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Plasma Concentration of Pigment Epithelium-Derived Factor in Patients with Diabetic Retinopathy

Departments of Ophthalmology (N.O., M.Matsuo., K.Ma., C.S., K.Mi., H.M., T.T.) and Public Health (A.T., T.N.), Kansai Medical University, Moriguchi, Osaka 570-8507, Japan; and Department of Ophthalmology (M.W., N.J., A.H., M.Matsum.), Kansai Medical University, Hirakata, Osaka 573-1136, Japan

Address all correspondence and requests for reprints to: Nahoko Ogata, Department of Ophthalmology, Kansai Medical University, Fumizono-cho 10-15, Moriguchi, Osaka 570-8507, Japan. E-mail: ogata{at}takii.kmu.ac.jp.

	Abstract

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Context: Pigment epithelium-derived factor (PEDF) is a strong inhibitor of angiogenesis. Eyes with diabetic retinopathy have low levels of ocular PEDF; however, the PEDF levels in the blood of diabetics have still not been determined.

Objectives: Our objective was to determine the plasma levels of PEDF in diabetic patients and to determine the relationship with the stage of the diabetic retinopathy.

Design and Setting: This study was designed as a cross-sectional, institutional study.

Patients or Other Participants: A total of 145 Japanese were studied; 112 had type 2 diabetes mellitus, and 33 were healthy controls.

Intervention: There was no intervention.

Main Outcome Measures: The plasma level of PEDF was measured by ELISA, and the stage of diabetic retinopathy was determined by ophthalmic examinations. Clinical systemic status of diabetic patients was also examined.

Results: The plasma PEDF level in diabetic patients (6.68 ± 0.54 µg/ml; mean ± SEM) was significantly higher than that in controls (4.38 ± 0.59 µg/ml, P = 0.03), and the level was especially high in patients with proliferative diabetic retinopathy (7.78 ± 0.98 µg/ml; n = 45; P = 0.005). The gender (P = 0.03), blood urea nitrogen (P = 0.005), and triglycerides (P = 0.04) were significant and independent determinants of plasma PEDF levels in diabetic patients.

Conclusions: The PEDF level in the plasma was significantly elevated in diabetic patients, especially those with proliferative diabetic retinopathy. High levels of PEDF in the plasma may be related to the progression of diabetic retinopathy.

	Introduction

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PIGMENT EPITHELIUM-DERIVED factor (PEDF) is a 50-kDa protein first identified in the conditioned medium of human retinal pigment epithelial cells as a neurotrophic factor (1, 2). Sequence analysis of the human PEDF gene showed that it is a member of the serine protease inhibitor (serpin) gene family (3). PEDF inhibited the migration of endothelial cells in vitro in a dose-dependent manner and was more effective than angiostatin, thrombospondin-1, and endostatin (4). These results placed PEDF among the most potent natural inhibitors of angiogenesis.

Diabetic retinopathy is a serious microvascular complication of diabetes mellitus and is the major cause of adult blindness when it progresses to the proliferative diabetic retinopathy (PDR) stage. It is characterized by microvascular damage and capillary nonperfusion resulting in retinal neovascularization. The level of intraocular PEDF has been shown to decrease with advancing stages of diabetic retinopathy (5, 6, 7, 8). Although the PEDF levels in the eyes of patients with diabetic retinopathy have been reported to be low, the PEDF levels in the blood of patients with diabetes mellitus have still not been determined.

The aim of this study was to determine the plasma levels of PEDF in patients with diabetes mellitus and to determine whether the level was correlated with the stage of diabetic retinopathy.

	Subjects and Methods

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Subjects

The procedures used in this study conformed to the tenets of the Declaration of Helsinki and were carried out after receiving approval from the Institutional Review Committee of Kansai Medical University. An informed consent was obtained from all patients and volunteers.

A total of 145 Japanese were studied. Blood samples were collected from 112 type 2 diabetic patients (54 men and 58 women), ages 18–87 yr (60.9 ± 0.9 yr, mean ± SEM) and 33 healthy volunteers (19 men and 14 women), ages 17–92 yr (55.3 ± 3.7 yr) at the Kansai Medical University.

Diabetic status

Whether the diabetic patients were receiving or not receiving insulin was recorded.

The levels of glycosylated hemoglobin (HbA_1c), blood urea nitrogen, and triglycerides were determined by standard laboratory procedures.

Examination of diabetic retinopathy

All diabetic patients and healthy volunteers underwent a standard ophthalmic examination at the time of the blood sample collection. The stage of diabetic retinopathy was determined by ophthalmoscopy and fluorescein angiography, and the patients were classified according to the severity scale of diabetic retinopathy (9): no apparent diabetic retinopathy (NDR), mild to moderate nonproliferative diabetic retinopathy (M-NPDR), severe nonproliferative diabetic retinopathy (S-NPDR), and PDR. Patients with other ocular diseases such as uveitis, glaucoma, age-related macular degeneration, retinal degeneration, and retinal vascular thrombosis were excluded. However, diabetic patients with simple cataracts were included.

PEDF level in plasma

The PEDF level in the plasma was measured with an ELISA kit (Chemikine PEDF Sandwich ELISA Kit; Chemicon International, Temecula, CA). Briefly, blood samples were collected in tubes containing EDTA, and platelet-poor plasma was prepared by centrifugation (3000 rpm for 20 min) and stored at –80 C before use. Before the assay, the samples were thawed on ice, and urea was added to the samples to a final concentration of 8 M. After incubation on ice for 1 h, the samples were diluted in assay diluent and then immediately applied to the assay plates and measured according to the manufacturer’s protocol.

Statistical analyses

The results are expressed as means ± SEM, and the data were analyzed statistically using unpaired t tests and one-way ANOVA with the Bonferroni’s test when comparing groups. The association between PEDF and age was examined by Pearson’s product moment correlation coefficient (r). The relationships between the plasma PEDF level and clinical parameters were determined by multiple regression analysis to evaluate the factors that have an effect on plasma PEDF levels. The statistical analysis package SPSS 14.0 for Windows was used for statistical processing. A P value of <0.05 was accepted as significant.

	Results

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Plasma PEDF levels in controls

The mean plasma level of PEDF in the controls was 4.38 ± 0.59 µg/ml (range, 0–15.1 µg/ml; n = 33; Fig. 1A). The PEDF levels in the plasma increased significantly with increasing age (r = 0.62; P < 0.001).

View larger version (8K): [in this window] [in a new window]   FIG. 1. PEDF levels in diabetic patients and controls. A, PEDF level in diabetic patients (DM) is significantly higher than that in controls; B, PEDF levels and the stage of diabetic retinopathy. *, P < 0.05. Bars indicate mean ± SEM.

The mean plasma level of PEDF in all diabetic patients (6.68 ± 0.54 µg/ml; range, 0–32.2 µg/ml; n = 112) was significantly higher than that in controls (P = 0.03; Fig. 1A).

Plasma PEDF levels and stage of diabetic retinopathy

The level of plasma PEDF was 5.84 ± 1.72 µg/ml (range, 0–18.7 µg/ml) in eyes with NDR (n = 12, 66.0 ± 2.5 yr), 6.05 ± 1.02 µg/ml (range, 0.2–13.0 µg/ml) with M-NPDR (n = 16, 63.3 ± 2.9 yr), 5.95 ± 0.80 µg/ml (range, 0.2–20.5 µg/ml) with S-NPDR (n = 39, 62.8 ± 1.2 yr), and 7.79 ± 0.98 µg/ml (range, 0.2–32.2 µg/ml) with PDR (n = 45, 57.1 ± 1.4 yr) (Fig. 1B). The plasma level of PEDF in the PDR group was significantly higher than that of controls (P = 0.005, Bonferroni’s test), but it was not significantly different from that in the other diabetic retinopathy groups.

Plasma PEDF levels and clinical systemic status of diabetic patients

Data regarding age, gender, treatment with or without insulin, and levels of HbA_1c, blood urea nitrogen, and triglycerides were collected from all diabetic patients (n = 112). HbA_1c was selected as a representative parameter of glucose control, blood urea nitrogen as a representative parameter of renal function, and the triglyceride level as a representative parameter of lipid metabolism. All of these are important parameters and were considered to be representative of the systemic diabetic condition.

This analysis model showed that these parameters (age, gender, insulin treatment, HbA_1c, blood urea nitrogen, and triglycerides) were significantly different parameters (P < 0.0001), and each variance inflation factor varied from 1.012–1.166. Thus, this analysis was statistically significant.

Multiple regression analysis showed that the gender (men, 7.67 ± 0.86 µg/ml, n = 54; women, 5.86 ± 0.69 µg/ml, n = 58; P < 0.03), blood urea nitrogen (21.3 ± 1.1 mg/dl, P < 0.001), and triglycerides (147. 0 ± 10.0 mg/dl, P = 0.04) were independent and significant determinants of plasma PEDF levels in diabetic patients, but insulin treatment (P = 0.91), age (P = 0.84), and HbA_1c (7.5 ± 0.2%, P = 0.83) were not significantly related (Table 1).

	Discussion

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PEDF is also called EPC-1 (early population doubling level cDNA-1), reflecting its up-regulation during cell cycle arrest (G0) in young but not senescent cells in vitro (12). Thus, PEDF is considered to be a marker for cell senescence. Our results showed that the plasma PEDF levels increased with aging in controls but not in the diabetic group. We had reported that the intraocular PEDF level in normal controls decreased with increasing age (13), which is different from the PEDF level in the plasma of the controls. This difference may be because the retina is isolated from the blood circulation by the blood-retinal barrier, and the PEDF in the eye is synthesized mainly by the retinal pigment epithelial cells. The synthesis of PEDF may decrease with increasing age in normal eyes.

We found that the plasma level of PEDF was significantly higher in the diabetic group than that in controls. HbA_1c and insulin treatment were not significantly related to the PEDF levels, and the reason for this remains unknown. The PEDF level in men was higher than that in women in diabetic patients, and the reason for this difference is also still unknown, but the hormonal environment may affect the level. It is very interesting that blood urea nitrogen and triglycerides were significantly and independently correlated with plasma PEDF levels in diabetic patients. A recent study reported high levels of PEDF in the serum of patients with end-stage renal disease (14), which is compatible with our results.

PEDF is synthesized in adipose tissue, and its level is down-regulated during the differentiation of cells to mature adipocytes (15). This expression pattern is opposite to that of adiponectin, and therefore, the PEDF expression in adipocytes was suggested to increase with obesity or insulin resistance in contrast to that of adiponectin (16). In addition, Yamagishi et al. (17) reported that the PEDF levels were higher in proportion to the number of components of the metabolic syndrome. From these observations, they suggest that the serum PEDF concentrations may increase as a system to counteract against the coronary risk factors in the metabolic syndrome.

Considering that PEDF was high in diabetic patients and, together with these earlier results, similar mechanisms may be present in diabetes mellitus. Our findings indicate that PEDF is most likely associated with the metabolism in patients with diabetes mellitus and may be associated with vascular damage. Vascular endothelial growth factor (VEGF) is a strong angiogenic factor, and many studies have demonstrated that VEGF induces the progression of diabetic retinopathy. Advanced glycation end products (AGEs) in diabetic patients are also involved in the leukostasis and microthrombosis that result in PDR. It has been suggested that PEDF counteracts the effects of VEGF (18), and it also been suggested that PEDF significantly inhibits AGE activity (19). Thus, increased levels of PEDF in the blood of patients with the PDR may be a response to counteract the activity of VEGF and AGEs.

Previous studies demonstrated that the level of PEDF was lower in eyes with diabetic retinopathy, especially in eyes with PDR (5, 6, 7, 8). These findings indicated that the decrease of PEDF in the eyes might be involved in the progression of diabetic retinopathy and the degree of retinal neovascularization. However, the results of our present study showed that the plasma PEDF levels were significantly higher in patients with PDR. Furthermore, studies with paired sets of plasma and ocular PEDF levels in diabetic patients may reveal the correlation more clearly.

In summary, the PEDF level in the blood is elevated in diabetic patients, especially in those with PDR.

	Footnotes

 
This study was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education in Japan and by the Japan Association for Inhibition of Blindness.

Disclosure Summary: The authors have nothing to disclose.

First Published Online January 9, 2007

Abbreviations: AGE, Advanced glycation end product; HbA_1c, glycosylated hemoglobin; M-NPDR, mild to moderate non-PDR; NDR, no apparent diabetic retinopathy; PDR, proliferative diabetic retinopathy; PEDF, pigment epithelium-derived factor; S-NPDR, severe non-PDR; VEGF, vascular endothelial growth factor.

Received October 17, 2006.

Accepted December 28, 2006.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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