Department of Cell and Molecular Pharmacology and Experimental Therapeutics and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29403
Submitted 15 December 2003 ; accepted in final form 5 May 2004
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ABSTRACT |
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angiogenesis; hypoxia-inducible factor 1; hypoxia; insulin-like growth factor binding protein; vascular endothelial growth factor
VEGF is a potent angiogenic and permeability-enhancing peptide causally linked to neovascularization of the retina and iris (1, 2, 26, 29, 47). In choroidal neovascularization (CNV), a condition that develops in 10% of age-related macular degeneration (AMD) sufferers, newly formed choroidal blood vessels enter the subretinal space, where leakage and bleeding lead to retinal detachment and photoreceptor death (10, 1820, 32, 37). The ineffectiveness of the principal treatments for CNV, namely argon laser photocoagulation and photodynamic therapy, underscores the reason why this disease represents the most common cause of severe vision loss in elderly patients in developed countries (31, 32). Identification of the mediators of ocular angiogenesis would provide important targets for the development of selective inhibitors of CNV (23, 31). Of particular interest to our studies is the retinal pigment epithelium (RPE), a monolayer of highly specialized epithelial cells interposed between the retinal photoreceptors and the choroid (6, 68). Central to photoreceptor survival and function, the RPE is the major source of angiogenic (e.g., VEGF) and antiangiogenic [e.g., pigment epithelium-derived factor (PEDF)] factors and may therefore play a central role in the modulation and progression of CNV (4, 11, 24, 28, 30, 40, 56, 62, 65).
A number of animal models support a role for increased RPE VEGF secretion in the progression of CNV (9, 27, 50). In addition to elevated VEGF levels in the vitreous (62), the RPE and surrounding subretinal membranes express increased levels of VEGF and its receptor kinase insert domain receptor (KDR)/fetal liver kinase receptor-1 (Flk-1) in CNV (3, 48, 58); these increased levels have been attributed to the cellular hypoxic response (59). A number of factors regulate VEGF production; among them, insulin-like growth factor (IGF)-I has been demonstrated to stimulate VEGF expression. Punglia and coworkers (40) showed that increased serum and vitreous IGF-I levels correlate with a wide variety of ischemic retinal disorders linked to neovascularization of the retina and iris. Examination of dissected postmortem RPE-choroid as well as cultured RPE cell lines has found transcription and cell membrane localization of the IGF-I and IGF-II receptors (34, 38, 53, 54, 59, 63) as well as transcription and secretion of IGF-I and IGF-II (34, 36, 38, 53, 63), along with IGF binding proteins (IGFBPs) 16 and the IGFBP-related protein IGFBP-rP1 (36, 38, 53, 63, 64). Because IGFs bind with higher affinity to IGFBPs than to the IGF-I receptor, IGFBPs are capable of acting as antagonists by reducing IGF bioavailability through sequestration (25, 42). Thus the RPE provides the necessary components for a subretinal autocrine-paracrine IGF system capable of modulating retinal function as well as contributing to the pathogenesis of CNV (60, 67).
On the basis of a growing body of evidence demonstrating that IGF-I can induce HIF-1 activity and the secretion of VEGF and IGFBP-3 in RPE cells in vivo and in vitro (1517, 22, 35, 41, 43, 45), we used the spontaneously transformed RPE cell line ARPE-19 (12) to examine the effect of IGF-I on HIF-1 protein expression, VEGF and IGFBP-3 secretion, and the autocrine effects of VEGF and IGFBP-3. Immunoblot analysis revealed IGF-I-induced upregulation of total HIF-1
protein, whereas luciferase reporter assays of HIF-1 transcriptional activity demonstrated accumulation of HIF-1
correlated with the formation of functional HIF-1 heterodimers. In contrast, addition of exogenous VEGF had no significant effect on HIF-1
protein levels in control or IGF-I-stimulated cells. Western and ligand blot analyses of conditioned medium confirmed that IGF-I induced VEGF and IGFBP-3 secretion, recombinant human (rh)VEGF induced IGFBP-3 secretion, and rhIGFBP-3 attenuated IGF-I-stimulated VEGF release. These findings demonstrate that, as seen for VEGF, IGF-I-induced stimulation of IGFBP-3 secretion in RPE cells correlates with increased HIF-1
expression and nuclear localization. We have also identified a unique autocrine function of VEGF in inducing the secretion of IGFBP-3 in control and IGF-I-stimulated ARPE-19 cells without affecting HIF-1 protein expression. Finally, our study demonstrates the negative-feedback role of IGFBP-3 in sequestering and thereby attenuating IGF-I-induced VEGF secretion.
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EXPERIMENTAL PROCEDURES |
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Tissue culture. ARPE-19 cells were incubated in a 1-to-1 ratio of Dulbecco's modified Eagle's medium Base D-5030 and Nutrient Mixture F-12 (Ham) N-6760 with 10% FBS and 10 µl/ml penicillin-streptomycin solution. Unless otherwise stated, cells were maintained at 37°C in a humidified 5% CO2-95% air incubator.
IGF-I, IGFBP-3, CoCl2, tunicamycin, and VEGF treatments. ARPE-19 cells were seeded at a density of 8.6 x 105/well in six-well (9.6-cm2 area) plates. Confluent cells were serum starved (FBS was eliminated in all experiments) for 24 h, to remove known stimulatory growth factors (including IGF-I), before the indicated treatment in fresh, serum-free, medium.
Immunoblot and ligand blot analysis. Confluent serum-starved cells were treated with IGF-I or CoCl2 as indicated, and whole cell lysates were prepared with a modified RIPA buffer containing (in mM) 50 Tris·HCl pH 7.4, 150 NaCl, 10 EDTA, 1 PMSF, 2 sodium orthovanadate, and 10 NaF with 1% Triton X-100 and 10 µg/ml aprotinin and leupeptin. Protein content was determined by BCA assay (Pierce), and 100-µg aliquots were solubilized in SDS sample buffer. VEGF and IGFBP-3 in conditioned medium were quantified after precipitation in 10% trichloroacetic acid (TCA), washing of the pellet with acetone, and solubilization in SDS sample buffer. Proteins so collected were resolved on 12.5% nonreducing polyacrylamide gels, transferred to nitrocellulose (Osmonics, Westborough MA) with a TE-70 SemiPhor apparatus (Hoefer Scientific Instruments, San Francisco, CA), and subjected to ligand or immunoblot analysis. For ligand blot analysis, protein-containing nitrocellulose membranes were washed for 10 min at 23°C in Tris-buffered saline (TBS) containing 3% Triton X-100 and blocked for 1 h with TBS containing 0.2% gelatin. Blots were probed overnight at 4°C with 10 ng/ml tetrabiotinylated IGF-I (Robinson SA and Rosenzweig SA, unpublished data), followed by a 2-h incubation at 23°C with 200 ng/ml Neutravidin-HRP in TBS containing 0.1% Tween 20 and 0.1% BSA. Blots were visualized with the ECL reagent (Amersham Biosciences) on Biomax film (Kodak). Films were subsequently digitized to tiff format, and band intensity was quantified with NIH Image, version II.
For immunoblots, nitrocellulose membranes were blocked for 1 h in bovine lacto transfer technique optimizer (BLOTTO), a TBS solution containing 0.1% Tween 20 and 5% milk protein (reviewed in Ref. 51), before being probed with 1 µg/ml VEGF polyclonal antibody or 1 µg/ml HIF-1 monoclonal antibody, 1 µg/ml HIF-1
monoclonal antibody, or 1:10,000
-actin monoclonal antibody in BLOTTO. HRP-linked secondary antibodies diluted 1:5,000 in BLOTTO were subsequently added for 2 h. To reprobe HIF-1
immunoblots for HIF-1
or
-actin levels, antibodies were removed from the nitrocellulose via the application of Chemicon light stripping solution according to the manufacturer's instructions. Blots were visualized with the ECL reagent as described above.
Luciferase assays. To assay the transcriptional activity of HIF-1, we used the pGL2 basic p2.1 enolase 1 (ENO1) promoter vector, which contains a 68-bp ENO1 promoter fragment encompassing a HIF-1 binding site downstream from the luciferase gene (46). Each well of subconfluent ARPE-19 cells was transiently cotransfected with 100 ng of reporter plasmid and 50 ng of pRL-SV40 Renilla as a control for transfection efficiency. After 24 h, cells were treated with 100 nM IGF-I or 100 µM CoCl2 in 500 µl/well fresh serum-free medium. After an 18-h incubation, cells were lysed in 100 µl/well passive lysis buffer provided with the Dual-Luciferase Reporter Assay System. Cells were scraped and centrifuged for 10 min at 18,890 g, and 20 µl of supernatant per sample was loaded on a 96-well plate and processed for luciferase activity on the Victor2 1420 Multilabel Counter (PerkinElmer Life Sciences, Downers Grove, IL) with the firefly and Renilla luciferase buffers provided with the Dual-Luciferase kit.
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RESULTS |
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DISCUSSION |
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Although the signaling cascade leading to IGF-I-induced HIF-1 expression is still intensely debated, it is well established that the VEGF promoter contains HREs, activation of which results from the binding of HIF-1 (reviewed in Ref. 45). Similarly, a connection, although tenuous, has been established between HIF-1 activity and IGFBP-3 protein expression. Work by Feldser and colleagues (16) demonstrated that although the IGFBP-3 promoter lacks an obvious HRE, IGFBP-3 gene expression was markedly reduced in HIF-1
-deficient cells under hypoxic conditions. These findings suggest some other level of regulation, possibly an indirect effect. In addition, there is still controversy in the literature as to whether IGF-I also upregulates the translation of HIF-1
. Irrespective of the mechanisms by which HIF-1 levels are increased, the present results demonstrate that IGF-I stimulates VEGF and IGFBP-3 secretion in a time- and dose-dependent manner. Together, these findings extend the initial work of Randolph et al. (41) and Punglia et al. (40) demonstrating IGF-I-induced increases in VEGF and IGFBP-3, respectively.
Elevated subretinal levels of VEGF can act to trigger the progression of CNV in animal models (4, 24, 27, 50, 62). VEGF and its receptors, including Flk-1 and feline sarcoma virus-like tyrosine receptor-1 (Flt-1), colocalize to RPE cells (8, 21). Accordingly, we examined whether ARPE-19 cells respond to VEGF. Whereas rhVEGF addition did not alter HIF-1 expression, it did stimulate secretion of IGFBP-3, suggesting that VEGF may regulate RPE cell function in an autocrine manner.
Low oxygen tension (12%) significantly promotes angiogenesis by stimulating VEGF secretion and the upregulation of KDR (7, 61). These conditions also promote the formation of oxygen radicals through a mechanism involving the electron transport chain. It has been reported that reactive oxygen species (ROS) increase the DNA binding activity of HIF-1 (7). It is tempting to speculate that VEGF stimulation of ROS leads to greater HIF-1 binding to the HRE in the IGFBP-3 promoter, leading to increased expression of IGFBP-3. This may serve to explain the observed VEGF stimulation of IGFBP-3 secretion in the absence of detectable alterations in HIF-1
expression.
IGFBP-3 release by RPE cells may have important implications in the regulation of IGF-I/IGF-II autocrine and/or paracrine functions at the RPE and photoreceptor layers, given that IGF action may be inhibited (42) or enhanced (5, 13) by IGFBP-3. Our results demonstrate that IGF-I stimulates IGFBP-3 secretion in a time- and dose-dependent manner. Furthermore, IGF-I-induced VEGF secretion was attenuated by rhIGFBP-3 addition. In light of the fact that IGF-I upregulates the secretion of IGFBP-3 and VEGF in ARPE-19 cells and that their secretion occurs at the apical pole in polarized RPE cells (33, 49), the ability of IGFBP-3 to reduce the bioavailability of IGF-I may play a major role in modulating VEGF secretion by RPE cells in the subretinal space (25, 33, 49). As such, fluctuations in IGF-I, IGF-II, or IGFBPs may have significant implications on RPE cell proliferation and migration after choroidal capillary invasion and the subsequent leakage of circulatory IGFs from choroidal vessels (41, 48, 52, 58, 66). Consequently, dysregulation of the IGF-I system at the level of the subretina may contribute to changes in RPE morphology and increases in angiogenic factor secretion, consistent with CNV.
In summary, we have shown that IGF-I stimulates the expression of HIF-1 and the formation of functional HIF-1 dimers as well as the secretion of VEGF and IGFBP-3 in a time- and dose-dependent manner. In contrast, VEGF enhances the secretion of IGFBP-3 both in the absence and presence of IGF-I without affecting HIF-1
protein expression. Although it had no effect alone, IGFBP-3 attenuated IGF-I-induced VEGF secretion to control levels when present in 10-fold molar excess of exogenously added IGF-I. Together, these results provide further evidence for a role of an IGF-I autocrine/paracrine system in the retina, both in terms of normal ocular physiology as well as in the progression of CNV. The ability of rhVEGF to enhance IGFBP-3 expression, which in turn attenuates IGF-I-stimulated VEGF secretion, constitutes a novel negative autocrine loop regulating this potent angiogenic factor. Furthermore, the ability of rhIGFBP-3 to attenuate IGF-I stimulation of VEGF to constitutive levels presents a tempting avenue in the development of peptide mimetics that retain the IGF-I antagonistic properties of IGFBP-3. Such antagonists may be helpful in the treatment of a wide variety of ischemic retinal disorders linked to neovascularization of the retina and iris where serum and vitreous IGF-I levels are elevated (40). Although HIF-1
is primarily maintained at low levels under normoxic conditions by a degradation process involving the ubiquitin-proteasome system, several cytokines have been found to increase HIF-1 activity (17, 22, 35, 43, 45, 57). Van Obberghen and colleagues (57) reported that insulin stimulates HIF-1
translation via a phosphatidylinositol 3-kinase (PI3-kinase)-dependent signaling pathway in ARPE-19 cells. They also reported that insulin and IGF-I stimulate VEGF expression via different signaling pathways in NIH 3T3 cells (35). Whereas insulin stimulates PI 3-kinase/protein kinase B (PKB), induction by IGF-I involves ERK/mitogen-activated protein kinase (MAPK). In contrast, Semenza and colleagues (17) reported that IGF-I induces HIF-1
synthesis through both PI 3-kinase and MAPK pathways in HCT116 human colon cancer cells. We propose to carry out studies designed to elucidate the roles of reduced oxygen tension and retinal cytokines on HIF-1
expression and VEGF and IGFBP-3 secretion in the RPE and their influence on the progression of CNV. Studies at the cellular level will provide important insights into the mechanisms underlying the pathologies observed in the animal models of CNV. This will lead to a better understanding of the pathogenesis of this disease and to better treatments for this leading cause of blindness.
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GRANTS |
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ACKNOWLEDGMENTS |
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A portion of this work was presented at the 85th Annual Meeting of the Endocrine Society, June 2002, San Francisco, CA; the 1st joint symposium of the Growth Hormone Research Society and the International Society for Insulin-like Growth Factor Research, October 2002, Boston, MA; and the Association for Research in Vision and Ophthalmology Annual Meeting, May 2003, Fort Lauderdale, FL.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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