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Department of Medicine, Department of Cell Biology, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd., Oklahoma City, Oklahoma, USA
1 Department of Biochemistry, Zhongshan University, Guangzhou, China
(Requests for offprints should be addressed to J-x Ma; Email: jian-xing-ma{at}ouhsc.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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VEGF is a major angiogenic factor, playing a crucial role in normal and pathological angiogenesis (Pe’er et al. 1995, Aiello 1996, Aiello & Wong 2000, Dvorak 2000). VEGF is also referred to as vascular permeability factor (VPF) based on its ability to induce vascular hyper-permeability (Dvorak et al. 1995). In the early stage of streptozotocin (STZ)-induced diabetes, significant increases in retinal VEGF levels have been found to correlate with retinal vascular permeability (Antonetti et al. 1998). This early BRB breakdown can be successfully prevented by VEGF TrapA40, a soluble VEGF receptor fms-like tyrosine kinase (Flt)/Fc chimera (Qaum et al. 2001). In the oxygen-induced retinopathy (OIR), a commonly used model for proliferative diabetic retinopathy (PDR), retinal VEGF levels are significantly elevated, which correlates with the retinal NV progression (Gao et al. 2002b). The VEGF levels decline to the normal level when the regression of NV occurs (Gao et al. 2002b). These findings suggest that VEGF is a key mediator of both vascular hyper-permeability and retinal NV.
PEDF is a neurotrophic factor and a potent angiogenic inhibitor, originally identified from cultured retinal pigment epithelial (RPE) cells (Tombran--Tink et al. 1991, Dawson et al. 1999, Tombran-Tink & Barnstable 2003a). Previously, we have demonstrated that, in OIR rats, PEDF levels in the retina are significantly decreased in contrast to the increased VEGF levels, which leads to an increased retinal VEGF/PEDF ratio (Gao et al. 2001). This disturbed balance correlates with the formation and progression of retinal NV (Gao et al. 2001, 2002b). The decrease of PEDF is also observed in the vitreous and aqueous humor of the patients with active DR and diabetic macular edema (DME) (Ogata et al. 2001b, 2002, Boehm et al. 2003). After successful laser treatment, the PEDF levels in the vitreous were elevated, correlating with the regression of retinal NV (Ogata et al. 2001a).
Although the functions of PEDF and VEGF have been well studied in the eye, the interactions between these two counteracting angiogenic factors are largely unclear. Based on the solid evidence showing the inverse correlation between PEDF and VEGF levels in a variety of vascular diseases, we hypothesize that there exists a reciprocal regulation between these two major angiogenic regulators (Gao et al. 2001, Ohno-Matsui et al. 2001, Doll et al. 2003, Kim et al. 2003). In this study, we tested this hypothesis and explored the mechanisms by which PEDF regulates VEGF in retinal cells.
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Materials and methods |
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Brown Norway (BN) rats were purchased from Charles River Laboratories (Wilmington, MA, USA). Care, use and treatment of all animals in this study were in strict agreement with the guidelines in the Care and Use of Laboratory Animals set forth by the University of Oklahoma.
Experimental reagents
Recombinant human PEDF was purchased from BioProducts MD, LLC (Middletown, MD, USA). Recombinant human VEGF165 was purchased from PeproTech Inc. (Rocky Hill, NJ, USA). CoCl2 and BSA were purchased from Sigma.
Development of OIR rat model
OIR rat model was established as described previously (Gao et al. 2001, Zhang et al. 2004, 2005b). Briefly, newborn BN rats were exposed to 75% oxygen at postnatal day 7 (P7) for 5 days and returned to room air to develop retinopathy. The intravitreal injection was given at P14 and the eyeball was enucleated at P16.
Cell culture
Primary bovine retinal capillary endothelial cells (RCEC) were isolated and cultured in DMEM supplied with 10% human serum as described previously (Gao et al. 2003). A rat Müller cell line (rMC-1) was also used in the experiments. After reaching 80% confluence, cells were exposed to serum-free medium for 12 h before the treatments with desired reagents.
Western blot analysis of VEGF and PEDF
Western blot analysis was performed as described previously (Gao et al. 2002a). Briefly, 50 µg total protein from each sample were blotted with primary antibodies: anti-VEGF (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-PEDF (1:1000, Chemicon Inc., Temecula, CA, USA). The same membrane was stripped and reblotted with an anti-ß-actin antibody for normalization.
Quantification of VEGF and PEDF by ELISA
The protein concentration was measured with the BioRad DC protein assay. The concentrations of VEGF and PEDF in the cell culture medium were measured using ELISA kits specific for VEGF (R&D Systems, Minneapolis, MN, USA) and PEDF (Chemicon Inc.) according to the manufacturers’ instructions.
siRNA transfection in rat retinal Müller cells
PEDF siRNA was synthesized using the silencer siRNA construction kit (Ambion, Austin, TX, USA). The following DNA templates were used to synthesize double-stranded PEDF, siRNA: sense, GGA UUU CUA CUU GGA UGA ATT, and anti-sense, UUC AUC CAA GUA GAA AUC. Transfection was performed with the siPORT lipid transfection reagent (Ambion) following the manufacturer’s instructions.
Semi-quantitative RT-PCR analysis
Total RNA was extracted using the RNeasy mini-isolation kit (Qiagen). PCR was performed as described previously (Shao et al. 2004), using PEDF-specific primers: forward primer, 5'-GTC ACT GCC CGC CAG AAG AAC-3', and reverse primer, 5'-TGT TAC CCA CTG CCC CTT GAA-3'. Specific PCR products were examined by electrophoresis in 1% agarose gel.
Real time RT-PCR analysis
Primers specific for VEGF (VEGF-F, 5'-ggccaactacaacttcttcc-3', VEGF-R, 5'-ccatcatggattcgagaaaa-3') were used for real-time RT-PCR. The PCR was performed using GeneAmp RNA PCR kit and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The average CT (threshold cycle) of fluorescence units was used to analyze the mRNA levels. The VEGF mRNA levels were normalized by 18 S ribosomal RNA levels. Quantification was calculated as: mRNA levels (percent of control) = 2
(CT) with CT = CT, VEGF–CT, 18S RNA and (CT) = CT, control–CT, treatment.
Transient transfection and dual luciferase assay
D407 cells, a cell-line derived from human RPE cells, were seeded onto 12-well culture plates at a density of 1.0 x 105 cells/well and grown for 24 h. The VEGF promoter-luciferase reporter in the pGal3 vector was transfected into the cells using Fugene 6 transfection reagent (Roche) according to the manufacturer’s instructions. In the case of co-transfection experiments, pRL-TK and pGal3-VEGF promoter constructs were co-transfected at a ratio of 3:2. Constructs of pGal3 (+) and pGal3 (–) were used as positive and negative control respectively. Eighteen hours following the transfection, the cells were exposed to 200 µM CoCl2 in the presence or absence of 160 nM PEDF for 6 h. Then the cells were washed with PBS, and lysed by passive lysis buffer (Promega). The cell lysate was collected and incubated with the luciferase assay reagent (Promega). Luminescence was measured by a microplate luminometer (Berthold Detection Systems, Pforzheim, Germany). The promoter activity was normalized by the Renilla reniformis luciferase from pRL-TK.
Analysis of hypoxia-induced factor-
(HIF-1) nuclear translocation
Cultured RCEC were trypsinized, counted and washed three times in PBS. Nuclear and cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic extraction kit (Pierce, Rockford, IL, USA) according to the manufacturer’s protocol. The same amount of nuclear and cytoplasmic proteins was applied to Western blot analysis using an anti-HIF-1 antibody (Santa Cruz Biotechnology, Inc.) at a 1:500 dilution.
Determination of MAP kinase activation
The total proteins from the cultured RCEC or dissected retinas from OIR rats were isolated on 12% SDS-PAGE. The phosphorylation of p42/p44 was measured using a phosphorylation-specific antibody (1:1000, Santa Cruz Biotechnology, Inc.) and normalized by the total p42/p44 levels.
VEGF-binding assay in retinal endothelial cells
VEGF (PeproTech) was labeled with 125I using the Chloromine T 125I labeling kit (ICN Pharmaceuticals, Inc., Costa Mesa, CA, USA) following the manufacturer’s protocol as described previously (Gotte et al. 2002, Gao et al. 2003). The same amount of 125I-VEGF (4 nM) was incubated with primary RCEC for 1 h. Unbound 125I-VEGF was removed by four washes. The cells were then lysed, and the 125I-VEGF bound to the cells was quantified by a 
-counter, calculated to molar concentration based on VEGF standards.
In vitro VEGF receptor-binding assay
Microtiter plates (Corning Inc., Corning, NY, USA) with 96 wells were coated with 5 nM carrier-free human recombinant VEGF receptor (KDR)/IgG Fc (R&D Systems) in PBS overnight at 4 °C. The wells were then washed three times with PBS containing 0.1% (v/v) Tween 20 (PBS-T) and blocked with 1% BSA in PBS for 2 h. The plates were then incubated for 2 h with 1 nM recombinant human VEGF165 in the presence of various concentrations of PEDF or BSA, ranging from 0.1 to 1 000 nM as control. After three washes, a biotinylated anti-VEGF antibody (R&D Systems) was added to the wells and incubated for another 2 h, followed by incubation with horseradish peroxidase-conjugated streptavidin for 20 min. Then the color reagents were added, and the results were measured by a microplate reader (Perkin-Elmer Wallac, Inc., Boston, MA, USA). All the experiments were carried out in triplicate and repeated three times.
Statistical analysis
Statistical analysis employed the Student’s t-test. Statistical difference was considered significant at P<0.05.
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Results |
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). In RCEC exposed to hypoxia, PEDF has also shown a potent inhibitory effect on VEGF expression in a PEDF concentration-dependent manner (Fig. 1B).
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Recombinant PEDF was added to and incubated with retinal Müller cells for 24 h in the presence of 200 µM cobalt chloride (CoCl2) or hypoxia (<5% O2 concentration). The cells were harvested by trypsinization and thoroughly washed by PBS. The intracellular PEDF levels were determined by western blot analysis. The results showed that, after the CoCl2 (Fig. 2A) or hypoxia (Fig. 2B) treatment, the PEDF expression was drastically decreased. Treatment with exogenous PEDF significantly upregulated the expression of endogenous PEDF in a concentration-dependent manner (Fig. 2A, B). Western blot analysis showed that the PEDF treatment downregulated VEGF expression in the same samples (Fig. 2B).
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The effect of PEDF on the expression of endogenous PEDF and VEGF was determined in the retina of rats with OIR. The PEDF used for intravitreal injection was expressed and purified from Escherichia coli, which has a molecular weight of 45 kDa, lower than native PEDF (50 kDa) due to the lack of glycosylation. At P14, PEDF was injected into the vitreous of one eye at 1 µg/eye and PBS was injected into the contralateral eye as control. At 48 h after the injection, PEDF and VEGF levels in the retina were determined by western blot analysis. The results showed that endogenous PEDF levels in the retina were significantly higher in the PEDF-injected eyes than in the control eyes (Fig. 3A). Endogenous PEDF had a different molecular weight compared with the recombinant PEDF for the injection, indicating that the elevated retinal PEDF levels are indeed from endogenous expression. The VEGF levels, in contrast, were drastically decreased in the PEDF-injected eyes compared with the contralateral control eyes (Fig. 3B).
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). The PEDF protein levels in the culture medium were also decreased as shown by ELISA (Fig. 4B). In the same cells, the mRNA and protein levels of VEGF were determined by quantitative real-time RT-PCR and ELISA respectively. The results showed that, after silencing of PEDF, the expression of VEGF was significantly increased at both the mRNA (Fig. 4C) and protein levels (Fig. 4D), suggesting that PEDF is an endogenous, negative regulator of VEGF expression in retinal Müller cells.
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P,<0.0001). PEDF at 160 nM completely blocked the increase in VEGF promoter activity induced by CoCl2, suggesting that the downregulation of VEGF by PEDF in hypoxia is at least partially through the inhibition of VEGF gene transcription (Fig. 5A P,<0.0001). Moreover, under normoxic conditions, PEDF had no effect on the VEGF promoter activity (Fig. 5A P,>0.15), suggesting that PEDF plays an important role in maintaining the normal level of VEGF in the retina.
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activation and protein kinase phosphorylation. HIF-1 is the major transcriptional factor mediating the regulation of VEGF expression (Hata et al. 1995). In cultured RCEC exposed to hypoxia with or without PEDF treatment for 24 h, the nuclear and cytoplasmic proteins were isolated from the treated cells. Western blot analysis showed that, under normal culture conditions, HIF-1 is almost undetectable in the nucleus (Fig. 5B
). Exposure to hypoxia for 24 h resulted in a significant increase in the nuclear HIF-1 level, indicating activation of HIF-1 induced by hypoxia (Fig. 5B). PEDF at 40 and 160 nM dramatically inhibited the hypoxia-induced HIF-1 nuclear translocation (Fig. 5B).
As the MAP kinase pathway is known to play a role in the VEGF regulation, we have determined the effect of PEDF on the activation of MAP kinase. Serum-deprived RCEC were stimulated with bFGF, and phosphorylation of p42/p44 was determined by western blot analysis. A 5-min challenge with basic fibroblast growth factor (bFGF) resulted in a two-fold increase in p42/p44 phosphorylation when compared with those in the unchallenged cells (data not shown). PEDF decreased the bFGF-induced p42/p44 phosphorylation by fourfold in RCEC (Fig. 5C). Similarly, we have also determined the effect of PEDF on MAP kinase activation in the retina. PEDF was injected into the vitreous of OIR rats at P14, and the retinas were dissected and pooled for MAP kinase phosphorylation assay at 48 h after the injection of PEDF. Consistent with the results in cultured RCEC, PEDF decreased p42/p44 phosphorylation two-fold in the retina of the rats with OIR (Fig. 5D).
PEDF competes with VEGF for binding to RCEC. Incubation of 125I-VEGF (4 nM) with RCEC for 1 h resulted in specific binding of VEGF to VEGF receptor (VEGFR) on RCEC. To determine the competition between PEDF and VEGF for VEGFR binding, the same amount of 125I-VEGF was added to RCEC together with increasing concentrations (40, 400, and 4000 nM) of unlabeled PEDF or plasminogen kringle 5 (K5), another potent angiogenic inhibitor. Unlabeled VEGF (400 nM) was used as a positive control. In the presence of excess amounts of unlabeled PEDF, VEGF bound to RCEC was decreased in a PEDF concentration-dependent manner (Fig. 6A). In contrast, K5 in the same concentration range did not inhibit VEGF binding with VEGFR, suggesting different mechanisms of action between PEDF and K5 (Fig. 6A), although they both reduce vascular permeability and inhibit retinal NV.
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). Moreover, the binding was not affected by the same concentrations of albumin (negative control) and heat-inactivated PEDF protein, suggesting that the competition of PEDF with VEGF on VEGFR2 binding is a specific effect (Fig. 6B).
VEGF decreased PEDF expression and secretion in RCEC but not in Müller cells. The effect of VEGF on the expression of PEDF was investigated in cultured RCEC and Müller cells. Western blot analysis showed that after 24-h incubation of RCEC with 40 ng/ml VEGF, the PEDF expression was drastically decreased (Fig. 7A). In retinal Müller cells, however, the same concentration of VEGF had no effect on the expression of PEDF (Fig. 7A). The same membrane was stripped and reblotted with an anti-VEGF antibody, which showed that VEGF induced a significant increase of endogenous VEGF expression in RCEC, but not in Müller cells (Fig. 7A and B), consistent with the cell type-specificity in its effect on PEDF expression. The effect of VEGF on the regulation of PEDF was further confirmed by measuring the PEDF secretion into the culture medium of RCEC. The ELISA result showed that VEGF decreased PEDF secreted from RCEC in a VEGF concentration-dependent manner (Fig. 7B). These results suggest that the effect of VEGF on the regulation of PEDF production and its autoregulation is endothelial cell-specific.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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VEGF is a potent VPF and a major angiogenic stimulator with endothelial cell-specific mitogenic activity and plays a crucial role in both normal and pathological angiogenesis (Dvorak et al. 1995, Pierce et al. 1995, Aiello 1996, Aiello et al. 1997). In diabetic patients with proliferative retinopathy, VEGF levels are significantly increased in the vitreous and the retina, and successful laser treatment decreased vitreous VEGF levels by 75% (Adamis et al. 1994, Aiello et al. 1994, Pe’er et al. 1995). In patients with DME, significantly elevated VEGF levels in the aqueous humor are correlated with the severity of macular edema (Funatsu et al. 2003). These findings suggest that over-production of VEGF in the retinal diabetics is a major cause of DME as well as retinal NV. In the retina, VEGF is produced by multiple cell types, including the RPE, pericytes, endothelial, glial, Müller, and ganglion cells (Pe’er et al. 1995, Dorey et al. 1996, Lu & Adamis 2002). Among them, Müller cells and RPE are believed to be the major source of VEGF in the retina, and endothelial cells to be the primary target of VEGF (Pierce et al. 1995, Dorey et al. 1996). Cultured vascular endothelial cells also express high levels of VEGF in an autocrine manner (Simorre--Pinatel et al. 1994). In the present study, we have investigated the effect of PEDF on the regulation of VEGF expression in cultured retinal Müller cells and RCEC. Our results demonstrated that, under normoxic and hypoxic conditions, PEDF significantly downregulated VEGF expression in both RCEC and Müller cells as well as in the retina of the OIR rat model. Moreover, in cultured RPE cells, PEDF inhibited the hypoxia-induced increase in VEGF promoter activity, but had no effect on VEGF promoter activity under normoxic conditions. These results suggested that PEDF is a negative regulator of VEGF expression in the retina. In the normal eye, the high levels of PEDF in the vitreous and the retina inhibits the over-expression of VEGF, which, in turn, maintains the quiescence of retinal vascular endothelial cells and the integrity of BRB. Under hypoxia and other pathological conditions, in addition to the upregulation of VEGF induced directly by the primary insults, the decreased PEDF levels also contribute to the over-production of VEGF. The fact that silencing of PEDF gene expression alone is sufficient to increase VEGF expression further supports the role of PEDF as an endogenous inhibitor of VEGF expression and suggests that the reduced PEDF levels contribute to over-production and subsequent activation of vascular endothelial cells.
HIF-1 is a major transcriptional factor regulating VEGF expression under hypoxia (Forsythe et al. 1996, Ozaki et al. 1999). The present study demonstrated that the nuclear HIF-1 level was elevated significantly in the retinal endothelial cells insulted by hypoxia, correlating with increased VEGF expression. Moreover, the total amount of HIF-1 was drastically increased under hypoxic conditions (data not shown). Incubation with PEDF significantly reduced the nuclear HIF-1 levels, indicating an inhibited HIF-1 nuclear translocation. These results suggest that inhibiting HIF-1 activation is responsible, at least partially, for the downregulated VEGF expression by PEDF. Furthermore, we determined if p42/p44 MAP kinase activation, an important signaling pathway in VEGF expression, is involved in the regulation of VEGF by PEDF (Milanini et al. 1998). Our results demonstrated that PEDF significantly inhibited the activation of p42/p44 in the retinal endothelial cells as well as in OIR retina, suggesting that the downregulation of VEGF by PEDF may be, at least in part, via inhibition of the MAP kinase activation. It is noteworthy that we have used a 24-h time scale in the experiments, as we found that the nuclear HIF-1 level was significantly higher at this time point. Although our results showed that PEDF inhibited MAPK activation and, thus, may directly block the downstream activation of HIF-1, we cannot exclude the possibility that this inhibitory effect of PEDF may be secondary, i.e. via blocking the positive feedback regulation of VEGF.
In addition to the regulation of VEGF expression, the present study, for the first time, demonstrated that PEDF effectively inhibited VEGF binding to VEGFR2 on RCEC and in isolated VEGFR2 in vitro. VEGFR2 is the major type of receptor responsible for the activity of VEGF on vascular permeability and angiogenesis (Aiello et al. 1995, Ozaki et al. 2000, Witmer et al. 2002). Several studies have shown that blockade of the activation of VEGFR2 induced by VEGF binding successfully abrogated retinal NV (Aiello et al. 1995, Ozaki et al. 2000). In the past decade, although PEDF has been well studied as a potent angiogenic inhibitor, the molecular mechanisms for its anti-angiogenic and anti-permeability activities are still uncertain. Our results demonstrated that PEDF significantly competed with the VEGF binding to VEGFR2, while the competitive capacity was lost in heat-inactivated PEDF. In endothelial cells, we have used an excess amount of PEDF to ensure the competitive effect of PEDF (Gotte et al. 2002). The structural basis responsible for the competition between PEDF and VEGF for binding to VEGFR2 remains to be elucidated.
PEDF is a multifaceted factor with neurotropic and anti-angiogenic functions (Tombran-Tink & Barnstable 2003b, Tombran-Tink et al. 1991). Although PEDF as a major endogenous angiogenic inhibitor has been extensively studied, the regulation of PEDF is not yet well understood (Tombran-Tink & Barnstable 2003b). Our study demonstrated that PEDF is secreted from RCEC and Müller cells. Under hypoxic conditions, PEDF secretion was significantly decreased along with the increase in VEGF expression. This result is consistent with the recent study reported by Eichler et al.(2004), which shows that hypoxia downregulates PEDF expression at the RNA level by 1.7-fold and at the protein level by two-fold in retinal Müller cells. Although hypoxia did induce a significant increase in VEGF expression and secretion, the addition of exogenous VEGF did not cause any change in the expression of PEDF in Müller cells, suggesting that hypoxia-induced downregulation of PEDF expression in Müller cells cannot be ascribed to the elevated VEGF levels. On the other hand, VEGF significantly downregulates PEDF expression in retinal endothelial cells, but not in Müller cells, suggesting that the regulation of PEDF by VEGF is dependent on VEGF receptor, which is expressed in endothelial cells but not in Müller cells.
Another important finding of the current study is that PEDF positively autoregulated endogenous PEDF expression in cultured Müller cells and in the retina of diabetic rats. PEDF is a major endogenous angiogenic inhibitor in the retina. Moreover, PEDF has recently been identified as a potent anti-inflammatory factor and anti-permeability factor (Liu et al. 2004, Zhang et al. 2005a). Furthermore, a decrease in ocular PEDF levels is believed to contribute to the pathogenesis of DR. Therefore, upregulation of endogenous PEDF levels in the retina will be beneficial in diabetic patients. The mechanisms by which PEDF is regulated by VEGF and by PEDF itself via positive feedback regulation remain to be determined in the future.
In summary, our study demonstrated that there exists a reciprocal regulation between PEDF and VEGF in the retina, which may represent an important mechanism for maintaining the balance between the two angiogenic regulatory systems. PEDF downregulates VEGF expression via inhibition of MAPK-mediated HIF-1 activation and competes with VEGF binding to VEGFR2. The decrease in PEDF levels is, at least partially, responsible for the increase in VEGF expression and the subsequent BRB breakdown and retinal NV in diabetic retinopathy. This study suggests that the angiogenic stimulator system and angiogenic inhibitor system have complex interactions, which may represent an important mechanism for maintaining the homeostasis of angiogenesis. The detailed mechanism responsible for these interactions remains to be elucidated.
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Acknowledgements |
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References |
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Received in final form 22 March 2006
Accepted 30 March 2006
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