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(Investigative Ophthalmology and Visual Science. 2001;42:1617-1625.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Angiopoietin-1 Upregulation by Vascular Endothelial Growth Factor in Human Retinal Pigment Epithelial Cells

Masanori Hangai1,2, Toshinori Murata1,2, Nobuaki Miyawaki1,2, Christine Spee3, Jennifer I. Lim1,2, Shikun He2,3, David R. Hinton1,2,3 and Stephen J. Ryan1,2

1 From the Departments of Ophthalmology and 3 Pathology, and 2 Doheny Eye Institute, Keck School of Medicine at the University of Southern California, Los Angeles.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine whether vascular endothelial growth factor (VEGF) regulates angiopoietin (Ang)-1 and -2 expression in retinal pigment epithelial (RPE) cells.

METHODS. Expression of VEGF, Ang1, and Ang2 in surgically removed human choroidal neovascular membranes (CNVMs) was analyzed by double-label confocal immunofluorescence microscopy. Total RNA was extracted from cultured human RPE cells treated with VEGF for mRNA analysis. Northern blot analysis was performed to examine the time course and dose response of Ang1 and Ang2 mRNA expression. mRNA stability and nuclear run-on analyses were performed. Secreted Ang1 and Ang2 protein levels in conditioned media from RPE cells were examined by Western blot analysis.

RESULTS. Ang1 and Ang2 immunostaining colocalized with VEGF-positive stromal cells in human CNVMs. Ang1 and Ang2 mRNAs were expressed by cultured serum-starved RPE cells. VEGF upregulated Ang1 mRNA in a time- and dose-dependent manner without a significant change in Ang2 mRNA. Ang1 and Ang2 mRNAs in RPE cells were as stable as that of S18. VEGF stimulation further increased the half-life of Ang1 mRNA, but did not alter its transcription rate. VEGF increased the amount of Ang1, but not Ang2, protein secreted into the medium.

CONCLUSIONS. The colocalization of Ang1 and Ang2 with VEGF in CNVM stromal cells and the upregulation of Ang1 expression by VEGF in cultured RPE cells suggest that VEGF may selectively modulate Ang expression during CNV.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Choroidal neovascularization (CNV) is a major blinding complication of age-related macular degeneration (ARMD).1 2 Increasing evidence suggests that retinal pigment epithelial (RPE) cells play a key role in the pathogenesis of CNV.3 4 In established CNV, multiple angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-1 and -2, have been detected in a variety of cells, including RPE cells.5 6 7 8 9 10 Furthermore, in normal eyes, polarized secretion of VEGF to the choriocapillaris by RPE and polarized localization of VEGF receptors on the inner choriocapillaris have been reported, suggesting that under resting conditions, RPE-derived VEGF plays a role in the maintenance of the choriocapillaris.11 In addition, several angiogenesis inhibitors, including thrombospondin-1 and pigment epithelium–derived factor, have also been detected in the RPE monolayer.12 13 14 Furthermore, there is evidence that Fas ligand on RPE cells inhibits CNV by Fas-mediated apoptotic killing of choroidal endothelial cells.15 Thus, it appears that the choriocapillaris is maintained by a delicate balance of angiogenic activators and inhibitors produced by RPE cells and that CNV may result from a loss of this balance within the subretinal space.

VEGF, a primary ligand for the endothelial tyrosine kinase receptors Flt-1 and KDR, has been strongly implicated in neovascularization within the eye in clinical and experimental studies.3 16 17 18 19 20 21 22 23 24 25 Ang1 is an agonistic ligand for another endothelial tyrosine kinase receptor, tyrosine kinase with immunoglobulin-like loops and epidermal growth factor homology domains (Tie2).26 In contrast, Ang2 also binds to Tie2, but inhibits Ang1-mediated Tie2 phosphorylation.27 The spectrum of roles that Ang1 and Ang2 play in angiogenesis is quite different from that of VEGF, but recent studies have demonstrated the importance of cooperative interaction of Ang1 and Ang2 with VEGF in physiologic and pathologic angiogenesis.27 28 29 30 31 Based on disrupted angiogenesis in mice deficient in Ang1 and Tie2, it has been suggested that Ang1 is an important regulator of angiogenesis at its later stages, including vessel stabilization and maturation.28 32 In contrast, Ang2 probably cooperates with VEGF in actively sprouting vessels by blocking the stabilizing or maturing function of Ang1, thus allowing vessels to revert to, and remain in, a plastic state where they may respond better to a sprouting signal from VEGF.27 Furthermore, in VEGF-induced neovascularization assayed using the corneal micropocket, Ang1 and Ang2 stimulation results in distinct angiogenic phenotypes. Ang1 with VEGF enlarges the vascular lumen and recruits smooth muscle {alpha}-actin–positive cells to the vascular wall, whereas Ang2 markedly enhances VEGF-induced neovascularization.30 Thus, it appears that sequential regulation of Ang1 and Ang2 in association with VEGF expression within the microenvironment of angiogenic blood vessels is important to ensure precise and stage-appropriate angiogenic signals to endothelial cells.

RPE cells express both VEGF and VEGF receptors and respond to VEGF stimulation in vitro.33 34 35 In experimental choroidal neovascular membranes (CNVMs), RPE cells express mRNAs for VEGF and its receptors.36 Also, RPE cells increase their VEGF expression in response to pathogenic stimuli.37 38 We considered the possibility that VEGF may control production of Ang1 and Ang2 in RPE cells in an autocrine manner to achieve efficient temporal and spatial cooperation between the VEGF and Ang systems during CNV. In the present study, Ang1 and Ang2 expression colocalized with that of VEGF in human choroidal neovascular membranes. VEGF upregulated Ang1 mRNA and protein levels in human RPE cells by increasing its mRNA stability.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Collection of Choroidal Neovascular Membranes
Surgical excision of ARMD-related, subfoveal CNVMs was performed in 12 eyes of 12 patients. All specimens were obtained by one of the authors (JIL, five specimens) during the course of the patients’ treatments, or the specimens were obtained from our frozen archives (seven specimens). The tenets of the Declaration of Helsinki were followed, informed consent was obtained after explanation of the nature and possible consequences of this study, and institutional review board (University of Southern California) approval was granted for this study. Each of the fresh, surgically excised CNVMs was placed in isotonic saline at 4°C, then snap frozen in optimum cutting temperature compound (Ames/Miles Inc., Elkhart, IN) within 1 hour. Each specimen was serially sectioned on a cryostat into 6-µm frozen sections on glass slides. The sections were fixed in reagent-grade acetone for 5 minutes at room temperature and stored at -80°C. Sections from 7 of the 12 specimens had been evaluated previously for expression of other growth factors.7

Double-Label Confocal Immunofluorescence Studies
Thawed sections were air dried, fixed with reagent-grade acetone for 5 minutes, and washed with Tris buffer (pH 7.4). Sections were blocked for 15 minutes with 1% bovine serum albumin (Sigma, St. Louis, MO) in Tris buffer after endogenous peroxide was blocked by 0.3% hydrogen peroxide. For double labeling, sections were incubated first with a polyclonal antibody for 30 minutes and rhodamine-labeled anti-rabbit or anti-goat secondary antibody. Sections were then washed profusely with Tris buffer, followed by monoclonal antibody immunohistochemistry using the FITC-labeled anti-mouse secondary antibody. Negative controls included omission of primary antibody or an irrelevant polyclonal or isotype-matched monoclonal primary antibody. In all cases negative control specimens showed only faint, insignificant staining. Rabbit polyclonal antibodies against human Ang1 and Ang2 (1:500 dilution) were provided by George D. Yancopoulos of Regeneron Pharmaceuticals, Inc. (Tarrytown, NY) and purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Specificity of the Regeneron Ang1 and Ang2 antibodies was confirmed by an absorption test involving an excess of the peptides used to raise the antibodies (Ang1: N-terminal peptide, NQRRSPENSGRRYNRIQHGQ; Ang2: N-terminal peptide, NFRKSMDSIGKKQYQVQHGS). Mouse monoclonal antibody against human VEGF (1:100 dilution) was obtained from Santa Cruz Biotechnology and mouse monoclonal antibody against pancytokeratin from Dako Corp. (Carpinteria, CA). Stained sections were observed with a high-resolution laser-scanning microscope (model LSM210; Carl Zeiss, Jena, Germany).

Cell Culture
Human RPE cells were isolated from human fetal eyes (Anatomic Gift Foundation) or from adult (>60 years) donor eyes without evidence of macular disease, as previously described,39 and cultured in Dulbecco’s modified Eagle’s medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal bovine serum (Gemini Bioproducts, Calabasas, CA), 2 mM L-glutamine (Omega Scientific, Tarzana, CA), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were grown at 37°C in a humidified atmosphere of 5% CO2. The purity of primary cultured RPE cells was determined by counting the number of the cells that were immunoreactive for cytokeratin (Dako). Only cultures that had more than 98% cytokeratin-positive cells were used for analysis (data not shown).

Northern Blot
Total RNA was extracted from RPE cells using the Trizol reagent (Gibco BRL, Gaithersburg, MD) according to the manufacturer’s instructions. Equal amounts of total RNA (10–20 µg per lane) were fractionated on 1% (wt/vol) agarose/6.3%formaldehyde gels and blotted on a nylon membrane (Nylon Duralon-UV; Stratagene, La Jolla, CA), by using the traditional capillary system, in 10x SSPE (1.5 M NaCl, 100 mM sodium phosphate, and 10 mM Na2 EDTA). Filters were cross-linked (UV Stratalinker 1800; Stratagene) and hybridized at 68°C for 2 hours (ExpressHyb Hybridization Solution; Clontech Laboratories, Palo Alto, CA) containing 0.1 mg/ml denatured salmon sperm DNA with 2 to 3 x 106 counts per minute (cpm)/ml 32P-labeled Ang1 (a 0.57-kb SpeI-EcoRI fragment of human Ang1 cDNA), Ang2 (a 0.64-kb EcoRI-HindIII fragment of human Ang2 cDNA), and VEGF (a 0.6-kb BamHI fragment of human VEGF cDNA). Ang1, Ang2, and VEGF cDNAs were kindly provided by Regeneron Pharmaceuticals, Inc. The cDNAs were labeled (specific activity of 1 x 109 cpm/µg) using a random primer labeling kit (Rediprime; Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions. After hybridization, filters were washed three times in 2x SSPE/0.1% SDS for 15 minutes at room temperature and then in 0.1x SSPE/0.1% SDS for 30 minutes at 60°C. To correct for differences in RNA loading, the filters were stripped and rehybridized with a human S18 rRNA probe (Ambion, Austin, TX). The filters were scanned, and radioactivity was quantified by computer imaging (PhosphoImager with ImageQuant software; Molecular Dynamics, Sunnyvale, CA).

mRNA Stability Analysis
RPE cells were exposed to vehicle or VEGF (10 ng/ml) for 8 hours and then incubated with actinomycin D (10 µg/ml) to stop RNA synthesis. Total RNA was isolated at the indicated time points and used for Northern hybridization, as described.

Nuclear Run-On Analysis
RPE cells were treated with vehicle or VEGF (10 ng/ml) for 8 hours. The cells were washed twice with ice-cold phosphate-buffered saline, scraped off the dish in ice-cold SSC (150 mM sodium chloride and 15 mM sodium citrate) and collected in a 15-ml tube by centrifugation at 500g for 5 minutes at 4°C. Subsequent steps were performed at 4°C. The cells were resuspended in 4 ml lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40) and then were disrupted with homogenizers (10 strokes; Dounce; Kontes Glass, Vineland, NJ). Nuclei were pelleted by centrifugation at 500g for 5 minutes, resuspended in 100 µl of glycerol storage buffer (10 mM Tris-HCl, [pH 8.3], 40% [vol/vol] glycerol, 5 mM MgCl2, and 0.1 mM EDTA), and frozen in liquid N2. The nuclear suspension was mixed with 0.1 ml 2x reaction buffer (100 mM HEPES [pH 8.0]; 10 mM MgCl2; 300 mM KCl; 200 U/ml RNasin [Roche Molecular Biochemicals, Indianapolis, IN]; 1 mM each ATP, GTP, and CTP; and 150 µCi (1 µCi = 37 kBq) [32P]UTP [3000 Ci/mmol; Amersham Pharmacia Biotech]) and incubated for 30 minutes at 30°C. Transcription was stopped by adding 20 µg DNase I, followed by 80 µg proteinase K. The 32P-labeled RNA was purified by extraction with phenol-chloroform and two sequential precipitations with ammonium acetate. Equal amounts of 32P-labeled RNA were hybridized in 50% formamide, 5x SSC, 5x Denhardt’s solution, 1% SDS (1x SSC, pH 7.0) at 42°C for 72 hours. Filters contained 0.1 to 5 µg each of linearized plasmids immobilized on membranes (Zeta-Probe GT; Bio-Rad Laboratories, Hercules, CA) after blotting in 12x SSPE with a microfiltration apparatus (Bio-Dot SF; Bio-Rad). Filters were washed three times with 2x SSC and 0.1% SDS at 42oC for 5 minutes, followed by two washes with 0.2x SSC and 0.1% SDS at 65°C for 15 minutes, and then analyzed as for Northern blot analysis (PhosphoImager; Molecular Dynamics).

Western Blot
Confluent RPE cells were incubated with serum-free medium for 24 hours, whereupon the cells were exposed to vehicle or VEGF (10 ng/ml). The conditioned medium was collected, and cell debris was removed by centrifugation at 14,000g. Total protein concentrations of the cell-free supernatant were measured by a Bradford-based assay kit (Bio-Rad), and equal amounts of protein were fractionated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore Corp., Bedford, MA) and then probed with polyclonal rabbit anti-human Ang1 (Regeneron, 0.45 µg/ml) or anti-human Ang2 antibodies (Regeneron, 0.22 µg/ml). Bound antibodies were developed using a chemiluminescence kit, as described by the manufacturer (ECL; Amersham Pharmacia Biotech). The membranes were scanned, and blue fluorescence intensity was quantified (Storm PhosphoImager with ImageQuant software; Molecular Dynamics).

Statistical Analysis
Experiments were performed in triplicate at least three times. Values are expressed as mean ± SEM. Factorial ANOVA was performed followed by Fisher’s least-significant difference test. Statistical significance was defined as {alpha} < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunofluorescence Analysis of Human CNVMs
To determine whether Ang1 and Ang2 expression colocalizes with that of VEGF in human CNVMs, we performed confocal double-label immunofluorescent analysis of human CNVMs. These membranes have strong autofluorescence derived from lipofuscin, causing some false-positive staining when immunofluorescent methods are used. Therefore, in this study, we observed unstained adjacent sections to confirm that the positive staining was not derived from the autofluorescence. As previously reported, there were numerous cells positive for VEGF, Ang1, and Ang2, to a varying extent, in all surgically excised CNVMs.10 In the stromal regions, a majority of Ang1- or Ang2-positive cells were immunoreactive for pancytokeratin, indicating that migrating RPE cells within the CNVMs produce Ang1 and Ang2 (Fig. 1A) . Only a minor population of cytokeratin-negative cells were immunoreactive for Ang1 and Ang2. In each CMVM, VEGF immunostaining highly colocalized with that of Ang1 and Ang2 (Fig. 1B) . There was no apparent difference in the degree of colocalization between VEGF-Ang1 and VEGF-Ang2. Similar results were obtained, whether using the Ang1 and Ang2 antibodies obtained from Regeneron or from Santa Cruz Biotechnology.



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  		Figure 1. Colocalization of Ang1 and Ang2 with VEGF in human CNVMs. Double-labeled confocal immunofluorescence experiments were performed using the anti-VEGF monoclonal antibody (red), the anti-pancytokeratin antibody (red), and the anti-Ang1 or anti-Ang2 polyclonal antibodies (green; Regeneron, Tarrytown, NY). Results of a representative CNV membrane and the respective combined images are shown. Colocalization produces a yellow signal. Magnification, x50.

 
Time- and Dose-Dependent Regulation of mRNA Expression for Ang1 and Ang2 by VEGF in Fetal RPE Cells
Northern blot analysis revealed that Ang1 and Ang2 mRNAs were expressed in serum-starved RPE cells. Stimulation of these cells with 10 ng/ml VEGF increased Ang1 mRNA levels gradually to a maximum of 2.8-fold at 12 hours (P < 0.01; Fig. 2 ). In contrast, Ang2 mRNA levels were unchanged over 24 hours. Ang1 mRNA was expressed at low levels in retinal and choroidal endothelial cells, but the same VEGF stimulation did not change its level (data not shown). Thus, the upregulation of Ang1 in response to VEGF was specific to RPE cells in the cell types examined. To confirm that the effects of VEGF on the induction of Ang1 mRNA were dose dependent, cells were stimulated with various concentrations of VEGF for 12 hours. The upregulation of Ang1 mRNA was dose dependent with a maximal 2.5-fold increase (P < 0.01; Fig. 3 ).



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  		Figure 2. Time-course of mRNA induction of Ang1 and Ang2 by VEGF in human fetal RPE cells. VEGF upregulated Ang1 mRNA levels. Total RNA was extracted from RPE cells at the indicated times after stimulation with VEGF (10 ng/ml) and subjected to Northern blot analysis (15 µg total RNA/lane). Results were quantified by computer imaging. Differences in loading were normalized by using the signal intensity of S18. The corrected density was plotted in comparison with the 0-hour value. Results are mean ± SEM from three separate experiments for each time point. A representative autoradiogram is shown (*P < 0.05, **P < 0.01).

 


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  		Figure 3. Dose–response of mRNA induction of Ang1 by VEGF in human fetal RPE cells. Induction of Ang1 mRNA by VEGF was dose dependent. RPE cells were stimulated with the indicated concentration of VEGF. Total RNA was extracted at 12 hours after the stimulation and analyzed as described in Figure 2 , with results expressed as in Figure 2 (*P < 0.05, **P < 0.01).

 
Increase in Stability of Ang1 mRNA by VEGF
To elucidate the mechanism by which VEGF upregulates Ang1 mRNA, we measured mRNA stability. Unexpectedly, Ang1 and Ang2 mRNA levels normalized by those of S18 remained unchanged for 8 hours after actinomycin-D treatment, suggesting that Ang1 and Ang2 mRNAs in RPE cells are as stable as that of S18 (Figs. 4A 4B 4C) . The approximate half-life of mRNA decay usually can be calculated based on the decay curves for the mRNA levels normalized by a stable housekeeping gene such as S18. Because ribosomal RNA primarily represents total RNA and is more stable than other housekeeping genes, such as the ß-actin and tubulin genes, the use of S18 as an internal control seems to be appropriate to examine stability of stable genes.40 41



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  		Figure 4. Effect of VEGF on Ang1, Ang2, and VEGF mRNA half-lives in human fetal RPE cells. Ang1 (B) and Ang2 (C) mRNAs were very stable compared with that of VEGF (D). VEGF increased the stability of Ang1 mRNA, but not of Ang2 and VEGF mRNAs at the time points examined. RPE cells were exposed to vehicle or VEGF (10 ng/ml) for 8 hours before the addition of actinomycin D (10 µg/ml). Total RNA was extracted from the cells at the indicated times after actinomycin D treatment, and Northern blot analysis was performed as described in Figure 2 . The mean values of the corrected signal intensity were plotted as a percentage of the 0-hour value in logarithmic scale. Results are mean ± SEM from three separate experiments for each time point (*P < 0.05).

 
As a reference, under the same experimental conditions, VEGF mRNA levels decreased rapidly, and the half-life of VEGF mRNA (4.8 hours) was obtained by using the decay curve of VEGF mRNA levels normalized to S18 mRNA levels on a logarithmic scale (Figs. 4A 4D) . However, Ang1 and Ang2 mRNAs were so stable that reliable measurements of their half-lives could not be obtained by using the decay curves of the mRNA levels normalized to S18. Nonetheless, it is clear that the stability of Ang1 and Ang2 mRNAs in RPE cells was extremely high compared with that in bovine choroidal endothelial cells (Ang1 half-life, 3.7 hours; Ang2 half-life, 1.7 hours; data not shown). Addition of VEGF (10 ng/ml) further increased the stability only of Ang1 mRNA (Fig. 4) . In contrast, the same VEGF stimulation had little if any effects on Ang2 mRNA stability and decreased VEGF mRNA stability.

Nuclear run-on analysis was also performed. Although in parallel assays prostaglandin E2 increased the transcription rate of the VEGF gene in RPE cells and tumor necrosis factor-{alpha} increased transcription rate of Ang1 and Ang2 genes in choroidal endothelial cells (data not shown), VEGF treatment (10 ng/ml) of RPE cells did not change the transcription rate of the tested angiogenic growth factors and their receptors, including Ang1 (Fig. 5) , except for a decrease in the transcription rate of the VEGF gene. Taken together, these experiments demonstrate that Ang1 mRNA induction by VEGF in RPE cells was due to an increase in Ang1 mRNA stability.



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  		Figure 5. Effect of VEGF on the Ang1 transcription rate in human fetal RPE cells. VEGF did not alter the transcription rate for Ang1. RPE cells were exposed to vehicle or VEGF (10 ng/ml) for 8 hours (Ang1). Nuclei were isolated, and in vitro transcription was allowed to resume in the presence of [32P] UTP. Equal amounts of 32P-labeled RNA probes were hybridized to nylon membrane on which each cDNA (5 µg) was immobilized. Reduced glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 5 µg) and ß-actin (0.1 µg) were blotted as internal controls. The band intensities were measured by computer imaging, and the values of each factor were normalized using the signal intensity of GAPDH. Results are expressed as in Figure 2 .

 
Effects of VEGF on Ang1 and Ang2 Protein Levels in Fetal RPE Cells
In preliminary experiments, Ang1 and Ang2 proteins were detected primarily in conditioned media collected from serum-starved RPE cells, but were found at much lower levels in cell lysates. Therefore, the effects of VEGF on Ang1 and Ang2 secretion into conditioned media were examined by Western blot analysis. Stimulation of RPE cells with 10 ng/ml of VEGF for 24 to 48 hours did not change the cell number but increased Ang1 protein levels by 1.8-fold (P < 0.01) at 24 hours (Fig. 6 and by 1.5-fold (P < 0.05) at 48 hours. The same stimulation did not change Ang2 protein levels over the same time course. These results indicate that, after VEGF stimulation, an increase in Ang1 mRNA was associated with an increase in Ang1 protein in RPE cells.



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  		Figure 6. Western blot analysis of Ang1 and Ang2 proteins in conditioned media from VEGF-treated human fetal RPE cells. VEGF increased Ang1 (A), but not Ang2 (B), protein secretion. RPE cells were exposed to vehicle or VEGF (10 ng/ml), and the conditioned media were collected 24 and 48 hours after the stimulation. Approximately 5 µg protein per lane was resolved by 10% polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, and immunoblotted using polyclonal antibodies to Ang1 or Ang2. Bound antibodies were detected using a horseradish peroxidase–based chemoluminescence method and quantified by computer imaging. The intensity was plotted in comparison to the vehicle value (C). Results are mean ± SEM from three separate experiments (*P < 0.01; **P < 0.05); representative bands are shown in (A) and (B).

 
Increase of Ang1 mRNA in Adult RPE Cells by VEGF
Experiments were performed using fetal RPE because of the ease of obtaining the large numbers of cells that are required for such experiments. To demonstrate that these data are applicable to adult cells, we also examined human adult RPE cells obtained from eyes of donors older than 60 years, for the induction of Ang1 mRNA by VEGF. Stimulation of the cultured adult cells with 10 ng/ml VEGF increased Ang1 mRNA levels by 3.1-fold at 8 hours (P < 0.05; Fig. 7 ), whereas Ang2 mRNA levels were steady. These data suggest that VEGF upregulates Ang1 not only in fetal RPE cells but also in adult RPE cells.



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  		Figure 7. Induction of Ang1 mRNA by VEGF in human adult RPE cells. VEGF upregulated Ang1 mRNA levels in RPE cells from adult donor eyes. Total RNA was extracted from RPE cells at the indicated times after stimulation with VEGF (10 ng/ml) and analyzed, with results expressed as in Figure 2 (*P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Ang-Tie2 system is unique, in that a natural antagonist for Ang1 exists and that both Ang1 and Ang2 contribute to angiogenesis at distinct stages.27 29 Ang1 is constitutively expressed primarily by nonendothelial (mesenchymal, smooth muscle, and tumor) cells associated with blood vessels during physiologic and pathologic angiogenesis.28 31 42 Tie2 is expressed in quiescent blood vessels throughout the body and in angiogenic blood vessels.43 Increasing evidence suggests that Ang1-mediated Tie2 activation is required for angiogenesis, especially at its later stages, to produce structurally and functionally mature blood vessels.28 29 30 44 45 In contrast, Ang2 is induced in endothelial cells within actively sprouting vessels or zones of vessel regression, suggesting that Ang2-mediated autocrine Tie2 inactivation or other unknown effects of Ang2 are required for active sprouting or vessel regression.27 29 31 Thus, the relative levels of Ang1 and Ang2 are regulated, depending on the stage of angiogenesis. However, it has been shown that both Ang1 and Ang2 are expressed to a similar extent in the same human CNVMs.10 CNVMs consist of a mixture of variously vascularized and fibrotic regions. It is possible that relative levels of Ang1 and Ang2 are delicately regulated depending on the regional microenvironment of CNV. Our results indicate that RPE cells are capable of producing Ang1 and Ang2 and that VEGF can upregulate RPE cell expression of Ang1 without a significant change in Ang2 expression. This regulation may help induce an Ang1-dominant local microenvironment in CNVMs.

Fetal RPE cells were used in this study, because they divide rapidly and thus are suitable to provide the large number of cells required for the extensive analyses reported. Fetal human RPE cells are frequently used as a source of cultured RPE cells for in vitro experiments, and they appropriately express a wide range of cytokine and growth factor receptors including receptors for VEGF.46 47 Because morphologic and functional aging changes in the RPE are well described, it was necessary to determine whether the effects of VEGF on Ang expression are similar in fetal and adult cells.48 In the present study, VEGF upregulated Ang1 but not Ang2 in both fetal and adult RPE, suggesting that aging changes do not interfere with the ability of RPE to respond appropriately to VEGF. Whether RPE cells derived from patients with ARMD respond in a similar way was not determined in this study, because of the extreme difficulty in culturing large numbers of cells from these eyes.

Our previous observation of experimental CNV in monkeys revealed the importance of RPE cells in the development and maturation of CNV.49 Newly formed vessels are leaky with abundant fenestrated vascular walls and open (permeable) interendothelial junctions.50 This early active stage is followed by maturating stages during which newly formed vessels become less leaky with decreased fenestrations and acquire a dense basement membrane and tightly closed interendothelial junctions. During the maturation processes, RPE cells migrate and proliferate around the new vessels and finally envelop them.49 Thus, RPE cells are believed to play an essential role in the maturation and/or suppression of newly formed subretinal vessels. However, little is known about the molecular mechanisms involved in interactions of RPE and endothelial cells during the maturation processes. It has been shown that a combination of Ang1 and VEGF can increase luminal diameter of angiogenic blood vessels and recruit smooth muscle {alpha}-actin–positive cells to vascular walls, suggesting cooperative roles between Ang1 and VEGF in vessel maturation.30 Furthermore, it has recently been shown that Ang1 plays a key role in establishing leakage-resistant blood vessels and that Ang1 can protect blood vessels against VEGF-induced plasma leakage.45 51 RPE-derived Ang1 and VEGF in CNVMs may collaborate to induce structural and functional maturation in such enveloped new vessels.

In a previous report using bovine retinal microvascular endothelial cells, Ang2 expression was upregulated by VEGF and hypoxia, whereas Ang1 mRNA expression was sustained.52 It is interesting that regulation of Ang1 and Ang2 expression by VEGF differed between endothelial cells and vessel-associated nonendothelial cells in the eye. This distinction may be due to the different contributions of endothelial and RPE cells in the neovascularization processes. Of importance, in our results, Ang1 and Ang2 mRNAs in RPE cells were much more stable than Ang1 and Ang2 mRNAs in endothelial cells, including retinal (Ang2 half-life, 3.8 hours)52 and choroidal (Ang1 half-life, 3.7 hours; Ang2 half-life, 1.7 hours) endothelial cells. In our study, VEGF further increased the stability of Ang1 mRNA. In contrast, upregulation of Ang2 in retinal endothelial cells was rapid and short lived and was due to an increase in transcription rate. Evidence has been provided that active sprouting requires prompt and short Ang2-mediated autocrine blockade of Tie2 activation to allow the endothelial cells to better respond to a sprouting signal by VEGF. For this purpose, the rapid and short-lived induction of endothelial Ang2 and its autocrine action to endothelial cells is advantageous. In contrast, at the later stage of neovascularization, endothelial cells may require sustained Tie2 activation for vessel stabilization, maturation, and survival. For this purpose, stable expression of Ang1 by RPE cells is advantageous. At this later stage of CNV, VEGF may shift the balance of Ang1 and Ang2 expression in RPE cells to an Ang1-dominant state by stabilizing Ang1 mRNA and may achieve more effective cooperation with Ang1 to establish structurally and functionally mature blood vessels.

Human CNVMs show strong expression of both Fas and Fas ligand and contain numerous apoptotic cells.53 A functional study revealed that Fas ligand on RPE cells controls experimental CNV by Fas-mediated killing of choroidal endothelial cells.15 Thus, apoptotic endothelial cell death may be one important mechanism by which the retina controls CNV. There is increasing evidence that Ang1 and VEGF can protect endothelial cells against apoptotic death.31 54 55 The sustained expression of Ang1 by RPE cells may promote stabilization of CNV by protecting choroidal endothelial cells against apoptosis in cooperation with VEGF. Upregulation of Ang1 by VEGF in RPE cells could enhance these antiapoptotic activities to further stabilize CNV.

The sustained presence of CNVMs beneath the retina causes damage to the retina. On the one hand, actively leaking CNV results in macular edema and subretinal hemorrhage, which are serious causes of vision loss. On the other hand, less leaky, fibrotic CNVMs block various supporting activities of the RPE-choriocapillaris for photoreceptors. Accordingly, it is important to identify the factors that maintain newly formed blood vessels as well as those that stimulate CNV to initiate rational therapeutic interventions. Although RPE-derived Ang1 and VEGF may be the important factors that maintain CNV, it has been shown that Ang1 also plays a key role in establishing leakage-resistant blood vessels and preventing VEGF-induced plasma leakage.45 51 Thus, RPE-derived Ang1 could be both beneficial and detrimental to the retina. Whether blocking Ang1 in CNVMs would promote or inhibit the development of retinal damage remains an important question to be resolved.


   Acknowledgements
 
The authors thank Susan Clarke for editorial assistance.


   Footnotes
 
Supported by National Institutes of Health Grants EY01545 and EY03040 and grants from the JG Foundation and Research to Prevent Blindness. MH was supported by Kyoto University Foundation, Japan National Society for the Prevention of Blindness, and Nippon Eye Bank Association.

Submitted for publication August 15, 2000; revised January 12, 2001; accepted February 7, 2001.

Commercial relationships policy: F.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Stephen J. Ryan, Department of Ophthalmology, Doheny Eye Institute, Keck School of Medicine at the University of Southern California, 1450 San Pablo Street, DEI#5600, Los Angeles, CA 90033. sryan{at}hsc.usc.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. . Macular Photocoagulation Study Group (1991) Argon laser photocoagulation for neovascular maculopathy: five-year results from randomized clinical trials Arch Ophthalmol 109,1109-1114[Medline][Order article via Infotrieve]
  2. D’Amato, RJ, Adamis, AP (1995) Angiogenesis inhibition in age-related macular degeneration Ophthalmology 102,1261-1262[Medline][Order article via Infotrieve]
  3. D’Amore, PA (1994) Mechanisms of retinal and choroidal neovascularization Invest Ophthalmol Vis Sci 35,3974-3979[Free Full Text]
  4. Campochiaro, PA, Soloway, P, Ryan, SJ, Miller, JW (1999) The pathogenesis of choroidal neovascularization in patients with age-related macular degeneration Mol Vis 5,34[Medline][Order article via Infotrieve]
  5. Frank, RN, Amin, RH, Eliott, D, Puklin, JE, Abrams, GW (1996) Basic fibroblast growth factor and vascular endothelial growth factor are present in epiretinal and choroidal neovascular membranes Am J Ophthalmol 122,393-403[Medline][Order article via Infotrieve]
  6. Kvanta, A, Algvere, PV, Berglin, L, Seregard, S. (1996) Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor Invest Ophthalmol Vis Sci 37,1929-1934[Abstract/Free Full Text]
  7. Lopez, PF, Sippy, BD, Lambert, HM, Thach, AB, Hinton, DR (1996) Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes Invest Ophthalmol Vis Sci 37,855-868[Abstract/Free Full Text]
  8. Ishibashi, T, Hata, Y, Yoshikawa, H, Nakagawa, K, Sueishi, K, Inomata, H. (1997) Expression of vascular endothelial growth factor in experimental choroidal neovascularization Graefes Arch Clin Exp Ophthalmol 235,159-167[Medline][Order article via Infotrieve]
  9. Kliffen, M, Sharma, HS, Mooy, CM, Kerkvliet, S, de Jong, PT (1997) Increased expression of angiogenic growth factors in age-related maculopathy Br J Ophthalmol 81,154-162[Abstract/Free Full Text]
  10. Otani, A, Takagi, H, Oh, H, Koyama, S, Matsumura, M, Honda, Y. (1999) Expressions of angiopoietins and Tie2 in human choroidal neovascular membranes Invest Ophthalmol Vis Sci 40,1912-1920[Abstract/Free Full Text]
  11. Blaauwgeers, HG, Holtkamp, GM, Rutten, H, et al (1999) Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris: evidence for a trophic paracrine relation Am J Pathol 155,421-428[Abstract/Free Full Text]
  12. Steele, FR, Chader, GJ, Johnson, LV, Tombran-Tink, J. (1993) Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family Proc Natl Acad Sci USA 90,1526-1530[Abstract/Free Full Text]
  13. Dawson, DW, Volpert, OV, Gillis, P, et al (1999) Pigment epithelium-derived factor: a potent inhibitor of angiogenesis Science 285,245-248[Abstract/Free Full Text]
  14. Miyajima-Uchida, H, Hayashi, H, Beppu, R, et al (2000) Production and accumulation of thrombospondin-1 in human retinal pigment epithelial cells Invest Ophthalmol Vis Sci 41,561-567[Abstract/Free Full Text]
  15. Kaplan, HJ, Leibole, MA, Tezel, T, Ferguson, TA (1999) Fas ligand (CD95 ligand) controls angiogenesis beneath the retina Nat Med 5,292-297[Medline][Order article via Infotrieve]
  16. Adamis, AP, Miller, JW, Bernal, MT, et al (1994) Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy Am J Ophthalmol 118,445-450[Medline][Order article via Infotrieve]
  17. Aiello, LP, Avery, RL, Arrigg, PG, et al (1994) Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders N Engl J Med 331,1480-1487[Abstract/Free Full Text]
  18. Miller, JW, Adamis, AP, Shima, DT, et al (1994) Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model Am J Pathol 145,574-584[Abstract]
  19. Aiello, LP, Pierce, EA, Foley, ED, et al (1995) Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins Proc Natl Acad Sci USA 92,10457-10461[Abstract/Free Full Text]
  20. Pierce, EA, Avery, RL, Foley, ED, Aiello, LP, Smith, LE (1995) Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization Proc Natl Acad Sci USA 92,905-909[Abstract/Free Full Text]
  21. Robinson, GS, Pierce, EA, Rook, SL, Foley, E, Webb, R, Smith, LE (1996) Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy Proc Natl Acad Sci USA 93,4851-4856[Abstract/Free Full Text]
  22. Aiello, LP (1997) Vascular endothelial growth factor: 20th-century mechanisms, 21st-century therapies Invest Ophthalmol Vis Sci 38,1647-1652[Free Full Text]
  23. Neely, KA, Gardner, TW (1998) Ocular neovascularization: clarifying complex interactions Am J Pathol 153,665-670[Free Full Text]
  24. Ozaki, H, Seo, MS, Ozaki, K, et al (2000) Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization Am J Pathol 156,697-707[Abstract/Free Full Text]
  25. Spilsbury, K, Garrett, KL, Shen, WY, Constable, IJ, Rakoczy, PE (2000) Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization Am J Pathol 157,135-144[Abstract/Free Full Text]
  26. Davis, S, Aldrich, TH, Jones, PF, et al (1996) Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning Cell 87,1161-1169[Medline][Order article via Infotrieve]
  27. Maisonpierre, PC, Suri, C, Jones, PF, et al (1997) Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis Science 277,55-60[Abstract/Free Full Text]
  28. Suri, C, Jones, PF, Patan, S, et al (1996) Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis Cell 87,1171-1180[Medline][Order article via Infotrieve]
  29. Hanahan, D. (1997) Signaling vascular morphogenesis and maintenance Science 277,48-50[Free Full Text]
  30. Asahara, T, Chen, D, Takahashi, T, et al (1998) Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularization Circ Res 83,233-240[Abstract/Free Full Text]
  31. Holash, J, Maisonpierre, PC, Compton, D, et al (1999) Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF Science 284,1994-1998[Abstract/Free Full Text]
  32. Sato, TN, Tozawa, Y, Deutsch, U, et al (1995) Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation Nature 376,70-74[Medline][Order article via Infotrieve]
  33. Adamis, AP, Shima, DT, Yeo, KT, et al (1993) Synthesis and secretion of vascular permeability factor/vascular endothelial growth factor by human retinal pigment epithelial cells Biochem Biophys Res Commun 193,631-638[Medline][Order article via Infotrieve]
  34. Guerrin, M, Moukadiri, H, Chollet, P, et al (1995) Vasculotropin/vascular endothelial growth factor is an autocrine growth factor for human retinal pigment epithelial cells cultured in vitro J Cell Physiol 164,385-394[Medline][Order article via Infotrieve]
  35. Hoffmann, S, Masood, R, Zhang, Y, et al (2000) Selective killing of RPE with a vascular endothelial growth factor chimeric toxin Invest Ophthalmol Vis Sci 41,2389-2393[Abstract/Free Full Text]
  36. Wada, M, Ogata, N, Otsuji, T, Uyama, M. (1999) Expression of vascular endothelial growth factor and its receptor (KDR/flk-1) mRNA in experimental choroidal neovascularization Curr Eye Res 18,203-213[Medline][Order article via Infotrieve]
  37. Lu, M, Kuroki, M, Amano, S, et al (1998) Advanced glycation end products increase retinal vascular endothelial growth factor expression J Clin Invest 101,1219-1224[Medline][Order article via Infotrieve]
  38. Punglia, RS, Lu, M, Hsu, J, et al (1997) Regulation of vascular endothelial growth factor expression by insulin-like growth factor I Diabetes 46,1619-1626[Abstract]
  39. Sheu, SJ, Sakamoto, T, Osusky, R, et al (1994) Transforming growth factor-ß regulates human retinal pigment epithelial cell phagocytosis by influencing a protein kinase C-dependent pathway Graefes Arch Clin Exp Ophthalmol 232,695-701[Medline][Order article via Infotrieve]
  40. Volloch, V, Housman, D. (1981) Stability of globin mRNA in terminally differentiating murine erythroleukemia cells Cell 23,509-514[Medline][Order article via Infotrieve]
  41. Krowczynska, A, Yenofsky, R, Brawerman, G. (1985) Regulation of messenger RNA stability in mouse erythroleukemia cells J Mol Biol 181,231-239[Medline][Order article via Infotrieve]
  42. Stratmann, A, Risau, W, Plate, KH (1998) Cell type-specific expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis Am J Pathol 153,1459-1466[Abstract/Free Full Text]
  43. Wong, AL, Haroon, ZA, Werner, S, Dewhirst, MW, Greenberg, CS, Peters, KG (1997) Tie2 expression and phosphorylation in angiogenic and quiescent adult tissues Circ Res 81,567-574[Abstract/Free Full Text]
  44. Papapetropoulos, A, Garcia-Cardena, G, Dengler, TJ, Maisonpierre, PC, Yancopoulos, GD, Sessa, WC (1999) Direct actions of angiopoietin-1 on human endothelium: evidence for network stabilization, cell survival, and interaction with other angiogenic growth factors Lab Invest 79,213-223[Medline][Order article via Infotrieve]
  45. Thurston, G, Suri, C, Smith, K, et al (1999) Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1 Science 286,2511-2514[Abstract/Free Full Text]
  46. Sippy, BD, Hofman, FM, He, S, et al (1995) SV40-immortalized and primary cultured human retinal pigment epithelial cells share similar patterns of cytokine-receptor expression and cytokine responsiveness Curr Eye Res 14,495-503[Medline][Order article via Infotrieve]
  47. Hoffmann, S, Masood, R, Zhang, Y, He, S, Ryan, SJ, Gill, P, Hinton, DR (2000) Selective killing of RPE with a vascular endothelial growth factor chimeric toxin Invest Ophthalmol Vis Sci 41,2389-2393
  48. Hjelmeland, LM, Cristofolo, VJ, Funk, W, Rakoczy, E, Katz, ML (1999) Senescence of the retinal pigment epithelium Mol Vis 5,33[Medline][Order article via Infotrieve]
  49. Miller, H, Miller, B, Ryan, SJ (1986) The role of retinal pigment epithelium in the involution of subretinal neovascularization Invest Ophthalmol Vis Sci 27,1644-1652[Abstract/Free Full Text]
  50. Ishibashi, T, Miller, H, Orr, G, Sorgente, N, Ryan, SJ (1987) Morphologic observations on experimental subretinal neovascularization in the monkey Invest Ophthalmol Vis Sci 28,1116-1130[Abstract/Free Full Text]
  51. Thurston, G, Rudge, JS, Ioffe, E, et al (2000) Angiopoietin-1 protects the adult vasculature against plasma leakage Nat Med 6,460-463[Medline][Order article via Infotrieve]
  52. Oh, H, Takagi, H, Suzuma, K, Otani, A, Matsumura, M, Honda, Y. (1999) Hypoxia and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in bovine microvascular endothelial cells J Biol Chem 274,15732-15739[Abstract/Free Full Text]
  53. Hinton, DR, He, S, Lopez, PF (1998) Apoptosis in surgically excised choroidal neovascular membranes in age-related macular degeneration Arch Ophthalmol 116,203-209[Abstract/Free Full Text]
  54. Gerber, HP, Dixit, V, Ferrara, N. (1998) Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells J Biol Chem 273,13313-13316[Abstract/Free Full Text]
  55. Kwak, HJ, So, JN, Lee, SJ, Kim, I, Koh, GY (1999) Angiopoietin-1 is an apoptosis survival factor for endothelial cells FEBS Lett 448,249-253[Medline][Order article via Infotrieve]



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