OBJECTIVE

To identify and isolate cDNAs for the alternatively spliced vascular endothelial growth factor (VEGF) mRNAs present in retina and to compare the relative levels of the splice variants and localization of VEGF mRNA in nonischemic and ischemic adult simian retinas.

METHODS

Retinas of cynomolgous monkeys were made ischemic by laser occlusion of the main branch retinal veins. Reverse transcription-polymerase chain reaction was used to amplify the VEGF coding region of RNA from ischemic and control retinas, and amplification products were analyzed by agarose gel electrophoresis, Southern blot, and nucleotide sequencing. Analysis of VEGF mRNA expression was accomplished by in situ hybridization.

RESULTS

Control and ischemic retinas produce mRNAs that correspond to the 121 and 165 amino acid diffusible isoforms of VEGF, and relatively low levels of VEGF189, the heparin-binding isoform. There was no significant difference in the levels of the alternatively spliced VEGF transcripts between control and ischemic retinas. Cloning and sequencing revealed that simVEGF cDNAs are 99% identical to human VEGFs and are predicted to encode proteins identical to their respective human homologues. In situ hybridization of nonischemic retinas revealed a low level of VEGF mRNA in retinal ganglion cells and in the inner nuclear layer. VEGF mRNA levels were elevated in ischemic retinas as early as 1 day after laser vein occlusion, when there was a small but reproducible increase in signal. The expression peaked at approximately 13 days, coincident with maximal iris neovascularization, and was significantly reduced by 28 days, when the iris vessels largely regressed.

CONCLUSIONS

The elevation of simVEGF121 and VEGF165 in ischemic retinas is consistent with a role for diffusible, retina-derived angiogenic factors in the development of ocular neovascularization.

OBJECTIVE

Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) play important roles during neurovascular repair after stroke. In this study, we imaged VEGFR expression with positron emission tomography (PET) to noninvasively analyze poststroke angiogenesis.

METHODS

Female Sprague-Dawley rats after distal middle cerebral artery occlusion surgery were subjected to weekly MRI, (18)F-FDG PET, and (64)Cu-DOTA-VEGF(121) PET scans. Several control experiments were performed to confirm the VEGFR specificity of (64)Cu-DOTA-VEGF(121) uptake in the stroke border zone. VEGFR, BrdU, lectin staining, and (125)I-VEGF(165) autoradiography on stroke brain tissue slices were performed to validate the in vivo findings.

RESULTS

T2-weighed MRI correlated with the "cold spot" on (18)F-FDG PET for rats undergoing distal middle cerebral artery occlusion surgery. The (64)Cu-DOTA-VEGF(121) uptake in the stroke border zone peaked at approximately 10 days after surgery, indicating neovascularization as confirmed by histology (VEGFR-2, BrdU, and lectin staining). VEGFR specificity of (64)Cu-DOTA-VEGF(121) uptake was confirmed by significantly lower uptake of (64)Cu-DOTA-VEGF(mutant) in vivo and intense (125)I-VEGF(165) uptake ex vivo in the stroke border zone. No appreciable uptake of (64)Cu-DOTA-VEGF(121) was observed in the brain of sham-operated rats.

CONCLUSIONS

For the first time to our knowledge, we successfully evaluated the VEGFR expression kinetics noninvasively in a rat stroke model. In vivo imaging of VEGFR expression could become a significant clinical tool to plan and monitor therapies aimed at improving poststroke angiogenesis.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and matrix metalloproteinases (MMP) regulate each other, contributing to tumor progression. We have previously reported that MMP9 induces the release of tumor VEGF, promoting ascites formation in human ovarian carcinoma xenografts. The aim of this study was to investigate whether tumor-derived VEGF regulated the expression of gelatinase by the stroma, influencing the invasive properties of ovarian tumors. Tumor variants derived from 1A9 human ovarian carcinoma, stably expressing VEGF(<em>121</em>) in the sense (1A9-VS-1) and antisense orientations (1A9-VAS-3), were used. In vivo, zymographic analysis of tumors from 1A9-VS-1 implanted in the peritoneal cavity of nude mice showed higher levels of gelatinases, particularly murine MMP9, indicating that VEGF stimulates host expression of the matrix-degrading enzyme. Murine MMP9 expression was also high in the ovaries of mice bearing 1A9-VS-1 tumors. The effect on host MMP9 activity was organ-specific. The levels of host pro-MMP9 in ovaries correlated with the plasma levels of tumor VEGF and with the selective invasion of the ovaries. Induction of host MMP9 expression in tumors and ovaries was independent of the site of tumor <em>growth</em> as it was seen in mice carrying both intraperitoneal and subcutaneous tumors. The anti-VEGF antibody bevacizumab (Avastin) inhibited MMP9 expression and tumor invasion in the ovaries of mice bearing 1A9-VS-1 tumors. These findings point to a complex cross-talk between VEGF and MMPs in the progression of ovarian tumor and suggest the possibility of using VEGF inhibitors to affect MMP-dependent tumor invasion.

Angiogenesis is a multistep complex phenomenon critical for several inflammatory and neoplastic disorders. Basophils, normally confined to peripheral blood, can infiltrate the sites of chronic inflammation. In an attempt to obtain insights into the mechanism(s) underlying human basophil chemotaxis and its role in inflammation, we have characterized the expression and function of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factors</em> (VEGFs) and their receptors in these cells. Basophils express mRNA for three isoforms of VEGF-A (<em>121</em>, 165, and 189) and two isoforms of VEGF-B (167 and 186). Peripheral blood and basophils in nasal polyps contain VEGF-A localized in secretory granules. The concentration of VEGF-A in basophils was 144.4 +/- 10.8 pg/10(6) cells. Immunologic activation of basophils induced the release of VEGF-A. VEGF-A (10-500 ng/ml) induced basophil chemotaxis. Supernatants of activated basophils induced an angiogenic response in the chick embryo chorioallantoic membrane that was inhibited by an anti-VEGF-A Ab. The tyrosine kinase VEGFR-2 (VEGFR-2/KDR) mRNA was expressed in basophils. These cells also expressed mRNA for the soluble form of VEGFR-1 and neuropilin (NRP)1 and NRP2. Flow cytometric analysis indicated that basophils express epitopes recognized by mAbs against the extracellular domains of VEGFR-2, NRP1, and NRP2. Our data suggest that basophils could play a role in angiogenesis and inflammation through the expression of several forms of VEGF and their receptors.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is well characterized for its role in <em>endothelial</em> cell differentiation and <em>vascular</em> tube formation. Alternate splicing of the VEGF gene in mice results in various VEGF-A isoforms, including VEGF-<em>121</em> and VEGF-165. VEGF-165 is the most abundant isoform in the kidney and has been implicated in glomerulogenesis. However, its role in the tubular epithelium is not known. We demonstrate that VEGF-165 but not VEGF-<em>121</em> induces single-cell branching morphogenesis and multicellular tubulogenesis in mouse renal tubular epithelial cells and that these morphogenic effects require activation of the phosphatidylinositol 3-kinase (PI 3-K) and, to a lesser degree, the extracellular signal-regulated kinase and protein kinase C signaling pathways. Further, VEGF-165-stimulated sheet migration is dependent only on PI 3-K signaling. These morphogenic effects of VEGF-165 require activation of both VEGF receptor 2 (VEGFR-2) and neuropilin-1 (Nrp-1), since neutralizing antibodies to either of these receptors or the addition of semaphorin 3A (which blocks VEGF-165 binding to Nrp-1) prevents the morphogenic response and the phosphorylation of VEGFR-2 along with the downstream signaling. We thus conclude that in addition to <em>endothelial</em> vasculogenesis, VEGF can induce renal epithelial cell morphogenesis in a Nrp-1-dependent fashion.

A comprehensive, biophysically accurate, computational model of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) family member interactions with <em>endothelial</em> cell surface receptors was developed to study angiogenesis. Neuropilin-1 (NRP1) and the signaling VEGF receptor, VEGFR2, do not interact directly but are bridged by one VEGF isoform, VEGF(165). Using the model and published experimental data, we estimated the kinetic rate of this VEGFR2-NRP1 coupling in vitro. With the use of this rate, our model gives predictions in good quantitative agreement with several independent in vitro experiments involving VEGF(<em>121</em>) and VEGF(165) isoforms, confirming that VEGFR2-NRP1 coupling through VEGF(165) can fully explain the observed differences in receptor binding and phosphorylation in response to these isoforms. Model predictions also determine the mechanism of action of a commonly used NRP1 antibody and predict the results of potential future experiments. This is the first model to include VEGF isoforms or NRPs, and it is a necessary step toward a quantitative molecular level description of VEGF that can be extended to in vivo situations. The model has applications for both proangiogenic and antiangiogenic therapies, such as for heart disease and cancer, as well as in tissue engineering.

OBJECTIVE

To summarize the 6-month follow-up of a cohort of patients with clinically significant coronary artery disease who received direct myocardial injection of an E1-E3- adenovirus (Ad) gene transfer vector (Ad(GV)VEGF<em>121</em>.10) expressing the human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) <em>121</em> cDNA to induce therapeutic angiogenesis.

BACKGROUND

Therapeutic angiogenesis describes a novel approach to the treatment of vascular occlusive disease that uses the administration of growth factors known to induce neovascularization of ischemic tissues.

METHODS

Direct myocardial injection of Ad(GV)VEGF<em>121</em>.10 into an area of reversible ischemia was carried out in 21 patients as an adjunct to conventional coronary artery bypass grafting (group A, n = 15) or as sole therapy using a minithoracotomy (group B, n = 6).

RESULTS

No evidence of systemic or cardiac-related adverse events related to vector administration was observed up to 6 months after therapy. Trends toward improvement in angina class and exercise treadmill testing at 6-month follow-up in the sole therapy group suggest the effects of this therapy are persistent for>> or =6 months.

CONCLUSIONS

This study suggests that direct myocardial administration of Ad(GV)VEGF<em>121</em>.10 appears to be well tolerated in patients with clinically significant coronary artery disease. Initiation of phase II evaluation of this therapy appears warranted.

OBJECTIVE

Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) signaling pathway plays pivotal roles in regulating tumor angiogenesis. Quantitative positron emission tomography (PET) imaging of VEGFR will facilitate the planning of whether, and when, to start anti-angiogenic treatment and enable more robust and effective monitoring of such treatment.

METHODS

VEGF(121) was conjugated with DOTA (1,4,7,10-tetra-azacylododecane N,N',N'',N'''-tetraacetic acid) and then labeled with (64)Cu for PET imaging of mice bearing different-sized human glioblastoma U87MG tumors (n = 15). Western blotting and immunofluorescence staining of tumor tissue was carried out to correlate with/validate the imaging results.

RESULTS

The specific activity of (64)Cu-DOTA-VEGF(121) was 3.2 GBq/mg. The uptake of (64)Cu-DOTA-VEGF(121) in the tumor peaked when the tumor size was about 100-250 mm(3). Both small and large tumors had lower tracer uptake indicating a narrow range of tumor size with high VEGFR-2 expression. All tumors had similarly low VEGFR-1 expression. Most importantly, the tumor uptake value obtained from PET imaging had good linear correlation with the relative tumor tissue VEGFR-2 expression as measured by Western blot, where r (2) equals 0.68 based on the PET uptake at 4 h post-injection. Histology of the frozen tumor tissue corroborates well with the imaging results.

CONCLUSIONS

The tumor uptake of (64)Cu-DOTA-VEGF(121) measured by small-animal PET imaging reflects tumor VEGFR-2 expression level in vivo. Such correlation may facilitate future treatment planning and treatment monitoring of cancer and potentially other angiogenesis-related diseases.

Angiogenesis, a prerequisite for tumor <em>growth</em> and progression, results from a shift in the equilibrium between angiogenic <em>factors</em> and angiogenic inhibitors. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) has been identified as one of the most important <em>factors</em> mediating angiogenesis in physiological and pathological conditions. Through alternative splicing, four isoforms of VEGF are formed, consisting of 206, 189, 165, and <em>121</em> amino acids, respectively. VEGF206 and VEGF189 differ from VEGF165 and VEGF<em>121</em> in their bioavailability, with the longer forms being matrix-bound and the shorter forms freely diffusible. To investigate the relative importance of the VEGF isoforms in neoplastic transformation, we studied the pattern of splice variant expression by reverse transcription polymerase chain reaction (RT-PCR) in 18 lung and 11 colonic carcinomas and their corresponding normal tissues, respectively. The findings showed a significant upregulation of VEGF in both carcinomas, with VEGF165 and VEGF<em>121</em> being the predominant forms; VEGF189 was significantly expressed in normal lung but not colon; and VEGF206 was not detected in any specimen. The findings indicate that during malignant progression, an angiogenic switch favoring the shorter diffusible isoforms is likely to confer on the tumor a <em>growth</em> advantage. From the differential expression of VEGF isoforms in normal lung and colonic tissues, different functional roles of the splice variants is suggested. In particular, VEGF189 may be important for the maintenance of the <em>vascular</em> integrity of the lung.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) plays an important role in both physiological and pathological angiogenesis. Our previous studies showed a differential role of VEGF isoforms in retinal physiological angiogenesis. We also demonstrated that non-selective inhibition of VEGF by bevacizumab had a beneficial effect on surgical outcome after glaucoma filtration surgery by reducing angiogenesis as well as fibrosis. However, the function of the VEGF isoforms in pathological angiogenesis and wound healing in the eye still remains unidentified. This study was designed to elucidate the differential roles of VEGF isoforms in scar formation after trabeculectomy. Furthermore, we also investigated whether pegaptanib (Macugen™, Pfizer), an aptamer which specifically blocks VEGF(165), could improve surgical outcome by reducing postoperative scarring. VEGF-R2 and neuropilin-1 (NRP-1) expression was analyzed in vitro by RT-PCR, and were found to be expressed at higher levels in human umbilical vein <em>endothelial</em> cells (HUVEC) as compared to Tenon fibroblasts (TF). The effect of the different VEGF isoforms (VEGF(<em>121</em>), VEGF(165) and VEGF(189)) and pegaptanib on cell proliferation was determined via WST-1 assay. <em>Endothelial</em> cell proliferation was stimulated after addition of VEGF(<em>121</em>) and VEGF(165), whereas VEGF(<em>121</em>) and VEGF(189) increased fibroblast <em>growth</em>. These effects on proliferation were associated with an activation of the ERK pathway, as revealed using the TransAM c-Myc assay. Inhibition of the ERK pathway, by PD98059 administration, significantly reduced VEGF isoform induced cell <em>growth</em>. A dose-dependent reduction of <em>endothelial</em> cell proliferation was observed after pegaptanib administration, while only the highest dose was able to inhibit fibroblast <em>growth</em>. Next, the in vivo effect of pegaptanib was investigated in a rabbit model of trabeculectomy. The surgical outcome was evaluated by performing clinical investigations (IOP, bleb area, height and survival), as well as histomorphometric analyses of angiogenesis (CD31), inflammation (CD45) and fibrosis (Sirius Red). A single postoperative application of pegaptanib had a beneficial impact on surgical outcome, mainly by reducing angiogenesis, but not inflammation or collagen deposition. Repeated injections slightly improved surgical outcome, but again solely by reducing angiogenesis. In summary, our results revealed that the VEGF isoforms play a differential role in ocular wound healing: VEGF(165) and VEGF(<em>121</em>) predominantly affect blood vessel <em>growth</em>, whereas VEGF(189) is rather involved in fibrosis, an important process in wound healing.

Tumor oxygen status is a reliable prognostic marker that impacts malignant progression and outcome of tumor therapy. However, tumor oxygenation is heterogeneous and cannot be sufficiently described by a single parameter. It is influenced by several <em>factors</em> including microvessel density (MVD), blood flow (BF), blood volume (BV), blood oxygen saturation, tissue pO(2), oxygen consumption rate, and hypoxic fraction. The goal of this investigation was to integrate these measurements to obtain a comprehensive profile of tumor oxygenation. Platelet/<em>endothelial</em> cell adhesion molecule immunohistochemistry, the recessed oxygen microelectrode, color and power Doppler ultrasound (DUS), and diffuse light spectroscopy (DLS) were used to measure tumor oxygen status using <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-transfected hyper<em>vascular</em> human melanoma xenografts and their nontransfected counterparts as a model. NIH1286 human melanoma cells were transfected with a retroviral vector +/- a 720-bp fragment of human VEGF(<em>121</em>). High VEGF-producing clones were selected by ELISA. Oxygen consumption rate was measured in NIH1286/VEGF+ [VEGF-transfected cells (VEGF+ cells)] and NIH1286/Vec cells [cells transfected with vector alone (Vec cells)] using a standard Clark oxygen electrode. Athymic nude 6-8-week-old mice received s.c. injection in the right flank with 5 x 10(6) VEGF+ or Vec cells. When tumors were 10-14 mm in maximum dimension, serum was analyzed for VEGF by ELISA. Cryopreserved tumor tissue sections were immunostained for platelet/<em>endothelial</em> cell adhesion molecule, and MVD measurements were made. Tumor-bearing mice were anesthetized, and pO(2) measurements were made using Eppendorf pO(2) histograph or the recessed oxygen microelectrode. Tumor BF and BV were measured by quantitative analysis of DUS images. DLS was used to measure tumor BF and blood oxygen saturation variation. VEGF+ cell supernatants had 15,500 pg/ml VEGF, and Vec cells had 10 pg/ml. VEGF+ and Vec cells had equivalent oxygen consumption rates. VEGF+ tumors had a faster <em>growth</em> rate than Vec tumors. Serum from VEGF+ tumor-bearing mice showed 4,211 pg/ml VEGF, whereas VEGF was undetectable in the serum of control mice. MVD values were 74 +/- 11 in VEGF+ tumors and 39 +/- 4 in control tumors at x200 magnification/0.95-mm(2) area. The median pO(2) values were 3.5-fold higher in VEGF+ tumors than in Vec tumors by the recessed oxygen microelectrode and 18-fold higher by Eppendorf pO(2) histograph. DUS showed a 3.3-fold higher mean BF and a 5.5-fold higher BV in VEGF+ tumors than in Vec tumors. DLS showed a 3.2-fold higher mean BF and 1.7-fold higher oxygen saturation in the hyper<em>vascular</em> tumors as compared with the control tumor type, consistent with increased BF and BV data by DUS. An integrated approach that yields a comprehensive and consistent profile of oxygen status in tumors could potentially provide critical information for prognosis and treatment.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) occurs in at least five different isoforms because of alternative splicing of the gene. To investigate the roles of different VEGF isoforms in tumor blood vessel formation and tumorigenicity, the three major isoforms (VEGF(<em>121</em>), VEGF(165), and VEGF(189)) were overexpressed in an early-stage human melanoma cell line (WM1341B), which is VEGF-negative and nontumorigenic in immunodeficient mice. Although overexpression of VEGF(<em>121</em>) and VEGF(165) resulted in aggressive tumor <em>growth</em>, WM1341B cells transfected with VEGF(189) remained nontumorigenic and dormant on injection. Although tumor <em>growth</em> rate depended on the level and not the isoform of VEGF expressed, striking isoform-specific differences in <em>vascular</em> patterning were associated with VEGF(<em>121</em>)- versus VEGF(165)-dependent tumorigenic conversion of human melanoma. Thus, tumors overexpressing VEGF(165) generated dense, highly heterogeneous vessel networks that were distinctly different from those of tumors expressing VEGF(<em>121</em>) (poorly <em>vascular</em>ized and necrotic). Paradoxically, although VEGF(165) expression appears to result in the most effective tumor perfusion, it is the expression of VEGF(<em>121</em>) that is observed during human malignant melanoma progression. Indeed, unbiased selection of spontaneously tumorigenic variants of WM1341B (by coinjection with Matrigel) led to predominant expression of the VEGF(<em>121</em>) isoform. The <em>vascular</em> patterning in these tumors (1341-P3N1, 1341-P3N2) resembled that of the VEGF(<em>121</em>)-transfected WM1341B tumors. These results suggest that, for reasons that remain to be elucidated, a "minimal" program of tumor <em>vascular</em>ization may be favored during melanoma progression.

Bronchial <em>vascular</em> remodeling is an important feature of the pathology of chronic asthma, but the responsible mechanisms and main sources of angiogenic <em>factors</em> are unclear. Here we report that human airway smooth muscle cells express <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)<em>121</em>, 165, 189, 206 splice variants and secrete VEGF protein constitutively. VEGF protein secretion was increased by the proinflammatory asthma mediator bradykinin through post-transcriptional mechanisms. Bradykinin-induced VEGF secretion was dependent on the B2 bradykinin receptor, activation of protein kinase C, and generation of endogenous prostanoids. This is the first report that bradykinin can increase VEGF secretion in any biological system and the first to show that airway smooth muscle cells produce VEGF. Our results suggest a novel role for human airway smooth muscle in contributing to bronchial mucosal angiogenesis in chronic asthma by secretion of VEGF and suggest a wider role for mesenchymal cell products in mediating angiogenesis in inflammatory and allergic diseases.

The principal extrarenal manifestation of autosomal dominant polycystic kidney disease (ADPKD) involves formation of liver cysts derived from intrahepatic bile ducts. Autocrine and paracrine <em>factors</em> secreted into the cyst would be positioned to modulate the rate of hepatic cyst <em>growth</em>. The aim of this study was to identify potential <em>growth</em> <em>factors</em> present in human ADPKD liver cyst fluid. Cytokine array and enzyme-linked immunosorbent assay analysis of human ADPKD liver cyst fluid detected epithelial neutrophil attractant 78, interleukin (IL)-6 (503 +/- <em>121</em> pg/mL); and IL-8 (4,488 +/- 355 pg/mL); and elevated levels of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> compared with non-ADPKD bile (849 +/- 144 pg/mL vs. 270 pg/mL maximum concentration). ADPKD liver cyst cell cultures also released IL-8 and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>, suggesting that cystic epithelial cells themselves are capable of secreting these <em>factors</em>. Western blotting of cultured cyst cells and immunostaining of intact cysts demonstrate that cysteine-X-cysteine receptor 2, an epithelial neutrophil attractant 78 and IL-8 receptor, is expressed at the apical domain of cyst lining epithelial cells. Suggesting the cystic epithelial cells may exist in hypoxic conditions, electron microscopy of the ADPKD liver cyst epithelium revealed morphological features similar to those observed in ischemic bile ducts. These features include elongation, altered structure, and diminished abundance of apical microvilli. In conclusion, IL-8, epithelial neutrophil attractant 78, IL-6, and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> may serve as autocrine and paracrine <em>factors</em> to direct errant <em>growth</em> of ADPKD liver cyst epithelia. Interruption of these signaling pathways may provide therapeutic targets for inhibiting liver cyst expansion.

Locally produced <em>growth</em> <em>factors</em> may have important modulatory roles in final ovarian follicular <em>growth</em>. The aim of this study was to investigate the possible participation of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and basic fibroblast <em>growth</em> <em>factor</em> (FGF2) in bovine follicles during final <em>growth</em>. Ovaries were collected from a slaughterhouse within 10-20 min after exsanguination. A classification of follicles into five groups (<0.5; >0.5-5; >5-20; >20-180; >180 ng/ml) was performed according to the follicular fluid (FF) oestradiol-17 beta content. For a better characterisation of classes the mRNA expressions of FSH receptor, LH receptor and aromatase cytochrome P450 in theca interna (TI) and granulosa cells (GC) were determined. Analysis of VEGF transcript by RT-PCR showed that GC and theca cells express predominantly the smallest isoforms (VEGF(<em>121</em>) and VEGF(165)). VEGF mRNA expression in both tissues (TI and GC) and VEGF protein concentration in total follicle tissue increased significantly (and correlated) with developmental stages of follicle <em>growth</em>. The expression of mRNA for VEGF receptor (VEGFR)-1 and VEGFR-2 was very weak in GC, without any regulatory change during final follicle <em>growth</em>. In contrast, TI showed strong expression of mRNA for both receptors in all follicle classes examined. VEGF protein concentrations in FF increased significantly and continuously to maximum levels in preovulatory follicles. As shown by immunohistochemistry, VEGF protein was clearly localised in TI and GC of preovulatory follicles. FGF2 and FGF receptor (FGFR) mRNA expression in TI increased significantly during final <em>growth</em> of follicles. In contrast, the FGF2 and FGFR mRNA expression in GC was very weak and without any regulatory change during follicle <em>growth</em>. Histological observation revealed that FGF2 protein was localised in theca tissue (cytoplasm of <em>endothelial</em> cells and pericytes) but not in GC. Our results suggest that VEGF and FGF families are involved in the proliferation of capillaries that accompanies the selection of the preovulatory follicle resulting in an increased supply of nutrients and precursors, and therefore supporting the <em>growth</em> of the dominant follicle.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) plays an essential role in angiogenesis in the <em>growth</em> plate and ultimately in regulating endochondral ossification. Since longitudinal bone <em>growth</em> is often disturbed in children who are treated with glucocorticoids, we investigated the effects of dexamethasone on VEGF expression by epiphyseal chondrocytes. Cells were cultured from tibial <em>growth</em> plates of neonatal piglets. Using Northern blotting and RT-PCR techniques, the chondrocyte-specific markers aggrecan, collagen II and CD-RAP were detected. Also the glucocorticoid receptor (GR) was expressed. VEGF protein secreted from these cells was examined by ELISA and Western immunoblotting. The VEGF(<em>121</em>) and VEGF(165) isoforms were detected in the supernatant. As determined by RT-PCR, all three major mRNA splice variants were produced, including the species encoding VEGF(189). Dexamethasone (100 nM) inhibited both protein and mRNA expression by approximately 45%. Hydrocortisone (cortisol) and prednisolone also inhibited VEGF secretion, but they were less active than dexamethasone. The inhibitory actions of dexamethasone were almost completely blocked by the GR antagonist Org34116, indicating that the GR mediates these actions. Degradation of the VEGF mRNA was not accelerated by dexamethasone. Therefore, a transcriptional mechanism seems likely. Downregulation of this important <em>growth</em> <em>factor</em> could lead to disruption of the normal invasion of blood vessels in the <em>growth</em> plate, which could contribute to disturbed endochondral ossification and <em>growth</em>.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a pleiotropic <em>factor</em> that regulates embryonal vasculogenesis, tumor angiogenesis and <em>vascular</em> permeability. Among eight differential isoforms, VEGF <em>121</em> mainly regulates <em>vascular</em> permeability, while VEGF 165 induces angiogenesis. Our previous studies have suggested that VEGF <em>121</em> and VEGF 165 are mainly detected in the lesions of psoriasis and atopic dermatitis, especially VEGF <em>121</em>. VEGF <em>121</em> is the most predominant isoform, which plays a major role in the increased <em>vascular</em> permeability in the aforementioned skin lesions. Thus, the differential expression of VEGF isoforms may be critical in determining either an angiogenic or a hyper-permeable state. However, the distinct VEGF signaling pathways that induce angiogenesis and <em>vascular</em> hyper-permeability in <em>endothelial</em> cells have never been demonstrated. To clarify the differential effects elicited by VEGF <em>121</em> and VEGF 165, we compared the biological responses and the signaling pathways activated by VEGF <em>121</em> and VEGF 165 in human umbilical vein <em>endothelial</em> cells (HUVEC). VEGF 165 significantly increased the level of phosphorylation in the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, whereas VEGF <em>121</em> had little to no effect. In contrast, VEGF <em>121</em> induced rapid activation of the Src pathway, while VEGF 165 showed a less pronounced and delayed activation of the Src pathway. Furthermore, the VEGF-induced hyper-permeability and cell proliferation of HUVEC were inhibited by a Src inhibitor (PP2) and a MEK inhibitor (PD98059), respectively. These results indicate that distinct signaling pathways confer different <em>vascular</em> responses to VEGF <em>121</em> and VEGF 165.

We previously reported that <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) increases <em>vascular</em> permeability through the synthesis of <em>endothelial</em> platelet-activating <em>factor</em> (PAF), while others reported the contribution of nitric oxide (NO). Herein, we addressed the contribution of VEGF receptors and the role played by PAF and NO in VEGF-induced plasma protein extravasation. Using a modified Miles assay, intradermal injection in mice ears of VEGF-A(165), VEGF-A(<em>121</em>), and VEGF-C (1 microM) which activate VEGFR-2 (Flk-1) receptor increased <em>vascular</em> permeability, whereas a treatment with VEGFR-1 (Flt-1) analogs; PlGF and VEGF-B (1 microM) had no such effect. Pretreatment of mice with PAF receptor antagonist (LAU8080) or <em>endothelial</em> nitric oxide synthase (eNOS) inhibitor (L-NAME) abrogated protein extravasation mediated by VEGF-A(165). As opposed to PAF (0.01-1 microM), treatment with acetylcholine (ACh; up to 100 microM; inducer of NO synthesis) or sodium nitroprusside (SNP; up to 1 microM; NO donor) did not induce protein leakage. Simultaneous pretreatment of mice with eNOS and protein kinase A (PKA) inhibitors restored VEGF-A(165) <em>vascular</em> hyperpermeability suggesting that endogenous NO synthesis leads to PKA inhibition, which support maintenance of <em>vascular</em> integrity. Our data demonstrate that VEGF analogs increase <em>vascular</em> permeability through VEGFR-2 activation, and that both endogenous PAF and NO synthesis contribute to VEGF-A(165)-mediated <em>vascular</em> permeability. However, PAF but not NO directly increases <em>vascular</em> permeability per se, thereby, suggesting that PAF is a direct inflammatory mediator, whereas NO serves as a co<em>factor</em> in VEGF-A(165) proinflammatory activities.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is one of the key regulators of tumor neoangiogenesis. It acts through two types of high-affinity tyrosine kinase receptors (VEGF receptor-1 [VEGFR-1]/fms-related tyrosine kinase 1 [Flt-1] and VEGFR-2/kinase domain receptor [KDR]) expressed on <em>endothelial</em> cells. VEGFRs have also been detected on cancer cells, suggesting a possible autocrine effect of VEGF on their <em>growth</em>. We studied the expression of VEGF, VEGFR-1, and VEGFR-2 in human medulloblastoma cell lines (DAOY, D283Med, and D341Med) and investigated the possible autocrine mechanisms of VEGF on medulloblastoma cell proliferation. Reverse transcriptase PCR analysis showed the presence of VEGF and VEGFR mRNAs in all cell lines studied. Of the three VEGF isoforms, VEGF(<em>121</em>) and VEGF(189) were detected by Western blot analysis in all three medulloblastoma cell lines, whereas VEGF(165) was identified only in DAOY cells. Medulloblastoma cell lines expressed both VEGFR-1 and VEGFR-2. We also demonstrated expression of VEGF and its receptors in medulloblastoma tumor specimens. Exogenous VEGFR-2 inhibitor reduced the VEGF-dependent cell proliferation of DAOY and D283Med cells. In DAOY cells, VEGF(165) induced phosphorylation of VEGFR-2/KDR and of downstream proteins in the signal transduction pathway. These data suggest a possible autocrine role for VEGF in medulloblastoma <em>growth</em>. Targeting VEGF signaling may represent a new therapeutic option in the treatment of medulloblastoma.

We hypothesized that respiratory syncytial virus (RSV)-induced pathologies could be mediated, in part, by <em>vascular</em> active cytokines elaborated during virus infection. To address this hypothesis, we determined whether RSV stimulated <em>vascular</em> <em>endothelial</em> cell <em>growth</em> <em>factor</em> (VEGF)/<em>vascular</em> permeability <em>factor</em> (VPF) elaboration in vitro. Supernatants from unstimulated A549 cells and normal human bronchial epithelial cells contained modest levels of VEGF. In contrast, supernatants from RSV-infected cells contained elevated levels of VEGF/VPF. This stimulation was seen after as little as 2 h, was still prominent after 48 h, and, by immunoblot, was specific for the 165- and <em>121</em>-amino acid isoforms of VEGF/VPF. It was not associated with significant cell cytotoxicity or alterations in VEGF messenger RNA. It did, however, require new protein biosynthesis. In accordance with these findings, the 165- and <em>121</em>-amino acid isoforms of VEGF/VPF were also found in the nasal washings from patients with RSV infections. These studies demonstrate that RSV is a potent stimulator of VEGF/VPF elaboration and that, in vitro, this stimulation is mediated via a noncytotoxic translational and/or post-translational biosynthetic mechanism. VEGF/VPF may play an important role in the pathogenesis of RSV-induced disorders.

BACKGROUND

Adenovirus (Ad) vector-mediated gene therapy strategies have emerged as promising modalities for the "biological re<em>vascular</em>ization" of tissues. We hypothesized that direct intramyocardial, as opposed to intracoronary, administration of an Ad vector coding for the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> cDNA (Ad(GV)VEGF<em>121</em>.10) would provide highly focal Ad genome levels, and increases in VEGF, ideal for inducing localized therapeutic angiogenesis.

METHODS

Persistence and regional distribution of the vector were assessed by TaqMan real-time quantitative polymerase chain reaction technology and enzyme-linked immunosorbent assay, after intramyocardial Ad(GV)VEGF<em>121</em>.10 in the rat, and either intramyocardial or intracoronary (circumflex territory) vector in Yorkshire swine. Based on these results, we assessed the focal nature of the improved cardiac blood flow in a previously reported porcine myocardial ischemia model.

RESULTS

Intramyocardial delivery of Ad(GV)VEGF<em>121</em>.10 in the rat resulted in local persistence of the Ad genome that decreased 1,000-fold over 3 weeks, with peak myocardial VEGF expression 24 to 72 h after vector delivery. After intramyocardial Ad(GV)VEGF<em>121</em>.10 in the circumflex distribution of pigs, Ad vector genome and VEGF protein levels were more than 1,000-fold and more than 90-fold higher, respectively, in this distribution than in other myocardial regions. In comparison, intracoronary injection yielded maximum myocardial Ad genome and VEGF levels 33-fold and 9-fold lower, respectively, than that after intramyocardial delivery. Angiograms obtained 28 days after intramyocardial Ad(GV)VEGF<em>121</em>.10 demonstrated rapid circumflex reconstitution via collaterals localized to the region of vector administration.

CONCLUSIONS

These studies demonstrate that direct intramyocardial administration of Ad(GV)VEGF<em>121</em>.10 results in focal genome and VEGF levels, including focal angiogenesis, sufficient to normalize blood flow to the ischemic myocardium, findings that are relevant to designing human trials of gene therapy-mediated cardiac angiogenesis.

Angiogenesis is the formation of new blood vessels from the existing vasculature. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is an endothelium-specific angiogenic <em>factor</em> strongly implicated in pathological angiogenesis. In this study, the mRNA and protein expression of the four alternatively spliced VEGF isoforms (<em>121</em>, 165, 189 and 206 amino acids) were examined in normal and malignant breast tissues. Three VEGF transcripts were detected in both (<em>121</em>)165>189), whereas only VEGF165 protein was detected. The tumours expressed more VEGF mRNA (P = 0.02) and protein (P < 0.0001), with eight-fold more VEGF protein generated per mRNA unit (P = 0.009). To examine this further, the expression of eIF-4E, a translation initiation <em>factor</em>, was examined. Increased eIF-4E mRNA levels were detected in the tumours (P < 0.0001) that correlated with VEGF mRNA (P = 0.0002), implying co-regulation of these genes. VEGF mRNA expression was elevated in tumours expressing the epidermal <em>growth</em> <em>factor</em> receptor (P < 0.01), but there was no difference according to oestrogen receptor status (P = 0.9), node status (P = 0.09) or between differing histologies (P = 0.4). These data suggest that elevated VEGF protein expression, by both enhanced transcription and translation, is a potential means by which tumour angiogenesis is induced in breast carcinomas. VEGF expression is also significantly associated with <em>factors</em> correlating with a poor outcome, implying a role in progression of this disease.

A key mechanism underlying physiological angiogenesis of the human endometrium is its ability to regenerate the <em>vascular</em> capillary network and to perform <em>vascular</em> remodeling (i.e., development of spiral arteries). <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is associated with angiogenesis and capillary permeability in this tissue. VEGF is expressed as several spliced variants, its main human isoforms contain <em>121</em> and 165 aa; 17beta-estradiol (E(2)) increases endometrial VEGF, possibly in all isoforms. Here we show that progesterone (P) selectively increases the expression of the VEGF(189) (V(189)) isoform in the human uterus. V(189) is identified in the conditioned medium of stromal cells treated with E(2) + P; its presence in this in vitro model of decidual stromal cells is detected after 6-8 days, using ELISA, and after 8-10 days, using Western blot analysis with different antibodies, including one specific for V(189). The secretion pattern of V(189) parallels that of the decidual protein IGFBP-1. V(189) is secreted as a native isoform, as compared with the migration of recombinant V(189) by SDS/PAGE. In situ hybridization and immunocytochemistry(,) performed on the same biopsies, suggest that decidual cells express V(189) during the mid-late secretory phase of the menstrual cycle and early gestation. Finally, using an in vivo permeability assay, we show that native V(189) increases capillary permeability. These observations demonstrate that P regulates V(189) expression in decidual cells, which could have important implications for understanding uterine <em>vascular</em> remodeling and implantation, and may be relevant in a range of disease states such as edema and irregular bleeding.

OBJECTIVE

Vascular endothelial growth factor (VEGF) is a cytokine participating in inflammation with potent endothelial cell effects. It is produced by macrophages, neutrophils and vascular endothelial cells and can alter vessel permeability. Behçet's syndrome is a systemic inflammatory disorder with unknown etiology. Vascular endothelial dysfunction is one of the prominent features of the disease. We previously demonstrated the possible involvement of proinflammatory cytokines [tumor necrosis factor (TNF)-alpha, soluble interleukin-2 receptor (sIL-2R), interleukin (IL)-6 and IL-8], nitric oxide (NO) and adrenomedullin in the etiopathogenesis of Behçet's syndrome. Since VEGF expression is induced by these cytokines and VEGF itself is a potent stimulator of NO production with endothelial cell effects, this study aimed to investigate whether VEGF was affected during the course of Behçet's syndrome. We also assessed the possible involvement of VEGF in ocular Behçet's syndrome or in disease activity.

METHODS

This multicenter case-control study included a total of 39 patients with active (n = 22) or inactive (n = 17) Behçet's syndrome (mean age, 38.1 +/- 10.4 years; 21 men and 18 women) satisfying International Study Group criteria, and 15 healthy hospital-based control volunteers (mean age, 39.2 +/- 9.3 years; eight men and seven women) matched for age and gender from a similar ethnic background. Patients were examined by a dermatologist and an ophthalmologist with an interest in Behçet's syndrome. Plasma VEGF concentrations were measured using a newly established enzyme-linked immunosorbent assay. Clinical findings and acute-phase reactant parameters such as erythrocyte sedimentation rate, alpha1-antitrypsin, alpha2-macroglobulin, and neutrophil count were used to classify the disease in Behçet's patients as active or inactive. The Wilcoxon test or the Mann-Whitney U-test was used for statistical analysis as indicated and the results were expressed as mean +/- SD, with range.

RESULTS

The mean plasma VEGF level in patients with Behçet's syndrome (291.9 +/- 97.1 pg/mL; range 121-532 pg/mL) was higher than that in control subjects (103.0 +/- 43.6 pg/mL; range 25-187 pg/mL) and the difference was significant (P < 0.001). Patients with active disease had significantly (P < 0.001) higher VEGF levels than patients with inactive disease (347.6 +/- 87.1 vs. 219.9 +/- 51.6 pg/mL). In addition, ocular Behçet's patients (n = 23) had higher VEGF levels (315.7 +/- 92.1 pg/mL) than nonocular patients (n = 16, 257.8 +/- 96.6 pg/mL) and the difference was of borderline significance (P = 0.041). The levels of all acute-phase reactant parameters were significantly higher in the active stage than in the inactive stage (for each, P < 0.01) or in control subjects (for each, P < 0.001).

CONCLUSIONS

VEGF may participate in the course of Behçet's syndrome, especially in the active stage, and elevated levels of VEGF may be an additional risk factor for the development of ocular disease, contributing to poor visual outcome.