Table 1.
Characteristics of the study population.
Table 2.
Polymorphisms in the ARMS2 gene lesion: Distribution and Genotypes in neovascular Age-Related Macular Degeneration (nAMD), Polypoidal Choroidal Vasculopathy, and Controls in the northern Chinese Population.
Table 3.
Polymorphisms in the ARMS2 gene lesion: Distribution and Allele in neovascular Age-Related Macular Degeneration (nAMD), Polypoidal Choroidal Vasculopathy, and Controls in the northern Chinese Population.
Table 4.
Polymorphisms in the HTRA1 gene lesion: Distribution and Genotypes in neovascular Age-Related Macular Degeneration (nAMD), Polypoidal Choroidal Vasculopathy, and Controls in the northern Chinese Population.
Table 5.
Polymorphisms in the HTRA1 gene lesion: Distribution and Allele in neovascular Age-Related Macular Degeneration (nAMD), Polypoidal Choroidal Vasculopathy, and Controls in the northern Chinese Population.
Figure 1.
Analysis of pair-wise LD across ARMS2 and HTRA1 SNPs in northern Chinese PCV and nAMD cohort.
A) Analysis of pair-wise LD across the eight ARMS2 and HTRA1 SNPs in northern Chinese PCV cohort. B) Analysis of pair-wise LD across the eight ARMS2 and HTRA1 SNPs in northern Chinese nAMD cohort. The relative physical position of each SNP is given in the upper diagram. The pairwise D′ between all SNPs is given below each SNP combination. And when D′ = 1.0, no number is given inside the square. Bright red squares indicate D′≥0.90 and LOD≥2. Bright red squares indicate D′<0.90 and LOD≥2. White squares indicate D′<0.90 and LOD<2.
Table 6.
Haplotype analysis of ARMS2 and HTRA1 polymorphisms in exudative AMD and PCV.
Table 7.
Logistic regression analysis of SNPs in ARMS2 and HTRA1 between AMD and PCV.
Figure 2.
Rs10490924 (A69S in a coding change) does not affect mRNA, protein, or surface expression of ARMS2.
A)Direct sequencing verified the wild-type (WT) and G270T ARMS2 plasmids. B) RT-PCR showed no significant difference between wild-type and G270T ARMS2 mRNA expression. C) Real-time RT-PCR confirmed the mRNA level finding. D) Western blot showed that the levels and migrations of the SNP mutant proteins were comparable with wild type. All experiments were repeated >3 times.
Figure 3.
Effect of wild-type ARMS2 and rs10490924 on the proliferation of RF/6A and human RPE cells.
RF/6A (A) and ARPE-19 (B) cell proliferation was measured with an MTT assay at 24 h, 48 h, 72 h, 96 h. Values are the means±SD of at least three independent experiments. Asterisks denote values significantly different from those of cells treated with wild-type ARMS2 and rs10490924 compared to negative control (p<0.01). (C)The time course of the ARMS2 protein expression profile mirrored that for ARMS2 protein expression levels after transfection in ARPE-19 cells. Abbreviations: wild-type ARMS2 plasmid-treated cells (WT); rs10490924 plasmid -treated cells (G270T); pReceiver-M29-Basic plasmid-treated cells (Vector) (*P<0.05, **P<0.01).
Figure 4.
Effects of wild-type ARMS2 and rs10490924 on the attachment of RF/6A and RPE cells.
Cell attachment was assessed after 6 h incubation and subsequent MTT assay. Values are the means±SD of at least three independent experiments. Asterisks denote values significantly different from those of cells treated with wild-type ARMS2 and rs10490924 compared to negative control (p<0.01). Abbreviations: wild-type ARMS2 plasmid-treated cells (WT); rs10490924 plasmid -treated cells (G270T); pReceiver-M29-Basic plasmid-treated cells (Vector) (*P<0.05, **P<0.01).
Figure 5.
Effect of wild-type ARMS2 and rs10490924 on the migration of RF/6A and human RPE cells.
The migratory activity of both cell lines was estimated based on the number of cells that had migrated through the filter of the chamber. A) Migrated cells of pReceiver-M29-Basic plasmid-treated RF/6A cells. B) Migrated cells of wild-type ARMS2 plasmid-treated RF/6A cells. C) Migrated cells of rs10490924 plasmid -treated RF/6A cells. Values are the means±SD of at least three independent experiments. D) The results showed that the number of migrating cells in the wild-type ARMS2 plasmid -treated group was the most during the three groups(p<0.01). Abbreviations: wild-type ARMS2 plasmid-treated cells (WT); rs10490924 plasmid -treated cells (G270T); pReceiver-M29-Basic plasmid-treated cells (Vector) (*P<0.05, **P<0.01).
Figure 6.
Effect of wild-type ARMS2 and rs10490924 on the tube formation of RF/6A cells.
PReceiver-M29-Basic plasmid-treated RF/6A cells (A), wild-type ARMS2 plasmid-treated cells RF/6A cells (B) and rs10490924 plasmid -treated RF/6A cells (C) were plated on Matrigel as described in Methods. After 24 h of incubation, the three groups cells formed well organized capillary-like structures. Values are the means±SD of at least three independent experiments. There are no significant differenence during the three groups (D, p>0.05). Abbreviations: wild-type ARMS2 plasmid-treated cells (WT); rs10490924 plasmid -treated cells (G270T); pReceiver-M29-Basic plasmid-treated cells (Vector).
Figure 7.
Effect of wild-type ARMS2 and rs10490924 on the apoptosis of human RPE cells.
Apoptosis was quantified by flow cytometry measured by Annexin V and PI staining. Data are presented as mean±SEM.Each experiment was repeated at least three independent times. DMEM+10%FBS control was set to 100%.*P<0.05. UR: late apoptotic cells; LR: early apoptotic cells, UR+LR: apoptotic cells. Abbreviations: wild-type ARMS2 plasmid-treated cells (WT); rs10490924 plasmid -treated cells (G270T); pReceiver-M29-Basic plasmid-treated cells (Vector).
Table 8.
Summary of flow cytomery data of apoSptosis measured by Annexin V and PI.
Figure 8.
Effects of wild-type ARMS2 and rs10490924 on the cell cycles of human RPE cells.
A) Cell cycle of pReceiver-M29-Basic plasmid-treated ARPE-19 cells. B) Cell cycle of wild-type ARMS2 plasmid-treated ARPE-19 cells. C) Cell cycle of rs10490924 plasmid-treated ARPE-19 cells. D) Data from the ARPE-19 cell cycle distribution of the control group, wild-type ARMS2 and rs10490924 group. Flow cytometric analysis demonstrates the effects of wild-type ARMS2 and rs10490924 on the human RPE cell cycle. The x-axis represents fluorescence intensity on a logarithmic scale and the y-axis represents the number of events. The results show that the fraction of cells in the G1 phase has decreased and the proportion of cells in the S phase has increased in the presence of wild-type ARMS2 and rs10490924-treated cells(*P<0.05, **P<0.01). Values are the mean±SD from three independent experiments. Abbreviations: wild-type ARMS2 plasmid-treated cells (WT); rs10490924 plasmid -treated cells (G270T); pReceiver-M29-Basic plasmid-treated cells (Vector).