Cerebral Vascular Endothelial Cells Research Articles

Cerebral small vessel injury in mice with damage to ACE2-expressing cerebral vascular endothelial cells and post COVID-19 patients.

The angiotensin-converting enzyme 2 (ACE2), which is expressed in cerebral vascular endothelial cells (CVECs), has been currently identified as a functional receptor for SARS-CoV-2. We specifically induced injury to ACE2-expressing CVECs in mice and evaluated the effects of such targeted damage through magnetic resonance imaging (MRI) and cognitive behavioral tests. In parallel, we recruited a single-center cohort of COVID-19 survivors and further assessed their brain microvascular injury based on cognition and emotional scales, cranial MRI scans, and blood proteomic measurements. Here, we show an array of pathological and behavioral alterations characteristic of cerebral small vessel disease (CSVD) in mice that targeted damage to ACE2-expressing CVECs, and COVID-19 survivors. These CSVD-like manifestations persist for at least 7 months post-recovery from COVID-19. Our findings suggest that SARS-CoV-2 may induce cerebral small vessel damage with persistent sequelae, underscoring the imperative for heightened clinical vigilance in mitigating or treating SARS-CoV-2-mediated cerebral endothelial injury throughout infection and convalescence. Cerebral small vessel disease-associated changes were observed after targeted damage to angiotensin-converting enzyme 2-expressing cerebral vascular endothelial cells. SARS-CoV-2 may induce cerebral small vessel damage with persistent sequelae. Clinical vigilance is needed in preventing SARS-CoV-2-induced cerebral endothelial damage during infection and recovery.

Taraxacum coreanum (Korean Dandelion) Extract Protects against Lipopolysaccharide-Induced Blood-Brain Barrier Destruction via Regulation of Tight Junctions and Inflammatory Responses in bEnd.3 Cells.

Dysfunction of the blood-brain barrier (BBB) is closely related to neuroinflammation-mediated neurodegenerative disorders. Lipopolysaccharide (LPS), an endotoxin, can cause inflammation by impairing the brain endothelial barrier function and increasing the BBB permeability. Although Taraxacum coreanum NAKAI extract (TC), a traditional medicine widely used in Korea, has antioxidant and anti-inflammatory properties, the protective effects on neuroinflammation and BBB dysfunction are not fully understood. In the present study, bEnd.3 cerebral vascular endothelial cells were treated with TC followed by LPS exposure, and the effects on transendothelial electrical resistance (TEER) values, pro-inflammatory cytokine production, and expression of proteins related to inflammatory responses and tight junction integrity were assessed. The TC-treated group exhibited elevated TEER values in bEnd.3 monolayer compared to LPS-only treated group. In addition, TC treatment increased the expression of proteins involved in the tight junctions, such as ZO-1, claudin-5, and occludin. Furthermore, the TC-treated group suppressed the proteins expression-related to nuclear factor-κB (NF-κB) pathway. Taken together, TC attenuates LPS-induced neuroinflammatory responses by regulating NF-κB activation, which may contribute to protecting against BBB disruption. These findings suggest that TC may have the potential to be used as a material for functional foods to prevent neuroinflammation-related brain diseases.

Invasive metastatic tumor-camouflaged ROS responsive nanosystem for targeting therapeutic brain injury after cardiac arrest

Drug transmission through the blood-brain barrier (BBB) is considered an arduous challenge for brain injury treatment following the return of spontaneous circulation after cardiac arrest (CA-ROSC). Inspired by the propensity of melanoma metastasis to the brain, B16F10 cell membranes are camouflaged on 2-methoxyestradiol (2ME2)-loaded reactive oxygen species (ROS)-triggered “Padlock” nanoparticles that are constructed by phenylboronic acid pinacol esters conjugated D-a-tocopheryl polyethylene glycol succinate (TPGS-PBAP). The biomimetic nanoparticles (BM@TP/2ME2) can be internalized, mainly mediated by the mutual recognition and interaction between CD44v6 expressed on B16F10 cell membranes and hyaluronic acid on cerebral vascular endothelial cells, and they responsively release 2ME2 by the oxidative stress microenvironment. Notably, BM@TP/2ME2 can scavenge excessive ROS to reestablish redox balance, reverse neuroinflammation, and restore autophagic flux in damaged neurons, eventually exerting a remarkable neuroprotective effect after CA-ROSC in vitro and in vivo. This biomimetic drug delivery system is a novel and promising strategy for the treatment of cerebral ischemia-reperfusion injury after CA-ROSC.

Role of Gpr124 in the Migration and Proliferation of Retinal Microvascular Endothelial Cells and Microangiopathies in Diabetic Retinopathy.

Retinal microangiopathies, such as neovascularization and preretinal and vitreous hemorrhages, are the primary pathological features of diabetic retinopathy (DR). These conditions can worsen visual impairment and may result in blindness. Furthermore, multiple metabolic pathways are associated with microangiopathy in DR. However, the specific underlying pathological mechanisms remain unclear. Several studies have demonstrated the important role of G protein-coupled receptor 124 (Gpr124) in cerebral vascular endothelial cells, but its effect on the retinal endothelium has not been elucidated. In this study, we found that Gpr124 is expressed in both pathological retinal fibrous vascular membranes of DR patients and retinal blood vessels of mice, with elevated protein expression specifically observed in the retinas of DR model mice. Furthermore, Gpr124 expression was elevated after high-glucose treatment of human retinal microvascular endothelial cells (HRMECs). Inhibition of Gpr124 expression affected the high glucose-induced proliferation, migration, and tube-forming ability of HRMECs. We concluded that Gpr124 expression was upregulated in DR and promoted HRMECs angiogenesis in a high-glucose environment. This finding may help to elucidate the pathogenesis of DR and provide a critical research basis for identifying effective treatments.

Modular Satellite Nanoparticles for Remedying Primary and Secondary Injury in Cerebral Ischemia‐Reperfusion

AbstractReperfusion therapy, employed in the treatment of acute stroke, frequently proves to be inadequate in addressing the primary brain tissue injury and may even give rise to secondary damage. The study introduces a satellite nanoparticle platform named MEps, which combine the neural repair properties of bone marrow mesenchymal stem cell exosomes (Exos) with the inflammatory site‐targeting abilities of macrophage membranes (MMs). MMs and Exos in MEps act like satellites, ensuring precise positioning and information transmission. MEps rapidly form a protective barrier on the damaged cerebral vascular endothelial cells through the interaction of adhesion molecules with their receptors, blocking the infiltration of neutrophils. Subsequently, repair factors in Exos repair the damaged cells and initiate neurogenesis. The results indicate that this innovative approach effectively mitigates ischemic‐reperfusion injury at multiple levels and demonstrates strong biocompatibility. This strategy holds promise for clinical applications in alleviating ischemic‐reperfusion injury.

Plasma-derived exosomes contributes to endothelial-to-mesenchymal transition in Moyamoya disease

BackgroundMoyamoya disease (MMD) is a cerebrovascular disease with a high disability rate; however, its pathogenesis remains unknown. Endothelial-mesenchymal transition (EndMT) is the pathological basis of many vascular diseases; however, the key role of EndMT in MMD has not yet been reported. MethodWe collected vascular tissues from three control samples and six patients with MMD to detect the expression of EndMT-related genes. To elucidate the mechanism of EndMT in MMD, we performed in vitro cell experiments. Plasma-derived exosomes (PDEs) can transmit information between cells and tissues and are of considerable importance in several disease studies. PDEs were used to stimulate EndMT phenotype in cerebrovascular endothelial cells. ResultsMultiplex fluorescent immunohistochemistry staining confirmed that CD31, VE-cadherin and E-cadherin down-regulated, whereas α-SMA and vimentin were significantly up-regulated in moyamoya vascular endothelial cells than in control samples. PDEs from MMD patients significantly promoted cell proliferation and migration, resulting in slender cells. PDEs induce EndMT-related phenotype changes in cerebral vascular endothelial cells, including decreased endothelial cell marker expression and increased mesenchymal cell marker expression. We demonstrated that EndMT phenotypic alterations are mediated, in part, by microRNA(miRNAs). ConclusionThis study was the first to propose that EndMT may exist in the vessels of patients with MMD. PDEs induce the EndMT phenotype to promote the development of MMD. This study aimed to provide a new theoretical basis for elucidating the pathogenesis of MMD.

Self-Assembled nanoparticles of natural bioactive molecules enhance the delivery and efficacy of paclitaxel in glioblastoma.

Glioblastoma (GBM) is the most common primary malignant tumor in the central nervous system. Paclitaxel (PTX) is a well-established and highly effective anti-cancer drug for peripheral solid tumors. However, the application of PTX in GBM is hindered by several limitations, including poor water solubility, restricted entry across the blood-brain barrier (BBB), and enhanced excretion by efflux transporters. P-glycoprotein (P-gp) is a crucial efflux transporter that is abundantly present in cerebral vascular endothelial cells and GBM cells. It plays a significant role in the exocytosis of PTX within tumor tissues. Recently, we have developed a novel technique for creating self-assembled nanoparticles utilizing a range of natural bioactive molecules. These nanoparticles can encapsulate insoluble drugs and effectively cross the BBB. In additional, we revealed that certain nanoparticles have the potential to act as P-gp inhibitors, thereby reducing the excretion of PTX. In this study, we conducted a screening of bioactive molecular nanoparticles to identify those that effectively inhibit the function of P-gp transporters. Among the candidates, we identified ursolic acid nanoparticles (UA NPs) as the P-gp inhibitors. Furthermore, we prepared co-assembled UA NPs embedded with paclitaxel, referred to as UA-PTX NPs. Our results demonstrate that UA-PTX NPs can enhance the blood concentration of PTX, facilitate its entry into the BBB, and inhibit the function of P-gp, resulting in a decrease in the excretion of PTX. This discovery effectively addressed the above three issues associated with the use of PTX in glioma treatment. UA-PTX NPs demonstrate strong anti-tumor effects and show great potential for treating GBM.

Shedding Light on Lysosomal Malondialdehyde Affecting Vitamin B12 Transport during Cerebral Ischemia/Reperfusion Injury.

Cerebral ischemia-reperfusion injury (CIRI) is often accompanied by upregulation of homocysteine (Hcy). Excessive Hcy damages cerebral vascular endothelial cells and neurons, inducing neurotoxicity and even neurodegeneration. Normally, supplementation of vitamin B12 is an ideal intervention to reduce Hcy. However, vitamin B12 therapy is clinically inefficacious for CIRI. Considering oxidative stress is closely related to CIRI, the lysosome is the pivotal site for vitamin B12 transport. Lysosomal oxidative stress might hinder the transport of vitamin B12. Whether lysosomal malondialdehyde (lysosomal MDA), as the authoritative biomarker of lysosomal oxidative stress, interferes with the transport of vitamin B12 has not been elucidated. This is ascribed to the absence of effective methods for real-time and in situ measurement of lysosomal MDA within living brains. Herein, a fluorescence imaging agent, Lyso-MCBH, was constructed to specifically monitor lysosomal MDA by entering the brain and targeting the lysosome. Erupting the lysosomal MDA level in living brains of mice under CIRI was first observed using Lyso-MCBH. Excessive lysosomal MDA was found to affect the efficacy of vitamin B12 by blocking the transport of vitamin B12 from the lysosome to the cytoplasm. More importantly, the expression and function of the vitamin B12 transporter LMBD1 were proved to be associated with excessive lysosomal MDA. Altogether, the revealing of the lysosomal MDA-LMBD1 axis provides a cogent interpretation of the inefficacy of vitamin B12 in CIRI, which could be a prospective therapeutic target.

Thyrotropin receptor autoantibody (TRAb) enhance the expression of thyrotropin receptor on mouse brain vascular endothelial cells: in vivo and in vitro.

The possibility that thyrotropin receptor (TSHR) expression in non-thyroid tissue is well-documented. However, there is insufficient data on the expression of TSHR in medulla oblongata regions, particularly when focusing on the background of encephalopathy associated with hyperthyroidism. In this study, we explored the expression of the functional TSHR in Graves' disease (GD) mouse cerebral vascular endothelial cells and the effects of thyrotropin receptor autoantibody (TRAb) on its expression. A mouse model of GD was constructed with an adenovirus overexpressing TSHR289. The location and expression of the TSHR gene and protein in vivo were determined via RT-qPCR, Western blot, and immunofluorescence techniques. The effect of TRAb on the expression of functional TSHR in vitro was investigated using bEnd.3 cells. Our results show that medulla oblongata vascular endothelial cells from GD mice expressed higher levels of TSHR compared to control mice. In an in vitro experiment, novel results demonstrated that after treatment with a monoclonal TSHR-specific agonistic antibody (M22), the expression of TSHR on the bEnd.3 cells increased at both the protein and mRNA levels. Furthermore, compared with bEnd.3 cells were treated with IBMX only, those treated with M22 showed increased cAMP production. This study suggested that TSHR is expressed and functionally active in the mouse medulla oblongata and in vitro-cultured bEnd.3 cells and TRAb (M22) increased the expression of TSHR on bEnd.3 cells.

PS-B01-3: IS IT POSSIBLE TO ACCELERATE SENESCENCE IN THE VASCULAR ENDOTHELIAL CELL BY MODULATING SEVERAL MICRORNAS?

Backgrounds: Many factors are involved in cellular aging, and senescence induction requires complex regulation of various signaling networks and processes. Specifically, in the area of aging-related vascular cognitive impairment, laboratory-based findings have not yet yielded agents of practical use for clinical settings. One possible reason is that the physiologic elements of aging have been insufficiently considered such as in a drug screening experiments (particular in the vitro-blood brain barrier model). It is difficult to age primary human cerebral vascular endothelial cells for the experiments using standard methods because of their limited ability to survive in vitro. To establish techniques to mimic cellular aging by modulation of microRNAs, we aimed to identify key microRNAs capable of fine-tuning aging-associated genes, and thereby regulating the senescence of vascular endothelial cells. Methods: To identify candidate microRNAs, expression microRNA array was performed between control and senescent vascular endothelial cells. Thereafter, bioinformatic analysis was used to select key microRNAs. These candidates were then modulated in vitro using microRNA mimics and inhibitors in vascular endothelial cells, and senescence-associated gene expression patterns were evaluated by qPCR. Results: Seventeen microRNAs were significantly increased more than 2-fold in senescent cells. Of those, bioinformatic analysis concluded that miR-181a-5p, miR-30a-5p, miR-30a-3p, miR-100-5p, miR-21-5p, and miR-382-5p were likely associated with regulation of cellular senescence. We evaluated the potential targets of these six microRNAs by comparing them with cell-cycling and apoptosis-related genes from published mRNA transcriptional array data from aged tissues, and found that miR-181a-5p, miR-30a-5p and miR-30a-3p were enriched in overlapping targets compared with the other candidates. When modulating and comparing these microRNAs in vascular endothelial cells with several patterns, we noticed that over-expression of miR-30a-5p, and inhibition of both miR-30a-3p and miR-181a-5p, induce senescence. Conclusion: Simultaneous upregulation of miR-30a-5p, and downregulation of both miR-30a-3p and miR-181a-5p, mimic cellular senescence in vascular endothelial cells. This single step induction, as opposed to the time-consuming process of replicative passage senescence, would be useful for mimicking cell senescence vitro model.

Human albumin aggravates cerebral edema by disrupting the blood‑brain barrier in a rat model of ischemic stroke.

Cerebral edema and elevated intracranial pressure (ICP) are common complications observed following ischemic stroke. Osmotherapy has been used as a foundation to manage ICP induced by cerebral edema, and albumin is one of the most commonly used osmotic agents. The present study aimed to explore whether albumin lowered ICP by reducing cerebral edema when albumin elevated the colloid osmotic pressure (COP) of plasma. Sprague‑Dawley rats that underwent middle cerebral artery occlusion were used to assess COP and ICP. Magnetic resonance imaging measurements were performed to evaluate cerebral edema and infarct size. Evans blue was used to assess the blood‑brain barrier (BBB) permeability. Western blotting was used to determine the expression levels of the tight junction proteins in cerebral vascular endothelial cells. The results showed that 25% albumin treatment (1.25 g/kg) by intravenous injection elevated the COP of plasma but did not reduce the ICP in rats that had undergone ischemic stroke. Additionally, albumin did not reduce the infarct size and instead aggravated cerebral edema. Furthermore, the BBB permeability was increased by albumin. Concomitantly, albumin treatment significantly downregulated the expression of tight junction proteins (ZO‑1, occludin, and claudin‑5) in cerebral vascular endothelial cells. Tight junction protein expression was significantly upregulated when the cells were treated with an MMP‑9 inhibitor (GM6001). These results suggest that albumin aggravates cerebral edema in rats with ischemic stroke by increasing BBB permeability.

The role of LRP1 in Aβ efflux transport across the blood-brain barrier and cognitive dysfunction in diabetes mellitus

BackgroundThe incidence of cognitive dysfunction in diabetes is increasing yearly, which severely affects the quality of life of patients and places a heavy burden on families and society. It has been demonstrated that impaired clearance of cerebral amyloid β-protein (Aβ) is a central event in the initiation and progression of Aβ deposition and cognitive impairment in diabetic patients. However, until now, the molecular mechanism by which diabetes mellitus induces impaired clearance of Aβ has remained unclear. ObjectiveTo investigate the role and mechanism of lipoprotein receptor-related protein 1 (LRP1) in Aβ clearance impairment and cognitive function damage caused by diabetes. MethodsSPF male C57BL/6 mice were bred, and streptozotocin (STZ) (60 mg/kg/d) was intraperitoneally injected for 5 days to establish a diabetes model. The novel object recognition test and fear conditioning test were used to assess the cognitive function of mice in each group. Western blotting, qRT-PCR, ELISAs, and immunofluorescence staining were used to detect the expression levels of Aβ and Aβ clearance-related proteins in mouse brains. HBMECs were cultured in vitro to establish the blood-brain barrier model. The clearance rate of Aβ and the expression levels of LRP1 were measured under different glucose concentration culture conditions. HBMECs were transfected with lentivirus to overexpress or knock down the LRP1, and then, the changes in Aβ clearance were detected again. We injected adeno-associated virus AAV9-SP-A-LRP1 shRNA into the tail vein of DM mice to selectively knock down LRP1 gene expression in cerebral vascular endothelial cells. Then, the cognitive function and the expression levels of Aβ and Aβ clearance-related proteins in the brains of normal, DM and LRP1 knockdown mice were detected. ResultsCompared with the controls, diabetic mice showed impaired cognitive performance, increased deposition of Aβ in the brain and decreased expression of LRP1 in the brain microvasculature. In vitro experiments showed that high glucose can downregulate the expression of LRP1 in HBMECs and damage the Aβ clearance across the blood-brain barrier (BBB). The reduction in the clearance rate of Aβ induced by high glucose was reversed by LRP1 overexpression but further substantially decreased when LRP1 was knocked down. ConclusionHyperglycemia can impair Aβ efflux in the brain by downregulating the expression of LRP1 in the brain microvasculature, eventually resulting in cognitive impairment.

Free-cell therapeutics and mechanism of exosomes from adipose-derived stem cells in promoting wound healing: current understanding and future applications.

Free-cell therapeutics and mechanism of exosomes from adipose-derived stem cells in promoting wound healing: current understanding and future applications.

A Cu(II)-based coordination polymer: catalytic properties and treatment activity on stroke

ABSTRACT A fresh Cu(II) coordination polymer, i.e., [Cu3(Hmbdc)2(mbdc)2(dmphen)2]n (1, H2mbdc = isophthalic acid, dmphen = 4,7-dimethyl-1,10-phenanthroline), has been generated with the hydrothermal reactions between Cu salts and the mixed ligands of 4,7-dimethyl-1,10-phenanthroline and isophthalic acid. Moreover, the catalytic activity of 1 was evaluated via degrading the Congo red with a method of Fenton with an excellent degradation efficiency of 95.8% at 100 min. Next, the application value of compound on stroke was assessed, and the related mechanisms were explored at the same time. First of all, the tumor necrosis factor-α and recombinant rat IL-1β content released into the plasma were determined with enzyme-linked immunosorbent assay detection kit. Besides, the activation of the HMGB1/TLR4 signaling pathway activation in cerebral vascular endothelial cells was also determined with real-time reverse transcription–polymerase chain reaction assay.

The Cold Receptor TRPM8 Mediates the Antioxidant and Cytoprotective Effects of Mild Hypothermia in Cerebral Endothelial Cells During Inflammation

Cerebral vascular endothelium, the key component of the neurovascular unit, is most vulnerable to oxidative stress injury. Our previous reports in newborn pigs indicated that brain hypothermia prevents cerebral endothelial injury by oxidative stress caused by neonatal seizures and asphyxia. We used primary cerebral vascular endothelial cells (CVEC) from newborn pigs as an in vitro model of oxidative stress condition in the brain caused by inflammation. During normothermia (37˚C), CVEC respond to a pro‐inflammatory cytokine TNF‐alpha (30 ng/ml) by excessive formation of reactive oxygen species (ROS) leading to cell death by apoptosis (caspase‐3 activation, DNA fragmentation, loss of cell‐cell contacts). Remarkably, mild hypothermia (24.5°C) reduced ROS and prevented apoptosis in CVEC exposed to TNF‐alpha. We tested the hypothesis that the cold sensing menthol receptor TRPM8 mediates the cytoprotective effects of mild hypothermia in cerebral endothelium. TRPM8 (mRNA and protein) is expressed in CVEC. The TRPM8 agonist icilin (5 µM) reduced ROS and prevented apoptosis in CVEC exposed to TNF‐alpha at 37˚C, thus mimicking the cytoprotective effects of mild cooling. The TRPM8 antagonist AMTB (20 µM) reversed the icilin‐mediated cytoprotective effects during inflammation. The cell surface biotinylation technique suggested that mild hypothermia and icilin (at 37˚C) promote a time‐dependent TRPM8 translocation from an intracellular compartment to the plasma membrane concomitantly with the receptor activation. We used the in vitro model of the blood‐brain barrier (BBB) to test the functional consequences of TRPM8‐mediated cytoprotection. Confluent monolayers of CVEC cultured on transwell inserts with semipermeable membranes were exposed to TNF‐alpha during normothermia and mild hypothermia conditions. The BBB properties were detected by transendothelial electrical resistance (TEER) and the permeability to 3 kDa fluorescent Dextran. During a 2‐6 h exposure to TNF‐alpha at 37°C, TEER was time‐dependently reduced, and the Dextran permeability was elevated indicating the BBB opening. Mild hypothermia (24.5°C) and icilin (at 37˚C) largely preserved the BBB properties during inflammatory conditions. Overall, our findings demonstrate that mild hypothermia, via a TRPM8‐mediated mechanism, reduces oxidative stress, promotes cell survival, and prevents BBB damage during inflammation. The TRPM8 targeting approaches may provide a promising therapeutic strategy for cerebrovascular disease caused by brain inflammation.

Acute Antioxidant and Cytoprotective Effects of Caffeine in the Model of Neurovascular Injury by Oxidative Stress

Caffeine is commonly used in the neonatal intensive care units for respiratory disorders, but the effects of this medication in the neonatal cerebral circulation remain largely unexplored. We used newborn pigs as a clinically relevant model of the neonatal human brain to investigate the effects of caffeine on cerebral arterioles and primary neurovascular cells. First, we examined dose‐dependent cerebrovascular and systemic effects of water‐soluble anhydrous caffeine in anesthetized newborn pigs equipped with closed cranial windows. Caffeine administered systemically at therapeutic concentrations (20‐40 mg/kg i.v.) had no significant effects on diameter of pial arterioles, mean arterial blood pressure (MABP), and heart rate within 10‐60 min after the injection. When used at a higher concentration (70 mg/kg i.v.), caffeine produced dilation of pial arterioles (15‐20%) accompanied by an increased heart rate, without any effect on MABP. Topical application of caffeine (10‐5‐10‐3M) under the cranial window produced a dose‐dependent dilation of pial cerebral arterioles (15‐30%) that was fully reversed by a brief flushing with artificial cerebrospinal fluid. These in vivo data indicate that cerebral vascular cells are potential targets for caffeine. Next, we tested the hypothesis that caffeine exhibits cytoprotective properties in cerebral circulation during oxidative stress conditions. We used an in vitro model of acute oxidative stress‐induced neurovascular injury caused by excitotoxic and inflammatory conditions concomitant with epileptic seizures, asphyxia, and cerebral ischemia. Endothelial cells and cortical astrocytes are the key components of the neurovascular unit that are most vulnerable to oxidative stress injury. Primary cultures of cerebral vascular endothelial cells (CVEC) and cortical astrocytes from newborn pigs responded to pro‐inflammatory cytokine TNF‐alpha (30 ng/ml) and excitotoxic glutamate (1‐2 mM) by a surge in reactive oxygen species (ROS) production leading to apoptosis. NADPH oxidase is the major ROS‐generating system in the neurovascular unit during inflammation and excitotoxicity. We examined the acute effects of caffeine on oxidative stress‐induced neurovascular cell death caused by TNF‐alpha and excitotoxic glutamate. Caffeine (0.1‐10 µM) inhibited NADPH oxidase activation, reduced overall ROS generation, and attenuated major key events of apoptosis (caspase‐3 activation, DNA fragmentation, and loss of cell‐cell contacts) in CVEC and astrocytes exposed to TNF‐a and glutamate. These data demonstrate, for the first time, that caffeine exhibits acute antioxidant and cytoprotective effects in cerebral vascular endothelial cells and astrocytes. Overall, endothelial and astrocytic components of the neurovascular unit represent target cells for caffeine cytoprotective therapy in the neonatal cerebral circulation during oxidative stress.

Interleukin-35 exhibits protective effects in a rat model of hypoxic-ischemic encephalopathy through the inhibition of microglia-mediated inflammation.

BackgroundHypoxic-ischemic encephalopathy (HIE) brain damage is related to inflammatory responses and oxidative stress. Interleukin (IL)-35 is an antioxidant and anti-inflammatory cytokine. Thus, the effect of IL-35 treatment on neonatal rats with hypoxic-ischemic brain injury was investigated.MethodsA total of 96 7-day-old Sprague Dawley rats were randomly divided into three groups: sham group, HIE group, and IL-35 group. After left common carotid occlusion and 2.5 h hypoxia (HI injury), IL-35 (20 µg/g) was intraperitoneally (i.p.) administered to the pups. In vitro, BV2 cells were treated with or without IL-35 6 h before oxygen-glucose deprivation (OGD) insult and the microglia culture medium (MCM) was co-cultured with b.End3 cerebral vascular endothelial cells. Microglial polarization and activation were assessed by real-time quantitative polymerase chain reaction (RT-qPCR), Western blot, and enzyme-linked immunosorbent assay (ELISA). Endothelial cell dysfunction was measured by cell counting kit-8 and Western blot assays.ResultsAdministration of IL-35 alleviated neurological deficiencies, decreased brain edema, ameliorated cerebral infarction, and limited M1 microglial polarization in HI-injured pups. Meanwhile, IL-35 decreased pro-inflammatory cytokines, tumor necrosis factor-α, IL-1β, and reactive oxygen species generation in OGD-induced bEnd.3 cells. Furthermore, IL-35 treatment could reverse the vascular endothelial cell injury induced by microglial polarization. Finally, IL-35 markedly suppressed the activation of hypoxia-inducible factor-1α (HIF-1α) and the nuclear factor-κB (NF-κB) signaling pathway in vivo and in vitro.ConclusionsIL-35 relieved hypoxic-ischemic-induced brain injury and inhibited the inflammatory response by suppressing microglial polarization and activation. These results suggest that IL-35 might have potential applications for the treatment of HIE.

Evaluation of anisodamine-mediated amelioration of hypoxic injury in brain microvascular endothelial cells

Purpose: To investigate the therapeutic effect of anisodamine on hypoxic injury of brain microvascular endothelial cells, and the underlying mechanism of action.
 Methods: A hypoxic injury model of rat primary brain microvascular endothelial cells was established. Cell viability, proliferation, percent survival and apoptosis were assessed using MTT, EdU staining, trypan blue staining and TUNEL staining assays, respectively. The activities of lactate dehydrogenase (LDH) and matrix metalloproteinase-9 (MMP-9) were measured using colorimetry and enzyme-linked immunosorbent assay (ELISA), respectively. Expression levels of key proteins in the PI3K/Akt signal route were determined by Western blotting.
 Results: Anisodamine treatment significantly reduced apoptosis and LDH leakage in the rat cerebral vascular endothelial cell hypoxia injury model group (p < 0.001). At doses of 3 and 10 mM, anisodamine markedly reduced the cellular level of MMP-9 in rat cerebral vascular endothelial cell hypoxia injury model group (p < 0.001). Treatment of rat brain microvascular endothelial cells with anisodamine significantly reduced levels of caspase-3 and Bax, but up-regulated the expression levels of Bcl2, p-Akt and PI3K (p < 0.001).
 Conclusion: Anisodamine exerts significant protective effect against hypoxic injury in rat brain microvascular endothelial cells by modulating PI3K/Akt signaling pathway. This finding provides a scientific basis for the clinical application of anisodamine in the treatment and prevention of ischemic cerebrovascular disease.

A new mechanism of POCD caused by sevoflurane in mice: cognitive impairment induced by cross-dysfunction of iron and glucose metabolism.

Sevoflurane (Sev) is a commonly used anesthetic in hospitals that can cause neurotoxicity. Postoperative cognitive dysfunction (POCD) is a common clinical problem induced by some anesthetics. However, the exact mechanism of neurotoxicity induced by Sev is unclear. Here we studied a new mechanism of POCD induced by Sev. We treated 15-month-old mice with 2% Sev for 6 hours, and we had found that Sev causes POCD. Using isobaric tags for relative and absolute quantitation (iTRAQ), we found that the transporter and the metabolism of carbohydrates and inorganic ions were involved in the cognitive impairment induced by Sev. Using synchrotron radiation micro-X-ray fluorescence (μ-XRF), we showed that Sev caused the iron overload in the brain of 15-month-old mice. Subsequently, excessive iron led to oxidative stress and impaired mitochondrial function that further led to glucose metabolism disorder and reduced ATP production by regulating the expression of key enzyme genes or proteins including G6Pase, Pck1, and Cs. Meanwhile, Sev also inhibited the oxygen consumption rate and glucose absorption by downregulating the expression of glucose transporter 1 in cerebral vascular endothelial cells. The cross-dysfunction of iron and glucose metabolism caused the apoptosis in the cortex and hippocampus through Bcl2/Bax pathway. In conclusion, the data here showed a new mechanism that Sev caused apoptosis by cross-dysregulation of iron and glucose metabolism and induced energy stress in mice. Maintaining iron and glucose metabolism homeostasis may play an important role in cognitive impairment induced by Sev.

Expression of IFN-induced transmembrane protein 1 in glomerular endothelial cells.

Glomerular endothelial cells (GECs) are directly exposed to circulating viral particles in the glomerulus. Although viral infections may trigger the development of acute kidney injury or the worsening of pre-existing chronic kidney disease, the specific molecular mechanisms underlying antiviral reactions via the activation of endothelial Toll-like receptor 3 signaling in the kidney remain to be determined. Interferon (IFN)-induced transmembrane protein 1 (IFITM1), a member of interferon-stimulated gene protein family, is involved in the prevention of viral entry into cerebral vascular endothelial cells, respiratory epithelial cells, and endometrium. However, as far as we are aware, the implication of IFITM1 associated with viral infections in GECs has not been investigated to date. Cultured, normal human GECs were treated with polyinosinic-polycytidylic acid (poly IC), a synthesized viral double-stranded RNA, then the expression of IFITM1 was examined by quantitative real-time reverse transcription-polymerase chain reaction and western blotting. To further elucidate the poly IC-induced signaling pathway, the cells were applied to RNA interference against IFN-β, nuclear factor-κB p65, and IFN regulatory factor 3. We also conducted an immunofluorescence study to examine endothelial IFITM1 expression in biopsy specimens from patients with chronic kidney disease. We found that the activation of Toll-like receptor 3 induced endothelial expression of IFITM1, and that this involved IFN regulatory factor 3 and IFN-β, but not nuclear factor-κB. Intense endothelial IFITM1 immunoreactivity was observed in biopsy specimens from patients with lupus nephritis. Antiviral reaction-related endothelial expression of IFITM1 may be involved, at least in part, in the development of particularly in lupus nephritis. Further detailed studies of the implication of interferon stimulated genes, including IFITM1 in GECs are needed.