HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 20/2/323 05-4313fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, S. X. Articles by Ma, J.-x. Search for Related Content PubMed PubMed Citation Articles by Zhang, S. X. Articles by Ma, J.-x.

Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor

Sarah X. Zhang^*, Joshua J. Wang^*, Guoquan Gao, Chunkui Shao, Robert Mott^* and Jian-xing Ma^*,1

^* Department of Medicine Endocrinology, Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA; and
Department of Biochemistry, Zhongshan University, Guangzhou, China

¹ Correspondence: 941 Stanton L. Young Blvd., BSEB 328B, Oklahoma City 73104, OK, USA. E-mail: jian-xing-ma{at}ouhsc.edu

SPECIFIC AIMS

Diabetic retinopathy (DR) is a common microvascular complication of diabetes and a leading cause of adult blindness, characterized by diabetic macular edema (DME) and retinal neovascularization (NV). Pigment epithelium-derived factor (PEDF) is a neurotrophic factor and potent angiogenic inhibitor. In diabetic patients with active retinopathy and DME, decrease of PEDF in ocular fluid predicts the progression of DR, suggesting that PEDF is involved in the formation of DR. However, the mechanism is not yet understood. In recent yeas, accumulating evidence shows that inflammation plays an important role in the pathogenesis of DR. We hypothesize that inflammation could be a possible link between the decrease of PEDF and its implication in DR. The aim of the present study is to determine the antiinflammatory effect of PEDF and its therapeutic potential in DR.

PRINCIPAL FINDINGS

1. Decrease of PEDF levels in the retina and plasma in a rat model of endotoxin-induced uveitis (EIU)
Using Western blot analysis and ELISA, we demonstrated that PEDF levels in the retina were drastically decreased in EIU rats at 24 h after lipopolysaccharide injection, coincident with the increase of retinal MCP-1 and TNF- (P<0.05, n=8–11). PEDF level in the plasma was also significantly decreased in EIU rats when compared with that in control rats (P<0.05, n=6), while plasma MCP-1 and TNF- levels (P<0.05, n=6) were significantly elevated. These findings suggest that PEDF is a negative acute phase protein.

2. Reduction of retinal vascular permeability by PEDF in rats with oxygen-induced retinopathy (OIR) and diabetes
Using the Evans blue method, we demonstrated that intravitreal injection PEDF at doses of 3, 1.5, 0.75, and 0.375 µg/eye significantly reduced retinal vascular permeability in a PEDF dose-dependent manner in rats with OIR (paired t test, P<0.05, n=4). After a single intravitreal injection, the effect of PEDF on permeability peaked at 48 h and disappeared after 3 days. Western blot analysis showed that the retinal level of occludin, one of the major tight junction proteins was increased in PEDF-injected eyes, suggesting PEDF is protective for the tight junction. In 2- and 4-wk streptozotocin-induced diabetic rats, retinal vascular permeability was significantly reduced in PEDF-injected eyes, coincident with the increase of occludin levels in the retina.

3. PEDF inhibited VEGF-induced endothelial monolayer hyperpermeability
In cultured retinal capillary endothelial cells (RCEC), monolayer endothelial permeability was measured after incubation with VEGF (100 ng/mL) in presence or absence of PEDF (100 nM) for 24 h. The result showed that permeability was significantly increased by VEGF and blocked by PEDF (P<0.05, n=3). Immunocytochemistry analysis showed that in endothelial cells exposed to VEGF, the tight junction proteins occludin and ZO-1 were drastically decreased and redistributed from the plasma membrane to the cytoplasm. PEDF dramatically ameliorated VEGF-induced tight junction protein dysregulation. These results suggested that PEDF inhibited VEGF-induced endothelial permeability and BRB breakdown, at least partially, by blocking dysregulation of tight junction protein.

4. Decrease of retinal cytokine levels and VEGF, VEGF receptor 2 levels by PEDF in OIR and diabetes
As inflammation is recognized as a key process in vascular permeability, we hypothesized that the effect of PEDF on reducing permeability was through its antiinflammatory effect. To test this hypothesis, we first determined PEDF’s effect on the regulation of retinal TNF-, MCP-1 and sICAM-1, which have been shown as the important inflammatory factors inducing leukocyte-endothelial interaction and subsequent vascular permeability increase. In both OIR and diabetic rat models, at 48 h after treatment, retinal MCP-1, TNF-, and sICAM-1 levels were significantly decreased in PEDF-injected eyes when compared with that in PBS-injected eyes (Fig. 1 , paired t test, P<0.05, n=6–11). Retinal levels of VEGF, a potent vascular permeability factor and a proinflammatory factor, as analyzed by Western blot and immunohistochemistry, were drastically decreased by PEDF. The retinal VEGF receptor 2 (KDR) levels were also significantly reduced in PEDF-treated eyes (paired t test, P<0.05, n=6–10). These results suggest that the effect of PEDF on reducing retinal vascular permeability is mediated, at least in part, by inhibition of retinal inflammation.

View larger version (24K): [in this window] [in a new window]   Figure 1. Retinal levels of MCP-1, TNF- and ICAM-1 after intravitreal injection of PEDF in rats with OIR and diabetes. In rats with OIR, intravitreal injection of PEDF (3 µg/eye) was given at P14 and the retinas were dissected at P16. PBS injection into the contralateral eye was used as the control. Retinal MCP-1 (A), TNF- (B) and ICAM-1 (C) levels were measured by ELISA. In 2-wk diabetic rats, retinas were dissected at 48 h after intravitreal injection of PEDF (3 µg/eye) and retinal MCP-1 (D), TNF- (E), and ICAM-1 (F) levels were measured by ELISA. Results were normalized by total protein concentration in the retinas and expressed as pg/mg protein (mean±SD, n=6–11). *P < 0.05, **P < 0.01.

We determined the effects of PEDF on inflammation in cultured RCEC. Using Western blot analysis, we demonstrated that ICAM-1 expression was significantly up-regulated after exposure to hypoxia for 24 h. PEDF at doses of 40 and 160 nM drastically inhibited up-regulation of ICAM-1. TNF- secretion determined by ELISA was also increased under hypoxia condition. PEDF treatment at doses of 10, 40, and 160 nM significantly inhibited the increase of TNF- secretion in a dose-dependent manner (P<0.05, n=3). These results confirmed the antiinflammatory effects of PEDF and suggested that PEDF directly inhibited that inflammatory response to hypoxia.

5. Silencing of PEDF increased VEGF and TNF- secretion in cultured retinal Müller cells
Because PEDF is an endogenous protein in the retina, we investigated the effect of decreased PEDF expression on the regulation of inflammatory factors. Retinal Müller cells were used in this experiment as they are the major source of VEGF and also produce a variety of inflammatory factors such as TNF- and MCP-1. Results showed that after silencing of PEDF, which was confirmed by the dramatic decrease of PEDF level, secretion of TNF- (Fig. 2 , P<0.05, n=3) and VEGF (Fig. 2 , P<0.01, n=3) was significantly increased when compared with controls. These results suggest that down-regulation of PEDF expression alone is sufficient to result in overexpression of proinflammatory factors, and thus, PEDF is an endogenous antiinflammatory factor.

View larger version (20K): [in this window] [in a new window]   Figure 2. Down-regulation of PEDF expression by siRNA in rat retinal Müller cells. Rat retinal Müller cells were transfected with PEDF siRNA. Protein levels of PEDF (A), TNF- (B), and VEGF (C) in the culture medium were measured by ELISA 24 h after the transfection. Values statistically different from the controls: *P < 0.05, **P < 0.01. Results represent 3 independent experiments.

CONCLUSIONS AND SIGNIFICANCE

PEDF is a major endogenous angiogenic inhibitor in the eye and has been well studied in ocular neovascular diseases, such as DR, OIR, and ptyregia. Recently, PEDF was shown to reduce VEGF-induced vascular leakage, implying its involvement in the regulation of vascular permeability. However, the mechanisms underlying the effects of PEDF on the retinal NV and permeability are largely unclear. The present study for the first time demonstrated that PEDF is a negative acute phase protein. In the rat retina and cultured retinal cells, PEDF operated as a regulator of inflammatory factors and suppressed endothelial permeability by protecting tight junction proteins. These findings suggest that PEDF is an endogenous antiinflammatory factor that may be actively involved in the pathogenesis of DR.

Hypoxia-induced retinal vascular leakage or blood-retinal barrier (BRB) breakdown are important pathophysiological features in DR and other retinal neovascular diseases, such as OIR. Accumulating evidence has shown that inflammation is the key factor mediating BRB breakdown in DR and OIR, as well as EIU. Our results showed that an intravitreal injection of low dose of PEDF significantly reduced retinal vascular permeability in both DR and OIR, with a coincide increase of endothelial tight junction proteins. Retinal inflammatory factors, including ICAM-1, TNF-, and MCP-1 levels were significantly lower in PEDF-injected eyes. In cultured retinal endothelial cells, PEDF also drastically decreased hypoxia-induced ICAM-1 expression and TNF- production, suggesting that PEDF directly inhibited inflammatory responses in endothelial cells. In retinal Müller cells, silencing of PEDF using siRNA resulted in significant increase of TNF- production, indicating that PEDF is a negative regulator of retinal inflammatory factors. These results suggest that PEDF is a potent endogenous inflammatory inhibitor. The decrease of PEDF in diabetic and hypoxic retina contributes to the inflammatory status of retina.

VEGF is a potent vascular permeability factor and a proinflammatory factor mediating hypoxia-and diabetes-induced inflammation. In the present study, we demonstrated that PEDF not only reduced retinal VEGF levels, but also decreased VEGFR-2 levels in OIR and DR, suggesting that the effect of PEDF on reducing inflammation and permeability may be related to VEGF inhibition. Our in vitro studies showed that in retinal Müller cells, silencing of PEDF resulted in the significant increase of VEGF production. In cultured retinal endothelial cells, PEDF decreased VEGF-induced permeability by inhibiting dysregulation of tight junction proteins. These findings suggest that PEDF, as a counterpart of VEGF, may inhibit VEGF expression and function at multiple levels.

Our findings suggest that there are endogenous inhibitors of inflammation in the retina and that decreased levels of these antiinflammatory proteins contribute to retinal inflammation in diabetes, which represent a new pathogenic mechanism for diabetic retinopathy (Fig. 3 ). This study also revealed a new target for drug intervention of diabetic retinopathy (i.e., boosting endogenous antiinflammatory factors).

View larger version (37K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating endogenous antiinflammatory factors and diabetic retinopathy. This diagram depicts the existence of endogenous antiinflammatory factors, such as PEDF. In diabetes, proinflammatory factors such as VEGF increase; and antiinflammatory factors decrease. This imbalance leads to dysregulation of inflammatory factors, including TNF-, MCP-1, and ICAM-1, which further cause the leukostasis and subsequent vascular damage, such as the increase of vascular permeability and capillary loss.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4313fje;

This article has been cited by other articles:

				A. Nehme and J. Edelman Dexamethasone Inhibits High Glucose-, TNF-{alpha}-, and IL-1{beta}-Induced Secretion of Inflammatory and Angiogenic Mediators from Retinal Microvascular Pericytes Invest. Ophthalmol. Vis. Sci., May 1, 2008; 49(5): 2030 - 2038. [Abstract] [Full Text] [PDF]

				J. J. Wang, S. X. Zhang, R. Mott, Y. Chen, R. R. Knapp, W. Cao, and J.-x. Ma Anti-inflammatory effects of pigment epithelium-derived factor in diabetic nephropathy Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1166 - F1173. [Abstract] [Full Text] [PDF]

				Y. Behl, P. Krothapalli, T. Desta, A. DiPiazza, S. Roy, and D. T. Graves Diabetes-Enhanced Tumor Necrosis Factor-{alpha} Production Promotes Apoptosis and the Loss of Retinal Microvascular Cells in Type 1 and Type 2 Models of Diabetic Retinopathy Am. J. Pathol., May 1, 2008; 172(5): 1411 - 1418. [Abstract] [Full Text] [PDF]

				Y. Yoshida, S.-i. Yamagishi, T. Matsui, K. Nakamura, T. Imaizumi, K. Yoshimura, and R. Yamakawa Positive correlation of pigment epithelium-derived factor and total antioxidant capacity in aqueous humour of patients with uveitis and proliferative diabetic retinopathy Br. J. Ophthalmol., September 1, 2007; 91(9): 1133 - 1134. [Abstract] [Full Text] [PDF]

				S. Mizuno, A. Nishiwaki, H. Morita, T. Miyake, and Y. Ogura Effects of Periocular Administration of Triamcinolone Acetonide on Leukocyte-Endothelium Interactions in the Ischemic Retina Invest. Ophthalmol. Vis. Sci., June 1, 2007; 48(6): 2831 - 2836. [Abstract] [Full Text] [PDF]

				Y. Yoshida, S.-i. Yamagishi, T. Matsui, K. Nakamura, T. Imaizumi, K. Yoshimura, and R. Yamakawa Positive correlation between pigment epithelium-derived factor and monocyte chemoattractant protein-1 levels in the aqueous humour of patients with uveitis Br. J. Ophthalmol., June 1, 2007; 91(6): 737 - 738. [Abstract] [Full Text] [PDF]

				Y. Yoshida, S.-i. Yamagishi, T. Matsui, K. Nakamura, T. Imaizumi, K. Yoshimura, and R. Yamakawa Increased levels of pigment epithelium-derived factor in aqueous humor of patients with uveitis Br. J. Ophthalmol., February 1, 2007; 91(2): 149 - 150. [Abstract] [Full Text] [PDF]

				C. M. C. Batista, T. E. Kippin, S. Willaime-Morawek, M. K. Shimabukuro, W. Akamatsu, and D. van der Kooy A Progressive and Cell Non-Autonomous Increase in Striatal Neural Stem Cells in the Huntington's Disease R6/2 Mouse J. Neurosci., October 11, 2006; 26(41): 10452 - 10460. [Abstract] [Full Text] [PDF]

				P. Zamiri, S. Masli, J. W. Streilein, and A. W. Taylor Pigment epithelial growth factor suppresses inflammation by modulating macrophage activation. Invest. Ophthalmol. Vis. Sci., September 1, 2006; 47(9): 3912 - 3918. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 20/2/323 05-4313fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, S. X. Articles by Ma, J.-x. Search for Related Content PubMed PubMed Citation Articles by Zhang, S. X. Articles by Ma, J.-x.