| Part 1: Oxidized low-density lipoprotein promotes laser-induced choroidal neovascularization in miceBackground:Age-related macular degeneration(AMD)is the most common blindness causing disease in the elderly.One of the earliest signs of it is drusen formation,which highly enriched with cholesterol,lipoproteins and polyunsaturated fatty acid phospholipids.The macular is in a high oxidative stress environment.These phospholipids are highly susceptible to oxidative stress and can be modified to oxidized phospholipids(Ox PLs).Ox PLs strongly activate downstream inflammatory cascades,which can promote the release of inflammatory factors,chemokines and growth factors,as well as the inflammatory cells infiltration like macrophage.Meanwhile,triggering upon ligand binding with transmembrane receptors,it is phagocytosed by retinal pigment epithelium(RPE)cells and macrophages,leading to the accumulation of intracellular cholesterol,which further affects the function of RPE cells.Multiple effects eventually lead to the death of RPE cells and the formation of choroidal neovascularization(CNV),which means AMD occurrence.Previous study has confirmed that subretinal injection of oxidized low-density lipoprotein(Ox LDL)could induce CNV formation,but it has the disadvantages of difficulty in operation,poor reproducibility,and inconvenience of quantification.The most commonly used CNV animal model is caused by induced rupture of Bruch’s membrane via laser photocoagulation,which is relatively easier to operate,higher repeatability,and easier for CNV quantification.However,the biggest disadvantage is that it is built on healthy mice that some pathological factors such as oxidative damage and inflammation are different from patients.Intravitreal injection is one of the commonly used ophthalmic treatment methods,and the operation is relatively simpler.Therefore,we observed the effects of Ox LDL vitreous injection on laser photocoagulation-induced CNV formation,hoping to establish a new CNV model for further research,especially the study of inflammation in AMD.Objective:To investigate whether Ox LDL intravitreal injection can promote the formation of laser-induced CNV and its mechanism,so as to establish a new CNV model.Method:CNV was induced in C57BL/6J mice by 532 nm laser photocoagulator.Immediately after it,intravitreal injection was performed.The experiments were divided into three groups: Control,Ox LDL and LDL.After 7 days,the animals were sacrificed to obtain eyeballs.Frozen sections and retinal flatmounts were generated for immunofluorescence staining using antibodies against markers of Ox LDL(T15)/macrophages(F4/80)/VEC(CD31)and FITC-isolectin staining to observe lipid deposition,macrophage aggregation and CNV formation.ARPE19 cells were treated with 25μg/ml Ox LDL,with PBS and the same concentration of LDL as controls.Different times later,the Oil red O staining was used to detect intracellular lipid deposition.Western blotting(WB)and quantitative real-time polymerase chain reaction(q RT-PCR)were used to detect changes in expression of inflammatory factors,chemokines and proangiogenic factors,Ox LDL receptor inhibitor was used to explore mechanisms.Results:1.Immunofluorescence staining showed that after 7d,when compared with the LDL group and the Control group,T15-labeled Ox LDL was mainly deposited around the CNV area,the F4/80-labeled macrophages,the CD31-labeled vascular endothelial cells number and CNV area were increased in the Ox LDL group(P<0.05).Meanwhile,WB results showed that VEGF expression was increased at d7(P<0.05)when compared with the LDL group and the Control group.2.q RT-PCR results showed that after 5d,the m RNA expression of CC chemokine receptor2(CCR2),interleukin-6(IL-6),IL-1β,and matrix metalloproteinase9(MMP9)in the choroid of Ox LDL group were higher than that of Control and LDL groups(P<0.05).WB showed the similar results as q RT-PCR in CCR2,IL-6 and IL-1β protein expression(P<0.05).3.Both q RT-PCR and WB results showed that Ox LDL can significantly promote the expression of lectin-like oxidized low-density lipoprotein receptor1(LOX1).At the same time,RPE cells’ intracellular lipid deposition in Ox LDL group was significantly higher than that in LDL and Control group(P<0.05).4.LOX1 was activated by Ox LDL.Protein expression of IL-1β,IL-6,and vascular endothelial growth factor(VEGF)was increased by Ox LDL in a dose-dependent manner.LOX1 inhibitors significantly reduced expression of inflammatory factors IL-1β and VEGF(P<0.05).Conclusion:The intravitreal injected Ox LDL could diffuse to the subretinal space through the laser-damaged retina,then enter the RPE cells through LOX1,resulting in increased expression of CCR2,IL-6,VEGF,IL-1β,MMP9,and thereby promoted macrophages accumulation and CNV formation.In addition to Bruch membrane destruction,this modified CNV model includes lipid metabolism disorders,oxidative stress,and inflammation,which are involed in the CNV formation,therefore it is consider as an ideal CNV model.Part 2: Liver X receptor agonist inhibits the Ox LDL promoted CNV formation in miceBackground:The first part confirmed that intravitreal injection of Ox LDL can promote the formation of laser-induced CNV.This pathological process involves factors such as lipid metabolism disorders,oxidative stress,and inflammation.Liver X receptor(LXR)is a ligand-activated nuclear transcription factor,which belongs to the nuclear receptor family.After activation,it can play a role in regulating lipid metabolism,promoting cholesterol transport,reducing inflammation and inhibiting neovascularization.LXR has two subtypes,LXRα and LXRβ.The LXR agonist TO901317(TO)can non-selectively activate both subtypes,and it may be used for the treatment of abnormal lipid metabolism and inflammation related diseases,such as atherosclerosis.AMD and atherosclerosis are age-related diseases;Ox LDL plays a similar role in both diseases.However,the effect and role of LXR agonists on CNV formation are unclear.Objective:To investigate the effect of LXR agonist on CNV formation that Ox LDL promoted and its mechanism.Method:C57BL/6J mice were used to establish a laser-induced CNV model with Ox LDL intravitreal injection(like part 1).TO was applied with a single injection at a concentration of 10 μM.The experiment was divided into four groups: Control,Ox LDL,Ox LDL+TO and TO.Animals were sacrificed at d3,d5,and d7 and eyeballs were harvested,samples were collected and tested as the first part.Cultured ARPE19 cells were divided into Control,Ox LDL,Ox LDL+TO and TO groups.The Oil red O staining was used to detect intracellular lipid deposition.The conditioned medium of each group was collected and added to cultured monkey choroid-retinal vascular endothelial(RF/6A)cells to observe the effect on neovascularization.WB and q RT-PCR were used to detect the expression of inflammatory factors,chemokines and proangiogenic factors.Finally,RPE cells were transfected with si LXR to explore the specific mechanism of action.Results:1.Immunofluorescence staining showed that after 7d,when compared with the LDL group,the area of CNV in the Ox LDL+TO group decreased and the difference was statistically significant(P<0.05).In the Ox LDL group,T15-labeled Ox LDL,F4/80-labeled macrophages,and CD31-labeled VEC mainly located around the CNV area.Compared with the Ox LDL group,angiogenesis,lipid deposition and macrophage accumulation in the Ox LDL+TO group were significantly lesser(P<0.05).2.Compared with Ox LDL group,the expression of IL-6,CCR2 and VEGF m RNA in Ox LDL+TO group were reduced(P<0.05).The m RNA expression of these factors at d5 was higher than that at d3(P<0.05).3.The results of tube formation showed that the Ox LDL group derived conditioned medium increased the tube formation significantly when compared with the Control group,while the Ox LDL+TO group derived mudium decreased the numbers of tube formation(P<0.05).4.Compared with the Ox LDL group,the amount of intracellular lipid deposition in the Ox LDL+TO group was significantly reduced.At the same time,the expression of ABCG1 increased(P<0.05),but there was no significant change in ABCA1 expression.5.TO activated LXR gene with reducing the expression of CCR2,IL-6,VEGF and IL-1β protein;after LXR gene knockdown,the expression of VEGF and IL-1β protein in cells increased(P<0.05).6.The choroidal experiments demonstrated that Ox LDL promoted AKT and NF-κB activation,and the p-AKT and NF-κB p65 expressions at d5 are higher than that of d3,while TO inhibited the activation of these pathways(P<0.05).Studies of the RPE cells demonstrated that TO can reduce the protein expression of tumor necrosis factor-α(TNF-α),p-IκBα,IL-1β and NF-κB p65;And the NF-κB p65 protein expression increased after LXR gene knockdown(P<0.05).7.Immunofluorescence staining showed that the NF-κB p65 protein in the Ox LDL group expressed in the nucleus,and most of them expressed in the cytoplasm after adding of TO.WB results showed that Ox LDL increased the expression of IκB protein in the cytoplasm and NF-κB p65 protein in the nucleus,while TO decreased these expressions(P<0.05).Conclusion:LXR agonist promotes cholesterol efflux in RPE cells by increasing the transporter ABCG1,reduces the expression of inflammatory factors and pro-angiogenic factors,thereby inhibiting the formation of Ox LDL-induced CNV.The inflammatory response is primarily regulated by inhibiting the NF-κB p65 signaling pathway.The results provide a theoretical basis for the LXR agonists’ application in the prevention and treatment of AMD.Part 3: Ox LDL promotes CNV formation by regulating vascular endothelial cellBackground:Vascular endothelial cell(VEC)activation is the beginning of angiogenesis.It is activated by proangiogenic factors,undergo differentiation,pseudopod formation,migration,and secrete proteases to degrade the basement membrane(BM)and extracellular matrix(ECM),thereby promoting new blood vessel formation.Endothelial mesenchymal transition(End MT)is a process in which endothelial cells lose some cellular characteristics and acquire characteristics of mesenchymal cells,including loss of adhesion,enhanced cell migration ability,and increased ECM secretion.End MT is involved in angiogenesis.The first part of this study confirmed that Ox LDL can promote the formation of CNV in mice,and the second part confirmed that the conditioned medium derived from Ox LDL treated RPE cells could promote the tube formation of VEC.However,what is the direct effect of Ox LDL on VEC? whether End MT is involved in this process is unknown? The following experiments were conducted for these questions.Objective:To explore the effect of Ox LDL on VEC and its mechanism.Method:Firstly,different concentrations of Ox LDL were added to the cultured RF/6A cells to detect the effect on cell viability.Secondly,Ox LDL of lower concentration that had no significant damage to cell viability was used to study its influence on RF/6A cell migration and tube formation,and WB and q RT-PCR were used to detect the expression of cytokines at the same time.Finally,according to changes in cytokine expression,specific antibodies and inhibitors of cell signaling pathways were selected to explore the molecular mechanisms involved.Results:1.Low concentrations(25 or 50 μg/ml)of Ox LDL had no effect on the viability of RF/6A cells,while Ox LDL at concentrations of 100 or 200 μg/ml could reduce cell viability significantly.Transwell results showed that at 24 h,the number of cell migration in the Ox LDL group at 25 μg/ml and 50 μg/ml were significantly higher than that in the Control group.The tube formation results showed that at 6h and 18 h,the number of Ox LDL group composed tubes of 25 μg/ml and 50 μg/ml were significantly higher than that of the Control group.2.Ox LDL significantly increased the m RNA expression of transforming growth factor-β2(TGF-β2),VEGF,VEGFR2,and CCR2,and slightly reduced the m RNA expression of insulin-like growth factor-1(IGF-1).WB results showed that Ox LDL increased protein expression of TGF-β2,VEGF,and VEGFR2,while decreased IGF-1 protein expression.Anti-VEGF antibody decreased Ox LDL’s pro-angiogenic ability in the early stage(6h),while anti-TGF-β2 antibody worked mainly in the late stage(18h).The results showed that Ox LDL strongly increased the End MT protein markers expression of Smad2,Smad3,and α-smooth muscle actin(α-SMA).3.Studies of signal pathways found that the total protein expression of AKT,ERK,and p38 MAPK did not change significantly after Ox LDL treatment,while the protein expression of p-AKT and p-ERK increased,and p-p38 decreased significantly.4.MEK inhibitor PD98059 reduced p-ERK activation and VEGF protein expression,and PI3 K inhibitor LY294002 reduced p-AKT activation and TGF-β2 protein expression.Migration analysis results showed that PD98059 and LY294002 both inhibit the migration of RF/6A cells.Tube formation results demonstrated that the ERK inhibitor and PI3 K inhibitor had a similar inhibitory effect on angiogenic capacity of RF/6A cells at 6h,while the effect of PI3 K inhibitor was stronger than ERK inhibitor at 18 h.Conclusion:Our results indicate that Ox LDL promotes CNV formation by increasing VEGF expression in the early stage,with activation of the MEK/ERK pathway.And Ox LDL induces the TGF-β2/Smad signaling axis,which leads to End MT,to affects the later stage of CNV formation by activating the PI3K/AKT pathway. |
