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Coexpression of VEGF Receptors VEGF-R2 and Neuropilin-1 in Proliferative Diabetic Retinopathy

From the Departments of ¹ Pathology and ² Ophthalmology, Keio University School of Medicine, Tokyo, Japan.

	Abstract

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PURPOSE. To elucidate vascular endothelial growth factor (VEGF)-mediated pathogenesis of fibrovascular proliferation in diabetic retinopathy.

METHODS. Fibrovascular tissues were obtained at vitrectomy from 22 cases with proliferative diabetic retinopathy. The half-divided tissues were processed for reverse transcription–polymerase chain reaction (RT–PCR) analysis to examine the expression of VEGF isoforms and their receptors. Paraffin sections of the other half were used for immunohistochemistry for CD34, glial fibrillary acidic protein and VEGF, and in situ hybridization for VEGF.

RESULTS. RT–PCR analysis demonstrated the expression of VEGF receptors VEGF-R1, VEGF-R2, and neuropilin-1 in 12, 14, and 14 of 22 cases, respectively. Notably, VEGF-R2 and neuropilin-1 were simultaneously expressed in the identical 14 tissues. The isoform VEGF₁₂₁ was constitutively expressed in all the tissues examined, whereas the expression of VEGF₁₆₅ was confined to the 7 tissues that also expressed VEGF-R2 and neuropilin-1. The vascular density of fibrovascular tissues evaluated by immunohistochemistry for CD34 was significantly higher in the cases with the expression of VEGF-R2 and neuropilin-1 than in those without their expression (P < 0.01), whereas VEGF-R1 expression had no such relationship with the vascular density. The fibrovascular tissues that expressed VEGF₁₆₅ together with VEGF-R2 and neuropilin-1 were found in significantly younger patients (P < 0.01). In situ hybridization and immunohistochemical studies demonstrated that glial cells in the fibrovascular tissues express and produce VEGF.

CONCLUSIONS. Coexpression of VEGF-R2 and neuropilin-1 is suggested to facilitate fibrovascular proliferation in diabetic retinopathy.

	Introduction

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Diabetic retinopathy is a major cause of adult blindness when it progresses to the stage of proliferative diabetic retinopathy characterized by fibrovascular proliferation. However, molecular mechanisms of angiogenesis in proliferative diabetic retinopathy are not fully understood, although several growth factors including vascular endothelial growth factor (VEGF) have been suggested to be involved.^{1 2 3 4 5 6}

VEGF is an angiogenic mitogen secreted from various types of cells^{7 8} and plays a major role in the angiogenesis under physiologic and pathologic conditions such as normal embryonic development,^{9 10 11} wound healing,¹² and solid tumor growth.^{13 14} VEGF has five major isoforms generated by alternative splicing from a single gene (i.e., VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆). Two high-affinity tyrosine–kinase receptors for VEGF (i.e., fms-like tyrosine kinase-1, Flt-1,¹⁵ and kinase insert domain–containing receptor, KDR^{16 17} ) have been cloned and characterized. Flt-1 and KDR are now known as VEGF-R1 and VEGF-R2, respectively. The recent study has reported an additional receptor for VEGF (i.e., neuropilin-1¹⁸ ), which was originally recognized as a molecule involved in axonal guidance in the developing nervous tissue.¹⁹ Increasing evidence indicates that the signal transduction mediated by VEGF-R2 is different from that by VEGF-R1^{20 21 22} and the bioactivities of VEGF differ between the isoforms.^{23 24} Neuropilin-1 has been shown to enhance the bioactivity of VEGF₁₆₅, but not that of VEGF₁₂₁, by increasing the binding affinity of VEGF₁₆₅ to VEGF-R2.¹⁸ Thus, neuropilin-1 is considered to function as a VEGF₁₆₅-specific receptor when coexpressed with VEGF-R2.

Involvement of VEGF in proliferative diabetic retinopathy has been suggested by previous findings that the vitreous fluid from eyes with proliferative diabetic retinopathy contains a large amount of VEGF,^{1 2} which is produced by the ischemic retinal cells,^{4 5} and that the fibrovascular proliferative tissue expresses VEGF.³ However, VEGF receptors involved in the fibrovascular proliferation have not been examined previously, and the relationship between the histopathology of the fibrovascular tissue and the expression pattern of VEGF isoforms and their receptors is a matter of concern. In addition, the cells in the fibrovascular tissue responsible for VEGF production remain unknown.

In the present study, we examined the expression of VEGF receptors VEGF-R1, VEGF-R2, and neuropilin-1, together with that of VEGF isoforms, in the fibrovascular tissues excised at vitrectomy and also analyzed the histopathology of the tissues. We demonstrate for the first time that coexpression of VEGF-R2 and neuropilin-1 correlates with the degree of angiogenesis in proliferative diabetic retinopathy and that glial cells in the fibrovascular tissue express and produce VEGF.

	Methods

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Fibrovascular Tissues
Fibrovascular tissues were obtained from 22 eyes of 22 patients with proliferative diabetic retinopathy during vitrectomy performed at Keio University Hospital between July 1998 and April 1999. Operative indication included persistent vitreous hemorrhage and traction retinal detachment involving or threatening the macula. All the patients gave their informed consent to our study, which followed the tenets of the Declaration of Helsinki. Clinical information of the present cases is shown in Table 1 . For histopathologic evaluation, half-divided surgical specimen of each case was fixed in 4% paraformaldehyde at 4°C immediately after removal, and embedded in paraffin for light microscopic analyses. The other half was processed for RNA extraction for reverse transcription–polymerase chain reaction (RT–PCR) analysis.

Morphometric Analysis
The degree of angiogenesis was evaluated by the morphometric analysis of the tissue sections immunostained for CD34. In each section, the entire tissue was photographed to measure the whole area of the tissue, and the number of CD34-immunoreactive vessels with a distinct lumen was counted. Two of the authors independently counted the number of vessels without any clinical information on each patient, and the average number of vessels per square millimeter was calculated as vascular density (vessels per square millimeter) of the section. In each case, two sections 80- to 100-µm apart were subjected to calculation of vascular density, and the mean of these two values was used as the final vascular density for statistical analyses (Mann–Whitney U test, Spearman rank correlation, Kruskal–Wallis test, and Dunn procedure).

RT–PCR and Sequencing Analysis
Total cellular RNA was prepared using ISOGEN (NIPPON GENE, Toyama, Japan) according to the manufacturer’s protocol. In brief, surgical materials were homogenized in 1 ml of ISOGEN, and 200 µl of chloroform was added. After centrifugation at 4°C, the aqueous phase was collected, and total RNA was precipitated with an equal volume of isopropanol. RNA was then dissolved in 10 µl of water treated with diethyl pyrocarbonate.

We calculated the relative amount of RNA in each case by quantifying the amplified ß-actin cDNA fragment because total amount of RNA extracted in each case was below the limit of the ordinary measurement with a UV photometer due to the minute size of the tissue. Two µl of the solution containing total RNA was reverse-transcribed with a First-Strand cDNA Synthesis Kit (Pharmacia Biotech, Uppsala, Sweden) at 37°C for 1 hour in a 15-µl reaction volume containing random hexadeoxynucleotide primer and Moloney Murine Leukemia Virus reverse transcriptase. A 2-µl aliquot of the reaction product was subjected to 30 cycles of PCR for amplification of ß-actin cDNA. Density of the band of amplified ß-actin cDNA was measured in each case, and the relative amount of total RNA extracted from each tissue was calculated. Based on the above results, we adjusted the starting amount of RNA for further RT–PCR analysis on the expression of VEGF, VEGF-R1, VEGF-R2, neuropilin-1, and ß-actin. RNA was reverse-transcribed as described above, and PCR was performed at 30 cycles in a 50-µl reaction volume containing 800 nM of each primer, 100 µM of dNTP, and 5 U Taq DNA polymerase (TOYOBO, Tokyo, Japan) in a thermal controller (Mini Cycler; MJ Research, Watertown, MA). The thermal cycle was 1 minute at 94°C; 2 minutes at either 64°C (VEGF), 64°C (VEGF-R1), 63°C (VEGF-R2), 63°C (neuropilin-1), or 67°C (ß-actin); and 3 minutes at 72°C, followed by 3 minutes at 72°C. The nucleotide sequences of the PCR primers were 5'-TGC CTT GCT GCT CTA CCT CC-3' (forward, on exon 1) and 5'-TCA CCG CCT CGG CTT GTC AC-3' (reverse, on exon 8) for VEGF; 5'-GAT GTT GAG GAA GAG GAG GAT T-3' (forward) and 5'-AAG CTA GTT TCC TGG GGG TAT A-3' (reverse) for VEGF-R1; 5'-GAT GTG GTT CTG AGT CCG TCT-3' (forward) and 5'-CAT GGC TCT GCT TCT CCT TTG-3' (reverse) for VEGF-R2; 5'-CAA CGA TAA ATG TGG CGA TAC T-3' (forward) and 5'-TAT ACT GGG AAG AAG CTG TGA T-3' (reverse) for neuropilin-1; 5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3' (forward) and 5'-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG-3' (reverse) for ß-actin. The above RT–PCR analysis enabled us to discriminate each isoform of VEGF by the difference in size of each amplified DNA fragment. The expected sizes of the amplified cDNA fragments of VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₉, VEGF₂₀₆, VEGF-R1, VEGF-R2, neuropilin-1, and ß-actin were 0.41, 0.48, 0.54, 0.61, 0.66, 1.1, 0.56, 0.82, and 0.66 kb, respectively. An aliquot of the PCR product was electrophoresed in a 1.5% agarose gel and stained with ethidium bromide.

To confirm the specific amplification from the target mRNAs, the RT–PCR products were subcloned into the pBluescript KS vector (Stratagene, La Jolla, CA) and were analyzed by sequencing with fluorescent T7 primer (Amersham Pharmacia Biotech, Buckinghamshire, UK) using a Thermo Sequenase fluorescence-labeled primer cycle sequencing kit (Amersham Pharmacia Biotech) and ALF DNA sequencer II (Amersham Pharmacia Biotech).

In Situ Hybridization
Serial paraffin sections, 4-µm-thick, were treated with proteinase K (5 µg/ml; Sigma Chemical, St. Louis, MO) in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA at 37°C for 30 minutes, and post-fixed in 4% paraformaldehyde at room temperature for 10 minutes. They were then rinsed in 0.1 M phosphate buffer, incubated in 0.2 M HCl at room temperature for 10 minutes, and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine–HCl (pH 8.0) at room temperature for 10 minutes. After being washed in 0.1 M phosphate buffer, they were dehydrated in ethanol and air-dried.

Single-strand sense and antisense digoxigenin-labeled RNA probes were generated by in vitro transcription of the cDNA with T3 or T7 RNA polymerase using the DIG RNA Labeling Kit (Boehringer Mannheim, Mannheim, Germany) following the protocol from the manufacturer. Template DNA was a 517-bp cDNA encoding human VEGF₁₂₁, which was cloned in pBluescript KS vector. This cDNA clone was kindly provided by Herbert A. Weich (Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany).

Hybridization with the digoxigenin-labeled RNA probes was performed at 50°C for 16 hours in 40 µl of buffer containing 50% formamide, 10 mM Tris–HCl (pH 7.6), 0.2 µg/µl tRNA, 1x Denhardt’s solution (0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 10% dextran sulfate, 600 mM NaCl, 0.25% sodium dodecyl sulfate (SDS) and 1 mM EDTA. After hybridization, the sections were washed in a buffer containing 50% formamide and 2x SSC at 50°C for 30 minutes, followed by digestion with ribonuclease A (10 µg/ml; Sigma Chemical) at 37°C for 30 minutes. After being washed in 2x SSC at 50°C for 20 minutes and twice in 0.2x SSC at 50°C for 20 minutes, they were treated with 0.3% hydrogen peroxide and 0.1% sodium azide in distilled water for 30 minutes at room temperature to block endogenous peroxidase activity. After blocking nonspecific binding with 10% normal horse serum, they were incubated with mouse anti-digoxigenin antibody (1/750 dilution; Boehringer Mannheim) at room temperature for 90 minutes, then incubated with biotinylated horse antibodies against mouse immunoglobulins (1/200 dilution; Vector Laboratories, Burlingame, CA) for 30 minutes, and, finally, reacted with a solution containing the complex of avidin (1/100 dilution; DAKO A/S) and biotinylated horseradish peroxidase (1/100 dilution; DAKO A/S) for 30 minutes. Color was developed with 3,3'-diaminobenzidine tetrahydrochloride (0.2 mg/ml) in 0.05 M Tris–HCl (pH 7.6) containing 0.003% hydrogen peroxide, and the sections were counterstained with methyl green.

	Results

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Vascular Density of Fibrovascular Tissues
The degree of angiogenesis in fibrovascular tissues was different among the patients (Fig. 1) , and, thus, the vascular density was measured by morphometric analysis of the sections immunostained for CD34. Vascular structures were clearly detected in the immunostained sections as shown in Figure 1 , and the vascular density of the tissue was readily evaluated. The vascular density of each case is shown in Table 1 .

View larger version (129K): [in this window] [in a new window]   Figure 1. Histology of the fibrovascular tissues in proliferative diabetic retinopathy. Two representative cases are presented. (A, B) Highly vascularized fibrovascular tissue (157.2 vessels/mm2) from patient 17. (C, D) Sparsely vascularized tissue (32.1 vessels/mm2) from patient 7. (A, C) Hematoxylin–eosin stain; (B, D) immunohistochemistry for CD34 counterstained with methyl green. Scale bars, 200 µm.

View larger version (28K): [in this window] [in a new window]   Figure 2. RT–PCR for the expression of VEGF isoforms and their receptors (VEGF-R1, VEGF-R2, and neuropilin-1) in the fibrovascular tissue. Patients who express VEGF165, VEGF-R1, VEGF-R2, or neuropilin-1 are indicated with "+" under each panel. Note that VEGF-R2 and neuropilin-1 are expressed in the identical cases.

View larger version (21K): [in this window] [in a new window]   Figure 3. Relationship between the vascular density and the expression of VEGF-R1, VEGF-R2, and neuropilin-1. (A) The vascular density shows no significant difference between the tissues with VEGF-R1 expression and those without its expression (P = 0.74, Mann–Whitney U test). (B) The vascular density of the tissues with the coexpression of VEGF-R2 and neuropilin-1 is significantly higher than that without their coexpression (P < 0.01, Mann–Whitney U test). (C) There is a reverse correlation between the age of cases and the vascular density of fibrovascular tissues (P < 0.01, Spearman rank correlation). (D) Analysis after age restriction shows the significant difference in vascular density between the patients with the coexpression of VEGF-R2 and neuropilin-1 and those without the coexpression (P < 0.01, Mann–Whitney U test).

View larger version (15K): [in this window] [in a new window]   Figure 4. Difference in vascular density and age among group I [VEGF165 (+), VEGF-R2 (+), and neuropilin-1 (+)]; group II [VEGF165 (-), VEGF-R2 (+), and neuropilin-1 (+)]; and group III [VEGF165 (-), VEGF-R2 (-), and neuropilin-1 (-)]. Significant differences are shown in vascular density (A) between groups I and III (P < 0.05, Dunn procedure) and between groups II and III (P < 0.05, Dunn procedure), and in age (B) between groups I and III (P < 0.01, Dunn procedure). Differences were examined by Kruskal–Wallis test and subsequent Dunn procedure as a post-hoc test.

To see the influence of preoperative panretinal photocoagulation on the expression of VEGF₁₆₅ and its receptors VEGF-R2 and neuropilin-1 in fibrovascular tissues, the total amount of the therapy was compared among groups I, II, and III. However, the difference was not statistically significant (P = 0.43, Table 2 ).

Other clinical factors including duration of diabetes and HbA1c were also compared among groups I, II, and III. However, no difference was shown in duration of diabetes (P = 0.69) or in HbA1c (P = 0.55, Table 2 ), and, thus, the vascular density was not confounded by these clinical factors or the preoperative laser therapy.

Tissue Localization of VEGF
In situ hybridization was performed to identify the VEGF-expressing cells in fibrovascular tissues. As shown in Figure 5A , VEGF mRNA was detected with the antisense probe mainly in the cells located at the margin of the tissues and frequently in the cluster-forming cells, whereas the sense probe gave only a background signal (Fig. 5B) . Immunohistochemistry for GFAP on the serial section revealed that the cells expressing VEGF mRNA were also positive for GFAP (Fig. 5C) , indicating that they are glial cells. No or negligible immunostaining was found with nonimmune antibodies (Fig. 5D) . GFAP-positive cells were found in all the fibrovascular tissues examined, although such cells in each tissue varied in number. VEGF protein was immunolocalized not only to the glial cells but also to the endothelial cells (Fig. 5E) . The positive immunostaining for VEGF was abolished with the antibodies absorbed with the antigen (Fig. 5F) .

View larger version (78K): [in this window] [in a new window]   Figure 5. In situ hybridization for VEGF and immunohistochemistry for GFAP and VEGF in the serial sections of the fibrovascular tissue from patient 17: VEGF165 (+), VEGF-R2 (+), neuropilin-1 (+). (A, B) In situ hybridization for VEGF. Note that the cells located at the margin of the tissue and the cluster-forming cells (arrows) are labeled with the antisense probe (A), whereas only a background signal is shown with the sense probe (B). (C) Immunohistochemistry for GFAP. Note that the cells expressing VEGF mRNA (arrows in A) are also positive for GFAP (arrows). (D) Negative control with nonimmune immunoglobulins. (E) Immunohistochemistry for VEGF. VEGF protein is immunolocalized not only to the GFAP-positive cells (large arrows) but also to the endothelial cells (small double arrows). (F) Immunostaining with the VEGF antibodies absorbed with the antigen. Methyl green counterstain for all. Scale bars, 100 µm.

	Discussion

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In the present study, the isoform VEGF₁₂₁ was constitutively expressed in the fibrovascular tissues, which varied in vascularity. Thus, this isoform may not be a molecule controlling the activity of fibrovascular proliferation. On the other hand, the expression of VEGF₁₆₅ was confined to some of the patients with highly vascularized tissue that coexpressed VEGF-R2 and neuropilin-1. It has been reported that VEGF₁₆₅ is a more potent endothelial cell mitogen than VEGF₁₂₁^{23 24} and that neuropilin-1 is an isoform-specific receptor that enhances the bioactivity of VEGF₁₆₅ through VEGF-R2.¹⁸ Taking these findings into consideration, it is conceivable that the expression of VEGF₁₆₅ in the fibrovascular tissue that expresses VEGF-R2 and neuropilin-1 facilitates angiogenesis by means of self-sufficient interaction between the ligand and the receptors inside the growing tissue. However, the present study did not show any difference in the vascular density between group I, VEGF₁₆₅ (+), VEGF-R2 (+), neuropilin-1 (+) and group II, VEGF₁₆₅ (-), VEGF-R2 (+), neuropilin-1 (+) (120.8 ± 73.4 versus 108.8 ± 53.0 vessels/mm²). VEGF-mediated angiogenesis is for the most part hypoxia-induced^{25 26} and ischemic retinal cells in diabetic retinopathy^{4 5} secrete the soluble forms of VEGF that can act remotely in the closed space of the eye, resulting in an intraocular pathologic angiogenic process such as preretinal fibrovascular proliferation and iris neovascularization. Thus, the fibrovascular tissue can readily receive VEGF₁₆₅ secreted from the ischemic retina once the tissue coexpresses VEGF-R2 and neuropilin-1. This idea supports our result showing that the fibrovascular tissues with the coexpression of VEGF-R2 and neuropilin-1 were highly vascularized despite the absence of VEGF₁₆₅ expression inside the tissue.

Our study showed that the isoform VEGF₁₆₅ was expressed inside the fibrovascular tissues of significantly younger patients (group I). We clinically encounter proliferative diabetic retinopathy, which takes a rapid course especially in young patients. This age distribution leads to the idea that the production of VEGF₁₆₅ inside the fibrovascular tissue with its receptors may contribute to rapid growth of the tissue in a self-proliferation system.

Panretinal photocoagulation is a well-established therapy to reduce angiogenesis in diabetic retinopathy. However, the present study showed that the amount of the therapy had no relationship to the expression of VEGF-R2, neuropilin-1, or VEGF₁₆₅ in the fibrovascular tissue. Thus, other mechanisms, such as the destruction of VEGF-producing cells in the retina, are suggested to mediate the effect of the therapy.

Glial cells are known to migrate from the retina to the preretinal tissues of various pathologic conditions such as idiopathic epiretinal membrane,²⁷ proliferative vitreoretinopathy,²⁸ and proliferative diabetic retinopathy²⁹ and are suggested to be involved in the pathogenesis. During normal development of retinal vasculature, VEGF-secreting glial cells in the retina play a key role in the formation of new vessels.^{30 31} Our present immunohistochemical and in situ hybridization studies demonstrated for the first time that glial cells inside the fibrovascular tissue express and produce VEGF. In addition, VEGF protein was immunolocalized to the endothelial cells in which VEGF mRNA expression was not detected by in situ hybridization. These suggest the possibility that VEGF secreted from glial cells reached its receptors in the endothelial cells. Furthermore, the isoform VEGF₁₆₅ expression inside the fibrovascular tissue of group I patients, which was demonstrated by our RT–PCR analysis, must be attributed to glial cells inside the tissue. In these young patients, VEGF₁₆₅ produced by glial cells inside the fibrovascular tissues could activate the isoform-specific signaling via VEGF-R2 and neuropilin-1 in endothelial cells. Thus, rapid progression of retinopathy in young diabetic patients may be explained by this glial cell–mediated self-proliferation mechanism. Further studies are necessary to substantiate this hypothesis.

	Acknowledgements

 
We thank Yuichirou Akiyama and Hitoshi Abe for their excellent technical assistance, Toru Takebayashi and Yuji Nishiwaki (Department of Preventive Medicine and Public Health, Keio University School of Medicine, Tokyo, Japan) for their advice on the statistical analyses, and Shingo Kato (Department of Microbiology, Keio University School of Medicine, Tokyo, Japan) for his assistance in RT–PCR analysis.

	Footnotes

 
Submitted for publication August 5, 1999; revised December 17, 1999; accepted January 11, 2000.

Commercial relationships policy: N.

Corresponding author: Eiji Ikeda, Department of Pathology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. eikeda{at}med.keio.ac.jp

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Adamis, AP, Miller, JW, Bernal, MT, et al (1994) Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy Am J Ophthalmol 118,445-450[Medline][Order article via Infotrieve]
2. Aiello, LP, Avery, RL, Arrigg, PG, et al (1994) Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders N Engl J Med 331,1480-1487[Abstract/Free Full Text]
3. Malecaze, F, Clamens, S, Simorre–Pinatel, V, et al (1994) Detection of vascular endothelial growth factor messenger RNA and vascular endothelial growth factor-like activity in proliferative diabetic retinopathy Arch Ophthalmol 112,1476-1482[Abstract]
4. Pe’er, J, Shweiki, D, Itin, A, Hemo, I, Gnessin, H, Keshet, E. (1995) Hypoxia-induced expression of vascular endothelial growth factor by retinal cells is a common factor in neovascularizing ocular diseases Lab Invest 72,638-645[Medline][Order article via Infotrieve]
5. Pe’er, J, Folberg, R, Itin, A, Gnessin, H, Hemo, I, Keshet, E. (1996) Upregulated expression of vascular endothelial growth factor in proliferative diabetic retinopathy Br J Ophthalmol 80,241-245[Abstract/Free Full Text]
6. Shinoda, K, Ishida, S, Kawashima, S, et al (1999) Comparison of the levels of hepatocyte growth factor and vascular endothelial growth factor in aqueous fluid and serum with grades of retinopathy in patients with diabetes mellitus Br J Ophthalmol 83,834-837[Abstract/Free Full Text]
7. Keck, PJ, Hauser, SD, Krivi, G, et al (1989) Vascular permeability factor, an endothelial cell mitogen related to PDGF Science 246,1309-1312[Abstract/Free Full Text]
8. Leung, DW, Cachianes, G, Kuang, WJ, Goeddel, DV, Ferrara, N. (1989) Vascular endothelial growth factor is a secreted angiogenic mitogen Science 246,1306-1309[Abstract/Free Full Text]
9. Breier, G, Albrecht, U, Sterrer, S, Risau, W. (1992) Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation Development 114,521-532[Abstract]
10. Millauer, B, Wizigmann–Voos, S, Schnurch, H, et al (1993) High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis Cell 72,835-846[Medline][Order article via Infotrieve]
11. Carmeliet, P, Ferreira, V, Breier, G, et al (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele Nature 380,435-439[Medline][Order article via Infotrieve]
12. Frank, S, Hubner, G, Breier, G, Longaker, MT, Greenhalgh, DG, Werner, S. (1995) Regulation of vascular endothelial growth factor expression in cultured keratinocytes: implications for normal and impaired wound healing J Biol Chem 270,12607-12613[Abstract/Free Full Text]
13. Plate, KH, Breier, G, Weich, HA, Risau, W. (1992) Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo Nature 359,845-848[Medline][Order article via Infotrieve]
14. Millauer, B, Shawver, LK, Plate, KH, Risau, W, Ullrich, A. (1994) Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant Nature 367,576-579[Medline][Order article via Infotrieve]
15. Shibuya, M, Seetharam, L, Ishii, Y, et al (1994) Possible involvement of VEGF-FLT tyrosine kinase receptor system in normal and tumor angiogenesis Princess Takamatsu Symp 24,162-170[Medline][Order article via Infotrieve]
16. Terman, BI, Carrion, ME, Kovacs, E, Rasmussen, BA, Eddy, RL, Shows, TB (1991) Identification of a new endothelial cell growth factor receptor tyrosine kinase Oncogene 6,1677-1683[Medline][Order article via Infotrieve]
17. Terman, BI, Dougher–Vermazen, M, Carrion, ME, et al (1992) Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor Biochem Biophys Res Commun 187,1579-1586[Medline][Order article via Infotrieve]
18. Soker, S, Takashima, S, Miao, HQ, Neufeld, G, Klagsbrun, M. (1998) Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor Cell 92,735-745[Medline][Order article via Infotrieve]
19. He, Z, Tessier–Lavigne, M. (1997) Neuropilin is a receptor for the axonal chemorepellent Semaphorin III Cell 90,739-751[Medline][Order article via Infotrieve]
20. Waltenberger, J, Claesson–Welsh, L, Siegbahn, A, Shibuya, M, Heldin, CH (1994) Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor J Biol Chem 269,26988-26995[Abstract/Free Full Text]
21. Shalaby, F, Rossant, J, Yamaguchi, TP, et al (1995) Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice Nature 376,62-66[Medline][Order article via Infotrieve]
22. Fong, GH, Rossant, J, Gertsenstein, M, Breitman, ML (1995) Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium Nature 376,66-70[Medline][Order article via Infotrieve]
23. Keyt, BA, Nguyen, HV, Berleau, LT, et al (1996) Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors: generation of receptor-selective VEGF variants by site-directed mutagenesis J Biol Chem 271,5638-5646[Abstract/Free Full Text]
24. Soker, S, Gollamudi–Payne, S, Fidder, H, Charmahelli, H, Klagsbrun, M. (1997) Inhibition of vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation by a peptide corresponding to the exon 7-encoded domain of VEGF165 J Biol Chem 272,31582-31588[Abstract/Free Full Text]
25. Shweiki, D, Itin, A, Soffer, D, Keshet, E. (1992) Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis Nature 359,843-845[Medline][Order article via Infotrieve]
26. Ikeda, E, Achen, MG, Breier, G, Risau, W. (1995) Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells J Biol Chem 270,19761-19766[Abstract/Free Full Text]
27. Smiddy, WE, Maguire, AM, Green, WR, et al (1989) Idiopathic epiretinal membranes: ultrastructural characteristics and clinicopathologic correlation Ophthalmology 96,811-821[Medline][Order article via Infotrieve]
28. Wiedemann, P, Weller, M, Heimann, K. (1990) Proliferative vitreoretinopathy: new discoveries in pathophysiology and therapy Klin Monatsbl Augenheilkd 197,355-361[Medline][Order article via Infotrieve]
29. Ohira, A, de Juan, E, Jr (1990) Characterization of glial involvement in proliferative diabetic retinopathy Ophthalmologica 201,187-195[Medline][Order article via Infotrieve]
30. Stone, J, Itin, A, Alon, T, et al (1995) Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia J Neurosci 15,4738-4747[Abstract]
31. Provis, JM, Leech, J, Diaz, CM, Penfold, PL, Stone, J, Keshet, E. (1997) Development of the human retinal vasculature: cellular relations and VEGF expression Exp Eye Res 65,555-568[Medline][Order article via Infotrieve]

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				T. Nakagawa, W. Sato, Y. Y. Sautin, O. Glushakova, B. Croker, M. A. Atkinson, C. C. Tisher, and R. J. Johnson Uncoupling of Vascular Endothelial Growth Factor with Nitric Oxide as a Mechanism for Diabetic Vasculopathy J. Am. Soc. Nephrol., March 1, 2006; 17(3): 736 - 745. [Abstract] [Full Text] [PDF]

				K. Noda, S. Ishida, H. Shinoda, T. Koto, T. Aoki, K. Tsubota, Y. Oguchi, Y. Okada, and E. Ikeda Hypoxia Induces the Expression of Membrane-Type 1 Matrix Metalloproteinase in Retinal Glial Cells Invest. Ophthalmol. Vis. Sci., October 1, 2005; 46(10): 3817 - 3824. [Abstract] [Full Text] [PDF]

				T. Gustafsson, H. Ameln, H. Fischer, C. J. Sundberg, J. A. Timmons, and E. Jansson VEGF-A splice variants and related receptor expression in human skeletal muscle following submaximal exercise J Appl Physiol, June 1, 2005; 98(6): 2137 - 2146. [Abstract] [Full Text] [PDF]

				S. Miyahara, J. Kiryu, K. Yamashiro, K. Miyamoto, F. Hirose, H. Tamura, H. Katsuta, K. Nishijima, A. Tsujikawa, and Y. Honda Simvastatin Inhibits Leukocyte Accumulation and Vascular Permeability in the Retinas of Rats with Streptozotocin-Induced Diabetes Am. J. Pathol., May 1, 2004; 164(5): 1697 - 1706. [Abstract] [Full Text] [PDF]

				T. Usui, S. Ishida, K. Yamashiro, Y. Kaji, V. Poulaki, J. Moore, T. Moore, S. Amano, Y. Horikawa, D. Dartt, et al. VEGF164(165) as the Pathological Isoform: Differential Leukocyte and Endothelial Responses through VEGFR1 and VEGFR2 Invest. Ophthalmol. Vis. Sci., February 1, 2004; 45(2): 368 - 374. [Abstract] [Full Text] [PDF]

				S. Ishida, T. Usui, K. Yamashiro, Y. Kaji, E. Ahmed, K. G. Carrasquillo, S. Amano, T. Hida, Y. Oguchi, and A. P. Adamis VEGF164 Is Proinflammatory in the Diabetic Retina Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 2155 - 2162. [Abstract] [Full Text] [PDF]

				K. Noda, S. Ishida, M. Inoue, K.-i. Obata, Y. Oguchi, Y. Okada, and E. Ikeda Production and Activation of Matrix Metalloproteinase-2 in Proliferative Diabetic Retinopathy Invest. Ophthalmol. Vis. Sci., May 1, 2003; 44(5): 2163 - 2170. [Abstract] [Full Text] [PDF]

				G. Hashimoto, I. Inoki, Y. Fujii, T. Aoki, E. Ikeda, and Y. Okada Matrix Metalloproteinases Cleave Connective Tissue Growth Factor and Reactivate Angiogenic Activity of Vascular Endothelial Growth Factor 165 J. Biol. Chem., September 20, 2002; 277(39): 36288 - 36295. [Abstract] [Full Text] [PDF]

				J. L. Yu, J. W. Rak, G. Klement, and R. S. Kerbel Vascular Endothelial Growth Factor Isoform Expression as a Determinant of Blood Vessel Patterning in Human Melanoma Xenografts Cancer Res., March 1, 2002; 62(6): 1838 - 1846. [Abstract] [Full Text] [PDF]

				A. M. Matthies, Q. E. H. Low, M. W. Lingen, and L. A. DiPietro Neuropilin-1 Participates in Wound Angiogenesis Am. J. Pathol., January 1, 2002; 160(1): 289 - 296. [Abstract] [Full Text] [PDF]

				L. E. Benjamin Glucose, VEGF-A, and Diabetic Complications Am. J. Pathol., April 1, 2001; 158(4): 1181 - 1184. [Full Text] [PDF]

				G. B. Whitaker, B. J. Limberg, and J. S. Rosenbaum Vascular Endothelial Growth Factor Receptor-2 and Neuropilin-1 Form a Receptor Complex That Is Responsible for the Differential Signaling Potency of VEGF165 and VEGF121 J. Biol. Chem., June 29, 2001; 276(27): 25520 - 25531. [Abstract] [Full Text] [PDF]

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