Multifaceted roles of lymphatic and blood endothelial cells in the tumor microenvironment of hepatocellular carcinoma:A comprehensive review

The tumor microenvironment is a complex network of cells,extracellular matrix,and signaling molecules that plays a critical role in tumor progression and metastasis.Lymphatic and blood vessels are major routes for solid tumor metastasis and essential parts of tumor drainage conduits.However,recent studies have shown that lymphatic endothelial cells(LECs)and blood endothelial cells(BECs)also play multifaceted roles in the tumor microenvironment beyond their structural functions,particularly in hepatocellular carcinoma(HCC).This comprehensive review summarizes the diverse roles played by LECs and BECs in HCC,including their involvement in angiogenesis,immune modulation,lymphangiogenesis,and metastasis.By providing a detailed account of the complex interplay between LECs,BECs,and tumor cells,this review aims to shed light on future research directions regarding the immune regulatory function of LECs and potential therapeutic targets for HCC.

Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway

Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.

Gamma-glutamyl transferase 5 overexpression in cerebrovascular endothelial cells improves brain pathology,cognition,and behavior in APP/PS1 mice

In patients with Alzheimer’s disease,gamma-glutamyl transferase 5(GGT5)expression has been observed to be downregulated in cerebrovascular endothelial cells.However,the functional role of GGT5 in the development of Alzheimer’s disease remains unclear.This study aimed to explore the effect of GGT5 on cognitive function and brain pathology in an APP/PS1 mouse model of Alzheimer’s disease,as well as the underlying mechanism.We observed a significant reduction in GGT5 expression in two in vitro models of Alzheimer’s disease(Aβ_(1-42)-treated hCMEC/D3 and bEnd.3 cells),as well as in the APP/PS1 mouse model.Additionally,injection of APP/PS1 mice with an adeno-associated virus encoding GGT5 enhanced hippocampal synaptic plasticity and mitigated cognitive deficits.Interestingly,increasing GGT5 expression in cerebrovascular endothelial cells reduced levels of both soluble and insoluble amyloid-βin the brains of APP/PS1 mice.This effect may be attributable to inhibition of the expression ofβ-site APP cleaving enzyme 1,which is mediated by nuclear factor-kappa B.Our findings demonstrate that GGT5 expression in cerebrovascular endothelial cells is inversely associated with Alzheimer’s disease pathogenesis,and that GGT5 upregulation mitigates cognitive deficits in APP/PS1 mice.These findings suggest that GGT5 expression in cerebrovascular endothelial cells is a potential therapeutic target and biomarker for Alzheimer’s disease.

Inhibition of viability of human retinal microvascular endothelial cells by vialinin A under high glucose condition

AIM:To investigate the effects of vialinin A on viability of human retinal endothelial cells(HRECs)under high glucose condition and its potential mechanism.METHODS:The HRECs were divided into four groups:normal glucose control group(NG,5 mmol/L D-glucose),high glucose group(HG,30 mmol/L D-glucose),HG+1μmol/L vialinin A group,and HG+5μmol/L vialinin A group.The cell viabilities were measured with cell counting kit-8(CCK-8)assay for proliferation,with scratch assay for migration,and tube formation,for evaluation of the impact of vialinin A on cellular behaviour.Real-time PCR and Western blotting were used to determine the expression level of vascular endothelial growth factor(VEGF).RESULTS:The proliferative capacity and migration of HRECs was reduced by 5μmol/L vialinin A in high glucose environment(both P<0.05).Vialinin A also inhibited highglucose-induced tube formation of HRECs.The expression level of VEGF and PI3K in HRECs was also significantly decreased by vialinin A(P<0.05).CONCLUSION:Vialinin A inhibits the cell viability of HRECs.It may serve as a potential target for anti-angiogenic therapy.

Neural stem cell-derived exosomes regulate cell proliferation,migration,and cell death of brain microvascular endothelial cells via the miR-9/Hes1 axis under hypoxia

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.

Small extracellular vesicles derived from cerebral endothelial cells with elevated microRNA 27a promote ischemic stroke recovery

Axonal remodeling is a critical aspect of ischemic brain repair processes and contributes to spontaneous functional recovery.Our previous in vitro study demonstrated that exosomes/small extracellular vesicles(sEVs)isolated from cerebral endothelial cells(CEC-sEVs)of ischemic brain promote axonal growth of embryonic cortical neurons and that microRNA 27a(miR-27a)is an elevated miRNA in ischemic CEC-sEVs.In the present study,we investigated whether normal CEC-sEVs engineered to enrich their levels of miR-27a(27a-sEVs)further enhance axonal growth and improve neurological outcomes after ischemic stroke when compared with treatment with non-engineered CEC-sEVs.27a-sEVs were isolated from the conditioned medium of healthy mouse CECs transfected with a lentiviral miR-27a expression vector.Small EVs isolated from CECs transfected with a scramble vector(Scra-sEVs)were used as a control.Adult male mice were subjected to permanent middle cerebral artery occlusion and then were randomly treated with 27a-sEVs or Scra-sEVs.An array of behavior assays was used to measure neurological function.Compared with treatment of ischemic stroke with Scra-sEVs,treatment with 27a-sEVs significantly augmented axons and spines in the peri-infarct zone and in the corticospinal tract of the spinal grey matter of the denervated side,and significantly improved neurological outcomes.In vitro studies demonstrated that CEC-sEVs carrying reduced miR-27a abolished 27a-sEV-augmented axonal growth.Ultrastructural analysis revealed that 27a-sEVs systemically administered preferentially localized to the pre-synaptic active zone,while quantitative reverse transcription-polymerase chain reaction and Western Blot analysis showed elevated miR-27a,and reduced axonal inhibitory proteins Semaphorin 6A and Ras Homolog Family Member A in the peri-infarct zone.Blockage of the Clathrin-dependent endocytosis pathway substantially reduced neuronal internalization of 27a-sEVs.Our data provide evidence that 27a-sEVs have a therapeutic effect on stroke recovery by promoting axonal remodeling and improving neurological outcomes.Our findings also suggest that suppression of axonal inhibitory proteins such as Semaphorin 6A may contribute to the beneficial effect of 27a-sEVs on axonal remodeling.

Crosstalk among Oxidative Stress,Autophagy,and Apoptosis in the Protective Effects of Ginsenoside Rb1 on Brain Microvascular Endothelial Cells:A Mixed Computational and Experimental Study

Objective Brain microvascular endothelial cells (BMECs) were found to shift from their usually inactive state to an active state in ischemic stroke (IS) and cause neuronal damage. Ginsenoside Rb1 (GRb1),a component derived from medicinal plants,is known for its pharmacological benefits in IS,but its protective effects on BMECs have yet to be explored. This study aimed to investigate the potential protective effects of GRb1 on BMECs. Methods An in vitro oxygen-glucose deprivation/reperfusion (OGD/R) model was established to mimic ischemia-reperfusion (I/R) injury. Bulk RNA-sequencing data were analyzed by using the Human Autophagy Database and various bioinformatic tools,including gene set enrichment analysis (GSEA),Gene Ontology (GO) classification and enrichment analysis,Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis,protein-protein interaction network analysis,and molecular docking. Experimental validation was also performed to ensure the reliability of our findings. Results Rb1 had a protective effect on BMECs subjected to OGD/R injury. Specifically,GRb1 was found to modulate the interplay between oxidative stress,apoptosis,and autophagy in BMECs. Key targets such as sequestosome 1 (SQSTM1/p62),autophagy related 5 (ATG5),and hypoxia-inducible factor 1-alpha (HIF-1α) were identified,highlighting their potential roles in mediating the protective effects of GRb1 against IS-induced damage. Conclusion GRbl protects BMECs against OGD/R injury by influencing oxidative stress,apoptosis,and autophagy. The identification of SQSTM1/p62,ATG5,and HIF-1α as promising targets further supports the potential of GRb1 as a therapeutic agent for IS,providing a foundation for future research into its mechanisms and applications in IS treatment.

Anti-vascular endothelial growth factor drugs combined with laser photocoagulation maintain retinal ganglion cell integrity in patients with diabetic macular edema: study protocol for a prospective, non-randomized, controlled clinical trial

The integrity of retinal ganglion cells is tightly associated with diabetic macular degeneration that leads to damage and death of retinal ganglion cells,affecting vision.The major clinical treatments for diabetic macular edema are anti-vascular endothelial growth factor drugs and laser photocoagulation.However,although the macular thickness can be normalized with each of these two therapies used alone,the vision does not improve in many patients.This might result from the incomplete recovery of retinal ganglion cell injury.Therefore,a prospective,non-randomized,controlled clinical trial was designed to investigate the effect of anti-vascular endothelial growth factor drugs combined with laser photocoagulation on the integrity of retinal ganglion cells in patients with diabetic macular edema and its relationship with vision recovery.In this trial,150 patients with diabetic macular edema will be equally divided into three groups according to therapeutic methods,followed by treatment with anti-vascular endothelial growth factor drugs,laser photocoagulation therapy,and their combination.All patients will be followed up for 12 months.The primary outcome measure is retinal ganglion cell-inner plexiform layer thickness at 12 months after treatment.The secondary outcome measures include retinal ganglion cell-inner plexiform layer thickness before and 1,3,6,and 9 months after treatment,retinal nerve fiber layer thickness,best-corrected visual acuity,macular area thickness,and choroidal thickness before and 1,3,6,9,and 12 months after treatment.Safety measure is the incidence of adverse events at 1,3,6,9,and 12 months after treatment.The study protocol hopes to validate the better efficacy and safety of the combined treatment in patients with diabetic macula compared with the other two monotherapies alone during the 12-month follow-up period.The trial is designed to focus on clarifying the time-effect relationship between imaging measures related to the integrity of retinal ganglion cells and best-corrected visual acuity.The trial protocol was approved by the Medical Ethics Committee of the Affiliated Hospital of Beihua University with approval No.(2023)(26)on April 25,2023,and was registered with the Chinese Clinical Trial Registry(registration number:ChiCTR2300072478,June 14,2023,protocol version:2.0).

Analyzing the pharmacological substances and targets of Xuefu Zhuyu decoction in hypertensive vascular endothelial cells

Background:Xuefu Zhuyu decoction(XFZY)could significantly improve the function of hypertensive vascular endothelial cells,but the targets and mechanism are not clear.This study is to analyze the pharmacological substances and targets of Xuefu Zhuyu decoction in hypertensive vascular endothelial cells.Methods:This study used Xuefu Zhuyu decoction to intervene human umbilical vein endothelial cells incubated by hypertensive patients’serum,then detected the function of vascular endothelial cells.The aqueous extract of XFZY was analyzed and validated by liquid chromatography-mass spectrometry technology;Finally,macromolecular docking technology was used to analyze the potential active substances and targets of XFZY in the prevention and treatment of hypertension.Results:Compared with the model group,the XFZY group showed a significant increase in NO expression(P<0.01)and a significant decrease in ET-1 expression(P<0.001);and the expression of BIP,P-JNK,CHOP,and BAX in XFZY group cells was significantly decreased(P<0.001),while the expression of JNK and BCL2 was significantly increased(P<0.001).19 main compounds were identified in XFZY and there were 3 pairs of molecular complexes with high affinity for markers of the endoplasmic reticulum stress,including BIP-Hesperidin complex,BIP-HSYA complex and JNK-Naringin complex.Conclusion:This study analyzed the potential pharmacodynamic substance and targets of Xuefu Zhuyu decoction in improving the function of hypertensive vascular endothelial cells,which could provide a scientific basis for the future molecular mechanism of XFZY in treating hypertension.

β-Estradiol 17-acetate enhances the in vitro vitality of endothelial cells isolated from the brain of patients subjected to neurosurgery

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.

Destructive effects on endothelial cells of grafts in cytomegalovirus DNA-positive patients after keratoplasty

AIM:To investigate corneal graft survival rate and endothelial cell density(ECD)loss after keratoplasty in cytomegalovirus(CMV)positive patients.METHODS:This was a retrospective cohort study.We analyzed the clinical data of patients who underwent viral DNA detection in aqueous humor/corneal tissue collected during keratoplasty from March 2015 to December 2018 at the Peking University Third Hospital,Beijing,China.To further evaluate the effect of CMV on graft survival rate and ECD loss,patients were divided into three groups:1)CMV DNA positive(CMV+)group;2)viral DNA negative(virus-)group,comprising virus-group eyes pairwise matched to eyes in the CMV+group according to ocular comorbidities;3)control group,comprising virus-group eyes without ocular comorbidities.The follow-up indicators including graft survival rate,ECD,ECD loss,and central corneal thickness(CCT),were analyzed by Tukey honestly significant difference(HSD)test.RESULTS:Each group included 29 cases.The graft survival rate in CMV+group were lowest among the three groups(P=0.000).No significant difference in donor graft ECD was found among three groups(P=0.54).ECD in the CMV+group was lower than the virus-group at 12(P=0.009),and 24mo(P=0.002)after keratoplasties.Furthermore,ECD loss was higher in the CMV+group than in the virus-group in the middle stage(6-12mo)postkeratoplasty(P=0.017),and significantly higher in the early stage(0-6mo)in the virus-group than in the control group(P=0.000).CONCLUSION:CMV reduces the graft survival rate and exerts persistent detrimental effects on the ECD after keratoplasty.The graft ECD loss associate with CMV infection mainly occurrs in the middle stage(6-12mo postoperatively),while ocular comorbidities mainly affects ECD in the early stage(0-6mo postoperatively).

Trimethylamine N-oxide aggravates vascular permeability and endothelial cell dysfunction under diabetic condition:in vitro and in vivo study

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.

Human umbilical cord mesenchymal stem cells derivedexosomes on VEGF-A in hypoxic-induced mice retinal astrocytes and mice model of retinopathy of prematurity

AIM:To observe the effect of human umbilical cord mesenchymal stem cells(hUCMSCs)secretions on the relevant factors in mouse retinal astrocytes,and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A(VEGF-A)and to observe the therapeutic effect on the mouse model of retinopathy of prematurity(ROP).METHODS:Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group.MTT assay,flow cytometry,reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were used to detect related indicators.Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored.At last,the efficacy of exosomes of UCMSCs in a mouse ROP model was explored.Graphpad6 was used to comprehensively process data information.RESULTS:The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation.Reactive oxygen species(ROS)and hypoxia inducible factor-1α(HIF-1α)of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased,and the ROP cell model was established after 6h of hypoxia.The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α,the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent.Compared with the ROP cell model group,the expression of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signal pathway related factors in the hUCMSCs exocrine group is significantly decreased.The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1αin ROP model tissues.HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion.CONCLUSION:In a hypoxia induced mouse retinal astrocyte model,hUCMSCs exosomes are found to effectively reduce the expression of HIF-1αand VEGF-A,which are positively correlated with the concentration of hUCMSCs exosomes.HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1αand VEGF-A proteins in ROP mice,and are positively correlated with drug dosage.Besides,they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.

Ferroptosis inhibition protects vascular endothelial cells and maintains integrity of the blood-spinal cord barrier after spinal cord injury

Maintaining the integrity of the blood-spinal cord barrier is critical for the recove ry of spinal cord injury.Ferro ptosis contributes to the pathogenesis of spinal cord injury.We hypothesized that ferroptosis is involved in disruption of the blood-s pinal cord barrier.In this study,we administe red the ferroptosis inhibitor liproxstatin-1 intraperitoneally after contusive spinal co rd injury in rats.Liproxstatin-1 improved locomotor recovery and somatosensory evoked potential electrophysiological performance after spinal cord inju ry.Liproxstatin-1 maintained blood-spinal cord barrier integrity by upregulation of the expression of tight junction protein.Liproxstatin-1 inhibited ferroptosis of endothelial cell after spinal cord injury,as shown by the immunofluorescence of an endothelial cell marker(rat endothelium cell antigen-1,RECA-1) and fe rroptosis markers Acyl-CoA synthetase long-chain family member 4 and 15-lipoxygenase.Liproxstatin-1reduced brain endothelial cell ferroptosis in vitro by upregulating glutathione peroxidase 4 and downregulating Acyl-CoA synthetase long-chain family member4 and 15-lipoxygenase.Furthermore,inflammatory cell recruitment and astrogliosis were mitigated after liproxstatin-1 treatment.In summary,liproxstatin-1im proved spinal cord injury recovery by inhibiting ferroptosis in endothelial cells and maintaining blood-s pinal co rd barrier integrity.

Thinking outside the black box:are the brain endothelial cells the new main target in Alzheimer's disease?

The blood-brain barrier is the interface through which the brain interacts with the milieu and consists mainly of a sophisticated network of brain endothelial cells that forms blood vessels and selectively moves molecules inside and outside the brain through multiple mechanisms of transport.Although brain endothelial cell function is crucial for brain homeostasis,their role in neurodegenerative diseases has historically not been considered with the same importance as other brain cells such as microglia,astroglia,neurons,or even molecules such as amyloid beta,Tau,or alpha-synuclein.Alzheimer's disease is the most common neurodegenerative disease,and brain endothelial cell dysfunction has been reported by several groups.However,its impairment has barely been considered as a potential therapeutic target.Here we review the most recent advances in the relationship between Alzheimer's disease and brain endothelial cells commitment and analyze the possible mechanisms through which their alterations contribute to this neurodegenerative disease,highlighting their inflammatory phenotype and the possibility of an impaired secretory pattern of brain endothelial cells that could contribute to the progression of this ailment.Finally,we discuss why shall brain endothelial cells be appreciated as a therapeutic target instead of solely an obstacle for delivering treatments to the injured brain in Alzheimer's disease.

The role of liver sinusoidal endothelial cells in liver remodeling after injury

Liver transplantation is the optimal treatment for patients with end-stage liver disease,metabolic liver diseases,and hepatic malignancies that are not amenable to resection.Hepatic ischemia-reperfusion injury(IRI)is the main problem in liver transplantation and liver resection,leading to parenchymal cell injury and organ dysfunction.The damage of liver sinusoidal endothelial cells(LSECs)is a critical event in IRI.LSECs work as an important regulating factor of liver regeneration after partial hepatectomy.This review primarily describes the mechanisms of LSECs injury in IRI and explores the roles of LSECs in liver regeneration,and briefly introduces the protective strategies targeting LSECs damaged in IRI.

Role of apigenin in high glucose-induced retinal microvascular endothelial cell dysfunction via regulating NOX4/p38 MAPK pathway in vitro

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.

Overexpression of mitogen-activated protein kinase phosphatase-1 in endothelial cells reduces blood-brain barrier injury in a mouse model of ischemic stroke

Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.

Integrin binding peptides facilitate growth and interconnected vascular-like network formation of rat primary cortical vascular endothelial cells in vitro

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.

Inhibition of VEGF-A expression in hypoxia-exposed fetal retinal microvascular endothelial cells by exosomes derived from human umbilical cord mesenchymal stem cells

Objective:This study aimed to investigate the potential of human umbilical cord mesenchymal stem cell(hucMSC)-derived exosomes(hucMSC-Exos)in inhibiting hypoxia-induced cell hyper proliferation and overexpression of vascular endothelial growth factor A(VEGF-A)in immature human fetal retinal microvascular endothelial cells(hfRMECs).Methods:Exosomes were isolated from hucMSCs using cryogenic ultracentrifugation and characterized through various techniques,including transmission electron microscopy,nanoparticle tracking analysis,bicinchoninic acid assays,and western blotting.The hfRMECs were identified using von Willebrand factor(vWF)co-staining and divided into four groups:a control group cultured under normoxic condition,a hypoxic model group,a hypoxic group treated with low-concentration hucMSC-Exos(75μg/mL)and a hypoxic group treated with high-concentration hucMSC-Exos(100μg/mL).Cell viability and proliferation were assessed using Cell Counting Kit-8(CCK-8)assay and EdU(5-ethynyl-2′-deoxyuridine)assay respectively.Expression levels of VEGF-A were evaluated using RT-PCR,western blotting and immunofluorescence.Results:Hypoxia significantly increased hfRMECs’viability and proliferation by upregulating VEGF-A levels.The administration of hucMSC-Exos effectively reversed this response,with the high-concentration group exhibiting greater efficacy compared to the lowconcentration group.Conclusion:In conclusion,hucMSC-Exos can dose-dependently inhibit hypoxia-induced hyperproliferation and VEGF-A overexpression in immature fetal retinal microvascular endothelial cells.