Cells bEnd Research Articles

Glycine Receptor Beta Subunit (GlyR-β) Promotes Potential Angiogenesis and Neurological Regeneration during Early-Stage Recovery after Cerebral Ischemia Stroke/Reperfusion in Mice.

Ischemic stroke is mainly caused by cerebral artery thrombosis. This study investigated the role of glycine receptor beta subunit (GlyR-β) in the recovery from cerebral ischemia stroke/reperfusion. The oxygen glucose deprivation and recovery (OGD/R) bEnd3 cell model and the middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model were used in this study. Expression of both the GlyR-β gene and vascular endothelial growth factor (Vegf), cell proliferation, and tube formation ability was decreased in bEnd3 cells after OGD/R, and was reversed by overexpression of GlyR-β. Neurological function, asindicated by Zea Longa scores, area of cerebral ischemia, and pathological changes were increased in mice after MCAO/R, and were ameliorated by overexpression of the glycine receptor beta (Glrb) gene at 24 h and 7 d after MCAO/R. Expression of GlyR-β and Gap-43 was decreased, and the expression of CD34, Vegf, and Bdnf, and cell growth as determined by a bromodeoxyuridine (BrdU) assay, increased in the affected brain tissue of MCAO/R mice in a time-dependent manner. GlyR-β overexpression resulted in enhanced expression of CD34, Vegf, Growth association protein 43 (Gap-43), and brain-derived neurotrophic factor (Bdnf) and cell growth in affected brain tissue of MCAO/R mice in a time-dependent manner. GlyR-β promoted potential angiogenesis and neurological regeneration in affected brain tissue, thus promoting recovery from cerebral ischemia stroke/reperfusion.

The protective effects of Axitinib on blood-brain barrier dysfunction and ischemia-reperfusion injury in acute ischemic stroke

Background and purposeThe pathophysiological features of acute ischemic stroke (AIS) often involve dysfunction of the blood-brain barrier (BBB), characterized by the degradation of tight junction proteins (Tjs) leading to increased permeability. This dysfunction can exacerbate cerebral injury and contribute to severe complications. The permeability of the BBB fluctuates during different stages of AIS and is influenced by various factors. Developing effective therapies to restore BBB function remains a significant challenge in AIS treatment. High levels of vascular endothelial growth factor (VEGF) in the early stages of AIS have been shown to worsen BBB breakdown and stroke progression. Our study aimed to investigate the protective effects of the VEGF receptor inhibitor Axitinib on BBB dysfunction and cerebral ischemia/reperfusion-induced injury. MethodsBEnd3 cell exposed to oxygen–glucose deprivation (OGD) model was constructed to estimate pharmacological activity of Axitinib (400 ng/ml) on anti-apoptosis and pathological barrier function recovery. In vivo, rats were subjected to a 1 h transient middle cerebral artery occlusion and 23 h reperfusion (tMCAO/R) to investigate the permeability of BBB and cerebral tissue damage. Axitinib was administered through the tail vein at the beginning of reperfusion. BBB integrity was assessed by Evans blue leakage and the expression levels of Tjs claudin-5 and occludin. ResultsOur research revealed that co-incubation with Axitinib enhanced the cell viability of OGD-insulted bEnd3 cells, decreased LDH leakage rate, and suppressed the expression of apoptosis-related proteins cytochrome C and Bax. Axitinib also mitigated the damage to Tjs and facilitated the restoration of transepithelial electrical resistance in OGD-insulted bEnd.3 cells. In vivo, Axitinib administration reduced intracerebral Evans blue leakage and up-regulated the expression of Tjs in the penumbra brain tissue in tMCAO/R rats. Notably, 10 mg/kg Axitinib exerted a significant anti-ischemic effect by decreasing cerebral infarct volume and brain edema volume, improving neurological function, and reducing pro-inflammatory cytokines IL-6 and TNF-α in the brain. ConclusionsOur study highlights Axitinib as a potent protectant of blood-brain barrier function, capable of promoting pathological blood-brain barrier recovery through VEGF inhibition and increased expression of tight junction proteins in AIS. This suggests that VEGF antagonism within the first 24 h post-stroke could be a novel therapeutic approach to enhance blood-brain barrier function and mitigate ischemia-reperfusion injury.

Study on the role of CCM3 gene and lead exposure induced neurotoxicity through neurovascular units

This study aimed to determine the toxic effects of vascular CCM3 gene deficiency and lead (Pb) exposure on the nervous system. Lentiviral transfection was performed to generate a stable strain of brain microvascular endothelial cells with low CCM3 expression. MTT assay assessed the survival rate of cells exposed to Pb, determining the dose and duration of Pb exposure in vitro. Proteomic analysis was performed on the differentially expressed proteins in bEnd3 and HT22 cells and flow cytometry was used to detect cell apoptosis. Finally, urine samples from pregnant and postpartum women were subjected to ICP-MS to detect Pb levels and HPLC to detect neurotransmitter metabolites. Based on the proteomic analysis of bEnd3 (CCM3-/-) cells co-cultured with HT22 cells, it was determined that HT22 cells and CCM3 genes interfered with bEnd3 cell differential proteins,22Abbreviations: SNP, FGF2, EGF, AD including apoptosis and ferroptosis pathways. Electron microscopy observation, ICP-MS iron ion loading detection, and WB determination of protein GPX4 expression confirmed that HT22 cells undergo apoptosis, while bEnd3 cells undergo multiple pathways of iron death and apoptosis regulation. Furthermore, a linear regression model showed the interaction between maternal urine Pb levels, the rs9818496 site of the CCM3 SNP in peripheral blood DNA, and the concentration of the neurotransmitter metabolite 5-HIAA in maternal urine (F=4.198, P < 0.05). bEnd3 cells with CCM3 gene deficiency can induce HT22 cell apoptosis through iron death and apoptosis pathways under Pb exposure in a combined cell culture Pb exposure model, and CCM3 gene deficiency in endothelial cells and Pb exposure interacts with neural cell HT22. Epidemiological studies on maternal and newborn infants further confirmed the interaction between urine Pb levels in mothers and the SNP rs9818496 site of the CCM3 gene in peripheral blood DNA.

Kv7 channel activation reduces brain endothelial cell permeability and prevents kainic acid-induced blood-brain barrier damage.

Ion channels in the blood-brain barrier (BBB) play a main role in controlling the interstitial fluid composition and cerebral blood flow, and their dysfunction contributes to the disruption of the BBB occurring in many neurological diseases such as epilepsy. In this study, using morphological and functional approaches, we evaluated the expression and role in the BBB of Kv7 channels, a family of voltage-gated potassium channels including five members (Kv7.1-5) that play a major role in the regulation of cell excitability and transmembrane flux of potassium ions. Immunofluorescence experiments showed that Kv7.1, Kv7.4, and Kv7.5 were expressed in rat brain microvessels (BMVs), as well as brain primary- and clonal (BEND-3) endothelial cells (ECs). Kv7.5 localized at the cell-to-cell junction sites, whereas Kv7.4 was also found in pericytes. The Kv7 activator retigabine increased transendothelial electrical resistance (TEER) in both primary ECs and BEND-3 cells; moreover, retigabine reduced paracellular dextran flux in BEND-3 cells. These effects were prevented by the selective Kv7 blocker XE-991. Exposure to retigabine also hyperpolarized cell membrane and increased tight junctions (TJs) integrity in BEND-3 cells. BMVs from rats treated with kainic acid (KA) showed a disruption of TJs and a selective reduction of Kv7.5 expression. In BEND-3 cells, retigabine prevented the increase of cell permeability and the reduction of TJs integrity induced by KA. Overall, these findings demonstrate that Kv7 channels are expressed in the BBB, where they modulate barrier properties both in physiological and pathological conditions.NEW & NOTEWORTHY This study describes for the first time the expression and the functional role of Kv7 potassium channels in the blood-brain barrier. We show that the opening of Kv7 channels reduces endothelial cell permeability both in physiological and pathological conditions via the hyperpolarization of cell membrane and the sealing of tight junctions. Therefore, activation of endothelial Kv7 channels might be a useful strategy to treat epilepsy and other neurological disorders characterized by blood-brain barrier dysfunction.

Effect of Phase-Change Nanodroplets and Ultrasound on Blood-Brain Barrier Permeability In Vitro.

Phase-change nanodroplets (PCND;NDs) are emulsions with a perfluorocarbon (PFC) core that undergo acoustic vaporisation as a response to ultrasound (US). Nanodroplets change to microbubbles and cavitate while under the effect of US. This cavitation can apply forces on cell connections in biological barrier membranes, such as the blood-brain barrier (BBB), and trigger a transient and reversible increased permeability to molecules and matter. This study aims to present the preparation of lipid-based NDs and investigate their effects on the brain endothelial cell barrier in vitro. The NDs were prepared using the thin-film hydration method, followed by the PFC addition. They were characterised for size, cavitation (using a high-speed camera), and PFC encapsulation (using FTIR). The bEnd.3 (mouse brain endothelial) cells were seeded onto transwell inserts. Fluorescein with NDs and/or microbubbles were applied on the bEND3 cells and the effect of US on fluorescein permeability was measured. The Live/Dead assay was used to assess the BBB integrity after the treatments. Size and PFC content analysis indicated that the NDs were stable while stored. High-speed camera imaging confirmed that the NDs cavitate after US exposure of 0.12 MPa. The BBB cell model experiments revealed a 4-fold increase in cell membrane permeation after the combined application of US and NDs. The Live/Dead assay results indicated damage to the BBB membrane integrity, but this damage was less when compared to the one caused by microbubbles. This in vitro study shows that nanodroplets have the potential to cause BBB opening in a similar manner to microbubbles. Both cavitation agents caused damage on the endothelial cells. It appears that NDs cause less cell damage compared to microbubbles.

Hyperglycemia Aggravates the Cerebral Ischemia Injury via Protein O-GlcNAcylation.

At least one-third of Alzheimer's disease (AD) patients have cerebrovascular abnormalities, micro- and macro-infarctions, and ischemic white matter alterations. Stroke prognosis impacts AD development due to vascular disease. Hyperglycemia can readily produce vascular lesions and atherosclerosis, increasing the risk of cerebral ischemia. Our previous research has demonstrated that protein O-GlcNAcylation, a dynamic and reversible post-translational modification, provides protection against ischemic stroke. However, the role of O-GlcNAcylation in the exacerbation of cerebral ischemia injury due to hyperglycemia remains to be elucidated. In this study, we explored the role and underlying mechanism of protein O-GlcNAcylation in the exacerbation of cerebral ischemia injury caused by hyperglycemia. High glucose-cultured brain microvascular endothelial (bEnd3) cells were injured by oxygen-glucose deprivation. Cell viability was used as the assay result. Stroke outcomes and hemorrhagic transformation incidence were assessed in mice after middle cerebral artery occlusion under high glucose and streptozotocin-induced hyperglycemic conditions. Western blot estimated that O-GlcNAcylation influenced apoptosis levels in vitro and in vivo. In in vitro analyses showed that Thiamet-G induces upregulation of protein O-GlcNAcylation, which attenuates oxygen-glucose deprivation/R-induce injury in bEnd3 cells cultured under normal glucose conditions, while aggravated it under high glucose conditions. In in vivo analyses, Thiamet-G exacerbated cerebral ischemic injury and induced hemorrhagic transformation, accompanied by increased apoptosis. While blocking protein O-GlcNAcylation with 6-diazo-5-oxo-L-norleucine alleviated cerebral injury of ischemic stroke in different hyperglycemic mice. Overall, our study highlights the crucial role of O-GlcNAcylation in exacerbating cerebral ischemia injury under conditions of hyperglycemia. O-GlcNAcylation could potentially serve as a therapeutic target for ischemic stroke associated with AD.

Protective effects of KLF4 on blood–brain barrier and oxidative stress after cerebral ischemia–reperfusion in rats through the Nrf2/Trx1 pathway

PurposeTo investigate the role of KLF4 in CI/R injury and whether Nrf2/Trx1 axis acted as a downstream pathway of KLF4 to exert the protective role in blood–brain barrier destruction after CI/R. MethodsThe tMCAO rat model in vivo was constructed and received the intracerebroventricular injection of 5 μg/kg and 10 μg/kg rhKLF4 before operation. TTC, brain water content, neurological function, ELISA, behavioral tests, HE, TUNEL, and qRT-PCR were performed to detect the protective role of KLF4 on CIR. Double-fluorescence staining and western blot were performed to determine the localization and spatiotemporal expression in brain tissues. Furthermore, we also analyzed the effect of KLF4 on the blood–brain barrier (BBB) and related mechanisms in vivo and in vitro. Nrf2 inhibitor tretinoin was applied, which was intraperitoneally injected into CIR rat. Evans blue staining was conducted. In vitro OGD/R models of bEnd.3 cells were also established, and received KLF4 overexpressed transfection and 12.5 µM tretinoin incubation. The permeability of bEnd.3 cells was evaluated by TEER and FITC-dextran leakage. BBB-related factors and oxidative stress were also analyzed, respectively. The tubular ability of KLF4 on OGD/R bEnd3 cells was also evaluated. ResultsIn vivo study confirmed that KLF4 was expressed in astrocyte, and its content increased with time. KLF4 protected against brain injury caused by cerebral ischemia–reperfusion, reduced cerebral infarction area and oxidative stress levels, and promoted the recovery of behavioral ability in rats. Simultaneously, mechanism experiments confirmed that the repair effect of KLF4 on cerebral ischemia–reperfusion injury was closely related to the Nrf2/Trx1 pathway. KLF4 exerted the neuroprotective effect through upregulating Nrf2/Trx1 pathway. Consistent with in vivo animal study, in vitro study also confirmed the effect of KLF4 on the permeability of bEnd.3 cells after OGD/R injury through Nrf2/Trx1 pathway. ConclusionCollectively, KLF4 played neuroprotective role in CIR induced MCAO and OGD/R, and the beneficial effects of KLF4 was partly linked to Nrf2/Trx1 pathway.

Neutrophil-derived cathelicidin promotes cerebral angiogenesis after ischemic stroke.

Neutrophils play critical roles in the evolving of brain injuries following ischemic stroke. However, how they impact the brain repair in the late phase after stroke remain uncertain. Using a prospective clinical stroke patient cohort, we found significantly increased cathelicidin antimicrobial peptide (CAMP) in the peripheral blood of stroke patients compared to that of healthy controls. While in the mouse stroke model, CAMP was present in the peripheral blood, brain ischemic core and significantly increased at day 1, 3, 7, 14 after middle cerebral artery occlusion (MCAO). CAMP-/- mice exhibited significantly increased infarct volume, exacerbated neurological outcome, reduced cerebral endothelial cell proliferation and vascular density at 7 and 14 days after MCAO. Using bEND3 cells subjected to oxygen-glucose deprivation (OGD), we found significantly increased angiogenesis-related gene expression with the treatment of recombinant CAMP peptide (rCAMP) after reoxygenation. Intracerebroventricular injection (ICV) of AZD-5069, the antagonist of CAMP receptor CXCR2, or knockdown of CXCR2 by shCXCR2 recombinant adeno-associated virus (rAAV) impeded angiogenesis and neurological recovery after MCAO. Administration of rCAMP promoted endothelial proliferation and angiogenesis and attenuated neurological deficits 14 days after MCAO. In conclusion, neutrophil derived CAMP represents an important mediator that could promote post-stroke angiogenesis and neurological recovery in the late phase after stroke.

New Indole-3-Propionic Acid and 5-Methoxy-Indole Carboxylic Acid Derived Hydrazone Hybrids as Multifunctional Neuroprotectors.

In light of the known neuroprotective properties of indole compounds and the promising potential of hydrazone derivatives, two series of aldehyde-heterocyclic hybrids combining those pharmacophores were synthesized as new multifunctional neuroprotectors. The obtained derivatives of indole-3-propionic acid (IPA) and 5-methoxy-indole carboxylic acid (5MICA) had good safety profiles: Hemolytic effects < 5% (200 μM) and IC50 > 150 µM were found in the majority of the SH-SY5Y and bEnd3 cell lines. The 2,3-dihydroxy, 2-hydroxy-4-methoxy, and syringaldehyde derivatives of 5MICA exhibited the strongest neuroprotection against H2O2-induced oxidative stress in SH-SY5Y cells and 6-OHDA-induced neurotoxicity in rat-brain synaptosomes. All the compounds suppressed the iron-induced lipid peroxidation. The hydroxyl derivatives were also the most active in terms of deoxyribose-degradation inhibition, whereas the 3,4-dihydroxy derivatives were able to decrease the superoxide-anion generation. Both series of compounds showed an increased inhibition of hMAO-B, with greater expression detected in the 5MICA hybrids. The in vitro BBB model with the bEnd3 cell line showed that some compounds increased the permeability of the endothelial monolayer while maintaining the tight junctions. The combined results demonstrated that the derivatives of IPA and 5MICA showed strong neuroprotective, antioxidant, MAO-B inhibitory activity and could be considered as prospective multifunctional compounds for the treatment of neurodegenerative disorders.

Transmission of NLRP3-IL-1β Signals in Cerebral Ischemia and Reperfusion Injury: from Microglia to Adjacent Neuron and Endothelial Cells via IL-1β/IL-1R1/TRAF6.

The pyrin domain-containing protein 3 (NLRP3) inflammasome drives the profound cerebral ischemia and reperfusion injury (I/R) and mediates the secretion of IL-1β (interleukin-1β), which exerts a subsequent cascade of inflammatory injury. The NLRP3-activated-microglial manipulation in adjacent neuronal and endothelial NLRP3 activation has been confirmed in our previous studies. In the present study, we extended the cognition of how microglia mediated neuronal and endothelial NLRP3-IL-1β signaling during cerebral ischemia and reperfusion injury. In vitro, Neuro-2a and bEND3 cells were cultured alone or co-cultured with BV2 cells and oxygen-glucose deprivation/reoxygenation (OGD/R) was performed. In vivo, transient middle cerebral artery occlusion (tMCAO) rat models and lentiviral silencing targeting IL-1R1 were performed. The NLRP3 inflammasome activation was evaluated by enzyme-linked immunosorbent assay, western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence. In the co-culture system after OGD/R treatment, NLRP3 inflammasomes in neurons and endothelial cells were activated by microglial IL-1β via IL-1β/IL-1R1/TRAF6 signaling pathway, with the basal protein level of NLRP3. In addition, ruptured lysosomes engulfing ASC specks which were possibly secreted from microglia triggered the enhanced NLRP3 expression. In cortices of tMCAO rats at 24h of reperfusion, silencing IL-1R1, mainly presented in neurons and endothelial cells, was efficient to block the subsequent inflammatory damage and leukocyte brain infiltration, leading to better neurological outcome. Neuronal and endothelial NLRP3 inflammasomes were activated by microglia in cerebral ischemia and reperfusion injury mainly via IL-1β/IL-1R1/TRAF6 signaling, which might be therapeutically targetable.

Four Chemotherapeutic Compounds That Limit Blood-Brain-Barrier Invasion by Toxoplasma gondii

Background: Toxoplasma gondii, an intracellular protozoan parasite, exists in the host brain as cysts, which can result in Toxoplasmic Encephalitis (TE) and neurological diseases. However, few studies have been conducted on TE, particularly on how to prevent it. Previous proteomics studies have showed that the expression of C3 in rat brains was up-regulated after T. gondii infection. Methods: In this study, we used T. gondii to infect mice and bEnd 3 cells to confirm the relation between T. gondii and the expression of C3. BEnd3 cells membrane proteins which directly interacted with C3a were screened by pull down. Finally, animal behavior experiments were conducted to compare the differences in the inhibitory ability of TE by four chemotherapeutic compounds (SB290157, CVF, NSC23766, and Anxa1). Results: All chemotherapeutic compounds in this study can inhibit TE and cognitive behavior in the host. However, Anxa 1 is the most suitable material to inhibit mice TE. Conclusion: T. gondii infection promotes TE by promoting host C3 production. Anxa1 was selected as the most appropriate material to prevent TE among four chemotherapeutic compounds closely related to C3.

Endothelial‐specific nanodelivery of Rho kinase inhibitors: Targeting tumor angiogenesis and metastasis

ObjectiveThe limitations of current vascular endothelial growth factor (VEGF)‐centered anti‐angiogenic therapy include the inhibition of physiological angiogenesis, development of evasive resistance, and increased vascular leakage, which drives the need for a novel therapy for tumor angiogenesis and metastasis. RhoA pathway inhibition blocks VEGF‐ and sphingosine‐1 phosphate (S1P)‐induced angiogenesis and vascular leakage without hindering endogenous angiogenesis. Fasudil, a clinically relevant Rho‐associated kinase (ROCK) inhibitor, blocks the RhoA downstream signaling, but adverse events (hypotension, skin reactions) exist. Here, we propose endothelial ROCK targeting with Fasudil‐containing immune‐related nanodelivery.HypothesisFasudil‐encapsulated CD31‐targeting liposomal formulation is a potential tumor antiangiogenic therapy by blocking RhoA signaling via ROCK inhibition in the endothelial cells.MethodsFasudil‐liposomal nanoparticles were prepared by DPPC, cholesterol, 16:0 PEG2000PE, DSPE‐PEG2000‐mal, DSPE‐PEG‐FITC, and CHCl3: MeOH (4:1) combination, thin‐film formation, downsizing after Fasudil incorporation, filtering, and conjugation with IgG and CD31‐targeting antibodies. We used dynamic light scattering (DLS) for the liposomal characterization, dose‐response experiments on primary Human Umbilical Vein Endothelial Cells (HUVEC) and Bend3 cells for in vitro toxicity evaluation, antibody (Ab)‐to‐liposome binding evaluation and pathway inhibition evaluation by western blot, and UPLC analysis (EE)% for the evaluation of encapsulation efficiency. The binding affinity of the CD31‐conjugated liposomes and their time‐dependent cell incorporation were evaluated by competitive binding assay via immunohistochemistry (IHC) and flow cytometry. Morphometric analysis was assessed by Livecyte imaging analysis.ResultsSize and PDI measurement by DLS confirmed the stability of our formulation, which presented limited toxicity (concentration: 0.625‐5120 µM; IC50 at 43.05 µM [HUVECs over 24 hours of treatment]) with a significantly higher overall survival rate than Fasudil. Efficient CD31 and mouse IgG Ab conjugation to Fasudil liposomes and blockade of the RhoA downstream signaling in endothelial cells were confirmed. The specific binding of CD31‐targeting liposomes on endothelial cells was verified in the presence and absence of excess CD31 Ab. The time‐dependent study confirmed the kinetics of liposome incorporation in endothelial cells and the enhanced uptake of CD31 liposomes compared to IgG. Cell morphometric and physiological analysis revealed comparable alterations caused by Fasudil‐ and endothelial‐specific Fasudil‐containing liposomes.ConclusionWe have successfully developed Fasudil‐encapsulated, CD31‐targeting liposomes. Future studies will be directed to study the formulation’s effect on endothelial physiology in vitro and in vivo, tumor growth, angiogenesis, and metastasis.

NLRP3 Inhibition Reduces rt-PA Induced Endothelial Dysfunction under Ischemic Conditions.

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is a mainstay of acute ischemic stroke treatment but is associated with bleeding complications, especially after prolonged large vessel occlusion. Recently, inhibition of the NLRP3 inflammasome led to preserved blood–brain barrier (BBB) integrity in experimental stroke in vivo. To further address the potential of NLRP3 inflammasome inhibition as adjunct stroke treatment we used immortalized brain derived endothelial cells (bEnd5) as an in vitro model of the BBB. We treated bEnd5 with rt-PA in combination with the NLRP3 specific inhibitor MCC950 or vehicle under normoxic as well as ischemic (OGD) conditions. We found that rt-PA exerted a cytotoxic effect on bEnd5 cells under OGD confirming that rt-PA is harmful to the BBB. This detrimental effect could be significantly reduced by MCC950 treatment. Moreover, under ischemic conditions, the Cell Index—a sensible indicator for a patent BBB—and the protein expression of Zonula occludens 1 stabilized after MCC950 treatment. At the same time, the extent of endothelial cell death and NLRP3 expression decreased. In conclusion, NLRP3 inhibition can protect the BBB from rt-PA-induced damage and thereby potentially increase the narrow time window for safe thrombolysis in stroke.

Treponema pallidum Tp0751 alters the expression of tight junction proteins by promoting bEnd3 cell apoptosis and IL-6 secretion

BackgroundNeurosyphilis is a serious complication caused by the invasion of the central nervous system by Treponema pallidum subsp. pallidum (T. pallidum). However, the molecular mechanism by which T. pallidum crosses the blood-brain barrier has not been fully elucidated. ObjectivesThe primary purpose of this experimental design was to explore the effect of the T. pallidum adhesion protein Tp0751 on the blood-brain barrier and cerebrovascular endothelial cells. MethodsBEnd3 cells were used to construct a monolayer blood-brain barrier model in vitro. The integrity of blood-brain barrier model was evaluated by a transendothelial cell resistance meter and transmission electron microscope after the stimulation of recombinant protein TP0751. Hoechst 33258 staining and flow cytometry were used to detect the apoptosis rate. Western blotting assay was used to measure the expression of tight junction proteins and apoptosis-related proteins. The enzyme activity detection kit was responsible for detecting the enzyme activities of Caspase 3, Caspase 8 and Caspase 9. The expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 at the transcription and translation levels were detected by qRT-PCR and ELISA, respectively. ResultsThe results showed that, the tight junction structures between cells showed no obvious fragmentation, but the levels of the tight junction proteins zonula occludens-1 and occludin were reduced by the effects of Tp0751 on bEnd3 cells. In addition, further research demonstrated that after incubation with bEnd3 cells, Tp0751 induced cell apoptosis in a concentration- and time-dependent manner via the caspase 8/caspase 3 pathway. These apoptotic processes may have contributed to the changes in tight junction proteins expression. Furthermore, the Tp0751 protein may be involved in the pathogenic process by which T. pallidum crosses the blood-brain barrier by promoting secretion of the proinflammatory factor interleukin-6. ConclusionsOn the whole, this study is the first to reveal and highlight that Tp0751 may affect the expression of tight junction proteins by inducing apoptosis and promoting the secretion of the inflammatory cytokine IL-6, thus playing a role in the progression of neurosyphilis caused by T. pallidum.

Angiotensin-(1-7) ameliorates high glucose-induced vascular endothelial injury through suppressing chloride channel 3

ABSTRACT Diabetes Mellitus (DM) is a significant risk factor for cardiovascular disease (CVD), which is leading cause of deaths in DM patients. However, there are limited effective medical therapies for diabetic CVD. Vascular endothelial injury caused by DM is a critical risk factor for diabetic CVD. Previous study has indicated that Angiotensin-(1-7) (Ang-(1-7)) may prevent diabetic CVD, whereas it is not clear that Ang-(1-7) whether attenuates diabetic CVD through suppressing vascular endothelial injury. In this study, we found that Ang-(1-7) alleviated high glucose (HG)-induced endothelial injury in bEnd3 cells. Moreover, Ang-(1-7) ameliorated HG-induced endothelial injury through downregulating chloride channel 3 (CIC-3) via Mas receptor. Furthermore, HG-induced CIC-3 enhanced reactive oxygen species (ROS) and cytokine production and reduced the level of nitric oxide (NO), while Ang-(1-7) preserved the impact of HG-induced CIC-3 on productions of ROS, cytokine and NO through inhibiting CIC-3 via Mas receptor. Summarily, the present study revealed that Ang-(1-7) alleviated HG-induced vascular endothelial injury through the inhibition of CIC-3, suggested that Ang-(1-7) may preserve diabetic CVD through suppressing HG-induced vascular endothelial injury.

The Role of Cytoskeletal Proteins in the Formation of a Functional In Vitro Blood-Brain Barrier Model.

The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain’s microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB.

Proteome-wide analysis reveals molecular pathways affected by AgNP in a ROS-dependent manner

The use of mass spectrometry-based proteomics has been increasingly applied in nanomaterials risk assessments as it provides a proteome-wide overview of the molecular disturbances induced by its exposure. Here, we used this technique to gain detailed molecular insights into the role of ROS as an effector of AgNP toxicity, by incubating Bend3 cells with AgNP in the absence or presence of an antioxidant N-acetyl L-cystein (NAC). ROS generation is a key player in AgNP-induced toxicity, as cellular homeostasis was kept in the presence of NAC. By integrating MS/MS data with bioinformatics tools, in the absence of NAC, we were able to pinpoint precisely which biological pathways were affected by AgNP. Cells respond to AgNP-induced ROS generation by increasing their antioxidant pool, via NRF2 pathway activation. Additionally, cell proliferation-related pathways were strongly inhibited in a ROS-dependent manner. These findings reveal important aspects of the AgNP mechanism of action at the protein level.

CRH/CRHR1 modulates cerebrovascular endothelial cell permeability in association with S1PR2 and S1PR3 under oxidative stress

Corticotrophin-releasing hormone (CRH) has been demonstrated to participate in vascular inflammation and permeability. Our previous studies have shown that blockade of S1PR2 or CRHR1 inhibited H2O2-induced brain endothelial hyperpermeability via inhibiting cPLA2 phosphorylation. However, little is known about the linkage between S1PRs and CRHR1 in oxidative stress-induced cerebrovascular endothelial hyperpermeability. Here we observed the opposite effects of S1PR2 to those of S1PR3 on the monolayer permeability of bEnd3 cells in response to H2O2. Interestingly, activation of CRHR1 was found to reverse the effects resulting from blockade/silencing of both S1PR2 and S1PR3. In bEnd3 monolayer, blockade/knockdown of S1PR2 reduced the endothelial hyperpermeability and suppressed the tight junction protein ZO-1 redistribution caused by H2O2, along with the inhibition of p38, ERK and cPLA2 phosphorylation. On the contrary, suppression/silencing of S1PR3 further promoted H2O2-induced endothelial hyperpermeability and ZO-1 redistribution, accompanied by the increased phosphorylation of p38, ERK and cPLA2. In the presence of CRH, the effects resulting from the suppression of both S1PR2 and S1PR3 were abolished. Our results elucidate a possible linkage between CRHR1 and S1PR2/S1PR3 involving in the regulation of endothelial monolayer permeability under oxidative stress condition.

Tofacitinib Ameliorates Retinal Vascular Leakage in a Murine Model of Diabetic Retinopathy with Type 2 Diabetes.

We have previously reported that inhibition of the Janus kinase 1 (JAK1) signaling ameliorates IL-17A-mediated blood-retinal barrier (BRB) dysfunction. Higher levels of IL-17A have been observed in the blood and intraocular fluids in patients with diabetic retinopathy (DR), in particular those with diabetic macular oedema. This study aimed to understand whether JAK1 inhibition could prevent BRB dysfunction in db/db mice, a model of type 2 diabetes (T2D). An in vitro study showed that high glucose treatment disrupted the junctional distribution of claudin-5 in bEnd3 cells and ZO-1 in ARPE19 cells and that tofacitinib citrate treatment prevented high glucose-mediated tight junction disruption. Albumin leakage, accompanied by increased levels of the phosphorylated form of JAK1 (pJAK1), was observed in three-month-old db/db mice. Treatment of two-and-a-half-month-old db/db mice with tofacitinib citrate for two weeks significantly reduced retinal albumin leakage and reduced pJAK1 expression. pJAK1 expression was also detected in human DR retina. Our results suggest that JAK1 inhibition can ameliorate BRB dysfunction in T2D, and JAK1 inhibitors such as tofacitinib citrate may be re-purposed for the management of diabetic macular oedema.

Effects of Lipotoxicity in Brain Microvascular Endothelial Cells During Sirt3 Deficiency-Potential Role in Comorbid Alzheimer's Disease.

Silence information regulator 3 (SIRT3) is an NAD+ dependent deacetylase enzyme that enhances the function of key mitochondrial proteins. We have earlier demonstrated that deletion of Sirt3 gene leads to downregulation of metabolic enzymes, mitochondrial dysfunction and neuroinflammation in the brain, the major causes of Alzheimer’s disease (AD). We also reported recently that Sirt3 gene deletion in Alzheimer’s transgenic mice leads to exacerbation of neuroinflammation, amyloid plaque deposition and microglial activation. AD often coexists with other brain lesions caused by comorbidities which can exert their deleterious effects through the neurovascular unit. This unit consists of brain microvascular endothelial cells (BMECs), end feet of astrocytes, and pericytes. BMECs are uniquely different from other vascular endothelial cells because they are glued together by tight-junction proteins. BMECs are in constant contact with circulating factors as they line the luminal side. Therefore, we hypothesized that vascular endothelial injury caused by comorbidities plays a significant role in neuroinflammation. Herein, we investigated the effects of lipotoxicity in BMECs and how Sirt3 deficiency facilitate the deleterious effects of lipotoxicity on them using in vivo and in vitro models. We observed decreases in the levels of SIRT3 and tight junction proteins in the brain samples of western diet-fed APP/PS1 mice. Similar observations were obtained with Alzheimer’s post-mortem samples. Exposure of BEND3 cells, mouse brain-derived Endothelial cells3, to a combination of high glucose and palmitic acid resulted in significant (P < 0.01-P < 0.001) decreases in the levels of SIRT3, claudin-5 and ZO-1. Induction of inflammatory mediators, including Cox-2, CXCL1, RANTES, and GADD45β was also observed in these treated cells. Interestingly, the induction was more with Sirt3-silenced BEND3 cells, suggesting that Sirt3 deficiency exacerbates inflammatory response. Palmitic acid was more potent in inducing the inflammatory mediators. Significant cytotoxicity and changes in microglial morphology were observed when cocultures of Sirt3-silenced BEND3 and Sirt3-silenced BV2 cells were exposed to palmitic acid. Transendothelial electrical resistance measurement with these cocultures suggested decreased barrier integrity. The findings of this study suggest that hyperlipidemia in comorbidities can compromise blood brain barrier integrity by inducing inflammatory mediators and decreasing tight junction proteins in the vascular endothelial cells of the AD brain, leading to activation of microglia.