CI-1023 (AdGVVEGF<em>121</em>.10) is a replication-deficient adenovirus vector (complete E1a-, partial E1b-, partial E3-) delivering human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-<em>121</em> gene. Previous studies from this group have established that CI-1023 can successfully transfer human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-<em>121</em> gene resulting in local tissue expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> protein. The purpose of this study was to evaluate neo<em>vascular</em>ization-promoting potency and efficacy of CI-1023 in a wide dose range. In a rat hindlimb ischaemic model, we measured neo<em>vascular</em>ization-promoting effect of CI-1023 using three end-points: post mortem angiography, immuno-histochemistry and Laser Doppler scanning of tissue blood perfusion. Neo<em>vascular</em>ization-promoting activity of CI-1023 over the dose range of 4 x 10(6) pu-4 x 10(10) pu was evaluated. Our data demonstrated an obvious dose-dependent effect between 4 x 10(6) pu-4 x 10(8) pu. The neo<em>vascular</em>izing effect is somewhat plateaued at the levels between 4 x 10(8) pu and 4 x 10(10) pu. We conclude CI-1023 is a potent neo<em>vascular</em>ization-promoting compound, with a dose-dependent effect between 4 x 10(6) pu-4 x 10(8) pu in the rat hindlimb ischaemic model.

OBJECTIVE

To investigate the effect of antisense <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)(<em>121</em>) cDNA transfection on the <em>growth</em> of K562 cells in nude mice.

METHODS

K562 cells transfected with the antisense (AS) or sense (S) VEGF(<em>121</em>) cDNA, and the vector (V, pcDNA3) alone were transplanted subcutaneously into nude mice and the <em>growth</em> of the transfected cells in vivo was investigated. The effects of transfected K562 cells on human bone marrow <em>endothelial</em> cells (BMEC) were analyzed by MTT assay, the microvessel density (MVD) in tumor mass by vWF immunohistochemistry stain.

RESULTS

K562/V tumor grew more slowly [(207.5 +/- 192.9) mm(3) vs (445.0 +/- 150.9) mm(3), P < 0.05] and K562/S tumor more rapidly than K562/V tumor did [(1 174.6 +/- 508.7)/mm(3) vs (445.0 +/- 150.9) mm(3), P < 0.01]. K562/S cell culture supernatant was more strongly in promoting the proliferation of BMEC than K562/V supernatant did, but K562/AS supernatant resulted in a marked decrease of the promoting effect as compared with K562/V's. The MVDs in K562/AS, K562/S, and K562/V tumors were [(11.0 +/- 7.6)/0.72 mm(2) vs (50.8 +/- 11.7)/0.72 mm(2) vs (18.9 +/- 7.0)/0.72 mm(2)], respectively.

CONCLUSIONS

Antisense VEGF(<em>121</em>) cDNA transfected K562 cells show <em>growth</em> retardation in transplanted nude mice, decrease of tumor MVD, and decrease of promoting BMEC proliferation capacity.

UNASSIGNED

Angiogenesis which occurs mandatory in solid tumors, is a critical step in malignancy progression. Vascular endothelial growth factor (VEGF) is mainly responsible for angiogenesis process and facilitates the formation of new vessels. Distribution of monoclonal antibodies against VEGF or VEGF receptor (VEGFR) into the solid tumors is limited because of their huge dimensions. Moreover, many investigations have demonstrated the usefulness of immunotoxins to halt angiogenesis in solid tumors.

UNASSIGNED

We designed, expressed and evaluated the cytotoxicity of a novel nano-immunotoxin composed of VEGF splice variant containing 121 amino acids (VEGF121) and truncated the exotoxin A of Pseudomonas aeruginosa (PE38-KDEL). The fusion protein VEGF121-PE38 was successfully cloned and expressed in Escherichia coli, purified by Ni+ 2 affinity chromatography. The fusion protein was subsequently subjected to refolding using the reduced and oxidized glutathione.

UNASSIGNED

The expression level of the fusion protein reached to 1 mg/ml. The VEGF121-PE38 immunotoxin showed a 59 KDa MW which had cytotoxic effect on HUVEC and 293/KDR cells as low and high expressing VEGFR2 cells, respectively. But the cytotoxicity on 293/KDR was 100 folds more than that of VEGFR2 low expressing cell HUVEC.

UNASSIGNED

The designed immunotoxin showed more selectivity for higher VEGFR2 expressing cells in vitro.

OBJECTIVE

We hypothesized that gene transfer of select growth factors to the placenta may enhance placental and fetal growth. Thus, we examined the effect of 8 growth factor transgenes on murine placenta.

METHODS

Adenoviral-mediated site-specific intraplacental gene transfer of 8 different <em>growth</em> <em>factor</em> transgenes at embryonic day (e) 14 was performed. Transgenes included angiopoietin-1, angiopoietin-2 (Ang-2), basic fibroblast <em>growth</em> <em>factor</em>, hepatocyte <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1), placenta <em>growth</em> hormone, platelet-derived <em>growth</em> <em>factor</em>-B (PDGF-B), and <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>(<em>121</em>). Fetuses and placentas were harvested at e17 and assessed for survival, gene transfer efficiency, placenta area, and fetal and placental weights.

RESULTS

Efficient gene transfer to the placenta was detected with minimal dissemination to the fetus. Overexpression of IGF-1, PDGF-B, and Ang-2 resulted in an increase in placenta cross-sectional area. Only Ang-2 gene transfer resulted in increased fetal weight, and only Ang-2 and basic fibroblast growth factor resulted in a change in placental weight.

CONCLUSIONS

Site-specific placental gene transfer results in efficient gene transfer with minimal dissemination to the fetus. Adenoviral-mediated IGF-1, adenoviral-mediated PDGF-B, and adenoviral-mediated Ang-2 significantly increase placenta growth.

OBJECTIVE

To investigate the feasibility of intramuscular gene therapy for acute arterial ischemic diseases by use of plasmid pcDNA3-VEGF121 and to evaluate therapeutic efficiency of vascular endothelial growth factor (VEGF) by different routes of administration.

METHODS

Fifty New Zealand White rabbits were randomly assigned to either gelation sponge carrying-pcDNA3-VEGF121 (n = 18), intramuscular injection-pcDNA3-VEGF121 (n = 18), or pcDNA3 (as control group, n = 14). After ligation of the external iliac artery and complete excision of the femoral artery, 500 micrograms of the plasmid pcDNA3-VEGF121 were transfected into the muscles of the ischemic limb by gelation sponge carrying or direct intramuscular-injection. Immediately after gene transfection, blood flow of the internal iliac artery were measured. VEGF121 gene expression was detected by RT-PCR after 2 days, 1 week, 2 weeks, 3 weeks and 4 weeks of transfection. After 30 days, blood flow of the internal iliac artery, angiographic score and histological vessels of ischemic hindlimbs were measured respectively.

RESULTS

In the two VEGF-treated groups, VEGF121 mRNA expressed in the transfected ischemic muscles after 2 days and lasted 2 weeks. Immediately after gene transfection, blood flow of the internal iliac artery had no significant difference between three groups. After 30 days, blood flow of the internal iliac artery, angiographic score and capillary density were significantly greater in both VEGF-treated groups than in control group. Complexity of vascular branching and vessel density of gelation sponge-VEGF treated limbs were significantly greater when compared with the intramuscular-injection limbs.

CONCLUSIONS

These findings suggest the feasibility of employing gene therapy of pcDNA3-VEGF121 could augment collateral development and tissue perfusion in an animal model of hindlimb ischemia, and gelation sponge carrying VEGF gene may respect a potential therapy methods.

OBJECTIVE: To study the expression of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) mRNA isoforms in ovarian carcinoma and to explore their role in tumorigenesis and development of ovarian carcinoma. METHODS: The types and levels of VEGF mRNA isoforms of surgical samples from 30 patients with ovarian carcinoma were determined by relatively quantative RT-PCR, nest PCR and sequence analysis. RESULTS: VEGF(<em>121</em>), VEGF(145), VEGF(165) and VEGF(189)mRNA were detected in normal ovaries and ovarian carcinoma tissues. The expression level of VEGF(<em>121</em>) was significantly higher than that of VEGF(145), VEGF(165) and VEGF(189) (P<0.001, respectively). The expression of all 4 isoforms in carcinoma tissues was increased significantly compared with that in normal ovaries (P<0.05). CONCLUSION: Overexpression of VEGF(<em>121</em>), VEGF(145), VEGF (165) and VEGF(189) mRNA, especially VEGF(<em>121</em>), was found in varian carcinoma tissues. This findings suggest that all 4 VEGF isoforms may be involved in the tumorigenesis and development of ovarian carcinoma and VEGF(<em>121</em>) may play a key role.

Poor prognosis associated with the dysregulated expression of activin A in a number of malignancies has been related to with numerous aspects of tumorigenesis, including angiogenesis. The present study investigated the prognostic significance of activin A immunoexpression in blood vessels and cancer cells in a number of oral squamous cell carcinoma (OSCC) cases and applied in vitro strategies to determine the impact of activin A on angiogenesis. In a cohort of 95 patients with OSCC, immunoexpression of activin A in both blood vessels and tumor cells was quantified and the association with clinicopathological parameters and survival was analyzed. Effects of activin A on the tube formation, proliferation and migration of human umbilical vein <em>endothelial</em> cells (HUVECs) were evaluated in gain‑of‑function (treatment with recombinant activin A) or loss‑of‑function [treatment with activin A‑antagonist follistatin or by stable transfection with short hairpin RNA (shRNA) targeting activin A] conditions. Conditioned medium from an OSCC cell line with shRNA‑mediated depletion of activin A was also tested. The profile of pro‑ and anti‑angiogenic <em>factors</em> regulated by activin A was assessed with a human angiogenesis quantitative PCR (qPCR) array. <em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> A (VEGFA) and its major isoforms were evaluated by reverse transcription‑qPCR and ELISA. Activin A expression in blood vessels demonstrated an independent prognostic value in the multivariate analysis with a hazard ratio of 2.47 [95% confidence interval (CI), 1.30‑4.71; P=0.006) for disease‑specific survival and 2.09 (95% CI, 1.07‑4.08l: P=0.03) for disease‑free survival. Activin A significantly increased tubular formation of HUVECs concomitantly with an increase in proliferation. This effect was validated by reduced proliferation and tubular formation of HUVECs following inhibition of activin A by follistatin or shRNA, as well as by treatment of HUVECs with conditioned medium from activin A‑depleted OSCC cells. Activin A‑knockdown increased the migration of HUVECs. In addition, activin A stimulated the phosphorylation of SMAD2/3 and the expression and production of total VEGFA, significantly enhancing the expression of its pro‑angiogenic isoform <em>121</em>. The present findings suggest that activin A is a predictor of the prognosis of patients with OSCC, and provide evidence that activin A, in an autocrine and paracrine manner, may contribute to OSCC angiogenesis through differential expression of the isoform <em>121</em> of VEGFA.

OBJECTIVE

To construct the adenoviral vector bringing hVEGF(<em>121</em>) cDNA for evaluation of the possibility of VEGF gene therapy in ischemic bone disease.

METHODS

Human vascular endothelial growth factor (hVEGF(<em>121</em>)) cDNA obtained from the plasmid pCDI/VEGF(<em>121</em>) was cloned into plasmid pshuttle and further cloned to Adeno-X Viral DNA. The recombinant adenoviral plasmid was identified and then transferred to the adenoviral packaging cell HEK293 by lipofectamine mediated gene transfer method to pack the virus. After titilating the virus, the mouse bone marrow stromal cells (MSC) were transfected by the adenovirus and the expression of VEGF gene was detected.

RESULTS

The recombinant Adeno-VEGF(<em>121</em>) was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. After MSCs were tranfected by the virus, RT-PCR showed that hVEGF(<em>121</em>) mRNA was transcripted from the hVEGF(<em>121</em>) gene. Western blot and immune histochemistry showed VEGF(<em>121</em>) protein was expressed in transgene MSCs.

CONCLUSIONS

The recombinant adenoviral vector bringing hVEGF(<em>121</em>) cDNA was successfully constructed and the transgene MSC expressed hVEGF gene in vitro, it provided the further foundation of VEGF gene therapy for bone ischemic diseases.

Alterations in peripheral <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) levels were observed in major depressive disorder and relative treatments and were shown to be influenced by genetic variants. The study objective was to explore, at a genome-wide level, possible interplaying effects between the genetic background and major depressive disorder in regulating VEGF levels. Moreover, we aimed to investigate the association between these variants and response to electroconvulsive therapy. A genome-wide association study was carried out both on controls and patients with major depressive disorder (n = 145; n = <em>121</em>) in correlation with serum VEGF levels determined by ELISA. Five SNPs not included in SNP arrays were additionally genotyped. Seventy-one patients with treatment-resistant depression underwent electroconvulsive therapy and were evaluated as responders/nonresponders. An association between VEGF levels and a locus in 6p21.1, downstream the VEGF gene, was evidenced both in controls (best SNP: FDR-corrected p = 2.4 × 10^-5 ) and in patients with major depressive disorder (best SNP: FDR-corrected p = 2.6 × 10^-3 ). The alleles associated with lower VEGF concentrations in patients were also associated with nonresponse to electroconvulsive therapy (p = .01). These results confirm a role of SNPs in 6p21.1 locus as major influencers of circulating VEGF levels also in patients affected by major depressive disorder and indicate a possible implication in response to electroconvulsive therapy.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) plays an important role in both physiological and pathological angiogenesis. VEGF receptor‑1 (VEGFR‑1) acts as a decoy VEGF receptor that enables the regulation of VEGF on the <em>vascular</em> endothelium. In the present study, the recombinant human VEGFR1D1‑3/Fc (rhVEGFR‑1), which contains key domains for VEGF binding, was cloned and expressed in Chinese hamster ovary (CHO) cells. The rhVEGFR‑1 protein was purified using protein‑A affinity chromatography. The molecular weight of rhVEGFR‑1 was found to be ~162 and 81 kD in non‑reducing and reducing SDS‑PAGE, respectively. The majority of the final protein products were in the dimeric conformation. Western blot analysis revealed that rhVEGFR‑1 was only capable of binding to the full glycan form of rhVEGF‑165 and rhVEGF‑<em>121</em>. The dissociation constant for the binding of rhVEGFR‑1 to VEGF‑165, detected using Biacore, was 285 pM. In addition, rhVEGFR‑1 inhibited the proliferation and migration of human micro<em>vascular</em> <em>endothelial</em> cells. In vivo experiments also demonstrated that rhVEGFR‑1 inhibited chicken chorioallantoic membrane neo<em>vascular</em>ization and angiogenesis in nude mice. In conclusion, an anti‑angiogenic recombinant soluble VEGFR was expressed (up to 5 mg/l) in CHO cells and was shown to be capable of inhibiting neo<em>vascular</em>ization in vivo and in vitro.

The <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is an essential <em>factor</em> to pathologic angiogenesis. Disruption of VEGF/VEGF receptor interaction in cancer patients inhibits the development of new and pre-existing tumor blood vessels. Consequently, VEGF becomes an important therapeutic target for handling solid tumors. In this work, human VEGF was produced in the culture supernatant of SiHa cells transduced with a replication-defective adenoviral vector (pAdhVEGF_<em>121</em>) encoding this molecule. The 35 kDa VEGF_<em>121</em> homodimer was obtained from clarified culture media as a glycosylated protein. VEGF_<em>121</em> expression levels were strictly dependent on the adenoviral viral load used. VEGF_<em>121</em> was produced with purity over 98% after a single step chromatography by immobilized metal affinity chromatography. Additionally, VEGF_<em>121</em> binds Bevacizumab antibody with a K_D of 7 nM. Biological characterization by mitogenic assay in HUVEC and ECV-304 cells showed that VEGF_<em>121</em> stimulates cell proliferation in a dose-dependent manner in both cells. Finally, the neo<em>vascular</em>ization activity of VEGF_<em>121</em> was demonstrated by <em>vascular</em> permeability assays in matrigel plug-bearing mice, showing significantly increased vasculature leakage after treatment with VEGF_<em>121</em>. Consequently, transduction of SiHa cells with adenovirus is a suitable alternative for manufacture heterologous proteins of therapeutic interest.

Objective: Despite the current immunotherapy era, VEGFR inhibitors maintain effectiveness in metastatic renal cell cancer. Real-world data concerning pazopanib are limited. The aim of this study is to add information about efficacy and safety of pazopanib as first-line treatment in metastatic renal cell cancer patients not enrolled into clinical trials.

Methods: Retrospective analysis (the PAMERIT study) of first-line pazopanib in real-world metastatic renal cell cancer patients among 39 Centers in Italy. Outcomes were progression-free survival, overall survival, objective response rate and treatment-related adverse events. Kaplan-Meier curves, log-rank test and multivariable Cox's models were used and adjusted for age, histology, previous renal surgery, International Metastatic RCC Database Consortium score and pazopanib initial dose.

<strong class="sub-title"> Results: </strong> Among 474 patients, 87.3% had clear cell metastatic renal cell cancer histology. Most of them (84.6%) had upfront renal surgery. Median progression-free survival and overall survival were 15.8 and 34.4 months, respectively, significantly correlating with International Metastatic RCC Database Consortium's good prognosis (P < 0.001), ECOG PS 0 (P < 0.001), age (<75 years, P = 0.005), surgery (P < 0.001) and response to pazopanib (P < 0.001). After 3 months of pazopanib, overall disease control rate have been observed in 76.6% patients. Among International Metastatic RCC Database Consortium's favorable group patients, 57/<em>121</em> (47%) showed complete/partial response. No unexpected AEs emerged.

Conclusions: In this real-world study, metastatic renal cell cancer patients treated with first-line pazopanib reached greater progression-free survival and overall survival than in pivotal studies and had high response rates when belonging to International Metastatic RCC Database Consortium's favorable group, without new toxicities. Pazopanib has been confirmed a valid first-line option for International Metastatic RCC Database Consortium's good prognosis metastatic renal cell cancer patients who cannot be submitted to immunotherapy.

Keywords: first-line therapy; metastatic renal cell carcinoma; pazopanib; real-world effectiveness; vascular endothelial growth factor receptor tyrosine kinase inhibitors.

The anti-fibrotic properties of ranibizumab have been well documented. As an antagonist to <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF), ranibizumab works by binding and neutralizing all active VEGF-A, thus limiting progressive cell <em>growth</em> and proliferation. Ranibizumab application in ocular diseases has shown remarkable desired effects; however, to date, its antifibrotic mechanism is not well understood. In this study, we identified metabolic changes in ranibizumab-treated human Tenon's fibroblasts (HTFs). Cultured HTFs were treated for 48 h with 0.5 mg/mL of ranibizumab and 0.5 mg/mL control IgG antibody which serves as a negative control. Samples from each group were injected into Agilent 6520 Q-TOF liquid chromatography/mass spectrometer (LC/MS) system to establish the metabolite expression in both ranibizumab treated cells and control group. Data obtained was analyzed using Agilent Mass Hunter Qualitative Analysis software to identify the most regulated metabolite following ranibizumab treatment. At <i>p</i>-value < 0.01 with the cut off value of two-fold change, 31 identified metabolites were found to be significantly upregulated in ranibizumab-treated group, with six of the mostly upregulated having insignificant role in fibroblast cell cycle and wound healing regulations. Meanwhile, <em>121</em> identified metabolites that were downregulated, and seven of the mostly downregulated are significantly involved in cell cycle and proliferation. Our findings suggest that ranibizumab abrogates the tissue scarring and wound healing process by regulating the expression of metabolites associated with fibrotic activity. In particular, we found that vitamin Bs are important in maintaining normal folate cycle, nucleotide synthesis, and homocysteine and spermidine metabolism. This study provides an insight into ranibizumab's mechanism of action in HTFs from the perspective of metabolomics.

The molecular mechanisms of pre-eclampsia are being increasingly clarified in animals and humans. With the uncovering of these mechanisms, preventive therapy strategies using chronic infusion of adrenomedullin, <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em>-<em>121</em> (VEGF-<em>121</em>), losartan, and sildenafil have been proposed to block narrow spiral artery formation in the placenta by suppressing related possible <em>factors</em> for pre-eclampsia. However, although such preventive treatments have been partly successful, they have failed in ameliorating fetal <em>growth</em> restriction and carry the risk of possible side-effects of drugs on pregnant mothers. In this study, we attempted to develop a new symptomatic treatment for pre-eclampsia by directly rescuing placental ischemia with artificial oxygen carriers (hemoglobin vesicles: HbV) since previous data indicate that placental ischemia/hypoxia may alone be sufficient to lead to pre-eclampsia through up-regulation of sFlt-1, one of the main candidate molecules for the cause of pre-eclampsia. Using a rat model, the present study demonstrated that a simple treatment using hemoglobin vesicles for placental ischemia rescues placental and fetal hypoxia, leading to appropriate fetal <em>growth</em>. The present study is the first to demonstrate hemoglobin vesicles successfully decreasing maternal plasma levels of sFlt-1 and ameliorating fetal <em>growth</em> restriction in the pre-eclampsia rat model (p < 0.05, one-way ANOVA). In future, chronic infusion of hemoglobin vesicles could be a potential effective and noninvasive therapy for delaying or even alleviating the need for Caesarean sections in pre-eclampsia.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)-A is known to play key biological roles in angiogenesis and <em>vascular</em> permeability. We previously identified VEGF-A as an accurate biomarker of benign pancreatic cystic lesions known as serous cystic neoplasms (SCN). In the present study, we seek to further characterize the expression of VEGF-A and its splice isoforms in different pancreatic cysts including SCN. Patients undergoing surgery were consented for the collection of pancreatic cystic lesion tissue (SCN, pseudocysts, mucinous cysts) and normal adjacent pancreas as well as pancreatic cyst fluid. Following RNA isolation from the tissues, relative VEGF-A gene expression was quantitatively analyzed using real-time PCR (qPCR), and VEGF-A isoform expression was evaluated by reverse transcriptase (RT)-PCR. Relative VEGF-A gene expression was significantly increased in SCN, demonstrating transcriptional upregulation in SCN compared to other pancreatic cyst tissues (P < 0.0001). VEGF-189, -165, -145, and -<em>121</em> splice variants were detected in both normal adjacent pancreas and pancreatic cystic lesions; the novel VEGF-111 isoform was variably expressed in normal and cyst tissues. Finally, VEGF isoform levels in pancreatic cyst fluid were measured by isoform-specific ELISAs. VEGF-165, -145, and -<em>121</em> proteins were present in pancreatic cyst fluids; VEGF-165 levels were significantly higher in SCN cyst fluid. Thus, multiple VEGF isoforms were expressed in normal pancreas and pancreatic cysts. Of particular interest are VEGF-145 and -111, which have not previously been described in human pancreas where they may exhibit unique biological activities in health and/or disease.

Keywords: Isoform; Pancreatic cyst; Serous cystic neoplasm; VEGF; VEGF-111; VEGF-145.

OBJECTIVE

To select anti-VEGF (vascular endothelial growth factor) antibodies (Fab fragments) from human phage display antibody library and to identify their specification and activities.

METHODS

Human immunoglobulin heavy chain and light chain genes were separately amplified by RT-PCR from human peripheral lymphocytes using family specific primers and signal sequences of immunoglobulin. Human antibody library was constructed by phage display technology, and phage Fab antibodies to VEGF were screened from this library. ELISA, Western blot and (3)H-thymidine incorporation assays were used for the specification and neutralization activities of these Fab antibodies. Sequencing analysis was carried out for further identification of the antibodies.

RESULTS

The repertoire of human phage display Fab library was 1.5 x 10(8). After 4 round panning with VEGF(121), 280 clones were checked for their binding activities with ELISA and 12 clones could bind to VEGF(121) specifically. Western blot demonstrated that bacterially expressed soluble Fab could specifically recognize VEGF. Results of (3)H- thymidine incorporation showed that one clone of soluble Fab could neutralize the mitogenic activity of VEGF(165) on HUVEC. Sequencing analysis showed that the obtained V(H) gene belonged to human VH6 subgroup and the light chain was VJC rearranged human VL4 gene.

CONCLUSIONS

Human anti-VEGF antibodies can be obtained from human phage display library, which provides a basis for preparation of high affinity human anti-VEGF monoclonal antibodies through antibody engineer technique.

OBJECTIVE

To investigate the optimal high cell density fermentation conditions of recombinant E.coli BL21/pET- 24a/hVEGF(<em>121</em>) expressing recombinant human <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (rhVEGF(<em>121</em>)).

METHODS

The effects of the composition of the fermentation medium, induction time and fed-batch carbon sources on the expression level of rhVEGF(<em>121</em>) and cell output were analyzed.

CONCLUSIONS

When cultured in modified M9 medium and induced for 4 h in the presence of 0.5 mmol/L IPTG at 37 degrees celsius; with glycerol as the carbon sources by continuous fed-batch mode, the recombinant E.coli expressed rhVEGF(<em>121</em>) at the level up to 23% of the total proteins and the yield reached 68 g/L. The optimized fermentation condition for recombinant E.coli enables high expression level of rhVEGF(<em>121</em>).

UNASSIGNED

Hepatocellular carcinoma (HCC) is the most common and aggressive type of malignant liver tumor. HCC progression depends significantly on its vascularization and formation of new blood vessels. Vascular endothelial growth factor A (VEGFA) is a crucial regulator of tumor vascularization and components of VEGF-induced cell signaling pathways are important targets of therapeutical drugs that demonstrated the highest efficiency in case of advanced HCC (sorafenib and regorafenib). VEGFA is expressed as a set of isoforms with different functional properties, thus VEGFA isoform expression pattern may affect tumor sensitivity to anti-angiogenic drugs. However, information about VEGFA isoforms expression in HCC is still incomplete and contradictory. The present study aims to quantitatively investigate VEGFA isoform expression aberrations in HCC tissue.

UNASSIGNED

A total of 50 pairs of HCC and non-tumor tissue samples were used to evaluate the VEGFA isoform spectrum using RT-PCR and quantitatively estimate changes in isoform expression using RT-qPCR. Correlations between these changes and tumor clinicopathological characteristics were analyzed.

UNASSIGNED

We identified VEGFA-189, VEGFA-165, and VEGFA-121 as predominant isoforms in liver tissue. Anti-angiogenic VEGFA-xxxb variants constituted no more than 5% of all mature VEGFA transcripts detected and their expression was not changed significantly in HCC tissue. We demonstrated for the first time that the least active variant VEGFA-189 is frequently repressed in HCC (p < 0.001), while no uniform changes were detected for potent angiogenesis stimulators VEGFA-165 and VEGFA-121. Isoform balance in HCC shifts from VEGFA-189 towards VEGFA-165 or VEGFA-121 in the majority of cases (p < 0.001). Changes in fractions, but not expression levels, of VEGFA-189 (decrease) and VEGFA-121 (increase) correlated with advanced Tumor-Node-Metastasis (TNM) and Barcelona Clinic Liver Cancer (BCLC) tumor stages (p < 0.05), VEGFA-189 fraction reduction was also associated with poor tumor differentiation (p < 0.05).

UNASSIGNED

A distinct shift in VEGFA isoform balance towards more pro-angiogenic variants occurs in HCC tissue and may modulate overall impact of VEGFA signaling. We suppose that the ratio between VEGFA isoforms is an important parameter governing HCC angiogenesis that may affect HCC progression and be used for optimizing the strategy of HCC therapy by predicting the response to anti-angiogenic drugs.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) is a specific mitogen for <em>vascular</em> <em>endothelial</em> cells. It has been associated with angiogenesis, <em>growth</em>, metastasis and poor prognosis in solid tumors. Lately, it has been known that VEGF expression is higher in bone marrow from chronic myeloid leukemia (CML) patients than that in normal subjects. However, it is not clarified that the effect of VEGF on the abnormal proliferation of CML cells. In order to explore the effect of autocrine VEGF on CML cells, K562 cells were transfected with the VEGF(<em>121</em>) cDNA sense vector (K562/S) or with the VEGF(<em>121</em>) cDNA antisense vector (K562/As). K562 cells were transfected with the pcDNA(3) vector (K562/V) as the control. Cell proliferation was determined by MTT and colony forming assay in vitro. Flow cytometric Annexin-V-FITC/PI dual labeling technique was performed to observe the effect of VEGF(<em>121</em>) cDNA transfection on apoptosis of K562 cells. Results indicated that K562/S transfectants exhibited a 3-fold increase in VEGF secretion, and K562/As transfectants exhibited a 49% reduction in VEGF secretion. K562/As showed a reduced <em>growth</em> rate and colony forming efficiency as compared to K562/V. K562/S showed an increasing <em>growth</em> rate and colony forming efficiency as compared to K562/V. K562/As had more apoptotic cells than K562/V and K562/S in the same culture condition. These data suggest that VEGF plays an important role in the abnormal proliferation and apoptosis in CML cells through an autocrine mechanism.

<em>Vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) and such plasminogen activation system components as uPA, PAI-1 and tPA were determined by enzyme immunoassay methods in endometrial tumors from <em>121</em> patients and 18 samples of endometrial hyperplasia of varying degree. Endometrial carcinoma concentrations of uPA vs. PAI-1 were significantly higher than those in hyperplasia. Significant direct correlations--uPA vs. VEGF, uPA vs. PAI-1 and PAI-1 vs. VEGF--were established in endometrial tumors, and inverse ones for tPA vs. uPA and tPA vs. VEGF. A marked correlation with prognostic <em>factors</em> was found for PAI-1 and VEGF: levels of these proteins were relatively higher in cases of tumor progression (FIGO stage and deeper myometrial invasion), poor cell differentiation, and loss of hormone sensitivity. Higher uPA expression was associated with deeper myometrial invasion while, in endometrial tumors with unfavorable prognosis, it was VEGF level alone that was significantly higher.

The correlation between <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF)<em>121</em> mRNA expression and microvessel density was examined in 42 cases of primary lung cancer. Microvessels in tumors were highlighted by staining <em>endothelial</em> cells for <em>factor</em> VIII, CD31, and BNH9. The microvessel density in a high VEGF<em>121</em> expressing group was significantly greater than that in the low expressing group in the assessment with anti-CD31 antibody. The difference of survival stratified by the vessel density was significant in patients. with stage I disease. The results suggest a relationship between neomicrovessel density and VEGF<em>121</em>. VEGF<em>121</em> may be a useful marker of the tumor neo<em>vascular</em>ization in lung cancer.

Background Circulating levels of sFLT-1 (soluble fms-like tyrosine kinase 1), the extracellular domain of <em>vascular</em> <em>endothelial</em> <em>growth</em> <em>factor</em> (VEGF) receptor 1, and its ratio to levels of placental <em>growth</em> <em>factor</em> are markers of the occurrence and severity of preeclampsia. Methods and Results C57BL/6 pregnant mice on embryonic day 14.5 (E14.5), male, and non-pregnant female mice were exposed to air or to Br₂ at 600 ppm for 30 minutes and were treated with vehicle or with VEGF-<em>121</em> (100 μg/kg, subcutaneously) daily, starting 48 hours post-exposure. Plasma, bronchoalveolar lavage fluid, lungs, fetuses, and placentas were collected 120 hours post-exposure. In Br₂-exposed pregnant mice, there was a time-dependent and significant increase in plasma levels of sFLT-1 which correlated with increases in mouse lung wet/dry weights and bronchoalveolar lavage fluid protein content. Supplementation of exogenous VEGF-<em>121</em> improved survival and weight gain, reduced lung wet/dry weights, decreased bronchoalveolar lavage fluid protein levels, enhanced placental development, and improved fetal <em>growth</em> in pregnant mice exposed to Br₂. Exogenous VEGF-<em>121</em> administration had no effect in non-pregnant mice. Conclusions These results implicate inhibition of VEGF signaling driven by sFLT-1 overexpression as a mechanism of pregnancy-specific injury leading to lung edema, maternal mortality, and fetal <em>growth</em> restriction after bromine gas exposure.

OBJECTIVE

To detect the expression of the vascular endothelial growth factor (VEGF) and its receptors, the fms-like tyrosine (flt-1), kinase insert domain-containing receptor (KDR) in normal human endometrium during menstrual cycle and investigate the functions of VEGF and its receptors for development and differentiation of human endometrium.

METHODS

Immunohistochemistry and in situ hybridization techniques were used to measure the level of VEGF, flt-1 and KDR protein and mRNA in normal endometrium from 50 women. Few samples of endometrium respectively in the proliferative and secretory phase were homogenized for Western Blot analysis of VEGF subtypes. The blood vessel density was estimated by counting staining with a endothelial marker VIII factor.

RESULTS

The VEGF and its receptors mainly expressed in endometrial endothelial cells and gland epithelium. The level of VEGF, protein and mRNA, as well as flt-1 in mid-secretory and menstrual phase were highest (P < 0.05). The expression in early proliferative phase was lowest. KDR was with more intense expression from mid-proliferative phase to westrual phase. The acreage of the blood vessel wall and cavity was larger in the secretory than that in proliferative phase (P < 0.05), but those number of the blood vessels didn't alter through the menstrual cycle (P>> 0.05). Western Blot analysis showed stained VEGF bands in 34,000 (VEGF(121)), 46,000 (VEGF(165)), 54,000 (VEGF(189)), 68,000 (VEGF(206)), predominantly in 34,000 and 46,000.

CONCLUSIONS

VEGF, flt-1 and KDR, including both protein and mRNA, showed a pronounced menstrual cycle-dependent expression in cycling endometrium. VEGF and its receptors expressing higher in secretory phase and menstruation probably be involved in embryonic implantation and endometrial shedding.

OBJECTIVE

To discover the potential role of recombinant VEGF121 (rVEGF121) injection for the prevention of fetal growth restriction in a preeclampsia (PE) mouse model (Mus musculus).

METHODS

This is an experimental study of 30 pregnant mice that were randomly divided into three groups: normal, PE, and PE with rVEGF121 injection. The PE mouse model was created by injecting anti Qa-2 10 ng iv, which is deleterious to Qa-2 expression (homologous to HLA-G), from the first to the fourth day of gestation. PE was validated by measuring serum levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor(PIGF) and also by kidney histopathology. Recombinant VEGF121 was given on the ninth day until the 11th day of pregnancy; mice were terminated on the 16th day. Fetal weights were acquired with a Denver analytical balance. Serum levels of sFlt-1 and PlGF were measured using enzyme-linked immunosorbent assay (ELISA). The data were statistically analyzed via analysis of variance (ANOVA).

RESULTS

On average, fetal birth weight was 0.7150 g in the normal group, 0.4936 g in the PE group, and 0.6768 g in the PE with rVEGF121 injection group. ANOVA showed significant growth restriction in the PE group (P=0.006), confirming the use of anti Qa-2 as a suitable PE model. Kidney histopathology results, sFlt-1 levels, and PlGF levels also demonstrated that anti Qa-2 consistently conferred hallmarks of PE in mice. Vascular endothelial growth factor (VEGF) injection prevented fetal growth restriction; comparable fetal weights were observed between the PE model with VEGF treatment and the normal group (P=0.610) but differed from the untreated PE group (P=0.021).

CONCLUSIONS

Injection of rVEGF121 has the potential to prevent fetal growth restriction in a newly proposed PE mouse model.