ABSTRACT
Objective:To investigate the molecular expression and pathological features of endothelial cell (EC) in a murine model of choroidal neovascularization (CNV) based on single-cell RNA sequencing (scRNA-seq).Methods:Six C57BL/6 mice aged 6-8 weeks were randomly divided into two groups, with 3 mice in each group.Bilateral eyeballs were enucleated.The choroidal tissues from the two groups were isolated by shearing the complex and scraping the choroid, respectively.Single-cell suspension was prepared by continuous digestion with trypsin/type Ⅰ collagenase at 37 ℃, and the cell viability and EC ratio were detected by flow cytometry to determine the preparation method of single-cell suspension.Another 6 mice were randomly assigned into the control group and the CNV group, with 3 mice in each group.The CNV model was induced by laser photocoagulation and single-cell suspensions were prepared 7 days after modeling.Gene expression library construction was performed using the Chromi-um (10x Genomics) instrument.High throughput sequencing was performed using the Illumina Novaseq6000 to obtain the expression matrix.The EC subpopulations were classified according to previous researches and the Cellmarker database.Pseudo-time analysis was performed in EC, revealing the gene expression matrix of different states.CNV-EC were further selected with preliminary analysis of the expression characteristics.Another 6 mice were selected to establish the CNV model and eyeball frozen sections were prepared 7 days after modeling.Expression and distribution as well as the area percentage of EC marker Pecam1, mitochondrial outer membrane proteins Tomm20 and mt-Co1, and capillary markers Kdr and Plvap were observed by immunofluorescence staining, and the vascular diameter was calculated.The use and care of animals followed the ARVO statement.This study protocol was approved by the Experimental Animal Welfare and Ethics Committee of Air Force Military Medical University (No.20200181).Results:The cell viability of the single-cell suspension prepared from choroidal-scleral fragments and choroidal scrapings was 99.4% and 99.1%, respectively, both of which met the sequencing requirements.The percentage of EC detected by flow cytometry was approximately 1.58%.The scRNA-seq result revealed that both the normal control and CNV groups contained 13 choroidal cell clusters.Compared with the normal control group, the proportions of rod/cone photoreceptor cells, EC and hematopoietic cells all increased, while the retinal pigment epithelium (RPE) and Schwan cells reduced in the CNV group.Among all clusters, EC constituted 18.4%.The pseudo-time analysis demonstrated that EC could be further divided into 4 states.The percentage of state 2 EC was 29.1% in the CNV group, which was significantly higher than 9.5% in the normal control group.Differentially expressed gene analysis showed that the expression of mitochondrion-related genes, including mt-Nd4 and mt-Atp6, were upregulated in state 2 EC, while capillary-related genes, including Kdr and Esm1, were downregulated.Immunofluorescent staining revealed that the area of Tomm20 and mt-Co1 in Pecam1-positive EC in the CNV area was (19.50±4.68)% and (4.64±2.82)%, respectively, which were both higher than (3.00±2.09)% and (0.18±0.34)% in normal area ( t=7.88, 3.84; both at P<0.01). The area of Kdr and Plvap in Pecam1-positive EC in the CNV area was (1.50±0.29)% and (0.79±0.97)%, respectively, which were both lower than (31.30±5.44)% and (10.43±2.28)% in the normal area ( t=13.40, 9.48; both at P<0.01). The vascular diameter in the CNV area was (5.52±1.85)μm, which was larger than (4.21±1.84)μm in the normal area ( t=9.57, P<0.001). Conclusions:When CNV occurs, the proportion of EC in choroid increases, and CNV-EC shows pathologic features of mitochondrial metabolic activation and loss of capillary properties, suggesting the mitochondrial activation of EC may play a role in the formation of CNV.
ABSTRACT
Objective To investigate whether vascular endothelial cells in choroidal neovascularization whether hypoxia condition can up-regulate SNAI1 and activate matrix metalloproteinase (MMP)2 and MMP9 therefore to participate in choroidal neovascularization(CNV).Methods Sixteen SPF male C57 mice aged 6-8 weeks were divided into control group and model group.CNV models were induced by retinal laser photocoagulation,and flatmount and frozen sections of retinal pigment epithelium (RPE)-choroid-sclera compound were prepared at 7 days after modeling.The CNV in flat-mount was examined by Isolectin B4 staining,and the location of SNAI1,MMP2 and MMP9 in frozen sections was determined by immunofluorescence technology.The expression of SNAI1,MMP2 and MMP9 at mRNA level in CNV was detected by real-time fluorescence quantitative PCR (real-time PCR).The use and care of experimental animals complied with Statement for the Use of Animals in Ophthalmic and Visual Research.The RF/6A cells were divided into normoxia group and hypoxia group and cultured for 24 hours in 5％ CO2condition and mix condition of 94％ N2,5％ CO2 and 1％ O2,respectively.The expression of SNAI1,MMP2 and MMP9 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay,respectively.Small interfering RNA of SNAI1 (siSNAI1) was transfected into the cells,and then the expression of MMP2 in the cells at mRNA and protein levels was detected by real-time PCR and Western blot assay,respectively,and the migrating number of the cells was assayed by Transwell chamber assay.Results CD31 and SNAI1 positive-response cells were seen in RPE-choroid-sclera flat-mounts under the laser scanning confocal microscope.The relative expression levels of SNAI1mRNA and MMP2 mRNA in RPE-choroid-sclera tissues were higher in the model group than those in the control group (SNAI1 mRNA:1.291 ±0.060 vs.0.759±0.074,P =0.001;MMP2 mRNA:1.610±0.424 vs.0.772 ±0.080,P =0.044).The expression of MMP9 mRNA was not significantly elevated between model group and control group (P＞0.05).The relative expression level of MMP2 mRNA was higher in comparison with MMP9 mRNA in the model group (P＜0.01).The relative expressions of hypoxic induced factor 1α (HIF-1α),SNAI1 and MMP2 at mRNA level and protein level in RF/6A cells were significantly higher in the hypoxia group than those in the normoxia group (all at P＜0.05) and no considerable difference was seen in MMP9 mRNA expression between the two groups (P＞0.05).The relative expressions of MMP2 mRNA in the cells were 0.217±0.036 and 0.818±0.105,and those of MMP2 protein in the cells were 0.236±0.009 and 1.043±0.120 in the hypoxia+siSNAI1 group and only hypoxia group,respectively,with significant differences between them (P =0.002,0.003).The migrating number of the cells was (254.60 ±71.31)/field in the hypoxia+siSNAI1 group,which was significantly less than (534.10±96.21) /field in the control group (P =0.029).Conclusions The hypoxic environment at CNV can activate MMP2 by up-regulating the expression of SNAI1,which promotes the migration of vascular endothelial cells and therefore participates in CNV formation,and the intervention of SNAI1 activation under the hypoxic condition can inhibit this process.