Mast cells accumulate in large numbers at angiogenic sites, where they have been shown to express a number of proangiogenic <em>factors</em>, including <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF-A). PGE(2) is known to strongly promote angiogenesis and is found in increased levels at sites of chronic inflammation and around solid tumors. The expression pattern of VEGF and the regulation of VEGF-A by PGE(2) were examined in cord blood-derived human mast cells (CBMC). CBMC expressed mRNA for five isoforms of VEGF-A and other members of the VEGF family (VEGF-B, VEGF-C, and VEGF-D) with strong expression of the most potent secretory isoforms. PGE(2) was a very strong inducer of VEGF-A(<em>121</em>/165) production by CBMC and also elevated VEGF-A mRNA expression. The amount of VEGF-A(<em>121</em>/165) protein production induced by PGE(2) was 4-fold greater than that induced by IgE-mediated activation of CBMC. Moreover, the response to PGE(2) as well as to other cAMP-elevating agents such as forskolin and salbutamol was observed under conditions that were not associated with mast cell degranulation. CBMC expressed substantial levels of the EP(2) receptor, but not the EP(4) receptor, when examined by flow cytometry. In contrast to other reported PGE(2)-mediated effects on mast cells, VEGF-A(<em>121</em>/165) production occurred via activation of the EP(2) receptor. These data suggest a role for human mast cells as a potent source of VEGF(<em>121</em>/165) in the absence of degranulation, and may provide new opportunities to regulate angiogenesis at mast cell-rich sites.

BACKGROUND

The expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-A isoforms (<em>121</em>, 165, 189, 206), VEGF-B, VEGF-C and VEGF-D in both experimental and clinical models of head and neck squamous cell carcinoma (HNSCC) was determined and correlated with conventional clinicopathologic parameters, with particular reference to cervical nodal metastasis.

METHODS

The mRNA expression of VEGFs in 14 HNSCC cell lines was compared with 4 normal keratinocyte cultures and 10 fibroblast cultures using a semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay. Protein levels were determined by Western blotting and enzyme-linked immunosorbent assay (ELISA). The authors then examined the expression of VEGFs in tissues from 54 patients including histologically normal epithelium (n = 32), early invasive squamous cell carcinomas (SCCs) (n = 23), advanced primary SCCs (n = 31), and lymph node metastases (n = 27).

RESULTS

Increased levels of VEGF-A (all four isoforms) and VEGF-C were found in tumor cell lines compared with normal cells, whereas no differences in VEGF-B levels were found. VEGF-D expression, however, was lower in HNSCC cells. Studies in clinical samples showed highly significant increases in mRNA expression of all four isoforms of VEGF-A and VEGF-C in tumors versus normal epithelium. In contrast, the levels of VEGF-D were significantly decreased in tumors, and VEGF-B expression appeared similar in both normal and malignant tissues. Multivariate analysis demonstrated that an infiltrative mode of invasion and enhanced expression of VEGF-A (isoforms <em>121</em> and 165) and VEGF-C had predictive value for the presence of cervical nodal metastases.

CONCLUSIONS

Up-regulation of VEGF-A (two isoforms) and VEGF-C and down-regulation of VEGF-D have been common features in HNSCC. Thus VEGF-A and VEGF-C appeared to play a vital role in the metastatic process of HNSCC.

Alternative splicing of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) mRNA results in three distinct molecular forms of <em>121</em> or 165 (V165) amino acids that are released in the conditioned medium of cultured cells and one longer isoform of 189 amino acids (V189) that remains cell-associated. V189 has been expressed in wild type CHO-K1 cells and in glycosaminoglycan-deficient pgsA-745 Chinese hamster ovary (CHO) mutant cells. It could be released from CHO-K1 cell membranes by heparin or a synthetic peptide designed on the sequence encoded by exon 6 but was freely released from CHO mutant cells. In both cases, the immunoreactive V189 was mainly released as a 40-kDa cleaved form, provided that the serine protease urokinase, but not plasmin, was active. Recombinant V189 was purified from insect cells infected with a recombinant baculovirus as a nonmitogenic 50-kDa precursor that binds to the receptor Flt-1 but not to Flk-1. It could be matured by urokinase as a 38-kDa fragment able to bind to Flk-1 and to trigger cell proliferation. V165 and V189, however, could be cleaved by plasmin as 34-kDa fragments that exhibit a decreased mitogenic activity. These findings indicate that the carboxyl-terminal domain of V189 masks its binding domain to Flk-1.

Human papillomavirus (HPV) 16 is involved in causing cervical cancer. The E6 and E7 proteins of HPV 16 immortalize human keratinocytes and this is due, at least in part, to inactivation of the tumor suppressor proteins p53 and pRB. These tumor suppressor proteins also regulate the expression of pro- and antiangiogenic <em>factors</em> by cells. For this reason, experiments were conducted to determine whether the expression of E6 and E7 in primary keratinocytes alters the phenotype of these cells such that they express diminished levels of antiangiogenic <em>factors</em> and/or increased levels of proangiogenic <em>factors</em>. To avoid variances in experimental observations, pools of human foreskin keratinocytes from multiple sources were infected with recombinant retrovirus expressing HPV 16 E6 and E7 or control retrovirus. Gene array analysis, RT-PCR, ELISAs and Western blotting showed that in cells expressing HPV 16 E6 and E7, expression levels of two potent angiogenesis inhibitors, thrombospondin-1 and maspin, were lower compared to controls. Additionally, major angiogenesis inducers, interleukin-8 and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), were increased relative to controls. VEGF can be produced as multiple splice variants, all of which are required for the formation of patent blood vessels. The expression of HPV 16 E6 and E7 in keratinocytes augmented expression of VEGF <em>121</em>, 145, 165 and 189. These observations show that HPV 16 E6 and E7 alter the phenotype of primary keratinocytes, diminishing expression of inhibitors and increasing expression of inducers of angiogenesis. This altered phenotype may permit keratinocytes infected by HPV 16 to play a role in the progression of cancer by permitting tumors to acquire a blood supply permissive of <em>growth</em> and spread.

OBJECTIVE

To investigate the vitreous levels of bevacizumab and vascular endothelial growth factor-A (VEGF-A) after intravitreal injection of the drug in patients with choroidal neovascularization (CNV).

METHODS

Interventional case series.

METHODS

Eleven eyes of 11 patients with submacular hemorrhage and CNV due to age-related macular degeneration (n = 10) or angioid streaks (n = 1).

METHODS

All patients were treatment naïve except for a single dose of intravitreal injection of bevacizumab (1.25 mg/50 muL dose) and subsequent vitrectomy after various intervals (1-101 days) because of active and progressive lesion. Intravitreal free bevacizumab and VEGF-A levels were measured using enzyme-linked immunosorbent assay and microsphere-based immunoassay, respectively. Vitreous VEGF-A isoforms were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting.

METHODS

Intravitreal bevacizumab and VEGF-A levels were measured and pharmacokinetic parameters were calculated.

RESULTS

Pharmacokinetics of intravitreal bevacizumab followed a 2-compartment model with initial and terminal half-lives of 0.5 and 6.7 days, respectively. Bevacizumab could be detected in all cases, ranging from 2.63 ng/ml to 165 microg/ml. The peak concentration was observed on the second day after intravitreal bevacizumab injection. Vitreous free VEGF-A levels ranged from 0.2 to 33.9 pg/ml and showed a negative correlation with the bevacizumab concentration (P<0.001; r = -0.955) and a positive correlation with time (P<0.001; r = 0.964). However, the percentage expression of VEGF-A(165) exhibited a positive correlation with the bevacizumab concentration (P = 0.032, r = 0.645) and a negative correlation with time (P = 0.007, r = -0.755). A time-dependent increase was found for the percentage expression of VEGF-A(189) (P = 0.023, r = 0.673). Neither bevacizumab- nor time-related alterations were found for VEGF-A(121).

CONCLUSIONS

Based on pharmacokinetics, the interval of 6-7 weeks would be appropriate for efficacy, although clinical trials should guide dosing recommendations. Vitreous levels of free VEGF-A showed a negative correlation with the bevacizumab concentration, which confirmed the in vivo binding affinity of bevacizumab to VEGF-A. The analysis of the VEGF-A isoforms suggests differences of interaction between bevacizumab and individual VEGF-A isoforms.

The <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> is produced by a large variety of human tumors, including melanoma, in which it appears to play an important role in the process of tumor-induced angiogenesis. Little information is available on the role of placenta <em>growth</em> <em>factor</em>, a member of the <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> family of cytokines, in tumor angiogenesis, even though placenta <em>growth</em> <em>factor</em>/<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> heterodimers have been recently isolated from tumor cells. To investigate the role of placenta <em>growth</em> <em>factor</em> and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> homodimers and heterodimers in melanoma angiogenesis and <em>growth</em>, 19 human melanoma cell lines derived from primary or metastatic tumors were characterized for the expression of these cytokines and their receptors. Release of placenta <em>growth</em> <em>factor</em> and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> polypeptides into the supernatant of human melanoma cells was demonstrated. Reverse transcriptase polymerase chain reaction analysis showed the presence of mRNAs encoding at least three different <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> isoforms (VEGF(<em>121</em>), VEGF(165), and VEGF(189)) and transcripts for two placenta <em>growth</em> <em>factor</em> isoforms (PlGF-1 and PlGF-2) in human melanoma cells. In addition, placenta <em>growth</em> <em>factor</em> expression in human melanoma in vivo was detected by immunohistochemical staining of tumor specimens. Both primary and metastatic melanoma cells were found to express the mRNAs encoding for <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> and placenta <em>growth</em> <em>factor</em> receptors (KDR, Flt-1, neuropilin-1, and neuropilin-2), and exposure of melanoma cells to these cytokines resulted in a specific proliferative response, supporting the hypothesis of a role of these angiogenic <em>factors</em> in melanoma <em>growth</em>. J Invest Dermatol 115:1000-1007 2000

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a central regulator of both physiological and pathological angiogenesis. Pegaptanib, a 28-nucleotide RNA aptamer specific for the VEGF(165) isoform, binds to it in the extracellular space, leaving other isoforms unaffected, and inhibits such key VEGF actions as promotion of <em>endothelial</em> cell proliferation and survival, and <em>vascular</em> permeability. Pegaptanib already has been examined as a treatment for two diseases associated with ocular neo<em>vascular</em>ization, age-related macular degeneration (AMD) and diabetic macular edema (DME). Preclinical studies have shown that VEGF(165) alone mediates pathological ocular neo<em>vascular</em>ization and that its inactivation by pegaptanib inhibits the choroidal neo<em>vascular</em>ization observed in patients with neo<em>vascular</em> AMD. In contrast, physiological <em>vascular</em>ization, which is supported by the VEGF(<em>121</em>) isoform, is unaffected by this inactivation of VEGF(165). In addition, animal model studies have shown that intravitreous injection of pegaptanib can inhibit the breakdown of the blood-retinal barrier characteristic of diabetes and even can reverse this damage to some degree. These preclinical findings formed the basis for randomized controlled trials examining the efficacy of pegaptanib as a therapy for AMD and DME. The VEGF Inhibition Study in Ocular Neo<em>vascular</em>ization (VISION) trial comprising two replicate, pivotal phase 3 studies, demonstrated that intravitreous injection of pegaptanib resulted in significant clinical benefit, compared with sham injection, for all prespecified clinical end points, irrespective of patient demographics or angiographic subtype, and led to pegaptanib's approval as a treatment for AMD. A phase 2 trial has provided support for the efficacy of intravitreous pegaptanib in the treatment of DME.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> is a potent direct-acting angiogenic <em>factor</em>. Early in hepatocarcinogenesis, hepatocellular carcinomas do not show hyper<em>vascular</em>ity; at later stages, they require abundant arterial blood flow. We investigated the role of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> in hepatocellular carcinoma arterialization. We studied 51 patients with hepatocellular carcinoma. All patients had undergone hepatic arteriography. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> expression was investigated by immunohistochemistry (n = 51) and in situ hybridization (n = 13), and the changes in <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> expression were evaluated in relation to tumor differentiation and changes in tumor <em>vascular</em>ity. The expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> isoforms in hepatocellular carcinomas was also analyzed by reverse transcriptase-polymerase chain reaction (n = 10). <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> expression was detected in hepatoma cells and hepatic stellate cells, and increased <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> expression was associated with tumor dedifferentiation. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> expression in hyper<em>vascular</em> hepatocellular carcinomas was greater than in those not showing hyper<em>vascular</em>ity. The major <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> isoforms expressed in hepatocellular carcinoma were <em>121</em> and 165. These findings indicate that <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factors</em> <em>121</em> and 165 play a critical role in the process of angiogenesis in hepatocellular carcinomas.

BACKGROUND

Fatty acid synthase (FASN) is overexpressed and associated with poor prognosis in several human cancers. Here, we investigate the effect of FASN inhibitors on the metastatic spread and angiogenesis in experimental melanomas and cultured melanoma cells.

METHODS

The lung colonisation assay and cutaneous melanomas were performed by the inoculation of mouse melanoma B16-F10 cells in C57BL6 mice. Blood vessel endothelial cells (RAEC and HUVEC) were applied to determine cell proliferation, apoptosis, and the formation of capillary-like structures. Vascular endothelial growth factor A (VEGFA) expression was evaluated by quantitative RT-PCR and ELISA in B16-F10, human melanoma (SK-MEL-25), and human oral squamous carcinoma (SCC-9) cells. Conditioned media from these cancer cell lines were used to study the effects of FASN inhibitors on endothelial cells.

RESULTS

B16-F10 melanoma-induced metastases and angiogenesis were significantly reduced in orlistat-treated mice. Fatty acid synthase inhibitors reduced the viability, proliferation, and the formation of capillary-like structures by RAEC cells, as well as the tumour cell-mediated formation of HUVEC capillary-like structures. Cerulenin and orlistat stimulated the production of total VEGFA in B16-F10, SK-MEL-25, and SCC-9 cells. Both drugs also enhanced VEGFA(121), (165), (189,) and (165b) in SK-MEL-25 and SCC-9 cells.

CONCLUSIONS

FASN inhibitors reduce metastasis and tumour-induced angiogenesis in experimental melanomas, and differentially modulate VEGFA expression in B16-F10 cells.

OBJECTIVE

To report the incidence of endophthalmitis in association with different antibiotic prophylaxis strategies after intravitreal injections of anti-vascular endothelial growth factors and triamcinolone acetonide.

METHODS

Retrospective, comparative case series.

METHODS

Fifteen thousand eight hundred ninety-five intravitreal injections (9453 ranibizumab, 5386 bevacizumab, 935 triamcinolone acetonide, 121 pegaptanib sodium) were reviewed for 2465 patients between January 5, 2005, and August 31, 2010. The number of injections was determined from billing code and patient records.

METHODS

The indications for injection included age-related macular degeneration, diabetic macular edema, central and branch retinal vein occlusion, and miscellaneous causes. Three strategies of topical antibiotic prophylaxis were used by the respective surgeons: (1) antibiotics given for 5 days after each injection, (2) antibiotics given immediately after each injection, and (3) no antibiotics given.

METHODS

The primary outcome measures were the incidence of culture-positive endophthalmitis and culture-negative cases of suspected endophthalmitis.

RESULTS

Nine eyes of 9 patients with suspected endophthalmitis after injection were identified. Three of the 9 cases had culture-positive results. The overall incidence of endophthalmitis was 9 in 15 895. The incidence of culture-negative cases of suspected endophthalmitis and culture-proven endophthalmitis after injection was 6 in 15 895 and 3 in 15 895, respectively. Taking into account both culture-positive endophthalmitis and culture-negative cases of suspected endophthalmitis, the incidence per injection was 5 in 8259 for patients who were given antibiotics for 5 days after injection, 2 in 2370 for those who received antibiotics immediately after each injection, and 2 in 5266 who received no antibiotics. However, if considering culture-proven endophthalmitis alone, the use of topical antibiotics, given immediately or for 5 days after injection, showed lower rates of endophthalmitis compared with those without postinjection antibiotics. The risk of endophthalmitis after intravitreal injection varied among agents that were used. Among the 9 cases of clinically suspected endophthalmitis, regardless of prophylactic strategies used, the incidence of endophthalmitis per injection was 2 in 935 for triamcinolone acetonide, 3 in 9453 for ranibizumab, and 4 in 5386 for bevacizumab.

CONCLUSIONS

The overall rate of intravitreal injection-related endophthalmitis is greater with the use of topical antibiotics, given immediately or for 5 days after the injection, compared with no antibiotics.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) plays a key role in the <em>growth</em> and metastasis of solid tumors. We generated a fusion protein containing VEGF(<em>121</em>) linked by a flexible G(4)S tether to the toxin gelonin (rGel) and expressed this as a soluble protein in bacteria. Purified VEGF(<em>121</em>)/rGel migrated as an 84-kDa homodimer under nonreducing conditions. VEGF(<em>121</em>)/rGel bound to purified, immobilized Flk-1, and the binding was competed by VEGF(<em>121</em>). Both VEGF(<em>121</em>)/rGel and VEGF(<em>121</em>) stimulated cellular kinase insert domain receptor (KDR) phosphorylation. The VEGF(<em>121</em>)/rGel fusion construct was highly cytotoxic to <em>endothelial</em> cells overexpressing the KDR/Flk-1 receptor. The IC(50) of the construct on dividing <em>endothelial</em> cells expressing 10(5) or more KDR/Flk-1 receptors per cell was 0.5-1 nM, as compared with 300 nM for rGel itself. Dividing <em>endothelial</em> cells overexpressing KDR were approximately 60-fold more sensitive to VEGF(<em>121</em>)/rGel than were nondividing cells. <em>Endothelial</em> cells overexpressing FLT-1 were not sensitive to the fusion protein. Human melanoma (A-375) or human prostate (PC-3) xenografts treated with the fusion construct demonstrated a reduction in tumor volume to 16% of untreated controls. The fusion construct localized selectively to PC-3 tumor vessels and caused thrombotic damage to tumor vessels with extravasation of red blood cells into the tumor bed. These studies demonstrate the successful use of VEGF(<em>121</em>)/rGel fusion construct for the targeted destruction of tumor vasculature in vivo.

We determined the molecular mechanisms that regulate the pathogenesis of malignant pleural effusion (PE) associated with advanced stage of human, non-small-cell lung cancer. Intravenous injection of human PC14 and PC14PE6 (adenocarcinoma) or H226 (squamous cell carcinoma) cells into nude mice yielded numerous lung lesions. PC14 and PC14PE6 lung lesions invaded the pleura and produced PE containing a high level of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-localized <em>vascular</em> hyperpermeability. Lung lesions produced by H226 cells were confined to the lung parenchyma with no PE. The level of expression of VEGF mRNA and protein by the cell lines directly correlated with extent of PE formation. Transfection of PC14PE6 cells with antisense VEGF165 gene did not inhibit invasion into the pleural space but reduced PE formation. H226 cells transfected with either sense VEGF 165 or sense VEGF <em>121</em> genes induced localized <em>vascular</em> hyperpermeability and produced PE only after direct implantation into the thoracic cavity. The production of PE was thus associated with the ability of tumor cells to invade the pleura, a property associated with expression of high levels of urokinase-type plasminogen activator and low levels of TIMP-2. Collectively, the data demonstrate that the production of malignant PE requires tumor cells to invade the pleura and express high levels of VEGF/VPF.

BACKGROUND

A method for identifying tissue experiencing hypoxic stress due to atherosclerotic <em>vascular</em> disease would be clinically useful. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-<em>121</em> (VEGF<em>121</em>) is an angiogenic protein secreted in response to hypoxia that binds to VEGF receptors overexpressed by ischemic microvasculature. We tested the hypothesis that VEGF receptors could serve as markers for ischemic tissue and hence provide a target for imaging such tissue with radiolabeled human VEGF<em>121</em>.

RESULTS

A rabbit model of unilateral hindlimb ischemia was created by femoral artery excision (n=14). Control rabbits (n=5) underwent identical surgery without femoral excision. On postoperative day 10, rabbits were intravenously administered 100 microCi of 111In-labeled recombinant human VEGF<em>121</em>, and biodistribution studies and planar imaging were conducted at 3, 24, and 48 hours. On postmortem gamma counting, there was greater accumulation of 111In-labeled VEGF<em>121</em> in ischemic than in control tissue (P<0.02). Differential uptake of isotope by ischemic muscle was not seen in rabbits injected with 125I-labeled human serum albumin (n=6). Radioactivity imaged in hindlimb regions of interest was significantly higher in ischemic muscle than in sham-operated and contralateral nonoperated hindlimb at 3 hours (P<0.02). Immunohistochemical staining confirmed upregulation of VEGF receptors in ischemic skeletal muscle.

CONCLUSIONS

Identification of the ischemic state via targeted radiolabeling of hypoxia-induced angiogenic receptors is possible. This approach could be useful for monitoring the efficacy of revascularization strategies such as therapeutic angiogenesis.

BACKGROUND

Direct transfection of ischemic myocardium with naked plasmid DNA encoding for vascular endothelial growth factor-165 (VEGF165) has been shown to mobilize endothelial progenitor cells (EPCs). This study examined the kinetics of circulating EPCs isolated from peripheral blood mononuclear cells after gene transfer, and their role in neovascularization of ischemic myocardium.

METHODS

The mononuclear cell population was isolated from peripheral venous blood samples of patients with functional class III or IV angina receiving intramyocardial VEGF165 gene transfer. Peripheral blood mononuclear cells were examined by an in vitro EPC culture assay and fluorescent-activated cell sorting. The data were compared with a control group consisting of patients who had undergone off-pump coronary artery bypass grafting without receiving gene transfer.

RESULTS

Coinciding with a rise in VEGF levels, mobilization of EPCs increased significantly over base line for 9 weeks after the treatment (121+/-14 cells/mm2 versus 36.8+/-8 cells/mm2, p < 0.0005), followed by a subsequent decrease. Fluorescent-activated cell sorting analysis confirmed culture assay data, with a statistically significant rise in cells expressing vascular endothelial-cadherin, CD51/61 [alphavbeta3], CD62E [E-selectin], CD34, and KDR. The control group failed to show significant mobilization of EPCs.

CONCLUSIONS

Mobilization of EPCs with resultant postnatal vasculogenesis, may play a role in revascularizing ischemic myocardium following human gene transfer with VEGF165.

Using plants as production <em>factor</em>ies for therapeutic proteins requires modification of their N-glycosylation pattern because of the immunogenicity of plant-specific sugar residues. In an attempt towards such humanization, we disrupted the genes for alpha1,3-fucosyltransferase and beta1,2-xylosyltransferase in Physcomitrella patens by homologous recombination. The single Deltafuc-t and Deltaxyl-t plants, as well as the double knockout, lacked transcripts of the corresponding genes, but did not differ from the wild-type moss in morphology, <em>growth</em>, development, and ability to secrete a recombinant protein, the human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> VEGF(<em>121</em>), into the culture medium. N-Glycan analysis, however, revealed the absence of 1,3-fucosyl and/or 1,2-xylosyl residues, respectively. Therefore, the modifications described here represent the key step towards the generation of moss lines suitable for the production of plant-made glycosylated biopharmaceuticals with nonallergenic N-glycans.

Expression of <em>vascular</em> permeability <em>factor</em>/<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VPF/VEGF) is markedly increased in the epidermis of lesional psoriatic skin and in healing skin wounds. In this study, we characterized the effects of several cytokines and <em>growth</em> <em>factors</em> on the expression and secretion of VPF/VEGF mRNA and protein by cultured human epidermal keratinocytes, as well as the effect of VPF/VEGF on the <em>growth</em> of cultured human dermal micro<em>vascular</em> <em>endothelial</em> cells. Transforming <em>growth</em> <em>factor</em>-alpha, epidermal <em>growth</em> <em>factor</em>, and phorbol myristate acetate markedly stimulated VPF/VEGF mRNA expression by cultured keratinocytes; as in psoriatic skin, the three most common VPF/VEGF isoforms (encoding proteins of <em>121</em>, 165, and 189 amino acids) were upregulated to an equal extent. Transforming <em>growth</em> <em>factor</em> (TGF)-alpha, epidermal <em>growth</em> <em>factor</em>, and phorbol myristate acetate also enhanced the secretion of VPF/VEGF by keratinocytes; in contrast, a number of other cytokines including interleukin (IL)-1, IL-6, IL-8, tumor necrosis <em>factor</em>-alpha, interferon-gamma, and transforming <em>growth</em> <em>factor</em>-beta did not induce VPF/VEGF secretion. The VPF/VEGF secreted by keratinocytes was biologically active in that, like recombinant human VPF/VEGF, it potently stimulated dermal <em>endothelial</em> cell proliferation. Scatchard analysis revealed two high-affinity VPF/VEGF binding sites on dermal <em>endothelial</em> cells with dissociation constants of 51 pM and 2.9 pM. These results suggest that the a<em>vascular</em> epidermis has the capacity to regulate dermal angiogenesis and micro<em>vascular</em> permeability by a paracrine mechanism involving the secretion of VPF/VEGF. Similar mechanisms may be anticipated in a variety of inflammatory and neoplastic skin diseases characterized by micro<em>vascular</em> hyperpermeability, edema, and angiogenesis.

The <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) family of cytokines is involved in the maintenance of existing adult blood vessels as well as in angiogenesis, the sprouting of new vessels. To study the proangiogenic activation of VEGF receptors (VEGFRs) by VEGF family members in skeletal muscle, we develop a computational model of VEGF isoforms (VEGF(<em>121</em>), VEGF(165)), their cell surface receptors, and the extracellular matrix in in vivo tissue. We build upon our validated model of the biochemical interactions between VEGF isoforms and receptor tyrosine kinases (VEGFR-1 and VEGFR-2) and nonsignaling neuropilin-1 coreceptors in vitro. The model is general and could be applied to any tissue; here we apply the model to simulate the transport of VEGF isoforms in human vastus lateralis muscle, which is extensively studied in physiological experiments. The simulations predict the distribution of VEGF isoforms in resting (nonexercising) muscle and the activation of VEGFR signaling. Little of the VEGF protein in muscle is present as free, unbound extracellular cytokine; the majority is bound to the cell surface receptors or to the extracellular matrix. However, interstitial sequestration of VEGF(165) does not affect steady-state receptor binding. In the absence of neuropilin, VEGF(<em>121</em>) and VEGF(165) behave similarly, but neuropilin enhances the binding of VEGF(165) to VEGFR-2. This model is the first to study VEGF tissue distribution and receptor activation in human muscle, and it provides a platform for the design and evaluation of therapeutic approaches.

A number of <em>vascular</em> therapies could benefit from advanced methods for presentation of angiogenic <em>growth</em> <em>factors</em>, including <em>growth</em> of endothelium on small caliber <em>vascular</em> grafts and re<em>vascular</em>ization of ischemic tissue through induction of collateral vessels and microvessels. To explore methods to optimize the presentation and release of angiogenic <em>factors</em> in such applications in device integration and tissue repair, we studied three variant forms of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF<em>121</em>), each with differential susceptibility to local cellular proteolytic activity, formulated within fibrin matrices. (1) The prototypic variant alpha2PI(1-8)-VEGF<em>121</em> remains immobilized in fibrin matrices until its liberation by cell-associated enzymes, such as plasmin, that degrade the fibrin network [slow, cell-demanded release; J. Control. Release 72 (2001) 101-113]; the alpha2PI(1-8) domain serves as a site for covalent attachment to fibrin during coagulation. (2) We created a new VEGF variant, alpha2PI(1-8)-Pla-VEGF<em>121</em> that couples to fibrin via a plasmin-sensitive sequence (Pla). Cleavage of this target site by plasmin enables direct release of alpha2PI(1-8)-Pla-VEGF<em>121</em> from bulk matrix degradation (accelerated, cell-demanded release). (3) Native VEGF<em>121</em> (burst, passive release) was considered as a reference. VEGF release profiles were determined experimentally as well as mathematically, alpha2PI(1-8)-Pla-VEGF<em>121</em> being released ca. fourfold more quickly than alpha2PI(1-8)-VEGF<em>121</em>, both being retained compared to native VEGF<em>121</em>; the differences in release could be accounted for based on knowledge of the plasmin sensitivity of the bound <em>growth</em> <em>factor</em> and the structure of the fibrin network. The bound <em>factors</em> were competent in inducing <em>endothelial</em> cell proliferation, the matrix-bound forms being more effective than native VEGF<em>121</em>; as well as competent in inducing <em>endothelial</em> progenitor cell maturation into <em>endothelial</em> cells. These matrix-bound variants of VEGF<em>121</em> may be particularly useful where retention in locally applied surgical sites is desired, such as prevention of washout from <em>vascular</em> graft coatings and slowing loss from tissue in<em>growth</em> matrices used in local tissue re<em>vascular</em>ization and repair.

We previously reported that <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-A(165) inflammatory effect is mediated by acute platelet-activating <em>factor</em> synthesis from <em>endothelial</em> cells upon the activation of VEGF receptor-2 (VEGFR-2) and its coreceptor, neuropilin-1 (NRP-1). In addition, VEGF-A(165) promotes the release of other <em>endothelial</em> mediators including nitric oxide and prostacyclin (PGI(2)). However, it is unknown whether VEGF-A(165) is mediating PGI(2) synthesis through VEGF receptor-1 (VEGFR-1) and/or VEGF receptor-2 (VEGFR-2) activation and whether the coreceptor NRP-1 potentiates VEGF-A(165) activity. In this study, PGI(2) synthesis in bovine aortic <em>endothelial</em> cells (BAEC) was assessed by quantifying its stable metabolite (6-keto prostaglandin F(1alpha), 6-keto PGF(1alpha)) by enzyme-linked immunosorbent assay. Treatment of BAEC with VEGF analogs, VEGF-A(165) (VEGFR-1, VEGFR-2 and NRP-1 agonist) and VEGF-A(<em>121</em>) (VEGFR-1 and VEGFR-2 agonist) (up to 10(-9) m), increased PGI(2) synthesis by 70- and 40-fold within 15 min. Treatment with VEGFR-1 (placental <em>growth</em> <em>factor</em> and VEGF-B) or VEGFR-2 (VEGF-C) agonist did not increase PGI(2) synthesis. The combination of VEGFR-1 and VEGFR-2 agonists did not increase PGI(2) release. Pretreatment with a VEGFR-2 inhibitor abrogated PGI(2) release mediated by VEGF-A(165) and VEGF-A(<em>121</em>), and pretreatment of BAEC with antisense oligomers targeting VEGFR-1 or VEGFR-2 mRNA reduced PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(<em>121</em>) up to 79%. In summary, our data demonstrate that the activation of VEGFR-1 and VEGFR-2 heterodimer (VEGFR-1/R-2) is essential for PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(<em>121</em>), which cannot be reproduced by the parallel activation of VEGFR-1 and VEGFR-2 homodimers with corresponding agonists. In addition, the binding of VEGF-A(165) to NRP-1 potentiates its capacity to promote PGI(2) synthesis.

BACKGROUND

Angiogenesis has been implicated in the pathogenesis of idiopathic interstitial pneumonia (IIP). The aim of this study was to examine the relationship between plasma concentrations of the angiogenic cytokines interleukin 8 (IL-8), vascular endothelial growth factor (VEGF), and endothelin-1 (ET-1) and clinical parameters of disease progression over a 6 month period to identify potential aetiological mediators and prognostic markers of disease activity in patients with IIP.

METHODS

Forty nine patients with IIP (40 men) were recruited to the study. Plasma cytokine measurements, pulmonary function tests, and high resolution computed tomography (HRCT) scans were performed on recruitment and after 6 months. Plasma cytokine measurements were also performed in 15 healthy volunteers for control purposes.

RESULTS

Patients with IIP had significantly higher median (IQR) baseline concentrations of IL-8 and ET-1 than controls (155 (77-303) pg/ml v 31 (0-100) pg/ml, p<0.001) and (1.21 (0.91-1.88) pg/ml v 0.84 (0.67-1.13) pg/ml, p<0.01), respectively. Baseline concentrations of IL-8, ET-1, and VEGF were significantly related to the baseline HRCT fibrosis score (r = 0.42, p<0.005; r = 0.39, p<0.01; and r = 0.42, p<0.005, respectively). Patients with IIP who developed progressive disease had significantly higher baseline levels of IL-8 (345 (270-497) pg/ml v 121 (73-266) pg/ml, p = 0.001) and VEGF (1048 (666-2149) pg/ml v 658 (438-837) pg/ml, p = 0.019). Over 6 months the change in VEGF was significantly related to the change in HRCT fibrosis score (r = 0.565, p = 0.035) and negatively related to the change in forced vital capacity (r = -0.353, p = 0.035).

Patients with previously untreated squamous cell lung carcinomas were evaluated to see if combining the expression of molecular and cellular <em>factors</em> with the most important clinical prognostic <em>factors</em> could improve the diagnostic ability to predict prognosis. For this reason, immunohistochemistry was used to examine the squamous cell lung carcinomas from <em>121</em> patients for their expression of ERBB-1, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), cyclin A, FOS, JUN and MYC. Median survival was shorter for patients with ERBB-1-, VEGF-, cyclin A-, FOS-, or JUN-positive tumours. For those patients with positive lymph node involvement, the survival times were also shorter in the VEGF-positive, cyclin A-positive and FOS-positive groups. Multivariate analysis independently demonstrated a significant prognostic value for lymph node involvement, VEGF and FOS.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is essential for the induction of angiogenesis and drives both <em>endothelial</em> cell (EC) proliferation and migration. It has been suggested that VEGF also regulates vessel diameter, although this has not been tested explicitly. The two most abundant isoforms, VEGF(<em>121</em>) and VEGF(165), both signal through VEGF receptor 2 (VEGFR-2). We recently optimized a three-dimensional in vitro angiogenesis assay using HUVECs <em>growing</em> on Cytodex beads and embedded in fibrin gels. Fibroblasts provide critical <em>factors</em> that promote sprouting, lumen formation, and vessel stability. Using this assay, we have examined the role of VEGF in setting vessel diameter. Low concentrations of both VEGF(<em>121</em>) and VEGF(165) promote <em>growth</em> of long, thin vessels, whereas higher concentrations of VEGF remarkably enhance vessel diameter. Placental <em>growth</em> <em>factor</em>, which binds to VEGFR-1 but not VEGFR-2, does not promote capillary sprouting. Moreover, specific inhibition of VEGFR-2 signaling results in a dramatic reduction of EC sprouting in response to VEGF, indicating the critical importance of this receptor. The increase in vessel diameter is the result of cell proliferation and migration, rather than cellular hypertrophy, and likely depends on MEK1-ERK1/2 signaling. Both phosphatidylinositol 3-kinase and p38 activity are required for cell survival. We conclude that the diameter of new capillary sprouts can be determined by the local concentration of VEGF and that the action of VEGF on angiogenic EC in this assay is critically dependent on signaling through VEGFR-2.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) has five isoforms (VEGF206, 189, 165, 145 and <em>121</em>). Increased VEGF expression in renal cell carcinoma (RCC) is associated with angiogenesis, but, it is not apparent which isoform is involved in this effect. We examined the isoform patterns of VEGF by reverse transcription-polymerase chain reaction (RT-PCR) in 47 RCCs. All showed increased VEGF expression as compared with extraneoplastic renal tissue. Four of the 47 RCCs showed VEGF<em>121</em> alone, 10 showed VEGF<em>121</em> + 165, and 33 showed the VEGF<em>121</em> + 165 + 189 pattern. Patients with pathological stage pT3-4 RCC showed the VEGF<em>121</em> + 165 + 189 isoform pattern at a significantly higher incidence (10/10, 100%) than those with pT0-2 (23/37, 62%) (P < 0.022). The VEGF<em>121</em> + 165 + 189 isoform pattern was also significantly associated with high vessel counts and density (P = 0.0002, Mann-Whitney U test). These observations suggested that the VEGF189 mRNA isoform is closely associated with angiogenesis and results in the <em>growth</em> of RCC.

A major challenge for therapeutic delivery of angiogenic agents such as <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is to achieve sustained, low dose signaling leading to durable neovessel formation. To this end, we recently created a variant of VEGF(<em>121</em>), TG-VEGF(<em>121</em>) that directly binds to fibrin and gets released locally in proteolysis-triggered manner. Here we combined noninvasive biophotonic monitoring of VEGF receptor 2 gene activation in transgenic VEGFR2-luc mice and histomorphometry to compare <em>endothelial</em> activation and long-term neo<em>vascular</em>ization by actively released TG-VEGF(<em>121</em>)versus passively released, diffusible wild-type VEGF(<em>121</em>) in subcutaneous fibrin implants. Monitoring in real-time over 3 weeks of luciferase signal driven by the VEGFR2 promoter revealed <em>endothelial</em> activation in skin exposed to wild-type VEGF(<em>121</em>), but no detectable elevation over fibrin alone by TG-VEGF(<em>121</em>). Histology at 3 weeks, however, demonstrated that TG-VEGF(<em>121</em>) promoted vessel <em>growth</em> significantly more effectively and reliably than wild-type VEGF(<em>121</em>). The majority of vessels surviving to 3 weeks contained stabilizing smooth muscle cells. Yet, by 6 weeks, no extra vessels induced by exogenous VEGF were left. In conclusion, release of fibrin-conjugated variant TG-VEGF(<em>121</em>) elicited lower VEGFR2-luc activation than wild-type VEGF(<em>121</em>) yet significantly more <em>vascular</em>ization. In the absence of true physiological demand, even stabilized vessels are ultimately regressed.