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.

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.

Adenoviral 15-lipoxygenase-1 gene transfer inhibits hypoxia-induced proliferation of retinal microvascular endothelial cells in vitro

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/BL63 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-LS-LOX-for Ad-vectorvia 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 VEGFR(2) could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV.

Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia

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.

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.

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.