Alteration of intraocular pigment epithelium-derived factor and vascular endothelial growth factor i

    作者：Lei Cui，Hai Lu   

    作者单位：Department of Ophthalmology, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China

    【摘要】To determine the aqueous, vitreous, serum levels of pigment epithelium-derived factor（PEDF）and vascular endothelial growth factor（VEGF）in patients with proliferative diabetic retinopathy（PDR）, and to speculate on the source of the change in concentration and to discuss its clinical significance. METHODS：Forty-one eyes with proliferative diabetic retinopathy were included in the study, 16 of which were complicated by neovascularization of iris (NVI).Twenty-one eyes with idiopathic macular hole（MH）were as controls. The aqueous, vitreous, serum levels of PEDF and VEGF of all the groups were determined with ELISA. PEDF, VEGF and the levels in the three groups were compared with analysis of variance（ANOVA）. The PEDF, VEGF concentrations in aqueous, vitreous and serum were analyzed with Pearson correlation test,and the correlation of PEDF and VEGF levels was also analyzed with Pearson correlation test. RESULTS：The aqueous levels of PEDF decreased significantly in sequence in groups of control, PDR without NVI, PDR with NVI. VEGF levels increased coordinately. The similar findings existed in vitreous samples. The PEDF,VEGF levels in aqueous were not correlated significantly with those in serum, but correlated positively with those in vitreous. The intraocular levels of PEDF had a negative correlation to those of VEGF. CONCLUSION：The reduction of intraocular PEDF level and elevation of intraocular VEGF level may play an important role in the occurrence and progression of PDR. In the development of PDR , the PEDF,VEGF levels in aqueous may be mainly effected by local pathological changes, as anti-angiogenic and pro-angiogenic factors, their unbalanced intraocular distribution may promote the angiogenesis of the iris and retina.

    【关键词】  pigment epithelium-derived factor；vascular endothelial growth factor； rubeosis iridis；diabetic retinopathy；angiogenesis

    INTRODUCTION

    Neovascular eye diseases, like age-related macular degeneration, diabetic retinopathy, neovascular glaucoma, are among the leading causes of blindness. The routine therapies usually cannot achieve satisfying results. It was found that some anti-angiogenic and pro-angiogenic cytokines, represented by pigment epithelium-derived factor（PEDF）and vascular endothelial growth factor（VEGF）, opened a new field for therapeutic modalities of neovascular eye diseases. Impaired levels of PEDF and VEGF were found to be related significantly to choroidal and retinal neovascularization . Several experiments showed that, compared to controls and nonproliferative retinopathy（NPDR）, the aqueous PEDF levels in patients with proliferative retinopathy（PDR）were reduced and VEGF elevated . In our report, the aqueous, vitreous, serum levels of PEDF and VEGF were measured in patients with or without neovascularization of iris (NVI) secondary to PDR. The possible sources of the change in concentration were speculated and its clinical significance was discussed.

    MATERIALS AND METHODS

    Samples The patients were admitted in the eye centre of Beijing Tongren Hospital between November 2005 and July 2006. Undiluted aqueous, vitreous, serum samples were collected from 62 eyes of 62 individuals who underwent pars plana vitrectomy for proliferative diabetic retinopathy and idiopathic macular hole(MH). Specimens were collected in sterile tubes, placed immediately on ice, centrifuged to separate the cell contents, and stored at -80° C until they were analyzed. Table 1 shows baseline characteristics of the subjects.

    PEDF Detection The PEDF levels of various samples were measured with enzyme-linked immunosorbent assay（ELISA）kit( Chemicon International ). The urea-treated samples were incubated on ice for one hour and another hour at 37° C in 96-well microplates coated by a mouse monoclonal anti-PEDF antibody. Then the plates were washed. 100μL/well biotinylated mouse anti-human PEDF monoclonal antibody was added and incubated. After washing, streptavidin peroxidase-labeled polyclonal antibody was used, and a TMB/E substrate was added. After stopped with the stop solution, the plate was read at 450nm using a microplate reader.

    VEGF Detection The concentrations of VEGF were quantitated using an ELISA kit ( R&D Systems ). 200μL diluted samples were added into each well and incubated. Then washing of the plates and adding of streptavidin peroxidase-labeled polyclonal antibody. After incubation and washing, a substrate solution was used and protected from light for 20 minutes at room temperature. The plate was read at 450nm and corrected at 540nm immediately after stopped by stop solution.

    Statistical Analysis Statistical calculations were performed using SPSS 11.5. One-way analysis of variance(ANOVA) was used for the camparison of PEDF and VEGF levels in various groups. Correlations beween groups were studied by Pearson correlation test. P values less than 0.05 were accepted as statistically significant.

    RESULTS

    Aqueous PEDF and VEGF The aqueous levels of PEDF decreased significantly in sequence in groups of macular hole(MH), proliferative diabetic retinopathy(PDR) without neovascularization of iris(NVI), PDR with NVI. The aqueous levels VEGF increased significantly in sequence in groups of MH, PDR without neovascularization of NVI, PDR with NVI. Significant differences were found between any two groups (P﹤0.01,Figure 1).

    Figure 1 Aqueous PEDF and VEGF

    Vitreous PEDF and VEGF The vitreous levels of PEDF decreased significantly in sequence in groups of MH, PDR without NVI, PDR with NVI. The vitreous levels of VEGF increased significantly in sequence in groups of MH, PDR without NVI, PDR with NVI. There were significant differences between any two groups (P﹤0.01,Figure 2).

    Figure 2 Vitreous PEDF and VEGF

    Serum PEDF and VEGF There was no statistical difference of serum PEDF concentrations among the three groups ( P>0.05 ). The serum VEGF concentration in group PDR with NVI corresponded to that in group PDR without NVI (P > 0.05 ). The serum VEGF level in either of the two groups was much higher than that in the control group (P <0.01 ,Figure 3)

    Figure 3 Serum PEDF and VEGF

    Correlations of PEDF and VEGF between Aqueous and Serum/ vitreous In the three groups, the PEDF, VEGF levels in aqueous humor were not found to correlate significantly to those in serum (P﹥0.05). Similar results were found between aqueous humor and vitreous in MH group. In PDR without NVI group, there was a positive correlation of PEDF, VEGF levels in aqueous humor and in vitreous are represented in Figures 4,and 5. In the PDR group with NVI, the aqueous PEDF levels correlated positively to the ones of the vitreous (Figure 4), and the aqueous VEGF levels were not significantly correlated to the vitreous levels (Figure 5). In patients with PDR﹢/﹣NVI, the aqueous PEDF concentraton increased with the vitreous level. (r =0.76, P<0.05; r =0.53, P<0.05); no significant correlation was found in the control group.( r =0.22, P>0.05). In group PDR without NVI, a positive correlation was found between the aqueous and vitreous VEGF levels ( r =0.76, P<0.01). No significant correlation was found in the PDR group with NVI and in the control group(r =0.60, P>0.05; r =-0.304, P>0.05).

    Figure 4 Relationship of PEDF between aqueous humor and vitreous

    Figure 5 Relationship of VEGF between aqueous humor and vitreous

    Correlation of PEDF and VEGF A negative correlation between PEDF levels and VEGF levels in aqueous was found in the three groups (Figure 6). We also found a a negative correlation between PEDF levels and VEGF levels in vitreous in the three groups (Figure 7). No statistical correlation between PEDF levels and VEGF levels was found in serum. A negative correlation between PEDF levels and VEGF levels in aqueous was measured in the three groups: r = -0.71, P<0.01 in patients with NVI, r = -0.57, P<0.05 in patients without NVI, r = -0.69, P<0.05 in control group. A negative correlation between PEDF levels and VEGF levels in the vitreous was present in the three groups: r = -0.65, P<0.05 in patients with NVI, r = -0.66, P<0.01 in patients without NVI, r = -0.74, P<0.01 in control group.

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