March 2007
Volume 48, Issue 3
Free
Retinal Cell Biology  |   March 2007
RAGE Ligand Upregulation of VEGF Secretion in ARPE-19 Cells
Author Affiliations
  • Wanchao Ma
    From the Departments of Ophthalmology and
  • Song Eun Lee
    From the Departments of Ophthalmology and
  • Jiancheng Guo
    Surgery, Columbia University College of Physicians and Surgeons, New York, New York.
  • Wu Qu
    Surgery, Columbia University College of Physicians and Surgeons, New York, New York.
  • Barry I. Hudson
    Surgery, Columbia University College of Physicians and Surgeons, New York, New York.
  • Ann Marie Schmidt
    Surgery, Columbia University College of Physicians and Surgeons, New York, New York.
  • Gaetano R. Barile
    From the Departments of Ophthalmology and
Investigative Ophthalmology & Visual Science March 2007, Vol.48, 1355-1361. doi:10.1167/iovs.06-0738
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      Wanchao Ma, Song Eun Lee, Jiancheng Guo, Wu Qu, Barry I. Hudson, Ann Marie Schmidt, Gaetano R. Barile; RAGE Ligand Upregulation of VEGF Secretion in ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(3):1355-1361. doi: 10.1167/iovs.06-0738.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. The importance of VEGF in stimulating neovascular age-related macular degeneration (AMD) is well-recognized, but the initiating factors that induce local upregulation of VEGF remain unclear. The current study was conducted to test the hypothesis that activation of RAGE (receptor for advanced glycation end products [AGEs]) by its ligands, including AGEs, amyloid-β peptide (Aβ), and S100B/calgranulins, some of which are known components of drusen and Bruch’s membrane deposits, modulate secretion of VEGF by retinal pigment epithelial (RPE) cells.

methods. ARPE-19 cells were used for all experiments. The cells were transfected with constructs encoding a signal transduction mutant of human RAGE to assess the RAGE-dependence of intracellular signaling. VEGF secretion and gene expression were assessed by ELISA and quantitative real-time PCR. SDS-PAGE and size exclusion chromatography were performed to analyze the structural changes of S100B after oxidation of its thiol groups under denaturing and nondenaturing conditions, respectively. NF-κB activation was assessed via electrophoretic mobility shift assay (EMSA). The impact of the NF-κB inhibition was assessed by using parthenolide.

results. ARPE-19 cells basally secreted VEGF under normal cell culture conditions. Immobilized ligands of RAGE increased VEGF secretion in a RAGE-dependent manner. In contrast, soluble AGE-BSA, fresh Aβ, and S100B were less effective in increasing VEGF secretion. Studies with Aβ demonstrated that oligomeric and surface-immobilized forms of Aβ, but not soluble monomeric forms of Aβ, were effective upregulators of VEGF secretion via RAGE. Oxidation of S100B’s thiol groups resulted in the formation of oligomers that displayed distinct RAGE biological activity compared with the simple dimeric form. RAGE-mediated upregulation of VEGF secretion by ARPE-19 cells was largely dependent on NF-κB, as indicated by studies with parthenolide.

conclusions. Immobilized or oligomerized ligands for RAGE induce RPE cells to increase VEGF secretion. NF-κB plays a central role in RAGE-dependent RPE secretion of VEGF. In AMD, activation of the RAGE axis in RPE cells may contribute to upregulation of VEGF, potentially inciting or propagating neovascular macular disease.

Neovascularization and vascular leakage are major causes of visual loss in retinal vascular disorders and age-related macular degeneration (AMD). In neovascular AMD, RPE cellular dysfunction associated with production of VEGF is an important component of angiogenesis, as experimentally induced VEGF overexpression in the RPE is sufficient to induce choroidal neovascularization. 1 2 3 In the normal eye, VEGF receptors are localized to the choriocapillaris endothelium abutting RPE cells, and RPE cells secrete a basal level of VEGF that may maintain the physiologic function of the choriocapillaris. VEGF levels are significantly higher in patients with neovascular AMD than in healthy control subjects, 4 but the exact triggers of VEGF expression and secretion and subsequent neovascularization in AMD remain unclear. 
Increased protein cross-linking and the accumulation of advanced glycation end-products (AGEs) develop in aging human Bruch’s membrane. 5 AGEs may directly impact the structure of Bruch’s membrane and impede its physiologic function. Increased protein cross-linking and AGEs are also identified in drusen of eyes with AMD. 6 7 In addition to their direct impact on structural integrity of the basement membrane, AGEs may also exert pathogenic effects by engagement of cellular receptors, the best characterized of which is RAGE (receptor for AGEs). RAGE is a member of the immunoglobulin superfamily of cell surface molecules and has been implicated in long-term diabetic complications, neurodegenerative diseases, acute and chronic inflammatory disorders, and cancer. 8 RAGE contains an extracellular domain, a single transmembrane-spanning domain and a 43-amino acid highly negatively charged cytosolic tail. The cytosolic tail is essential for RAGE-mediated intracellular signaling, and introduction of RAGE molecules with deletion of the cytosolic domain into RAGE-expressing cells imparts a dominant-negative (DN) effect on RAGE, with consequent loss of receptor activation. 9 10  
RAGE is a multiligand receptor. In addition to binding AGEs, RAGE also is a signal transduction receptor for amyloid-β (Aβ) and S100/calgranulins. Amyloid is a component of drusen, with its deposition thought to be AMD-specific, and several studies have addressed the molecular contribution of amyloid to the development of AMD. 11 12 As RAGE is known to be present in RPE 13 and is upregulated in AMD, 14 we tested the hypothesis that ligand-RAGE interaction contributes to the development of neovascular macular disease, in part via modulation of VEGF production by RPE cells. 
Methods
Cell Culture and Transfection
ARPE-19 cells, generously provided by Janet Sparrow (Columbia University), were used throughout the investigation. The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all products from Invitrogen-Gibco, Rockville, MD). Cells were incubated at 37°C in a 5% CO2 incubator and subcultured with 0.05% trypsin-EDTA (Invitrogen-Gibco). Confluent cultures, except where noted, were trypsinized to prepare dissociated cells for all experiments. 
The human dominant-negative RAGE (DN-RAGE) gene, subcloned into the pcDNA3 plasmid, pcDNA3 (DN-RAGE), 15 was transfected into the ARPE-19 cells with transfection reagent (Lipofectamine 2000; Invitrogen) according to the manufacturer’s protocol. Mock-transfectants expressed vector alone. 
Western Immunoblot Analysis
Confluent ARPE-19 cells in 60-mm dishes were incubated in 2 mL of cell culture medium containing 10 μg/mL of anti-human RAGE IgG, 16 at 37°C in 5% CO2 incubator for 60 minutes. After the cells were washed three times with phosphate-buffered saline (PBS), they were collected and lysed with 0.2 mL of 0.5% Triton X-100 in PBS at 0°C. The complex of RAGE and its antibody in the supernatant was pulled down with protein A agarose beads (Sigma, St. Louis, MO) and eluted with 30 μL of 1× sample buffer for nonreducing SDS-PAGE (4%–20% Tris-glycine gel, Invitrogen-Novex, Carlsbad, CA). After the contents of the gels were transferred to nitrocellulose membrane (Invitrogen-Novex), Western blot was performed using rabbit anti-human RAGE IgG (2 μg/mL) and the enhanced chemiluminescence (ECL) detection system (GE Healthcare, Piscataway, NJ). Stably transfected C6 glioma cells expressing full-length human RAGE were used as a positive control. 15  
Preparation of RAGE Ligands
To prepare surface immobilized ligands, the wells of 96-well ELISA plate (Maxisorp; Nunc, Paisley, UK) were coated with 0.1 mL of freshly prepared solutions of AGE-BSA (200 μg/mL; Sigma-Aldrich, St. Louis, MO), S100B (10 μg/mL; Calbiochem, La Jolla, CA), and Aβ (Aβ 1-42, 10 μg/mL; Sigma-Aldrich) in 0.05 M carbonate buffer (pH 9.6) overnight at 4°C and then washed with PBS. Nonbound sites of the wells including those without coating were blocked with either 2% bovine serum albumin (BSA) in PBS or cell culture medium (blocking with either BSA or cell culture medium yielded identical results), and then they were washed with cell culture medium before the cells were plated. 
Oligomeric Aβ was generated by incubating freshly prepared 100 μM of Aβ in cold F12 medium (Invitrogen, Rockville, MD) at 4°C to 8°C for 24 hours and centrifuging at 20,000g for 10 minutes. Oxidized S100B was generated with copper-catalyzed autoxidation of its sulfhydryl groups. 17 Briefly, 10 μM of freshly prepared S100B in 20 mM Tris-HCl buffer (pH7.6) was first dialyzed against the buffer to rid the product of 1,4-dithiothreitol (DTT). Copper sulfate was then added to a final concentration of 80 μM. The solution was incubated at 37°C for 2 hours and then placed at 4°C for variable times. To transform the oxidized S100B to its final calcium-binding forms, a series of dialyses were performed at 4°C, first against 2 mM EDTA in calcium-free PBS, then in calcium-free PBS, and finally in PBS with calcium and magnesium (Invitrogen). A fast protein liquid chromatography (FPLC) system (with a Superdex 75 FPLC column; 10 × 300 mm, GE Healthcare) and PBS containing calcium and magnesium as running buffer was used for the separation and molecular weight determination of S100B oligomers. The column was calibrated by a gel filtration molecular weight markers kit (MW-GF-70; Sigma-Aldrich), and the elution of S100B was monitored continuously at 254 nm. Peaks of S100B oligomers were collected and concentrated (Ultrafree-MC Filter; Millipore, Bedford, MA). Protein levels were determined with protein assay (Bio-Rad Laboratories, Hercules, CA). 
ELISA for Determination of VEGF Levels
ARPE-19 cells (2 × 104) in 0.1 mL of cell culture medium were plated onto the wells of 96-well plates with the indicated protein coating as described above. Treatment with soluble RAGE ligands was also initiated at the time of plating of cells. Media was collected after 24 hours of incubation at 37°C in 5% CO2 and assessed for levels of VEGF by ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. 
Quantitative Real-Time PCR
Total RNA was isolated from ARPE-19 cells (RNeasy Plus Mini kit; Qiagen, Inc., Valencia, CA) and reverse transcribed to cDNA (TaqMan Reverse Transcription Reagents kit; Applied Biosystems [ABI], Foster City, CA), according to the manufacturer’s protocol. Quantitative real-time PCR (RT-PCR) was performed on a sequence-detection system (Prism 7900HT; ABI) according to the manufacturer’s instruction. Probe and primer sets for VEGF (Hs00173626_m1) and ribosomal RNA (4308329), as internal control, were used for quantitative VEGF expression assay and results were analyzed by the 2−ΔΔCT method. 18  
Electrophoretic Mobility Shift Assays of NF-κB Activation
ARPE-19 cells (3 × 106) in 4 mL of medium, with or without RAGE ligands, were plated into 60-mm cell culture dishes, without or with S100B coating, respectively, and incubated at 37°C in 5% CO2 for 2 hours. Nuclear proteins were prepared by using the isolation kit from Pierce Biotechnology (Rockford, IL), and 10 μg was used for NF-κB DNA binding activity assay with an EMSA kit (Promega, Madison, WI). The DNA-protein complexes were resolved by 6% polyacrylamide gel. For competition experiments, an excess (10×) of specific unlabeled double-stranded probe was added to the binding mixture. 
Statistical Analysis
Student’s t-test was used to analyze the statistical significance of the results. P < 0.01 was considered statistically significant. 
Results
Expression of Endogenous RAGE Gene and Exogenous DN-RAGE Gene in ARPE-19 Cells
We first sought to confirm that ARPE-19 cells express RAGE. Anti-human RAGE IgG was incubated with intact ARPE-19 cells, and the RAGE-anti-RAGE IgG complex was isolated from ARPE-19 cell lysate and used for Western blot analysis. As shown in Figure 1 , lane 1, a single plasma membrane protein with a molecular mass of 55 kDa was demonstrated by Western blot using anti-RAGE IgG. Stably transfected C6 glioma cells, expressing full-length human RAGE, were used as positive control for detection of RAGE antigen (Fig. 1 , lane 4). 
Because deletion of the cytosolic domain of RAGE exerts a dominant negative effect on RAGE function, the expression of exogenous DN-RAGE gene in ARPE-19 cells was used throughout this study to test if the increase in VEGF secretion by RAGE ligands was via activation of RAGE signal transduction. As shown in Figure 1 , lane 3, a human DN-RAGE gene that was integrated into pCDNA3 plasmid and introduced into ARPE-19 cells by transient transfection was highly expressed. Note that two bands are evident in lane 3. The upper band represents endogenous full-length RAGE and the lower, slightly smaller band, represents the transfected DN-RAGE construct in which the cytoplasmic domain was deleted. Note that in Figure 1 , lane 2 represents RAGE expression in mock (empty vector)-transfected ARPE-19 cells. 
Effect of AGE-BSA-RAGE Interaction on Secretion of VEGF in ARPE-19 Cells
Once we established that ARPE-19 cells expressed RAGE, we then tested the premise that ligand-RAGE interaction stimulated secretion of VEGF. Under normal cell culture conditions, ARPE-19 cells released basal level of VEGF into cell culture medium (Fig. 2) . Treatment of ARPE-19 cells with soluble AGE-BSA in cell culture medium failed to increase VEGF secretion in medium. Once coated onto the cell culture dish, however, AGE-BSA resulted in significantly increased levels of VEGF in the culture medium. Consistent with an important role for RAGE signaling in mediating the effects of immobilized AGE-BSA, when ARPE-19 cells were transfected with DN-RAGE, no enhanced secretion of VEGF was noted (Fig. 2) . These experiments demonstrate that immobilized AGE-BSA enhances VEGF secretion in RPE cells in a RAGE-dependent manner. 
Effect of Aβ-RAGE Interaction on Secretion of VEGF in ARPE-19 Cells
To examine the impact of distinct forms of Aβ on VEGF secretion via RAGE, experiments were performed with monomeric, oligomeric, and surface-immobilized Aβ preparations. ARPE-19 cells cultured in a 96-well plate were tested with these different forms of Aβ. Freshly prepared Aβ solutions bear the most monomeric features. Addition of monomeric Aβ into cell culture medium failed to increase VEGF secretion (Fig. 3) . When Aβ was immobilized on cell culture dish or oligomerized at 4°C for 24 hours, these forms of Aβ significantly enhanced VEGF secretion in a manner dependent on RAGE. Compared to mock-transfected ARPE-19 cells, those cells transfected with DN-RAGE failed to show increased secretion of VEGF after stimulation with coated Aβ (Fig. 3)
To confirm that Aβ upregulates mRNA transcripts of VEGF in ARPE-19 cells, as has been previously reported in primary human RPE culture, 12 and to demonstrate that the increased secretion of VEGF is the result of upregulation of VEGF gene expression, probe and primer sets designed for detection of all the splice variants of VEGF gene expression were used in quantitative real-time PCR experiments. Consistent with increased secretion of VEGF protein by RAGE ligands, VEGF mRNA expression was significantly upregulated by immobilized Aβ, whereas there was no change when monomeric Aβ was applied into cell culture medium (Fig. 4A) . Time-dependent studies revealed that Aβ-induced VEGF mRNA upregulation occurred after 2 hours, reaching statistically significant elevation at 6 hours and 16 hours, VEGF mRNA expression was increased nearly threefold in comparison to control cells (Fig. 4B)
Effect of S100B-RAGE Interaction on Secretion of VEGF in ARPE-19 Cells
Extracellular S100B protein mediates neurite extension and neuronal survival in neuronal cells, in a manner sensitive to reduction of disulfide bonds. 19 When either of the two cysteines within S100B was altered by site-directed mutagenesis, the resultant proteins were reported to lose both the neurite extension and neuronal survival properties. 20 21 Commercial preparations of S100B protein contain DTT and hence are reduced dimeric forms of S100B. Once coated onto cell culture wells, this form of S100B was effective in stimulating secretion of VEGF, in a manner dependent on intact RAGE signal transduction (Fig. 5)
Based on these observations, we hypothesized that the disulfide bonds necessary for S100B extracellular activity are also necessary for S100B oligomer formation. To assess potential structural changes of S100B after oxidation of its thiol groups, we performed SDS-PAGE (Fig. 6A)and size-exclusion chromatography (Fig. 6B) , to analyze S100B properties under denaturing and nondenaturing conditions, respectively. As shown in Figure 6A , lane 1, fresh S100B is a noncovalently linked dimer under physiological solutions that eluted as a single protein peak with molecular weight of 28 to 31 kDa (Superdex G-75 column; Invitrogen; Fig. 6B ), and was separated into monomers by denaturing SDS-PAGE under nonreducing conditions. SDS-PAGE analyses of S100B oxidation products confirmed that disulfide formation between S100B monomers was completed after 2 hours’ reaction with cupric sulfate at 37°C. All S100B formed disulfide-linked dimers, and no higher cross-linking products could be detected even with extension of the reaction time (Figs. 6A , lanes 2–4). When the S100B oxidation products were subjected to size exclusion chromatography, dramatic structural changes could be detected with respect to the oligomeric state of S100B. As shown in Figure 6B 6aS100B tetramer, as a minor peak, appeared after 2 hours of oxidation at 37°C and a higher oligomer, an octamer, eluted as a major component after extended reaction at 4°C. These results demonstrate that reconfigurations of S100B after intermolecular disulfide formation occurred in a time-dependent manner. Oxidized S100B dimer, tetramer, and octamer were then collected, concentrated, and tested for their ability to activate RAGE. The only active soluble form of S100B was its oxidized octameric form. Other forms did not induce RAGE-dependent secretion of VEGF (Fig. 6D)
Effect of NF-κB Inhibition on RAGE-Dependent VEGF Secretion in ARPE-19 Cells
Ligand-RAGE interaction may trigger activation of a range of signaling pathways, including activation of NF-κB. 15 22 23 As shown in Figure 7 , lane 4, activation of RAGE by immobilized S100B induced NF-κB nuclear translocation indicating increased binding of NF-κB to its consensus sequence that may regulate a variety of cellular gene expression changes. To elucidate whether VEGF expression is a downstream consequence of ligand-RAGE stimulated NF-κB activation, a specific inhibitor of NK-κB activation, parthenolide, 24 was incubated with ARPE-19 cells before exposure to RAGE ligands. As demonstrated in Figure 8 , addition of parthenolide into cell culture medium suppressed the increased secretion of VEGF from ARPE-19 cells treated with active forms of RAGE ligands, including immobilized AGE-BSA, S100B, and Aβ. 
Discussion
The successful clinical application of the anti-VEGF compounds bevacizumab, ranibizumab, and pegaptanib in AMD strongly supports the importance of VEGF in progression of neovascular AMD. Hypoxia is a major inducer of VEGF gene transcription, and localized hypoxia overlying regions of drusen deposition and thickened Bruch’s membrane may increase the risk of VEGF upregulation and neovascular AMD in the aging macula with drusen accumulation. In addition to hypoxia, other growth factors, inflammatory cytokines, and “upstream” stimuli can also upregulate VEGF expression. 25 In this study, we show for the first time that RAGE ligands, known to be present in drusen and increased in aging Bruch’s membrane, can directly induce ARPE-19 cells to secrete VEGF via activation of RAGE. The most effective trigger is a specific deposit present in drusen from eyes with AMD-amyloid-β peptide, a major proinflammatory component of Alzheimer’s disease plaques. Amyloid-β increases VEGF mRNA and protein levels in ARPE-19 cells, confirming previous experiments with primary cultures of human RPE that have also implicated this peptide in the pathogenesis of AMD. 12 We demonstrate that two other ligands for RAGE, AGEs and S100/calgranulin, are also capable of increasing VEGF secretion by RPE cells. Each of these ligands increases VEGF production in a RAGE-dependent manner, as the introduction of a dominant-negative gene construct of RAGE lacking the cytosolic domain, known to abrogate RAGE activation, prevented upregulation of VEGF secretion by RPE cells in the setting of RAGE ligand stimulation. Inhibition of NF-κB activation by parthenolide effectively eliminates RAGE-dependent RPE secretion of VEGF, consistent with key roles for this transcription factor in mediating the impact of RAGE ligands. 
RAGE is expressed at low levels in homeostasis in a range of cell types, including endothelial cells, macrophages, vascular smooth muscle cells, Müller cells, and RPE cells. In embryonic development and in biological stresses including hyperglycemia, oxidative stress, inflammation, and neurodegenerative disorders, RAGE expression increases, with its ligands further upregulating receptor expression to amplify local cellular responses. In the case of RAGE in RPE cells, this mechanism of activation potentially can augment the response of RPE cells to RAGE ligands known to be present in the aging macula and early AMD, including AGEs and Aβ. Previous work has identified upregulated RAGE mRNA in RPE cells overlying basal deposits, 7 also known to contain AGEs, consistent with the known biology of RAGE activation. One consequence of this localized activation of the RAGE axis in RPE cells appears to be increased secretion of VEGF, which may target closely apposed choriocapillaris endothelium. Though the local levels of VEGF necessary to elicit a pathologic response are yet to be elucidated, increased RPE secretion of VEGF in some settings appears to be sufficient to induce vascular leakage and/or angiogenesis in the neovascular progression of macular degeneration. 
The RPE monolayer is exposed to Bruch’s membrane, an extracellular matrix that, with collagen as its major component, is a prime target for AGE modifications over long periods, such as with increasing age. A characteristic feature of collagen is its extensive posttranslational modifications, most of which are unique to collagen protein. Such modifications include hydroxylation of proline and lysine residues, glycation, glyco-oxidation, oxidative deamination of the ε-amino groups of lysine and hydroxylysine, and subsequent intra- and intermolecular cross-linking. AGE modifications increase with aging, hyperglycemia, inflammation, and oxidative stress. Extensive evidence suggests that AGEs may alter cell–matrix interactions by inducing changes in cellular adhesion and receptor activation. The heterogeneic chemical structures of AGEs prevent AGE-BSA from being uniform in composition, as different preparations have various levels of modifications with distinct chemical structures. Although it is not surprising that some AGE-BSA preparations have failed to activate RAGE, 26 the direct evidence for heterogeneities within AGE-BSA was reported recently, with only highly modified and cross-linked species preferentially bound to RAGE. 27 For surface immobilized AGEs, the formation of AGE clusters suitable for RAGE activation will increase exponentially with the density of AGEs (i.e., mathematically, in the simplest dimer model, when AGE density increases twofold, the opportunity to form an AGE-dimer increases approximately fourfold). In the aging macula, with increasing AGE-mediated structural clustering in Bruch’s membrane and with accumulation of drusen in early AMD, it is tempting to speculate that a certain threshold of disease is necessary to trigger localized neovascular or atrophic macular degeneration. Though the precise pathophysiologic levels of such triggers in vivo are unknown, the multiligand nature of RAGE raises the intriguing possibility that AGEs may provide the “seed” for further generation and augmentation of RAGE which, once set in motion, renders RPE cells more susceptible to other RAGE ligands, including S100B and Aβ. Indeed, the local environment of the RPE may render this cell unique, compared with other RAGE-bearing cells, with respect to its cellular response to ligands for RAGE. It appears that only when these ligands are sufficiently immobilized, cross-linked, or oligomerized is a threshold for activation of RAGE in RPE cells achieved, thereby effectively resulting in increased VEGF production. 
In the case of S100B, we further implicate the higher-order oxidized octameric form as the only soluble form of S100B capable of RAGE activation in RPE cells. The possible role of S100B in AMD has not been reported and is the subject of future study. Finally, we demonstrate that the inhibition of NF-κB effectively abrogated the response of RPE cells to RAGE ligands under conditions that would normally upregulate VEGF secretion. In this regard, we used parthenolide, a sesquiterpene lactone compound and an active substance in the medical herb Feverfew (Tanacetum parthenium) traditionally used in the treatment of inflammation. The NF-κB-dependent nature of RAGE-stimulated VEGF secretion in RPE cells renders this pathway, along with the RAGE axis itself, a potential therapeutic target for the reduction of the neovascular stimulus in eyes at high risk for progression of macular degeneration. Further studies are necessary to examine the clinical implications of these observations and to continue to define the role of RAGE and its ligands in the progression of AMD. 
 
Figure 1.
 
Expression of endogenous RAGE and exogenous DN-RAGE by Western blot analysis. Confluent normal ARPE-19 cells (lane 1), mock-transfectants (lane 2), and DN-RAGE transfectants (lane 3) were pretreated with anti-human RAGE IgG for 1 hour at 37°C in 5% CO2. RAGE molecules were isolated and detected. Total protein (5 μg) from human RAGE stably transfected C6 glioma cells was used as the RAGE-positive control (lane 4).
Figure 1.
 
Expression of endogenous RAGE and exogenous DN-RAGE by Western blot analysis. Confluent normal ARPE-19 cells (lane 1), mock-transfectants (lane 2), and DN-RAGE transfectants (lane 3) were pretreated with anti-human RAGE IgG for 1 hour at 37°C in 5% CO2. RAGE molecules were isolated and detected. Total protein (5 μg) from human RAGE stably transfected C6 glioma cells was used as the RAGE-positive control (lane 4).
Figure 2.
 
Effect of soluble and surface-immobilized AGE-BSA on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with 200 μg/mL of AGE-BSA in cell culture medium or coated onto the wells of cell culture plates. VEGF levels in medium were determined by ELISA after 24 hours incubation at 37°C in 5% CO2. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 2.
 
Effect of soluble and surface-immobilized AGE-BSA on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with 200 μg/mL of AGE-BSA in cell culture medium or coated onto the wells of cell culture plates. VEGF levels in medium were determined by ELISA after 24 hours incubation at 37°C in 5% CO2. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 3.
 
Impact of Aβ oligomers on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with freshly prepared Aβ in medium or coated onto the wells of 96-well plates, and oligomerized Aβ, all at 10 μg/mL. VEGF secretion was assayed as described and expressed in comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 3.
 
Impact of Aβ oligomers on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with freshly prepared Aβ in medium or coated onto the wells of 96-well plates, and oligomerized Aβ, all at 10 μg/mL. VEGF secretion was assayed as described and expressed in comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 4.
 
Upregulation of VEGF gene expression by Aβ-induced RAGE activation in ARPE-19 cells. (A) Normal ARPE-19 cells (5 × 105) in 2 mL of cell culture medium, with or without 20 μg of freshly prepared Aβ, were plated onto 35-mm cell culture dishes, without and with Aβ coating, respectively, and cultured at 37°C in 5% CO2 for 16 hours. (B) Time-dependent VEGF gene expression was performed with immobilized Aβ in 35-mm cell culture dishes. Results were expressed as comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 4.
 
Upregulation of VEGF gene expression by Aβ-induced RAGE activation in ARPE-19 cells. (A) Normal ARPE-19 cells (5 × 105) in 2 mL of cell culture medium, with or without 20 μg of freshly prepared Aβ, were plated onto 35-mm cell culture dishes, without and with Aβ coating, respectively, and cultured at 37°C in 5% CO2 for 16 hours. (B) Time-dependent VEGF gene expression was performed with immobilized Aβ in 35-mm cell culture dishes. Results were expressed as comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 5.
 
Effect of S100B on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with freshly prepared 10 μg/mL of S100B in cell culture medium or coated onto cell culture plates. VEGF secretion was assayed as described and expressed as comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 5.
 
Effect of S100B on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with freshly prepared 10 μg/mL of S100B in cell culture medium or coated onto cell culture plates. VEGF secretion was assayed as described and expressed as comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 6.
 
Preparation, isolation, and activities of distinct forms of S100B. (A) Nonreducing (lanes 1–4) and reducing (lanes 6–9) SDS-PAGE analyses of oxidized S100B, 3 μg/lane, Coomassie blue staining. Lanes 1 and 6: fresh S100B; lanes 2 and 7: S100B after 2 hours oxidation with copper sulfate at 37°C; lanes 3 and 8: S100B oxidized as lanes 2 and 7 with an additional 10 days’ incubation at 4°C; lanes 4 and 9: S100B similar to lanes 3 and 8, but with additional 14 days’ incubation at 4°C; lane 5: protein molecular mass marker. (B) Separation of oxidized S100B by FPLC. Short-term oxidation represents 2 hours’ oxidation at 37°C; and long-term oxidation represents short-term oxidation with an additional 24 days’ incubation at 4°C. Three oligomers with molecular masses 84 to 88, 49 to 52, and 28 to 31 kDa were separated and collected. Based on molecular masses and comparison to standards, these represent S100B octamer, tetramer, and dimer, respectively. (C) Elution profile of protein molecular weight standards on a separation column. BSA, 2 mg/mL, carbonic anhydrase (CA), 1 mg/mL, and cytochrome c (CytC), 0.5 mg/mL, were used to predict molecular mass of the distinct S100B oligomers shown in (B). (D) Effect of distinct S100B oligomers on VEGF secretion. ARPE-19 cells (2 × 104) were treated with 10 μg/mL of the S100B oligomers in cell culture medium. VEGF levels in medium were determined by ELISA after 24 hours’ incubation at 37°C in 5% CO2 and presented as the mean ± SD of at least three replicates (*P < 0.01).
Figure 6.
 
Preparation, isolation, and activities of distinct forms of S100B. (A) Nonreducing (lanes 1–4) and reducing (lanes 6–9) SDS-PAGE analyses of oxidized S100B, 3 μg/lane, Coomassie blue staining. Lanes 1 and 6: fresh S100B; lanes 2 and 7: S100B after 2 hours oxidation with copper sulfate at 37°C; lanes 3 and 8: S100B oxidized as lanes 2 and 7 with an additional 10 days’ incubation at 4°C; lanes 4 and 9: S100B similar to lanes 3 and 8, but with additional 14 days’ incubation at 4°C; lane 5: protein molecular mass marker. (B) Separation of oxidized S100B by FPLC. Short-term oxidation represents 2 hours’ oxidation at 37°C; and long-term oxidation represents short-term oxidation with an additional 24 days’ incubation at 4°C. Three oligomers with molecular masses 84 to 88, 49 to 52, and 28 to 31 kDa were separated and collected. Based on molecular masses and comparison to standards, these represent S100B octamer, tetramer, and dimer, respectively. (C) Elution profile of protein molecular weight standards on a separation column. BSA, 2 mg/mL, carbonic anhydrase (CA), 1 mg/mL, and cytochrome c (CytC), 0.5 mg/mL, were used to predict molecular mass of the distinct S100B oligomers shown in (B). (D) Effect of distinct S100B oligomers on VEGF secretion. ARPE-19 cells (2 × 104) were treated with 10 μg/mL of the S100B oligomers in cell culture medium. VEGF levels in medium were determined by ELISA after 24 hours’ incubation at 37°C in 5% CO2 and presented as the mean ± SD of at least three replicates (*P < 0.01).
Figure 7.
 
The effect of S100B on activation of NF-κB in ARPE-19 cells. ARPE-19 cells (3 × 106) were treated with soluble S100B or immobilized S100B for 2 hours. Isolation of nuclear protein and EMSA were performed as described. Lane 1: negative control (without nuclear proteins); lane 2: control cells; lane 3: soluble S100B-treated cells; lane 4: immobilized S100B-treated cells; lane 5: same as lane 4 but with excess unlabeled double-stranded probe.
Figure 7.
 
The effect of S100B on activation of NF-κB in ARPE-19 cells. ARPE-19 cells (3 × 106) were treated with soluble S100B or immobilized S100B for 2 hours. Isolation of nuclear protein and EMSA were performed as described. Lane 1: negative control (without nuclear proteins); lane 2: control cells; lane 3: soluble S100B-treated cells; lane 4: immobilized S100B-treated cells; lane 5: same as lane 4 but with excess unlabeled double-stranded probe.
Figure 8.
 
RAGE-dependent secretion of VEGF requires NF-κB activation in ARPE-19 cells. Normal ARPE-19 cells (2 × 104), with or without 5 μM parthenolide, were plated onto the wells of the indicated coated 96-well plate and cultured at 37°C in 5% CO2 for 24 hours, and then cell culture medium was tested for VEGF antigen by ELISA. The mean of results in at least three replicate experiments ± SD is shown. (*P < 0.01).
Figure 8.
 
RAGE-dependent secretion of VEGF requires NF-κB activation in ARPE-19 cells. Normal ARPE-19 cells (2 × 104), with or without 5 μM parthenolide, were plated onto the wells of the indicated coated 96-well plate and cultured at 37°C in 5% CO2 for 24 hours, and then cell culture medium was tested for VEGF antigen by ELISA. The mean of results in at least three replicate experiments ± SD is shown. (*P < 0.01).
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Figure 1.
 
Expression of endogenous RAGE and exogenous DN-RAGE by Western blot analysis. Confluent normal ARPE-19 cells (lane 1), mock-transfectants (lane 2), and DN-RAGE transfectants (lane 3) were pretreated with anti-human RAGE IgG for 1 hour at 37°C in 5% CO2. RAGE molecules were isolated and detected. Total protein (5 μg) from human RAGE stably transfected C6 glioma cells was used as the RAGE-positive control (lane 4).
Figure 1.
 
Expression of endogenous RAGE and exogenous DN-RAGE by Western blot analysis. Confluent normal ARPE-19 cells (lane 1), mock-transfectants (lane 2), and DN-RAGE transfectants (lane 3) were pretreated with anti-human RAGE IgG for 1 hour at 37°C in 5% CO2. RAGE molecules were isolated and detected. Total protein (5 μg) from human RAGE stably transfected C6 glioma cells was used as the RAGE-positive control (lane 4).
Figure 2.
 
Effect of soluble and surface-immobilized AGE-BSA on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with 200 μg/mL of AGE-BSA in cell culture medium or coated onto the wells of cell culture plates. VEGF levels in medium were determined by ELISA after 24 hours incubation at 37°C in 5% CO2. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 2.
 
Effect of soluble and surface-immobilized AGE-BSA on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with 200 μg/mL of AGE-BSA in cell culture medium or coated onto the wells of cell culture plates. VEGF levels in medium were determined by ELISA after 24 hours incubation at 37°C in 5% CO2. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 3.
 
Impact of Aβ oligomers on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with freshly prepared Aβ in medium or coated onto the wells of 96-well plates, and oligomerized Aβ, all at 10 μg/mL. VEGF secretion was assayed as described and expressed in comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 3.
 
Impact of Aβ oligomers on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with freshly prepared Aβ in medium or coated onto the wells of 96-well plates, and oligomerized Aβ, all at 10 μg/mL. VEGF secretion was assayed as described and expressed in comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 4.
 
Upregulation of VEGF gene expression by Aβ-induced RAGE activation in ARPE-19 cells. (A) Normal ARPE-19 cells (5 × 105) in 2 mL of cell culture medium, with or without 20 μg of freshly prepared Aβ, were plated onto 35-mm cell culture dishes, without and with Aβ coating, respectively, and cultured at 37°C in 5% CO2 for 16 hours. (B) Time-dependent VEGF gene expression was performed with immobilized Aβ in 35-mm cell culture dishes. Results were expressed as comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 4.
 
Upregulation of VEGF gene expression by Aβ-induced RAGE activation in ARPE-19 cells. (A) Normal ARPE-19 cells (5 × 105) in 2 mL of cell culture medium, with or without 20 μg of freshly prepared Aβ, were plated onto 35-mm cell culture dishes, without and with Aβ coating, respectively, and cultured at 37°C in 5% CO2 for 16 hours. (B) Time-dependent VEGF gene expression was performed with immobilized Aβ in 35-mm cell culture dishes. Results were expressed as comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 5.
 
Effect of S100B on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with freshly prepared 10 μg/mL of S100B in cell culture medium or coated onto cell culture plates. VEGF secretion was assayed as described and expressed as comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 5.
 
Effect of S100B on RAGE-dependent secretion of VEGF in ARPE-19 cells. Normal ARPE-19 cells (2 × 104; Normal), mock-transfected cells (Mock), and DN-RAGE–transfected cells (DN-RAGE) were treated with freshly prepared 10 μg/mL of S100B in cell culture medium or coated onto cell culture plates. VEGF secretion was assayed as described and expressed as comparison to normal ARPE-19 cells without treatment. The mean of results in at least three replicate experiments ± SD is shown (*P < 0.01).
Figure 6.
 
Preparation, isolation, and activities of distinct forms of S100B. (A) Nonreducing (lanes 1–4) and reducing (lanes 6–9) SDS-PAGE analyses of oxidized S100B, 3 μg/lane, Coomassie blue staining. Lanes 1 and 6: fresh S100B; lanes 2 and 7: S100B after 2 hours oxidation with copper sulfate at 37°C; lanes 3 and 8: S100B oxidized as lanes 2 and 7 with an additional 10 days’ incubation at 4°C; lanes 4 and 9: S100B similar to lanes 3 and 8, but with additional 14 days’ incubation at 4°C; lane 5: protein molecular mass marker. (B) Separation of oxidized S100B by FPLC. Short-term oxidation represents 2 hours’ oxidation at 37°C; and long-term oxidation represents short-term oxidation with an additional 24 days’ incubation at 4°C. Three oligomers with molecular masses 84 to 88, 49 to 52, and 28 to 31 kDa were separated and collected. Based on molecular masses and comparison to standards, these represent S100B octamer, tetramer, and dimer, respectively. (C) Elution profile of protein molecular weight standards on a separation column. BSA, 2 mg/mL, carbonic anhydrase (CA), 1 mg/mL, and cytochrome c (CytC), 0.5 mg/mL, were used to predict molecular mass of the distinct S100B oligomers shown in (B). (D) Effect of distinct S100B oligomers on VEGF secretion. ARPE-19 cells (2 × 104) were treated with 10 μg/mL of the S100B oligomers in cell culture medium. VEGF levels in medium were determined by ELISA after 24 hours’ incubation at 37°C in 5% CO2 and presented as the mean ± SD of at least three replicates (*P < 0.01).
Figure 6.
 
Preparation, isolation, and activities of distinct forms of S100B. (A) Nonreducing (lanes 1–4) and reducing (lanes 6–9) SDS-PAGE analyses of oxidized S100B, 3 μg/lane, Coomassie blue staining. Lanes 1 and 6: fresh S100B; lanes 2 and 7: S100B after 2 hours oxidation with copper sulfate at 37°C; lanes 3 and 8: S100B oxidized as lanes 2 and 7 with an additional 10 days’ incubation at 4°C; lanes 4 and 9: S100B similar to lanes 3 and 8, but with additional 14 days’ incubation at 4°C; lane 5: protein molecular mass marker. (B) Separation of oxidized S100B by FPLC. Short-term oxidation represents 2 hours’ oxidation at 37°C; and long-term oxidation represents short-term oxidation with an additional 24 days’ incubation at 4°C. Three oligomers with molecular masses 84 to 88, 49 to 52, and 28 to 31 kDa were separated and collected. Based on molecular masses and comparison to standards, these represent S100B octamer, tetramer, and dimer, respectively. (C) Elution profile of protein molecular weight standards on a separation column. BSA, 2 mg/mL, carbonic anhydrase (CA), 1 mg/mL, and cytochrome c (CytC), 0.5 mg/mL, were used to predict molecular mass of the distinct S100B oligomers shown in (B). (D) Effect of distinct S100B oligomers on VEGF secretion. ARPE-19 cells (2 × 104) were treated with 10 μg/mL of the S100B oligomers in cell culture medium. VEGF levels in medium were determined by ELISA after 24 hours’ incubation at 37°C in 5% CO2 and presented as the mean ± SD of at least three replicates (*P < 0.01).
Figure 7.
 
The effect of S100B on activation of NF-κB in ARPE-19 cells. ARPE-19 cells (3 × 106) were treated with soluble S100B or immobilized S100B for 2 hours. Isolation of nuclear protein and EMSA were performed as described. Lane 1: negative control (without nuclear proteins); lane 2: control cells; lane 3: soluble S100B-treated cells; lane 4: immobilized S100B-treated cells; lane 5: same as lane 4 but with excess unlabeled double-stranded probe.
Figure 7.
 
The effect of S100B on activation of NF-κB in ARPE-19 cells. ARPE-19 cells (3 × 106) were treated with soluble S100B or immobilized S100B for 2 hours. Isolation of nuclear protein and EMSA were performed as described. Lane 1: negative control (without nuclear proteins); lane 2: control cells; lane 3: soluble S100B-treated cells; lane 4: immobilized S100B-treated cells; lane 5: same as lane 4 but with excess unlabeled double-stranded probe.
Figure 8.
 
RAGE-dependent secretion of VEGF requires NF-κB activation in ARPE-19 cells. Normal ARPE-19 cells (2 × 104), with or without 5 μM parthenolide, were plated onto the wells of the indicated coated 96-well plate and cultured at 37°C in 5% CO2 for 24 hours, and then cell culture medium was tested for VEGF antigen by ELISA. The mean of results in at least three replicate experiments ± SD is shown. (*P < 0.01).
Figure 8.
 
RAGE-dependent secretion of VEGF requires NF-κB activation in ARPE-19 cells. Normal ARPE-19 cells (2 × 104), with or without 5 μM parthenolide, were plated onto the wells of the indicated coated 96-well plate and cultured at 37°C in 5% CO2 for 24 hours, and then cell culture medium was tested for VEGF antigen by ELISA. The mean of results in at least three replicate experiments ± SD is shown. (*P < 0.01).
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