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tight junction [keywords]
- Blood brain barrier dysfunction and delayed neurological deficits in mild traumatic brain injury induced by blast shock waves. [Journal Article, Review]
- Front Cell Neurosci 2014.:232.
Mild traumatic brain injury (mTBI) resulting from exposure to blast shock waves (BSWs) is one of the most predominant causes of illnesses among veterans who served in the recent Iraq and Afghanistan wars. Such mTBI can also happen to civilians if exposed to shock waves of bomb attacks by terrorists. While cognitive problems, memory dysfunction, depression, anxiety and diffuse white matter injury have been observed at both early and/or delayed time-points, an initial brain pathology resulting from exposure to BSWs appears to be the dysfunction or disruption of the blood-brain barrier (BBB). Studies in animal models suggest that exposure to relatively milder BSWs (123 kPa) initially induces free radical generating enzymes in and around brain capillaries, which enhances oxidative stress resulting in loss of tight junction (TJ) proteins, edema formation, and leakiness of BBB with disruption or loss of its components pericytes and astrocyte end-feet. On the other hand, exposure to more intense BSWs (145-323 kPa) causes acute disruption of the BBB with vascular lesions in the brain. Both of these scenarios lead to apoptosis of endothelial and neural cells and neuroinflammation in and around capillaries, which may progress into chronic traumatic encephalopathy (CTE) and/or a variety of neurological impairments, depending on brain regions that are afflicted with such lesions. This review discusses studies that examined alterations in the brain milieu causing dysfunction or disruption of the BBB and neuroinflammation following exposure to different intensities of BSWs. Furthermore, potential of early intervention strategies capable of easing oxidative stress, repairing the BBB or blocking inflammation for minimizing delayed neurological deficits resulting from exposure to BSWs is conferred.
- An all human 3d in vitro model of the blood brain barrier in nanoparticle delivery and cancer metastasis studies. [Journal Article]
- Neuro Oncol 2014 Jul.:iii33.
The blood-brain barrier (B-BB) is a dynamic functional unit consisting of endothelial cells, astrocytic endfeet and pericytes embedded in a specialized basement membrane. Most in vitro studies examine the ability of therapeutics to cross the B-BB using monolayer cultures or mixed species co-cultures and rely on Trans-Endothelial Electrical Resistance (TEER) measurements without verification of tight-junction (TJ) formation. More reliable high throughput in vitro models are therefore essential in brain tumour drug delivery and metastatic tumour behavioural studies. We are developing and testing a 3-dimensional in vitro model incorporating aspects of known B-BB in vivo characteristics.A 3D model of the B-BB was constructed using human brain endothelial cells (hCMEC/D3), human astrocytes (SC-1800/UP010) and human brain vascular pericytes (HBVP) in mono-, co-, tri- cultures. TEER values were measured using a cell monitoring system- cellZscope(®). TJ proteins (ZO-1 and occludin) were examined by Western blotting and immunocytochemistry. Effects of extracellular matrix (ECM) molecules (laminin, fibronectin, collagen type IV, agrin, perlecan) were assessed on endothelial cell proliferation, adhesion and TEER changes using an Electric Cell-Substrate Impedance Sensing (ECIS) system. Modified chitosyme nanoparticles and metastatic lung cancer cells were tested for effects on B-BB integrity.Co-cultures and tri-cultures resulted in significantly higher TEER values compared to endothelial cells alone (p < 0.05). TEER values for co-culture of ECs and astrocytes were 4620 Ω/cm2, and for tri-cultivation including pericytes, 4141.50 Ω/cm2 compared to single EC cultures (2244 Ω/cm2). Western blot revealed higher expression of ZO-1 and occludin in co- and tri-cultures. The individual optimal ECM concentrations for highest and longest TEER values differed depending on ECM molecule and concentration. Cell adhesion (%) correlated with TEER values. Recovery time of B-BB integrity following chitosyme nanoparticle/metastatic cell treatment, differed in mono-, co-, tri-cultures.Astrocytes contribute to B-BB formation in this in vitro model. Motile pericytes may positively or negatively influence barrier formation or TJ tightness. ECM has varied and profound effects on TEER values. Multiple measures are needed to confirm TJ formation in addition to TEER values as these may not reflect 'true' B-BB integrity. Using compounds with known low B-BB penetration and analysing TJ protein expression in combination with novel therapeutics will aid in providing meaningful data to bridge the gap between drug discovery and pre-clinical studies. In addition, our brain metastatic research group utilizes this model to test effect of CD15 knockdown in lung metastatic cells on endothelial cell adhesion and B-BB passage.Tumor Biology.
- Lack of interleukin-10-mediated anti-inflammatory signals and upregulated interferon gamma production are linked to increased intestinal epithelial cell apoptosis in pathogenic simian immunodeficiency virus infection. [JOURNAL ARTICLE]
- J Virol 2014 Aug 27.
Interleukin (IL)-10 is an immunomodulatory cytokine that is important for maintenance of epithelial cell (EC) survival and anti-inflammatory responses (AIR). The majority of HIV infections occur through the mucosal route despite mucosal epithelium acting as a barrier to HIV. Therefore understanding the role of IL-10 in maintenance of intestinal homeostasis during HIV infection is of interest for better characterization of the pathogenesis of HIV-mediated enteropathy. Here we demonstrated changes in mucosal IL-10 signaling during SIV infection in rhesus macaques. Disruption of the epithelial barrier was manifested by EC apoptosis and loss of tight junction protein ZO-1. Multiple cell types, including a limited number of ECs, produced IL-10. SIV infection resulted in increased levels of IL-10, however this was associated with increased production of mucosal IFNγ and TNFα, suggesting that IL-10 was not able to regulate AIR. This observation was supported by downregulation of STAT3, which is necessary to inhibit production of IFNγ and TNFα, and upregulation of SOCS1 and SOCS3, which are important regulatory molecules in the IL-10-mediated AIR. We also observed internalization of the IL-10 receptor (IL-10R) in mucosal lymphocytes, which could limit cellular availability of IL-10 for signaling and contribute to the loss of a functional AIR. Collectively, these findings demonstrate that internalization of IL-10R with the resultant impact on IL-10 signaling and dysregulation of the IL-10-mediated AIR might play a crucial role in EC damage and subsequent SIV/HIV pathogenesis.Interleukin-10 (IL-10), an important immunomodulatory cytokine plays a key role to control inflammatory function and homeostasis of the gastro-intestinal mucosal immune system. Despite recent advancements in the study of IL-10 and its role in HIV infection, the role of mucosal IL-10 in SIV/HIV infection in inducing enteropathy is not well understood. Here we demonstrated changes in mucosal IL-10 signaling during SIV infection in rhesus macaques. Disruption of the intestinal epithelial barrier was evident along with the increased levels of mucosal IL-10 production. Increased production of mucosal IFNγ and TNFα during SIV infection suggested that the increased level of mucosal IL-10 was not able to regulate anti-inflammatory responses. Our findings demonstrate that internalization of IL-10R with the resultant impact on IL-10 signaling and dysregulation of the IL-10-mediated anti-inflammatory responses might play a crucial role in epithelial cell damage and subsequent SIV/HIV pathogenesis.
- Analysis of aquaporin 9 expression in human epidermis and cultured keratinocytes. [Journal Article]
- FEBS Open Bio 2014.:611-6.
Aquaporin 9 (AQP9) is a member of the aquaglyceroporin family that transports glycerol, urea and other small solutes as well as water. Compared to the expression and function in epidermal keratinocytes of AQP3, another aquaglyceroporin, our knowledge of epidermal AQP9 remains elusive. In this study, we investigated the expression of AQP9 in the human epidermis and cultured keratinocytes. Immunofluorescence studies revealed that AQP9 expression is highly restricted to the stratum granulosum of the human epidermis, where occludin is also expressed at the tight junctions. Interestingly, the AQP3 staining decreased sharply below the cell layers in which AQP9 is expressed. In cultured normal human epidermal keratinocytes (NHEK), knock-down of AQP9 expression in the differentiated cells induced by RNA interference reduced glycerol uptake, which was not as pronounced as was the case with AQP3 knock-down cells. In contrast, similar reduction of urea uptake was detected in AQP9 and AQP3 knock-down cells. These findings suggested that AQP9 expression in NHEK facilitates at least the transport of glycerol and urea. Finally, we analyzed the effect of retinoic acid (RA), a potent stimulator of keratinocyte proliferation, on AQP3 and AQP9 mRNA expression in differentiated NHEK. Stimulation with RA at 1 μM for 24 h augmented AQP3 expression and down-regulated AQP9 expression. Collectively, these results indicate that AQP9 expression in epidermal keratinocytes is regulated in a different manner from that of AQP3.
- Viral Pathogen-Associated Molecular Patterns Regulate Blood-Brain Barrier Integrity via Competing Innate Cytokine Signals. [Journal Article]
- MBio 2014; 5(5)
Pattern recognition receptor (PRR) detection of pathogen-associated molecular patterns (PAMPs), such as viral RNA, drives innate immune responses against West Nile virus (WNV), an emerging neurotropic pathogen. Here we demonstrate that WNV PAMPs orchestrate endothelial responses to WNV via competing innate immune cytokine signals at the blood-brain barrier (BBB), a multicellular interface with highly specialized brain endothelial cells that normally prevents pathogen entry. While Th1 cytokines increase the permeability of endothelial barriers, type I interferon (IFN) promoted and stabilized BBB function. Induction of innate cytokines by pattern recognition pathways directly regulated BBB permeability and tight junction formation via balanced activation of the small GTPases Rac1 and RhoA, which in turn regulated the transendothelial trafficking of WNV. In vivo, mice with attenuated type I IFN signaling or IFN induction (Ifnar(-/-) Irf7(-/-)) exhibited enhanced BBB permeability and tight junction dysregulation after WNV infection. Together, these data provide new insight into host-pathogen interactions at the BBB during neurotropic viral infection.West Nile virus (WNV) is an emerging pathogen capable of infecting the central nervous system (CNS), causing fatal encephalitis. However, the mechanisms that control the ability of WNV to cross the blood-brain barrier (BBB) and access the CNS are unclear. In this study, we show that detection of WNV by host tissues induces innate immune cytokine expression at the BBB, regulating BBB structure and function and impacting transendothelial trafficking of WNV. This regulatory effect is shown to happen rapidly following exposure to virus, to occur independently of viral replication within BBB cells, and to require the signaling of cytoskeletal regulatory Rho GTPases. These results provide new understanding of host-pathogen interactions at the BBB during viral encephalitis.
- The complete functional recovery of chitosan-treated biomimetic hyperplastic and normoplastic urothelial models. [JOURNAL ARTICLE]
- Histochem Cell Biol 2014 Aug 27.
The urinary tract is exposed to a variety of possible injures that may lead to organ damage or loss, and thus, the establishment of valid in vitro urothelial models to study the mechanism of drug candidates is necessary. This study is the first to investigate the effect of chitosan on urothelia in vitro and to evaluate whether chitosan-treated urothelial models can regenerate in vitro and reestablish a functional urothelium. Biomimetic hyperplastic and normoplastic urothelial models were used to test the effect of chitosan (0.05 %) on partially and highly differentiated urothelial cells (UCs) by monitoring their molecular, ultrastructural, and physiological changes for 3 weeks. Chitosan caused an immediate and complete loss of transepithelial resistance (TER), tight junction disruption, cytopathological changes of UCs, and consequently enhanced the permeability of partially and highly differentiated urothelial models. However, 3 weeks after chitosan treatment, TER was reestablished, tight junctions resealed, permeability decreased, and progressive differentiation stages of newly exposed superficial UCs expressing uroplakins and tight junction protein claudin-8 were found. The in vitro models regenerated and reestablished urothelia with a tight barrier. The biomimetic urothelial models represent appropriate in vitro models for studying urothelial drug candidates as well as evaluating drug permeabilities and their intracellular function. Understanding the possible intracellular function of chitosan could significantly advance approaches to treating urothelial-specific diseases.
- Bubble technique for Descemet membrane endothelial keratoplasty tissue preparation in an eye bank: air or liquid? [JOURNAL ARTICLE]
- Acta Ophthalmol 2014 Aug 27.
To compare the big-bubble method using air and liquid as medium of separation for Descemet membrane endothelial keratoplasty (DMEK) lenticule preparation in an eye bank.Donor corneas (n = 20) were immersed in liquid [tissue culture medium (TCM)]. Air and liquid was injected using a 25-gauge needle in the posterior stroma or as near to the stroma-Descemet membrane (DM) phase as possible to create a complete bubble of larger diameter. The endothelial cell density and mortality were checked pre- and postbubble after deflating the tissue. Four pairs of tissues were used to analyse the intracellular tight junctions and three pairs for histological examination and DNA integrity studies, respectively.The yield obtained using air was 80%, whereas that with liquid was 100%. Single injection was required in six cases; twice in two cases; three and four times in one case each with air bubble, whereas seven cases required single injection; twice in two cases; and thrice in just one case with liquid bubble. The average diameter of the final lenticule was 9.12 (± 1.71) mm for air bubble and 9.78 (± 1.75) mm for liquid bubble with p = 0.4362 (no statistical significance). Endothelial cell mortality postbubble preparation was 8.9 (± 12.38) % for air and 6.25 (± 9.57) % for liquid (p = 0.6268).DM and endothelium could be separated exclusively using air or liquid bubble. However, liquid bubble seems to have certain advantages over air such as the generation of yield, larger diameter and higher maintenance of endothelial cell density and integrity.
- Fascin 1 is an actin filament bundling protein that regulates ectoplasmic specialization dynamics in the rat testis. [JOURNAL ARTICLE]
- Am J Physiol Endocrinol Metab 2014 Aug 26.
In the testis, spermatids are polarized cells with their heads pointing toward the basement membrane during maturation. This polarity is crucial to pack the maximal number of spermatids in the seminiferous epithelium so that millions of sperms can be produced daily. A loss of spermatid polarity is detected after rodents are exposed to toxicants (e.g., cadmium) or nonhormonal male contraceptives (e.g., adjudin), associated with a disruption on the expression and/or localization of polarity proteins. In the rat testis, fascin 1, an actin bundling protein found in mammalian cells, was expressed by Sertoli and germ cells. Fascin 1 was a component of the ectoplasmic specialization (ES), a testis-specific anchoring junction known to confer spermatid adhesion and polarity. Its expression in the seminiferous epithelium was stage-specific. Fascin 1 was localized to the basal ES at the Sertoli cell-cell interface of the blood-testis barrier in all stages of the epithelial cycle except diminished considerably at late stage VIII. Fascin 1 was highly expressed at the apical ES at stage VII-early stage VIII and restricted to the step 19 spermatids. Its knockdown by RNAi that silenced fascin 1 by ~70% in Sertoli cells cultured in vitro was found to perturb the tight junction (TJ)-permeability via a disruption of F-actin organization. Knockdown of fascin 1 in vivo by ~60-70% induced defects in spermatid polarity, mediated by a mis-localization and/or down-regulation of actin-bundling proteins Eps8 and palladin, thereby impeding F-actin organization and disrupting spermatid polarity. In summary, these findings provide insightful information on spermatid polarity regulation.
- Matrix metalloproteinase-9 leads to blood-brain barrier leakage in mice with eosinophilic meningoencephalitis caused by Angiostrongylus cantonensis. [JOURNAL ARTICLE]
- Acta Trop 2014 Aug 22.
Blood-brain barrier (BBB) disruption is associated with tight junction protein degradation, basal membrane disruption, and astrocyte damage. This study aims to investigate the role of matrix metalloproteinase (MMP)-9 in BBB disruption during Angiostrongylus cantonensis infection. We used mice infected with A. cantonensis, in which parasite-induced eosinophilia and inflammation might induce MMP-9 elevation. MMP-9 could cause claudin-5 degradation in endothelium tight junction, collagen type IV degradation in basal membranes, and S100B degradation in astrocytes of wild-type mice. BBB permeability was significantly attenuated in MMP-9 knockout mice than in wild-type mice in angiostrongyliasis meningoencephalitis. Immune cell aggregates were also more attenuated in the brains of MMP-9 knockout mice than in the brains of wild-type mice. Results suggest that MMP-9 activities are significant in BBB disruption in angiostrongyliasis meningoencephalitis. This study improves understanding of molecular mechanisms that underlie brain invasion by A. cantonensis, which is a key step in the pathogenesis of meningoencephalitis, and can offer a new strategy to reduce mortality.
- Modulation of intestinal epithelial cell proliferation, migration, and differentiation in vitro by astragalus polysaccharides. [Journal Article]
- PLoS One 2014; 9(8):e106674.
Previous studies have shown that Astragalus polysaccharides (APS) can be used to treat general gastrointestinal disturbances including intestinal mucosal injury. However, the mechanism by which APS mediate this effect is unclear. In the present study, the effects of APS on proliferation, migration, and differentiation of intestinal epithelial cells (IEC-6) were assessed using an in vitro wounding model and colorimetric thiazolyl blue (MTT) assays. The effect of APS on IEC-6 cell differentiation was observed using a light microscope and scanning electron microscope, and the expression of differentiation-specific markers of IEC-6 cells, such as cytokeratin 18 (CK18), alkaline phosphatase (ALP), tight junction protein ZO-2, and sucrase-isomaltase (SI), was determined by immunofluorescence assay (IFA) and real-time PCR. In addition, APS-induced signaling pathways in IEC-6 cells were characterized. Our results indicated that APS significantly enhance migration and proliferation of IEC-6 cells in vitro. APS-treated IEC-6 cells have numerous microvilli on their apical surface and also highly express CK18, ALP, ZO-2, and SI. Moreover, APS-treated IEC-6 cells, in which the activity and expression level of ornithine decarboxylase (ODC) were significantly elevated, also exhibited an increase in cellular putrescine, whereas no significant increase in TGF-β levels was observed. These findings suggest that APS may enhance intestinal epithelial cell proliferation, migration, and differentiation in vitro by stimulating ODC gene expression and activity and putrescine production, independent of TGF-β. Exogenous administration of APS may provide a new approach for modulating intestinal epithelial wound restitution in vivo.