<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGF) is one of the major regulators of angiogenesis. It plays an important role during the process of physiological and pathological neo<em>vascular</em>ization. Variant VEGF isoforms are generated as a result of alternative pre-mRNA splicing. To determine the expression of VEGF isoforms in angioma and head and neck squamous cell carcinoma (HNSCC), both being dependent on pathological neo<em>vascular</em>ization, we included 11 HNSCC cell lines, 4 hemangiomas and 5 <em>vascular</em> malformations (VMs) in the study. Tonsil mucosa served as normal control. Using reverse transcription polymerase chain reaction (RT-PCR) sequencing, the VEGF isoforms VEGF(189), VEGF(165) and VEGF(<em>121</em>) were regularly detected in all tested samples. VEGF(<em>121</em>) was the most abundant isoform in all tested tissues, whereas VEGF(165) exhibited lower levels, and VEGF(189) only very small amounts of transcript. Interestingly, VMs expressed significantly higher (p=0.0286) amounts of VEGF(<em>121</em>) compared with hemangiomas, which had levels similar to normal control mucosa. HNSCC cell lines demonstrated on-average higher levels of all three isoforms compared with the controls. Consistent with the clinical staging, a trend for VEGF overexpression was observed in tumor cells derived from N+ tumors compared to those derived from N0 tumors. One drawback of this study was the small number of specimens available, particularly since VMs and hemangiomas are relatively rare diseases. Future studies need to follow-up on these observations and further evaluate the potential role of specific VEGF isoforms in the pathogenesis of hemangioma and VM.

BACKGROUND

Perioperative high-dose oxygen (O2 ) exposure can cause hyperoxia. While the effect of constant hyperoxia on the vascular endothelium has been investigated to some extent, the impact of cyclic hyperoxia largely remains unknown. We hypothesized that cyclic hyperoxia would induce more injury than constant hyperoxia to human umbilical vein endothelial cells (HUVECs).

METHODS

HUVECs were exposed to cyclic hyperoxia (5-95% O2 ) or constant hyperoxia (95% O2 ), normoxia (21% O2 ), and hypoxia (5% O2 ). Cell growth, viability (Annexin V/propidium iodide and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) lactate dehydrogenase (LDH), release, cytokine (interleukin, IL and macrophage migration inhibitory factor, MIF) release, total antioxidant capacity (TAC), and superoxide dismutase activity (SOD) of cell lysate were assessed at baseline and 8, 24, and 72 h. A signal transduction pathway finder array for gene expression analysis was performed after 8 h.

RESULTS

Constant and cyclic hyperoxia-induced gradually detrimental effects on HUVECs. After 72 h, constant or cyclic hyperoxia exposure induced change in cytotoxic (LDH +12%, P = 0.026; apoptosis +121/61%, P < 0.01; alive cells -15%, P < 0.01; MTT -16/15%, P < 0.01), inflammatory (IL-6 +142/190%, P < 0.01; IL-8 +72/43%, P < 0.01; MIF +147/93%, P < 0.01), or redox-sensitive (SOD +278%, TAC-25% P < 0.01) markers. Gene expression analysis revealed that constant and cyclic hyperoxia exposure differently activates oxidative stress, nuclear factor kappa B, Notch, and peroxisome proliferator-activated receptor pathways.

CONCLUSIONS

Extreme hyperoxia exposure induces inflammation, apoptosis and cell death in HUVECs. Although our findings cannot be transferred to clinical settings, results suggest that hyperoxia exposure may cause vascular injury that could play a role in determining perioperative outcome.

Chronic decubital lesions have a limited potential to heal. Evidence suggests that a lack of local re<em>vascular</em>ization is involved in this aetiology. The present study investigated the expression of one of the most important angiogenic <em>factors</em>, the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), in different regions of sacral chronic decubital lesions and in normal skin by immunohistochemical, biochemical, molecular, and cell biology methods. To elucidate some of the <em>factors</em> responsible for the induction of VEGF in chronic skin ulcers, cultured fibroblasts were exposed to hypoxia and/or <em>growth</em> <em>factors</em>. In the central part (zone I) of chronic ulcers and in normal skin, immunostaining for VEGF remained largely negative. However, VEGF could be immunostained in cells in the granulation tissue adjacent to central necrosis (zone II). VEGF receptor 2 (VEGFR-2; KDR) could also be identified in microvessels. High VEGF levels were present in homogenates from granulation tissue by enzyme-linked immunosorbent assay (ELISA) and western blot experiments: low concentrations were found in areas of central necrosis and negligible amounts were present in normal skin. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that only the splice variants VEGF(<em>121</em>) and VEGF(165) were expressed. In cultured fibroblasts, hypoxia or platelet-derived <em>growth</em> <em>factor</em> (PDGF) raised VEGF production. The angiogenic peptide VEGF is present in all zones of chronic decubital ulcers. Its strong expression within the adjacent granulation tissue supports the view that there is no deficiency of VEGF. VEGF may be involved in the healing process of chronic skin lesions, but it seems that loss of another <em>factor</em> may be responsible for the poor healing response.

OBJECTIVE

The purpose of this study was to investigate the effects of gene transfection of endothelial cells with vascular endothelial growth factor (VEGF) on re-endothelialization and inhibiting in-stent restenosis.

METHODS

Stents coated with human umbilical vein endothelial cells (HUVECs) transfected with VEGF(121) were studied both in vitro and in vivo. In vitro studies were performed using a homemade extracorporeal circulation system. In vivo studies were performed using the rabbit abdominal aorta model.

RESULTS

In vitro studies confirmed that VEGF(121)-transfected cells adhered on the surface of stainless steel stents with over 90% of the surface covered within 24 hours of seeding. In vivo results showed that VEGF(121)-transfected HUVECs-coated stents were covered with seeding cells after implanting, and almost completely covered with cells after stent implantation for 1 week. In contrast, the non-endothelialized areas of bare metal stents and glutin/poly-L-lysine-coated stents were covered at 4 weeks, and the monolayers of cells were not observed, but fragile neointima was found on the surface. After 12 weeks, VEGF(121)-transfected HUVECs-coated stents significantly reduced the neointima area (0.78 ± 0.03 mm(2)) and stenosis (15.69 ± 2.61%) as compared with those for bare metal stents (neointima area = 2.26 ± 0.67 mm(2); the percentage of stenosis = 47.55 ± 7.10%;P < .01) and glutin/poly-L-lysine-coated stents (neointima area = 1.40 ± 0.37 mm(2); the percentage of stenosis = 31.37 ± 8.18%;P < .01).

CONCLUSIONS

In this small animal study, VEGF transfected human endothelial cells, when coated on stainless steel stents, reduce neointimal hyperplasia, promote endothelialization, and reduce in-stent restenosis. Additional studies with this technology are necessary to determine its ultimate utility in improving stents performance.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>/<em>vascular</em> permeability <em>factor</em> (VEGF/VPF) is a multifunctional cytokine that is expressed as four isoforms having 206, 189, 165, and <em>121</em> amino acids in humans. We constructed a system that produces the shortest isoform of human VEGF/VPF in Saccharomyces cerevisiae (yVEGF/VPF<em>121</em>). Active yVEGF/VPF<em>121</em> was secreted from the yeast cells as a glycosylated dimeric protein. Various biological activities of the purified yVEGF/VPF<em>121</em> were examined. It bound to cell surface receptor(s) and stimulated the <em>growth</em> of human umbilical vein <em>endothelial</em> cells in culture at a dose similar to that of native VEGF/VPF. Purified yVEGF/VPF<em>121</em> also induced angiogenesis in mice, and promoted the extravasation of plasma proteins from the blood vessels. These observations demonstrated that the shortest isoform of VEGF/VPF with an amino acid sequence of <em>121</em> residues contains enough information necessary to trigger both angiogenesis and the induction of <em>vascular</em> permeability upon binding to its cognate receptor(s).

BACKGROUND

Angiogenic gene therapy has been demonstrated to enhance perfusion to ischemic tissues, but it is unknown whether the administration of angiogenic <em>growth</em> <em>factors</em> will increase blood flow to nonischemic tissues. This study investigates whether enhanced myocardial perfusion can be mediated by adenovirus-mediated transfer of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> cDNA to nonischemic myocardium.

METHODS

New Zealand White rabbits received adenovirus (5 x 10(10) particle units) encoding for <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (n = 14) or a control vector without a transgene (n = 13) or saline solution (n = 9) via direct myocardial injection. Fluorescent microsphere perfusion studies and histologic analyses were performed 4 weeks later. In a parallel study, exercise treadmill testing was performed to assess the functional effects of this therapy in Sprague-Dawley rats.

RESULTS

Microsphere assessment of myocardial perfusion in rabbits 4 weeks after adenovirus-encoding vascular endothelial growth factor administration was greater than that for rats injected with control vector without a transgene or saline solution (3.2 +/- 0.5 vs 2.7 +/- 0.7 and 2.4 +/- 0.4, respectively; P <.03). The endothelial cell count per high power field was increased in animals injected with adenovirus-encoding vascular endothelial growth factor versus animals injected with control vector without a transgene or saline solution (147 +/- 27 vs 123 +/- 14 and 125 +/- 16 cells, respectively), although this did not reach statistical significance (P =.12). Rats treated with adenovirus-encoding vascular endothelial growth factor also demonstrated prolonged exercise tolerance compared with rats injected with control vector without a transgene or saline solution (exhaustion time: 26 +/- 5 minutes vs 19 +/- 2 minutes and 20 +/- 3 minutes, respectively; P =.006).

CONCLUSIONS

Adenovirus encoding-mediated transfer of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> induces an enhancement in regional perfusion in nonischemic myocardium that corresponds to changes in exercise tolerance. Adenovirus-encoding <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> therapy may be useful for inducing angiogenesis in the nonischemic state, such as for prophylactic therapy of early coronary artery disease.

Chronic unilateral ureteral obstruction (UUO) in the neonatal rat causes delayed renal maturation, tubular apoptosis, and interstitial inflammation. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) acts as a survival <em>factor</em> for tubular cells and reduces renal injury in several models of renal disease. To determine whether exogenous VEGF attenuates renal injury from UUO, rats were subjected within the first 48 h of life to sham operation, partial UUO, or complete UUO. Saline vehicle or VEGF(<em>121</em>) (50 mg/kg) was injected twice daily for 7 days, after which kidneys were harvested for histological study. The density of peritubular capillaries was measured with platelet-<em>endothelial</em> cell adhesion molecule-1 immunostaining, proliferating nuclei were detected by proliferating-cell nuclear antigen staining, apoptosis by the transferase-mediated dUTP nick end-labeling technique, macrophages by ED-1 immunostaining, and collagen by Sirius red staining. Glomerular number and maturation index were also determined in each group. Following chronic complete UUO in the neonatal rat, peritubular capillary density was significantly decreased. Cortical capillary density was further reduced by exogenous VEGF in the partially obstructed kidney. While UUO also decreased glomerular number and delayed glomerular maturation, exogenous VEGF exerted no additional effects. Cellular proliferation and tubular apoptosis increased in proportion to the severity of obstruction, but exogenous VEGF had no additional effects on proliferation, tubular apoptosis, or macrophage infiltration. However, VEGF reduced interstitial apoptosis in the kidney with partial UUO. We conclude that VEGF does not have salutary effects on the renal lesions caused by chronic UUO in the neonatal rat and may actually worsen obstructive nephropathy by aggravating the interstitial lesions.

With widespread applications in biosensors, diagnostics and therapeutics, unraveling the mechanism of the interactions between aptamers and their targets has become extremely important. In this study, the interaction forces between an aptamer and its protein target were successfully measured via AFM-based force spectroscopy at the molecular level. The angiogenic protein, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF(165)) covalently tethered to a mixed self-assembled monolayer surface and an anti-VEGF(165) aptamer attached to an AFM cantilever was used to probe this interaction. By comparing the binding probability and the force distribution of this system in a series of experiments, the interaction between the aptamer and the protein was confirmed and the effect of loading rate on the rupture force was studied. The specificity of the aptamer was investigated by measuring interactions with VEGF(<em>121</em>), an isoform of VEGF missing a critical binding domain, and VEGF(165) isoform blocked with heparin. The lower frequency of binding events compared with that of VEGF(165) reflected that despite a high affinity to its preferential target, the selectivity of this aptamer is impaired to some extent due to the flexible structure of aptamers. By changing the concentration of Mg(2+) ion in the binding buffer, we could verify the effect of metal ions as stabilizers of aptamer conformation. The results provide evidence at the molecular level that the structural stability of aptamer is closely related to higher binding force and that rigid aptamer tertiary structures require higher forces to unbind the aptamer/protein complex.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) plays a pivotal role in tumor progression via angiogenesis. Recently, gene transduction of wild-type p16INK4A, tumor suppressor gene, has been shown to result in downregulation of VEGF expression in p16INK4A-deleted glioma cells. Because expression of p16INK4A is regulated by methylation of the p16INK4A gene, we examined whether demethylation of the p16INK4A gene by 5-aza-2'-deoxycytidine (5-azadC) could cause the protein expression of VEGF as well as of p16INK4A in human lung cancer cells. For this, five different lung cancer cell lines with or without loss of p16 activity were used. H841 and Ma-10 cells had the methylated p16INK4A gene without expression of p16INK4A protein, whereas Ma-1 and H209 cells had the unmethylated p16INK4A gene with constitutive expression of p16INK4A protein. Neither the p16INK4A gene nor p16INK4A protein was detected in A549 cells. Treatment with 5-azadC caused demethylation of the p16INK4A gene with reexpression of p16INK4A protein in H841 and Ma-10 (methylated p16INK4A gene dominant) cell, but not in other cell lines such as Ma-1, H209 (unmethylated p16INK4A gene dominant), or A549 (p16INK4A gene deleted). In a parallel experiment, 5-azadC inhibited production of VEGF protein by H841 and Ma-10 cells, especially in the later hypermethylated cells, but not Ma-1, H209, or A549 cells. RT-PCR analysis showed that Ma-10 cells expressed VEGF isoforms <em>121</em>, 165, and 189, all of which were inhibited by 5-azadC. These findings indicate that the methylation status of the p16INK4A gene plays an important role in the regulation of angiogenesis associated with progression of lung cancer, through regulation of VEGF expression.

OBJECTIVE

Increased endothelin-1 (ET-1) is a hallmark of pulmonary arterial hypertension (PAH), and contributes to its pathogenesis. The factors controlling ET-1 in PAH are poorly understood. Combined with other stimuli, vascular endothelial growth factor (VEGF) blockade results in PAH-like lesions in animal models, and has been associated with PAH in humans. The effects of VEGF on ET-1 production by human lung blood microvascular endothelial cells (HMVEC-LBl) are unknown.

METHODS

We exposed HMVEC-LBl in-vitro to human VEGF-121 (40 ng/mL) in serum-free medium for 7h, in the absence or presence of the VEGF receptor antagonist, SU5416 (3 and 10 μM). ET-1 production was measured in the supernatant. Phosphorylation of VEGF receptor 2 (VEGFR2) was measured by Western blotting after exposure to VEGF without or with SU5416 for 5 and 10 min.

RESULTS

VEGF effectively caused VEGFR2 phosphorylation, which was blocked by SU5416. VEGF decreased ET-1 production by at least 29%. In the absence of VEGF, SU5416 increased ET-1 production, by 16% at 10 μM, and SU5416 was able to completely abolish the VEGF effect on ET-1 production.

CONCLUSIONS

VEGF may promote vascular health by decreasing ET-1 production in HVMEC-LBl. Blockade of VEGF signaling by SU5416 increases ET-1 levels. The role of VEGF in modulating endothelin production in PAH deserves further study.

Longitudinal bone <em>growth</em> is regulated in the <em>growth</em> plate. At the end of puberty, <em>growth</em> velocity diminishes and eventually ceases with the fusion of the <em>growth</em> plate through mechanisms that are not yet completely understood. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) has an important role in angiogenesis, but also in chondrocyte differentiation, chondrocyte survival, and the final stages of endochondral ossification. Estrogens have been shown to up-regulate VEGF expression in the uterus and bone of rats. In this study, we investigated the relation between estrogens and VEGF production in <em>growth</em> plate chondrocytes both in vivo and in vitro. The expression of VEGF protein was down-regulated upon ovariectomy and was restored upon estradiol (E(2)) supplementation in rat <em>growth</em> plates. In cultured rat chondrocyte cell line RCJ3.1C5.18, E(2) dose dependently stimulated <em>121</em> and 189 kDa isoforms of VEGF, but not the 164 kDa isoform. Finally, VEGF expression was observed at both protein and mRNA levels in human <em>growth</em> plate specimens. The protein level increased during pubertal development, supporting a link between estrogens and local VEGF production in the <em>growth</em> plate. We conclude that estrogens regulate VEGF expression in the epiphyseal <em>growth</em> plate, although the precise role of VEGF in estrogen-mediated <em>growth</em> plate fusion remains to be clarified.

Coronary artery disease (CAD) and peripheral arterial disease (PAD) are significant medical problems worldwide. Although substantial progress has been made in prevention as well as in the treatment, particularly of CAD, there are a large number of patients, who despite maximal medical treatment have substantial symptomatology and who are not candidates for mechanical re<em>vascular</em>ization. Therapeutic angiogenesis represents a novel, conceptually appealing treatment option. Ad(GV)VEGF<em>121</em>.10 (BIOBYPASS) is an adenovector, carrying the transgene encoding for human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF(<em>121</em>)). A number of preclinical studies have demonstrated angiogenic activity of BIOBYPASS, not only anatomically but also functionally. Phase I clinical studies have demonstrated that intramyocardial infection of BIOBYPASS in patients with severe CAD as well as intramuscular injections of BIOBYPASS in patients with severe peripheral <em>vascular</em> disease (PVD) was well tolerated; furthermore, these studies provided some intriguing indications of activity, which led to initiation of major randomized Phase II "proof-of-concept" studies. This paper provides a review of the rationale behind BIOBYPASS as well as a summary of pertinent preclinical and early clinical data.

Peroxisome Proliferator-Activated Receptor-β (PPARβ) is a ligand-inducible transcription <em>factor</em> activated by both natural (fatty acids and derivatives) and high affinity synthetic agonists. It is thought to play a role in angiogenesis development and <em>Vascular</em> <em>Endothelial</em> <em>Growth</em> <em>Factor</em> (VEGF) regulation but its contribution remains unclear. Until now, the PPARβ agonism effect on VEGF expression in cervical cancer cells was unknown. This led to our interest in assessing the effect of PPARβ activation on the regulation of different VEGF isoforms mRNA expression and the impact of E6 viral oncoprotein and its target p53 on this regulation in cervical cancer cells. Here, we showed that the PPARβ agonist L-165041 induces VEGF(<em>121</em>), VEGF(165) and VEGF(189) expression in HPV (Human Papillomavirus) positive HeLa cells but not in HPV negative cells. The underlying mechanisms did involve neither E6 oncoprotein nor p53. We highlighted a novel mode of PPARβ ligand action including a post-transcriptional regulation of VEGF mRNA expression through the p38 MAPK signaling pathway and the activation of the mRNA-stabilizing <em>factor</em> HuR. But most importantly, we clearly demonstrated that L-165041 acts independently of PPARβ since its effect was not reversed by a chemical inhibition with a specific antagonist and the siRNA-mediated knockdown of the nuclear receptor. As VEGF is crucial for cancer development, the impact of PPARβ ligands on VEGF production is of high importance. Thus, the molecular mechanism of their action has to be elucidated and as a result, PPARβ agonists currently in clinical trials should be carefully monitored.

BACKGROUND

The molecular merger of latest-generation transduction technologies with advanced transgene control modalities may foster decisive advances in therapeutic reprogramming of somatic cell phenotypes.

METHODS

We have engineered self-inactivating HIV-1-based lentiviral expression vectors for reversible macrolide-adjustable transgene expression.

RESULTS

Lentiviral particles engineered for macrolide-responsive human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF<em>121</em>) expression compared favourably with isogenic streptogramin- and tetracycline-responsive configurations and showed excellent <em>growth</em>-<em>factor</em> fine-tuning following transduction into a variety of mammalian cell lines and different human primary cells. Chicken embryos transduced for macrolide-controlled VEGF<em>121</em> production exhibited dose-dependent neo<em>vascular</em>ization and exemplified lentivector-delivered transgene transcription fine-tuning in vivo.

CONCLUSIONS

Macrolide-adjustable lentivectors enable robust and precise in vitro and in vivo transgene fine-tuning which may give future gene therapy trials a new impetus.

OBJECTIVE

To evaluate the prognostic value of melanocytic differentiation antigens and angiogenesis biomarkers in sentinel lymph nodes (SLNs) with melanoma micrometastases.

METHODS

Prognostic study of an inception cohort.

METHODS

Academic research. Patients Between July 1, 1999, and July 31, 2002, all patients who had primary cutaneous or mucosal melanomas that have a Breslow depth of 1.5 mm or greater, ulceration, or Clark level IV or V, or had SLN biopsies.

METHODS

By the use of quantitative reverse transcription-polymerase chain reaction, the expression of the following was analyzed in SLNs: 2 melanocytic differentiation antigens (tyrosinase [P17646] and melanoma antigen recognized by T cells [MART-1; Q16655]) and genes involved in angiogenesis (VEGF [NM_001025366] and VEGFR2 [AF035<em>121</em>]), lymphangiogenesis (VEGFC [NM_005429], VEGFR3 [X68203], LYVE1 [NM_016164], and PROX1 [002763]), and invasion (uPA [NM_002658], PAI1 [NM_00602], and EMMPRIN [L10240]). Outcome measures were the association of these melanocytic differentiation antigens and angiogenesis biomarkers with clinicopathologic characteristics of patients, and an evaluation of the prognostic value for relapse-free survival and overall survival.

RESULTS

Ninety-one patients were included, with a median follow-up period of 41 months. Micrometastases were present in 15% (14 of 91) of patients. Tyrosinase (P < .001), MART-1 (P < .001), <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF(<em>121</em>)) (P = .007), and PAI1 (P = .02) expression was significantly associated with micrometastasis. In univariate analysis, histologic findings and tyrosinase and MART-1 expression were significantly associated with relapse-free survival. Tyrosinase and MART-1 expression was associated with overall survival. A multiple Cox proportional hazards regression model identified negative histologic findings and tyrosinase expression that exceeded 27 copies/copy of TATA box-binding protein (third quartile) as significantly associated with an increased risk of relapse or death.

CONCLUSIONS

Quantitative assessment of melanocytic differentiation antigens in SLNs, which has prognostic value, is more specific than qualitative assessment. Prognosis may be more effectively predicted by the combination of quantitative assessment of melanocytic differentiation antigens in SLNs with histologic assessment. A significant association was found between the presence of micrometastases and the expression of angiogenesis biomarkers.

BACKGROUND

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), which affects tumor angiogenesis, is expressed as different splice variants, including the major isoforms VEGF(165) and VEGF(<em>121</em>), and can be cleaved by plasmin to generate VEGF(110). The amount of VEGF(<em>121</em>) and VEGF(110) in biological samples has not been well studied.

METHODS

We developed an ELISA that detects VEGF(165) and VEGF(<em>121</em>) equally, but does not detect VEGF(110). We used this ELISA together with 2 other ELISAs, one detecting VEGF(165) and the other detecting VEGF(165), VEGF(<em>121</em>), and VEGF(110) equally, to assess the concentrations of VEGF(<em>121</em>) and VEGF(110) in ovarian cancer tumors.

RESULTS

The median concentrations in ovarian cancer tumor lysates were 0.61 (range <0.055-74) fmol/mg protein for VEGF(165), 1.4 (range <0.20-500) fmol/mg protein for VEGF(165) plus VEGF(<em>121</em>), and 2.3 (range <0.079-520) fmol/mg protein for total VEGF including VEGF(110) (n = 248). VEGF concentrations measured by the 3 ELISAs were highly correlated (r = 0.91-0.94). Median estimated VEGF(<em>121</em>) and VEGF(110) concentrations were 0.77 and 0.58 fmol/mg protein, respectively. In lysates with measurable VEGF(165) and total VEGF concentrations, mean VEGF(165) was approximately 31% (SD 23%) of the total VEGF (n = 217). In contrast, VEGF(165) constituted approximately half of the total circulating VEGF.

CONCLUSIONS

VEGF(165), VEGF(<em>121</em>), and VEGF(110) may be present at significant amounts in ovarian cancer tumors.

Delta-like ligand 4 (Dll4)-Notch signaling is important in tumor angiogenesis; however, the prognostic value of D114 detection in patients with clear cell renal cell carcinoma (CCRCC) remains unclear. The present study aimed to determine whether the presence of high Dll4 expression levels was correlated with poor prognosis in CCRCC following curative resection. The D114 expression levels in four paired samples of CCRCC tissues and adjacent normal renal tissues were assayed by western blotting. Surgical specimens comprised <em>121</em> CCRCC tissue samples and 65 normal renal tissue samples, obtained from patients with CCRCC. The specimens were immunohistochemically assessed to determine Dll4 and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptor 2 (VEGFR-2) expression levels. The prognostic significance of Dll4 expression levels was evaluated by the Kaplan-Meier method and Cox regression analysis. The correlation between Dll4 expression levels and VEGFR-2 expression levels, tumor stage, tumor grade and metastasis, was examined by χ2 test and multivariate logistic regression. As determined by the western blotting results, Dll4 protein expression levels were significantly increased in CCRCC tissues compared with those in adjacent non-cancerous tissues. From the analysis of the surgical specimens, 53 (43.8%) CCRCC patients exhibited immunohistochemically high Dll4 expression levels and 68 (56.2%) patients exhibited low Dll4 expression levels. The survival curves revealed that the patients with high Dll4 expression levels had significantly shorter survival times than the patients with low Dll4 expression levels (P<0.001). Multivariate survival analysis demonstrated that the presence of high Dll4 expression levels was independently associated with reduced overall survival and progression-free survival times (P=0.021 and 0.034, respectively). A positive correlation was also identified between Dll4 and VEGFR-2 expression levels (P=0.001). In conclusion, the results show that the presence of high Dll4 expression levels was clearly associated with high VEGFR-2 expression levels, tumor grade, tumor stage and poor prognosis in CCRCC patients. Therefore, inhibition of Dll4 may exert potent <em>growth</em> inhibitory effects on tumors resistant to anti-VEGF therapies for CCRCC.

The aim of this study was to evaluate the potential effects of an insoluble dietary fiber from carob pod (IFC) (1 g ⋅ kg(-1) ⋅ d(-1) in the diet) on alterations associated with atherosclerosis in rabbits with dyslipidemia. Male New Zealand rabbits (n = 30) were fed the following diets for 8 wk: 1) a control diet (SF412; Panlab) as a control group representing normal conditions; 2) a control supplemented with 0.5% cholesterol + 14% coconut oil (DL) (SF302; Panlab) for 8 wk as a dyslipidemic group; and 3) a control containing 0.5% cholesterol + 14% coconut oil plus IFC (1 g ⋅ kg(-1) ⋅ d(-1)) (DL+IFC) for 8 wk. IFC was administered in a pellet mixed with the DL diet. The DL-fed group developed mixed dyslipidemia and atherosclerotic lesions, which were associated with <em>endothelial</em> dysfunction, inflammation, and fibrosis. Furthermore, sirtuin-1 (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) protein expression in the aorta were reduced to 77% and 63% of the control group, respectively (P < 0.05), in these rabbits. Administration of IFC to DL-fed rabbits reduced the size of the aortic lesion significantly (DL, 15.2% and DL+IFC, 2.6%) and normalized acetylcholine-induced relaxation (maximal response: control, 89.3%; DL, 61.6%; DL+IFC, 87.1%; P < 0.05) and <em>endothelial</em> nitric oxide synthase expression (DL, 52% and DL+IFC, 104% of the control group). IFC administration to DL-fed rabbits also reduced cluster of differentiation 36 (DL, 148% and DL+IFC, 104% of the control group; P < 0.05), plasminogen activator inhibitor-1 (DL, 141% and DL+IFC, 107% of the control group), tumor necrosis <em>factor</em>-α (DL, 166% and DL+IFC, 120% of the control group), <em>vascular</em> cell adhesion molecule-1 (DL, 153% and DL+IFC, 110% of the control group), transforming <em>growth</em> <em>factor</em>-β (DL, 173% and DL+IFC, 99% of the control group), and collagen I (DL, 157% and DL+IFC, 112% of the control group) in the aorta. These effects were accompanied by an enhancement of SIRT1 and PGC-1α (160% and <em>121</em>% of the control group, respectively; P < 0.05) <em>vascular</em> expression. In summary, we demonstrated for the first time, to our knowledge, that administration of IFC reduces the development of atherosclerosis in rabbits. This effect seems to be related to an improvement in <em>endothelial</em> function and a reduction of inflammation and fibrosis, most probably as a consequence of the reduction of serum concentrations of cholesterol and triglycerides. Increased expression of aortic SIRT1 and PGC-1α could play an important role in the observed effects of IFC in rabbits with dyslipidemia.

A gene therapy strategy involving direct myocardial administration of an adenovirus (Ad) vector encoding the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> cDNA (Ad(GV)VEGF<em>121</em>.10) has been shown to be capable of "biological re<em>vascular</em>ization" of ischemic myocardium in an established porcine model [Mack, C.A. (1998). J. Thorac. Cardiovasc. Surg. 115, 168-177]. The present study evaluates the local and systemic safety of this therapy in this porcine ischemia model and in normal mice. Myocardial ischemia was induced in Yorkshire swine with an ameroid constrictor 21 days prior to vector administration. Ad(GV)VEGF<em>121</em>.10 (10(9) or 10(10) PFU), Ad5 wild type (10(9) PFU), AdNull (control vector with no transgene; 10(9) PFU), saline, or no injection (naive) was administered in 10 sites in the ischemic, circumflex distribution of the myocardium. Toxicity was assessed by survival, serial echocardiography, blood analyses, and myocardial and liver histology at 3 and 28 days after vector administration. All pigs survived to sacrifice, except for one animal in the Ad(GV)VEGF<em>121</em>.10 (10(10) PFU) group, which died as a result of oversedation. Echocardiograms of Ad(GV)VEGF<em>121</em>.10-treated pigs demonstrated no differences in pericardial effusion, mitral valve regurgitation, or regional wall motion compared with control pigs. Intramyocardial administration of Ad(GV)VEGF<em>121</em>.10 included only minimal myocardial inflammation and necrosis, and no hepatic inflammation or necrosis. Only a mild elevation of the white blood cell count was encountered on day 3, which was transient and self-limited in the Ad(GV)VEGF<em>121</em>.10 group as compared with the saline-treated animals. As a measure of inadvertent intra<em>vascular</em> administration of vector, normal C57/BL6 mice received intravenous Ad(GV)VEGF<em>121</em>.10 (10(4), 10(6), 5 x 10(7), or 10(9) PFU), AdNull (5 x 10(7) or 10(9) PFU), or saline. Toxicity was assessed by survival, blood analyses, and organ histology at 3 and 7 days after vector administration. A separate group of C57/BL6 mice received intravenous AdmVEGF164 (Ad vector encoding the murine VEGF164 cDNA), Ad(GV)VEGF<em>121</em>.10, AdNull (10(8) PFU each group), or saline to assess duration of expression and safety of a homologous transgene. All mice survived to sacrifice except for 40% of the mice in the highest (10(9) PFU; a dose more than 10(3)-fold higher by body weight than the efficacious dose in pigs) Ad(GV)VEGF<em>121</em>.10 dose group, which died on days 5-6 after vector administration. The only differences seen in the blood analyses between treated and control mice were in the very high Ad(GV)VEGF<em>121</em>.10 dose group (10(9) PFU), which demonstrated an anemia as well as an increase in alkaline phosphatase when compared with all other treatment groups. Hepatic VEGF levels by ELISA in AdmVEGF164-treated mice did not persist beyond 14 days after vector administration, suggesting that persistent expression of a homologous VEGF gene transferred with an Ad vector is not a significant safety risk. Although this is not a chronic toxicity study, these data demonstrate the safety of direct myocardial administration of Ad(GV)VEGF<em>121</em>.10, and support the potential use of this strategy to treat human myocardial ischemia.

To determine the effect of 17beta-estradiol, raloxifene, progesterone, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), norethindrone (NET), tibolone and tibolone metabolites on <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) isoforms <em>121</em> and 165 and Thrombospondin-1 (TSp-1) messenger RNA (mRNA) in two breast cancer cell lines, MCF-7 and T47-D. MCF-7 and T47-D cells were cultured to 80% confluence, in vitro. After 24 h incubation in serum-free media, 1.0, 0.1, and 0.01 muM of 17beta-estradiol, raloxifene, raloxifene plus ICI 182780, tibolone, 3alpha-hydroxytibolone, and 3beta-hydroxytibolone were added to MCF-7 cells. Progesterone, MPA, LNG, NET, and Delta(4) tibolone at 1.0, 0.1, and 0.01 muM were added to T47-D cells. The cells plus steroids were incubated for a further 24 h. Total RNA was isolated using TRIZOL and reverse transcriptase-polymerase chain reaction was carried out using primers for VEGF, TSP-1, and cyclophilin, the latter as an internal control. Semiquantitative analysis was performed using 33P-CTP for radioactive labeling during the polymerase chain reaction. 17beta-estradiol, raloxifene, tibolone, 3alpha-hydroxytibolone, and 3beta-hydroxytibolone had no effect on VEGF mRNA in MCF-7 cells. Progesterone, MPA, LNG, and NET increased VEGF mRNA in T47-D cells. Delta(4) tibolone also increased VEGF mRNA but to a lesser extent than the progestogens. Raloxifene increased TSP-1 mRNA, this effect was not reversed by the addition of ICI 182780 to the media. 17beta-estradiol, raloxifene, tibolone and tibolone hydroxy-metabolites had no effect on VEGF mRNA in MCF-7 cells. Progesterone and progestins increased VEGF mRNA in T47-D breast cancer cells. Delta(4) tibolone was less effective than progestogens on this angiogenic gene in the T47-D cells. Raloxifene increased TSP-1. These differential effects may be related to breast cancer <em>growth</em>.

Human VEGF<em>121</em> (<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> isoform <em>121</em>) was produced as a recombinant fusion protein with GST (glutathione S-transferase) in Escherichia coli. After affinity purification with glutathione, the GST-VEGF<em>121</em> fusion protein preparation was used to obtain antibodies in mice against commercial hrVEGF (human recombinant VEGF) through immunization. It was also employed successfully to select specific antihuman VEGF antibody fragments of human origin employing phage-display technology. The fusion protein preparation was separated in monomeric, dimeric and oligomeric forms using size-exclusion chromatography. The dimers were recognized by a soluble VEGF receptor 2-Fc chimaera, and stimulated the <em>growth</em> of human umbilical-vein <em>endothelial</em> cells in vitro in a similar fashion to a commercial hrVEGF. The presence of GST in the fusion protein apparently did not affect the correct assembly of dimers and display of residues critical for receptor recognition. The two-step purification method reported in the present paper involves no laborious renaturalization methods, yields 10 mg/l of the mixture of different aggregation states after affinity chromatography, and 5 mg/l of the biologically active dimer after gel filtration, thus providing a source of material for the development of new anti-angiogenic therapeutic molecules.

The mechanisms involved in the blockade of proliferation in confluent <em>endothelial</em> cells are insufficiently understood. In this regard, the continuity of intercellular junctions appears to be critical to the regulation of <em>endothelial</em> monolayer cell <em>growth</em>. The present study examined the hypothesis that the disruption of the intercellular adherens junctions will trigger both <em>endothelial</em> cell proliferation and autocrine production of <em>growth</em> <em>factors</em>. With this purpose, we assessed the changes in <em>growth</em>, death resistance, and expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) under conditions of disruption of the intercellular junctions between <em>endothelial</em> cells. Disruption of cell junctions was produced by means of a specific anti-<em>vascular</em> <em>endothelial</em> cadherin monoclonal antibody, EGTA, or cytochalasin D. Our results disclosed that these maneuvers induce an increase in VEGF mRNA production, with transcription of the <em>121</em>-, 165-, and 189-amino acid isoforms of VEGF. Further evidence of the relationship between <em>endothelial</em> cells monolayer continuity and VEGF protein expression was obtained by the demonstration of an increase in VEGF protein, as determined by Western blot, induced by the aforementioned maneuvers, as well as by immunocytochemical detection of increased VEGF staining in the areas surrounding a mechanical <em>endothelial</em> injury and in <em>endothelial</em> cells at subconfluence. In functional terms, the autocrine expression of VEGF was associated with <em>growth</em>-promoting and cytoprotective effects, as assessed by [(3)H]thymidine uptake, (51)Cr release, and flow cytometry. In conclusion, our results reveal that disruption of homophilic inter<em>endothelial</em> junctions induces VEGF expression. Under these conditions, autocrine VEGF appears to have a relevant role in death inhibition and proliferation of <em>endothelial</em> cells.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is an <em>endothelial</em> cell-specific mitogen and a key mediator of aberrant <em>endothelial</em> cell proliferation and <em>vascular</em> permeability in a variety of human pathological situations such as tumor angiogenesis, diabetic retinopathy, or psoriasis. By amino-terminal deletion analysis and by site-directed mutagenesis we have identified a new domain within the amino-terminal alpha-helix that is essential for dimerization of VEGF. VEGF<em>121</em> variants containing amino acids 8 to <em>121</em> or 14 to <em>121</em>, respectively, either expressed in Escherichia coli and refolded in vitro, or expressed in Chinese hamster ovary cells, were in a dimeric conformation and showed full binding activity to VEGF receptors and stimulation of <em>endothelial</em> cell proliferation as compared with wild-type VEGF. In contrast, a VEGF<em>121</em> variant covering amino acids 18 to <em>121</em>, as well as a variant in which the hydrophobic amino acids Val14, Val15, Phe17, and Met18 within the amphipathic alpha-helix near the amino terminus were replaced by serine, failed to form biological active VEGF dimers. From these data we conclude that a domain between amino acids His12 and Asp19 within the amino-terminal alpha-helix is essential for formation of VEGF dimers, and we propose hydrophobic interactions between VEGF monomers to stabilize or favor dimerization.

Passive immunotherapy against soluble pro-angiogenic <em>factors</em> and/or their receptors in <em>endothelial</em> cells has become a promising approach in cancer therapeutics. There is also experimental evidence indicating that an active immunotherapy strategy directed towards these target molecules could also be effective. In this paper we show that it is possible to reduce tumor <em>growth</em> or increase the survival of tumor-bearing C57Bl/6 mice when animals are vaccinated with the human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) isoform <em>121</em> gene (hVEGF(<em>121</em>)), and later challenged with melanoma or lung carcinoma tumor cells. Immunization was done with 10 microg DNA doses of the hVEGF<em>121</em> gene, which is highly homologous to its mouse counterpart, administered on a weekly basis using a plasmid bearing 5 CpG bacterial motifs. Histopathology analyses of tumors of hVEGF(<em>121</em>) immunized animals showed a decrease in tumor cell density around vessels and in mitotic figures, as well as an increase in apoptotic tumor cells. A statistically significant cell cytotoxic response was found when spleen cells of immunized mice were co-cultured in vitro with mouse tumor VEGF-producing cells. Vaccination with an hVEGF<em>121</em> gene mutated to make it deficient for VEGF receptor binding, produced similar in vitro and in vivo results, and significantly reduced the number of spontaneous metastases produced by the mouse Lewis lung carcinoma. Our results indicate that human VEGF DNA can be employed for anti-angiogenic active immunotherapy in mice, and that direct cell cytotoxicity is a contributor mechanism to the overall anti-tumor effects seen in immunized animals.