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Elevated Serum Levels of Pigment Epithelium-Derived Factor in the Metabolic Syndrome

Department of Internal Medicine III (S.Y., H.A., M.E., K.F., A.H., Y.J., K.T., T.M., K.N., T.I.), Kurume University School of Medicine, Kurume 830-0011, Japan; and Cytokine Section (A.A., T.Y.), SRL Inc., Kanagawa 229-1125, Japan

Address all correspondence and requests for reprints to: Dr. Sho-ichi Yamagishi, Department of Internal Medicine III, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. E-mail: shoichi{at}med.kurume-u.ac.jp.

	Abstract

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Context: Pigment epithelium-derived factor (PEDF), a potent inhibitor of angiogenesis with neuronal differentiating activity, inhibits endothelial cell injury in vitro, thus suggesting the involvement of PEDF in atherosclerosis. Therefore, elucidating the relationship between serum levels of PEDF and coronary risk factors could provide a clue to understanding the pathophysiological role of PEDF in vivo.

Objective: We examined whether serum levels of PEDF were associated with risk factors for coronary artery disease.

Design: The study was designed as a cross-sectional study.

Setting: The study was set within the general community.

Patients or Other Participants: A total of 196 general Japanese residents (age 65.7 ± 9.3 yr; 71 males and 125 females) without clinical evidence of coronary or peripheral arterial occlusive diseases were enrolled in this study.

Results: PEDF showed a normal distribution, ranging from 8–24 µg/ml, with a mean of 14.6 ± 3.2 µg/ml. Multivariate analyses revealed that uric acid (P < 0.001), waist circumference (P = 0.009), insulin (P = 0.019), and triglycerides (P = 0.028) were significant independent determinants of serum PEDF levels. Age- and uric acid-adjusted PEDF levels were significantly higher (P = 0.048 for men and P = 0.007 for women) in proportion to the accumulation of the number of the components of the metabolic syndrome.

Conclusions: The present study reveals that serum levels of PEDF are strongly associated with the metabolic syndrome. Our results suggest that serum PEDF levels may be elevated as a counter-system in the metabolic syndrome.

	Introduction

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PIGMENT EPITHELIUM-DERIVED FACTOR (PEDF), a glycoprotein that belongs to the superfamily of serine protease inhibitors, was first purified from the conditioned media of human retinal pigment epithelial cells as a factor that possesses potent neuronal differentiating activity (1). Recently, PEDF has been shown to be a highly effective inhibitor of angiogenesis in cell culture and animal models; PEDF inhibited retinal endothelial cell (EC) growth and migration and suppressed ischemia-induced retinal neovascularization (2, 3). In addition, PEDF has been found in vitreous, and its levels were decreased in angiogenic eye diseases, suggesting that loss of PEDF in the eye is functionally important in the pathogenesis of proliferative diabetic retinopathy (4).

We have very recently found that PEDF inhibits TNF--induced nuclear factor-B activation and subsequent IL-6 expression in EC by suppressing nicotinamide adenine dinucleotide phosphate oxidase-mediated reactive oxygen species generation (5). PEDF also prevented advanced glycation end product- or angiotensin II-induced EC activation through its anti-oxidative properties (6, 7). These observations suggest that PEDF may play a protective role in the pathogenesis of atherosclerosis. However, because a commercially available kit is not suitable for the measurement of serum PEDF, serum levels of PEDF are not known in subjects with coronary risk factors.

In this study, we developed a simple, reliable, and specific ELISA system for human serum PEDF and then examined whether serum levels of PEDF were associated with risk factors in 196 subjects without overt cardiovascular diseases.

	Subjects and Methods

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Subjects

In 2004, in a fishing community in southwestern Japan (Uku town), a total of 204 general residents without cancers or inflammatory diseases received a health examination. Of them, eight rejected the blood test. Finally, a complete data set for 196 subjects (71 males and 125 females) on an overnight fast was available in this study.

Data collection

The medical history and use of tobacco and alcohol were ascertained by a questionnaire. Smoking and alcohol were classified as current habitual use or not. Height and weight were measured, and body mass index (BMI; kilograms per meter squared) was calculated as an index of obesity. Waist circumference was measured at the level of the umbilicus in the standing position. Blood pressure (BP) was measured in the sitting position (first) and supine position (second) at a 3-min interval using an upright standard sphygmomanometer. Vigorous physical activity and smoking were avoided for at least 30 min before BP measurement. The second BP measurement with the fifth phase diastolic pressure was used for analysis.

Blood was drawn from the antecubital vein for determinations of lipids [total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein cholesterol, and triglycerides], fasting plasma glucose, glycosylated hemoglobin, insulin, creatinine, uric acid, total protein, and PEDF levels in the morning after 12-h fast. Serum PEDF levels were measured with the ELISA as described below. Other chemistries (enzymatic assay method) were measured at a commercially available laboratory (The Kyodo Igaku Laboratory, Fukuoka, Japan). Creatinine clearance was estimated with the Cockcroft-Gault equation (8). The mayor and the welfare section of Uku town approved this study. Informed consent was obtained, and the study protocol was also approved by the Institutional Ethics Committee of Kurume University School of Medicine.

We defined the metabolic syndrome according to Adult Treatment Panel III (ATP III). ATP III identified five components of the metabolic syndrome [abdominal obesity, given as waist circumference (>101.6 cm, men; >88.9 cm, women), triglycerides (150 mg/dl), HDL cholesterol (<50 mg/dl, men; <40 mg/dl, women), BP (130/85 mm Hg), and fasting plasma glucose (110 mg/dl)] (9). However, Japanese are much smaller than Caucasians; therefore, it is not appropriate to use the criteria of abdominal obesity of ATP III (10). Because the International Diabetes Foundation recently published new criteria that modified the ATP III definition in Japanese, we adopted greater than 85 cm for men and greater than 90 cm for women for waist circumference, as proposed by the International Diabetes Foundation (11).

Assay for serum PEDF

Serum PEDF measurements were performed with the competitive ELISA on the following procedures. Briefly, 50 µl of serum was pretreated with 200 µl of 8 M urea for 1 h. Then 100-µl aliquots of standard recombinant human PEDF proteins (Chemicon International, Temecula, CA) or 50-fold diluted serum were added to the wells precoated with an anti-PEDF monoclonal antibody (Transgenic, Kumamoto, Japan). After 2 h, 50 µl of biotinylated antihuman PEDF polyclonal antibody (R&D Systems, Minneapolis, MN) was added to each well, and then the plate was read at 450 nm using a microplate reader. Interassay (n = 17) and intraassay (n = 14) coefficients of variation of the ELISA were 4.7 and 7.3%, respectively. Recovery of the added recombinant PEDF in serum was 94.2 ± 1.7% (mean ± SD). The assay linearity was shown intact with serial dilution of serum. We confirmed that concentrations of serum PEDF (n = 5) were similar to those of platelet-poor plasma (12.1 ± 3.3 vs. 11.2 ± 2.8 µg/ml) and that PEDF was actually present in such high levels by using Western blots (Fig. 1A).

View larger version (25K): [in this window] [in a new window]   FIG. 1. A, Western blots of recombinant PEDF protein or subject serum. Recombinant PEDF protein (control; 10 µg/ml) or subject serum (lanes 1–3) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The membrane was then treated with anti-PEDF monoclonal antibody. The resultant immunocomplexes were visualized with an enhanced chemiluminescence system. B, Age- and uric acid-adjusted means of PEDF levels stratified by the number of components of the metabolic syndrome.

Because of skewed distributions, the natural logarithmic transformations were performed for triglycerides, glucose, and insulin. Mean values with upper and lower 95% confidence intervals (CI) were exponentiated and presented as geometric mean ± SD. SD was approximated as the difference of the exponentiated CI divided by 3.92, which is the number of SD in a 95% CI where data are normally distributed. Results were presented as mean ± SD. The medications for hypertension, hyperlipidemia, and diabetes were coded as dummy variables. Univariate analysis was performed for determinants of serum PEDF levels. To determine final parameters of PEDF levels, multiple stepwise linear regression analysis was performed. Statistical significance was defined as P < 0.05.

	Results

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Demographic data

Demographical data of the subjects are presented in Table 1. Most of them were normotensive, nonobese, nondiabetic, and normolipidemic. The prevalence of alcohol intake and smoking was higher in men than in women. PEDF levels were normally distributed both in men and women. Average serum levels of PEDF in men were significantly higher than in women (15.4 ± 3.2 µg/ml vs. 14.1 ± 3.1 µg/ml).

Univariate analysis revealed significant correlations with BMI (P < 0.001), waist circumference (P < 0.001), HDL cholesterol (P < 0.001, inversely), triglycerides (P < 0.001), glucose (P = 0.013), insulin (P < 0.001), uric acid (P < 0.001), total protein (P = 0.015), and diabetes mellitus medication (P = 0.049). High sensitive C-reactive protein levels had a tendency to be correlated with serum PEDF levels (P = 0.075). For these significant factors, we performed a stepwise multiple regression analysis. Uric acid (P < 0.001), waist circumference (P = 0.009), insulin (P = 0.019), and triglycerides (P = 0.028) remained significant and were independently related to serum PEDF levels (R² = 0.239). Because serum levels of PEDF were significantly higher in men than in women, multivariate analyses were adjusted for gender. Triglycerides (P = 0.006) were independently related to PEDF levels in men, whereas uric acid (P < 0.001), waist circumference (P = 0.010), and insulin (P = 0.028) were related to PEDF levels in women. Mean PEDF levels stratified by the number of components of the metabolic syndrome (0, 1, 2, and more than 3) were compared using analysis of covariance adjusted for age and uric acid. A linear and significant trend (P = 0.048 for men and P = 0.007 for women) was demonstrated (Fig. 1B).

	Discussion

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The salient findings of this study are that PEDF levels were not only associated with the components of the metabolic syndrome but also higher in proportion to the accumulation of the number of the components.

Univariate analysis revealed that PEDF was associated with several coronary risk factors. However, it may be interesting to note that strong well-known risk factors such as age, BP, cholesterol, and smoking were not related to PEDF. And factors related to visceral obesity and insulin resistance such as BMI, waist circumference, HDL cholesterol, triglycerides, glucose, insulin, and uric acid are rather related to PEDF. Furthermore, multiple stepwise regression analysis revealed that uric acid, waist circumference, insulin, and triglycerides were significantly and independently related to serum PEDF levels. Although elevated serum uric acid is a common finding in patients with insulin resistance (12), it was not a component of the metabolic syndrome defined by ATP III (9). Therefore, we performed analysis of covariance after adjusting for uric acid (Fig. 1B). PEDF levels were significantly higher in both men and women in proportion to the accumulation of the number of the components of the metabolic syndrome. In the present study, we enrolled subjects without clinical evidence of coronary or peripheral arterial occlusive diseases to exclude the influence of tissue ischemia and hypoxia, factors to suppress PEDF production (2, 4). Therefore, it is unlikely that these factors could confound the association between PEDF and the metabolic syndrome in our subjects.

The mean PEDF level in the serum was 15 µg/ml in this study. Because the total protein in our population (Table 1) was 7.3 g/dl, PEDF was one of the most abundant circulating proteins in humans, accounting for 0.02% of total circulating proteins (13). The presence of PEDF transcripts in a broad range of adult human tissues, especially in liver, may account for such high levels of PEDF (14). Furthermore, recently, the expression level of PEDF in porcine liver has been shown to be associated with body muscularity and obesity (15). These observations suggest that liver may be a main origin of PEDF in circulation. In addition, it should be noted that PEDF is synthesized in adipose tissue and its level is down-regulated during the differentiation process to mature adipocytes (16). Because this expression pattern is opposite to that of adiponectin, adipocyte PEDF expression may be increased in obesity or insulin resistance in contrast to the case of adiponectin (17).

Although a commercially available PEDF ELISA kit can be obtained from Chemicon International, it is good for measuring PEDF of vitreous fluid, but not for serum. Therefore, in this study, we developed a modified ELISA method for measuring human serum PEDF. In this study, we demonstrated that our modified method was simple, specific, reliable, and suitable. In this assay, we found that pretreatment of serum with 8 M urea, whose process is lacking in a Chemicon kit, was indispensable for measuring human serum PEDF; serum PEDF levels detected by the commercial kit are three orders of magnitude lower (10–30 ng/ml) than those by our ELISA system. These observations suggest that most of circulating PEDF in serum may exist as a protein-bound form.

This study was cross-sectional and thus could not assess the question of whether elevation of PEDF was a cause or consequence of the metabolic syndrome. However, we have very recently found that administration of PEDF proteins could improve obesity in diabetic rats (Matsui, T., S. Yamagishi, K. Nakamura, and T. Imaizumi, unpublished data). Therefore, although clinical and pathophysiological significance of elevated serum PEDF levels in the metabolic syndrome remains to be clarified, given the fact that PEDF possesses antioxidant and antiinflammatory properties, serum PEDF levels may be elevated as a counter-system in the metabolic syndrome. Future longitudinal and/or interventional studies are needed to resolve these issues.

	Footnotes

 
This work was supported in part by Grants of Collaboration with Venture Companies Project from the Ministry of Education, Culture, Sports, Science and Technology, Japan (S.Y.) and by grants from the Kimura Memorial Heart Foundation, Kurume, Japan (S.Y. and H.A.).

First Published Online March 7, 2006

Abbreviations: BMI, Body mass index; BP, blood pressure; CI, confidence interval; EC, endothelial cell; HDL, high-density lipoprotein; PEDF, pigment epithelium-derived factor.

Received December 7, 2005.

Accepted March 1, 2006.

	References

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