Down-regulation of histone deacetylase 7 reduces biological activities of retinal microvascular endothelial cells under high glucose condition and related mechanism

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.

Inhibitory effects of safranal on laser-induced choroidal neovascularization and human choroidal microvascular endothelial cells and related pathways analyzed with transcriptome sequencing

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.

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.

Single-cell RNA-seq and bulk-seq identify RAB17 as a potential regulator of angiogenesis by human dermal microvascular endothelial cells in diabetic foot ulcers

Background:Angiogenesis is crucial in diabetic wound healing and is often impaired in diabetic foot ulcers(DFUs).Human dermal microvascular endothelial cells(HDMECs)are vital components in dermal angiogenesis;however,their functional and transcriptomic characteristics in DFU patients are not well understood.This study aimed to comprehensively analyse HDMECs from DFU patients and healthy controls and find the potential regulator of angiogenesis in DFUs.Methods:HDMECs were isolated from skin specimens of DFU patients and healthy controls via magnetic-activated cell sorting.The proliferation,migration and tube-formation abilities of the cells were then compared between the experimental groups.Both bulk RNA sequencing(bulk-seq)and single-cell RNA-seq(scRNA-seq)were used to identify RAB17 as a potential marker of angiogenesis,which was further confirmed via weighted gene co-expression network analysis(WGCNA)and least absolute shrink and selection operator(LASSO)regression.The role of RAB17 in angiogenesis was examined through in vitro and in vivo experiments.Results:The isolated HDMECs displayed typical markers of endothelial cells.HDMECs isolated from DFU patients showed considerably impaired tube formation,rather than proliferation or migration,compared to those from healthy controls.Gene set enrichment analysis(GSEA),fGSEA,and gene set variation analysis(GSVA)of bulk-seq and scRNA-seq indicated that angiogenesis was downregulated in DFU-HDMECs.LASSO regression identified two genes,RAB17 and CD200,as characteristic of DFU-HDMECs;additionally,the expression of RAB17 was found to be significantly reduced in DFU-HDMECs compared to that in the HDMECs of healthy controls.Overexpression of RAB17 was found to enhance angiogenesis,the expression of hypoxia inducible factor-1α and vascular endothelial growth factor A,and diabetic wound healing,partially through the mitogen-activated protein kinase/extracellular signal-regulated kinase signalling pathway.Conclusions:Our findings suggest that the impaired angiogenic capacity in DFUs may be related to the dysregulated expression of RAB17 in HDMECs.The identification of RAB17 as a potential molecular target provides a potential avenue for the treatment of impaired angiogenesis in DFUs.

Quercetin protects human brain microvascular endothelial cells from fibrillarβ-amyloid_(1–40)-induced toxicity

Amyloid beta-peptides(Aβ) are known to undergo active transport across the blood-brain barrier, and cerebral amyloid angiopathy has been shown to be a prominent feature in the majority of Alzheimer's disease. Quercetin is a natural flavonoid molecule and has been demonstrated to have potent neuroprotective effects, but its protective effect on endothelial cells under Aβ-damaged condition is unclear. In the present study, the protective effects of quercetin on brain microvascular endothelial cells injured by fibrillar Aβ_(1–40)(f Aβ_(1–40)) were observed. The results show that f Aβ_(1–40)-induced cytotoxicity in human brain microvascular endothelial cells(h BMECs) can be relieved by quercetin treatment. Quercetin increases cell viability, reduces the release of lactate dehydrogenase, and relieves nuclear condensation.Quercetin also alleviates intracellular reactive oxygen species generation and increases superoxide dismutase activity. Moreover, it strengthens the barrier integrity through the preservation of the transendothelial electrical resistance value, the relief of aggravated permeability, and the increase of characteristic enzyme levels after being exposed to f Aβ_(1–40). In conclusion, quercetin protects h BMECs from f Aβ_(1–40)-induced toxicity.

Genipin relieves diabetic retinopathy by down-regulation of advanced glycation end products via the mitochondrial metabolism related signaling pathway

BACKGROUND Glycation is an important step in aging and oxidative stress,which can lead to endothelial dysfunction and cause severe damage to the eyes or kidneys of diabetics.Inhibition of the formation of advanced glycation end products(AGEs)and their cell toxicity can be a useful therapeutic strategy in the prevention of diabetic retinopathy(DR).Gardenia jasminoides Ellis(GJE)fruit is a selective inhibitor of AGEs.Genipin is an active compound of GJE fruit,which can be employed to treat diabetes.AIM To confirm the effect of genipin,a vital component of GJE fruit,in preventing human retinal microvascular endothelial cells(hRMECs)from AGEs damage in DR,to investigate the effect of genipin in the down-regulation of AGEs expression,and to explore the role of the CHGA/UCP2/glucose transporter 1(GLUT1)signal pathway in this process.METHODS In vitro,cell viability was tested to determine the effects of different doses of glucose and genipin in hRMECs.Cell Counting Kit-8(CCK-8),colony formation assay,flow cytometry,immunofluorescence,wound healing assay,transwell assay,and tube-forming assay were used to detect the effect of genipin on hRMECs cultured in high glucose conditions.In vivo,streptozotocin(STZ)induced mice were used,and genipin was administered by intraocular injection(IOI).To explore the effect and mechanism of genipin in diabetic-induced retinal dysfunction,reactive oxygen species(ROS),mitochondrial membrane potential(MMP),and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose(2-NBDG)assays were performed to explore energy metabolism and oxidative stress damage in high glucose-induced hRMECs and STZ mouse retinas.Immunofluorescence and Western blot were used to investigate the expression of inflammatory cytokines[vascular endothelial growth factor(VEGF),SCG3,tumor necrosis factor-alpha(TNF-α),interleukin(IL)-1β,IL-18,and nucleotide-binding domain,leucine-rich-containing family,pyrin domain-containing 3(NLRP3)].The protein expression of the receptor of AGEs(RAGE)and the mitochondria-related signal molecules CHGA,GLUT1,and UCP2 in high glucose-induced hRMECs and STZ mouse retinas were measured and compared with the genipin-treated group.RESULTS The results of CCK-8 and colony formation assay showed that genipin promoted cell viability in high glucose(30 mmol/L D-Glucose)-induced hRMECs,especially at a 0.4μmol/L dose for 7 d.Flow cytometry results showed that high glucose can increase apoptosis rate by 30%,and genipin alleviated cell apoptosis in AGEs-induced hRMECs.A high glucose environment promoted ATP,ROS,MMP,and 2-NBDG levels,while genipin inhibited these phenotypic abnormalities in AGEs-induced hRMECs.Furthermore,genipin remarkably reduced the levels of the pro-inflammatory cytokines TNF-α,IL-1β,IL-18,and NLRP3 and impeded the expression of VEGF and SCG3 in AGEs-damaged hRMECs.These results showed that genipin can reverse high glucose induced damage with regard to cell proliferation and apoptosis in vitro,while reducing energy metabolism,oxidative stress,and inflammatory injury caused by high glucose.In addition,ROS levels and glucose uptake levels were higher in the retina from the untreated eye than in the genipin-treated eye of STZ mice.The expression of inflammatory cytokines and pathway protein in the untreated eye compared with the genipin-treated eye was significantly increased,as measured by Western blot.These results showed that IOI of genipin reduced the expression of CHGA,UCP2,and GLUT1,maintained the retinal structure,and decreased ROS,glucose uptake,and inflammation levels in vivo.In addition,we found that SCG3 expression might have a higher sensitivity in DR than VEGF as a diagnostic marker at the protein level.CONCLUSION Our study suggested that genipin ameliorates AGEs-induced hRMECs proliferation,apoptosis,energy metabolism,oxidative stress,and inflammatory injury,partially via the CHGA/UCP2/GLUT1 pathway.Control of advanced glycation by IOI of genipin may represent a strategy to prevent severe retinopathy and vision loss.

The relationship among amyloid-βdeposition,sphingomyelin level,and the expression and function of P-glycoprotein in Alzheimer’s disease pathological process

In Alzheimer’s disease,the transporter P-glycoprotein is responsible for the clearance of amyloid-βin the brain.Amyloid-βcorrelates with the sphingomyelin metabolism,and sphingomyelin participates in the regulation of P-glycoprotein.The amyloid cascade hypothesis describes amyloid-βas the central cause of Alzheimer’s disease neuropathology.Better understanding of the change of P-glycoprotein and sphingomyelin along with amyloid-βand their potential association in the pathological process of Alzheimer’s disease is critical.Herein,we found that the expression of P-glycoprotein in APP/PS1 mice tended to increase with age and was significantly higher at 9 and 12 months of age than that in wild-type mice at comparable age.The functionality of P-glycoprotein of APP/PS1 mice did not change with age but was significantly lower than that of wild-type mice at 12 months of age.Decreased sphingomyelin levels,increased ceramide levels,and the increased expression and activity of neutral sphingomyelinase 1 were observed in APP/PS1 mice at 9 and 12 months of age compared with the levels in wild-type mice.Similar results were observed in the Alzheimer’s disease mouse model induced by intracerebroventricular injection of amyloid-β1-42 and human cerebral microvascular endothelial cells treated with amyloid-β1-42.In human cerebral microvascular endothelial cells,neutral sphingomyelinase 1 inhibitor interfered with the changes of sphingomyelin metabolism and P-glycoprotein expression and functionality caused by amyloid-β1-42 treatment.Neutral sphingomyelinase 1 regulated the expression and functionality of P-glycoprotein and the levels of sphingomyelin and ceramide.Together,these findings indicate that neutral sphingomyelinase 1 regulates the expression and function of P-glycoprotein via the sphingomyelin/ceramide pathway.These studies may serve as new pursuits for the development of anti-Alzheimer’s disease drugs.

Piperlongumine Inhibits Zika Virus Replication In vitro and Promotes Up-Regulation of HO-1 Expression, Suggesting An Implication of Oxidative Stress

Owing to the widespread distribution of mosquitoes capable of transmitting Zika virus, lack of clinical vaccines and treatments, and poor immunity of populations to new infectious diseases, Zika virus has become a global public health concern. Recent studies have found that Zika virus can continuously infect human brain microvascular endothelial cells.These cells are the primary components of the blood–brain barrier of the cerebral cortex, and further infection of brain tissue may cause severe damage such as encephalitis and fetal pituitary disease. The present study found that a biologically active base, piperlongumine(PL), inhibited Zika virus replication in human brain microvascular endothelial cells, Vero cells, and human umbilical vein endothelial cells. PL also significantly increased heme oxygenase-1(HO-1) gene expression, while silencing HO-1 expression and using the reactive oxygen species scavenger, N-acetylcysteine, attenuated the inhibitory effect of PL on Zika virus replication. These results suggest that PL induces oxidative stress in cells by increasing reactive oxygen species. This, in turn, induces an increase in HO-1 expression, thereby inhibiting Zika virus replication. These findings provide novel clues for drug research on the prevention and treatment of Zika virus.