<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a multifunctional cytokine with distinct functions in angiogenesis, lymphangiogenesis, <em>vascular</em> permeability, and hematopoiesis. VEGF is a highly conserved, disulfide-bonded dimeric glycoprotein of 34 to 45 kDa produced by several cell types including fibroblasts, neutrophils, <em>endothelial</em> cells, and peripheral blood mononuclear cells, particularly T lymphocytes and macrophages. Six VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene, consisting of <em>121</em>, 145, 165, 183, 189, or 206 amino acids. VEGF<em>121</em>, VEGF145, and VEGF165 are secreted whereas VEGF183, VEGF189, and VEGF206 are cell membrane-bound. VEGF145 has a key role during the <em>vascular</em>ization of the human ovarian follicle and corpus luteum, in the placentation and embryonic periods, and in bone and wound healing, while VEGF165 is the most abundant and biologically active isoform. VEGF has been linked with a number of <em>vascular</em> pathologies including cardio<em>vascular</em> diseases such ischemic heart disease, heart failure, stroke, and diabetes and its related complications. In this review we aimed to present some important roles of VEGF in a number of clinical issues and indicate its involvement in several phenomena from the initial steps of the embryonic period to cardio<em>vascular</em> diseases.

This randomized phase II trial compared panitumumab plus fluorouracil, leucovorin, and irinotecan (FOLFIRI) with bevacizumab plus FOLFIRI as second-line chemotherapy for wild-type (WT) KRAS exon 2 metastatic colorectal cancer (mCRC) and to explore the values of oncogenes in circulating tumor DNA (ctDNA) and serum proteins as predictive biomarkers. Patients with WT KRAS exon 2 mCRC refractory to first-line chemotherapy containing oxaliplatin and bevacizumab were randomly assigned to panitumumab plus FOLFIRI or bevacizumab plus FOLFIRI. Of <em>121</em> randomly assigned patients, 117 were eligible. Median overall survival (OS) for panitumumab plus FOLFIRI and bevacizumab plus FOLFIRI were 16.2 and 13.4 months [hazard ratio (HR), 1.16; 95% CI, 0.76-1.77], respectively. Progression-free survival (PFS) was also similar (HR, 1.14; 95% CI, 0.78-1.66). KRAS, NRAS, and BRAF status using ctDNA was successfully examined in 109 patients, and mutations were identified in 19 patients (17.4%). Panitumumab plus FOLFIRI showed favorable survival compared with bevacizumab plus FOLFIRI in WT patients and unfavorable survival in those with mutations (P for interaction = 0.026 in OS and 0.054 in PFS). OS with bevacizumab plus FOLFIRI was better than panitumumab plus FOLFIRI in patients with high serum <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-A (VEGF-A) levels and worse in those with low levels (P for interaction = 0.016). Second-line FOLFIRI plus panitumumab and FOLFIRI plus bevacizumab showed a similar efficacy in patients with WT KRAS exon 2 mCRC. RAS and BRAF mutation in ctDNA could be a negative predictive marker for panitumumab.

Background and Aims: Intermediate stage hepatocellular carcinoma (HCC) can be treated by transarterial chemoembolization (TACE). However, there appear to be side effects, such as induction of proangiogenic <em>factors</em>, e.g. <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), which have been shown to be associated with a poor prognosis. This prospective study was designed to compare serum VEGF level response after TACE with different embolic agents in patients with HCC. Methods: Patients were assigned to one of three different TACE regimens: degradable starch microspheres (DSM) TACE, drug-eluting bead (DEBDOX) TACE or Lipiodol TACE (cTACE). All patients received 50 mg doxorubicin/m2 body surface area (BSA) during TACE. Serum VEGF levels were assessed before TACE treatment, 24 h post-treatment and 4 weeks later. Results: Twenty-two patients with 30 TACE treatments were enrolled. Compared to baseline VEGF levels, a marked increase was observed for 24 h post-TACE (164% of baseline level) and during the 4-week follow-up (170% of baseline level) only for the cTACE arm (p < 0.05). In contrast, the increase of serum VEGF levels were only 114% and 123% for DEBDOX and <em>121</em>% and 124% for DSM, respectively. Conclusions: Conventional TACE using Lipiodol shows marked increase in blood levels of the proangiogenic <em>factor</em> VEGF, while DEBDOX and DSM TACE induce only a moderate VEGF response.

<AbstractText>To identify maternal plasma protein markers for early preeclampsia (delivery <34 weeks of gestation) and to determine whether the prediction performance is affected by disease severity and presence of placental lesions consistent with maternal <em>vascular</em> malperfusion (MVM) among cases.</AbstractText><AbstractText>This longitudinal case-control study included 90 patients with a normal pregnancy and 33 patients with early preeclampsia. Two to six maternal plasma samples were collected throughout gestation from each woman. The abundance of 1,125 proteins was measured using high-affinity aptamer-based proteomic assays, and data were modeled using linear mixed-effects models. After data transformation into multiples of the mean values for gestational age, parsimonious linear discriminant analysis risk models were fit for each gestational-age interval (8-16, 16.1-22, 22.1-28, 28.1-32 weeks). Proteomic profiles of early preeclampsia cases were also compared to those of a combined set of controls and late preeclampsia cases (n = 76) reported previously. Prediction performance was estimated via bootstrap.</AbstractText><AbstractText>We found that 1) multi-protein models at 16.1-22 weeks of gestation predicted early preeclampsia with a sensitivity of 71% at a false-positive rate (FPR) of 10%. High abundance of matrix metalloproteinase-7 and glycoprotein IIbIIIa complex were the most reliable predictors at this gestational age; 2) at 22.1-28 weeks of gestation, lower abundance of placental <em>growth</em> <em>factor</em> (PlGF) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A, isoform <em>121</em> (VEGF-<em>121</em>), as well as elevated sialic acid binding immunoglobulin-like lectin 6 (siglec-6) and activin-A, were the best predictors of the subsequent development of early preeclampsia (81% sensitivity, FPR = 10%); 3) at 28.1-32 weeks of gestation, the sensitivity of multi-protein models was 85% (FPR = 10%) with the best predictors being activated leukocyte cell adhesion molecule, siglec-6, and VEGF-<em>121</em>; 4) the increase in siglec-6, activin-A, and VEGF-<em>121</em> at 22.1-28 weeks of gestation differentiated women who subsequently developed early preeclampsia from those who had a normal pregnancy or developed late preeclampsia (sensitivity 77%, FPR = 10%); 5) the sensitivity of risk models was higher for early preeclampsia with placental MVM lesions than for the entire early preeclampsia group (90% versus 71% at 16.1-22 weeks; 87% versus 81% at 22.1-28 weeks; and 90% versus 85% at 28.1-32 weeks, all FPR = 10%); and 6) the sensitivity of prediction models was higher for severe early preeclampsia than for the entire early preeclampsia group (84% versus 71% at 16.1-22 weeks).</AbstractText><AbstractText>We have presented herein a catalogue of proteome changes in maternal plasma proteome that precede the diagnosis of preeclampsia and can distinguish among early and late phenotypes. The sensitivity of maternal plasma protein models for early preeclampsia is higher in women with underlying <em>vascular</em> placental disease and in those with a severe phenotype.</AbstractText>

OBJECTIVE

Vascular endothelial growth factor (VEGF) gene expression has been linked to cancer progression. Here we hypothesise that the polymorphism and protein expression of VEGF are correlated with the pathogenesis and therapy response of pterygium.

METHODS

60 pterygial and 121 normal conjunctival samples were collected to determine the genotypes and protein expression of VEGF. Primary pterygium cells (PECs) were used to confirm the effect of the VEGF polymorphism on the angiogenesis of pterygium.

RESULTS

48 (83.3%) pterygial specimens tested positive for VEGF protein expression, which was significantly higher than in the control groups (16.7%, p<0.0001). The frequency of the 936 C>T variant, but not the -2578C>A variant, was significantly higher in the pterygium group compared with the control group. VEGF protein expression was significantly higher in the 936 C/C group than in the 936 C/T and T/T groups (p=0.001). The results of our cell model showed that PECs with the C/C genotype had a higher angiogenesis ability and higher response to the antiangiogenesis drug bevacizumab than cells with the C/T and T/T genotypes.

CONCLUSIONS

We suggest that VEGF could be used as a target for pterygium therapy in patients with the 936C>T genotype.

OBJECTIVE

To investigate the expression of vascular endothelial growth factor A (VEGF-A) and that its two receptors (VEGFR1, VEGFR2) in human preantral follicles.

METHODS

Immunohistochemical, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR) study of the expression of the VEGF-A system in human ovaries.

METHODS

Major tertiary-care academic center.

METHODS

Twenty-two patients who underwent pregnancy terminations at 21-35 gestational weeks and 29 girls/women aged 5-39 years who underwent ovarian laparoscopies.

METHODS

None.

METHODS

Laboratory analysis of human ovarian specimens.

RESULTS

Immunhistochemistry and in situ hybridization revealed the expression of the proteins and mRNA transcripts for VEGFR1 and VEGFR2 in oocytes, granulosa cells, and stroma cells from fetuses and girls/women. The protein for VEGF-A was detected immunohistochemically in oocytes, granulosa cells, and stroma cells from fetuses and girls. VEGF-A and VEGFR1 proteins were expressed more strongly than VEGFR2. VEGF-A(121), VEGF-A(165), and VEGF-A(189) isoforms were identified by RT-PCR in the ovarian samples from fetuses and women.

CONCLUSIONS

The presence of the VEGF-A receptors, particularly in the granulosa cells, suggests that VEGF-A might be involved in proliferation initiation of primordial follicles or play an as yet unknown role in human preantral follicles.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and placenta <em>growth</em> <em>factor</em> (PIGF) are members of a dimeric-<em>growth</em>-<em>factor</em> family with angiogenic properties. VEGF is a highly potent and specific mitogen for <em>endothelial</em> cells, playing a vital role in angiogenesis in vivo. The role of PIGF is less clear. We expressed the monomeric splice forms VEGF-165, VEGF-<em>121</em>, PIGF-1 and PIGF-2 as unfused genes in Escherichia coli using the pCYTEXP expression system. In vitro dimerization experiments revealed that both homo- and hetero-dimers can be formed from these monomeric proteins. The dimers were tested for their ability to promote capillary <em>growth</em> in vivo and stimulate DNA synthesis in cultured human <em>vascular</em> <em>endothelial</em> cells. Heterodimers comprising different VEGF splice forms, or combinations of VEGF/PIGF splice forms, showed mitogenic activity. The results demonstrate that four different heterodimeric <em>growth</em> <em>factors</em> are likely to have as yet uncharacterized functions in vivo.

The aim of this study was to investigate the role of ultrasonographic micro<em>vascular</em> imaging in the evaluation of prognosis of patients with invasive breast cancer treated by adjuvant therapies. A total of <em>121</em> patients with invasive breast cancer underwent ultrasonographic contrast-enhanced imaging, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) staining, and microvessel density (MVD) counts. The parameters of micro<em>vascular</em> imaging and the expression of VEGF and MVD in primary breast cancer were calculated. The correlation between these <em>factors</em> and the overall and progression-free survival rate were analyzed using the Kaplan-Meier method. Among <em>121</em> cases, the positive VEGF cases were 75 and negative ones were 46. The cut point of 52.3 was calculated by the regressive curve for MVD counts. The data showed the mean intensity (MI) was positively associated with both the MVD counts (r = .51, p < .001) and VEGF expression (r = .35, p < .001). For the prognosis of patients, high VEGF expression and MVD counts were associated with reduced progressive and survival times (PFS, p = .032 and p = .034; OS, p = .041 and p = .038, respectively). The correlation between parameters of micro<em>vascular</em> imaging, VEGF expressive status, and the MVD counts were established. The cut point of mean intensity (MI = 40) was used to investigate as an independent predictor for PFS (p = .021) and OS (p = .025), respectively, due to a strong correlation between MVD counts and VEGF expression in patients with invasive breast cancer. The micro<em>vascular</em> imaging could be a visual and helpful tool to predict the prognosis of patients with invasive breast cancer treated by adjuvant therapies.

OBJECTIVE

Different therapy strategies for coronary disease in conventionally untreatable patients have been developed, among them transmyocardial laser re<em>vascular</em>ization (TMLR) and the application of <em>growth</em> <em>factors</em>. The objective of our study was to determine whether a combined therapy of TMLR with a <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>(<em>121</em>) (VEGF(<em>121</em>)) plasmid is able to stimulate the development of sufficient collateral circulation and hereby to preserve cardiac function.

METHODS

A severe stenosis of the left anterior descending artery was created in healthy pigs. After 1 week, perfusion and regional contractility were assessed at baseline. Afterwards, the ischemic area was treated with TMLR (n=8), intramyocardial injection of naked plasmid DNA encoding VEGF(<em>121</em>) (n=7), or both (n=7). Control animals were left untreated (n=8). After 3 months, the animals were re-examined and underwent immunohistological analysis.

RESULTS

The number of capillaries increased only after injection of VEGF(<em>121</em>) plasmid alone compared to untreated ischemia and to the other therapy groups, whereas the number of arterioles was higher following TMLR treatment alone or in combination with VEGF(<em>121</em>) than it was in the case in untreated ischemic animals. However, only combined VEGF(<em>121</em>)+TLMR therapy resulted in an improvement in regional myocardial blood flow in comparison with 1 week ischemia, indicating the efficient development of collateral circulation. In contrast, better regional contractility compared to the 1-week baseline, as well as restoration of the pre-ischemic values, were achieved by both VEGF(<em>121</em>) and combined VEGF(<em>121</em>)+TLMR therapies.

CONCLUSIONS

This study of chronic myocardial ischemia with a porcine model indicates a synergistic action of TMLR and VEGF(<em>121</em>) gene therapy. Combined treatment alone achieved an increase of regional myocardial perfusion, which accompanied arteriogenesis and corresponded with the restoration of regional function.

BACKGROUND

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis. We hypothesized that a combination of recombinant angiogenic proteins might induce myocardial VEGF production and cause a shift in the mRNA signal produced.

METHODS

The left ventricles of New Zealand white rabbits were injected with 500 microL of saline, basic fibroblast growth factor (bFGF), platelet-derived growth factor-AB (PDGF(AB)), platelet-derived growth factor-BB (PDGF(BB)), bFGF + PDGF(AB), or bFGF + PDGF(BB). Myocardial VEGF production was analyzed by ELISA while mRNA splice variants were analyzed by RT-PCR 3 and 7 days after injection.

RESULTS

PDGF(BB) alone caused the most pronounced induction of VEGF. Three days after injection the induction of VEGF by PDGF(BB) was significant compared to all treatment groups, except the bFGF + PDGF(BB) group. Induction of VEGF by PDGF(BB) was associated with a decrease in mRNA production of VEGF(121) within the myocardium.

CONCLUSIONS

Injection of PDGF(BB) induces significant production of VEGF within the myocardium. This induction of VEGF production is associated with a shift toward other, less soluble forms of VEGF. These findings may allow more precise regulation of the myocardial response to therapeutic angiogenesis.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a powerful agent that causes hyperpermeability of blood vessels as well as <em>endothelial</em> cell proliferation. Recent investigations have revealed that production of VEGF increases in epidermis of psoriatic lesions, and the overproduced VEGF plays an important role in the pathogenesis of psoriasis. In this study, we used immunohistochemical staining as well as extraction of stratum corneum with physiological saline to further analyse VEGF produced in psoriatic lesions. Biological activity of VEGF in the psoriatic scales was assayed by cultured human umbilical vein <em>endothelial</em> cells in vitro. The immunohistochemical examination confirmed an increased production of VEGF in the keratinocytes of psoriatic lesions. In addition, we found that the content of VEGF contained in the psoriatic scales was approximately 50 times greater than that in normal stratum corneum. We also found that VEGF <em>121</em> isoform, which has an exclusive ability to cause hyperpermeability of blood vessels, was predominantly detected in psoriatic scales, suggesting a major role of VEGF <em>121</em> isoform on the altered structure of microvessels in psoriatic lesions.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-A (VEGF) exists as five different isoforms, which exert their <em>growth</em> stimulatory effects through interaction with the FLK and KDR receptors. The VEGF(<em>121</em>) isoform has been employed as a highly selective carrier of therapeutic agents to target tumor <em>endothelial</em> cells resulting in inhibition of tumor <em>growth</em> and metastasis. VEGF(<em>121</em>) and VEGF(<em>121</em>)/rGel fusion toxin containing hexa-histidine tags were expressed in Escherichia coli AD494 (DE3) pLysS. Media containing glycerol as a primary carbon source increased the specific expression levels of soluble VEGF(<em>121</em>) and VEGF(<em>121</em>)/rGel (mg/L/OD10) by more than two-fold over LB media when grown in a batchtype cultivation in a bioreactor. High cell densities over OD 40 were achieved using a fed-batch method and employing feeding medium containing glycerol and yeast extract. The overall production of the target proteins was improved 18-fold for VEGF(<em>121</em>) (59.2mg/L) and 27-fold for VEGF(<em>121</em>)/rGel (42.5mg/L), respectively, compared to the conventional flask cultivation method (3.3 and 1.6mg/L for VEGF(<em>121</em>) and VEGF(<em>121</em>)/rGel, respectively). The purified VEGF(<em>121</em>) and VEGF(<em>121</em>)/rGel fusion proteins were biologically active as assessed by phosphorylation of KDR receptors and cytotoxicity against KDR expressing cells.

Coexpression of a reporter gene and a therapeutic gene may allow for noninvasive monitoring of cardiac gene therapy. We sought to evaluate the usefulness of an adenoviral vector expressing mutant herpesviral thymidine kinase reporter gene (HSV1-sr39tk) and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) <em>121</em> in independent expression cassettes (Ad4tk).

METHODS

Accumulation of 14C-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FIAU) and 9-(4-18F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) as reporter probes, and secretion of VEGF into medium, were determined for Ad4tk-infected H9c2 rat cardiac cells in vitro.

RESULTS

In vitro tracer uptake increased with increasing vector concentration and over time. It was comparable to cells infected with adenovirus expressing only wild-type HSV1-tk (reporter probe: 14C-FIAU) or mutant HSV1-sr39tk (reporter probe: 18F-FHBG). No significant uptake was observed in cells infected with adenovirus expressing VEGF alone. With increasing vector concentration, Ad4tk-infected cells increasingly released VEGF into medium. VEGF production correlated significantly with cellular reporter probe uptake (r = 0.93; P = 0.0003).

CONCLUSIONS

The usefulness of a vector coexpressing HSV1-tk and VEGF for noninvasive imaging of expression of a therapeutic transgene has been demonstrated in vitro. This approach may allow for future in vivo monitoring of cardiac angiogenesis gene therapy.

Recent evidence points to the involvement of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) in mammalian reproductive physiology. Transgenic mice expressing VEGF (<em>121</em> isoform) under the control of the polyepithelial mucin-1 (muc-1) promoter showed a reduction in male fertility due to impaired spermiogenesis, and aberrant placentation leading to preferential rejection of male embryos. A skew in the sex ratio of the litters was seen (three females to two males), independently of whether the transgene was carried by the male or female parent. In-situ hybridization permitted distinction of expression of the human VEGF transgene from endogenous mouse VEGF, and confirmed expression of the transgene in a wide range of epithelial tissues. Expression of the transgene in spermatocytes and in the embryonic portion of placenta is thought to be responsible for the reduced fertility and embryonic resorptions respectively. Males showed either complete sperm maturation arrest or various gradations of partial fertility. Abnormally high or low VEGF in human semen has been reported to be correlated with a lack of pregnancy success following IVF. The muc1-VEGF (<em>121</em> isoform) transgenic mouse provides an animal model with which to further study this VEGF-induced pathology.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a <em>growth</em> <em>factor</em> for <em>vascular</em> <em>endothelial</em> cells in vitro. The present study was designed to determine whether serum VEGF levels increase in patients with acute myocardial infarction (AMI) compared with patients with stable exertional angina and control subjects, and to examine the serial changes of serum VEGF levels in patients with AMI. We examined serum VEGF levels by using antibody prepared from serum immunized with human VEGF(<em>121</em>). The serum VEGF level (pg/ml) was higher (p < 0. 0001) on admission in the patients with AMI (177 +/- 19) than in those with stable exertional angina (61 +/- 7) and control subjects (62 +/- 6). The serum VEGF level (pg/ml) of the patients with AMI was 177 +/- 19 on admission, 125 +/- 9 on day 3, 137 +/- 11 on day 5, 242 +/- 18 at 1 week, and 258 +/- 22 at 2 weeks after admission. The value was higher on admission than on day 3 after admission (p = 0.014), the values were higher at 1 week and 2 weeks than on admission, on day 3, and 5 (p < 0.01). Furthermore, there were correlations between peak VEGF levels at 1 week or 2 weeks after admission and peak creatine kinase levels. The increase of VEGF on admission in the patients with AMI may be due to the hypoxia of acute myocardial ischemia. The elevation at 1 week and 2 weeks from the onset may cause the development of collateral circulation in relation to the healing of the infarction site.

BACKGROUND

Tumor angiogenesis is a dynamic process that plays a major role in cancer progression. Vascular endothelial growth factor (VEGF) and its receptors play a pivotal role in angiogenesis. The expression of VEGF and its receptors VEGFR-1 and VEGFR-2 in renal cell carcinoma (RCC) was investigated in the perspective of anti-VEGF treatments.

METHODS

Total VEGF protein levels were quantified by enzyme-linked immunosorbent assay (ELISA) in tumor tissue samples from surgical specimens of 65 patients with clear cell RCC. At the cellular level the VEGF isoforms VEGFR-1 and VEGFR-2 mRNA were quantified by real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) in laser-microdissected tumoral epithelial as stromal cells and in corresponding normal tissue compartments. Colocalization of VEGF and VEGFR-1 proteins was studied by triple immunofluorescent labeling.

RESULTS

Protein VEGF in cytosolic extracts was significantly higher in tumoral than in nontumoral tissue (P< .0001). Event-free survival was significantly longer for patients with cytosolic VEGF lower than the cutoff (75th percentile of VEGF protein levels, P= .02). In laser-microdissected epithelial cells, VEGF(121) and VEGFR-1 mRNA expressions were higher in RCC than in corresponding nontumoral kidney (P= .007 and P= .002, respectively); they were also higher in stromal cells of RCC compared with nontumoral kidney (P= .02 and P= .003, respectively). There was no differential VEGFR-2 expression in epithelial or in stromal cells of tumoral or nontumoral kidney. By immunofluorescent labeling VEGF and VEGFR-1 colocalized on RCC tumor epithelial and stromal cells.

CONCLUSIONS

Combined laser microdissection and quantitative RT-PCR, as triple immunofluorescent labeling, underlined the preferential expression of the most soluble VEGF isoform, VEGF(121), and its receptor VEGFR-1, but not VEGFR-2, in epithelial and stromal cells of RCC.

The serine protease granzyme B (GrB; 25 kDa) is capable of inducing apoptosis through both caspase-dependent and caspase-independent mechanisms. We designed a novel <em>vascular</em>-targeting fusion construct designated as GrB/<em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)<em>121</em>, which is composed of a non-heparin-binding isoform of VEGF and the proapoptotic pathway enzyme GrB fused via a short, flexible tether (G4S). The chimeric fusion gene was then cloned into a bacterial vector, and the protein was expressed in Escherichia coli and purified by nickel-NTA metal affinity chromatography. Western blotting confirmed incorporation of both VEGF<em>121</em> and GrB proteins into the construct. GrB/VEGF<em>121</em> specifically bound (ELISA) to porcine aortic <em>endothelial</em> (PAE)/FLK-1 cells overexpressing the FLK-1/KDR receptor but not to cells overexpressing the FLT-1 receptor. Immunofluoresence studies showed that the GrB moiety of GrB/VEGF<em>121</em> was delivered efficiently and rapidly into the cytosol of PAE/FLK-1 cells but not into that of PAE/FLT-1 cells after 4 h treatment with GrB/VEGF<em>121</em>. Treatment of cells with GrB/VEGF<em>121</em> showed that the IC50 was approximately 10 nM against PAE/FLK-1 cells; however, there were no cytotoxic effects observed on PAE/FLT-1 cells at doses up to 200 nM. GrB/VEGF<em>121</em> induced apoptotic events specifically on PAE/FLK-1 as assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay, DNA laddering, and cytochrome c release from mitochondria. In addition, the fusion construct mediated the cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase in target <em>endothelial</em> cells within 4 h after treatment. In conclusion, delivery of the human proapoptotic pathway enzyme GrB to tumor <em>vascular</em> <em>endothelial</em> cells or to tumor cells may have significant therapeutic potential and represents a potent new class of targeted therapeutic agents with a unique mechanism of action.

BACKGROUND

Surgical cardiac revascularization produces a high degree of systemic inflammation and the secretion of several cytokines. Intensive postoperative inflammation may increase the incidence of postoperative atrial fibrillation and favor organ dysfunctions. No data documenting the anti-inflammatory properties of epicardial vagal ganglionated plexus stimulation are available.

OBJECTIVE

To verify the feasibility and safety of postoperative inferior vena cava-inferior atrial ganglionated plexus (IVC-IAGP) burst stimulation and the effectiveness of this approach in reducing serum levels of inflammatory cytokines.

METHODS

In 27 patients who were candidates for off-pump surgical revascularization, the IVC-IAGP was located during surgery, a temporary wire was inserted, and a negative atrioventricular node dromotropic effect was obtained in 20 patients on applying high-frequency burst stimulation. In 5 patients atrial fibrillation or phrenic nerve stimulation was induced, and the remaining 15 patients served as the experimental group. Twenty additional patients underwent off-pump surgical revascularization without IVC-IAGP stimulation and served as the control group. On arrival in the intensive care unit, the experimental group underwent IVC-IAGP stimulation for 6 hours. Blood samples were collected at different times.

RESULTS

The serum levels of cytokines were not statistically different at baseline and on arrival in the intensive care unit between the groups, while they proved statistically different after 6 hours of stimulation: interleukin-6 (EG: <em>121</em> ± 71 pg/mL vs CG: 280 ± 194 pg/mL; P = .004), tumor necrosis <em>factor</em>-α (EG: 2.68 ± 1.81 pg/mL vs CG: 5.87 ± 3.48 pg/mL; P = .003), <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (EG: 93 ± 43 pg/mL vs CG: 177 ± 86 pg/mL; P = .002), and epidermal <em>growth</em> <em>factor</em> (EG: 79 ± 48 pg/mL vs CG: 138 ± 76 pg/mL; P = .012).

CONCLUSIONS

Prolonged burst IVC-IAGP stimulation after surgical revascularization appears to be feasible and safe and significantly reduces inflammatory cytokines in the postoperative period.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is an intensively studied molecule that has significant potential, both in stimulating angiogenesis and as a target for antiangiogenic approaches. We utilised MCF-7 breast cancer cells transfected with either of two of the major VEGF isoforms, VEGF(<em>121</em>) or VEGF(165), or fibroblast <em>growth</em> <em>factor</em>-1 (FGF-1) to distinguish the effects of these <em>factors</em> on tumour <em>growth</em>, <em>vascular</em> function, and oxygen delivery. While each transfectant demonstrated substantially increased tumorigenicity and <em>growth</em> rate compared to vector controls, only VEGF(<em>121</em>) produced a combination of significantly reduced total and perfused vessel spacing, as well as a corresponding reduction in overall tumour hypoxia. Such pathophysiological effects are of potential importance, since antiangiogenic agents designed to block VEGF isoforms could in turn result in the development of therapeutically unfavourable environments. If antiangiogenic agents are also combined with conventional therapies such as irradiation or chemotherapy, microregional deficiencies in oxygenation could play a key role in ultimate therapeutic success.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is one of the most potent <em>factors</em> for stimulating angiogenesis, an essential process required for expansion of primary tumour and dissemination of malignant cells. To investigate the possible role of VEGF in facilitating metastasis of prostate cancer via stimulating angiogenesis, we have used Northern and slot blotting, reverse transcription polymerase chain reaction, nucleotide sequence analysis and enzyme-linked immunosorbent assay to compare the VEGF expression in series of human and rat cell lines with either benign or malignant characteristics. We have also employed the chick chorioallantoic membrane (CAM) assay to measure the angiogenic activity of the VEGF derived from both benign and malignant cells. The level of VEGF mRNA expressed in the seven malignant human and rat cell lines is 3.5- to 10-fold higher than that expressed in the benign cell lines. The three metastatic variants, generated by transfection of a benign cell line with DNA extracted from prostate carcinoma cells, expressed 2.5 to 5 times more VEGF mRNA than their parental benign cells. While VEGF <em>121</em> and 165 were predominantly expressed by both the benign and malignant cells, the transcript representing VEGF 189 isoform was only detected in the malignant cells. At protein level, three human malignant cell lines produced more VEGF (2.7-7.9 ng ml(-1)) than the benign cell line (1.3 ng ml(-1)). CAM assay detected a VEGF-dependent angiogenic activity in the medium from malignant cells, but only a relatively weak VEGF-independent activity in the medium from benign cells. These results demonstrated that malignant cells did over-express VEGF and only the VEGF derived from malignant cells was angiogenically active. Thus, we suggest that the VEGF produced by malignant cells might play an important role in facilitating metastasis of prostatic cancer.

The N-terminal region of parathyroid hormone (PTH) and PTH-related protein (PTHrP) interacts with a common PTH/PTHrP receptor in osteoblasts. These cells synthesize PTHrP, but its role in bone turnover is unclear. Intermittent treatment with N-terminal PTHrP or PTH stimulates bone <em>growth</em> in vivo, possibly by increasing local bone <em>factors</em>. In addition, C-terminal PTHrP (107-139), which does not bind to the PTH/PTHrP receptor, appears to affect bone resorption in vivo and in vitro, although its effect on bone formation in vivo remains controversial. Bone angiogenesis is an often overlooked but critical event in the process of bone remodeling. Recently, PTH (1-34) has been shown to induce gene expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), a potent angiogenic <em>factor</em>, by osteoblastic cells. However, no data are available on the effect of PTHrP (107-139) on VEGF expression in these cells. Using semiquantitative reverse transcription followed by PCR, we found that PTHrP (107-139), between 10 nM and 1 pM, increased VEGF mRNA in human osteoblastic (hOB) cells from trabecular bone. This effect of this agonist, at 10 nM, was maximal (fivefold for VEGF(165), and twofold for VEGF(<em>121</em>), compared to control) within 1 to 4 h. This effect was similar to that induced by PTHrP (1-34) in these cells, as well as in human osteosarcoma MG-63 cells, using Northern blot analysis. Moreover, the effect of both peptides, added together at 100 pM, was not higher than that observed with each peptide alone in hOB cells. The effects of PTHrP (107-139) and that of PTHrP (1-34) were abolished by actinomycin D in hOB cells. In these cells, the protein kinase C inhibitor staurosporine, but not the protein kinase A inhibitor H89, inhibited the increase in VEGF mRNA induced by 10 nM PTHrP (107-139). PTHrP (107-139), at 10 nM, also stimulated cytosolic VEGF immunostaining in hOB cells, and VEGF secretion into the medium conditioned by hOB or MG-63 cells for 24 h, which was (ng/mg protein): 10 +/- 1 or 5 +/- 3 (control), respectively, and 21 +/- 1 or 11 +/- 2 (PTHrP [107-139]-stimulated), respectively. Furthermore, medium conditioned by these cells for 24 h in the presence of 10 nM PTHrP (107-139), with or without 10 nM PTHrP (1-34), increased about 30% bovine aortic <em>endothelial</em> cell (BAEC) <em>growth</em> at 48 h. This effect was inhibited by adding a specific anti-VEGF antibody to the BAEC incubation medium. These findings demonstrate that the C-terminal domain of PTHrP induces expression and secretion of VEGF, a main angiogenic <em>factor</em>, in hOB cells and MG-63 cells. This relationship between PTHrP and VEGF has potential implications for both bone <em>vascular</em>ization and bone formation, and neoangiogenesis in PTHrP-producing tumors.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a potent angiogenic <em>factor</em> associated with the <em>growth</em> and metastasis of various cancers and plays a prominent role in vesical angiogenesis regulation. In this study, we investigated the effect of the phorbol 12-myristate 13-acetate (PMA) on the expression of VEGF in human bladder transitional carcinoma cells (RT4). RT4 cells expressed three VEGF isoforms (VEGF(189), VEGF(165), VEGF(<em>121</em>)). PMA increased VEGF mRNA expression time-dependently with a peak at 4 h. PMA increased the half-life of VEGF mRNA. The amount of VEGF protein in conditioned media was increased by PMA in a dose-dependent manner with a maximal effect at 10(-7) M. Staurosporine and calphostin C (PKC inhibitors) decreased PMA-induced VEGF mRNA expression as opposed to protein kinase A or cyclic nucleotide-dependent protein kinase inhibitors. Thus, in RT4 cells, VEGF expression is up-regulated by PMA via the PKC signalling pathway and according to a posttranscriptional mechanism.

OBJECTIVE

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> receptors (VEGFRs) are associated with tumor <em>growth</em> and induction of tumor angiogenesis and are known to be overexpressed in various human tumors. In the present study, we prepared and evaluated (68)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-benzyl (NOTA)-VEGF(<em>121</em>) as a positron emission tomography (PET) radioligand for the in vivo imaging of VEGFR expression.

METHODS

(68)Ga-NOTA-VEGF(<em>121</em>) was prepared by conjugation of VEGF(<em>121</em>) and p-SCN-NOTA, followed by radiolabeling with (68)GaCl(3) and then purification using a PD-10 column. Human aortic <em>endothelial</em> cell (HAEC) binding of (68)Ga-NOTA-VEGF(<em>121</em>) was measured as a function of time. MicroPET and biodistribution studies of U87MG tumor xenografted mice were performed at 1, 2, and 4 h after injection of (68)Ga-NOTA-VEGF(<em>121</em>). The tumor tissues were then sectioned and subjected to immunostaining.

RESULTS

The decay-corrected radiochemical yield of (68)Ga-NOTA-VEGF(<em>121</em>) was 40 ± 4.5 % and specific activity was 243.1 ± 104.6 GBq/μmol (8.6 ± 3.7 GBq/mg). (68)Ga-NOTA-VEGF(<em>121</em>) was avidly taken up by HAECs in a time-dependent manner, and the uptake was blocked either by 32 % with VEGF(<em>121</em>) or by 49 % with VEGFR2 antibody at 4 h post-incubation. In microPET images of U87MG tumor xenografted mice, radioactivity was accumulated in tumors (2.73±0.32 %ID/g at 2 h), and the uptake was blocked by 40 % in the presence of VEGF(<em>121</em>). In biodistribution studies, tumor uptake (1.84±0.14 %ID/g at 2 h) was blocked with VEGF(<em>121</em>) at a similar level (52 %) to that of microPET images. Immunostaining analysis of U87MG tumor tissues obtained after the microPET imaging showed high levels of VEGFR2 expression.

CONCLUSIONS

These results demonstrate that (68)Ga-NOTA-VEGF(<em>121</em>) has potential for the in vivo imaging of VEGFR expression. In addition, our results also suggest that the in vivo characteristics of radiolabeled VEGF depend on the properties of the radioisotope and the chelator used.

We have developed a <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> <em>121</em> (VEGF<em>121</em>)-based, dual positron emission tomography (PET)/optical imaging probe for monitoring VEGF receptor (VEGFR) expression using a streptavidin (SAv)-biotin platform. (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N″,N'″-tetraacetic acid (DOTA)-conjugated Alexa Fluor 680 (AF)-SAv/biotin-PEG-VEGF<em>121</em> ((64)Cu-labeled dual probe) was prepared with a radiochemical yield of 31.40 ± 3.30% and was stable for 24 h in serum. A human aortic <em>endothelial</em> cell binding study showed avid, time-dependent cellular uptake of the (64)Cu-labeled dual probe. MicroPET imaging of U87MG tumor-bearing mice injected with (64)Cu-labeled dual probe showed rapid, high accumulation of radioactivity in tumors, which reached 3.90 ± 0.17 %ID/g and 4.93 ± 0.80 %ID/g at 1 and 22 h after injection, respectively. Subsequent optical imaging of mice revealed strong fluorescence signals in tumors. Biodistribution studies performed after in vivo imaging demonstrated tumor uptake of 4.19 ± 0.14 %ID/g. Tumor uptake was blocked by 28% in the presence of VEGF<em>121</em>, confirming the VEGFR specificity of the (64)Cu-labeled dual probe. Ex vivo microPET images of major tissues showed the signal intensities consistent with optical images of the corresponding tissues. Moreover, it was shown that tumor uptake of the (64)Cu-labeled dual probe was not due to non-specific uptake by (64)Cu-DOTA-conjugated AF-SAv (tumor uptake; 1.57 ± 0.09 %ID/g). Taken together, these results suggest that the (64)Cu-labeled dual probe is a promising candidate for dual PET/optical imaging of VEGFR expression.