood–Tumor Barrier Permeability by Bradykinin

The purpose of this work is to investigate the potential for the small G protein RhoA to play a role in bradykinin (BK)-induced actin cytoskeleton rearrangement, tight junction (TJ) protein disassembly, and an increase in blood–tumor barrier (BTB) permeability in rat brain microvascular endothelial cells (RBMECs). Our study used primary RBMECs as an in vitro BTB model and a RhoA inhibitor (C₃ exoenzyme) to establish whether RhoA played a role in the process of TJ disassembly, stress fiber formation, and increasing BTB permeability by BK. Data from the HRP flux and TEER assays revealed that BTB permeability was increased by BK induction. C₃ exoenzyme could partially inhibit endothelial leakage and restored normal TEER values in RBMECs. An obvious shift in occludin distribution from insoluble to soluble fractions was observed as assessed by Western blot, which was prevented by C₃ exoenzyme. In addition, C₃ exoenzyme inhibited BK-induced relocation of occludin from cellular borders into the cytoplasm, as well as stress fiber formation in RBMECs. A time-dependent increase in RhoA activity by BK administration was observed, which was inhibited by C₃ exoenzyme. RhoA activation is important for BK-induced increase in BTB permeability and appears to involve the ability for RhoA to mediate occludin disassembly and stress fiber formation.     

Role of RhoA/ROCK signaling in endothelial-monocyte-activating polypeptide II opening of the blood-tumor barrier : role of RhoA/ROCK signaling in EMAP II opening of the BTB

The purpose of the present study was to determine the potential for RhoA/ROCK signaling to play a role in endothelial-monocyte-activating polypeptide (EMAP) II-induced increase in blood–tumor barrier (BTB) permeability in rat brain microvascular endothelial cells (RBMECs). In the present study, we used an in vitro BTB model, a RhoA inhibitor (C3 exoenzyme) and a ROCK inhibitor (Y27632) to determine whether RhoA/ROCK pathway play a role in the process of TJ disassembly, stress fiber formation, MLC and cofilin phosphorylation, as well as increase of BTB permeability induced by EMAP II. The results revealed that BTB permeability was increased by EMAP II induction, and C3 exoenzyme or Y27632 could partially inhibit the EMAP II-induced increase of BTB permeability. The significant down-regulations in tight junction (TJ)-associated proteins occludin, claudin-5 and ZO-1 and stress fiber formation by EMAP II administration were observed, which were partly prevented by C3 exoenzyme or Y27632 pretreatment. Moreover, the significant increases in RhoA activity, myosin light chain (MLC) and cofilin phosphorylation by EMAP II administration were observed, MLC and cofilin phosphorylation were partly inhibited by C3 exoenzyme or Y27632 pretreatment. The present study demonstrates that the activation of RhoA/ROCK signaling in RBMECs was required for the increase of BTB permeability and these effects are related with the ability for RhoA/ROCK to mediate TJ disassembly and stress fiber formation by phosphorylating cofilin and MLC.     

Differential distribution of tight junction proteins suggests a role for tanycytes in blood‐hypothalamus barrier regulation in the adult mouse brain

The median eminence is one of the seven so‐called circumventricular organs. It is located in the basal hypothalamus, ventral to the third ventricle and adjacent to the arcuate nucleus. This structure characteristically contains a rich capillary plexus and features a fenestrated endothelium, making it a direct target of blood‐borne molecules. The median eminence also contains highly specialized ependymal cells called tanycytes, which line the floor of the third ventricle. It has been hypothesized that one of the functions of these cells is to create a barrier that prevents substances in the portal capillary spaces from entering the brain. In this paper, we utilize immunohistochemistry to study the expression of tight junction proteins in the cells that compose the median eminence in adult mice. Our results indicate that tanycytes of the median eminence express occludin, ZO‐1, and claudin 1 and 5, but not claudin 3. Remarkably, these molecules are organized as a continuous belt around the cell bodies of the tanycytes that line the ventral part of the third ventricle. In contrast, the tanycytes at the periphery of the arcuate nucleus do not express claudin 1 and instead exhibit a disorganized expression pattern of occludin, ZO‐1, and claudin 5. Consistent with these observations, permeability studies using peripheral or central injections of Evans blue dye show that only the tanycytes of the median eminence are joined at their apices by functional tight junctions, whereas tanycytes located at the level of the arcuate nucleus form a permeable layer. In conclusion, this study reveals a unique expression pattern of tight junction proteins in hypothalamic tanycytes, which yields new insights into their barrier properties. J. Comp. Neurol. 518:943–962, 2010. © 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.     

The modulation of protein kinase A and heat shock protein 70 is involved in the reversible increase of blood-brain tumor barrier permeability induced by papaverine

Intra-arterial administration of papaverine has been revealed to cause an increase in the blood-brain tumor barrier (BTB) permeability. The exact mechanism of papaverine opening the BTB in chemotherapy of malignant cerebral tumors, however, has not been well described. We used a rat brain glioma (C6) model for studying how papaverine modulates the permeability of BTB by monitoring the activities of the tight junction (TJ)-associated protein occludin, claudin-5 and cytoskeletal protein filamentous actin (F-actin) and whether protein kinase A (PKA) and heat shock protein 70 (HSP70) were involved in the regulation of this biological process. The levels of occludin, claudin-5 and F-actin protein in the tumor tissues were down-regulated by papaverine via immunohistochemistry, immunofluorescence assays and Western blot, corresponding to the time-dependent change of the BTB permeability. The most obvious attenuation of occludin, claudin-5 and F-actin protein was observed at 1 h after papaverine perfusion, companied by a significant decrease in expression levels of PKA protein. The expression level of HSP70 in the tumor tissues was also progressively increased after papaverine perfusion and reached the maximum at 3 h. The results demonstrate that the reversible openning of BTB mediated by papaverine may be associated with the functional combination between PKA and HSP70. That is, BTB opening may be attributable to the down-regulation of occludin, claudin-5 and F-actin, and cAMP/PKA signaling pathway might be involved in this process. HSP70 is likely responsible for the BTB closing, which helping the repairment of injured TJ protein and the rebuilding of the BTB.     

The modulation of protein kinase A and heat shock protein 70 is involved in the reversible increase of blood–brain tumor barrier permeability induced by papaverine

Intra-arterial administration of papaverine has been revealed to cause an increase in the blood–brain tumor barrier (BTB) permeability. The exact mechanism of papaverine opening the BTB in chemotherapy of malignant cerebral tumors, however, has not been well described. We used a rat brain glioma (C6) model for studying how papaverine modulates the permeability of BTB by monitoring the activities of the tight junction (TJ)-associated protein occludin, claudin-5 and cytoskeletal protein filamentous actin (F-actin) and whether protein kinase A (PKA) and heat shock protein 70 (HSP70) were involved in the regulation of this biological process. The levels of occludin, claudin-5 and F-actin protein in the tumor tissues were down-regulated by papaverine via immunohistochemistry, immunofluorescence assays and Western blot, corresponding to the time-dependent change of the BTB permeability. The most obvious attenuation of occludin, claudin-5 and F-actin protein was observed at 1 h after papaverine perfusion, companied by a significant decrease in expression levels of PKA protein. The expression level of HSP70 in the tumor tissues was also progressively increased after papaverine perfusion and reached the maximum at 3 h. The results demonstrate that the reversible openning of BTB mediated by papaverine may be associated with the functional combination between PKA and HSP70. That is, BTB opening may be attributable to the down-regulation of occludin, claudin-5 and F-actin, and cAMP/PKA signaling pathway might be involved in this process. HSP70 is likely responsible for the BTB closing, which helping the repairment of injured TJ protein and the rebuilding of the BTB.     

Bradykinin regulates the expression of claudin‐5 in brain microvascular endothelial cells via calcium‐induced calcium release

To investigate the mechanism underlying the regulation of claudin‐5, a tight junction protein that participates primarily in the constitution of the blood–brain barrier by bradykinin (BK), we established a primary culture of rat brain microvascular endothelial cells (BMECs). BMECs were treated with 10^?5 M BK, and changes in the intracellular Ca²⁺ levels were measured by using the sensitive fluorescent dye fluo‐3; the expression and distribution of claudin‐5 were investigated by immunocytochemistry and Western blot analyses. We did not detect any expression of bradykinin B2 receptors in the BMECs or freshly isolated rat brain microvessels. We found that 10^?5 M BK triggered Ca²⁺ transients in BMECs, and further investigations revealed that inositol 1,4,5‐trisphosphate receptors (IP₃Rs) and ryanodine receptors (RyRs) on the endoplasmic reticulum (ER) were responsible for the Ca²⁺ fluctuation. Consequently, these intracellular Ca²⁺ changes that occur in response to BK application were identified as Ca²⁺‐induced Ca²⁺ release (CICR). Immunocytochemistry and Western blot results demonstrated that 10^?5 M BK could cause the internalization and a decrease in the expression of claudin‐5; agonists of IP₃Rs and RyRs, such as IP₃ and caffeine, enhanced the BK‐induced downregulation of claudin‐5, whereas antagonists of IP₃Rs and RyRs, such as 2‐APB and ryanodine, abrogated BK's effect on claudin‐5. In conclusion, the BK‐induced CICR in primary culture BMECs might be the mechanism by which BK modulates claudin‐5. © 2014 Wiley Periodicals, Inc.     

Mechanisms for Endothelial Monocyte-Activating Polypeptide-II-Induced Opening of the Blood–Tumor Barrier

Endothelial monocyte-activating polypeptide-II (EMAP-II) increases blood-tumor barrier (BTB) permeability by inducing alterations in the tight junction (TJ) complex between brain endothelial cells. In the present study, an in vitro BTB model was used to search for the interacting and functional cell surface molecule of EMAP-II as well as the signaling pathway involved in the EMAP-II-induced BTB hyperpermeability. Our results revealed that EMAP-II-induced increase in BTB permeability and down-regulation of TJ-related proteins occludin and ZO-1 were associated with its binding to ATP synthase α subunit (α-ATP synthase) on the surface of rat brain microvascular endothelial cells (BMECs). In addition, we observed that EMAP-II administration activated protein kinase C (PKC) and induced the translocation of PKC from the cytosolic to the membrane fraction of BMECs. The effects of EMAP-II on BTB permeability as well as expression levels of occludin and ZO-1 in BMECs were significantly diminished by H7, the inhibitor of PKC. In summary, these data suggest that EMAP-II increases BTB permeability through α-ATP synthase on the surface of BMECs, and PKC signaling pathway might be involved in this process.     

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.     

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.     

Low-frequency ultrasound irradiation increases blood-tumor barrier permeability by transcellular pathway in a rat glioma model

Low-frequency ultrasound (LFU) irradiation under certain acoustic intensity can increase blood-brain barrier permeability non-invasively and reversibly. The aim of this study was to find out the effect of LFU irradiation on blood-tumor barrier (BTB) permeability in rat C6 glioma model and the possible mechanism. In this research, Evans blue and H&E staining were used to evaluate the optimal parameter of LFU to open the BTB without damaging the normal brain tissue. Transmission electron microscopy was used to observe the changes of the number of pinocytotic vesicles in cerebral or glioma microvascular endothelial cells. The phosphorylation of tyrosine kinase Src, caveolin-1, and caveolin-2 was detected by western blot. The distribution and expressing levels of caveolae proteins, caveolin-1 and caveolin-2, were detected by immunohistochemical and immunofluorescent staining, RT-PCR, and western blot. Our research data showed that, in rat C6 glioma model, LFU irradiation at a frequency of 1 MHz, a power of 12 mW, and exposure time of 20 s induced the increase of BTB permeability temporally, which reached a peak at 1.5 h, then decreased and restored to normal level at 12 h after LFU irradiation. In the glioma microvascular endothelial cells of rat glioma model, LFU irradiation induced a significant increase of the pinocytotic vesicles' density. The phosphorylation of Src, caveolin-1, and caveolin-2 began to increase at 0.5 h and reached a maximum at 1 h. Immunohistochemical and immunofluorescent staining showed that caveolin-1 and caveolin-2 were co-localized in the glioma microvascular endothelial cells and glioma cells. The mRNA and protein expression levels of caveolin-1 and caveolin-2 were up-regulated, reached the peak value at 1.5 h, and re-normalized at 12 h after LFU irradiation. These results demonstrated that LFU irradiation increased BTB permeability by promoting transcellular transport in glioma microvascular endothelial cells. The phosphorylation of tyrosine kinase Src, caveolin-1, caveolin-2 and up-regulation of caveolin-1 and caveolin-2 were involved in LFU-induced caveolae-mediated endocytosis.     

Role of ATP synthase alpha subunit in low-dose endothelial monocyte-activating polypeptide-II-induced opening of the blood-tumor barrier

This study was performed to determine whether the α subunit of ATP synthase (α-ATP synthase) on brain microvascular endothelial cells (BMECs) serves as the functional target for endothelial monocyte-activating polypeptide-II (EMAP-II)-induced increase in blood-tumor barrier (BTB) permeability. Using a rat C6 glioma model, we found that low-dose (80 ng/kg) EMAP-II significantly decreased the mRNA and protein expression levels of tight junction (TJ)-related proteins claudin-5, occludin, and ZO-1 on BMECs. Meantime, radioimmunity and Western blot assay showed a significant decrease in the expression levels of cAMP and catalytic subunit of protein kinase A (PKAcs) of tumor tissues. Also, pretreatment with specific α-ATP synthase antibody significantly blocked the effects of EMAP-II on TJ-related proteins, cAMP, and PKAcs. In addition, double immunofluorescence assay identified that EMAP-II was co-localized with α-ATP synthase on BMECs. This in vivo study demonstrated that α subunit of ATP synthase on BMECs serves as the functional target for EMAP-II selective opening of the BTB, and that cAMP/PKA signaling transduction pathway might be involved in the modulating process.     

Ischemic Postconditioning Protects the Neurovascular Unit after Focal Cerebral Ischemia/Reperfusion Injury

Recently, cerebral ischemic postconditioning (Postcond) has been shown to reduce infarction volume in cerebral ischemia/reperfusion (I/R) injury. However, it is unclear if ischemic Postcond offers more extensive neuroprotection than current therapies. The aim of this study was to investigate the neuroprotective effects of ischemic Postcond on the neurovascular unit (NVU). A middle cerebral artery occlusion rat model was used; cerebral infarct volumes, neurologic scores, and transmission electron microscopy were evaluated 24 h after reperfusion. We used Evans blue extravasation, immunohistochemistry, and Western blot analyses to evaluate the integrity of the blood brain barrier (BBB) and the distribution and expression of the tight junction (TJ)-associated proteins of claudin-5 and occludin in brain microvessel endothelium. The Postcond group showed significantly reduced infarct volumes and decreased neurologic impairment scores compared to the I/R group. Also, injuries to the cerebral microvascular endothelial cells, astrocytes, and neurons were minimized in the Postcond group. The permeability of the BBB increased in both the I/R and Postcond groups, but the Postcond group showed a significant decrease in permeability than the I/R group. Expression of both claudin-5 and occludin were higher in the Postcond groups compared to the I/R group, but expression of both proteins decreased in the I/R and Postcond groups compared to the sham group. The results of our study suggest that ischemic Postcond is an effective way to reduce injury to neurons, astrocytes, and endothelial cells, to increase protein expressions of TJ-associated proteins, and to improve BBB intergrity affected by focal I/R. Ischemic Postcond could protect the NVU from I/R injury.