Title: Discovery of a Novel Control Element within the 5′-Untranslated Region of the Vascular Endothelial Growth Factor
Abstract: The regulation of vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis, is controlled primarily through the interactions of control elements located within the 5′- and 3′-untranslated regions, many of which are yet to be described. In this study we examined the 5′-untranslated region of human VEGF for control elements with the aim of regulating expression both in vitro and in vivo using oligonucleotide gene therapy. A potential control element was located, two sense oligonucleotides (S1 and S2) were designed based on its sequence, and a third oligonucleotide (S3) was designed as a control and mapped to the 16 base pairs immediately upstream. Retinal cells cultured in the presence of S1 and S2 resulted in a 2-fold increase of VEGF protein and a 1.5-fold increase in mRNA 24 h post-transfection whereas S3 had no significant effect (p > 0.05) compared with controls. Subsequent reporter gene studies confirmed the necessity of this element for up-regulation by S1. Further in vivo studies showed that S1 and S2 mediated an increase in VEGF protein in a rodent ocular model that resulted in angiogenesis. In addition to providing insight into the regulation of the vascular endothelial growth factor, the use of these oligonucleotides to stimulate vascular growth may prove useful for the treatment of ischemic tissues such as those found in the heart following infarct. The regulation of vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis, is controlled primarily through the interactions of control elements located within the 5′- and 3′-untranslated regions, many of which are yet to be described. In this study we examined the 5′-untranslated region of human VEGF for control elements with the aim of regulating expression both in vitro and in vivo using oligonucleotide gene therapy. A potential control element was located, two sense oligonucleotides (S1 and S2) were designed based on its sequence, and a third oligonucleotide (S3) was designed as a control and mapped to the 16 base pairs immediately upstream. Retinal cells cultured in the presence of S1 and S2 resulted in a 2-fold increase of VEGF protein and a 1.5-fold increase in mRNA 24 h post-transfection whereas S3 had no significant effect (p > 0.05) compared with controls. Subsequent reporter gene studies confirmed the necessity of this element for up-regulation by S1. Further in vivo studies showed that S1 and S2 mediated an increase in VEGF protein in a rodent ocular model that resulted in angiogenesis. In addition to providing insight into the regulation of the vascular endothelial growth factor, the use of these oligonucleotides to stimulate vascular growth may prove useful for the treatment of ischemic tissues such as those found in the heart following infarct. Vascular development is a fundamental requirement for all tissue growth, and the absence of adequate tissue vascularization results in cells becoming deprived of oxygen and nutrients. This fact provides the stimulus for cells to produce angiogenic factors, which function to recruit new blood vessels into the deprived tissue. The most important of the angiogenic factors involved in new blood vessel formation is vascular endothelial growth factor (VEGF), 1The abbreviations used are: VEGF, vascular endothelial growth factor; CFP, color fundus photography; ELISA, enzyme-linked immunosorbent assay; FA, fluorescein angiography; fg, femtogram; ODN, oligonucleotide; RPE, retinal pigment epithelial; UTR, untranslated region; RT-PCR, real time PCR. which is highly regulated and consists of four isoforms encoded by a single gene via alternate splicing (1Shima D.T. Kuroki M. Deutsch U. Ng Y.S. Adamis A.P. D'Amore P.A. J. Biol. Chem. 1996; 271: 3877-3883Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 2Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar). A characteristic of all four isoforms is the presence of an unusually long and GC-rich 5′- and 3′-untranslated region (UTR) (1Shima D.T. Kuroki M. Deutsch U. Ng Y.S. Adamis A.P. D'Amore P.A. J. Biol. Chem. 1996; 271: 3877-3883Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 2Tischer E. Mitchell R. Hartman T. Silva M. Gospodarowicz D. Fiddes J.C. Abraham J.A. J. Biol. Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar) that contains most of the important control and regulatory elements involved in the modulation of VEGF expression (reviews in Refs. 3Guhaniyogi J. Brewer G. Gene. 2001; 265: 11-23Crossref PubMed Scopus (555) Google Scholar and 4van der Velden A.W. Thomas A.A. Int. J. Biochem. Cell Biol. 1999; 31: 87-106Crossref PubMed Scopus (316) Google Scholar). These elements include several internal ribosomal entry sites (5Akiri G. Nahari D. Finkelstein Y. Le S.Y. Elroy-Stein O. Levi B.Z. Oncogene. 1998; 17: 227-236Crossref PubMed Scopus (223) Google Scholar, 6Miller D.L. Dibbens J.A. Damert A. Risau W. Vadas M.A. Goodall G.J. FEBS Lett. 1998; 434: 417-420Crossref PubMed Scopus (70) Google Scholar), hypoxia response elements (7Forsythe J.A. Jiang B.H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3234) Google Scholar), and a number of stabilizing and destabilizing sequences (8Dibbens J.A. Miller D.L. Damert A. Risau W. Vadas M.A. Goodall G.J. Mol. Biol. Cell. 1999; 10: 907-919Crossref PubMed Scopus (159) Google Scholar, 9Levy A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; 271: 25492-25497Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The importance of VEGF-mediated vascularization in disease states makes it an attractive target for gene therapies. Several methods of down-regulating VEGF for the treatment of tumors and ocular neovascularization are currently being explored (10Carrasquillo K.G. Ricker J.A. Rigas I.K. Miller J.W. Gragoudas E.S. Adamis A.P. Investig. Ophthalmol. Vis. Sci. 2003; 44: 290-299Crossref PubMed Scopus (163) Google Scholar, 11Im S.A. Kim J.S. Gomez-Manzano C. Fueyo J. Liu T.J. Cho M.S. Seong C.M. Lee S.N. Hong Y.K. Yung W.K. Br. J. Cancer. 2001; 84: 1252-1257Crossref PubMed Scopus (45) Google Scholar, 12Krzystolik M.G. Afshari M.A. Adamis A.P. Gaudreault J. Gragoudas E.S. Michaud N.A. Li W. Connolly E. O'Neill C.A. Miller J.W. Arch. Ophthalmol. 2002; 120: 338-346Crossref PubMed Scopus (556) Google Scholar). In addition, we have previously described a sense oligonucleotide (DS-085) that targets the 5′-UTR of the VEGF gene and has proven effective at down-regulating the transcription and subsequent translation of VEGF both in vitro and in vivo (13Garrett K.L. Shen W.Y. Rakoczy P.E. J. Gene Med. 2001; 3: 373-383Crossref PubMed Scopus (40) Google Scholar). The mechanism of action has been postulated to be due to Hoogsteen hydrogen bonding of the oligonucleotide (ODN) within the major groove of the duplex DNA, causing polymerase arrest (14Alunni-Fabbroni M. Manfioletti G. Manzini G. Xodo L.E. Eur. J. Biochem. 1994; 226: 831-839Crossref PubMed Scopus (29) Google Scholar, 15Baran N. Lapidot A. Manor H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 507-511Crossref PubMed Scopus (123) Google Scholar, 16Dayn A. Samadashwily G.M. Mirkin S.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11406-11410Crossref PubMed Scopus (91) Google Scholar, 17Krasilnikov A.S. Panyutin I.G. Samadashwily G.M. Cox R. Lazurkin Y.S. Mirkin S.M. Nucleic Acids Res. 1997; 25: 1339-1346Crossref PubMed Scopus (57) Google Scholar, 18Samadashwily G.M. Mirkin S.M. Gene. 1994; 149: 127-136Crossref PubMed Scopus (40) Google Scholar). Similar to the regulatory regions of other genes, DS-085 was found to be rich in GA purine residues (19Arnold R. Maueler W. Bassili G. Lutz M. Burke L. Epplen T.J. Renkawitz R. Gene. 2000; 253: 209-214Crossref PubMed Scopus (36) Google Scholar, 20Genuario R.R. Perry R.P. J. Biol. Chem. 1996; 271: 4388-4395Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). An examination of the 5′-UTR sequence was therefore made in an attempt to discover other potential homopurine regulatory sequences involved in VEGF expression. In this article we report on the discovery of a novel control element within the 5′-UTR of the VEGF gene that may represent the binding site of a destabilization protein. Oligonucleotide Design—The 5′-UTR sequence of human VEGF (GenBank™ accession number NM_003376) was examined for the presence of homopurine regions that may represent potential regulatory sites. Sense ODNs 1 and 2 (S1 and S2) were subsequently designed to recognize the first and final 16 bp, respectively, of a homopurinehomopyrimidine sequence identified from base pair -265 to -223 from the ATG start codon. In addition, a third sense ODN (S3) was designed and represented the 16 bp immediately 5′ to S1 and was used as a control. Oligo DS-085 has been described previously (13Garrett K.L. Shen W.Y. Rakoczy P.E. J. Gene Med. 2001; 3: 373-383Crossref PubMed Scopus (40) Google Scholar). Oligonucleotides were obtained from Proligo (Boulder, CO) and synthesized with a phosphorothioate (S) backbone. In Vitro Oligonucleotide VEGF Inhibition Assay—A human retinal pigment epithelial (RPE) cell line (RPE 51) was grown in culture and used to assess the effect that S1, S2, S3, and DS-085 have on the production of VEGF protein and mRNA. Cells were seeded into 2 × 6 well plates (35-mm diameter) at ∼4 × 105 cells per well and allowed to grow in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 0.5% streptomycin, and penicillin at 37 °C and 5% CO2 until 80% confluence was reached. Cytofectin (Gene Therapy Systems, San Diego, CA) was used per the manufacturer's instructions to deliver the ODNs into the cells at a final concentration of 1 μm. Control groups consisted of cells transfected with cytofectin alone and null treated cells, which were not manipulated in any way. Following transfection, one of the plates was transferred to a CO2 incubator and grown under normoxic condition at 5% CO2. The other plate was placed in a hypoxic incubator (2% O2 and 5% CO2), and each was grown for 24 h. After this time, the media were collected from both the normoxic and hypoxic grown cells for an enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (CYTELISA™; Cytimmune Sciences, College Park, MD) to determine the level of VEGF protein expression. The ELISA was performed per the manufacturer's instructions using 100 μl of undiluted culture media. The cells from each well were harvested by trypsinization and pelleted by centrifugation at 2000 × g for 5 min. The cell pellet was washed twice in isotonic saline and resuspended in 250 μl of the same. An A600 reading was taken to determine cell density, which was used to normalize the VEGF concentration. Levels of mRNA transcript were determined using RT-PCR. Cells were treated with 600 μl of Trizol (Qiagen, Clifton Hill, Victoria, Australia) directly in the culture wells 24 h post-transfection. The lysed suspension was transferred to a microfuge tube where chloroform (200 μl) was added, and the solution was subjected to a vortex to ensure complete mixing. The aqueous phase containing the total RNA was removed to a new microfuge tube, and 1 volume of isopropanol was added to precipitate the total RNA. The total RNA was pelleted by centrifugation at 20,000 × g for 10 min, and the pellet was washed in 70% ethanol. The ethanol was aspirated, the pellet was air-dried and resuspended in 200 μl of nuclease-free water, and the concentration was determined spectrometrically. An Omniscript™ RT kit (Qiagen) was used for the production of the first strand cDNA per the manufacturer's instructions starting with 200 ng of total RNA using an oligo(dT) primer in a final volume of 20 μl. Directly from this reaction, 1 μl was used as a template for the PCR of an internal VEGF fragment in addition to a β-actin fragment, which was used as an internal control. VEGF primers consisted of the sense 5′-CATCACGAAGTGGTGAAGTT-3′ and the antisense 5′-AACGCTCCAGGACTTATACC-3′. Primers used to amplify β-actin (GenBank™ accession number NM_007393) consisted of the sense 5′-AGGCACCAGGGCGTGAT-3′ and the antisense 5′-TTAATGTCACGCACGATTTC-3′. Both sets of primers (Proligo) were included with the following reaction components in a final volume of 25 μl: 2.5 μl of 10× reaction buffer, 2 mm MgCl2, 200 μm each dNTP, 6 pmol of each primer, and 1 unit of Tth+ polymerase (Fisher Biotech, Perth, Western Australia, Australia). A touchdown cycling reaction was used and consisted of an initial denaturing step of 94 °C for 2 min followed by seven cycles of 94 °C for 10 s, 65 °C for 10 s with a drop of 1 °C per cycle, and 72 °C for 30 s. This was then followed by 41 cycles of 94 °C for 10 s, 58 °C for 10 s, and 71 °C for 30 s. Sample Statistics—The transfections were performed in quadruplet sets for statistical analysis. Results were analyzed by one-way analysis of variance followed by a post hoc Fisher's least squares difference analysis with 95% confidence limits using the GB-Stat™ statistical software package (Dynamic Microsystems, Silver Springs, MD). Reporter Gene Analysis—Plasmids were constructed to examine the role of various sections of the 5′-UTR in regulating VEGF expression both in the presence and absence of up-regulating ODNs. Initially, the entire 1039 bp of the human VEGF 5′-UTR was RT-PCR amplified from mRNA extracted from cultured human RPE 51 cells and subcloned into pGEM T Easy (Promega, Madison, WI) to produce pGEM T-UTR. The integrity of this clone was confirmed by DNA sequencing. Subsequently, the entire UTR was removed by digestion with ApaI and SalI (all restriction enzymes were sourced from New England Biolabs, Beverly, MA) and subcloned into the ApaI and XhoI site of the reporter plasmid (pΔUTR) consisting of the cytomegalovirus strong promoter linked to the secreted alkaline phosphatase reporter gene to produce pUTR-W. Two further reporter plasmids were constructed by subcloning the proximal 703-bp ApaI-XmnI fragment and the distal 396-bp XmnIEcoRI fragment of pGEM T-UTR into the ApaI-EcoRV and the EcoRVEcoRI sites of pΔUTR, respectively, to produce pUTR-L and pUTR-S. To examine the effect on reporter gene activity, 0.8 μg of each construct was used per well to transiently transfect RPE 51 cells cultured in 24-well plates using the transfection agent LipofectAMINE (Invitrogen) per the manufacturer's instructions. Transfection was allowed to proceed for 5 h, at which time the growth media were replaced with fresh media both with and without supplementation with a 1 μm concentration of S1 conjugated to the transfection agent cytofectin (Gene Therapy Systems). Media were sampled 24 h later and analyzed for secreted alkaline phosphatase activity by chemiluminescent detection on a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA). Transfections were performed in quadruplet sets, and the results were expressed as a percentage of relative light units compared with the pΔUTR control. Analysis of the results consisted of analysis of variance with post hoc Fisher's least squares difference analysis with 95% confidence limits. Injections and in Vivo Analysis—All animal experiments were performed in accordance with the animal use guidelines of the Association for Research in Vision and Ophthalmology and were approved by the Animal Ethics Committee of the University of Western Australia. Oligonucleotide delivery to the anterior chamber was carried out on 6-8-week-old non-pigmented RCS/rdy+ rats that had been anesthetized by an intramuscular injection of ketamine (50 mg kg-1 body weight) and xylazine (8 mg kg-1 body weight), followed by topical application of proparacaine hydrochloride to the eye. Two and one-half microliters of a 1 mm oligonucleotide solution or vehicle (phosphate-buffered saline containing 10% glycerol) were injected into the anterior chamber of both eyes of each rat via the temporal limbus using a 32-gauge needle attached to a 5-μl Hamilton syringe after the same amount of aqueous humor was drained. Ophthalmologic examinations of the eyes were performed 7 days post-injection and photographed using a slit lamp camera. Sub retinal injections were performed on 8- to 9-week-old non-pigmented RCS/rdy+ rats and C57 BL/6J mice of the same age. The injection technique used has been described previously (21Axel D.I. Spyridopoulos I. Riessen R. Runge H. Viebahn R. Karsch K.R. J. Vasc. Res. 2000; 37 (303-304): 221-234Crossref PubMed Scopus (24) Google Scholar). Briefly, the conjunctiva was cut close to the limbus to expose the sclera, which was then punctured with a 30-guage needle. A 32-guage needle was passed through this hole in a tangential direction under an operating microscope. Two microliters of oligonucleotide were delivered into the subretinal space of each eye. The needle was kept in the subretinal space for 1 min, withdrawn gently, and antibiotic ointment was applied to the wound site. Ophthalmologic examinations were performed 7 days post-injection and consisted of color fundus photography (CFP) and fluorescein angiography (FA). A second group of similarly injected rats were euthanized 7 days post-injection, and the eyes were enucleated and placed into 200 μl of phosphate buffered saline containing protease inhibitors. The eyes were thoroughly homogenized and centrifuged at 3000 × g for 15 min at 4 °C. To determine the concentration of VEGF in the eyes, 50 μl of supernatant was used in an ELISA specific for mouse/rat VEGF (Quantikine; R&D Systems). The concentration was normalized against total protein concentrations of the supernatant. ELISA results were analyzed using analysis of variance with a post hoc Dunnnett's procedure (GB-Stat™). Regulation of VEGF Expression—S1,S2,S3, and DS-085 were transfected into the RPE 51 cell line, and the effects of VEGF translation and transcription were measured using ELISA and RT-PCR, respectively. ELISA (Fig. 1) revealed that both S1 (1073 ± 64 pg ml-1) and S2 (969 ± 60 pg ml-1) facilitated a statistically significant (p < 0.01) up-regulation of VEGF protein by ∼2-fold as compared with the non-transfected control (578 ± 63 pg ml-1). ODN S3 (593 ± 33 pg ml-1) had no significant effect (p > 0.05), whereas the transfection agent cytofectin mediated a slight decrease in VEGF expression (508 ± 38 pg ml-1). However, this result was not found to be significant (p > 0.05). To examine the effects on VEGF at the transcriptional level, total RNA was extracted from cells transfected with S1, S2, S3, and DS-085 and subsequently used as a template for RT-PCR (Fig. 2a). The profile for mRNA levels in the transfected cells, as determined by densitometry of the PCR products (Fig. 2b), reflected the protein concentration profile. Transfection with S1 and S2 mediated an increase in the levels of VEGF mRNA by a factor of 1.5 as compared with the non-transfected control 24 h after transfection. However, the increase in mRNA mediated by S1 and S2 was not found to be proportional to the increase in protein concentration. The previously described oligonucleotide DS-085 decreased the level of mRNA by 57.5%, which was directly proportional to the decrease in protein. This result indicated that the mechanism of down-regulation by DS-085 was separate to and distinct from the mechanism of up-regulation of protein by S1 and S2. Transfection with the S3 oligonucleotide resulted in no significant effect as compared with the effect on the control samples, which was the same in regard to protein concentration. Similarly, transfection with vehicle (cytofectin) alone produced a slight decrease (5%) in VEGF mRNA equivalent to that found for the protein reduction and may be reflective of the slight cytotoxic effect known to be associated with cytofectin (21Axel D.I. Spyridopoulos I. Riessen R. Runge H. Viebahn R. Karsch K.R. J. Vasc. Res. 2000; 37 (303-304): 221-234Crossref PubMed Scopus (24) Google Scholar). Reporter Gene Analysis—Retinal cells were transiently transfected with the reporter gene constructs and cultured in both the presence and the absence of S1. Media were sampled and tested for alkaline phosphatase activity using a chemiluminescent detection method, and the results are summarized in Fig. 3. Plasmid pΔUTR showed strong reporter gene activity and remained unaffected when cultured in the presence of S1 (data not shown). Transfection with plasmid pUTR-W, which contains the entire VEGF 5′-UTR, resulted in a significant (p < 0.01) decrease (65%) in reporter gene activity compared with pΔUTR. However, when cultured in the presence of S1, reporter gene activity significantly (p < 0.01) increased 1.79-fold to be 62.9 ± 5.4% of pΔUTR activity. When transfected with pUTR-L in which the proposed destabilizing element was absent, the decrease in reporter activity as compared with pΔUTR was significantly less (p < 0.01) than that of pUTR-W, indicating a more stable transcript. In addition, we only see a small increase in reporter activity when cultured in the presence of S1 (63.5 ± 4.3% to 73.9 ± 3.3% compared with pΔUTR), which was not found to be significant (p > 0.05). In cells transfected with pUTR-S, which comprises the 3′-distal end of the VEGF 5′-UTR and contains the destabilizing element, we again see reporter gene activity significantly (p < 0.01) increased by 1.39-fold when cultured in the presence of S1, indicating a stabilizing effect. In Vivo Analysis—To determine whether the in vitro observations would translate into an in vivo effect, S1, S2, and S3 were injected into the anterior chamber of rat eyes. Subsequent ophthalmologic examination showed strong neovascularization in the iris of rat eyes 7 days following injection with S1 and S2, (Fig. 4), but no effect was observed in rat eyes injected with S3 or vehicle. This indicates that the oligonucleotides were able to mediate the up-regulation of VEGF in the eye and produce an angiogenic response. The result observed in the iris was reflected in rats that were injected in the subretinal space with S1, S2, and S3. Eyes injected with S3 remained clear of angiogenesis when viewed using CFP and FA (Fig. 5, a and b, respectively) for the duration of the experiment. However, a strong angiogenic response was observed in eyes 7 days post-injection with S1 and S2 when viewed using CFP. Neovascularization occurred some distance from the injection site and appeared as a distinct red band of blood vessels extending across the retina (Fig. 5c). Further examination using fluorescein angiography confirmed the formation of new vessels, which appears as hyperfluorescence (Fig. 5d) due to the “leaky” nature that blood vessels possess during angiogenesis. In addition, FA revealed the presence of microaneurisms (not shown) in eyes injected with S1 and S2. Later examinations performed 14 days post-injection of S1 and S2 revealed the occurrence of intraretinal hemorrhage in eyes injected with S1 and S2 that appears as black spots using CFP (Fig. 5c) and as hypo-fluorescence using fluorescein angiography (Fig. 5f). The intraretinal hemorrhage increased in severity 21 days post-injection (Fig. 5g) and appears as a large hypo-fluorescent area using FA (Fig. 5h). The observations detailed above were closely paralleled in a similar mouse model following subretinal injection where leakiness, micro aneurysms, and intraretinal hemorrhage were also observed in eyes injected with S1 and S2 7 days post-injection, whereas eyes injected with S3 retained a normal appearance (photographs not shown). To correlate the angiogenic response seen in vivo to the results found in vitro, ELISA was used to assay the VEGF protein concentrations from the eyes of rats injected with S1, S2, and S3. The results (Fig. 6) show that both S1 and S2 mediated a significant (p < 0.05) increase in the concentration of VEGF protein within the eye (2952 ± 193 fg mg-1 and 2404 ± 124 fg mg-1 of total protein respectively) compared with the vehicle-injected control (1930 ± 92 fg mg-1 of total protein). In addition, consistent to the in vitro results and in vivo observations, S3 was unable to significantly change the concentration of ocular VEGF (1768 ± 45 fg mg-1 of total protein). Sequence Comparison—Cross-species comparisons of VEGF 5′-UTR sequences between bovine (GenBank™ accession number NM_174216), murine (GenBank™ accession number NM_009505), and human have revealed a high level of conservation for the S1 to S2 region between human and bovine, but the murine 5′-UTR revealed a complete lack of the S1 sequence (Fig. 7). No sequence information was available for the 5′-UTR of the rat and, therefore, no direct comparison could be made. Controlled regulation of VEGF in vivo is important in maintaining the health of many tissues and cells types. However, increased levels of VEGF associated with ischemic conditions leads to a variety of angiogenic ocular diseases, including diabetic retinopathy and retinopathy of prematurity (22Adamis A.P. Miller J.W. Bernal M.T. D'Amico D.J. Folkman J. Yeo T.K. Yeo K.T. Am. J. Ophthalmol. 1994; 118: 445-450Abstract Full Text PDF PubMed Scopus (1211) Google Scholar, 23Aiello L.P. Avery R.L. Arrigg P.G. Keyt B.A. Jampel H.D. Shah S.T. Pasquale L.R. Thieme H. Iwamoto M.A. Park J.E. Nguyen H.V. Aiello L.M. Ferrara N. King G.L. N. Engl. J. Med. 1994; 331: 1480-1487Crossref PubMed Scopus (3436) Google Scholar), in addition to promoting vasculogenesis in cancerous tissues (24Ding I. Liu W. Sun J. Paoni S.F. Hernady E. Fenton B.M. Okunieff P. Adv. Exp. Med. Biol. 2003; 530: 603-609Crossref PubMed Scopus (7) Google Scholar, 25Siemeister G. Martiny-Baron G. Marme D. Cancer Metastasis Rev. 1998; 17: 241-248Crossref PubMed Scopus (86) Google Scholar). Central to the regulation of VEGF is the presence of both a 5′-UTR and 3′-UTR, both of which contain many regulatory elements, including hypoxia and glucose response elements (26Iida K. Kawakami Y. Sone H. Suzuki H. Yatoh S. Isobe K. Takekoshi K. Yamada N. Life Sci. 2002; 71: 1607-1614Crossref PubMed Scopus (20) Google Scholar), in addition to stabilizing and destabilizing elements (8Dibbens J.A. Miller D.L. Damert A. Risau W. Vadas M.A. Goodall G.J. Mol. Biol. Cell. 1999; 10: 907-919Crossref PubMed Scopus (159) Google Scholar). In this study we report on the discovery of a novel control element within the 5′-UTR of the human VEGF gene that may act as a target site for a destabilizing protein in addition to providing further insight into its regulation. Two sense oligonucleotides (S1 and S2) were designed to resemble a potential regulatory region within the 5′-UTR of the VEGF gene. A third ODN (S3) was designed as a control and mapped to the 16 bp immediately 5′ to S1. Results from the in vitro studies demonstrated that S1 and S2 mediated a 2-fold increase in protein production and up to a 1.5-fold increase in the level of a mRNA transcript. This indicates that the sequences in the 5′-UTR represented by S1 and S2 contain regulatory elements involved in the modulation of VEGF production. Possible mechanisms for VEGF protein up-regulation by S1 and S2 include competitive inhibition of either a mRNA destabilizing protein or a transcriptional repressor protein. In the case of the latter, transcriptional repressor proteins have been described previously (19Arnold R. Maueler W. Bassili G. Lutz M. Burke L. Epplen T.J. Renkawitz R. Gene. 2000; 253: 209-214Crossref PubMed Scopus (36) Google Scholar, 20Genuario R.R. Perry R.P. J. Biol. Chem. 1996; 271: 4388-4395Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and share a common theme of recognizing the variations of a homopurine GA-type sequence consensus motif similar to the sequence found in S1 and S2. However, our data suggest that S1 and S2 are competing for the recognition site of an mRNA destabilizing protein. If the mechanism of up-regulation were mediated by increased mRNA production through the inhibition of a repressor protein, we would see a proportional increase between protein and mRNA. However, this was not the case for S1 and S2 where protein levels were increased by 2-fold compared with the control, whereas mRNA was only increased by 1.5 and 1.25 times, respectively. Levels of mRNA are determined by the equilibrium that exists between synthesis and degradation; therefore, an increase in stability will reduce degradation and cause an equilibrium shift resulting in higher levels of mRNA being present without an increase in mRNA transcription. The improved mRNA stability and, hence, the increased half-life will result in a proportionally greater amount of protein produced per molecule of mRNA. In addition, stabilization/destabilization of mRNA has been shown previously to be the mechanism associated with increases in VEGF protein during periods of hypoxia (27Shima D.T. Deutsch U. D'Amore P.A. FEBS Lett. 1995; 370: 203-208Crossref PubMed Scopus (281) Google Scholar) and has been well documented as playing a role in the regulation of other cellular elements such as transferrin receptors (28Koeller D.M. Horowitz J.A. Casey J.L. Klausner R.D. Harford J.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7778-7782Crossref PubMed Scopus (107) Google Scholar, 29Mullner E.W. Kuhn L.C. Cell. 1988; 53: 815-825Abstract Full Text PDF PubMed Scopus (375) Google Scholar), elastin (30Hew Y. Lau C. Grzelczak Z. Keeley F.W. J. Biol. Chem. 2000; 275: 24857-24864Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and resistin (31Kawashima J. Tsuruzoe K. Motoshima H. Shirakami A. Sakai K. Hirashima Y. Toyonaga T. Araki E. Diabetologia. 2003; 46: 231-240Crossref PubMed Scopus (52) Google Scholar). The possibility of destabilizing elements being present within the 5′-UTR of the VEGF mRNA transcript has been reported previously (8Dibbens J.A. Miller D.L. Damert A. Risau W. Vadas M.A. Goodall G.J. Mol. Biol. Cell. 1999; 10: 907-919Crossref PubMed Scopus (159) Google Scholar), but no definitive consensus sequence has yet been described. Reporter gene studies have been used to confirm that the activity of S1 and S2 was exerted at the transcriptional level. The presence of the many regulatory factors located within the 5′-UTR of VEGF makes it difficult to explain the overall effect that truncation has on reporter gene activity. However, we were able to show that removal of the 3′-distal end of the 5′-UTR (pUTR-L), which contained the proposed regulatory element, resulted in a reduced down-regulatory effect compared with the full UTR construct (pUTR-W) in addition to a loss of up-regulation of reporter product when cultured in the presence of S1. Conversely, cells transfected with reporter plasmids containing the complete 5′-UTR, and the fragment containing the proposed destabilizing element (pUTR-W and pUTR-S, respectively) showed significant increases in reporter gene activity when cultured in the presence of S1. It should be noted that the relative increase in reporter activity was less for pUTR-S (1.39-fold) than for the complete pUTR-W (1.79-fold), which was a comparable increase over that found in the in vitro ELISA assays. Use of computer modeling has shown the 5′-UTR of VEGF to possess a complex secondary structure that is crucial for normal functioning of the gene (5Akiri G. Nahari D. Finkelstein Y. Le S.Y. Elroy-Stein O. Levi B.Z. Oncogene. 1998; 17: 227-236Crossref PubMed Scopus (223) Google Scholar, 32Huez I. Creancier L. Audigier S. Gensac M.C. Prats A.C. Prats H. Mol. Cell. Biol. 1998; 18: 6178-6190Crossref PubMed Scopus (245) Google Scholar). It is therefore conceivable that truncation of the 5′-UTR would cause an alteration in the folding pattern and affect the normal functions of the various control elements, resulting in reduced functioning such as that described above. To study the effects of S1, S2, and S3 on VEGF regulation in vivo, a rodent ocular model was chosen. VEGF isoforms are the same for all tissues, and the eye makes an attractive organ to use because the effects on ocular vascularization by changes in VEGF levels have been well described (review in Ref. 33Witmer A.N. Vrensen G.F. Van Noorden C.J. Schlingemann R.O. Prog. Retin. Eye Res. 2003; 22: 1-29Crossref PubMed Scopus (800) Google Scholar). In addition, the vasculature of the eye can be readily studied through real time ophthalmologic examination. When introduced to the anterior chamber of the rat eye, a strong neovascular response in the iris was observed for both S1 and S2. Likewise, subretinal injection of S1 and S2 in both rats and mice resulted in a similar response in the retina in addition to the formation of microaneurysms and leakage associated with the growth of new blood vessels. This pattern of neovascularization can also be observed in a rodent model with an elevated expression of a VEGF transgene (34Baffi J. Byrnes G. Chan C.C. Csaky K.G. Investig. Ophthalmol. Vis. Sci. 2000; 41: 3582-3589PubMed Google Scholar) as well as in patients suffering from diabetic retinopathy (35Moore J. Bagley S. Ireland G. McLeod D. Boulton M.E. J. Anat. 1999; 194: 89-100Crossref PubMed Google Scholar). The injection of S3 resulted in no observable response as was seen in the in vitro study. This provided a strong indication that the presence of S1 and S2 mediated an increase in the level VEGF protein with the effect of stimulating neovascularization. To test this hypothesis, the concentration of VEGF in rat eyes 7 days post-injection with S1,S2, and S3 was calculated. Eyes injected with S1 and S2 showed significantly elevated VEGF protein levels (1.59- and 1.25-fold, respectively) compared with the vehicle-injected control when measured using ELISA, whereas no difference was recorded in S3-injected eyes. Although VEGF has been identified as a highly potent angiogenic factor, to date no data exists on the minimal increase required to promote an angiogenic response in the retina. Previous studies using RPE cells have shown that the up-regulation of VEGF due to hypoxic conditions to be 1.3- to 1.5-fold (36Blaauwgeers H.G. Holtkamp G.M. Rutten H. Witmer A.N. Koolwijk P. Partanen T.A. Alitalo K. Kroon M.E. Kijlstra A. van Hinsbergh V.W. Schlingemann R.O. Am. J. Pathol. 1999; 155: 421-428Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). In addition, VEGF was preferentially secreted to the basal side of the cell to yield a 2- to 7-fold higher accumulation of VEGF on the basal side as compared with the apical side, which may prove to be the more important factor involved in ocular angiogenesis. Comparisons of published sequences of the 5′-UTR show some variation in the sequence region proposed for the presence of a destabilizing element. However, S1 and S2 both mediate a response in the mouse model, which lacks the S1 sequence, thereby providing evidence that the inhibition was due to a shorter consensus sequence common to both S1 and S2. Responses to hypoxia are dependent on the presence of the hypoxia response element, which consists of a 6-bp core consensus sequence (37Su H. Arakawa-Hoyt J. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9480-9485Crossref PubMed Scopus (130) Google Scholar). Similarly, low levels of glucose can mediate and increase in VEGF through the glucose response element (26Iida K. Kawakami Y. Sone H. Suzuki H. Yatoh S. Isobe K. Takekoshi K. Yamada N. Life Sci. 2002; 71: 1607-1614Crossref PubMed Scopus (20) Google Scholar). S1 and S2 both contain the element (T/A)GGGG, which may represent the core recognition sequence of a destabilizing protein. This research provides the strongest evidence to date for the existence of a destabilizing element within the 5′-UTR of the VEGF gene. In addition, we have shown the potential location of these elements and discussed their importance in the regulation of VEGF protein production. Further research will evaluate the protein-mRNA relationship and how this may translate into a disease state. This understanding may lead to potential treatments for ischemic tissues such as that found in the heart following a myocardial infarct. We thank Dr. Reza Ghassemifar for donating the primers used in the RT-PCR.