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Antiangiogenic effects and transcriptional regulation of pigment epithelium-derived factor in diabetic retinopathy
Microvascular Research 80 (2010) 31–36
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Microvascular Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m v r e
Regular Article
Antiangiogenic effects and transcriptional regulation of pigment epithelium-derived factor in diabetic retinopathy Binhui Wang a, Philip Atherton b, Rekha Patel b, Gillian Manning a, Richard Donnelly a,⁎ a b
Vascular Medicine, School of Graduate-Entry Medicine and Health, University of Nottingham, UK Clinical Physiology, School of Graduate-Entry Medicine and Health, University of Nottingham, UK
a r t i c l e
i n f o
Article history: Received 30 July 2009 Revised 8 January 2010 Accepted 19 February 2010 Available online 26 February 2010 Keywords: Angiogenesis Diabetes Pigment epithelium-derived factor (PEDF) VEGF Retinopathy Vascular tubule formation Transcription
a b s t r a c t The effects of the antiangiogenic cytokine PEDF on key steps in retinal angiogenesis, specifically endothelial cell proliferation and vascular tubule formation, and the regulation of PEDF expression in retinal capillary endothelial cells were evaluated. HUVECs were co-cultured with fibroblasts to construct a model of angiogenesis using the Angiokit assay, and image analysis software was used to measure the effects of PEDF and VEGF on vascular tubule formation. Quantitative real-time PCR analysis was used to determine the expression of PEDF in microvascular endothelial cells exposed to glucose 20 mM, insulin 100 nM and VEGF 10 ng/ml. PEDF inhibited endothelial cell proliferation and significantly decreased the number of tubules (629 + 93 AU vs 311 + 31, p = 0.001), number of branching points (145 + 19 AU vs 46 + 5, p = 0.03) and total tubule length (4848 + 748 AU vs 11,172 + 2353, p = 0.001). In bovine retinal capillary endothelial cells (BRCECs), PEDF mRNA and protein expression was suppressed by insulin (22%) in a rapamycin-sensitive manner; wortmannin had no effect. PEDF mRNA expression was also significantly reduced in the presence of high glucose (23%) and VEGF (25%). In conclusion, PEDF inhibits key steps in the angiogenic response of BRCECs, including endothelial cell proliferation and vascular tubule formation. Gene expression of PEDF is negatively regulated by glucose, insulin (via an mTOR-dependent pathway) and VEGF. © 2010 Elsevier Inc. All rights reserved.
Introduction Angiogenesis – sprouting of new vessels from existing vascular structures – is a complex process which plays an important role in the development and progression of diabetes-related vascular complications (Carmeliet, 2000). Furthermore, angiogenesis is often abnormal in diabetes, either increased, as in proliferative diabetic retinopathy (Simo et al, 2006), or decreased, as in delayed wound healing and poor collateral vessel formation (Abaci et al, 1999). The underlying mechanisms are complex, but local differences in the formation and/ or clearance of pro- and antiangiogenic growth factors may create imbalances that favour or inhibit new vessel formation (Gao et al, 2001). Increased levels of vascular endothelial growth factor (VEGF) have been highlighted as a proangiogenic mediator in diabetic microangiopathy (Chiarelli et al, 2000), but few studies have investigated the regulation and mechanism of action of cytokines, such as pigment epithelium-derived factor (PEDF), that inhibit angiogenesis (Dawson
⁎ Corresponding author. School of Graduate-Entry Medicine and Health, University of Nottingham Medical School, Derby City General Hospital, Uttoxeter Road, Derby DE22 3DT, UK. Fax: + 44 1332 724619. E-mail address: [email protected] (R. Donnelly). 0026-2862/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2010.02.012
et al, 1999). PEDF is a 418-aminoacid, 50 kDa protein which is a member of the serine protease inhibitor (serpin) family (Dawson et al, 1999; Tombran-Tink and Barnstable, 2003). It is widely expressed throughout the body, especially in the nervous system and the retina, and circulating levels are increased in the metabolic syndrome (Yamagishi et al, 2006). PEDF has antiangiogenic, antioxidant and anti-inflammatory effects (Tombran-Tink and Barnstable, 2003; Barnstable and Tombran-Tink, 2004). There is increasing evidence that PEDF plays an important role in the pathogenesis of diabetic retinopathy (Barnstable and Tombran-Tink, 2004; King and Suzuma, 2000; Mori et al, 2001; Boehm et al, 2003; Iizuka et al, 2007) and nephropathy (Wang et al, 2005; Matsuyama et al, 2008). In the eye, PEDF inhibits retinal endothelial cell growth and migration, and attenuates ischemia-induced neovascularization (King and Suzuma, 2000; Mori et al, 2001). Intra-ocular levels of PEDF decrease with advancing stages of diabetic retinopathy (Ogata et al., 2001, 2002; Spranger et al, 2001), and experimental interventions to locally increase PEDF concentrations (either by over-expression of PEDF or administration of recombinant PEDF) inhibit blood vessel growth and attenuate renal and retinal tissue damage (Stellmach et al, 2001; Zhang et al, 2006; Wang et al, 2006). The aims of this study were to investigate the effects of PEDF on retinal endothelial cell proliferation and vascular tubule formation, and to investigate whether high glucose, insulin and the proangiogenic growth factor VEGF regulate PEDF expression in retinal capillary endothelial cells.
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B. Wang et al. / Microvascular Research 80 (2010) 31–36
Methods
Vascular tubule formation (Angiokit) assay
Cell lines
HUVECs were co-cultured with fibroblasts in a 24-well plate to construct an in vitro model of angiogenesis (AngioKit, TCS CellWorks, Buckingham, UK), as described previously (Bishop et al, 1999). Over 12–14 days, cellular organisation develops from small islands into anastomosing tubular networks. Co-cultures were exposed to rhVEGF165 10 ng/ml (R&D Systems), rhPEDF 2 nM and rhPEDF 10 nM (Upstate USA Lot 22439) in optimized media. Fresh treatments were changed on Days 1, 4, 7 and 9. After cell fixing on Day 11, vascular structures were visualized by labeling with mouse anti-CD31 and BCIP/NBT substrate according to the manufacturer instructions
Human umbilical vein endothelial cells (HUVECs) (Cambrex Corporation, UK) were cultured in endothelial growth media-2 (EGM-2) for use in the proliferation assay in passages 4–5. Bovine retinal capillary endothelial cells (BRCECs) were obtained from freshly slaughtered cattle and cultured according to previously described methods (Chibber et al, 1999; Ben-Mahmud et al, 2004). Plastic materials used for cell culture procedures were pre-coated with 2% gelatin type B (Sigma, UK).
Fig. 1. Effects of PEDF 2 nM (△), PEDF 10 nM (●) and VEGF 10 ng/ml (○), relative to control (▲), on proliferation of bovine retinal capillary endothelial cells (panel a) and human umbilical vein endothelial cells (panel c) after 24, 48 and 72 h. PEDF 2 nM attenuated VEGF 10 ng/ml induced endothelial cell proliferation (panel b: VEGF 10 ng/ml alone [□], PEDF 2 nM + VEGF 10 ng/ml [▲] vs control [♦]), and the inhibitory effect of PEDF on endothelial cell proliferation was evident under high glucose conditions (panel d: control [♦], glucose 20 mM [□], glucose 20 mM + PEDF 2 nM [△]).
B. Wang et al. / Microvascular Research 80 (2010) 31–36
(TCS CellWorks). Multiple photomicrographs were taken, and angiogenesis was quantified using image analysis software (AngioSys 1.0) to assess tubule number and tubule length, and the number of branching points (Bishop et al, 1999). Cell proliferation assay Six 96-well cell culture plates (Corning) were pre-coated with 2% gelatin type B, and left in the flow hood for 2 h before washing with sterile PBS to remove any excess gelatine. Cells were subcultured and counted. BRCECs were re-seeded onto the gelatin-pre-coated plates at 8000 cells/cm2 (2400 cells/well), and HUVECs were cultured in untreated plates at 4000 cells/cm2 (1200 cells/well). Cells were incubated with rhPEDF 2 nM, rhPEDF 10 nM and rhVEGF 10 ng/ml. Cell proliferation was measured after 24, 48 and 72 h incubation by adding MTS solution 20 μl/well (Promega UK) and measuring the absorbance at 490 nm.
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Primers used for human cells were: beta-2 microglobulin (B2M), sense 5′-GCT ATC CAG CGT ACT CCA AA-3′, anti-sense 5′-GAA AGA CCA GTC CTT GCT GA-3′; PEDF, sense 5′-ATC GTC TTT GAG AAG CTG-3′, antisense 5′-CAA ACT TTG TTA CCC ACT GC-3′. Primers used for bovine cells were: B2M, sense 5′-TTG CCC TTT GCC CTT TCC TC-3′, anti-sense 5′-CTG CCA ACA CAG ACC ACA CT-3′; and bovine PEDF, sense 5′-GAT GAG GAG AGG ACC GTG AAA G-3′, anti-sense 5′-TGG GCG ATC TTG CAG TTG AG-3′. Quantitative real-time PCR analysis was used to determine the relative levels of gene expression. Relative PEDF or VEGF gene expression was determined by the use of a mathematical method developed by Pfaffl (2001), normalizing expression to the housekeeping gene, beta-2 microglobulin (B2M). B2M was confirmed to be unchanged in these experiments (data not shown). SDS-PAGE followed by immunoblotting was used to measure the effects of insulin on PEDF protein expression in human umbilical artery smooth muscle cells. Following standard procedures, a mouse anti-human PEDF monoclonal antibody (Chemicon MAB1059) was used with chemiluminescent detection.
Real Time RT-PCR and immunoblotting Statistical analysis Cells were cultured in 6-well plates (Corns, UK). After reaching 80–90% confluence, the cells were serum-starved and quiesced for 24 h then exposed to insulin 100 nM (Sigma I0516); or insulin + inhibitors of PI-3-kinase (wortmannin 100 nM) or mTOR (rapamycin 100 nM); or rhVEGF165 10 ng/ml; or glucose 20 nM (D-(+)-glucose, Sigma G8769) for 24 h. The cells were then lysed, and for each sample total RNA was extracted with TRIzol Reagent (Sigma, T9424) according to the manufacturer's protocol. The yield of total RNA was measured by spectrophotometry at 260 nm. The quality and equality of loading of RNA samples were confirmed via agarose gel electrophoresis. A 1 μg aliquot of RNA was used for reverse transcription with the iScript™ cDNA synthesis kit (Bio-Rad, 170-8890). The following reagents were then added to create a 25 μl PCR reaction mix: 2 μl cDNA, 0.75 μl primers (3 pmol), 9 μl nuclease-free water and 12.5 μl supermix (Bio-Rad SYBR Green). The PCR conditions were 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 45 s. Amplification size and reaction specificity were confirmed by melt curve analysis and 1.5% agarose gel electrophoresis. All reactions were performed in duplicate.
All statistical calculations were undertaken using Smith's Statistical Package Software (SSPS, Version 2.80, CA, USA). Results are presented as mean+ standard deviation. Statistical significance was determined using student's t-test for paired comparisons. Data from the proliferation assay were analysed using repeated measures to compare the variances from a single treatment in the entire time length. At separate time points differences were evaluated by ANOVA. p-values b0.05 were considered statistically significant. Results Effects of PEDF and VEGF on endothelial cell proliferation VEGF 10 ng/ml increased proliferation of BRCECs (p = 0.015, n = 11) and HUVECs (p = 0.010, n = 11) (Fig. 1), whereas PEDF attenuated the proliferation of BRCECs (PEDF 2 nM [n = 11], p = 0.022; PEDF 10 nM [n = 11], p = 0.018) and HUVECs (PEDF 2 nM [n = 11], p = 0.035; PEDF 10 nM [n = 11], p = 0.017). The inhibitory effects of PEDF disappeared
Fig. 2. Example of the vascular tubule formation (Angiokit) assay under control conditions (top left panel), and in response to PEDF and VEGF.
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after 24 h (Fig. 1a). PEDF attenuated VEGF-induced proliferation of HUVECs (Fig. 1b) and had a negative effect on endothelial cell proliferation under conditions of high glucose (Fig. 1d). Effects of PEDF and VEGF on vascular tubule formation PEDF inhibited tubule formation whereas VEGF increased tubule formation (Fig. 2). The positive effect of VEGF was due to a significant increase in the number of branching points (298 + 96 AU [arbitrary units] per field of view [FOV] vs 145 + 19 AU per FOV, p b 0.001), the number of tubules (1094+281 AU per FOV vs 629 + 93 AU per FOV,
Fig. 3. Image-analysis software of vascular tubule formation shows the effects of PEDF 2 nM ( ), PEDF 10 nM ( ) and VEGF 10 ng/ml ( ) on the number of branching points (panel a), the number of tubules (panel b) and the total length of tubules (panel c).
p b 0.001) and average tubule length (18,938 + 4500 AU per FOV vs 11,171 + 2353 AU per FOV, p b 0.001) (Fig. 3). In contrast, PEDF 10 nM decreased the number of tubule branching points (145 + 19 AU per FOV vs 46 + 5, p = 0.03), the number of tubules (629 + 93 AU per FOV vs 311 + 31, p = 0.001) and average tubule length (11,172 + 2353 AU per FOV vs 4848 + 748, p = 0.001) (Fig. 3).
Fig. 4. (a): The effects of insulin on PEDF gene expression relative to beta-2 microglobulin in BRCEC, and (b) the inhibitory effect of insulin on PEDF protein expression relative to GAPDH. N = 5, *p = 0.01, ✝p = 0.004 vs control.
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Regulation of PEDF Expression in BRCEC In BRCECs, insulin 100 nM, with or without the PI3K/Akt inhibitor wortmannin, significantly reduced the expression of PEDF mRNA by 25% (n = 4, p = 0.004) and 22% (n = 4, p = 0.012), respectively. However, insulin had no significant effect on PEDF expression when combined with the mTOR inhibitor rapamycin (Fig. 4). Insulininduced down-regulation of PEDF mRNA also resulted in a significant reduction in PEDF protein (n = 5, Fig. 4). Glucose 20 mM inhibited the expression of PEDF by approximately 23% (n = 4, p = 0.038) (Fig. 5). VEGF 10 ng/ml also inhibited the expression of PEDF by approximately 25% (n = 4, p = 0.043), relative to controls (Fig. 5). Discussion PEDF is a multifunctional protein, coded by the serine proteinase inhibitor, clade F, member 1 (SERRPINF1) gene, which has both antiangiogenic and neurotrophic activities (Dawson et al, 1999; Tombran-Tink and Barnstable, 2003). The neuroprotective effects of PEDF include preservation of photoreceptors in the eye (Cao et al, 2001). Previous studies have shown that relative imbalances in the local availability of PEDF and VEGF, because of either changes in expression or clearance, e.g. in response to hypoxia, play an important role in the neovascularization associated with diabetic retinopathy and nephropathy (Gao et al, 2001; Wang et al, 2005; Ogata et al, 2002; Gao et al, 2002). Circulating levels and intra-ocular concentrations of PEDF, as well as polymorphisms of the PEDF gene, have been associated with susceptibility to, and progression of, diabetic eye disease (Boehm et al, 2003; Iizuka et al, 2007; Jenkins et al, 2007; Yoshida et al, 2007). To date, the antiangiogenic effects of PEDF have been largely attributed to reduced endothelial cell migration (Dawson et al, 1999) and apoptosis of activated endothelial cells (Stellmach et al, 2001), but the present study has highlighted the inhibitory effects of PEDF on
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retinal capillary endothelial cell proliferation and vascular tubule formation. The effects of PEDF on cell growth were modest and transient compared with previous work showing a more sustained inhibitory effect of PEDF on VEGF-induced proliferation (Duh et al, 2002). The concentrations of PEDF used in these experiments were broadly similar to those used previously (Dawson et al, 1999; Stellmach et al, 2001). It is unclear whether the transient effect on endothelial cell proliferation was due to cellular adaptation or tolerance. The co-culture model of vascular tubule formation using fibroblasts and HUVECs is well established (Bussolati et al, 2001; Simcock et al, 2008), and aims to recreate the in vivo environment where inter-cellular communication is important in the regulation of angiogenesis (Bishop et al, 1999). Furthermore, automated measurements of tubule formation in vitro seem to correlate well with results obtained using in vivo systems for measuring neovascularization (Donovan et al, 2001). In this study, the co-ordinated development of CD31-positive capillary-like structures was markedly attenuated by PEDF, in contrast to the stimulatory effects of VEGF. Vascular tubule formation is a critical step in angiogenesis, and PEDF reduced significantly the number and length of tubules as well as the number of branching points. The underlying mechanism is unclear, but PEDF is an antiinflammatory cytokine with effects on several pathways that regulate cellular organisation and structural differentiation. In particular, PEDF inhibits the migration of endothelial cells induced by VEGF and fibroblast growth factor (Dawson et al, 1999), and inhibits the expression of tumour necrosis factor-α (TNF-α), VEGF, monocyte chemoattractant protein-1 (MCP-1) and inter-cellular adhesion molecule-1 (ICAM-1) (Waltenberger, 2001). Diabetes is associated with abnormal angiogenesis, and Larger et al (2004) showed that hyperglycemia affects several key steps in new vessel formation without changing the expression levels of vascular growth factors (e.g. VEGF) or their receptors. However, these authors did not measure PEDF. Very little is known about the transcriptional control of PEDF, apart from hypoxia-induced down-regulation (Dawson et al, 1999), but the results of this study show that high glucose and VEGF have significant inhibitory effects on PEDF gene expression. Thus, under conditions of hyperglycemia in proliferative diabetic retinopathy there is not only a marked increase in local formation of VEGF but also a reduction in the antiangiogenic cytokine PEDF. However, the extent to which small changes in PEDF availability affect VEGF-induced neovascularisation in the diabetic eye in vivo is uncertain. That insulin also down-regulates PEDF has not been previously reported, an effect that appears to be at least partly dependent upon the mammalian target of rapamycin (mTOR) kinase. In addition to insulin-induced down-regulation of PEDF, angiogenic responses to hypoxia are also mediated, in part, via the mTOR pathway (Wouters and Koritzinsky, 2008). The effect of insulin on PEDF expression may be clinically relevant, because diabetic retinopathy can deteriorate in the short term if poor glycemic control is reversed quickly with intensive insulin therapy (Arun et al, 2004). A modest (25%) sustained reduction in PEDF expression, in the context of diabetes-induced upregulation of proangiogenic pathways, is likely to have a clinically meaningful effect of ocular complications. In summary, this study has shown that PEDF has potent inhibitory effects on vascular tubule formation, a key step in angiogenesis, and that PEDF gene expression is down-regulated by glucose, insulin and VEGF.
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Fig. 5. Glucose 20 mM (panel a) and VEGF 10 ng/ml (panel b) inhibited the expression of PEDF relative to beta-2 microglobulin in BRCEC.
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Contents lists available at ScienceDirect
Microvascular Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m v r e
Regular Article
Antiangiogenic effects and transcriptional regulation of pigment epithelium-derived factor in diabetic retinopathy Binhui Wang a, Philip Atherton b, Rekha Patel b, Gillian Manning a, Richard Donnelly a,⁎ a b
Vascular Medicine, School of Graduate-Entry Medicine and Health, University of Nottingham, UK Clinical Physiology, School of Graduate-Entry Medicine and Health, University of Nottingham, UK
a r t i c l e
i n f o
Article history: Received 30 July 2009 Revised 8 January 2010 Accepted 19 February 2010 Available online 26 February 2010 Keywords: Angiogenesis Diabetes Pigment epithelium-derived factor (PEDF) VEGF Retinopathy Vascular tubule formation Transcription
a b s t r a c t The effects of the antiangiogenic cytokine PEDF on key steps in retinal angiogenesis, specifically endothelial cell proliferation and vascular tubule formation, and the regulation of PEDF expression in retinal capillary endothelial cells were evaluated. HUVECs were co-cultured with fibroblasts to construct a model of angiogenesis using the Angiokit assay, and image analysis software was used to measure the effects of PEDF and VEGF on vascular tubule formation. Quantitative real-time PCR analysis was used to determine the expression of PEDF in microvascular endothelial cells exposed to glucose 20 mM, insulin 100 nM and VEGF 10 ng/ml. PEDF inhibited endothelial cell proliferation and significantly decreased the number of tubules (629 + 93 AU vs 311 + 31, p = 0.001), number of branching points (145 + 19 AU vs 46 + 5, p = 0.03) and total tubule length (4848 + 748 AU vs 11,172 + 2353, p = 0.001). In bovine retinal capillary endothelial cells (BRCECs), PEDF mRNA and protein expression was suppressed by insulin (22%) in a rapamycin-sensitive manner; wortmannin had no effect. PEDF mRNA expression was also significantly reduced in the presence of high glucose (23%) and VEGF (25%). In conclusion, PEDF inhibits key steps in the angiogenic response of BRCECs, including endothelial cell proliferation and vascular tubule formation. Gene expression of PEDF is negatively regulated by glucose, insulin (via an mTOR-dependent pathway) and VEGF. © 2010 Elsevier Inc. All rights reserved.
Introduction Angiogenesis – sprouting of new vessels from existing vascular structures – is a complex process which plays an important role in the development and progression of diabetes-related vascular complications (Carmeliet, 2000). Furthermore, angiogenesis is often abnormal in diabetes, either increased, as in proliferative diabetic retinopathy (Simo et al, 2006), or decreased, as in delayed wound healing and poor collateral vessel formation (Abaci et al, 1999). The underlying mechanisms are complex, but local differences in the formation and/ or clearance of pro- and antiangiogenic growth factors may create imbalances that favour or inhibit new vessel formation (Gao et al, 2001). Increased levels of vascular endothelial growth factor (VEGF) have been highlighted as a proangiogenic mediator in diabetic microangiopathy (Chiarelli et al, 2000), but few studies have investigated the regulation and mechanism of action of cytokines, such as pigment epithelium-derived factor (PEDF), that inhibit angiogenesis (Dawson
⁎ Corresponding author. School of Graduate-Entry Medicine and Health, University of Nottingham Medical School, Derby City General Hospital, Uttoxeter Road, Derby DE22 3DT, UK. Fax: + 44 1332 724619. E-mail address: [email protected] (R. Donnelly). 0026-2862/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2010.02.012
et al, 1999). PEDF is a 418-aminoacid, 50 kDa protein which is a member of the serine protease inhibitor (serpin) family (Dawson et al, 1999; Tombran-Tink and Barnstable, 2003). It is widely expressed throughout the body, especially in the nervous system and the retina, and circulating levels are increased in the metabolic syndrome (Yamagishi et al, 2006). PEDF has antiangiogenic, antioxidant and anti-inflammatory effects (Tombran-Tink and Barnstable, 2003; Barnstable and Tombran-Tink, 2004). There is increasing evidence that PEDF plays an important role in the pathogenesis of diabetic retinopathy (Barnstable and Tombran-Tink, 2004; King and Suzuma, 2000; Mori et al, 2001; Boehm et al, 2003; Iizuka et al, 2007) and nephropathy (Wang et al, 2005; Matsuyama et al, 2008). In the eye, PEDF inhibits retinal endothelial cell growth and migration, and attenuates ischemia-induced neovascularization (King and Suzuma, 2000; Mori et al, 2001). Intra-ocular levels of PEDF decrease with advancing stages of diabetic retinopathy (Ogata et al., 2001, 2002; Spranger et al, 2001), and experimental interventions to locally increase PEDF concentrations (either by over-expression of PEDF or administration of recombinant PEDF) inhibit blood vessel growth and attenuate renal and retinal tissue damage (Stellmach et al, 2001; Zhang et al, 2006; Wang et al, 2006). The aims of this study were to investigate the effects of PEDF on retinal endothelial cell proliferation and vascular tubule formation, and to investigate whether high glucose, insulin and the proangiogenic growth factor VEGF regulate PEDF expression in retinal capillary endothelial cells.
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Methods
Vascular tubule formation (Angiokit) assay
Cell lines
HUVECs were co-cultured with fibroblasts in a 24-well plate to construct an in vitro model of angiogenesis (AngioKit, TCS CellWorks, Buckingham, UK), as described previously (Bishop et al, 1999). Over 12–14 days, cellular organisation develops from small islands into anastomosing tubular networks. Co-cultures were exposed to rhVEGF165 10 ng/ml (R&D Systems), rhPEDF 2 nM and rhPEDF 10 nM (Upstate USA Lot 22439) in optimized media. Fresh treatments were changed on Days 1, 4, 7 and 9. After cell fixing on Day 11, vascular structures were visualized by labeling with mouse anti-CD31 and BCIP/NBT substrate according to the manufacturer instructions
Human umbilical vein endothelial cells (HUVECs) (Cambrex Corporation, UK) were cultured in endothelial growth media-2 (EGM-2) for use in the proliferation assay in passages 4–5. Bovine retinal capillary endothelial cells (BRCECs) were obtained from freshly slaughtered cattle and cultured according to previously described methods (Chibber et al, 1999; Ben-Mahmud et al, 2004). Plastic materials used for cell culture procedures were pre-coated with 2% gelatin type B (Sigma, UK).
Fig. 1. Effects of PEDF 2 nM (△), PEDF 10 nM (●) and VEGF 10 ng/ml (○), relative to control (▲), on proliferation of bovine retinal capillary endothelial cells (panel a) and human umbilical vein endothelial cells (panel c) after 24, 48 and 72 h. PEDF 2 nM attenuated VEGF 10 ng/ml induced endothelial cell proliferation (panel b: VEGF 10 ng/ml alone [□], PEDF 2 nM + VEGF 10 ng/ml [▲] vs control [♦]), and the inhibitory effect of PEDF on endothelial cell proliferation was evident under high glucose conditions (panel d: control [♦], glucose 20 mM [□], glucose 20 mM + PEDF 2 nM [△]).
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(TCS CellWorks). Multiple photomicrographs were taken, and angiogenesis was quantified using image analysis software (AngioSys 1.0) to assess tubule number and tubule length, and the number of branching points (Bishop et al, 1999). Cell proliferation assay Six 96-well cell culture plates (Corning) were pre-coated with 2% gelatin type B, and left in the flow hood for 2 h before washing with sterile PBS to remove any excess gelatine. Cells were subcultured and counted. BRCECs were re-seeded onto the gelatin-pre-coated plates at 8000 cells/cm2 (2400 cells/well), and HUVECs were cultured in untreated plates at 4000 cells/cm2 (1200 cells/well). Cells were incubated with rhPEDF 2 nM, rhPEDF 10 nM and rhVEGF 10 ng/ml. Cell proliferation was measured after 24, 48 and 72 h incubation by adding MTS solution 20 μl/well (Promega UK) and measuring the absorbance at 490 nm.
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Primers used for human cells were: beta-2 microglobulin (B2M), sense 5′-GCT ATC CAG CGT ACT CCA AA-3′, anti-sense 5′-GAA AGA CCA GTC CTT GCT GA-3′; PEDF, sense 5′-ATC GTC TTT GAG AAG CTG-3′, antisense 5′-CAA ACT TTG TTA CCC ACT GC-3′. Primers used for bovine cells were: B2M, sense 5′-TTG CCC TTT GCC CTT TCC TC-3′, anti-sense 5′-CTG CCA ACA CAG ACC ACA CT-3′; and bovine PEDF, sense 5′-GAT GAG GAG AGG ACC GTG AAA G-3′, anti-sense 5′-TGG GCG ATC TTG CAG TTG AG-3′. Quantitative real-time PCR analysis was used to determine the relative levels of gene expression. Relative PEDF or VEGF gene expression was determined by the use of a mathematical method developed by Pfaffl (2001), normalizing expression to the housekeeping gene, beta-2 microglobulin (B2M). B2M was confirmed to be unchanged in these experiments (data not shown). SDS-PAGE followed by immunoblotting was used to measure the effects of insulin on PEDF protein expression in human umbilical artery smooth muscle cells. Following standard procedures, a mouse anti-human PEDF monoclonal antibody (Chemicon MAB1059) was used with chemiluminescent detection.
Real Time RT-PCR and immunoblotting Statistical analysis Cells were cultured in 6-well plates (Corns, UK). After reaching 80–90% confluence, the cells were serum-starved and quiesced for 24 h then exposed to insulin 100 nM (Sigma I0516); or insulin + inhibitors of PI-3-kinase (wortmannin 100 nM) or mTOR (rapamycin 100 nM); or rhVEGF165 10 ng/ml; or glucose 20 nM (D-(+)-glucose, Sigma G8769) for 24 h. The cells were then lysed, and for each sample total RNA was extracted with TRIzol Reagent (Sigma, T9424) according to the manufacturer's protocol. The yield of total RNA was measured by spectrophotometry at 260 nm. The quality and equality of loading of RNA samples were confirmed via agarose gel electrophoresis. A 1 μg aliquot of RNA was used for reverse transcription with the iScript™ cDNA synthesis kit (Bio-Rad, 170-8890). The following reagents were then added to create a 25 μl PCR reaction mix: 2 μl cDNA, 0.75 μl primers (3 pmol), 9 μl nuclease-free water and 12.5 μl supermix (Bio-Rad SYBR Green). The PCR conditions were 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 45 s. Amplification size and reaction specificity were confirmed by melt curve analysis and 1.5% agarose gel electrophoresis. All reactions were performed in duplicate.
All statistical calculations were undertaken using Smith's Statistical Package Software (SSPS, Version 2.80, CA, USA). Results are presented as mean+ standard deviation. Statistical significance was determined using student's t-test for paired comparisons. Data from the proliferation assay were analysed using repeated measures to compare the variances from a single treatment in the entire time length. At separate time points differences were evaluated by ANOVA. p-values b0.05 were considered statistically significant. Results Effects of PEDF and VEGF on endothelial cell proliferation VEGF 10 ng/ml increased proliferation of BRCECs (p = 0.015, n = 11) and HUVECs (p = 0.010, n = 11) (Fig. 1), whereas PEDF attenuated the proliferation of BRCECs (PEDF 2 nM [n = 11], p = 0.022; PEDF 10 nM [n = 11], p = 0.018) and HUVECs (PEDF 2 nM [n = 11], p = 0.035; PEDF 10 nM [n = 11], p = 0.017). The inhibitory effects of PEDF disappeared
Fig. 2. Example of the vascular tubule formation (Angiokit) assay under control conditions (top left panel), and in response to PEDF and VEGF.
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after 24 h (Fig. 1a). PEDF attenuated VEGF-induced proliferation of HUVECs (Fig. 1b) and had a negative effect on endothelial cell proliferation under conditions of high glucose (Fig. 1d). Effects of PEDF and VEGF on vascular tubule formation PEDF inhibited tubule formation whereas VEGF increased tubule formation (Fig. 2). The positive effect of VEGF was due to a significant increase in the number of branching points (298 + 96 AU [arbitrary units] per field of view [FOV] vs 145 + 19 AU per FOV, p b 0.001), the number of tubules (1094+281 AU per FOV vs 629 + 93 AU per FOV,
Fig. 3. Image-analysis software of vascular tubule formation shows the effects of PEDF 2 nM ( ), PEDF 10 nM ( ) and VEGF 10 ng/ml ( ) on the number of branching points (panel a), the number of tubules (panel b) and the total length of tubules (panel c).
p b 0.001) and average tubule length (18,938 + 4500 AU per FOV vs 11,171 + 2353 AU per FOV, p b 0.001) (Fig. 3). In contrast, PEDF 10 nM decreased the number of tubule branching points (145 + 19 AU per FOV vs 46 + 5, p = 0.03), the number of tubules (629 + 93 AU per FOV vs 311 + 31, p = 0.001) and average tubule length (11,172 + 2353 AU per FOV vs 4848 + 748, p = 0.001) (Fig. 3).
Fig. 4. (a): The effects of insulin on PEDF gene expression relative to beta-2 microglobulin in BRCEC, and (b) the inhibitory effect of insulin on PEDF protein expression relative to GAPDH. N = 5, *p = 0.01, ✝p = 0.004 vs control.
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Regulation of PEDF Expression in BRCEC In BRCECs, insulin 100 nM, with or without the PI3K/Akt inhibitor wortmannin, significantly reduced the expression of PEDF mRNA by 25% (n = 4, p = 0.004) and 22% (n = 4, p = 0.012), respectively. However, insulin had no significant effect on PEDF expression when combined with the mTOR inhibitor rapamycin (Fig. 4). Insulininduced down-regulation of PEDF mRNA also resulted in a significant reduction in PEDF protein (n = 5, Fig. 4). Glucose 20 mM inhibited the expression of PEDF by approximately 23% (n = 4, p = 0.038) (Fig. 5). VEGF 10 ng/ml also inhibited the expression of PEDF by approximately 25% (n = 4, p = 0.043), relative to controls (Fig. 5). Discussion PEDF is a multifunctional protein, coded by the serine proteinase inhibitor, clade F, member 1 (SERRPINF1) gene, which has both antiangiogenic and neurotrophic activities (Dawson et al, 1999; Tombran-Tink and Barnstable, 2003). The neuroprotective effects of PEDF include preservation of photoreceptors in the eye (Cao et al, 2001). Previous studies have shown that relative imbalances in the local availability of PEDF and VEGF, because of either changes in expression or clearance, e.g. in response to hypoxia, play an important role in the neovascularization associated with diabetic retinopathy and nephropathy (Gao et al, 2001; Wang et al, 2005; Ogata et al, 2002; Gao et al, 2002). Circulating levels and intra-ocular concentrations of PEDF, as well as polymorphisms of the PEDF gene, have been associated with susceptibility to, and progression of, diabetic eye disease (Boehm et al, 2003; Iizuka et al, 2007; Jenkins et al, 2007; Yoshida et al, 2007). To date, the antiangiogenic effects of PEDF have been largely attributed to reduced endothelial cell migration (Dawson et al, 1999) and apoptosis of activated endothelial cells (Stellmach et al, 2001), but the present study has highlighted the inhibitory effects of PEDF on
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retinal capillary endothelial cell proliferation and vascular tubule formation. The effects of PEDF on cell growth were modest and transient compared with previous work showing a more sustained inhibitory effect of PEDF on VEGF-induced proliferation (Duh et al, 2002). The concentrations of PEDF used in these experiments were broadly similar to those used previously (Dawson et al, 1999; Stellmach et al, 2001). It is unclear whether the transient effect on endothelial cell proliferation was due to cellular adaptation or tolerance. The co-culture model of vascular tubule formation using fibroblasts and HUVECs is well established (Bussolati et al, 2001; Simcock et al, 2008), and aims to recreate the in vivo environment where inter-cellular communication is important in the regulation of angiogenesis (Bishop et al, 1999). Furthermore, automated measurements of tubule formation in vitro seem to correlate well with results obtained using in vivo systems for measuring neovascularization (Donovan et al, 2001). In this study, the co-ordinated development of CD31-positive capillary-like structures was markedly attenuated by PEDF, in contrast to the stimulatory effects of VEGF. Vascular tubule formation is a critical step in angiogenesis, and PEDF reduced significantly the number and length of tubules as well as the number of branching points. The underlying mechanism is unclear, but PEDF is an antiinflammatory cytokine with effects on several pathways that regulate cellular organisation and structural differentiation. In particular, PEDF inhibits the migration of endothelial cells induced by VEGF and fibroblast growth factor (Dawson et al, 1999), and inhibits the expression of tumour necrosis factor-α (TNF-α), VEGF, monocyte chemoattractant protein-1 (MCP-1) and inter-cellular adhesion molecule-1 (ICAM-1) (Waltenberger, 2001). Diabetes is associated with abnormal angiogenesis, and Larger et al (2004) showed that hyperglycemia affects several key steps in new vessel formation without changing the expression levels of vascular growth factors (e.g. VEGF) or their receptors. However, these authors did not measure PEDF. Very little is known about the transcriptional control of PEDF, apart from hypoxia-induced down-regulation (Dawson et al, 1999), but the results of this study show that high glucose and VEGF have significant inhibitory effects on PEDF gene expression. Thus, under conditions of hyperglycemia in proliferative diabetic retinopathy there is not only a marked increase in local formation of VEGF but also a reduction in the antiangiogenic cytokine PEDF. However, the extent to which small changes in PEDF availability affect VEGF-induced neovascularisation in the diabetic eye in vivo is uncertain. That insulin also down-regulates PEDF has not been previously reported, an effect that appears to be at least partly dependent upon the mammalian target of rapamycin (mTOR) kinase. In addition to insulin-induced down-regulation of PEDF, angiogenic responses to hypoxia are also mediated, in part, via the mTOR pathway (Wouters and Koritzinsky, 2008). The effect of insulin on PEDF expression may be clinically relevant, because diabetic retinopathy can deteriorate in the short term if poor glycemic control is reversed quickly with intensive insulin therapy (Arun et al, 2004). A modest (25%) sustained reduction in PEDF expression, in the context of diabetes-induced upregulation of proangiogenic pathways, is likely to have a clinically meaningful effect of ocular complications. In summary, this study has shown that PEDF has potent inhibitory effects on vascular tubule formation, a key step in angiogenesis, and that PEDF gene expression is down-regulated by glucose, insulin and VEGF.
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Fig. 5. Glucose 20 mM (panel a) and VEGF 10 ng/ml (panel b) inhibited the expression of PEDF relative to beta-2 microglobulin in BRCEC.
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