Background:Our previous study found that mouse embryonic neural stem cell(NSC)-derived exosomes(EXOs)regulated NSC differentiation via the miR-9/Hes1 axis.However,the effects of EXOs on brain microvascular endothelial...Background:Our previous study found that mouse embryonic neural stem cell(NSC)-derived exosomes(EXOs)regulated NSC differentiation via the miR-9/Hes1 axis.However,the effects of EXOs on brain microvascular endothelial cell(BMEC)dysfunction via the miR-9/Hes1 axis remain unknown.Therefore,the current study aimed to determine the effects of EXOs on BMEC proliferation,migration,and death via the miR-9/Hes1 axis.Methods:Immunofluorescence,quantitative real-time polymerase chain reaction,cell counting kit-8 assay,wound healing assay,calcein-acetoxymethyl/propidium iodide staining,and hematoxylin and eosin staining were used to determine the role and mechanism of EXOs on BMECs.Results:EXOs promoted BMEC proliferation and migration and reduced cell death under hypoxic conditions.The overexpression of miR-9 promoted BMEC prolifera-tion and migration and reduced cell death under hypoxic conditions.Moreover,miR-9 downregulation inhibited BMEC proliferation and migration and also promoted cell death.Hes1 silencing ameliorated the effect of amtagomiR-9 on BMEC proliferation and migration and cell death.Hyperemic structures were observed in the regions of the hippocampus and cortex in hypoxia-induced mice.Meanwhile,EXO treatment improved cerebrovascular alterations.Conclusion:NSC-derived EXOs can promote BMEC proliferation and migra-tion and reduce cell death via the miR-9/Hes1 axis under hypoxic conditions.Therefore,EXO therapeutic strategies could be considered for hypoxia-induced vascular injury.展开更多
In the current landscape of endothelial cell isolation for building in vitro models of the blood-brain barrier,our work moves towards reproducing the features of the neurovascular unit to achieve glial compliance thro...In the current landscape of endothelial cell isolation for building in vitro models of the blood-brain barrier,our work moves towards reproducing the features of the neurovascular unit to achieve glial compliance through an innovative biomimetic coating technology for brain chronic implants.We hypothesized that the autologous origin of human brain mic rovascular endothelial cells(hBMECs)is the first requirement for the suitable coating to prevent the glial inflammato ry response trigge red by foreign neuroprosthetics.Therefo re,this study established a new procedure to preserve the in vitro viability of hBMECs isolated from gray and white matter specimens taken from neurosurge ry patients.Culturing adult hBMECs is generally considered a challenging task due to the difficult survival ex vivo and progressive reduction in proliferation of these cells.The addition of 10 nMβ-estradiol 17-acetate to the hBMEC culture medium was found to be an essential and discriminating factor promoting adhesion and proliferation both after isolation and thawing,suppo rting the well-known protective role played by estrogens on microvessels.In particular,β-estradiol 17-acetate was critical for both freshly isolated and thawed female-derived hBMECs,while it was not necessary for freshly isolated male-derived hBMECs;however,it did countera ct the decay in the viability of the latter after thawing.The tumo r-free hBMECs were thus cultured for up to 2 months and their growth efficiency was assessed befo re and after two periods of cryopreservation.Des pite the thermal stress,the hBMECs remained viable and suitable for re-freezing and storage for several months.This approach increasing in vitro viability of hBMECs opens new perspectives for the use of cryopreserved autologous hBMECs as biomimetic therapeutic tools,offering the potential to avoid additional surgical sampling for each patient.展开更多
Brain microvascular endothelial cells form the interface between nervous tissue and circulating blood, and regulate central nervous system homeostasis. Brain microvascular endothelial cells differ from peripheral endo...Brain microvascular endothelial cells form the interface between nervous tissue and circulating blood, and regulate central nervous system homeostasis. Brain microvascular endothelial cells differ from peripheral endothelial cells with regards expression of specific ion transporters and receptors, and contain fewer fenestrations and pinocytotic vesicles. Brain microvascular endothelial cells also synthesize several factors that influence blood vessel function. This review describes the morphological characteristics and functions of brain microvascular endothelial cells, and summarizes current knowledge regarding changes in brain microvascular endothelial cells during stroke progression and therapies. Future studies should focus on identifying mechanisms underlying such changes and developing possible neuroprotective therapeutic interventions.展开更多
Drugs for the treatment and prevention of nervous system diseases must permeate the bloodbrain barrier to take effect.In vitro models of the blood-brain barrier are therefore important in the investigation of drug per...Drugs for the treatment and prevention of nervous system diseases must permeate the bloodbrain barrier to take effect.In vitro models of the blood-brain barrier are therefore important in the investigation of drug permeation mechanisms.However,to date,no unified method has been described for establishing a blood-brain barrier model.Here,we modified an in vitro model of the blood-brain barrier by seeding brain microvascular endothelial cells and astrocytes from newborn rats on a polyester Transwell cell culture membrane with 0.4-μm pores,and conducted transepithelial electrical resistance measurements,leakage tests and assays for specific bloodbrain barrier enzymes.We show that the permeability of our model is as low as that of the bloodbrain barrier in vivo.Our model will be a valuable tool in the study of the mechanisms of action of neuroprotective drugs.展开更多
Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on o...Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation-induced hypoxia inducible factor-1α expression.In this study,we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression.MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner.Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α.Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression;however,DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA.These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.展开更多
In this work, blank polylactic acid (PLA) nanoparticles with unstained surface were prepared by the nano-deposition method. On the basis of the preparation, the effect of surface modification on brain microvascular ...In this work, blank polylactic acid (PLA) nanoparticles with unstained surface were prepared by the nano-deposition method. On the basis of the preparation, the effect of surface modification on brain microvascular endothelial cells (BMECs) targeting was examined by in vivo experiments and fluorescence microscopy. The results showed that PLA nanoparticles are less toxic than PACA nanoparticles but their BMECs targeting is similar to PACA nanoparticles. The experiments suggest that drugs can he loaded onto the particles and become more stable through adsorption on the surface of PLA nanoparticles with high surface activity. The surface of PLA nanoparticles was obviously modified and the hydrophilicity was increased as well in the presence of non-ionic surfactants on PLA nanoparticles. As a targeting moiety, polysobate 80 (T-80) can facilitate BMECs targeting of PLA nanoparticles.展开更多
Objective:To investigate the protective effect of Cordyceps sinensis extract (CSE)on injury of primary cultured rat brain microvascular endothelial cells (rBMECs) induced by oxygen-glucose deprivation (OGD).Methods:We...Objective:To investigate the protective effect of Cordyceps sinensis extract (CSE)on injury of primary cultured rat brain microvascular endothelial cells (rBMECs) induced by oxygen-glucose deprivation (OGD).Methods:We isolated and cultured primary rBMECs in order to establish an in vitro OGD model.Cellular activity was detected using a cell counting kit to determine the appropriate dosage.The rBMECs were divided into control,model,low-,mid-,and high-dose (5,10,20 μg.mL-1) CSE groups under OGD for 6 hours.CSE was dissolved in cell culture medium to the appropriate concentration,passed through a 0.22 μm sterile filter,and administered for 12 hours before and during OGD.Cellular morphology was observed under a microscope.Lactate dehydrogenase level in cultural supernatant,superoxide dismutase activity,and the content of nitric oxide and malondialdehyde in cells were tested by colorimetric methods.Levels of tumor necrosis factor-α and interleukin-1 beta in cells were determined by enzyme-linked immunosorbent assay.Results:After 12-hour administration of CSE at the concentration of 5,10,20 iμg.mL-1 before and during OGD,compared with the model group,the CSE groups obviously alleviated the damage of rBMECs induced by OGD,inhibited the apoptosis and the necrosis of the cells,and improved cellular morphology of rBMECs.Additionally,compared with the model group,CSE also restrained lactate dehydrogenase leakage in hypoxic cells (P <.01),significantly increased superoxide dismutase activity (P <.05),and reduced the levels of nitric oxide,malondialdehyde,tumor necrosis factor-α,and interleukin-1 beta (P <.05).Conclusion:C.sinensis extract plays a significant role in protecting injured primary cultured rBMECs induced by OGD.The mechanism may be related with the increase of cellular antioxidative capacity and anti-inflammatory effect.展开更多
Objective: To investigate the role of human host heme-oxygenase-1(HO-1) in pathogenesis of cerebral malaria in the in vitro model,Methods: The effect of human host HO-1 [human brain microvascular endothelial cell(HBME...Objective: To investigate the role of human host heme-oxygenase-1(HO-1) in pathogenesis of cerebral malaria in the in vitro model,Methods: The effect of human host HO-1 [human brain microvascular endothelial cell(HBMEC)] on hemoglobin degradation in the co-culture model of HBMEC and ITG Plasmodium falciparum-infected red cells(i RBC) through measurement of the enzymatic products iron and bilirubin,Results: Following exposure to the HO-1 inducer Co PPIX at all concentrations,the HBMEC cells apoptosis occurred,which could be prominently observed at 15 μM of 3 h exposure,In contrast,there was no significant change in the morphology in the non-exposed i RBC at all concentrations and exposure time,This observation was in agreement with the levels of the enzymatic degradation products iron and bilirubin,of which the highest levels(106.03 and 1 753.54% of baseline level,respectively) were observed at 15 μM vs,20 μM at 3 h vs,24 h exposure,For the effect of the HO-1 inhibitor Zn PPIX,HBMEC cell morphology was mostly unchanged,but significant inhibitory effect on cell apoptosis was seen at 10 μM for the exposure period of 3 h(37.17% of baseline level),The degree of the inhibitory effect as reflected by the level of iron produced was not clearly observed(highest effect at 10 μM and 3 h exposure),Conclusions: Results provide at least in part,insight into the contribution of HO-1 on CM pathogenesis and need to be confirmed in animal model.展开更多
Objective:To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells,provide important carriers and tool cells for the research of related cerebrovascular diseases.Metho...Objective:To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells,provide important carriers and tool cells for the research of related cerebrovascular diseases.Methods:The cerebral cortexes of rabbits were collected aseptic and inoculated after cutting,passing through cell sieve,bovine serum albumin density gradient centrifugation,typeⅡcollagenase digestion,finally inoculated and cultured.The cultured cells were identified by cell morphological observation and angiogenesis experiment.Results:Under the inverted microscope,the cells were short fusiform or polygonal,and grew in clusters and adhere to the wall.After the cells were densely fused,they would be in a typical monolayer flat,“pebbled"mosaic arrangement.Tube formation test had the ability to form tubes structure.Conclusion:This method can successfully separate and cultivate primary rabbit brain microvascular endothelial cells.展开更多
Shuxuetong injection composed of leech(Hirudo nipponica Whitman) and earthworm(Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotect...Shuxuetong injection composed of leech(Hirudo nipponica Whitman) and earthworm(Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotective mechanism of Shuxuetong injection remains poorly understood. Here, cerebral microvascular endothelial cells(bEnd.3) were incubated in glucose-free Dulbecco's modified Eagle's medium containing 95% N_2/5% CO_2 for 6 hours, followed by high-glucose medium containing 95% O_2 and 5% CO_2 for 18 hours to establish an oxygen-glucose deprivation/reperfusion model. This in vitro cell model was administered Shuxuetong injection at 1/32, 1/64, and 1/128 concentrations(diluted 32-, 64-, and 128-times). Cell Counting Kit-8 assay was used to evaluate cell viability. A fluorescence method was used to measure lactate dehydrogenase, and a fluorescence microplate reader used to detect intracellular reactive oxygen species. A fluorescent probe was also used to measure mitochondrial superoxide production. A cell resistance meter was used to measure transepithelial resistance and examine integrity of monolayer cells. The fluorescein isothiocyanate-dextran test was performed to examine blood-brain barrier permeability. Real-time reverse transcription polymerase chain reaction was performed to analyze mRNA expression levels of tumor necrosis factor alpha, interleukin-1β, interleukin-6, and inducible nitric oxide synthase. Western blot assay was performed to analyze expression of caspase-3, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, occludin, vascular endothelial growth factor, cleaved caspase-3, B-cell lymphoma 2, phosphorylated extracellular signal-regulated protein kinase, extracellular signal-regulated protein kinase, nuclear factor-κB p65, I kappa B alpha, phosphorylated I kappa B alpha, I kappa B kinase, phosphorylated I kappa B kinase, claudin-5, and zonula occludens-1. Our results show that Shuxuetong injection increases bEnd.3 cell viability and B-cell lymphoma 2 expression, reduces cleaved caspase-3 expression, inhibits production of reactive oxygen species and mitochondrial superoxide, suppresses expression of tumor necrosis factor alpha, interleukin-1β, interleukin-6, inducible nitric oxide synthase mRNA, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, markedly increases transepithelial resistance, decreases blood-brain barrier permeability, upregulates claudin-5, occludin, and zonula occludens-1 expression, reduces nuclear factor-κB p65 and vascular endothelial growth factor expression, and reduces I kappa B alpha, extracellular signal-regulated protein kinase 1/2, and I kappa B kinase phosphorylation levels. Overall, these findings suggest that Shuxuetong injection has protective effects on brain microvascular endothelial cells after oxygen-glucose deprivation/reperfusion. Moreover, its protective effect is associated with reduction of mitochondrial superoxide production, inhibition of the inflammatory response, and inhibition of vascular endothelial growth factor, extracellular signal-regulated protein kinase 1/2, and the nuclear factor-κB p65 signaling pathway.展开更多
Long non-coding RNAs regulate brain microvascular endothelial cell death, the inflammatory response and angiogenesis during and after ischemia/reperfusion and oxygen-glucose deprivation/reoxygenation(OGD/R) insults....Long non-coding RNAs regulate brain microvascular endothelial cell death, the inflammatory response and angiogenesis during and after ischemia/reperfusion and oxygen-glucose deprivation/reoxygenation(OGD/R) insults. The long non-coding RNA, SNHG12, is upregulated after ischemia/reperfusion and OGD/R in microvascular endothelial cells of the mouse brain. However, its role in ischemic stroke has not been studied. We hypothesized that SNHG12 positively regulates ischemic stroke, and therefore we investigated its mechanism of action. We established an OGD/R mouse cell model to mimic ischemic stroke by exposing brain microvascular endothelial cells to OGD for 0, 2, 4, 8, 16 or 24 hours and reoxygenation for 4 hours. Quantitative real-time polymerase chain reaction showed that SNHG12 levels in brain microvascular endothelial cells increased with respect to OGD exposure time. Brain microvascular endothelial cells were transfected with pc DNA-control, pc DNA-SNHG12, si-control, or si-SNHG12. After exposure to OGD for 16 hours, these cells were then analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, trypan blue exclusion, western blot, and capillary-like tube formation assays. Overexpression of SNHG12 inhibited brain microvascular endothelial cell death and the inflammatory response but promoted angiogenesis after OGD/R, while SNHG12 knockdown had the opposite effects. miR-199a was identified as a target of SNHG12, and SNHG12 overexpression reversed the effect of miR-199a on brain microvascular endothelial cell death, the inflammatory response, and angiogenesis. These findings suggest that SNHG12 suppresses endothelial cell injury induced by OGD/R by targeting miR-199a.展开更多
Upregulation of vascular endothelial growth factor A/basic fibroblast growth factor(VEGFA/b FGF)expression in the penumbra of cerebral ischemia can increase vascular volume,reduce lesion volume,and enhance neural cell...Upregulation of vascular endothelial growth factor A/basic fibroblast growth factor(VEGFA/b FGF)expression in the penumbra of cerebral ischemia can increase vascular volume,reduce lesion volume,and enhance neural cell proliferation and differentiation,thereby exerting neuroprotective effects.However,the beneficial effects of endogenous VEGFA/b FGF are limited as their expression is only transiently increased.In this study,we generated multilayered nanofiber membranes loaded with VEGFA/b FGF using layer-by-layer self-assembly and electrospinning techniques.We found that a membrane containing 10 layers had an ideal ultrastructure and could efficiently and stably release growth factors for more than 1 month.This 10-layered nanofiber membrane promoted brain microvascular endothelial cell tube formation and proliferation,inhibited neuronal apoptosis,upregulated the expression of tight junction proteins,and improved the viability of various cellular components of neurovascular units under conditions of oxygen/glucose deprivation.Furthermore,this nanofiber membrane decreased the expression of Janus kinase-2/signal transducer and activator of transcription-3(JAK2/STAT3),Bax/Bcl-2,and cleaved caspase-3.Therefore,this nanofiber membrane exhibits a neuroprotective effect on oxygen/glucose-deprived neurovascular units by inhibiting the JAK2/STAT3 pathway.展开更多
Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating im...Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating important function of cells such as survival, growth and development during tissue organization, differentiation and organogenesis. In this study, we used an integrin-binding array platform to identify the important types of integrins and their binding peptides that facilitate adhesion, growth, development, and vascular-like network formation of rat primary brain microvascular endothelial cells. Brain microvascular endothelial cells were isolated from rat brain on post-natal day 7. Cells were cultured in a custom-designed integrin array system containing short synthetic peptides binding to 16 types of integrins commonly expressed on cells in vertebrates. After 7 days of culture, the brain microvascular endothelial cells were processed for immunostaining with markers for endothelial cells including von Willibrand factor and platelet endothelial cell adhesion molecule. 5-Bromo-2′-dexoyuridine was added to the culture at 48 hours prior to fixation to assess cell proliferation. Among 16 integrins tested, we found that α5β1, αvβ5 and αvβ8 greatly promoted proliferation of endothelial cells in culture. To investigate the effect of integrin-binding peptides in promoting neovascularization and angiogenesis, the binding peptides to the above three types of integrins were immobilized to our custom-designed hydrogel in three-dimensional(3 D) culture of brain microvascular endothelial cells with the addition of vascular endothelial growth factor. Following a 7-day 3 D culture, the culture was fixed and processed for double labeling of phalloidin with von Willibrand factor or platelet endothelial cell adhesion molecule and assessed under confocal microscopy. In the 3 D culture in hydrogels conjugated with the integrin-binding peptide, brain microvascular endothelial cells formed interconnected vascular-like network with clearly discernable lumens, which is reminiscent of brain microvascular network in vivo. With the novel integrin-binding array system, we identified the specific types of integrins on brain microvascular endothelial cells that mediate cell adhesion and growth followed by functionalizing a 3 D hydrogel culture system using the binding peptides that specifically bind to the identified integrins, leading to robust growth and lumenized microvascular-like network formation of brain microvascular endothelial cells in 3 D culture. This technology can be used for in vitro and in vivo vascularization of transplants or brain lesions to promote brain tissue regeneration following neurological insults.展开更多
目的探讨映山红花总黄酮(total flavones of rhododendra,TFR)促大鼠脑血管内皮细胞体外形成血管作用及与VEGFR_(2)和神经源性硫化氢(H_(2)S)的关系。方法采用大鼠脑血管内皮细胞单独培养及和与海马神经元共培养,分别采用不同的实验方...目的探讨映山红花总黄酮(total flavones of rhododendra,TFR)促大鼠脑血管内皮细胞体外形成血管作用及与VEGFR_(2)和神经源性硫化氢(H_(2)S)的关系。方法采用大鼠脑血管内皮细胞单独培养及和与海马神经元共培养,分别采用不同的实验方法检测细胞增殖、迁移、成管及H_(2)S含量和钙离子荧光强度,包括CCK-8法、细胞划痕法、Transwell法、基质胶成管、H_(2)S试剂盒及钙离子荧光探针法。结果在单独培养的大鼠脑血管内皮细胞上,H_(2)S供体NaHS(200μmol·L^(-1))和TFR(90、270、810 mg·L^(-1))对大鼠脑血管内皮细胞的增殖、迁移、成管及[Ca^(2+)]i荧光强度都有明显的促进作用。而VEGFR_(2)阻断剂SU5416(10μmol·L^(-1))可抑制TFR的促进内皮细胞增殖、迁移和形成血管及[Ca^(2+)]i荧光强度;在与海马神经元共培养的大鼠脑血管内皮细胞上,TFR显著地升高共培养中H_(2)S含量,并被CBS抑制剂AOAA(200μmol·L^(-1))抑制。与此同时,TFR明显地促进共培养中大鼠脑血管内皮细胞的形成血管作用,并可被AOAA和VEGFR_(2)阻断剂SU5416显著地抑制。结论TFR在体外可通过VEGFR_(2)升高[Ca^(2+)]i来促进脑血管内皮细胞形成血管,并可通过诱导神经元中CBS生成H_(2)S作用于大鼠脑血管内皮细胞的VEGFR_(2)来促进血管形成。展开更多
基金Program of Natural Science Foundation of Shanghai,Grant/Award Number:21ZR1453800 and 22ZR1452400Program of National Natural Science Foundation of China,Grant/Award Number:82370057+3 种基金Fundamental Research Funds for the Central Universities,Grant/Award Number:22120220562Program of Shanghai Municipal Health Commission,Grant/Award Number:20204Y0384Program of National Key Research and Development Project of China,Grant/Award Number:2023YFC2509500。
文摘Background:Our previous study found that mouse embryonic neural stem cell(NSC)-derived exosomes(EXOs)regulated NSC differentiation via the miR-9/Hes1 axis.However,the effects of EXOs on brain microvascular endothelial cell(BMEC)dysfunction via the miR-9/Hes1 axis remain unknown.Therefore,the current study aimed to determine the effects of EXOs on BMEC proliferation,migration,and death via the miR-9/Hes1 axis.Methods:Immunofluorescence,quantitative real-time polymerase chain reaction,cell counting kit-8 assay,wound healing assay,calcein-acetoxymethyl/propidium iodide staining,and hematoxylin and eosin staining were used to determine the role and mechanism of EXOs on BMECs.Results:EXOs promoted BMEC proliferation and migration and reduced cell death under hypoxic conditions.The overexpression of miR-9 promoted BMEC prolifera-tion and migration and reduced cell death under hypoxic conditions.Moreover,miR-9 downregulation inhibited BMEC proliferation and migration and also promoted cell death.Hes1 silencing ameliorated the effect of amtagomiR-9 on BMEC proliferation and migration and cell death.Hyperemic structures were observed in the regions of the hippocampus and cortex in hypoxia-induced mice.Meanwhile,EXO treatment improved cerebrovascular alterations.Conclusion:NSC-derived EXOs can promote BMEC proliferation and migra-tion and reduce cell death via the miR-9/Hes1 axis under hypoxic conditions.Therefore,EXO therapeutic strategies could be considered for hypoxia-induced vascular injury.
基金supported by EnTimeMent H2020-FETPROACT-824160(to LF)。
文摘In the current landscape of endothelial cell isolation for building in vitro models of the blood-brain barrier,our work moves towards reproducing the features of the neurovascular unit to achieve glial compliance through an innovative biomimetic coating technology for brain chronic implants.We hypothesized that the autologous origin of human brain mic rovascular endothelial cells(hBMECs)is the first requirement for the suitable coating to prevent the glial inflammato ry response trigge red by foreign neuroprosthetics.Therefo re,this study established a new procedure to preserve the in vitro viability of hBMECs isolated from gray and white matter specimens taken from neurosurge ry patients.Culturing adult hBMECs is generally considered a challenging task due to the difficult survival ex vivo and progressive reduction in proliferation of these cells.The addition of 10 nMβ-estradiol 17-acetate to the hBMEC culture medium was found to be an essential and discriminating factor promoting adhesion and proliferation both after isolation and thawing,suppo rting the well-known protective role played by estrogens on microvessels.In particular,β-estradiol 17-acetate was critical for both freshly isolated and thawed female-derived hBMECs,while it was not necessary for freshly isolated male-derived hBMECs;however,it did countera ct the decay in the viability of the latter after thawing.The tumo r-free hBMECs were thus cultured for up to 2 months and their growth efficiency was assessed befo re and after two periods of cryopreservation.Des pite the thermal stress,the hBMECs remained viable and suitable for re-freezing and storage for several months.This approach increasing in vitro viability of hBMECs opens new perspectives for the use of cryopreserved autologous hBMECs as biomimetic therapeutic tools,offering the potential to avoid additional surgical sampling for each patient.
基金supported by grants from the National Natural Science Foundation of ChinaNo.8117111281371272 to MCL
文摘Brain microvascular endothelial cells form the interface between nervous tissue and circulating blood, and regulate central nervous system homeostasis. Brain microvascular endothelial cells differ from peripheral endothelial cells with regards expression of specific ion transporters and receptors, and contain fewer fenestrations and pinocytotic vesicles. Brain microvascular endothelial cells also synthesize several factors that influence blood vessel function. This review describes the morphological characteristics and functions of brain microvascular endothelial cells, and summarizes current knowledge regarding changes in brain microvascular endothelial cells during stroke progression and therapies. Future studies should focus on identifying mechanisms underlying such changes and developing possible neuroprotective therapeutic interventions.
基金supported by the National Natural Science Foundation of China,No.81374005,30973979grant from the National Science and Technology Support Program during the Twelfth"Five-Year"Plan Period of China,No.2012BAI26B03
文摘Drugs for the treatment and prevention of nervous system diseases must permeate the bloodbrain barrier to take effect.In vitro models of the blood-brain barrier are therefore important in the investigation of drug permeation mechanisms.However,to date,no unified method has been described for establishing a blood-brain barrier model.Here,we modified an in vitro model of the blood-brain barrier by seeding brain microvascular endothelial cells and astrocytes from newborn rats on a polyester Transwell cell culture membrane with 0.4-μm pores,and conducted transepithelial electrical resistance measurements,leakage tests and assays for specific bloodbrain barrier enzymes.We show that the permeability of our model is as low as that of the bloodbrain barrier in vivo.Our model will be a valuable tool in the study of the mechanisms of action of neuroprotective drugs.
基金supported by the National Natural Science Foundation of China,No.30471917 and 30770766
文摘Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation-induced hypoxia inducible factor-1α expression.In this study,we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression.MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner.Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α.Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression;however,DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA.These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.
文摘In this work, blank polylactic acid (PLA) nanoparticles with unstained surface were prepared by the nano-deposition method. On the basis of the preparation, the effect of surface modification on brain microvascular endothelial cells (BMECs) targeting was examined by in vivo experiments and fluorescence microscopy. The results showed that PLA nanoparticles are less toxic than PACA nanoparticles but their BMECs targeting is similar to PACA nanoparticles. The experiments suggest that drugs can he loaded onto the particles and become more stable through adsorption on the surface of PLA nanoparticles with high surface activity. The surface of PLA nanoparticles was obviously modified and the hydrophilicity was increased as well in the presence of non-ionic surfactants on PLA nanoparticles. As a targeting moiety, polysobate 80 (T-80) can facilitate BMECs targeting of PLA nanoparticles.
基金This study was supported by the National Natural Science Foundation of China(81403319 and 81603453)the Beijing Excellent Talent Project(2014000020124G114).
文摘Objective:To investigate the protective effect of Cordyceps sinensis extract (CSE)on injury of primary cultured rat brain microvascular endothelial cells (rBMECs) induced by oxygen-glucose deprivation (OGD).Methods:We isolated and cultured primary rBMECs in order to establish an in vitro OGD model.Cellular activity was detected using a cell counting kit to determine the appropriate dosage.The rBMECs were divided into control,model,low-,mid-,and high-dose (5,10,20 μg.mL-1) CSE groups under OGD for 6 hours.CSE was dissolved in cell culture medium to the appropriate concentration,passed through a 0.22 μm sterile filter,and administered for 12 hours before and during OGD.Cellular morphology was observed under a microscope.Lactate dehydrogenase level in cultural supernatant,superoxide dismutase activity,and the content of nitric oxide and malondialdehyde in cells were tested by colorimetric methods.Levels of tumor necrosis factor-α and interleukin-1 beta in cells were determined by enzyme-linked immunosorbent assay.Results:After 12-hour administration of CSE at the concentration of 5,10,20 iμg.mL-1 before and during OGD,compared with the model group,the CSE groups obviously alleviated the damage of rBMECs induced by OGD,inhibited the apoptosis and the necrosis of the cells,and improved cellular morphology of rBMECs.Additionally,compared with the model group,CSE also restrained lactate dehydrogenase leakage in hypoxic cells (P <.01),significantly increased superoxide dismutase activity (P <.05),and reduced the levels of nitric oxide,malondialdehyde,tumor necrosis factor-α,and interleukin-1 beta (P <.05).Conclusion:C.sinensis extract plays a significant role in protecting injured primary cultured rBMECs induced by OGD.The mechanism may be related with the increase of cellular antioxidative capacity and anti-inflammatory effect.
基金supported by The Commission on Higher Education,Ministry of Education of Thailand,The National Research University Project of Thailand(NRU)Office of Higher Education Commission,Thammasat University(Center of Excellence in Pharmacology and Molecular Biology of Malaria and Cholangiocarcinoma)+1 种基金Liverpool School of Tropical Medicine,University of Liverpool,UKThe Royal Golden Jubilee PhD Programme,Thailand Research Fund-Thammasat University Joint Fund and Graduated Student Grant to P.Thongdee(Grant No.PHD/0365/2552)
文摘Objective: To investigate the role of human host heme-oxygenase-1(HO-1) in pathogenesis of cerebral malaria in the in vitro model,Methods: The effect of human host HO-1 [human brain microvascular endothelial cell(HBMEC)] on hemoglobin degradation in the co-culture model of HBMEC and ITG Plasmodium falciparum-infected red cells(i RBC) through measurement of the enzymatic products iron and bilirubin,Results: Following exposure to the HO-1 inducer Co PPIX at all concentrations,the HBMEC cells apoptosis occurred,which could be prominently observed at 15 μM of 3 h exposure,In contrast,there was no significant change in the morphology in the non-exposed i RBC at all concentrations and exposure time,This observation was in agreement with the levels of the enzymatic degradation products iron and bilirubin,of which the highest levels(106.03 and 1 753.54% of baseline level,respectively) were observed at 15 μM vs,20 μM at 3 h vs,24 h exposure,For the effect of the HO-1 inhibitor Zn PPIX,HBMEC cell morphology was mostly unchanged,but significant inhibitory effect on cell apoptosis was seen at 10 μM for the exposure period of 3 h(37.17% of baseline level),The degree of the inhibitory effect as reflected by the level of iron produced was not clearly observed(highest effect at 10 μM and 3 h exposure),Conclusions: Results provide at least in part,insight into the contribution of HO-1 on CM pathogenesis and need to be confirmed in animal model.
基金This study was supported by the National Natural Science Foundation of China(81072714)the Natural Science Foundation of Fujian Province,China(No.2017J01545)。
文摘Objective:To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells,provide important carriers and tool cells for the research of related cerebrovascular diseases.Methods:The cerebral cortexes of rabbits were collected aseptic and inoculated after cutting,passing through cell sieve,bovine serum albumin density gradient centrifugation,typeⅡcollagenase digestion,finally inoculated and cultured.The cultured cells were identified by cell morphological observation and angiogenesis experiment.Results:Under the inverted microscope,the cells were short fusiform or polygonal,and grew in clusters and adhere to the wall.After the cells were densely fused,they would be in a typical monolayer flat,“pebbled"mosaic arrangement.Tube formation test had the ability to form tubes structure.Conclusion:This method can successfully separate and cultivate primary rabbit brain microvascular endothelial cells.
基金supported in part by the National Natural Science Foundation of China,No.81573644(to LMH),81573733(to SWX)the Tianjin 131 Innovative Team Project,China(to HW)+5 种基金the National Major Science and Technology Project of China,No.2012ZX09101201-004(to SWX)the Science and Technology Plan Project of Tianjin of China,No.16PTSYJC00120(to LMH)the Applied Foundation and Frontier Technology Research Program of Tianjin of China(General Project),No.14JCYBJC28900(to SXW)the National International Science and Technology Cooperation Project of China,No.2015DFA30430(to HW)the Key Program of the Natural Science Foundation of Tianjin of China,No.16ICZDJC36300(to HW)the Scientific Research and Technology Development Plan Project of Guangxi Zhuang Autonomous Region of China,No.14125008-2-5(to SXW)
文摘Shuxuetong injection composed of leech(Hirudo nipponica Whitman) and earthworm(Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotective mechanism of Shuxuetong injection remains poorly understood. Here, cerebral microvascular endothelial cells(bEnd.3) were incubated in glucose-free Dulbecco's modified Eagle's medium containing 95% N_2/5% CO_2 for 6 hours, followed by high-glucose medium containing 95% O_2 and 5% CO_2 for 18 hours to establish an oxygen-glucose deprivation/reperfusion model. This in vitro cell model was administered Shuxuetong injection at 1/32, 1/64, and 1/128 concentrations(diluted 32-, 64-, and 128-times). Cell Counting Kit-8 assay was used to evaluate cell viability. A fluorescence method was used to measure lactate dehydrogenase, and a fluorescence microplate reader used to detect intracellular reactive oxygen species. A fluorescent probe was also used to measure mitochondrial superoxide production. A cell resistance meter was used to measure transepithelial resistance and examine integrity of monolayer cells. The fluorescein isothiocyanate-dextran test was performed to examine blood-brain barrier permeability. Real-time reverse transcription polymerase chain reaction was performed to analyze mRNA expression levels of tumor necrosis factor alpha, interleukin-1β, interleukin-6, and inducible nitric oxide synthase. Western blot assay was performed to analyze expression of caspase-3, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, occludin, vascular endothelial growth factor, cleaved caspase-3, B-cell lymphoma 2, phosphorylated extracellular signal-regulated protein kinase, extracellular signal-regulated protein kinase, nuclear factor-κB p65, I kappa B alpha, phosphorylated I kappa B alpha, I kappa B kinase, phosphorylated I kappa B kinase, claudin-5, and zonula occludens-1. Our results show that Shuxuetong injection increases bEnd.3 cell viability and B-cell lymphoma 2 expression, reduces cleaved caspase-3 expression, inhibits production of reactive oxygen species and mitochondrial superoxide, suppresses expression of tumor necrosis factor alpha, interleukin-1β, interleukin-6, inducible nitric oxide synthase mRNA, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, markedly increases transepithelial resistance, decreases blood-brain barrier permeability, upregulates claudin-5, occludin, and zonula occludens-1 expression, reduces nuclear factor-κB p65 and vascular endothelial growth factor expression, and reduces I kappa B alpha, extracellular signal-regulated protein kinase 1/2, and I kappa B kinase phosphorylation levels. Overall, these findings suggest that Shuxuetong injection has protective effects on brain microvascular endothelial cells after oxygen-glucose deprivation/reperfusion. Moreover, its protective effect is associated with reduction of mitochondrial superoxide production, inhibition of the inflammatory response, and inhibition of vascular endothelial growth factor, extracellular signal-regulated protein kinase 1/2, and the nuclear factor-κB p65 signaling pathway.
基金supported by the Natural Science Foundation of Hainan Province of China,No.817334
文摘Long non-coding RNAs regulate brain microvascular endothelial cell death, the inflammatory response and angiogenesis during and after ischemia/reperfusion and oxygen-glucose deprivation/reoxygenation(OGD/R) insults. The long non-coding RNA, SNHG12, is upregulated after ischemia/reperfusion and OGD/R in microvascular endothelial cells of the mouse brain. However, its role in ischemic stroke has not been studied. We hypothesized that SNHG12 positively regulates ischemic stroke, and therefore we investigated its mechanism of action. We established an OGD/R mouse cell model to mimic ischemic stroke by exposing brain microvascular endothelial cells to OGD for 0, 2, 4, 8, 16 or 24 hours and reoxygenation for 4 hours. Quantitative real-time polymerase chain reaction showed that SNHG12 levels in brain microvascular endothelial cells increased with respect to OGD exposure time. Brain microvascular endothelial cells were transfected with pc DNA-control, pc DNA-SNHG12, si-control, or si-SNHG12. After exposure to OGD for 16 hours, these cells were then analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, trypan blue exclusion, western blot, and capillary-like tube formation assays. Overexpression of SNHG12 inhibited brain microvascular endothelial cell death and the inflammatory response but promoted angiogenesis after OGD/R, while SNHG12 knockdown had the opposite effects. miR-199a was identified as a target of SNHG12, and SNHG12 overexpression reversed the effect of miR-199a on brain microvascular endothelial cell death, the inflammatory response, and angiogenesis. These findings suggest that SNHG12 suppresses endothelial cell injury induced by OGD/R by targeting miR-199a.
基金supported by the National Natural Science Foundation of China,Nos.81974207(to JH),82001383(to DW)the Special Clinical Research Project of Health Profession of Shanghai Municipal Health Commission,No.20204Y0076(to DW)。
文摘Upregulation of vascular endothelial growth factor A/basic fibroblast growth factor(VEGFA/b FGF)expression in the penumbra of cerebral ischemia can increase vascular volume,reduce lesion volume,and enhance neural cell proliferation and differentiation,thereby exerting neuroprotective effects.However,the beneficial effects of endogenous VEGFA/b FGF are limited as their expression is only transiently increased.In this study,we generated multilayered nanofiber membranes loaded with VEGFA/b FGF using layer-by-layer self-assembly and electrospinning techniques.We found that a membrane containing 10 layers had an ideal ultrastructure and could efficiently and stably release growth factors for more than 1 month.This 10-layered nanofiber membrane promoted brain microvascular endothelial cell tube formation and proliferation,inhibited neuronal apoptosis,upregulated the expression of tight junction proteins,and improved the viability of various cellular components of neurovascular units under conditions of oxygen/glucose deprivation.Furthermore,this nanofiber membrane decreased the expression of Janus kinase-2/signal transducer and activator of transcription-3(JAK2/STAT3),Bax/Bcl-2,and cleaved caspase-3.Therefore,this nanofiber membrane exhibits a neuroprotective effect on oxygen/glucose-deprived neurovascular units by inhibiting the JAK2/STAT3 pathway.
基金supported by NIH grant RO1 NS093985 (to DS, NZ, XW) and RO1 NS101955 (to DS)the VCU Microscopy Facility,supported,in part,by funding from NIH-NCI Cancer Center Support Grant P30 CA016059。
文摘Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating important function of cells such as survival, growth and development during tissue organization, differentiation and organogenesis. In this study, we used an integrin-binding array platform to identify the important types of integrins and their binding peptides that facilitate adhesion, growth, development, and vascular-like network formation of rat primary brain microvascular endothelial cells. Brain microvascular endothelial cells were isolated from rat brain on post-natal day 7. Cells were cultured in a custom-designed integrin array system containing short synthetic peptides binding to 16 types of integrins commonly expressed on cells in vertebrates. After 7 days of culture, the brain microvascular endothelial cells were processed for immunostaining with markers for endothelial cells including von Willibrand factor and platelet endothelial cell adhesion molecule. 5-Bromo-2′-dexoyuridine was added to the culture at 48 hours prior to fixation to assess cell proliferation. Among 16 integrins tested, we found that α5β1, αvβ5 and αvβ8 greatly promoted proliferation of endothelial cells in culture. To investigate the effect of integrin-binding peptides in promoting neovascularization and angiogenesis, the binding peptides to the above three types of integrins were immobilized to our custom-designed hydrogel in three-dimensional(3 D) culture of brain microvascular endothelial cells with the addition of vascular endothelial growth factor. Following a 7-day 3 D culture, the culture was fixed and processed for double labeling of phalloidin with von Willibrand factor or platelet endothelial cell adhesion molecule and assessed under confocal microscopy. In the 3 D culture in hydrogels conjugated with the integrin-binding peptide, brain microvascular endothelial cells formed interconnected vascular-like network with clearly discernable lumens, which is reminiscent of brain microvascular network in vivo. With the novel integrin-binding array system, we identified the specific types of integrins on brain microvascular endothelial cells that mediate cell adhesion and growth followed by functionalizing a 3 D hydrogel culture system using the binding peptides that specifically bind to the identified integrins, leading to robust growth and lumenized microvascular-like network formation of brain microvascular endothelial cells in 3 D culture. This technology can be used for in vitro and in vivo vascularization of transplants or brain lesions to promote brain tissue regeneration following neurological insults.
文摘目的探讨映山红花总黄酮(total flavones of rhododendra,TFR)促大鼠脑血管内皮细胞体外形成血管作用及与VEGFR_(2)和神经源性硫化氢(H_(2)S)的关系。方法采用大鼠脑血管内皮细胞单独培养及和与海马神经元共培养,分别采用不同的实验方法检测细胞增殖、迁移、成管及H_(2)S含量和钙离子荧光强度,包括CCK-8法、细胞划痕法、Transwell法、基质胶成管、H_(2)S试剂盒及钙离子荧光探针法。结果在单独培养的大鼠脑血管内皮细胞上,H_(2)S供体NaHS(200μmol·L^(-1))和TFR(90、270、810 mg·L^(-1))对大鼠脑血管内皮细胞的增殖、迁移、成管及[Ca^(2+)]i荧光强度都有明显的促进作用。而VEGFR_(2)阻断剂SU5416(10μmol·L^(-1))可抑制TFR的促进内皮细胞增殖、迁移和形成血管及[Ca^(2+)]i荧光强度;在与海马神经元共培养的大鼠脑血管内皮细胞上,TFR显著地升高共培养中H_(2)S含量,并被CBS抑制剂AOAA(200μmol·L^(-1))抑制。与此同时,TFR明显地促进共培养中大鼠脑血管内皮细胞的形成血管作用,并可被AOAA和VEGFR_(2)阻断剂SU5416显著地抑制。结论TFR在体外可通过VEGFR_(2)升高[Ca^(2+)]i来促进脑血管内皮细胞形成血管,并可通过诱导神经元中CBS生成H_(2)S作用于大鼠脑血管内皮细胞的VEGFR_(2)来促进血管形成。