Correlation between Ficolin-3 and Vascular Endothelial Growth Factor-to-Pigment Epithelium-Derived Factor Ratio in the Vitreous of Eyes with Proliferative Diabetic Retinopathy


To investigate the relationship of ficolin-3 with inflammatory and angiogenic factors, namely, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), in the vitreous of eyes with proliferative diabetic retinopathy (PDR).


Case-control study.


Vitreous fluid and serum samples were obtained at the time of vitreoretinal surgery from 21 eyes of 21 patients with PDR and from 16 eyes of 16 nondiabetic patients with idiopathic macular hole. Ficolin-3, VEGF, and PEDF concentrations were determined by enzyme-linked immunosorbent assay.


Vitreous ficolin-3 levels were significantly higher in eyes with PDR than in eyes with idiopathic macular hole. Similarly, VEGF levels also were significantly higher in eyes with PDR than in eyes with idiopathic macular hole. In contrast, vitreous PEDF concentrations were significantly lower in eyes with PDR than in eyes with idiopathic macular hole, and the VEGF-to-PEDF ratio was higher in eyes with PDR than in eyes with idiopathic macular hole. Furthermore, a significant correlation between ficolin-3 and the VEGF-to-PEDF ratio was observed in the vitreous of eyes with PDR. In addition, serum ficolin-3 levels in eyes with PDR were higher than the levels in eyes with idiopathic macular hole, and the vitreous ficolin-3 levels also correlated with serum ficolin-3 levels.


We found that ficolin-3 levels were elevated in the vitreous fluid of patients with PDR. Our results suggest that ficolin-3 may be used as a new therapeutic target for treatment of PDR.

Diabetic retinopathy (DR) is one of the very serious and potentially blinding complications of diabetes and is the leading cause of blindness in people of working age. The mechanisms underlying the development of DR have been completely elucidated. DR shares many characteristics of a low-grade inflammatory disease, such as increased levels of proinflammatory cytokines in the retina and vitreous and increased permeability of retinal capillaries that become nonperfused and ischemic. Moreover, the development of diabetic complications is related to the elevated level of complement and may be used as new therapeutic targets. Further, the mannose-binding lectin pathway, an overall regulatory component of the complement system, has been found to be directly involved in the development of diabetic angiopathy.

As members of the mannose-binding lectin, ficolins also are known to activate the lectin pathway of complement. The 3 ficolins that have been identified in humans are M-ficolin, L-ficolin, and H-ficolin (referred to as ficolin-1, ficolin-2, and ficolin-3, respectively). On recognition of the infectious agent, ficolins act through 2 distinct routes: they initiate the lectin pathway of complement activation through attached serine proteases and they act through a primitive opsonophagocytosis, thus limiting the infection and concurrently orchestrating the subsequent adaptive clonal immune response. The dysfunction or abnormal expression of ficolins may play a crucial role in the pathogenesis of human diseases, including infectious and inflammatory diseases such as recurrent respiratory infections, autoimmune disease resulting from apoptosis, systemic lupus erythematosus, immunoglobulin A nephropathy, and the clinical syndrome of pre-eclampsia. Recently, using a proteomic approach, we found that ficolin-3 was upregulated significantly in the serum of type 2 diabetic patients ( P = .012).

Vascular endothelial growth factor (VEGF) plays a major role in the initiation and development of DR. VEGF levels also are reported to be elevated in patients with proliferative diabetic retinopathy (PDR). Our previous study uncovered that VEGF itself produces many of the vascular abnormalities common to DR, such as the increase in cellular apoptosis and permeability. In contrast, pigment epithelium-derived factor (PEDF), a potent inhibitor of inflammatory and angiogenesis, has been found to be involved in the pathogenesis of PDR. Gao and Zheng and associates reported that the time course of the VEGF-to-PEDF ratio change correlated with the development and progression of retinal neovascularization; the VEGF-to-PEDF ratio represented a dynamic balance between angiogenic stimulators and inhibitors, and any disturbance of the balance seems to play a key role in the pathogenesis of DR.

These findings prompted us to examine whether ficolin-3 plays a role in the exacerbation of DR in cooperation with VEGF and PEDF. To the best of our knowledge, this is the first investigation of the involvement of ficolin-3 with inflammatory and angiogenic factors, VEGF and PEDF, in the vitreous of eyes with PDR. Analysis of the vitreous fluid allowed us to clarify the correlation between ficolin-3 and the VEGF-to-PEDF ratio in the pathogenesis of PDR.


The subjects were 37 patients (37 eyes) who had undergone vitreous surgery for treatment of retinal disorders. Some samples were collected from the eyes of type 2 diabetes patients (n = 21) with PDR. The control group comprised vitreous samples from 16 eyes of 16 patients who underwent vitrectomy for an idiopathic macular hole. Samples from eyes obtained during a repeat vitrectomy were excluded. None of the patients with an idiopathic macular hole had diabetes mellitus. The demographic details of the patients are shown in Tables 1 and 2 . In the present study, the patients who had undergone previous laser treatment before their current surgery were excluded. The glycosylated hemoglobin levels in all patients recruited into this study were less than 8 mmol/L.


Intravitreous Concentrations of Ficolin-3, Vascular Endothelial Growth Factor, and Pigment Epithelial-Derived Factor in Patients with Proliferative Diabetic Retinopathy and Patients with Idiopathic Macular Hole

Control PDR P Value
No. of cases 16 21
Gender (male:female) 3:5 4:6
Age (years) 53 ± 7.8 56 ± 8.5
HbAlc (%) 5.23 ± 0.26 8.69 ± 2.37 .001
VH 10
VH + TRD 7
Idiopathic macular hole 16
Level of ficolin-3 (ng/mL) 42.8 ± 12.4 118.6 ± 30.8 .003
Level of VEGF (pg/mL) <31.2 a 1078.9 ± 832.8 .001
Level of PEDF (μg/mL) 20.1 ± 15.0 5.8 ± 3.3 .002
VEGF-to-PEDF ratio (10 –6 ) 1.54 186.02 .001

HbAlc = glycosylated hemoglobin; PEDF = pigment epithelial-derived factor; PDR = proliferative diabetic retinopathy; TRD = tractional retinal detachment; VEGF = vascular endothelial growth factor; VH = vitreous hemorrhage.

a Vitreous level of VEGF regarded as 31.2 pg/mL in control participants.


Serum Concentrations of Ficolin-3 in Patients with Nondiabetic Retinopathy, Patients with Nonproliferative Diabetic Retinopathy, Patients with Proliferative Diabetic Retinopathy, and Controls

Control non-DR Diabetes non-PDR PDR
No. of cases 16 20 20 21
Sex (male:female) 3:5 1:1 9:11 4:6
Age (years) 53.00 ± 7.80 56.03 ± 10.15 55.69 ± 10.25 56.00 ± 8.50
HbA1c (%) 5.27 ± 0.25 8.34 ± 2.19 a 7.81 ± 2.03 a 8.47 ± 2.31 a
Ficolin-3 (μg/mL) 8.74 ± 3.89 10.72 ± 4.62 a 12.37 ± 5.46 a , b 14.03 ± 7.62 a , b , c

HbAlc = glycosylated hemoglobin; non-DR = nondiabetic retinopathy; non-PDR = nonproliferative diabetic retinopathy; PDR = proliferative diabetic retinopathy.

a P < .01 vs control.

b P < .05 vs non-DR.

c P < .01 vs non-PDR.

After patient consent was obtained, the vitreous fluid (0.6 to 1.0 mL) was aspirated through the vitreous cutter under the simultaneous inflation of the vitreous cavity with air through the infusion cannula; the fluid was frozen rapidly to −80 C until the assay was performed. Vitreous samples were used for measurement of ficolin-3, VEGF, and PEDF by enzyme-linked immunosorbent assay.

Ficolin-3 measurements were performed using a commercially available enzyme-linked immunosorbent assay kit (USCN Life Science & Technology Company, Wuhan, China) according to the manufacturer’s protocol. The detection limit of the human ficolin-3 assay was 0.156 ng/mL, and intra-assay and interassay variations were 3.5% and 8.5%, respectively. Duplicate measurements were obtained for all samples. Serial dilutions of recombinant ficolin-3 were included in all assays as a standard. Similarly, VEGF enzyme-linked immunosorbent assay was performed using a Quantikine VEGF or PEDF assay kit (R & D Systems, Inc, Minneapolis, Minnesota, USA) to quantify the levels of vitreous VEGF. The sensitivity of the assay was 31.2 pg/mL, and the intra-assay and interassay variations of the VEGF assay were 6.6% and 7.5%, respectively. The sensitivity of the PEDF assay was 0.9 μg/mL, and the intra-assay and interassay variations of the PEDF assay were 5.3% and 16.0%, respectively. Serial dilutions of recombinant human VEGF or PEDF were included in all assays to serve as standards.

Statistical Analysis

The experimental data were expressed as the mean ± standard deviation. Significant differences between the 2 groups were calculated by 1-way analysis of variance, followed by the Student t test using the statistical software program SPSS for Windows version 17.0 (SPSS, Inc, Chicago, Illinois, USA). Pearson correlation tests also were performed. P values < .05 were considered significant in all cases.

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Jan 16, 2017 | Posted by in OPHTHALMOLOGY | Comments Off on Correlation between Ficolin-3 and Vascular Endothelial Growth Factor-to-Pigment Epithelium-Derived Factor Ratio in the Vitreous of Eyes with Proliferative Diabetic Retinopathy

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