Clinical Chemistry Link to Randox Laboratories Web Site
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 47: 654-660, 2001;
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (55)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wellmann, S.
Right arrow Articles by Seeger, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wellmann, S.
Right arrow Articles by Seeger, K.
Related Collections
Right arrow Molecular Diagnostics and Genetics
Right arrow Endocrinology and Metabolism
(Clinical Chemistry. 2001;47:654-660.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Specific Reverse Transcription-PCR Quantification of Vascular Endothelial Growth Factor (VEGF) Splice Variants by LightCycler Technology

Sven Wellmanna,1, Tillmann Taube1, Krisztina Paal1, Hagen Graf v. Einsiedel1, Wilhelm Geilen1, Georg Seifert1, Cornelia Eckert1, Günter Henze1 and Karlheinz Seeger1

1 Department of Pediatric Oncology/Hematology, Charité Medical Center, Campus Virchow-Klinikum, Humboldt-University at Berlin, 13353 Berlin, Germany.
a Address correspondence to this author at: Otto-Heubner-Centrum für Kinder- und Jugendmedizin der Charité, Campus Virchow-Klinikum, Forschungshaus R 2.0412, Augustenburger Platz 1, 13353 Berlin, Germany. Fax 49-30-450-59911; e-mail sven.wellmann{at}charite.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Overexpression of vascular endothelial growth factor (VEGF) is associated with increased angiogenesis, growth and invasion in solid tumors, and hematologic malignancies. The expression of isoforms of VEGF, which mediate different effects, can be discriminated by splice-variant-specific quantitative reverse transcription-PCR (RT-PCR), but current methods have only modest sensitivity and precision and suffer from heteroduplex formation.

Methods: We used a real-time RT-PCR assay on the LightCycler system. Applicability for detection of different VEGF mRNAs and total VEGF message was tested on seven healthy tissues (each pooled from healthy donors) and seven correlated malignant tissues. Results were normalized to ß2-microglobulin mRNA. Amplification of VEGF splice variants was performed exclusively with variant-specific reverse primers, whereas forward primer and fluorescent probe were common to obtain similar RT-PCR kinetics.

Results: Highly specific detection of VEGF splice variants was achieved with minor intra- and interassay variation (<0.22 threshold cycle). Total VEGF expression was higher in malignant tissues. In healthy tissues, the mRNA encoding diffusible variants VEGF121 and VEGF165 constituted on average 78% (SD = 9.3%) of the total VEGF message, and the cell-adherent variant VEGF189 constituted on average 22% (SD = 5.4%). In contrast, in malignant tissues VEGF121 and VEGF165 accounted for 94% (SD = 7.6%) and VEGF189 only 6% (SD = 3.7%).

Conclusions: Because of the ability for quantification of VEGF splice variants with high specificity, sensitivity, and reproducibility, this new LightCycler assay is superior to conventional semiquantitative competitive RT-PCR and immunological assays and may contribute to better understanding of VEGF-mediated angiogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Vascular endothelial growth factor (VEGF),1 also known as vascular permeability factor, is an endothelial cell-specific mitogen and a significant mediator of angiogenesis during a variety of nonpathological and pathological processes (1)(2)(3). VEGF is expressed in response to hypoxia and other stimuli by healthy as well as neoplastic cells (4)(5)(6)(7). The concentrations of VEGF mRNA and the expression of VEGF protein in human solid tumors correlate positively with malignant progression (8)(9). Recent evidence indicates that increased angiogenesis mediated by VEGF also plays a pivotal role in hematopoietic tumors (10)(11)(12)(13)(14).

VEGF is a 34- to 42-kDa heparin-binding, dimeric, disulfide-bound glycoprotein existing in at least five homodimeric isoforms. The monomers consist of 121, 145, 165, 189 and 206 amino acids (VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206), respectively (15)(16). The precise functional differences among the isoforms are not yet known. Differences in solubility, receptor affinity, and mitogenic potency have been described [reviewed in Neufeld et al. (17)]. The primary VEGF transcript derives from a single VEGF gene coding for eight exons. Variable alternative mRNA splicing involves exons 6 and 7, whereas the amino acids encoded by exons 1–5 and 8 are conserved in all isoforms. Exons 6 and 7 encode two distinct heparin-binding domains. The presence or absence of these domains influences solubility and receptor binding. The heparin-binding domain encoded by exon 6 determines binding to the extracellular matrix. Thus, isoforms containing the domain encoded by exon 6 (VEGF145, VEGF189, and VEGF206) are bound tightly to cell surface heparin-containing proteoglycans in the extracellular matrix (18)(19), whereas isoforms lacking the domain encoded by exon 6 are diffusible. VEGF165, which contains only one heparin-binding region encoded by exon 7, is moderately diffusible, whereas VEGF121, which lacks the domains encoded by exons 6 and 7, is highly diffusible.

At present, little is known on the relative abundance of the different VEGF splice variants in healthy cells of different origin and in the malignant counterparts. Whereas the major splice variants VEGF121, VEGF165, and VEGF189 are expressed in nearly all investigated tissues in different ratios, the variants VEGF145 and VEGF206 (20) as well as the recently described variants VEGF148 (21) and VEGF183 (22) are restricted to only a very few cell types and seem to be of minor importance.

In recent investigations based on competitive reverse transcription (RT)-PCR assays, the increased expression of VEGF165 and VEGF189 in osteosarcoma (23), renal cell carcinoma (24), non-small-cell lung cancer (25), and colon cancer (26) was correlated with neovascularization, tumor progression, and poor prognosis. Cheung et al. (27) pointed out that during malignant progression, an angiogenic switch favoring the variants that encode for the shorter diffusible isoforms (VEGF121 and VEGF165) occurs. Furthermore, Cheng et al. (28) could show that intracerebral tumor-associated hemorrhage is caused by overexpression of the variants that encode for the short diffusible isoforms, VEGF121 and VEGF165. On the other hand, the cell-associated isoform VEGF189 contributes to successful xenotransplantability of various human solid tumors via augmentation of stromal vascularization (29).

In this study, a novel real-time RT-PCR was developed for specific quantification of VEGF splice variants to investigate the expression of VEGF splice variants and their ratio in healthy and malignant tissues, using the new LightCycler technology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
preparation of calibrators
Total cellular RNA was isolated from Jurkat cells by the Qiagen RNeasy Mini reagent set (Qiagen) according to the manufacturer’s recommendations. Reverse transcription was carried out as described elsewhere (30). The transcripts that encode for VEGF splice variants VEGF121, VEGF165, and VEGF189 as well as total VEGF (VEGFtotal) were amplified by RT-PCR using specific primers (sequences for all primers see below). The housekeeping gene ß2-microglobulin (B2M) served as internal control. PCR products were separated on a 3% agarose gel, and bands were excised, purified, cloned, and sequenced. For cloning, the TOPO TA Cloning reagent set pCRII (Invitrogen Corporation) was used according to the manufacturer’s recommendations. Plasmids were purified using the Qiagen Miniprep reagent set (Qiagen) and sequenced (Big Dye Terminator Cycle on ABI Prism 377 Automated Sequencer; both from PE Applied Biosystems).

quantitative real-time rt-pcr
Primer and probe design.
Quantification of the mRNA message coding for VEGF121, VEGF165, VEGF189, and VEGFtotal was performed using LightCycler technology (Roche Diagnostics). To reach high conformity of the PCR kinetics for the VEGF splice variants and VEGFtotal, a common forward primer, ex3fo (5'-CCCTGATGAGATCGAGTACATCTT-3'), and a common fluorescent hydrolyzation probe, VEGF-probe (5'-ATCCTGTGTGCCCCTGATGCGATGCGGT-3'), both located in exon 3 (part of the conserved region of all VEGF splice variants) were designed (Fig. 1 ). Specific amplification of each splice variant was performed exclusively with specific reverse primers spanning the variant specific exon boundaries: ex5/8re (5'-GCCTCGGCTTGTCACATTTT-3', 254-bp amplicon) for VEGF121, spanning the boundaries of exons 5 and 8; ex5/7re (5'-AGCAAGGCCCACAGGGATTT-3', 254-bp amplicon) for VEGF165, spanning the boundaries of exons 5 and 7; and ex6re (5'-AACGCTCCAGGACTTATACCG-3', 310-bp amplicon) for VEGF189, located in exon 6. For quantification of the complete VEGF message (VEGFtotal), the common forward primer, the common fluorescent probe, and a common reverse primer located in exon 8 (ex8re: 5'-ACCGCCTCGGCTTGTCAC-3') were used for simultaneous amplification of all variants by external PCR. For normalization, the published B2M primer/probe set (31) was adapted to our conditions: B2M forward primer (5'-GATGAGTATGCCTGCCGTGTG-3') and B2M reverse primer (5'-CAATCCAAATGCGGCATCT-3'), which produce a 114-bp amplicon; and B2M hydrolyzation probe (5'-CCTCCATGATGCTGCTTACATGTCTCGATCCC-3'). Hydrolyzation probes were labeled with a reporter dye (6-carboxy-fluorescein phosphoramidite) at the 5' end and a quencher dye (5-carboxy-tetramethylrhodamine) at the 3' end. The similar calculated melting temperatures for all primers enabled the use of the same cycling program for all samples. The melting temperatures for primers and probes were calculated using OLIGO 5.0 (MedProbe) or the JAVA OLIGO program (TIB Molbiol).



View larger version (36K):
[in this window]
[in a new window]
  		Figure 1. Position of primers and probe for quantification of VEGFtotal and specific quantification of VEGF splice variants (VEGF121, VEGF165, and VEGF189) on cDNA.

PCR conditions.
Quantitative PCR was performed in a total reaction volume of 20 µL per capillary for the LightCycler format. For conventional PCR, the amounts of reagents were scaled up to 30 µL. Bovine serum albumin (Sigma) was added only to the LightCycler reaction mixture to avoid nonspecific binding of reagents to the LightCycler glass capillary. The 20-µL reaction mixture contained 0.75 U of a temperature-release Taq DNA polymerase (Platinum DNA Polymerase; Gibco BRL, Life Technologies), 2 µL of the supplied 10x PCR buffer, 7.5 mM MgCl2, 0.2 mM dNTPs (Gibco BRL, Life Technologies), 3 µg of bovine serum albumin (Sigma), 0.25 µM each primer, 0.1 µM fluorescent probe (primers and probes; TIB Molbiol), and ± 0.4 ng of cDNA. The amplification conditions for LightCycler consisted of an initial 1.5-min denaturation step at 95 °C, followed by 40 cycles of denaturation at 95 °C for 8 s, annealing at 68 °C for 12 s, and extension at 72 °C for 20 s. Conventional PCR cycling conditions were as follows: 3-min denaturation step at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 68 °C for 15 s, and extension at 72 °C for 25 s.

Samples.
Fourteen cDNA samples from seven healthy and seven malignant tissues (Clontech Laboratories) were investigated by quantitative real-time PCR to demonstrate the applicability of the new method. Healthy tissues were pooled from several healthy Caucasians: lung, pancreas, kidney, liver, heart, skeletal muscle, and placenta. Malignant tissues were obtained from well-characterized tumors that had been explanted and propagated as xenografts in athymic nude mice, and used as model systems for cancer research: tumor cell lines LuCa1, a lung carcinoma explanted from a metastasis; LuCa2, a lung carcinoma explanted from a tumor; PaCa, a pancreatic adenocarcinoma; BrCa, a breast carcinoma; OvCa, an ovarian carcinoma; PrCa, a prostatic adenocarcinoma; and CoCa, a colon adenocarcinoma. Each provided cDNA preparation (± 0.2 ng/µL) was diluted 1:1 in water, and 4 µL of this dilutions was used per 20-µL real-time PCR reaction volume.

Normalization and quantification.
To determine the absolute copy number of the target transcripts, the cloned plasmid cDNAs for B2M and for the VEGF mRNA splice variants VEGF121, VEGF165, and VEGF189 were used to generate a calibration curve. Purified plasmid cDNA templates were measured in a photometer, and copy numbers were calculated from the absorbance at 260 nm (A260). Plasmid cDNA was serially diluted in log steps from 108 copies down to 10 copies in a 1-µL volume. A calibration curve was created by plotting the threshold cycle (Ct) vs the known copy number for each plasmid template in the dilutions. The copy numbers for all unknown samples were determined by LightCycler software 3.1, according to the calibration curve. To correct for differences in both RNA quality and quantity between samples, data were normalized by dividing the copy number of the target cDNA by the copy number of B2M. Quantitative results are presented as copies of target gene per 1000 copies of B2M. For each tissue, B2M and the target genes VEGF121, VEGF165, VEGF189, and VEGFtotal were quantified simultaneously in triplicate in one LightCycler run, together with dilutions of calibrators spanning four orders of magnitude from 4 x 106 to 4 x 102 copies per reaction mixture in duplicates and the appropriate non-template controls for each PCR. The range from 4 x 106 to 4 x 102 copies (corresponding to cycles 20–35) was chosen because the Cts for all measured target copies were within ± 2 cycles of this range.

To have comparable conditions for analyzing VEGF expression in all 14 tissues, we set the mean Ct value of the first dilution of the calibrators (4 x 106) to cycle 20 by adjusting the baseline. Statistics were performed using Excel 97 computer software (Microsoft Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
After extensive optimization of primer, MgCl2, and bovine serum albumin concentrations as well as reaction temperatures and times, we obtained a highly specific, sensitive, and reproducible quantitative real-time RT-PCR assay for specific detection of the three most abundant VEGF splice variants as well as the complete VEGF message (VEGFtotal).

specificity
Specific detection of different VEGF variants was achieved with the following primer pairs: for VEGF121, primer pair ex3fo-ex5/8re; for VEGF165, primer pair ex3fo-ex5/7re; and for VEGF189, primer pair ex3fo-ex6re. For the variants VEGF121, VEGF165, and VEGF189, 105 plasmid copies were detectable with the appropriate primer sets with a mean Ct of 24. Real-time RT-PCR with the "nonspecific" primer set for the same copy number did not generate any reporter fluorescence signal even after 40 PCR cycles, and did not show any PCR product in 3% agarose gel electrophoresis after ethidium bromide staining (Fig. 2 ).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 2. Agarose gel electrophoresis of PCR products showing specificity of real-time RT-PCR for VEGF splice variants.

Lanes 1–4, primers specific for VEGF189; lanes 5–8, primers specific for VEGF165; lanes 9–12, primer specific for VEGF121. Lanes 1, 5, and 9, plasmid template VEGF189; lanes 2, 6, and 10, plasmid template VEGF165; lanes 3, 7, and 11, plasmid template VEGF121. Lanes 4, 8, and 12, non-template controls. Lane M, 100-bp DNA size marker.

determination of pcr sensitivity and efficiency
The sensitivity of the PCR method using different primer/probe sets was determined from the Ct values obtained with known quantities of plasmid cDNA. All calibration curves for the plasmid cDNAs VEGF121, VEGF165, VEGF189, and for B2M cDNA (each diluted 10-fold from 4 x 106 to 4 x 102 copies of plasmid cDNA per reaction mixture) showed linearity over the entire quantification range with correlation coefficients >0.99, indicating a precise log-linear relationship. Because the amplicons of the three VEGF splice variants and B2M have different sizes (range, 114–310 bp), PCR efficiency may vary. However, for all dilutions of the four different plasmid calibrators, the mean SD of the Ct values for each dilution step was 0.21 cycles. This deviation was not significantly different from the SD for the dilution series for the individual plasmid calibrators (SD = 0.18 cycles). The slopes for the dilution series for all four plasmid were 3.34–3.48, indicating comparable PCR amplification efficiencies. Thus, for the different targets, all experiments were performed using one calibration curve for all targets, including B2M (Fig. 3 ).



View larger version (34K):
[in this window]
[in a new window]
  		Figure 3. Fluorescence data for all three VEGF splice variant calibrators together with the resulting calibration curve generated by the LightCycler software.

VEGF121, VEGF165, and VEGF189 diluted from 4 x 106 to 4 x 102 copy numbers in duplicate.

reproducibility
The intrarun variability, calculated from triplicate samples for all of the targets, showed an average SD for the Ct of 0.16 cycles. The difference in absolute Ct values for each set of triplicates was never >0.53 cycles. The interrun variability was assessed by comparing the results of 14 different runs (corresponding to 14 different tissues) using duplicates of the calibrator dilutions. When we compared the means of two replicate measurements at each dilution, the difference in the Ct was always <1.1 cycle, with a mean SD for all runs of 0.21 cycles. The slopes of the calibration curves of all 14 runs were 3.32–3.52.

vegf splice variants and vegftotal expression in healthy and malignant tissues
The measured copy numbers for VEGFtotal cDNA were, on average, 90% (SD = 5.6%) of the sum of the different VEGF splice variant measurements in the 14 cDNA samples investigated. The LightCycler PCR products of all 14 cDNA samples were collected, and gel electrophoresis was performed. The copy numbers for the VEGF splice variants in all 14 tissues are presented in Table 1 .


View this table:
[in this window]
[in a new window]
  		Table 1. Expression1 of VEGF splice variants in seven healthy and seven malignant tissue samples.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Increasing evidence indicates that the ratio of the VEGF isoforms rather than total VEGF is responsible for VEGF-mediated angiogenesis in physiological and pathological conditions (23)(24)(25)(26)(27). We present here for the first time a specific, sensitive, and reproducible real-time RT-PCR approach for absolute quantification of the most abundant VEGF splice variants (VEGF121, VEGF165, and VEGF189) as well as of the total amount of VEGF (VEGFtotal), using LightCycler technology to obtain better insight into the regulation and distribution of the VEGF isoform ratio in healthy and malignant tissues.

Common immunological assays allow quantification of VEGF but fail to discriminate among isoforms because of a lack of VEGF isoform-specific monoclonal antibodies. Therefore, at present, investigation of the impact of differential expression of the various VEGF isoforms is possible only on the mRNA level. Several publications have indicated that expression of VEGF is controlled at the mRNA level by regulation of transcription rate and/or by changing mRNA stability (32)(33). To date, competitive RT-PCR has been the only method for semiquantitative detection of variant mRNA transcripts. This approach has two major disadvantages: (a) PCR endpoint analysis permits only semiquantitative analysis at a low sensitivity; and (b) formation of heteroduplexes between the different variants (34)(35), especially for the extensively spliced VEGF variants (36), further impairs quantification. In contrast, real-time quantitative RT-PCR, where quantification is performed during the exponential phase of amplification, permits accurate quantification over a wide dynamic range without any post-PCR processing. Heteroduplex formation does not play any role in real-time RT-PCR amplification. In the assay described here, each splice variant was separately and specifically amplified.

As shown in Fig. 4 , expression of the different VEGF splice variants is detectable when the external primer pair for VEGFtotal is used, but absolute quantification of each variant as well as VEGFtotal from the gel is not exact. Therefore, statements concerning the absolute quantities of different splice variants obtained by competitive PCR should be judged critically.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. Agarose gel electrophoresis of amplification products generated by LightCycler RT-PCR (40 cycles) for VEGFtotal, allowing semiquantitative measurement of VEGF splice variants.

Lanes 1–7, healthy tissues (lung, pancreas, kidney, liver, heart, skeletal muscle, and placenta, respectively). Lanes 8–14, malignant tissues (lung carcinoma metastasis, lung carcinoma, pancreatic adenocarcinoma, breast carcinoma, ovarian carcinoma, prostatic adenocarcinoma, and colon adenocarcinoma, respectively). Lane M, 100-bp DNA size marker. A shift toward increased expression of shorter variants in malignant tissues is apparent.

The presented standardized VEGF real-time RT-PCR assay using variant-specific reverse primers in combination with a common hydrolyzation fluorescent probe and a common forward primer ensures highly specific quantification with very good sensitivity and enables comparison of results generated by different investigators.

In this study, the mRNA message of VEGF splice variants and VEGFtotal in 14 (7 healthy and 7 malignant) tissues of different origin was investigated. The total VEGF message was generally up-regulated in malignant tissues (20.5–728 copies/103 copies of B2M) compared with healthy tissues (4.2–81 copies/103 copies of B2M). Most remarkably, the copy number for VEGFtotal and the sum of the different VEGF splice variant copies showed excellent correlation (on average, 90%; SD = 5.6%). In all 14 tissues, the splice variants VEGF165 and VEGF121 showed higher expression than VEGF189. In healthy tissues, the mRNA encoding diffusible variants VEGF121 and VEGF165 constituted on average 78% (SD = 9.3%) of the total VEGF message, and the cell-adherent variant VEGF189 constituted on average 22% (SD = 5.4%). In contrast, in malignant tissues, VEGF121 and VEGF165 constituted 94% (SD = 7.6%) of the total VEGF message, whereas VEGF189 was 6% (SD = 3.7%). When we compared the absolute copy numbers for VEGF189 in all healthy and malignant tissues, no difference was found (Table 1Up ), whereas VEGF165 and VEGF121 were up-regulated in malignant tissues. Our data, indicating that overexpression of VEGF in tumors is caused by the more diffusible variants, support the observations that during malignant progression an angiogenic switch favoring the shorter diffusible isoforms (VEGF121 and VEGF165) occurs (27).

Because of the small number of correlated tissues investigated, differences in VEGF expression mark only a trend and allow no final conclusions. To verify the findings and to confirm the utility of the presented method, fresh malignant and healthy tissues from the same individuals should be measured. Furthermore, the prognostic significance of this assay could be demonstrated by examining malignant tissues from individuals with carcinomas in situ and by comparing with individuals with metastatic cancers.

In conclusion, the presented quantitative real-time RT-PCR assay clearly demonstrates many advantages over other existing methods for (semi)quantitative analysis of VEGF splice variants, including specificity, sensitivity, reduced carryover contamination, and rapid, accurate, and simultaneous quantification of multiple samples. Thus, we believe that this assay may be a promising tool for further investigations in the field of tumor angiogenesis and prognostic impact of VEGF in cancer.


   Acknowledgments
 
This work was supported by the Deutsche Kinderkrebsstiftung e.V. (Bonn, Germany), by Verein zur Förderung der Tagesklinik e.V. (Berlin, Germany), and by Olfert Landt, TIB Molbiol (Berlin, Germany). We appreciate the excellent technical assistance of Lucia Badiali and Gisela Götze.


   Footnotes
 
1 Nonstandard abbreviations: VEGF, vascular endothelial growth factor; RT-PCR, reverse transcription-PCR; B2M, ß2-microglobulin; and Ct, threshold cycle.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989;161:851-858.[ISI][Medline] [Order article via Infotrieve]
  2. Plouet J, Schilling J, Gospodarowicz D. Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT-20 cells. EMBO J 1989;8:3801-3806.[ISI][Medline] [Order article via Infotrieve]
  3. Gospodarowicz D, Abraham JA, Schilling J. Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proc Natl Acad Sci U S A 1989;86:7311-7315.[Abstract/Free Full Text]
  4. Ferrara N, Houck K, Jakeman L, Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev 1992;13:18-32.[ISI][Medline] [Order article via Infotrieve]
  5. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27-31.[ISI][Medline] [Order article via Infotrieve]
  6. Iruela Arispe ML, Dvorak HF. Angiogenesis: a dynamic balance of stimulators and inhibitors. Thromb Haemost 1997;78:672-677.[ISI][Medline] [Order article via Infotrieve]
  7. Risau W. Mechanisms of angiogenesis. Nature 1997;386:671-674.[Medline] [Order article via Infotrieve]
  8. Ohta Y, Endo Y, Tanaka M, Shimizu J, Oda M, Hayashi Y, et al. Significance of vascular endothelial growth factor messenger RNA expression in primary lung cancer. Clin Cancer Res 1996;2:1411-1416.[Abstract]
  9. Larcher F, Robles AI, Duran H, Murillas R, Quintanilla M, Cano A, et al. Up-regulation of vascular endothelial growth factor/vascular permeability factor in mouse skin carcinogenesis correlates with malignant progression state and activated H-ras expression levels. Cancer Res 1996;56:5391-5396.[Abstract/Free Full Text]
  10. Perez Atayde AR, Sallan SE, Tedrow U, Connors S, Allred E, Folkman J.. Spectrum of tumor angiogenesis in the bone marrow of children with acute lymphoblastic leukemia. Am J Pathol 1997;150:815-821.[Abstract]
  11. Fiedler W, Graeven U, Ergun S, Verago S, Kilic N, Stockschlader M, Hossfeld DK. Vascular endothelial growth factor, a possible paracrine growth factor in human acute myeloid leukemia. Blood 1997;89:1870-1875.[Abstract/Free Full Text]
  12. Aguayo A, Estey E, Kantarjian H, Mansouri T, Gidel C, Keating M, et al. Cellular vascular endothelial growth factor is a predictor of outcome in patients with acute myeloid leukemia. Blood 1999;94:3717-3721.[Abstract/Free Full Text]
  13. Bellamy WT, Richter L, Frutiger Y, Grogan TM. Expression of vascular endothelial growth factor and its receptors in hematopoietic malignancies. Cancer Res 1999;59:728-733.[Abstract/Free Full Text]
  14. Hussong JW, Rodgers GM, Shami PJ. Evidence of increased angiogenesis in patients with acute myeloid leukemia. Blood 2000;95:309-313.[Abstract/Free Full Text]
  15. Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, Abraham JA. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem 1991;266:11947-11954.[Abstract/Free Full Text]
  16. Anthony FW, Wheeler T, Elcock CL, Pickett M, Thomas EJ. Identification of a specific pattern of vascular endothelial growth factor mRNA expression in human placenta and cultured placental fibroblasts [Short Report]. Placenta 1994;15:557-561.[ISI][Medline] [Order article via Infotrieve]
  17. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999;13:9-22.[Abstract/Free Full Text]
  18. Poltorak Z, Cohen T, Sivan R, Kandelis Y, Spira G, Vlodavsky I, et al. VEGF145, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix. J Biol Chem 1997;272:7151-7158.[Abstract/Free Full Text]
  19. Cohen T, Gitay Goren H, Sharon R, Shibuya M, Halaban R, Levi BZ, Neufeld G. VEGF121, a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells. J Biol Chem 1995;270:11322-11326.[Abstract/Free Full Text]
  20. Burchardt T, Burchardt M, Chen MW, Buttyan R, de la Taille A, Shabsigh A, Shabsigh R. Expression of VEGF splice variants 144/145 and 205/206 in adult male tissues. IUBMB Life 1999;48:405-408.[ISI][Medline] [Order article via Infotrieve]
  21. Whittle C, Gillespie K, Harrison R, Mathieson PW, Harper SJ. Heterogeneous vascular endothelial growth factor (VEGF) isoform mRNA and receptor mRNA expression in human glomeruli, and the identification of VEGF148 mRNA, a novel truncated splice variant. Clin Sci (Colch) 1999;97:303-312.[Medline] [Order article via Infotrieve]
  22. Jingjing L, Xue Y, Agarwal N, Roque RS. Human Muller cells express VEGF183, a novel spliced variant of vascular endothelial growth factor. Invest Ophthalmol Vis Sci 1999;40:752-759.[Abstract/Free Full Text]
  23. Lee YH, Tokunaga T, Oshika Y, Suto R, Yanagisawa K, Tomisawa M, et al. Cell-retained isoforms of vascular endothelial growth factor (VEGF) are correlated with poor prognosis in osteosarcoma. Eur J Cancer 1999;35:1089-1093.
  24. Tomisawa M, Tokunaga T, Oshika Y, Tsuchida T, Fukushima Y, Sato H, et al. Expression pattern of vascular endothelial growth factor isoform is closely correlated with tumour stage and vascularisation in renal cell carcinoma. Eur J Cancer 1999;35:133-137.
  25. Oshika Y, Nakamura M, Tokunaga T, Ozeki Y, Fukushima Y, Hatanaka H, et al. Expression of cell-associated isoform of vascular endothelial growth factor 189 and its prognostic relevance in non-small cell lung cancer. Int J Oncol 1998;12:541-544.[ISI][Medline] [Order article via Infotrieve]
  26. Tokunaga T, Oshika Y, Abe Y, Ozeki Y, Sadahiro S, Kijima H, et al. Vascular endothelial growth factor (VEGF) mRNA isoform expression pattern is correlated with liver metastasis and poor prognosis in colon cancer. Br J Cancer 1998;77:998-1002.[ISI][Medline] [Order article via Infotrieve]
  27. Cheung N, Wong MP, Yuen ST, Leung SY, Chung LP. Tissue-specific expression pattern of vascular endothelial growth factor isoforms in the malignant transformation of lung and colon. Hum Pathol 1998;29:910-914.[ISI][Medline] [Order article via Infotrieve]
  28. Cheng SY, Nagane M, Huang HS, Cavenee WK. Intracerebral tumor-associated hemorrhage caused by overexpression of the vascular endothelial growth factor isoforms VEGF121 and VEGF165 but not VEGF189. Proc Natl Acad Sci U S A 1997;94:12081-12087.[Abstract/Free Full Text]
  29. Okamoto K, Oshika Y, Fukushima Y, Ohnishi Y, Tokunaga T, Tomii Y, et al. Xenografts of human solid tumors frequently express cellular-associated isoform of vascular endothelial growth factor (VEGF) 189. Oncol Rep 1999;6:1201-1204.[ISI][Medline] [Order article via Infotrieve]
  30. Seeger K, Adams HP, Buchwald D, Beyermann B, Kremens B, Niemeyer C, et al. TEL-AML1 fusion transcript in relapsed childhood acute lymphoblastic leukemia. The Berlin-Frankfurt-Munster Study Group. Blood 1998;91:1716-1722.[Abstract/Free Full Text]
  31. Pallisgaard N, Clausen N, Schroder H, Hokland P. Rapid and sensitive minimal residual disease detection in acute leukemia by quantitative real-time RT-PCR exemplified by t(12;21) TEL-AML1 fusion transcript. Genes Chromosomes Cancer 1999;26:355-365.[ISI][Medline] [Order article via Infotrieve]
  32. Punglia RS, Lu M, Hsu J, Kuroki M, Tolentino MJ, Keough K, et al. Regulation of vascular endothelial growth factor expression by insulin-like growth factor I. Diabetes 1997;46:1619-1626.[Abstract]
  33. Levy AP, Levy NS, Goldberg MA. Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 1996;271:2746-2753.[Abstract/Free Full Text]
  34. Henley WN, Schuebel KE, Nielsen DA. Limitations imposed by heteroduplex formation on quantitative RT-PCR. Biochem Biophys Res Commun 1996;226:113-117.[ISI][Medline] [Order article via Infotrieve]
  35. Robinson CA, Hayward Lester A, Hewetson A, Oefner PJ, Doris PA, Chilton BS. Quantification of alternatively spliced RUSH mRNA isoforms by QRT-PCR and IP-RP-HPLC analysis: a new approach to measuring regulated splicing efficiency. Gene 1997;198:1-4.[ISI][Medline] [Order article via Infotrieve]
  36. Eckhart L, Ban J, Ballaun C, Weninger W, Tschachler E. Reverse transcription-polymerase chain reaction products of alternatively spliced mRNAs form DNA heteroduplexes and heteroduplex complexes. J Biol Chem 1999;274:2613-2615.[Abstract/Free Full Text]



The following articles in journals at HighWire Press have cited this article:


Home page
 J. Immunol.Home page
H. Mouquet, S. Farci, P. Joly, B. Maillere, J. Leblond, L. Drouot, J. Leprince, M. C. Tonon, P. Loiseau, D. Charron, et al.
A Truncated Alternative Spliced Isoform of Human Desmoglein 1 Contains a Specific T Cell Epitope Binding to the Pemphigus Foliaceus-Associated HLA Class II DRbeta1*0102 Molecule
J. Immunol., November 1, 2006; 177(9): 6517 - 6526.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
A. Tropea, F. Miceli, F. Minici, F. Tiberi, M. Orlando, M. F. Gangale, F. Romani, S. Catino, S. Mancuso, P. Navarra, et al.
Regulation of Vascular Endothelial Growth Factor Synthesis and Release by Human Luteal Cells in Vitro
J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2303 - 2309.
[Abstract] [Full Text] [PDF]

Home page
 J. Mol. Diagn.Home page
S. Ogino, T. Kawasaki, M. Brahmandam, M. Cantor, G. J. Kirkner, D. Spiegelman, G. M. Makrigiorgos, D. J. Weisenberger, P. W. Laird, M. Loda, et al.
Precision and Performance Characteristics of Bisulfite Conversion and Real-Time PCR (MethyLight) for Quantitative DNA Methylation Analysis
J. Mol. Diagn., May 1, 2006; 8(2): 209 - 217.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
L. Milani, M. Fredriksson, and A.-C. Syvanen
Detection of Alternatively Spliced Transcripts in Leukemia Cell Lines by Minisequencing on Microarrays
Clin. Chem., February 1, 2006; 52(2): 202 - 211.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
E. Zygalaki, A. Stathopoulou, C. Kroupis, L. Kaklamanis, Z. Kyriakides, D. Kremastinos, and E. S. Lianidou
Real-Time Reverse Transcription-PCR Quantification of Vascular Endothelial Growth Factor Splice Variants
Clin. Chem., August 1, 2005; 51(8): 1518 - 1520.
[Full Text] [PDF]

Home page
 CirculationHome page
V. A.J. Kempf, M. Lebiedziejewski, K. Alitalo, J.-H. Walzlein, U. Ehehalt, J. Fiebig, S. Huber, B. Schutt, C. A. Sander, S. Muller, et al.
Activation of Hypoxia-Inducible Factor-1 in Bacillary Angiomatosis: Evidence for a Role of Hypoxia-Inducible Factor-1 in Bacterial Infections
Circulation, March 1, 2005; 111(8): 1054 - 1062.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
L. Ramon, J. Gilabert-Estelles, R. Castello, J. Gilabert, F. Espana, A. Romeu, M. Chirivella, J. Aznar, and A. Estelles
mRNA analysis of several components of the plasminogen activator and matrix metalloproteinase systems in endometriosis using a real-time quantitative RT-PCR assay
Hum. Reprod., January 1, 2005; 20(1): 272 - 278.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
R. Rej
Clinical Chemistry through Clinical Chemistry: A Journal Timeline
Clin. Chem., December 1, 2004; 50(12): 2415 - 2458.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
L. Jensen, H. Pilegaard, P. D. Neufer, and Y. Hellsten
Effect of acute exercise and exercise training on VEGF splice variants in human skeletal muscle
Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2004; 287(2): R397 - R402.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
S. Wellmann, C. Buhrer, E. Moderegger, A. Zelmer, R. Kirschner, P. Koehne, J. Fujita, and K. Seeger
Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism
J. Cell Sci., May 1, 2004; 117(9): 1785 - 1794.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. Mor, F. J. Quintana, and I. R. Cohen
Angiogenesis-Inflammation Cross-Talk: Vascular Endothelial Growth Factor Is Secreted by Activated T Cells and Induces Th1 Polarization
J. Immunol., April 1, 2004; 172(7): 4618 - 4623.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
Y. van Hensbergen, H. J. Broxterman, S. Rana, P. J. van Diest, M. C. A. Duyndam, K. Hoekman, H. M. Pinedo, and E. Boven
Reduced Growth, Increased Vascular Area, and Reduced Response to Cisplatin in CD13-Overexpressing Human Ovarian Cancer Xenografts
Clin. Cancer Res., February 1, 2004; 10(3): 1180 - 1191.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Kronke, V. N. Bochkov, J. Huber, F. Gruber, S. Bluml, A. Furnkranz, A. Kadl, B. R. Binder, and N. Leitinger
Oxidized Phospholipids Induce Expression of Human Heme Oxygenase-1 Involving Activation of cAMP-responsive Element-binding Protein
J. Biol. Chem., December 19, 2003; 278(51): 51006 - 51014.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
V. Richard, A. Luchin, R. M. Brena, C. Plass, and T. J. Rosol
Quantitative Evaluation of Alternative Promoter Usage and 3' Splice Variants for Parathyroid Hormone-related Protein by Real-Time Reverse Transcription-PCR
Clin. Chem., August 1, 2003; 49(8): 1398 - 1402.
[Full Text] [PDF]

Home page
 BloodHome page
F. Gruber, P. Hufnagl, R. Hofer-Warbinek, J. A. Schmid, J. M. Breuss, R. Huber-Beckmann, M. Lucerna, N. Papac, H. Harant, I. Lindley, et al.
Direct binding of Nur77/NAK-1 to the plasminogen activator inhibitor 1 (PAI-1) promoter regulates TNFalpha -induced PAI-1 expression
Blood, April 15, 2003; 101(8): 3042 - 3048.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Chem.Home page
R. Castello, A. Estelles, C. Vazquez, C. Falco, F. Espana, S. M. Almenar, C. Fuster, and J. Aznar
Quantitative Real-Time Reverse Transcription-PCR Assay for Urokinase Plasminogen Activator, Plasminogen Activator Inhibitor Type 1, and Tissue Metalloproteinase Inhibitor Type 1 Gene Expressions in Primary Breast Cancer
Clin. Chem., August 1, 2002; 48(8): 1288 - 1295.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
N. K. Ozaki, K. D. Beharry, K. C. Nishihara, Y. Akmal, J. G. Ang, R. Sheikh, and H. D. Modanlou
Regulation of Retinal Vascular Endothelial Growth Factor and Receptors in Rabbits Exposed to Hyperoxia
Invest. Ophthalmol. Vis. Sci., May 1, 2002; 43(5): 1546 - 1557.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. Loewe, W. Holnthoner, M. Groger, M. Pillinger, F. Gruber, D. Mechtcheriakova, E. Hofer, K. Wolff, and P. Petzelbauer
Dimethylfumarate Inhibits TNF-Induced Nuclear Entry of NF-{kappa}B/p65 in Human Endothelial Cells
J. Immunol., May 1, 2002; 168(9): 4781 - 4787.
[Abstract] [Full Text] [PDF]

<
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (55)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wellmann, S.
Right arrow Articles by Seeger, K.
Right arrow Search for Related Content
PubMed