Introduction Hyperglycaemia has been established as a major risk factor for diabetic reti- nopathy, but the precise mechanisms underlying the pathophysiology of dia- betic retinopathy remain unknown. It is widely accepted that the progression of this disease is mainly due to a local imbalance of pro- versus anti-angio- genic factors in the retina. A number of secreted factors, such as vascular endo- thelial growth factor (VEGF) (Aiello et al. 1994), angiopoietin2 (Watana- be et al. 2005a), IGF-1(insulin-like growth factor 1) (Miller et al. 2007), erythropoietin (Watanabe et al. 2005b) and pigment epithelium-derived factor (PEDF) (Funatsu et al. 2006), have been reported to be involved in the develop- ment of diabetic retinopathy, although the precise involvement of each of these factors in the pathogenesis of the disease remains unknown. Moreover, the origin of these factors is controversial, and it is possible that several types of cells in the retina contribute to the production of these molecules. Angiopoietin-like protein 4 (ANGPTL4) is induced by high glucose in retinal pigment epithelial cells and exhibits potent angiogenic activity on retinal endothelial cells Hirotaka Yokouchi, 1,2 , Koki Eto, 1 Wataru Nishimura, 1 Norio Takeda, 3 Yasushi Kaburagi, 1, * Shuichi Yamamoto 2 and Kazuki Yasuda 1 1 Department of Metabolic Disorder, Diabetes Research Center, Research Institute, Tokyo, Japan 2 Department of Ophthalmology and Visual Science, Chiba University Graduate School of Medicine, Chiba, Japan 3 Department of Ophthalmology, Hospital, National Center for Global Health and Medicine, Tokyo, Japan ABSTRACT. Purpose: Hyperglycaemia has been identified as major risk factor for diabetic retinopathy (DR). It is widely accepted that the progression of DR is mainly due to a local imbalance of pro- versus anti-angiogenic factors in the retina. In this study, we investigated whether retinal pigment epithe- lial (RPE) cells produced pro-angiogenic factors under high glucose (HG) conditions in vitro. Methods: Cultured human retinal endothelial (RE) cells were exposed to conditioned medium from retinal pigment epithelium cells (ARPE-19) grown in HG medium and assessed for tube for- mation. Based on the expression profiles of ARPE-19, we investigated whether ANGPTL4 was a major angiogenic factor released from ARPE-19 under HG conditions using cultured human RE cells as the test system for experiments with recombinant protein, conditioned medium from ARPE-19 and RNA interference (RNAi). Results: The conditioned medium from ARPE-19 cultured under HG conditions promoted tube for- mation of cultured human RE cells. GeneChip analysis showed that ANGPTL4 was one of the highest upregulated genes under HG conditions. In addition, recombinant ANGPTL4 promoted all of the elements of angiogenesis in human RE cells in vitro. The results of experiments using condi- tioned medium from ARPE-19 combined with RNAi demonstrated that ANGPTL4 was a major angiogenic factor released from ARPE-19 under HG conditions. Conclusions: ANGPTL4 was induced by high glucose in RPE cells and exhibited potent angio- genic activity on RE cells. Our results are unique and may potentially add a new candidate to the long list of molecules involved in diabetic retinopathy. Key words: ANGPTL4 – diabetic retinopathy – high glucose – retinal angiogenesis – retinal endothelial cell – retinal pigment epithelial cell Acta Ophthalmol. 2013: 91: e289–e297 ª 2013 The Authors Acta Ophthalmologica ª 2013 Acta Ophthalmologica Scandinavica Foundation doi: 10.1111/aos.12097 *Present address: Department of Diabetic Complications, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan. Acta Ophthalmologica 2013 e289
Transcript
Introduction
Hyperglycaemia has been establishedas a major risk factor for diabetic reti-nopathy, but the precise mechanismsunderlying the pathophysiology of dia-betic retinopathy remain unknown. Itis widely accepted that the progressionof this disease is mainly due to a localimbalance of pro- versus anti-angio-genic factors in the retina. A number ofsecreted factors, such as vascular endo-thelial growth factor (VEGF) (Aielloet al. 1994), angiopoietin2 (Watana-be et al. 2005a), IGF-1(insulin-likegrowth factor 1) (Miller et al. 2007),erythropoietin (Watanabe et al. 2005b)and pigment epithelium-derived factor(PEDF) (Funatsu et al. 2006), have beenreported to be involved in the develop-ment of diabetic retinopathy, althoughthe precise involvement of each of thesefactors in the pathogenesis of the diseaseremains unknown. Moreover, the originof these factors is controversial, and it ispossible that several types of cells in theretina contribute to the production ofthese molecules.
Angiopoietin-like protein 4(ANGPTL4) is induced by highglucose in retinal pigmentepithelial cells and exhibitspotent angiogenic activity onretinal endothelial cells
Hirotaka Yokouchi,1,2, Koki Eto,1 Wataru Nishimura,1
1Department of Metabolic Disorder, Diabetes Research Center, Research Institute,Tokyo, Japan2Department of Ophthalmology and Visual Science, Chiba University GraduateSchool of Medicine, Chiba, Japan3Department of Ophthalmology, Hospital, National Center for Global Health and
Medicine, Tokyo, Japan
ABSTRACT.Purpose: Hyperglycaemia has been identified as major risk factor for diabetic retinopathy (DR).
It is widely accepted that the progression of DR is mainly due to a local imbalance of pro- versus
anti-angiogenic factors in the retina. In this study, we investigated whether retinal pigment epithe-
lial (RPE) cells produced pro-angiogenic factors under high glucose (HG) conditions in vitro.
Methods: Cultured human retinal endothelial (RE) cells were exposed to conditioned medium
from retinal pigment epithelium cells (ARPE-19) grown in HG medium and assessed for tube for-
mation. Based on the expression profiles of ARPE-19, we investigated whether ANGPTL4 was a
major angiogenic factor released from ARPE-19 under HG conditions using cultured human RE
cells as the test system for experiments with recombinant protein, conditioned medium from
ARPE-19 and RNA interference (RNAi).
Results: The conditioned medium from ARPE-19 cultured under HG conditions promoted tube for-
mation of cultured human RE cells. GeneChip analysis showed that ANGPTL4 was one of the
highest upregulated genes under HG conditions. In addition, recombinant ANGPTL4 promoted all
of the elements of angiogenesis in human RE cells in vitro. The results of experiments using condi-
tioned medium from ARPE-19 combined with RNAi demonstrated that ANGPTL4 was a major
angiogenic factor released from ARPE-19 under HG conditions.
Conclusions: ANGPTL4 was induced by high glucose in RPE cells and exhibited potent angio-
genic activity on RE cells. Our results are unique and may potentially add a new candidate to
the long list of molecules involved in diabetic retinopathy.
Acta Ophthalmol. 2013: 91: e289–e297ª 2013 The Authors
Acta Ophthalmologica ª 2013 Acta Ophthalmologica Scandinavica Foundation
doi: 10.1111/aos.12097*Present address: Department of Diabetic Complications, Diabetes Research Center, Research
Institute, National Center for Global Health and Medicine, Tokyo, Japan.
Acta Ophthalmologica 2013
e289
Retinal pigment epithelial (RPE)cells are mono-layered cubical cellsthat lie in close proximity to the meta-bolically highly active photoreceptorcells. They have many important func-tions, including phagocytosis of theouter segment discs of rods and cones;the uptake, processing and release ofvitamin A; and mediation of the vec-torial transport of nutrients from thechoroidal blood to the photoreceptorcells (Hewitt & Adler 1997).
Retinal pigment epithelial cells havealso been shown to secrete growth fac-tors such as PEDF (Steele et al. 1992),fibroblast growth factor 5 (Dunn et al.1996) and platelet-derived growth fac-tor (Campochiaro et al. 1989). Thesefactors have been reported to stimu-late the proliferation of fibroblasts,glial cells and the RPE cells them-selves (Bryan & Campochiaro 1986).
Thus, RPE cells may be a goodcandidate as the putative origin ofdiabetic retinopathy-related secretoryfactors in the retina; however, fewstudies have investigated this possibil-ity (Sone et al. 1996; Young et al.2005; Hernandez et al. 2006; Yokoy-ama et al. 2006).
The gene for ANGPTL4 has beenidentified by several groups, and theprotein is also known as fasting-induced adipose factor (FIAF) (Ker-sten et al. 2000), hepatic fibrinogen ⁄ an-giopoietin-related protein (HFARP)(Kim et al. 2000) and PPARc angio-poietin-related protein (PGAR) (Yoonet al. 2000). Several previous reportshave suggested that the ANGPTL4protein may be involved in angiogene-sis (Ito et al. 2003; Le Jan et al. 2003;Hermann et al. 2005). Recently, it hasbeen reported that ANGPTL4 proteinmay be involved in the developmentand pathogenesis of retinal angiogene-sis in a model of oxygen-inducedretinopathy, but little is known aboutthe expression or the function of thisprotein in the eye (Perdiguero et al.2011).
In this study, we investigatedwhether RPE cells play a role in reti-nal angiogenesis under high glucoseconditions. Using human retinal endo-thelial (RE) cells as a model system,we demonstrated that RPE cells pro-duced tube-forming factors when cul-tured in medium containing a highglucose concentration and that thisactivity was mainly mediated byANGPTL4.
Materials and Methods
Cell cultures
The human RPE-derived cell line,ARPE-19, was purchased from theAmerican Type Culture Collection(Rockville, MA, USA). Human REcells were purchased from Cell Sys-tems Corporation (Kirkland, WA,USA).
Durgs and chemicals
Recombinant human ANGPTL4 andVEGF were purchased from Adipo-Gen (Seoul, Korea; AG-40A-0033,formerly designated as RP-AL4102)and R and D Systems (Minneapolis,MN, USA; 293-VE), respectively.CS-C medium and CS-C completemedium [containing 10% foetalbovine serum (FBS) and growth fac-tors] were purchased from Cell Sys-tems Corporation. ARPE-19 cells andRE cells were cultured at 37�C in anatmosphere containing 5% CO2.ARPE-19 cells were used at passages23–25 for all the experiments. Cellswere maintained in Dulbecco’s modi-fied Eagle’s medium with nutrientmixture F12 (Gibco BRL, GrandIsland, NY, USA) (DMEM ⁄F12) and15 mm HEPES buffer containing7.5 mm glucose supplemented with10% FBS (FBS; Gibco BRL),100 U ⁄ml of penicillin and 100 lg ⁄mlof streptomycin (Gibco BRL). For theglucose challenge, after the cells weregrown to 90% confluence, they werecultured for another 48 hr in the pres-ence of glucose at a concentration of5 mm, 7.5 mm, 12.5 mm or 17.5 mm inDMEM ⁄F12 supplemented with 10%FBS, which we referred to as very lowglucose (VLG), low glucose (LG),medium glucose (MG) or high glucose(HG) medium, respectively. Mediafrom ARPE-19 cells conditioned inthe presence of a high or low glucose(17.5 or 7.5 mm) were abbreviated ashigh glucose-conditioned media (HG-CM) or low glucose-conditionedmedia (LG-CM), respectively. ARPE-19 cells were cultured for 72 hr, exceptfor the knockdown experiments inwhich they were incubated for 48 hr.RE cells were used at passages 3–6 forall the experiments. They were grownin type-I collagen-coated dishes(Iwaki, Tokyo, Japan) and maintainedin CS-C complete medium.
In vitro tube formation assay
The tube formation assay was con-ducted using BD BioCoat AngiogenesisSystem Endothelial Cell Tube Forma-tion plates (BD Biosciences, Bedford,MA, USA) in accordance with the pro-tocol supplied by the manufacturer.Images of the tubules formed wereobtained under fluorescence micros-copy at 4· magnification. The tubelength in each well was measured fromone representative image per well usingthe WinRoof software (MITANI Cor-poration, Tokyo, Japan) and presentedas a percentage of the control.
Oligonucleotide microarray analysis
Total RNA was prepared from theARPE-19 cells cultured in each type ofmedium using TRIzol Reagent (Invitro-gen, Carlsbad, CA, USA). For transcrip-tome analysis, expression profiles ofARPE-19 cells cultured in LG or HGmedium containing 10% FBS were com-pared by the human HG-U133 Set Array(Affymetrix, Santa Clara, CA, USA).
Samples for the analysis were preparedin accordance with the manufacturer’sprotocol. Analysis was performed usingthe Affymetrix GeneChip� OperatingSoftware (GCOS). Probe sets werere-annotated using eldorado software(Genomatix, Munich, Germany), and thecellular components of the genes weredefined by Gene Ontology (GO) annota-tion.
Real-time RT-PCR analysis
Real-time RT-PCR analysis was per-formed using the TaqMan� probe withan ABI Prism 7900 sequence-detectionsystem (Applied Biosystems, FosterCity, CA, USA). The levels of the tar-get genes were normalized to the levelsof human ACTB (b-actin) using the com-parative threshold method. The identifica-tion numbers of the primers for humanANGPTL4 and human ACTB wereHs00211522_ml and Hs99999903_ml,respectively (Applied Biosystems).
The effect of glucose on ANGPTL4expression was further analysed inARPE-19 cells. After the cells reached80% confluence in LG medium, theywere further incubated in HG mediumfor 0, 30, 60, 90 or 120 min for atime-course analysis.
For concentration–response analy-sis, they were incubated for 48 hr inthe presence of various concentrations
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of glucose (VLG, LG, MG or HGmedium). The expression level ofANGPTL4 mRNA in the ARPE-19cells was determined by real-time RT-PCR as described previously.
Immunoprecipitation and immunoblot
analysis
ARPE-19 cells were collected andhomogenized in 25 mm Tris ⁄HCl pH7.5, 150 mm NaCl and 1% (w ⁄v) Triton-X, followed by centrifugation at 4�C.The supernatant or culture medium wasincubated with 10 ll of anti-ANGPTL4antibody (Abnova, Taipei City, Taiwan;H0051129-A01) fixed on protein GSepharose beads (Amersham PharmaciaBiotech, Piscataway, NJ, USA). The im-munoprecipitates were boiled for 10 minin the presence of b-mercaptoethanoland resolved by SDS-PAGE. Westernblots were performed usinganti-ANGPTL4 antibody as describedpreviously (Nishimura et al. 2002).
Retinal endothelial cell migration, inva-
sion and proliferation assays
For migration and invasion assays,1 · 105 RE cells were seeded on BDBioCoat Angiogenesis System Endo-thelial Cell Migration and Invasionplates (BD Bioscience) in CS-C Med-ium containing 0.4% FBS and eitherVEGF (5 ng ⁄ml) or ANGPTL4(100 ng ⁄ml), in accordance with theprotocols supplied by the manufac-turer. Cell migration was assayed bymotility through an unoccluded pore inthe presence of a thin layer of humanfibronectin, and cell invasion was mea-sured by the ability for endothelial cellsto degrade extracellular matrix andmigrate through the pores.
The proliferative activity of RE cellswas assessed based on cellular mito-chondrial dehydrogenase activity. Analiquot of 2 · 104 RE cells was addedto each well of a 96-well tray contain-ing CS-C complete medium. After star-vation, the cells were exposed to CS-Cmedium containing 0.4% FBS witheither VEGF (5 ng ⁄ml) or ANGPTL4(100 ng ⁄ml) for 48 hr, and then CellCount Reagent SF (Nacalai Tesque,Kyoto, Japan) was applied for 4 hr.
Knockdown of gene expression in ARPE-
19 cells by RNA interference (RNAi)
For RNAi experiments, ARPE-19 cellswere seeded at a density of 2 · 105
cells ⁄well into six-well culture platescontaining LG medium and incubatedfor 24 hr. The transfection was per-formed using Dharmafect� reagent(Dharmacon Inc., Lafayette, CO,USA) in LG medium and siRNA at afinal concentration of 20 nm. The siR-NAs used were for PPARc, HIF-1a,EGR1, ANGPTL4, VEGF or LaminA ⁄C as the control (siGENOMESMART pool M_003436-01, M_004018-02, M_006526-01, M_003550-01-0010,M_007807-01-0010, and D-001050-01-05; Dharmacon Inc.).
After 48-hr transfection, the med-ium was replaced by HG medium,and the incubation was continued foranother 48 hr.
Statistical analysis
The number of independent experi-ments is noted in the text and figurelegends. The results are expressed asmean values ± SD. StatView-5.0 soft-ware (SAS Institute Inc., Cary, NC,USA) was used for all statistical anal-yses. The Student’s t-test was carriedout to determine whether the differ-ences between groups were statisticallysignificant. Either p < 0.05 orp < 0.01 was considered significant.
Results
In vitro tube formation activity induced in
ARPE-19 cell-conditioned medium
To evaluate whether the factorssecreted by ARPE-19 cells promotedtube formation in RE cells in vitro,conditioned media from ARPE-19cells were overlaid onto tube forma-tion plates containing RE cells. Atfirst, we examined the effect of glucoseby using LG or HG medium. The HGmedium (Fig. 1C) increased tube for-mation in RE cells by 89.7 ± 53.0%(p < 0.05, n = 5), as compared withLG medium (Fig. 1A). Next, weexamined the effect of HG-CM. Thelength of the tubules formed in HG-CM(Fig. 1D) was 413.4 ± 78.2% (p < 0.01,n = 5) higher than that in LG-CM(Fig. 1B). A significant increase(602.0 ± 132.7%; p < 0.01, n = 5)in the length of the tubules formed byRE cells was also observed when theculture system was overlaid with HG-CM (Fig. 1D), as compared with thatin HG medium (Fig. 1C). Thus, theseresults suggest that HG-CM contained
high glucose-induced angiogenic fac-tors that promote the tube formationof RE cells (Fig. 1E).
Identification of ANGPTL4 as a candi-
date angiogenic factor
We next attempted to identify theangiogenic factors secreted by theARPE-19 cells cultured in the pres-ence of a high glucose concentration.Among the upregulated genes revealedby the gene expression analysis ofARPE-19 cells (Table 1), we focusedon ANGPTL4 as a strong candidatefor the high glucose concentration-induced angiogenic activity, as theexpression of this gene showed thehighest level of upregulation; itsexpression ranged from 2.46-fold [withprobe set 223333_s_at in experiment1] to 13.9-fold [with probe set221009_s_at in experiment 2].
Induction by glucose of ANGPTL4
expression in ARPE-19 cells
We next investigated the induction ofANGPTL4 expression by glucose infurther detail. The temporal profile ofANGPTL4 mRNA expression in theHGmedium is shown in Fig. 2A. Signif-icant induction of ANGPTL4 mRNAexpression in the presence of a highglucose concentration was observed at60 min (1.37 ± 0.14-fold, p < 0.05,n = 3); the expression level was furtherincreased at 90 min (2.35 ± 0.13-fold,p < 0.01, n = 3) and at 120 min(4.52 ± 0.54-fold, p < 0.01, n = 3),as compared with the control (0 min).In previous studies, both the full-lengthand the cleaved forms (fibrinogen-likedomain) of ANGPTL4 were reported,which resolved 55 and 38 kDa, respec-tively (Cazes et al. 2006). While thepredicted size of the full-length ANG-PTL4 is approximately 40 kDa, thepost-transcriptional modification mayexplain the apparent discrepancy.Immunoblot analyses using anti-ANG-PTL4 antibody demonstrated aapproximately 55-kDa protein both incell lysates and conditioned mediumfrom ARPE-19 cells cultured under thehigh glucose concentrations (Fig. 2B),suggesting the majority of ANGPTL4in HG-CM was the full-length protein.We also investigated the expression levelsof ANGPTL4 in ARPE-19 cells follow-ing exposure to different concentrationsof glucose. Figure 2C shows that the
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ANGPTL4 mRNA expression wasprogressively upregulated by increasingglucose concentrations in the medium.Cells cultured in MG and HG mediashowed an increase in ANGPTL4mRNA expression by 2.44 ± 0.37-fold(p < 0.01, n = 3) and 5.78 ± 0.87-fold (p < 0.01, n = 3), respectively, ascompared with cells cultured in LGmedium, which suggests that theexpression of ANGPTL4 mRNA canalso be significantly induced by moder-ately elevated glucose levels, corre-sponding to the condition that can beexpected in patients with fairly well-controlled diabetes. While the induc-
tion of ANGPTL4 in the liver and adi-pose tissue was demonstrated underfasting conditions (Kersten et al. 2000),ARPE-19 cells exposed to VLG med-ium showed a decrease in its expression(0.38 ± 0.18-fold, p < 0.05, n = 3).These results suggest the tissue specific-ity of the regulation of ANGPTL4 geneexpression.
Effect of siRNA inhibition of PPARc,
HIF-1a and EGR1 on the expression of
ANGPTL4 mRNA in ARPE-19 cells
To investigate the transcriptionalmachinery of ANGPTL4 expression
in the ARPE-19 cells more directly,we focused on three transcription fac-tors and performed RNAi experi-ments. ANGPTL4 has been reportedto be a downstream target of PPARc(Yoon et al. 2000). It has beenreported that the expression of ANG-PTL4 mRNA was highly upregulatedin cardiomyocytes under hypoxic con-ditions by HIF-1a (Belanger et al.2002). We also focused on EGR1,which was one of the genes upregulat-ed by high glucose in our GeneChipanalysis (about two to threefold,Table 1). EGR1 is an 80–82-kDa zincfinger transcription factor known tobe involved in cell proliferation anddifferentiation (Sukhatme et al. 1988)and has been shown to activate thetranscription of many genes in thepresence of high glucose, includingPDGF A and B, VEGF and insulin-like growth factor-2 (Silverman &Collins 1999). The effects of siRNAinhibition of PPARc, HIF-1a andEGR1 on the ANGPTL4 expressionare shown in Fig. 3. Expression of theANGPTL4 gene in ARPE-19 cellswas significantly reduced by89.8 ± 2.2% (p < 0.01, n = 3) and94.4 ± 3.5% (p < 0.01, n = 3) bythe siRNAs for HIF-1a and PPARc,respectively, as compared with cellstransfected with the control, but nosignificant change was noted aftertreatment with the siRNA for EGR1.These results suggest that bothPPARc and HIF-1a are essential forthe induction of ANGPTL4 by glu-cose in ARPE-19 cells.
Recombinant human ANGPTL4 exerts
angiogenic activity on RE cells
Angiogenesis by endothelial cellsinvolves multiple steps, namely cellinvasion, cell migration, cell prolifera-tion and tube formation (Folkman1986). Therefore, we studied the induc-tion of each of these steps in RE cellsby ANGPTL4 in vitro. Figure 4Ashows that treatment with recombinantfull-length ANGPTL4 (100 ng ⁄ml) orVEGF (5 ng ⁄ml) increased invasion by83.0 ± 20.7% (p < 0.01, n = 5) and139.5 ± 35.2% (p < 0.01, n = 5),respectively, as compared to control-treated cells. Figure 4B shows thattreatment with ANGPTL4 or VEGFincreased migration by 78.1 ± 27.2%(p < 0.01, n = 4) and 160.6 ± 35.7%(p < 0.01, n = 4), respectively, as
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Fig. 1. Tube formation activity of retinal endothelial (RE) cells in high glucose-conditioned
media (HG-CM) in vitro. The tube formation activity of the RE cells in low glucose-condi-
tioned media (LG-CM) or HG-CM was assessed by incubation for 16 hr on BD BioCoat
(E) Quantitative analysis of network structures. The total length of the network structures was
measured and the total length per field was calculated as a ratio of that in the control. n = 5.
Values are expressed as means ± SD. **p < 0.01, *p < 0.05 as compared with the result in
the control. Scale bars, 200 lm.
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compared to the control. Figure 4Cshows that treatment with ANGPTL4or VEGF increased the proliferativeactivity by 73.3 ± 10.0% (p < 0.01,n = 4) and 133.3 ± 36.6% (p < 0.01,n = 4), respectively, as compared withthe control. We also investigated the
effects of recombinant ANGPTL4 andVEGF on the tube formation by REcells (Fig. 5A–D). In this experiment,we used 15 ng ⁄ml rather than 5 ng ⁄mlVEGF, as 5 ng ⁄ml VEGF failed to
significantly induce tube formation.Figure 5D shows that treatment withANGPTL4 (100 ng ⁄ml) or VEGF(15 ng ⁄ml) increased the tube length by154.1 ± 43.3% (p < 0.01, n = 5) and
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Fig. 2. The effects of HG medium on ANGPTL4 mRNA and protein expression. (A) Time-course of the effects in HG medium (0–120 min).
n = 3. *p < 0.05, **p < 0.01 as compared with that at 0 min. (B) Immunoblot analyses using anti-ANGPTL4 antibody. Immunoprecipitates
from cell lysates (left, Lysate) and culture medium (right, CM) of ARPE-19 cells cultured in the high glucose medium were analysed by SDS-
PAGE followed by Western blotting. Expression of both the full-length form (bold arrowhead) and the short form of ANGPTL4 was observed
in the lysates, but the former is the predominant form in CM. The image is representative of three experiments. (C) Effects of elevated glucose
levels for 48 hrs’ stimulation. n = 3. The mRNA level of ANGPTL4 was normalized to that of ACTB, and the relative values were compared
with those at 0 min (A) and low glucose (LG) (C), respectively, and expressed as means ± SD. *p < 0.05, **p < 0.01 as compared with the
result in LG medium.
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ARPE-19 cells treated with siRNAs. ANG-
PTL4 mRNA expression in ARPE-19 cells
treated with 20 nm of siRNA for either HIF-
1a, PPARc or EGR1 and cultured in HG
medium for 48 hr was measured by real-time
RT-PCR and expressed as a ratio of that in
low glucose medium. n = 3. **p < 0.01 as
compared with the result in control, Lamin
A ⁄C. NS, no significance.
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Fig. 4. Retinal endothelial (RE) cell migration, invasion and proliferation assays. Either vascular
endothelial growth factor (5 ng ⁄ml) or ANGPTL4 (100 ng ⁄ml) human recombinant protein was
added to plates containing RE cells. (A) The invasion plate was incubated for 21 hr. n = 5. (B)
The migration plate was incubated for 27 hr. n = 4. (C) The proliferation assay was conducted
for 48 hr using Count Reagent SF. n = 4. Values are expressed as means ± SD and are calcu-
lated as a ratio of that in the control, CS-C Medium. **p < 0.01 compared with control.
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126.2 ± 34.4% (p < 0.01, n = 5),respectively, as compared with that inthe control. These results indicate thatANGPTL4 promotes each step ofangiogenesis in RE cells almost aspotently as VEGF in vitro.
Tube formation activity in HG-CM trea-
ted with siRNA
Next, we treated the ARPE-19 cellswith siRNA for ANGPTL4, VEGF orHIF-1a (Fig. 6) to determine whetherthese factors were involved in thetube-forming activity induced byARPE-19 cells cultured in HG med-ium. ANGPTL4, VEGF or HIF-1amRNA were reduced by more than80% by the corresponding siRNAtreatment (data not shown).
While the addition of HG-CM trea-ted with the siRNA for VEGF(Fig. 6B) shows reduction of the tubeformation activity by 42.1 ± 5.9%(p < 0.05, n = 5) in this analysis,HG-CM treated with 20 nm of thesiRNA for ANGPTL4 (Fig. 6C)showed reduction of the tube forma-tion activity by 68.6 ± 8.3%(p < 0.01) and those treated with thesiRNA for HIF-1a (Fig. 6D) showedreduction of the tube formation by93.6 ± 1.6% (p < 0.01, n = 5)
(Fig. 6E) as compared with the cellstreated with control siRNA for LaminA ⁄C (Fig. 6A).
In contrast, knockdown of theEGR1 gene did not reduce tube for-mation by RE cells in HG-CM (datanot shown), suggesting that the induc-tion of ANGPTL4 expression was notdependent on EGR1.
Discussion
In this study, we demonstrated thatARPE-19 cells produced factors withpotent angiogenic activity on humanRE cells in the presence of a high glu-cose concentration, and that thisangiogenic activity could be primarilyattributed to ANGPTL4. Contribu-tions from other angiogenic factorssuch as VEGF cannot be excluded,however; and the identification ofthese factors is under way. ANGPTL4belongs to the angiopoietin-like pro-tein family (ANGPTLs), members ofwhich are structurally similar to theangiopoietins, with N-terminal coiled-coiled domains and C-terminal fibrin-ogen-like domains, but do not bind toeither of the angiopoietin receptors,Tie1 or Tie2 (Kim et al. 2000).Although some ANGPTLs, includingANGPTL3 and ANGPTL6, exhibit
angiogenic activity (Camenisch et al.2002; Oike et al. 2004), the potentialangiogenic activity of other familymembers, including ANGPTL2(Dhanabal et al. 2002; Kubota et al.2005) and ANGPTL4, is rather con-troversial. Indeed, previous studieshave reported both pro-angiogenic(Le Jan et al. 2003; Hermann et al.2005; Perdiguero et al. 2011) and anti-angiogenic effects (Ito et al. 2003) ofANGPTL4. In this study, we demon-strated that recombinant humanANGPTL4 protein significantly pro-moted each of the four steps of angio-genesis on RE cells, namely invasion,migration, proliferation and tube for-mation (Auerbach et al. 2003) andthat its angiogenic activity was aspotent as that of VEGF, whoseinvolvement in the development ofdiabetic retinopathy has been wellestablished (Aiello et al. 1994).Although specific receptors for ANG-PTL4 have not been identified, itsbinding to integrins and other extra-cellular matrix proteins has beenreported in several cell types (Gohet al. 2010; Huang et al. 2011). In thisstudy, MatrigelTM and fibronectinwere included in the invasion and thetube formation assay systems and inthe migration assay system, respec-tively. Thus, the effects of this proteinmay be dependent on the assay systemor the origin of endothelial cells.However, as the angiogenic activity ofthis protein on RE cells was evidentin our system at least in vitro, it maybe interesting to note that the additiveeffects of VEGF and ANGPTL4 onangiogenesis have been reported inwhite adipose tissue (Gealekman et al.2008).
One unexpected finding in thisstudy was the induction of ANGPTL4expression by high glucose in theARPE-19 cells. Expression of ANG-PTL4 has been reported to bestrongly upregulated in liver and adi-pose tissue under fasting conditions(Kersten et al. 2000; Yoon et al.2000). Increased and decreased plasmalevels of ANGPTL4 in fasting condi-tions and under chronic high-fat feed-ing have been reported, indicatingthat ANGPTL4 may function as anendocrine signal involved in the regu-lation of metabolism (Kersten et al.2000; Yoon et al. 2000). The glucose-induced, rather than glucose-sup-pressed, expression of ANGPTL4 in
0
50
100
150
200
250
300
control VEGF15 ng/ml ANGPTL4-100 ng/ml
Tube
form
atio
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RE
cells
(% o
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trol)
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(A) (B)
(C) (D)
Fig. 5. In vitro tube formation assays. Either recombinant human ANGPTL4 or vascular endo-
thelial growth factor (VEGF) was added to plates containing retinal endothelial (RE) cells for
16 hr. (A) Control. CS-C Medium. (B) VEGF (15 ng ⁄ml). (C) ANGPTL4 (100 ng ⁄ml). (D)
Quantitative analysis of the tube formation activity of RE cells treated with either VEGF
(15 ng ⁄ml) or ANGPTL4 (100 ng ⁄ml). n = 5. Values are expressed as means ± SD.
**p < 0.01 as compared with that in the control. Scale bars, 200 lm.
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the ARPE-19 cells suggests that itsexpression may be regulated in a tis-sue-specific manner (Sango et al. 2006;Yamada et al. 2006; Dutton & Trayh-urn 2008).
The mechanisms by which high glu-cose induces ANGPTL4 expression inRPE cells are unknown, and multiplepathways may be involved. Amongthe three transcription factors weexamined, HIF-1a appeared to be oneof the essential factors for the glucose-induced ANGPTL4 expression inARPE-19 cells. The transcriptionalregulation of HIF-1a may be compli-cated, but our data may have a poten-tial therapeutic implication. Tissue orcellular hypoxia is often reported to
be involved in diabetes-related lesions,including atherosclerosis (Williamsonet al. 1993). It has reported that path-ological neovascularization, whichresults from tissue hypoxia, was alsostrongly inhibited in angptl4-deficientmice in a model of oxygen-inducedretinopathy, and ANGPTL4 controlshypoxia-driven angiogenesis in the ret-ina (Perdiguero et al. 2011). Also, ithas been reported that ANGPTL4gene expression is upregulated byHIF-1a in cardiomyocytes (Belangeret al. 2002). Although we did notdirectly demonstrate HIF-1a activa-tion in ARPE-19 cells, the dependenceof ANGPTL4 on HIF-1a may havenovel implications, as diabetic retinop-
athy is almost always associated withtissue ischaemia in the retina (Anto-netti et al. 2006).
ANGPTL4 was reported to beinduced by PPARc activation in adi-pose tissues (Yoon et al. 2000), andwe also showed that PPARc is essen-tial for glucose-induced ANGPTL4expression in ARPE-19 cells, althoughwe did not demonstrate direct PPARcactivation by glucose. Insulin has alsobeen reported to suppress ANGPTL4expression in adipocytes (Yamadaet al. 2006). While some patients oftype 2 diabetes mellitus exhibit hyper-insulinemia which compensates forinsulin resistance, others are insulin-deficient especially after a long clinicalcourse of the disease. It may interest-ing to speculate that ANGPTL4induction in the diabetic eye may becontext-dependent and affected by theambient insulin concentrations,although we did not test this hypothe-sis in our system.
Several recent lines of evidence indi-cate that ANGPTLs may play impor-tant roles in metabolic homoeostasis aswell as angiogenesis (Oike et al. 2005).Indeed, it has been shown that hepato-cyte-derived ANGPTL3 and ANG-PTL4 can regulate lipid metabolism(Koster et al. 2005), and variants in theANGPTL4 gene have been associatedwith lipid abnormalities in humans(Maxwell et al. 2010). Our study pro-vides further evidence, suggesting thatANGPTL4 may be an important linkbetween metabolism and angiogenesisin the retina. Of course, this does notreduce the importance of known fac-tors such as VEGF in diabetic retinop-athy, and although it remains to bedetermined in future studies whetherhuman RPE cells in vivo would alsoproduce ANGPTL4 under hypergly-caemic conditions, it would be interest-ing to see whether ANGPTL4 may beinvolved in some subtypes of diabeticretinopathy.
In summary, we demonstrated thatthe induction of angiogenic activity byglucose in ARPE-19 cells is mediated,at least in part, by the upregulation ofANGPTL4. Although our model,which is based on acute challenge byhigh glucose, does not precisely mimicthe long clinical course of diabetic ret-inopathy, and thus in vivo validationin diabetic animal models is necessary,our data suggest the novel possibilitythat RPE cells might contribute to the
**
**
Tube
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atio
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RE
cells
(% o
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)
*
(E)
(A) (B)
(C) (D)
Fig. 6. Effect of treatment of high glucose-conditioned media (HG-CM) treated with siRNAs
on the tube formation activity of retinal endothelial (RE) cells in vitro. The HG-CM treated
with 20 nm of siRNA for the control, Lamin A ⁄C (A), vascular endothelial growth factor
(VEGF) (B), ANGPTL4 (C) or HIF-1a (D) for 48 hr was added to the plates containing RE
cells for 16 hr. (E) Quantitative analysis of the tube formation activity of the RE cells overlaid
with HG-CM treated with 20 nm of either the control siRNA or the siRNA for VEGF, ANG-
PTL4 or HIF-1a and calculated as a ratio of that in HG medium. n = 5. Values are expressed
as means ± SD. *p < 0.05 **p < 0.01 as compared with the result in the control. Scale bars,
200 lm.
Acta Ophthalmologica 2013
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pathogenesis of diabetic retinopathyvia upregulated expression of ANG-PTL4.
Acknowledgements
We would like to thank Kazuo Ok-ubo, Dai Suzuki and Kazuko Nagasefor their technical Assistance, andToshiyuki Oshitari for discussions andcomments on this manuscript. Thiswork was supported by a Grant fromthe Ministry of Health, Labour andWelfare of Japan (K.Y.) and a grantfrom National Center for GlobalHealth and Medicine (K.Y.).
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Table 1. Genes (A) up- or (B) downregulated by high glucose in ARPE-19 cells.
Probe set Exp1 Exp 2 Gene symbol Cellular component(A) Upregulated genes
