AIM:To investigate the retinoprotective role of Apigenin(Api)against high glucose(HG)-induced human retinal microvascular endothelial cells(HRMECs),and to explore its regulatory mechanism.METHODS:HRMECs were stimulate...AIM:To investigate the retinoprotective role of Apigenin(Api)against high glucose(HG)-induced human retinal microvascular endothelial cells(HRMECs),and to explore its regulatory mechanism.METHODS:HRMECs were stimulated by HG for 48h to establish the in vitro cell model.Different concentrations of Api(2.5,5,and 10μmol/L)were applied for treatment.Cell counting kit-8(CCK-8),Transwell,and tube formation assays were performed to examine the effects of Api on the viability,migration,and angiogenesis in HG-induced HRMECs.Vascular permeability was evaluated by Evans blue dye.The inflammatory cytokines and oxidative stress-related factors were measured using their commercial kits.Protein expression of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase 4(NOX4)and p38 mitogen-activated protein kinase(MAPK)was measured by Western blot.RESULTS:Api prevented HG-induced HRMECs viability,migration,angiogenesis,and vascular permeability in a concentration-dependent manner.Meanwhile,Api also concentration-dependently inhibited inflammation and oxidative stress in HRMECs exposed to HG.In addition,HG caused an elevated expression of NOX4,which was retarded by Api treatment.HG stimulation facilitated the activation of p38 MAPK signaling in HRMECs,and Api could weaken this activation partly via downregulating NOX4 expression.Furthermore,overexpression of NOX4 or activation of p38 MAPK signaling greatly weakened the protective role of Api against HG-stimulated HRMECs.CONCLUSION:Api might exert a beneficial role in HGstimulated HRMECs through regulating NOX4/p38 MAPK pathway.展开更多
OBJECTIVE To investigate the regulatory effects of icariin(ICA)on cardiac micro⁃vascular endothelial cells(CMEC)after oxygenglucose deprivation reperfusion(OGD/R)injury.METHODS CMEC were subjected to OGD/R treatment t...OBJECTIVE To investigate the regulatory effects of icariin(ICA)on cardiac micro⁃vascular endothelial cells(CMEC)after oxygenglucose deprivation reperfusion(OGD/R)injury.METHODS CMEC were subjected to OGD/R treatment to construct a myocardial ischemiareperfusion model,and were divided into normal,model,low(10μmol·L^(-1)),medium(20μmol·L^(-1))and high(40μmol·L^(-1))ICA group,and high ICA+inhibitor group(40μmol·L^(-1)+20 nmol·L^(-1)).CCK-8 assay was used to assess the protective ability of ICA against CMEC,and cell migration assay and tube-formation assay were used to detect the migration and generation ability of CMEC.The TCMSP database,Swiss-Target database and literature mining methods were used to col⁃lect ICA-related targets,the GeneCards data⁃base was used to collect target genes related to myocardial ischemia/reperfusion,and Cytoscape 3.8.0 software was used to construct a"drug-tar⁃get-disease"network.The potential targets were imported into STRING 11.5 database to obtain the PPI network.GO and KEGG enrichment analyses were performed on the potential targets using the DAVID database.Molecular docking was performed using AutoDock-vina 1.1.2 soft⁃ware.Western blot detected the expression of related proteins.RESULTS After CMEC was subjected to OGD/R treatment,ICA had a protec⁃tive effect at 10^(-1)60μmol·L^(-1);the results of the cell migration assay showed that each group of ICA could promote the migratory effect of CMEC(P<0.01,P<0.01);and the results of tube-for⁃mation assay showed that each group of ICA could significantly promote the generation of branches(P<0.01)and the capillary length exten⁃sion(P<0.05).Network pharmacology collected a total of 23 ICA action targets,1500 disease tar⁃gets and 12 key targets.GO function enrichment analysis found 85 results.KEGG pathway enrich⁃ment analysis found 53 results,involving AGERAGE signaling pathway,sphingolipid signaling pathway and VEGF signaling pathway.Molecu⁃lar docking results showed that ICA had better binding with core targets PRKCB,PRKCA and PTGS2.Western blot results showed that ICA could regulate the expression of PRKCB,PRKCA and PTGS2 proteins.The results of cell migra⁃tion assay,tube-formation assay and protein expression were reversed after addition of PKC inhibitor.CONCLUSION The potential mecha⁃nism of action of ICA against myocardial isch⁃emia-reperfusion injury may be related to the reg⁃ulation of processes such as CMEC migration and angiogenesis,and it functions through the key target gene PKC.展开更多
AIM:To investigate the expression and effect of histone deacetylase 7(HDAC7)in human retinal microvascular endothelial cells(HRMECs)under high glucose condition and related mechanism,and the expression of HDAC7 in the...AIM:To investigate the expression and effect of histone deacetylase 7(HDAC7)in human retinal microvascular endothelial cells(HRMECs)under high glucose condition and related mechanism,and the expression of HDAC7 in the retinal tissue in diabetic rats.METHODS:The expression of HDAC7 in HRMECs under high glucose and the retinal tissue from normal or diabetic rats were detected with immunohistochemistry and Western blot.LV-shHDAC7 HRMECs were used to study the effect of HDAC7 on cell activities.Cell count kit-8(CCK-8),5-ethynyl2’-deoxyuridine(EdU),flow cytometry,scratch test,Transwell test and tube formation assay were used to examine the ability of cell proliferation,migration,and angiogenesis.Finally,a preliminary exploration of its mechanism was performed by Western blot.RESULTS:The expression of HDAC7 was both upregulated in retinal tissues of diabetic rats and high glucosetreated HRMECs.Down-regulation of HDAC7 expression significantly reduced the ability of proliferation,migration,and tube formation,and reversed the high glucose-induced high expression of CDK1/Cyclin B1 and vascular endothelial growth factor in high glucose-treated HRMECs.CONCLUSION:High glucose can up-regulate the expression of HDAC7 in HRMECs.Down-regulation of HDAC7 can inhibit HRMECs activities.HDAC7 is proposed to be involved in pathogenesis of diabetic retinopathy and a therapeutic target.展开更多
AIM:To provide the direct evidence for the crucial role of trimethylamine N-oxide(TMAO)in vascular permeability and endothelial cell dysfunction under diabetic condition.METHODS:The role of TMAO on the in vitro biolog...AIM:To provide the direct evidence for the crucial role of trimethylamine N-oxide(TMAO)in vascular permeability and endothelial cell dysfunction under diabetic condition.METHODS:The role of TMAO on the in vitro biological effect of human retinal microvascular endothelial cells(HRMEC)under high glucose conditions was tested by a cell counting kit,wound healing,a transwell and a tube formation assay.The inflammation-related gene expression affected by TMAO was tested by real-time polymerase chain reaction(RT-PCR).The expression of the cell junction was measured by Western blotting(WB)and immunofluorescence staining.In addition,two groups of rat models,diabetic and non-diabetic,were fed with normal or 0.1%TMAO for 16wk,and their plasma levels of TMAO,vascular endothelial growth factor(VEGF),interleukin(IL)-6 and tumor necrosis factor(TNF)-αwere tested.The vascular permeability of rat retinas was measured using FITC-Dextran,and the expression of zonula occludens(ZO)-1 and claudin-5 in rat retinas was detected by WB or immunofluorescence staining.RESULTS:TMAO administration significantly increased the cell proliferation,migration,and tube formation of primary HRMEC either in normal or high-glucose conditions.RT-PCR showed elevated inflammation-related gene expression of HRMEC under TMAO stimulation,while WB or immunofluorescence staining indicated decreased cell junction ZO-1 and occludin expression after high-glucose and TMAO treatment.Diabetic rats showed higher plasma levels of TMAO as well as retinal vascular leakage,which were even higher in TMAO-feeding diabetic rats.Furthermore,TMAO administration increased the rat plasma levels of VEGF,IL-6 and TNF-αwhile decreasing the retinal expression levels of ZO-1 and claudin-5.CONCLUSION:TMAO enhances the proliferation,migration,and tube formation of HRMEC,as well as destroys their vascular integrity and tight connection.It also regulates the expression of VEGF,IL-6,and TNF-α.展开更多
BACKGROUND Previous reports have suggested that the p38 mitogen-activated protein kinase signaling pathway is involved in the development of severe acute pancreatitis(SAP)-related acute lung injury(ALI).Inhibition of ...BACKGROUND Previous reports have suggested that the p38 mitogen-activated protein kinase signaling pathway is involved in the development of severe acute pancreatitis(SAP)-related acute lung injury(ALI).Inhibition of p38 by SB203580 blocked the inflammatory responses in SAP-ALI.However,the precise mechanism associated with p38 is unclear,particularly in pulmonary microvascular endothelial cell(PMVEC)injury.AIM To determine its role in the tumor necrosis factor-alpha(TNF-α)-induced inflammation and apoptosis of PMVECs in vitro.We then conducted in vivo experiments to confirm the effect of SB203580-mediated p38 inhibition on SAP-ALI.METHODS In vitro,PMVEC were transfected with mitogen-activated protein kinase kinase 6(Glu),which constitutively activates p38,and then stimulated with TNF-α.Flow cytometry and western blotting were performed to detect the cell apoptosis and inflammatory cytokine levels,respectively.In vivo,SAP-ALI was induced by 5%sodium taurocholate and three different doses of SB203580(2.5,5.0 or 10.0 mg/kg)were intraperitoneally injected prior to SAP induction.SAP-ALI was assessed by performing pulmonary histopathology assays,measuring myeloperoxidase activity,conducting arterial blood gas analyses and measuring TNF-α,interleukin(IL)-1βand IL-6 levels.Lung microvascular permeability was measured by determining bronchoalveolar lavage fluid protein concentration,Evans blue pulmonary cells was confirmed by performing a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis and examining the Bcl2,Bax,Bim and cle-caspase3 levels.The proteins levels of P-p38,NFκB,IκB,P-signal transducer and activator of transcription-3,nuclear factor erythroid 2-related factor 2,HO-1 and Myd88 were detected in the lungs to further evaluate the potential mechanism underlying the protective effect of SB203580.RESULTS In vitro,mitogen-activated protein kinase(Glu)transfection resulted in higher apoptotic rates and cytokine(IL-1βand IL-6)levels in TNF-α-treated PMVECs.In vivo,SB2035080 attenuated lung histopathological injury,decreased inflammatory activity(TNF-α,IL-1β,IL-6 and myeloperoxidase)and preserved pulmonary function.Furthermore,SB203580 significantly reversed changes in the bronchoalveolar lavage fluid protein concentration,Evans blue accumulation,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cell numbers,apoptosis-related proteins(cle-caspase3,Bim and Bax)and endothelial microstructure.Moreover,SB203580 significantly reduced the pulmonary P-p38,NFκB,P-signal transducer and activator of transcription-3 and Myd88 levels but increased the IκB and HO-1 levels.CONCLUSION p38 inhibition may protect against SAP-ALI by alleviating inflammation and the apoptotic death of PMVECs.展开更多
Objective:To investigate the protective effect of glucagon-like peptid-1(GLP-l) against cardiac microvascular endothelial cell(GTFCs) injured by high glucose.Methods:CMECs were isolated and cultured.Superoxide assay k...Objective:To investigate the protective effect of glucagon-like peptid-1(GLP-l) against cardiac microvascular endothelial cell(GTFCs) injured by high glucose.Methods:CMECs were isolated and cultured.Superoxide assay kit and dihydroethidine(DHE) staining were used to assess oxidative stress.TENEL staining and caspase 3 expression were used to assess the apoptosis of CMECs.H89 was used to inhibit eAMP/PKA pathway:fasudil was used to inhibit Rho/ROCK pathway.The protein expressions of Rho.ROCK uere examined by Western blol analysis.lesults:High glucose increased the production of ROS.the activity of NADPH.the apoptosis rate and the expression level of Rho/ROCK in CMECs.while GLP- 1 decreased high glucose-induced ROS production.the NADPH activity and the apoptosis rate and the expression level of Rho/ROCK in CMECs,the difference were statistically significant(P<0.05).Conclusions:GLP-1 could protect the cardiac microvessels against oxidative stress and apoptosis.The protective effects of GLP-1 are dependent on downstream inhibition of Rho through a cAMP/PKA-dependent manner,resulting in a subsequent decrease in the expression of NADPH oxidase.展开更多
AIM: To explore the state of autophagy and related mechanisms in the murine retinal microvascular endothelial cells(RMECs) under hypoxia stimulation.METHODS: The murine RMECs were primarily cultured and randomly divid...AIM: To explore the state of autophagy and related mechanisms in the murine retinal microvascular endothelial cells(RMECs) under hypoxia stimulation.METHODS: The murine RMECs were primarily cultured and randomly divided into three groups: hypoxia group(cultured in 1% O_2 environment), hypoxia+autophagy inhibition group [pretreated with 5 mmol/L 3-methyladenine(3-MA) for 4 h followed by incubation in 1% O_2] and control group(cultured under normoxic condition). The state of autophagy in RMECs was examined by assaying the turnover of light chain 3 B(LC3BB) and expression of Beclin-1, Atg3 and Atg5 proteins with Western blotting, by detecting formation of autophagosomes with transmission electron microscopy(TEM) and by counting the number of GFP+ puncta in RMECs. The protein levels of AMPK, P-AMPK, Akt, P-Akt, m-TOR and P-m TOR were also assayed by Western blotting.RESULTS: Primary murine RMECs were successfully cultured. Under hypoxic conditions, the ratio of LC3BB-Ⅱ/Ⅰ and the expression of Beclin-1, Atg3 and Atg5 proteins were increased when compared with the control group. In addition, the numbers of autophagosome and the GFP+ puncta were also increased under hypoxia. However, pretreatment with 3-MA obviously attenuated these changes in autophagy in RMECs under hypoxia. Protein expression of P-Akt and P-AMPK was increased but P-m TOR level was decreased in cells exposed to hypoxia. CONCLUSION: In murine RMECs autophagy is activated under hypoxia possibly through activation of the AMPK/mTOR signaling pathway.展开更多
AIM: To investigate biological effects of transthyretin(TTR) on the development of neovascularization under simulated diabetic retinopathy(DR) condition associated with high glucose and hypoxia. METHODS: Human retinal...AIM: To investigate biological effects of transthyretin(TTR) on the development of neovascularization under simulated diabetic retinopathy(DR) condition associated with high glucose and hypoxia. METHODS: Human retinal microvascular endothelial cells(h RECs) were cultured in normal and simulated DR environments with high glucose and hypoxia. The normal serum glucose concentration is approximately 5.5 mmol/L;thus, hyperglycemia was simulated with 25 mmol/L glucose, while hypoxia was induced using 200 μmol/L Co Cl2. The influence of TTR on h RECs and human retinal pigment epithelial cells(h RPECs) was determined by incubating the cells with 4 μmol/L TTR in normal and abnormal media. A co-culture system was then employed to evaluate the effects of h RPECs on h RECs. RESULTS: Decreased h RECs and h RPECs were observed under abnormal conditions, including high-glucose and hypoxic media. In addition, h RECs were significantly inhibited by 4 μmol/L exogenous TTR during hyperglycemic culture. During co-culture, h RPECs inhibited h RECs in both the normal and abnormal environments.CONCLUSION: h REC growth is inhibited by exogenous TTR under simulated DR environments with high-glucose and hypoxic, particularly in the medium containing 25 mmol/L glucose. h RPECs, which manufacture TTR in the eye, also represses h RECs in the same environment. TTR is predicted to inhibit the proliferation of h RECs and neovascularization.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
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
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
AIM:To determine the effects of safranal on choroidal neovascularization(CNV)and oxidative stress damage of human choroidal microvascular endothelial cells(HCVECs)and its possible mechanisms.METHODS:Forty-five rats we...AIM:To determine the effects of safranal on choroidal neovascularization(CNV)and oxidative stress damage of human choroidal microvascular endothelial cells(HCVECs)and its possible mechanisms.METHODS:Forty-five rats were used as a laser-induced CNV model for testing the efficacy and safety of safranal(0.5 mg/kg·d,intraperitoneally)on CNV.CNV leakage on fluorescein angiography(FA)and CNV thickness on histology was compared.HCVECs were used for a H_(2)O_(2)-induced oxidative stress model to test the effect of safranal in vitro.MTT essay was carried to test the inhibition rate of safranal on cell viability at different concentrations.Tube formation was used to test protective effect of safranal on angiogenesis at different concentrations.mRNA transcriptome sequencing was performed to find the possible signal pathway.The expressions of different molecules and their phosphorylation level were validated by Western blotting.RESULTS:On FA,the average CNV leakage area was 0.73±0.49 and 0.31±0.11 mm^(2)(P=0.012)in the control and safranal-treated group respectively.The average CNV thickness was 127.4±18.75 and 100.6±17.34μm(P=0.001)in control and safranal-treated group.Under the condition of oxidative stress,cell proliferation was inhibited by safranal and inhibition rates were 7.4%-35.4%at the different concentrations.For tube formation study,the number of new branches was 364 in control group and 35,42,and 17 in 20,40,and 80μg/mL safranal groups respectively(P<0.01).From the KEGG pathway bubble graph,the PI3K-AKT signaling pathway showed a high gene ratio.The protein expression was elevated of insulin receptor substrate(IRS)and the phosphorylation level of PI3K,phosphoinositide-dependent protein kinase 1/2(PDK1/2),AKT and Bcl-2 associated death promoter(BAD)was also elevated under oxidative stress condition but inhibited by safranal.CONCLUSION:Safranal can inhibit CNV both in vivo and in vitro,and the IRS-PI3K-PDK1/2-AKT-BAD signaling pathway is involved in the pathogenesis of CNV.展开更多
AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the ...AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism.METHODS: Primary RMVECs were isolated from the retinas of C57/BL6J mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot.RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-15-LOX-1 or Ad-vector via Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P<0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P<0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P<0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P<0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P>0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order.CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR2 could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV.展开更多
Various stem cells, including neural stem cells (NSCs), have been extensively studied in stroke models, but how to increase neuronal differentiation rate of NSCs remains unresolved, particu- larly in a damaged environ...Various stem cells, including neural stem cells (NSCs), have been extensively studied in stroke models, but how to increase neuronal differentiation rate of NSCs remains unresolved, particu- larly in a damaged environment. The purpose of this study was to investigate the effects of cerebral mi- crovascular endothelial cells (CMECs) on the neurogenesis of NSCs with or without oxygen-glucose deprivation (OGD). The NSCs acquired from primary culture were immunostained to prove cell purity. Survival and proliferation of NSCs were determined after the co-culture with CMECs for 7 days. After removing the CMECs, NSCs were randomly divided into two groups as follows:OGD and non-OGD groups. Both groups were maintained in differentiation culture for 4 days to evaluate the differentiation rate. Mouse embryo fibroblast (MEF) cells co-cultured with NSCs served as control group. NSCs co-cultured with CMECs had an increase in size (on the 7th day:89.80±26.12μm vs. 73.08±15.01 μm, P<0.001) (n=12) and number [on the 7th day:6.33±5.61/high power objective (HP) vs. 2.23±1.61/HP, P<0.001] (n=12) as compared with those co-cultured with MEF cells. After further differentiation culture for 4 days, NSCs co-cultured with CMECs had an increase in neuronal differentiation rate in OGD and non-OGD groups, but not in the control group (15.16% and 16.07% vs. 8.81%; both P<0.001) (n=6). This study provided evidence that OGD could not alter the effects of CMECs in promoting the neuronal differentiation potential of NSCs. These findings may have important implications for the development of new cell therapies for cerebral vascular diseases.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
We studied the effect of baicalin,an extract from Radix Scutellariae(a traditional Chinese medicine) in inducing mouse pulmonary microvascular endothelial cells(MPMVECs) to produce interferons(IFNs).MPMVECs were cultu...We studied the effect of baicalin,an extract from Radix Scutellariae(a traditional Chinese medicine) in inducing mouse pulmonary microvascular endothelial cells(MPMVECs) to produce interferons(IFNs).MPMVECs were cultured in vitro in the presence of different concentrations of baicalin(10,20,and 30 μg mL-1),and the cells and the culture media were harvested at various time intervals.The proteins and mRNA levels(relative to β-actin) of IFN-α/β and IFN-γ were analyzed by RT-PCR and enzyme-linked immunosorbent assay(ELISA).It was observed that baicalin substantially up-regulated the expression of IFN-α/β and IFN-γ.In all baicalin-treated groups,the relative levels of IFN-α/β and IFN-γ mRNAs peaked after 12 h of culturing,and IFN-α/β and IFN-γ proteins peaked after 24 h of culturing.These results suggest that baicalin can effectively induce the expression of IFNs in pulmonary microvascular endothelial cells,and thus potentially act as an antiviral compound.This study may provide background information for developing new antiviral drugs based on baicalin.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. T...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
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 en...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 be 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
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
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
文摘AIM:To investigate the retinoprotective role of Apigenin(Api)against high glucose(HG)-induced human retinal microvascular endothelial cells(HRMECs),and to explore its regulatory mechanism.METHODS:HRMECs were stimulated by HG for 48h to establish the in vitro cell model.Different concentrations of Api(2.5,5,and 10μmol/L)were applied for treatment.Cell counting kit-8(CCK-8),Transwell,and tube formation assays were performed to examine the effects of Api on the viability,migration,and angiogenesis in HG-induced HRMECs.Vascular permeability was evaluated by Evans blue dye.The inflammatory cytokines and oxidative stress-related factors were measured using their commercial kits.Protein expression of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase 4(NOX4)and p38 mitogen-activated protein kinase(MAPK)was measured by Western blot.RESULTS:Api prevented HG-induced HRMECs viability,migration,angiogenesis,and vascular permeability in a concentration-dependent manner.Meanwhile,Api also concentration-dependently inhibited inflammation and oxidative stress in HRMECs exposed to HG.In addition,HG caused an elevated expression of NOX4,which was retarded by Api treatment.HG stimulation facilitated the activation of p38 MAPK signaling in HRMECs,and Api could weaken this activation partly via downregulating NOX4 expression.Furthermore,overexpression of NOX4 or activation of p38 MAPK signaling greatly weakened the protective role of Api against HG-stimulated HRMECs.CONCLUSION:Api might exert a beneficial role in HGstimulated HRMECs through regulating NOX4/p38 MAPK pathway.
基金National Natural Science Foundation of China(82030124)National Natural Science Foundation of China(82174015)Science and Technology Innovation Project of China Academy of Traditional Chinese Medicine(CI2021A04609)。
文摘OBJECTIVE To investigate the regulatory effects of icariin(ICA)on cardiac micro⁃vascular endothelial cells(CMEC)after oxygenglucose deprivation reperfusion(OGD/R)injury.METHODS CMEC were subjected to OGD/R treatment to construct a myocardial ischemiareperfusion model,and were divided into normal,model,low(10μmol·L^(-1)),medium(20μmol·L^(-1))and high(40μmol·L^(-1))ICA group,and high ICA+inhibitor group(40μmol·L^(-1)+20 nmol·L^(-1)).CCK-8 assay was used to assess the protective ability of ICA against CMEC,and cell migration assay and tube-formation assay were used to detect the migration and generation ability of CMEC.The TCMSP database,Swiss-Target database and literature mining methods were used to col⁃lect ICA-related targets,the GeneCards data⁃base was used to collect target genes related to myocardial ischemia/reperfusion,and Cytoscape 3.8.0 software was used to construct a"drug-tar⁃get-disease"network.The potential targets were imported into STRING 11.5 database to obtain the PPI network.GO and KEGG enrichment analyses were performed on the potential targets using the DAVID database.Molecular docking was performed using AutoDock-vina 1.1.2 soft⁃ware.Western blot detected the expression of related proteins.RESULTS After CMEC was subjected to OGD/R treatment,ICA had a protec⁃tive effect at 10^(-1)60μmol·L^(-1);the results of the cell migration assay showed that each group of ICA could promote the migratory effect of CMEC(P<0.01,P<0.01);and the results of tube-for⁃mation assay showed that each group of ICA could significantly promote the generation of branches(P<0.01)and the capillary length exten⁃sion(P<0.05).Network pharmacology collected a total of 23 ICA action targets,1500 disease tar⁃gets and 12 key targets.GO function enrichment analysis found 85 results.KEGG pathway enrich⁃ment analysis found 53 results,involving AGERAGE signaling pathway,sphingolipid signaling pathway and VEGF signaling pathway.Molecu⁃lar docking results showed that ICA had better binding with core targets PRKCB,PRKCA and PTGS2.Western blot results showed that ICA could regulate the expression of PRKCB,PRKCA and PTGS2 proteins.The results of cell migra⁃tion assay,tube-formation assay and protein expression were reversed after addition of PKC inhibitor.CONCLUSION The potential mecha⁃nism of action of ICA against myocardial isch⁃emia-reperfusion injury may be related to the reg⁃ulation of processes such as CMEC migration and angiogenesis,and it functions through the key target gene PKC.
基金Supported by the Shaanxi Province Traditional Chinese Medicine Project(No.SZY-KJCYC-2023-028)。
文摘AIM:To investigate the expression and effect of histone deacetylase 7(HDAC7)in human retinal microvascular endothelial cells(HRMECs)under high glucose condition and related mechanism,and the expression of HDAC7 in the retinal tissue in diabetic rats.METHODS:The expression of HDAC7 in HRMECs under high glucose and the retinal tissue from normal or diabetic rats were detected with immunohistochemistry and Western blot.LV-shHDAC7 HRMECs were used to study the effect of HDAC7 on cell activities.Cell count kit-8(CCK-8),5-ethynyl2’-deoxyuridine(EdU),flow cytometry,scratch test,Transwell test and tube formation assay were used to examine the ability of cell proliferation,migration,and angiogenesis.Finally,a preliminary exploration of its mechanism was performed by Western blot.RESULTS:The expression of HDAC7 was both upregulated in retinal tissues of diabetic rats and high glucosetreated HRMECs.Down-regulation of HDAC7 expression significantly reduced the ability of proliferation,migration,and tube formation,and reversed the high glucose-induced high expression of CDK1/Cyclin B1 and vascular endothelial growth factor in high glucose-treated HRMECs.CONCLUSION:High glucose can up-regulate the expression of HDAC7 in HRMECs.Down-regulation of HDAC7 can inhibit HRMECs activities.HDAC7 is proposed to be involved in pathogenesis of diabetic retinopathy and a therapeutic target.
基金Supported by the National Natural Science Foundation in China(No.81671641)Jiangsu Provincial Medical Innovation Team(No.CXTDA2017039)Gusu Health Talents Program(No.GSWS 2022018).
文摘AIM:To provide the direct evidence for the crucial role of trimethylamine N-oxide(TMAO)in vascular permeability and endothelial cell dysfunction under diabetic condition.METHODS:The role of TMAO on the in vitro biological effect of human retinal microvascular endothelial cells(HRMEC)under high glucose conditions was tested by a cell counting kit,wound healing,a transwell and a tube formation assay.The inflammation-related gene expression affected by TMAO was tested by real-time polymerase chain reaction(RT-PCR).The expression of the cell junction was measured by Western blotting(WB)and immunofluorescence staining.In addition,two groups of rat models,diabetic and non-diabetic,were fed with normal or 0.1%TMAO for 16wk,and their plasma levels of TMAO,vascular endothelial growth factor(VEGF),interleukin(IL)-6 and tumor necrosis factor(TNF)-αwere tested.The vascular permeability of rat retinas was measured using FITC-Dextran,and the expression of zonula occludens(ZO)-1 and claudin-5 in rat retinas was detected by WB or immunofluorescence staining.RESULTS:TMAO administration significantly increased the cell proliferation,migration,and tube formation of primary HRMEC either in normal or high-glucose conditions.RT-PCR showed elevated inflammation-related gene expression of HRMEC under TMAO stimulation,while WB or immunofluorescence staining indicated decreased cell junction ZO-1 and occludin expression after high-glucose and TMAO treatment.Diabetic rats showed higher plasma levels of TMAO as well as retinal vascular leakage,which were even higher in TMAO-feeding diabetic rats.Furthermore,TMAO administration increased the rat plasma levels of VEGF,IL-6 and TNF-αwhile decreasing the retinal expression levels of ZO-1 and claudin-5.CONCLUSION:TMAO enhances the proliferation,migration,and tube formation of HRMEC,as well as destroys their vascular integrity and tight connection.It also regulates the expression of VEGF,IL-6,and TNF-α.
基金National Natural Science Foundation of China,No.81873107,No.82004154 and No.81573766Science and Technology Planning Program of Sichuan,No.2019YFS0259.
文摘BACKGROUND Previous reports have suggested that the p38 mitogen-activated protein kinase signaling pathway is involved in the development of severe acute pancreatitis(SAP)-related acute lung injury(ALI).Inhibition of p38 by SB203580 blocked the inflammatory responses in SAP-ALI.However,the precise mechanism associated with p38 is unclear,particularly in pulmonary microvascular endothelial cell(PMVEC)injury.AIM To determine its role in the tumor necrosis factor-alpha(TNF-α)-induced inflammation and apoptosis of PMVECs in vitro.We then conducted in vivo experiments to confirm the effect of SB203580-mediated p38 inhibition on SAP-ALI.METHODS In vitro,PMVEC were transfected with mitogen-activated protein kinase kinase 6(Glu),which constitutively activates p38,and then stimulated with TNF-α.Flow cytometry and western blotting were performed to detect the cell apoptosis and inflammatory cytokine levels,respectively.In vivo,SAP-ALI was induced by 5%sodium taurocholate and three different doses of SB203580(2.5,5.0 or 10.0 mg/kg)were intraperitoneally injected prior to SAP induction.SAP-ALI was assessed by performing pulmonary histopathology assays,measuring myeloperoxidase activity,conducting arterial blood gas analyses and measuring TNF-α,interleukin(IL)-1βand IL-6 levels.Lung microvascular permeability was measured by determining bronchoalveolar lavage fluid protein concentration,Evans blue pulmonary cells was confirmed by performing a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis and examining the Bcl2,Bax,Bim and cle-caspase3 levels.The proteins levels of P-p38,NFκB,IκB,P-signal transducer and activator of transcription-3,nuclear factor erythroid 2-related factor 2,HO-1 and Myd88 were detected in the lungs to further evaluate the potential mechanism underlying the protective effect of SB203580.RESULTS In vitro,mitogen-activated protein kinase(Glu)transfection resulted in higher apoptotic rates and cytokine(IL-1βand IL-6)levels in TNF-α-treated PMVECs.In vivo,SB2035080 attenuated lung histopathological injury,decreased inflammatory activity(TNF-α,IL-1β,IL-6 and myeloperoxidase)and preserved pulmonary function.Furthermore,SB203580 significantly reversed changes in the bronchoalveolar lavage fluid protein concentration,Evans blue accumulation,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cell numbers,apoptosis-related proteins(cle-caspase3,Bim and Bax)and endothelial microstructure.Moreover,SB203580 significantly reduced the pulmonary P-p38,NFκB,P-signal transducer and activator of transcription-3 and Myd88 levels but increased the IκB and HO-1 levels.CONCLUSION p38 inhibition may protect against SAP-ALI by alleviating inflammation and the apoptotic death of PMVECs.
基金supported by Shanghai Municipal Health Bureau Youth Subject(NO.20134y116)
文摘Objective:To investigate the protective effect of glucagon-like peptid-1(GLP-l) against cardiac microvascular endothelial cell(GTFCs) injured by high glucose.Methods:CMECs were isolated and cultured.Superoxide assay kit and dihydroethidine(DHE) staining were used to assess oxidative stress.TENEL staining and caspase 3 expression were used to assess the apoptosis of CMECs.H89 was used to inhibit eAMP/PKA pathway:fasudil was used to inhibit Rho/ROCK pathway.The protein expressions of Rho.ROCK uere examined by Western blol analysis.lesults:High glucose increased the production of ROS.the activity of NADPH.the apoptosis rate and the expression level of Rho/ROCK in CMECs.while GLP- 1 decreased high glucose-induced ROS production.the NADPH activity and the apoptosis rate and the expression level of Rho/ROCK in CMECs,the difference were statistically significant(P<0.05).Conclusions:GLP-1 could protect the cardiac microvessels against oxidative stress and apoptosis.The protective effects of GLP-1 are dependent on downstream inhibition of Rho through a cAMP/PKA-dependent manner,resulting in a subsequent decrease in the expression of NADPH oxidase.
基金Supported by the National Natural Science Foundation of China(No.81500726)
文摘AIM: To explore the state of autophagy and related mechanisms in the murine retinal microvascular endothelial cells(RMECs) under hypoxia stimulation.METHODS: The murine RMECs were primarily cultured and randomly divided into three groups: hypoxia group(cultured in 1% O_2 environment), hypoxia+autophagy inhibition group [pretreated with 5 mmol/L 3-methyladenine(3-MA) for 4 h followed by incubation in 1% O_2] and control group(cultured under normoxic condition). The state of autophagy in RMECs was examined by assaying the turnover of light chain 3 B(LC3BB) and expression of Beclin-1, Atg3 and Atg5 proteins with Western blotting, by detecting formation of autophagosomes with transmission electron microscopy(TEM) and by counting the number of GFP+ puncta in RMECs. The protein levels of AMPK, P-AMPK, Akt, P-Akt, m-TOR and P-m TOR were also assayed by Western blotting.RESULTS: Primary murine RMECs were successfully cultured. Under hypoxic conditions, the ratio of LC3BB-Ⅱ/Ⅰ and the expression of Beclin-1, Atg3 and Atg5 proteins were increased when compared with the control group. In addition, the numbers of autophagosome and the GFP+ puncta were also increased under hypoxia. However, pretreatment with 3-MA obviously attenuated these changes in autophagy in RMECs under hypoxia. Protein expression of P-Akt and P-AMPK was increased but P-m TOR level was decreased in cells exposed to hypoxia. CONCLUSION: In murine RMECs autophagy is activated under hypoxia possibly through activation of the AMPK/mTOR signaling pathway.
基金Supported by the National Natural Science Foundation of China (No. 81400415)
文摘AIM: To investigate biological effects of transthyretin(TTR) on the development of neovascularization under simulated diabetic retinopathy(DR) condition associated with high glucose and hypoxia. METHODS: Human retinal microvascular endothelial cells(h RECs) were cultured in normal and simulated DR environments with high glucose and hypoxia. The normal serum glucose concentration is approximately 5.5 mmol/L;thus, hyperglycemia was simulated with 25 mmol/L glucose, while hypoxia was induced using 200 μmol/L Co Cl2. The influence of TTR on h RECs and human retinal pigment epithelial cells(h RPECs) was determined by incubating the cells with 4 μmol/L TTR in normal and abnormal media. A co-culture system was then employed to evaluate the effects of h RPECs on h RECs. RESULTS: Decreased h RECs and h RPECs were observed under abnormal conditions, including high-glucose and hypoxic media. In addition, h RECs were significantly inhibited by 4 μmol/L exogenous TTR during hyperglycemic culture. During co-culture, h RPECs inhibited h RECs in both the normal and abnormal environments.CONCLUSION: h REC growth is inhibited by exogenous TTR under simulated DR environments with high-glucose and hypoxic, particularly in the medium containing 25 mmol/L glucose. h RPECs, which manufacture TTR in the eye, also represses h RECs in the same environment. TTR is predicted to inhibit the proliferation of h RECs and neovascularization.
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.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.
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.81760027,No.81860763)Youth Innovation Project of Affiliated Hospital of Inner Mongolia University for Nationalities(No.2018QNJJ01)Young and Middle-aged Ophthalmic Research Fund of Bethune-Lumitin(No.BJ-LM202005)。
文摘AIM:To determine the effects of safranal on choroidal neovascularization(CNV)and oxidative stress damage of human choroidal microvascular endothelial cells(HCVECs)and its possible mechanisms.METHODS:Forty-five rats were used as a laser-induced CNV model for testing the efficacy and safety of safranal(0.5 mg/kg·d,intraperitoneally)on CNV.CNV leakage on fluorescein angiography(FA)and CNV thickness on histology was compared.HCVECs were used for a H_(2)O_(2)-induced oxidative stress model to test the effect of safranal in vitro.MTT essay was carried to test the inhibition rate of safranal on cell viability at different concentrations.Tube formation was used to test protective effect of safranal on angiogenesis at different concentrations.mRNA transcriptome sequencing was performed to find the possible signal pathway.The expressions of different molecules and their phosphorylation level were validated by Western blotting.RESULTS:On FA,the average CNV leakage area was 0.73±0.49 and 0.31±0.11 mm^(2)(P=0.012)in the control and safranal-treated group respectively.The average CNV thickness was 127.4±18.75 and 100.6±17.34μm(P=0.001)in control and safranal-treated group.Under the condition of oxidative stress,cell proliferation was inhibited by safranal and inhibition rates were 7.4%-35.4%at the different concentrations.For tube formation study,the number of new branches was 364 in control group and 35,42,and 17 in 20,40,and 80μg/mL safranal groups respectively(P<0.01).From the KEGG pathway bubble graph,the PI3K-AKT signaling pathway showed a high gene ratio.The protein expression was elevated of insulin receptor substrate(IRS)and the phosphorylation level of PI3K,phosphoinositide-dependent protein kinase 1/2(PDK1/2),AKT and Bcl-2 associated death promoter(BAD)was also elevated under oxidative stress condition but inhibited by safranal.CONCLUSION:Safranal can inhibit CNV both in vivo and in vitro,and the IRS-PI3K-PDK1/2-AKT-BAD signaling pathway is involved in the pathogenesis of CNV.
基金National Natural Science Foundation of China (No. 81000393)
文摘AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism.METHODS: Primary RMVECs were isolated from the retinas of C57/BL6J mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot.RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-15-LOX-1 or Ad-vector via Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P<0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P<0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P<0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P<0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P>0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order.CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR2 could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV.
文摘Various stem cells, including neural stem cells (NSCs), have been extensively studied in stroke models, but how to increase neuronal differentiation rate of NSCs remains unresolved, particu- larly in a damaged environment. The purpose of this study was to investigate the effects of cerebral mi- crovascular endothelial cells (CMECs) on the neurogenesis of NSCs with or without oxygen-glucose deprivation (OGD). The NSCs acquired from primary culture were immunostained to prove cell purity. Survival and proliferation of NSCs were determined after the co-culture with CMECs for 7 days. After removing the CMECs, NSCs were randomly divided into two groups as follows:OGD and non-OGD groups. Both groups were maintained in differentiation culture for 4 days to evaluate the differentiation rate. Mouse embryo fibroblast (MEF) cells co-cultured with NSCs served as control group. NSCs co-cultured with CMECs had an increase in size (on the 7th day:89.80±26.12μm vs. 73.08±15.01 μm, P<0.001) (n=12) and number [on the 7th day:6.33±5.61/high power objective (HP) vs. 2.23±1.61/HP, P<0.001] (n=12) as compared with those co-cultured with MEF cells. After further differentiation culture for 4 days, NSCs co-cultured with CMECs had an increase in neuronal differentiation rate in OGD and non-OGD groups, but not in the control group (15.16% and 16.07% vs. 8.81%; both P<0.001) (n=6). This study provided evidence that OGD could not alter the effects of CMECs in promoting the neuronal differentiation potential of NSCs. These findings may have important implications for the development of new cell therapies for cerebral vascular diseases.
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 National Natural Science Foundation of China(30500368)the Beijing Natural Science Foundation,China(021001)+2 种基金the Funding Project for Academic Human Resources Development in Institutions of Higher Learning Under the Jurisdiction of Beijing Municipality,China(PHR201008422)the Scientific Research Program of Beijing Municipal Commission of Education,China(KM201110020009)the Beijing New Star Technology A Category Fund,China(2006A26)
文摘We studied the effect of baicalin,an extract from Radix Scutellariae(a traditional Chinese medicine) in inducing mouse pulmonary microvascular endothelial cells(MPMVECs) to produce interferons(IFNs).MPMVECs were cultured in vitro in the presence of different concentrations of baicalin(10,20,and 30 μg mL-1),and the cells and the culture media were harvested at various time intervals.The proteins and mRNA levels(relative to β-actin) of IFN-α/β and IFN-γ were analyzed by RT-PCR and enzyme-linked immunosorbent assay(ELISA).It was observed that baicalin substantially up-regulated the expression of IFN-α/β and IFN-γ.In all baicalin-treated groups,the relative levels of IFN-α/β and IFN-γ mRNAs peaked after 12 h of culturing,and IFN-α/β and IFN-γ proteins peaked after 24 h of culturing.These results suggest that baicalin can effectively induce the expression of IFNs in pulmonary microvascular endothelial cells,and thus potentially act as an antiviral compound.This study may provide background information for developing new antiviral drugs based on baicalin.
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.
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 be 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.
