Anti-human Vascular Endothelial Growth Factor Research Articles

Role of Thrombopoietin in Stimulating Megakaryopoiesis of ITP Patients Via Rescuing Vascular Niche

Background: Previous studies have shown that the vascular niche was damaged in patients with immune thrombocytopenia (ITP). It is mainly manifested that the migration and tube-formation ability of bone marrow endothelial progenitor cells (BM-EPCs) were significantly reduced. Endothelial cells as an important part of the vascular niche play an important role in regulation of megakaryocytes and platelet hematopoiesis. Our previous studies suggested that thrombopoietin (TPO) has a protective effect on human endothelial cells. This study aim to investigate whether TPO has a protective effect in EPCs of ITP patients and to explore its mechanism. Methods: BM-EPCs were isolated from ITP patients, and divided into two groups: TPO-treated group and TPO-free (control group). CCK8 was used to explore whether TPO has proliferative effect on BM-EPCs. BM-EPCs was identified by flow cytometry with co-expression of mouse anti-humanCD133, mouse anti-human CD34 and mouse anti-human vascular endothelial growth factor receptor (CD309) monoclonal antibodies. Moreover, the number of EPCs was evaluated with DiL-Ac-LDL uptake and FITC-UEA -I binding assay. Furthermore, the function of BM-EPCs was studied by using tube-formation and migration experiments. Results: The ITP patient-derived EPCs in TPO-treated group exhibited longer fusiform and elliptical shape after 5 days and cobblestone-like morphology after 7-10 days. The result of CCK-8 showed TPO could promote the proliferation of EPCs and the optional drug concentration is 100 ng/ml. Much higher expression of CD34/CD133/CD309 in TPO-treated group was detected by flow cytometry. The number of cells with immunofluorescence double staining in TPO group was significantly higher than of control group (n=4, p<0.05). The tube formation (n=5, p<0.05) and migration capacity (n=5, p<0.05) were significantly enhanced in TPO-treated group. Conclusion: Our studies indicates that TPO may exert megakaryopoiesis effect in ITP patients via protection of BM-EPCs. This may provide a supplemental explanation of how recombinant TPO and TPO receptor antagonist acts on ITP treatment. Keywords: thrombopoietin; vascular niche; endothelial progenitor cell, megakaryopoiesis; endothelial cell Disclosures No relevant conflicts of interest to declare.

In-vitro binding analysis of anti-human vascular endothelial growth factor antibodies bevacizumab and aflibercept with canine, feline, and equine vascular endothelial growth factor

PurposePromising results have been described for antibodies binding vascular endothelial growth factor (VEGF) in patients with corneal neovascularization. Whether veterinary patients would also benefit from this therapeutic approach has not been investigated yet. We examined binding properties of anti-human VEGF antibodies bevacizumab (Avastin®) and aflibercept (Zaltrap®) for canine, feline, and equine VEGF. MethodsHuman, equine, feline, and canine VEGF were analyzed for sequence similarity using the “Basic Local Alignment Search Tool” (BLAST). Western-blot analysis and ELISA were used to assess binding properties. ResultsBLAST analysis revealed a sequence homology of canine, feline, and equine VEGF to human VEGF-A of 93%, 92%, and 89%, respectively. Western-blot analysis showed immunoreactivity of bevacizumab with human, canine, and feline VEGF, but not with equine VEGF. Aflibercept recognized VEGF of all tested species. ELISA data indicated that bevacizumab and aflibercept bind canine VEGF in a dose-dependent manner. Feline VEGF was bound by bevacizumab and aflibercept in a dose-independent manner. ELISA study further confirmed the lack of bevacizumab binding to equine VEGF, and yielded also a dose-independent binding by aflibercept. ConclusionsBevacizumab and aflibercept turned out to bind VEGF with species-specific differences. Further studies are required to investigate their efficacy and safety under clinical conditions.

Adipose tissue-derived stem cells boost vascularization in grafted ovarian tissue by growth factor secretion and differentiation into endothelial cell lineages.

Adipose tissue-derived stem cells (ASCs) have multilineage differentiation potential, proangiogenic properties, and the ability to enhance vascularization in xenografted human ovarian tissue. The aim of the present study was to identify the mechanisms behind the proangiogenic effects of ASCs. For this purpose, severe combined immunodeficient (SCID) mice were grafted with frozen-thawed human ovarian tissue. ASCs were labeled by lentiviral transfection for expression of enhanced green fluorescent protein (eGFP), and human ovarian tissue was grafted using a previously described two-step procedure. In the control group, ovarian tissue was transplanted using the standard one-step approach. Samples were collected and analyzed after 7 days. Detection of the eGFP antigen by immunofluorescence showed ASCs surrounding and infiltrating ovarian tissue grafts. Significantly higher vessel density was observed in the ASC group (P = 0.0182 versus control) on Day 7. Co-expression of eGFP, CD34 and CD31 was demonstrated in human vessels, confirming ASC differentiation into human endothelial cell lineages. Increased gene expression of vascular endothelial growth factor (VEGF) was also shown in the ASC group (P = 0.0182 versus control). Immunohistochemistry targeting anti-human VEGF revealed significantly higher expression levels in the ASC group (P = 0.033 versus control), while VEGF and eGFP immunofluorescence showed greater growth factor expression in areas surrounding ASCs. In conclusion, ASCs differentiate into human vessels and promote secretion of VEGF when transplanted together with human ovarian tissue to SCID mouse peritoneum using a two-step ovarian tissue grafting procedure. This is a promising step towards potentially improving ovarian tissue quality and lifespan. Long-term studies should be conducted to investigate ASC safety and efficacy in the context of ovarian tissue transplantation.

High Glucose Promotes Epithelial-Mesenchymal Transition of Uterus Endometrial Cancer Cells by Increasing ER/GLUT4-Mediated VEGF Secretion

Background/Aims: Uterus endometrial cancer (UEC) is the common malignancy among gynecologic cancers, and most of them are type I estrogen-dependent UEC. Diabetes is well-known risk factor for the development of UEC. However, the underlying link between high glucose (HG) and the estrogen receptor in UEC remains unclear. Epithelial-mesenchymal transition (EMT) has also been shown to occur during the initiation of metastasis in cancer progression. The aim of this study was to determine the relationships and roles of HG, estrogen receptor and EMT in the growth and migration of UEC. Methods: The expression of glucose transport protein 4 (GLUT4) in the control endometrium and UEC tissues was detected by immunohistochemistry (IHC); the cell viability and invasion were analyzed through CCK-8 and Matrigel invasion assays; the transcriptional level of EMT-related genes was evaluated through real-time PCR; and the effect of HG and / or GLUT4 on estrogen receptors, vascular endothelial growth factor (VEGF) and its receptor VEGFR was analyzed through western blotting, ELISA and flow cytometry (FCM) assay, respectively. In addition, Ishikawa-xenografted nude mice were constructed and were used to analyze the effect of estrogen and GLUT4 on the growth of UEC in vivo. Results: Here, we found that exposure to HG led to a high level of viability and invasion of UEC cell lines (UECC, Ishikawa and RL95-2 cells). Compared with the normal endometrium, a higher level of GLUT4 was observed in UEC tissues. Silencing GLUT4 obviously inhibited the HG-promoted viability, invasion and expression of EMT-related genes (TWIST, SNAIL and CTNNB1) of UECC promoted by HG. Further analysis showed that HG and GLUT4 promoted the secretion of VEGF and expression of VEGFR in UECC. Treatment with HG led to the increase of estrogen receptor α (ERα) and β (ERβ) in UECC, blocking ERα or ERβ resulted in the decreases in GLUT4 expression, TWIST, SNAIL and CTNNB1 transcription, and VEGF and VEGFR expression in UECC. Treatment with anti-human VEGF neutralizing antibody restricted the viability and invasion of UECC that was induced by HG and estrogen. Exposure to estrogen accelerated growth, VEGF production, and TWIST and CTNNB1 expression in UEC in Ishikawa-xenografted nude mice, and silencing GLUT4 restricted these effects. Conclusion: These data suggest that HG increases GLUT4 and VEGF/VEGFR expression, further promotes EMT process and accelerates the development of UEC by up-regulating ER

Abstract A37: Dual wavelength near-infrared fluorescence imaging of VEGF-A and IGF-1R in ovarian cancer patient-derived xenografts

Abstract Background: Platinum-based chemotherapy in combination with surgery is the cornerstone of treatment for advanced high-grade serous ovarian cancer. Recurrence is common and additional treatments have been developed such as angiogenesis inhibition. Small subgroups of ovarian cancer patients may benefit from upfront selection based on tumor target expression. Here, we focus on near-infrared fluorescence (NIRF) dual imaging of the tumor secreted human vascular endothelial growth factor (VEGF-A) and the membrane-bound insulin-like growth factor 1 receptor (IGF-1R), simultaneously. The aim of our study is to determine tracer uptake and kinetics in relation to target expression in ovarian cancer patient-derived xenograft (PDX) models and the effect of cisplatin treatment on these parameters. Methods: Monoclonal antibody bevacizumab (anti-human VEGF-A) and MAB391 (anti-human IGF-1R) were coupled to NIRF dyes IRDye-800CW and IRDye-680RD, respectively. Specific tumor uptake was determined in a panel of subcutaneous ovarian cancer PDX models (established from 10 patients) in NOD SCID gamma mice during 1 week after intravenous co-injection of these tracers. In addition, two PDX models were treated weekly with cisplatin (4 mg/kg dose intraperitoneal administration) followed by NIRF dual imaging. Imaging results were compared with ELISA and immunostaining of VEGF-A and IGF1R. Results: We found large differences in average radiance for bevacizumab-800CW (range 2.53 - 23.3 x 106) and for MAB391-680RD (range 1.5 - 15.5 x 107) between PDX models at 24 hrs. In vivo kinetics of bevacizumab-800CW displayed a rapid tracer decline in PDX tumors 24 hrs after co-injection. MAB391-680RD, however, resided for over 6 days in PDX tumors depending on the IGF-1R positivity of tumors, indicating receptor-mediated tracer trapping. Moreover, IgG labeled with IRDye-800CW or IRDye-680RD showed similar kinetics as the bevacizumab-800CW. Elevated levels of both tracers were found in cisplatin-treated PDX tumors 24 hrs after co-injection. The higher levels of bevacizumab-800CW corresponded with higher intratumoral human VEGF-A levels in PDX tumors after cisplatin treatment. However, tracer decline kinetics in tumors did not change. Enhanced MAB391-680RD accumulation was related to higher IGF-1R tumor levels in the less responsive PDX model. A rapid decline of MAB391-680RD, similar to bevacizumab-800CW, was observed in the cisplatin-sensitive PDX model, which was related to reduced tumor proliferation and viability. Conclusions: These results indicate that levels and kinetics of targeted NIRF tracers can be used to dissect growth factor receptor-positive from -negative tumors. This encourages further exploration of multi-wavelength NIRF imaging to select targets for personalized medicine in small subgroups of ovarian cancer patients using PDX models. This work was supported by the Dutch Cancer Society (KWF) grants RUG 2010-4833 and RUG 2011-5231. Citation Format: Tushar Tomar, Nicolette G. Alkema, Jolanda A.L. Visser, Anton G. Terwisscha van Scheltinga, Gert Jan Meersma, Harry G. Klip, Evelien W. Duiker, Elisabeth G.E. De Vries, Ate G.J. Van der Zee, Steven De Jong. Dual wavelength near-infrared fluorescence imaging of VEGF-A and IGF-1R in ovarian cancer patient-derived xenografts. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A37.

Anti-Human VEGF Repebody Effectively Suppresses Choroidal Neovascularization and Vascular Leakage.

Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness among people over the age of 60. Vascular endothelial growth factor (VEGF) plays a major role in pathological angiogenesis in AMD. Herein, we present the development of an anti- human VEGF repebody, which is a small-sized protein binder consisting of leucine-rich repeat (LRR) modules. The anti-VEGF repebody selected through a phage-display was shown to have a high affinity and specificity for human VEGF. We demonstrate that this repebody effectively inhibits in vitro angiogenic cellular processes, such as proliferation and migration, by blocking the VEGF-mediated signaling pathway. The repebody was also shown to have a strong suppression effect on choroidal neovascularization (CNV) and vascular leakage in vivo. Our results indicate that the anti-VEGF repebody has a therapeutic potential for treating neovascular AMD as well as other VEGF-involved diseases including diabetic retinopathy and metastatic cancers.

Quantitative immunohistochemical assessment of blood and lymphatic microcirculation in cutaneous lichen planus lesions.

Latest advances have brought to light the hypothesis that angiogenesis and lymphangiogenesis are tightly connected to some chronic inflammatory diseases. The present study focuses on immunohistochemical assessment of the quantitative changes in the blood and lymphatic microcirculatory bed in common chronic dermatosis - cutaneous lichen planus. Double immunohistochemistry with CD34 and podoplanin antibodies was used to detect blood and lymphatic endothelium, while anti-human VEGF was used for the observation of a key angiogenesis and lymphangiogenesis inducer. Morphometric analysis was performed with QuickPhoto Micro image analysis software. Results confirmed statistically significant enlargement of both the blood and lymphatic microcirculatory beds. Compared to healthy skin, cutaneous lichen planus lesions revealed 1.6 times enlarged blood microcirculatory bed and 1.8 times enlarged lymphatic microcirculatory bed. Vascular endothelial growth factor (VEGF) expression in lesional skin was significantly higher in the epidermis (19.1 times increase) than in the dermis (10.3 times increase). These findings indicate a tight association of angiogenesis and lymphangiogenesis with the pathogenesis of cutaneous lichen planus.

Genetic modification of neurons to express bevacizumab for local anti-angiogenesis treatment of glioblastoma.

The median survival of glioblastoma multiforme (GBM) approximately 1 yr. Following surgical removal, systemic therapies are limited by the blood-brain barrier. To circumvent this, we developed a method to modify neurons with the genetic sequence for therapeutic monoclonal antibodies using adeno-associated virus (AAV) gene transfer vectors, directing persistent, local expression in the tumor milieu. The human U87MG GBM cell line or patient-derived early passage GBM cells were administered to the striatum of NOD/SCID immunodeficient mice.AAVrh.10BevMab, an AAVrh.10-based vector coding for bevacizumab (Avastin®), an anti-human vascular endothelial growth factor (VEGF) monoclonal antibody, was delivered to the area of the GBM xenograft. Localized expression of bevacizumab was demonstrated by quantitative PCR, ELISA and Western. Immunohistochemistry showed the bevacizumab was expressed in neurons. Concurrent administration of AAVrh.10BevMab with the U87MG tumor reduced tumor blood vessel density, and tumor volume and increased survival. Administration of AAVrh.10BevMab 1 wk after U87MG xenograft reduced growth and increased survival. Studies with patient-derived early passage GBM primary cells showed a reduction in primary tumor burden with an increased survival. This data supports the strategy of AAV-mediated CNS gene therapy to treat GBM, overcoming the blood-brain barrier through local, persistent delivery of an anti-angiogenesis monoclonal antibody.

Memristive Biosensors Under Varying Humidity Conditions

We attempt to examine the potential of silicon nanowire memristors in the field of nanobiosensing. The memristive devices are crystalline Silicon (Si) Nanowires (NWs) with Nickel Silicide (NiSi) terminals. The nanowires are fabricated on a Silicon-on-Insulator (SOI) wafer by an Ebeam Lithography Technique (EBL) process that allows high resolution at the nanoscale. A Deep Reactive Ion Etching (DRIE) technique is used to define free-standing nanowires. The close alignment between Silicon (Si) and Nickel-Silicide (NiSi) terminals forms a Schottky-barrier at their junction. The memristive effect of the fabricated devices matches well with the memristor theory. An equivalent circuit reproducing the memristive effect in current-voltage (I-V) characteristics of our silicon nanowires is presented too. The memristive silicon nanowire devices are then functionalized with anti-human VEGF (Vascular Endothelial Growth Factor) antibody and I-V characteristics are examined for the nanowires prior to and after protein functionalization. The uptake of bio-molecules linked to the surface of the memristive NWs is confirmed by the increased voltage gap in the hysteresis curve. The effects of varying humidity conditions on the conductivity of bio-modified memristive silicon nanowires are deeply investigated.

Expression and identification of recombinant chicken vascular endothelial growth factor in Pichia pastoris and its role in the pathogenesis of tibial dyschondroplasia

Vascular endothelial growth factor (VEGF) is an essential mediator of angiogenesis and endochondral ossification. To explore the role of VEGF in avian diseases such as tibial dyschondroplasia (TD), a typical disorder of endochondral ossification, we expressed and identified recombinant chicken VEGF (chVEGF) protein in Pichia pastoris and evaluated its effects on thiram-induced TD in broiler chickens. The SDS-PAGE showed that 2 recombinant proteins, with molecular weights of ˜46 and ˜70 kDa, were obtained. Western blot analysis indicated that the 2 proteins were recognized by rabbit anti-chicken and goat anti-human VEGF polyclonal antibodies. Moreover, the mixture of the proteins significantly stimulated angiogenesis in the chick chorioallantoic membrane. In 21-d-old broilers that had been fed a thiram-enriched diet (100 mg/kg of thiram for 2 d at 8 d old) to induce TD, intramuscular injection of the chVEGF proteins (at a dosage of 10 or 30 μg/kg) significantly reduced the severity of TD but had no effect on TD incidence or BW; decreased serum Ca and P concentrations and tartrate-resistant acid phosphatase activity and elevated serum alkaline phosphatase activity; enhanced the total antioxidant capacity, superoxide dismutase, and glutathione peroxidase activities in the liver and kidney; upregulated the expression of type X collagen, matrix metalloproteinase (MMP)-13, and Runx2; and downregulated the Bcl-2 expression in the growth plates. In thiram-treated broilers at 15 d old, the chVEGF proteins upregulated the expression of MMP-13 and Runx2, and had different effects on type X collagen and Bcl-2 expression at different dosages. Our results indicate that exogenous chVEGF proteins promoted the recovery of TD-affected growth plates by improving the antioxidant capacity in the liver and kidney and by regulating differential expression of genes relating to endochondral ossification at different stages of TD development; VEGF deficiency in the growth plates was involved in the pathogenesis of TD.

Abstract 5085: Less tumor engraftment after anti-VEGF therapy in pediatric low grade astrocytoma.

Abstract Introduction. Low grade astrocytomas are the most frequent brain tumors in children. Although children can benefit from treatments including neurosurgery, chemotherapy and radiotherapy, still children die due to tumor progression. Furthermore, morbidity can be serious. So new therapeutic strategies are warranted for these patients. Previously, we showed that angiogenesis is a characteristic of pediatric low grade astrocytoma (Sie et al, NAN 2010). Therefore, vascular endothelial growth factor (VEGF), the most critical angiogenic factor, may be a potential therapeutic target. The present study aimed to analyze the effect of VEGF inhibition in vitro and in vivo in pediatric low grade astrocytoma. Materials and methods. Three different pediatric low grade astrocytoma cell lines were used: Res-186 (WHO grade I astrocytoma), Res-259 and UW-467 (WHO grade II astrocytoma). Effects in vitro of the anti-VEGF monoclonal antibodies, bevacizumab (0 - 50 ug/ml, Avastin®, anti human VEGF) and B20-4.1.1 (0 - 200 ug/ml, anti human and mouse VEGF) were studied using a WST-1 cell viability assay and a cell proliferation assay with BRDU. Res-259 cells were orthotopically implanted in NOD-scid IL2Rgnull mice. After 6 weeks of treatment intraperitoneal twice weekly with bevacizumab (15 mg/kg), B20-4.1.1 (5 mg/kg) or phosphate buffered saline as control group, mice were euthanized and tumor engraftment was studied in brain slides using Aperio's imagescope viewer. Results. In vitro cell growth was not changed as measured by WST-1 and BRDU incorporation. In vivo results showed less tumor engraftment in bevacizumab (n = 10) and B20-4.1.1 (n = 9) treated mice compared with the control group (n = 10) (resp. 70%, 44%, 100%). In the bevacizumab and B20-4.1.1 treated mice tumor mass was lower as compared with controls (resp. P = 0.062, P = 0.001). Conclusion(s). This study showed less tumor engraftment after anti-VEGF therapy in a newly developed pediatric low grade astrocytoma mouse model. In these tumors anti-VEGF seems to work especially on tumor microenvironment. Suggesting that anti-VEGF could have a potential role in the therapeutic strategy for children with low grade astrocytoma, thoughtfulness on possible tumor escape mechanisms that may arise will be crucial. [W.F.A. den Dunnen and E.S.J.M. de Bont shared senior authorship.] Citation Format: Mariska Sie, Arend H. Sikkema, Frank J.G. Scherpen, Arja ter Elst, Kim R. Kampen, Eelco W. Hoving, Wilfred F.a. den Dunnen, Eveline S.j.m. de Bont. Less tumor engraftment after anti-VEGF therapy in pediatric low grade astrocytoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5085. doi:10.1158/1538-7445.AM2013-5085

P-0267 Correlation Between Serum Vascular Endothelial Growth Factor (Vegf) and Vegf-1 Expression in Patients with Colorectal Cancer. Preliminary Results

ABSTRACT Introduction In patients with colorectal cancer (CRC), several growth factors have been identified as regulator of angiogenesis, including vascular endothelial growth factor (VEGF), a pro-angiogenic factor which can be safely measured in the blood. Angiogenesis plays an important role in the progression of CRC, as well as in the development of regional lymph node and distant metastasis. VEGF is minimally expressed or not expressed in colorectal adenomas and in normal colonic mucosa, while it is overexpressed in inflammatory bowel diseases as well as in CRCs. It has been shown that high microvessels density, an indirect measure of the degree of angiogenesis, correlates with increased tumor aggressiveness and risk of metastasis development, and that overexpression of VEGF in cancer tissues represents a sensitive marker of angiogenesis. Because undetected metastases can contribute to the failure of primary treatment, their early identification has a substantial impact on adequate therapy. In this setting, several serum tumor markers and tissue-extracting prognostic factors have been tested, including endothelial cell growth factor, and angiogenic cytokines, such as VEGF. In several studies, preoperative serum VEGF was increased in patients with CRC compared to controls, and high VEGF serum concentration tended to occur with more advanced disease. However, these data are not confirmed, and exclusively VEGF overexpression in cancer tissues significantly correlates with tumor biology. The aim of this study was to evaluate the relationship between serum VEGF, and the expression of VEGF-1 in a group of patients with CRC. Download : Download full-size image Methods Archival paraffin-embedded tumor samples from 38 patients (26 men, 12 women, median age 64 years, range 39-76) with stage I-II colorectal adenocarcinoma who underwent intentionally curative surgical resection, were reviewed. None of the patients received preoperative adjuvant chemotherapy. Written informed consent was obtained from all the participants. Serum VEGF was measured with a commercially available human VEGF quantitative enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer's instructions. All analyses were made in duplicate, and the mean value was used for statistical calculations. Tissue microarray blocks for immunohistochemistry staining was obtained, serial 4 µm sections were cut, and then assessed using a commercial anti-human VEGF (clone VG-1). The Pearson's correlation coefficient calculation was used to evaluate results. Results The expression of VEGF was tested using a 4-grade scoring system, according to the degree of cytoplasmic staining, ranging from negative to intense staining. Overall, the mean serum VEGF concentration was 272.7±140.5 pg/mL (range 41-670 pg/mL), while the VEGF-1 grade was 2.8±1.0. There was a significant relationship between the stage of the disease and VEGF (R=0.43, p=0.006), and between serum VEGF and VEGF-1 grade (R=0.64, t=5.06, p Conclusion Tumor growth is dependent on angiogenesis and VEGF is potent growth factor with angiogenic activity. It can safely be tested by measurements of both serum VEGF levels and grade of tumor tissue expression of VEGF-1.

COX-2 Expression and Tumor Angiogenesis in Thyroid Carcinoma Patients among Northeast Chinese Population-Result of a Single-Center Study

Objective: Cyclooxygenase-2 (COX-2), one of the rate-limiting enzymes in the metabolism of arachidonic acid which is reported to be involved in the pathogenesis of many human tumors. As well, Vascular endothelial growth factor (VEGF) is well known to be involved in the infiltration and metastasis of many kinds of cancers. The aim of this study was to further elucidate the clinicopathologic significance of the immunohistochemical expressions of COX-2 and VEGF in thyroid carcinoma.Methods: Eighty-five patients with thyroid neoplasms were enrolled in our study from December 2003 to January 2010 from the authors' institution retrospectively. Their tumors were examined in the Department of Pathology, the First Bethune Hospital of Jilin University. Immunohistochemistry was performed on paraffin-embedded tissues sections using monoclonal anti-human COX-2 and VEGF antibodies. The tissues were classified into four types: papillary, follicular, medullary and undifferentiated. The patients ranged in age from 23 to 71 years. Breast cancer slides acted as control slides. The immunohistochemical stains were quantified by staining intensity and by the proportion of positively stained cells which were stained brown or yellow.Results: The results were analysed by χ2 test. COX-2 and VEGF expressions were stronger in thyroid carcinoma than in thyroid adenomas and normal tissues (P<0.01). COX-2 and VEGF expressions in thyroid carcinoma correlated with the tumor type and TNM stage.Conclusion: Our results suggest that expression of COX-2 and VEGF may promote angiogenesis of thyroid carcinoma, its infiltration, and metastasis.

99mTc-(CO)3 His-Annexin A5 Micro-SPECT Demonstrates Increased Cell Death by Irinotecan During the Vascular Normalization Window Caused by Bevacizumab

Colorectal tumors are dependent on angiogenesis for growth, and vascular endothelial growth factor (VEGF) is a key mediator of tumor angiogenesis. Antiangiogenic drugs can induce a transient normalization of the tumor vasculature with improved delivery of coadministered chemotherapy. The efficacy of antihuman VEGF antibody (bevacizumab) with or without irinotecan was evaluated in a colorectal cancer xenograft using (99m)Tc-(CO)(3) His-annexin A5. Colo205-bearing mice were treated with a single dose of bevacizumab (5 mg/kg) during 2, 4, or 6 d. Microvessel density, pericyte coverage (α-smooth-muscle actin immunostaining), collagen-covered tumor vessels (Masson trichrome staining), and tumor hypoxic fraction (pimonidazole staining) were determined at the 3 different time points after treatment with bevacizumab. To investigate the possible synergistic effects of combination therapy with bevacizumab and irinotecan, Colo205-bearing mice were treated with a single dose of bevacizumab 2, 4, or 6 d before administration of a single dose of irinotecan (100 mg/kg) or 0.9% NaCl. The apoptosis-detecting radiotracer (99m)Tc-(CO)(3) His-annexin A5 was injected (18.5 MBq) in mice 12, 24, and 48 h after the start of the irinotecan or NaCl treatment, and micro-SPECT was subsequently performed 3.5 h after injection of the radiotracer. Results were correlated to histologic analysis for apoptosis (caspase-3 activation). Four days after bevacizumab administration, microvessel density decreased significantly, and α-smooth-muscle actin and collagen-covered vessels, compared with control tumors, were increased, suggesting normalization of the tumor vasculature. Hypoxic fraction was slightly reduced 4 d after treatment with bevacizumab. SPECT analyses demonstrated a significant increase in tumoral (99m)Tc-(CO)(3) His-annexin A5 uptake 4 d after bevacizumab treatment and 24 h after irinotecan administration (232.78 ± 24.82 percentage injected dose/tumor weight [g]/body weight [kg], P < 0.05), compared with each monotherapy, indicating a synergistic effect of both therapies. (99m)Tc-(CO)(3) His-annexin A5 micro-SPECT demonstrates increased antitumor activity of irinotecan during the transient vascular normalization period caused by bevacizumab. Our data outline the importance of timing of combined anti-VEGF treatment with chemotherapy.

Vascular Endothelial Growth Factor (VEGF) and VEGF Receptor Expression in Biopsy Samples of Liver from Dogs with Congenital Portosystemic Shunts

Surgical attenuation of a congenital portosystemic shunt (CPSS) results in increased liver mass, development of intrahepatic portal vasculature and improved liver function. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. The aim of this study was to investigate the role of VEGF and its receptor in the hepatic response to CPSS surgery. The study included 99 dogs with CPSS treated with either partial or complete suture attenuation. Forty-four dogs with partial attenuation underwent a second surgery for complete attenuation. The expression of VEGF and VEGF receptor 2 (VEGFR2) in biopsy samples of liver was assessed by immunohistochemistry with rabbit anti-human VEGF polyclonal antibody and mouse anti-human VEGFR2 monoclonal antibody. Expression of these molecules was graded. The proportion of samples expressing VEGF was significantly greater in samples from dogs with CPSS compared with control samples (P=0.04) and the proportion of samples expressing VEGFR2 was significantly greater in control samples compared with samples from dogs with CPSS (P=0.04). VEGF labelling grade decreased significantly (P=0.038) and VEGFR2 increased significantly (P=0.046) between first and second surgery. The decrease in VEGF may reflect transient expression, preferential expression of other factors, reperfusion of existing vessels and/or increased angiogenesis before surgery in the form of arterialization and subsequent reduction due to improved portal blood flow. Partial suture attenuation was associated with a degree of 'normalization' of VEGF and VEGFR2 expression when compared with the control samples. Further investigation is needed to provide more information on the hepatic response to CPSS surgery.

Characteristics of human Ewing/PNET sarcoma models

Ewing/PNET (peripheral neuroepithelioma) tumors are rare aggressive bone sarcomas occurring in young people. Rare-disease clinical trials can require global collaborations and many years. In vivo models that as accurately as possible reflect the clinical disease are helpful in selecting therapeutics with the most promise of positive clinical impact. Human Ewing/PNET sarcoma cell lines developed over the past 45 years are described. Several of these have undergone genetic analysis and have been confirmed to be those of Ewing/PNET sarcoma. The A673 Ewing sarcoma line has proven to be particularly useful in understanding the biology of this disease in the mouse. The chromosomal translocation producing the EWS/FLI1 fusion transcript characterizes clinical Ewing sarcoma. Cell lines that express this genetic profile are confirmed to be those of Ewing sarcoma. The A673 Ewing sarcoma line grows in culture and as a xenograft in immunodeficient mice. The A673 model has been used to study Ewing sarcoma angiogenesis and response to antiangiogenic agents. Many Ewing sarcoma clinical specimens express the cell surface protein endosialin. Several Ewing sarcoma cell lines, including the A673 line, also express cell surface endosialin when grown as subcutaneous tumor nodules and as disseminated disease; thus the A673 is a useful model for the study of endosialin biology and endosialin-directed therapies. With the advent of tools that allow characterization of clinical disease to facilitate optimal treatment, it becomes imperative, especially for rare tumors, to develop preclinical models reflecting disease subsets. Ewing PNET sarcomas are a rare disease where models are available.

Vascular-endothelial growth factor and its high affinity receptor VEGFR-2 in the normal versus destructive lesions human forebrain during development: An immuno-histochemical comparative study

Vascular endothelial growth factor (VEGF) is an angiogenic inducer and neurotrophic factor both in adult and neonatal animal models. In the destructive lesions of the developing human brain, the role and expression of VEGF and of its mitogenic receptor VEGFR-2 have been hardly studied. The aim of the present work was to determine the immunohistochemical distribution of VEGF and VEGFR-2 in premature and full-term infants presenting with hypoxic/ischemic lesions, and to compare results with normal infant brains at similar developmental stages. Paraffin embedded brain tissue samples were assessed using anti-human VEGF and VEGFR-2 antibodies. In all undamaged forebrain areas, VEGF and VEGFR-2 displayed same expression patterns in control and pathologic brains, whatever the destructive lesion occurrence's time (before 25weeks of gestation (WG), between 25 and 34WG, perinatal period and infancy). In the destructive lesions, VEGF was always expressed in neurons, astrocytes and in neovessel walls, contrary to VEGFR-2 which was only expressed in dispersed astrocytes. VEGF was expressed in oligodendrocytes of prenatally damaged brains, whereas VEGF was expressed in these cells in undamaged areas from birth only, similarly to control brains. These data suggest that VEGF plays specific roles and may not be mediated by VEGFR-2 in human forebrain structures exposed to ischemia.

AAVrh.10-mediated genetic delivery of bevacizumab to the pleura to provide local anti-VEGF to suppress growth of metastatic lung tumors

SummaryVascular endothelial growth factor (VEGF) produced by tumor cells plays a central role in stimulating angiogenesis required for tumor growth. Humanized monoclonal anti-VEGF antibody (bevacizumab, Avastin®), approved as a treatment for non-squamous, non-small cell lung cancer, requires administration every 3 wk. We hypothesized that an intrapleural administration of an adeno-associated virus (AAV) vector expressing an anti-VEGF-A antibody equivalent of bevacizumab would result in sustained anti-VEGF-A localized expression within the lung and suppress metastatic tumor growth. The AAV vector AAVrh.10αVEGF encodes the light chain and heavy chain cDNAs of monoclonal antibody A.4.6.1, a murine antibody that specifically recognizes human VEGF-A with the same antigen-binding site as bevacizumab. A metastatic lung tumor model was established in SCID mice by intravenous administration of human DU145 prostate carcinoma cells. Intrapleural administration of AAVrh.10αVEGF directed long term expression of the anti-human VEGF-A antibody in lung, as demonstrated by sustained, high level anti-human VEGF titers in lung epithelial lining fluid for 40 wk, the duration of the study. In the AAVrh.10αVEGF-treated animals, tumor growth was significantly suppressed (p<0.05), the numbers of blood vessels and mitotic nuclei in the tumor was decreased (p<0.05), and there was increased survival (p<0.05). Thus, intrapleural administration of an AAVrh.10 vector encoding a murine monoclonal antibody equivalent of bevacizumab, effectively suppresses the growth of metastatic lung tumors, suggesting AAV-mediated gene transfer to the pleura to deliver bevacizumab locally to the lung as a novel alternative platform to conventional monoclonal antibody therapy.

VEGF blockade decreases the tumor uptake of systemic oncolytic herpes virus but enhances therapeutic efficacy when given after virotherapy

Effective therapies for metastatic sarcomas remain elusive. Oncolytic viruses have shown promise as anticancer agents, but their access to metastatic sites following systemic delivery is low. Because systemic delivery of small molecule chemotherapy is enhanced by prior treatment with anti-angiogenic agents due to changes in intravascular-to-tumor interstitial pressure, we sought to determine if anti-angiogenic pretreatment increases antitumor efficacy of systemic virotherapy by increasing virus uptake into tumor. Virus biodistribution and anti-tumor effects were monitored in tumor-bearing mice given anti-human VEGF or anti-mouse VEGFR2 before or after an intravenous injection of virus. Without pretreatment, the average virus titers in the tumor samples amplified 1700-fold over 48 hours but were undetectable in other organs. Following anti-angiogenic treatment, average virus titers in the tumor samples were unchanged or in some cases decreased by up to 100-fold. Thus, anti-angiogenic pretreatment failed to improve tumor uptake of systemic oncolytic HSV, in contrast to previously reported enhanced uptake of small molecules. Superior tumor control due to the combined effects of virus and anti-VEGF was seen most dramatically when anti-VEGF was given after virus. Our data suggest intravenous oncolytic HSV can treat distant sites of disease and be enhanced by anti-angiogenic therapy, but only when given in the proper sequence.

Effectiveness and Safety of Bevacizumab for Unresectable Non-Small-Cell Lung Cancer

Non-small-cell lung cancer (NSCLC) accounts for more than 85% of all cases of lung cancer. The 5-year survival of patients presenting with advanced stage NSCLC is less than 15%, indicating that additional treatment options are needed. Bevacizumab is a recombinant humanized version of the murine anti-human vascular endothelial growth factor (VEGF) monoclonal antibody with a high binding specificity for VEGF. The aim of this meta-analysis was to evaluate the effectiveness and safety of bevacizumab in patients with unresectable non-small-cell lung cancer (NSCLC) on the basis of evidence-based methodology. The electronic database PubMed was searched to identify randomized, controlled trials (RCTs) of bevacizumab for the treatment of unresectable NSCLC. Other databases such as the Cochrane Library Trials Register, the WHO Trial Registration, the National Cancer Institute, ClinicalTrials.gov, the European Organization for Research and Treatment of Cancer, the Southwest Oncology Group, the Eastern Cooperative Oncology Group, the European Society of Clinical Oncology and the American Society of Clinical Oncology were also searched. The meta-analysis was performed using Reviewer Manager Version 5.0 software provided by the Cochrane Collaboration. Outcome measures were overall survival rates, progression-free survival, tumour response rate, incidence of severe adverse events (SAEs) and treatment-related death. Four eligible studies that included 2101 patients were found; in these studies, bevacizumab was administered to 1237 patients. Neither high-dose (15 mg/kg) nor low-dose (7.5 mg/kg) bevacizumab increased 1-year overall survival rates compared with patients not treated with bevacizumab. However, high-dose bevacizumab, rather than low-dose, increased 2-year overall survival rate (risk ratio [RR] = 1.24; 95% confidence interval [CI] 1.04, 1.49) and tumour response rate (RR = 1.69; 95% CI 1.21, 2.35) compared with patients not treated with bevacizumab. Progression-free survival was also significantly improved in both the low- (hazard ratio [HR] = 0.76; 95% CI 0.64, 0.90) and high-dose groups (HR = 0.73; 95% CI 0.65, 0.81). There was a clear and significant increase in the rate of treatment-related death in the high-dose group (RR = 2.07; 95% CI 1.19, 3.59) compared with patients not treated with bevacizumab. No significant differences were noted in the rate of treatment-related death in the low-dose group or in the incidences of SAE in the low- or high-dose groups compared with patients not treated with bevacizumab. Neutropenia was easily induced in both the low- and high-dose bevacizumab groups. Patients who received high-dose bevacizumab tended to experience hypertension, neutropenia, haemoptysis, rash and headache more frequently than patients not treated with bevacizumab. Low-dose bevacizumab may significantly improve progression-free survival in patients with unresectable NSCLC, whereas high-dose bevacizumab may increase 2-year overall survival rates, prolong progression-free survival and improve tumour response rate but at the cost of higher treatment-related death. Larger well designed RCTs should be carried out in the future to clarify the role of bevacizumab in the treatment of NSCLC.