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In this study, cherry fruits and petioles from six ancient Italian Prunus avium L. varieties (Ferrovia, Capellina, Morellina, Ciambellana, Napoletana, and Bianca), were compared by chemical and bioinformatic analyses and evaluated for their antiangiogenic activity. The highest levels of total phenols and flavonoids were found in Napoletana petioles, and Morellina and Capellina fruits. HPLC-PDA-MS analyses showed similar phenolic profiles for all fruit extracts, with cyanidin-3-O-rutinoside, flavonols glycosides, and quinic acid derivatives as major components. Flavonoid glycosides were found in all petiole extracts, while proanthocyanidins B type were predominant in Capellina, Napoletana and Bianca. Accordingly to their higher polyphenolic content, petiole extracts exhibited stronger radical scavenging activity compared to the fruits. The best antiangiogenic response was exhibited by Morellina, Ferrovia, and Ciambellana petiole extracts, and by Ferrovia, Morellina, and Capellina fruit extracts; by bioinformatic studies rutin and cyanidin 3-O-rutinoside were recognised as the best candidate bioactive compounds. In conclusion, sweet cherry varietes were confirmed as valuable sources of phenols, showing also potential angiomodulator properties., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Genistein, a natural tyrosine kinase inhibitor, may act as an intraocular antiangiogenic agent. Its therapeutical use, however, is limited by its nonlinear pharmacokinetics. We aimed to determine genistein's kinetics and retinal tissue distributions in normal and diabetic rats. We developed an isocratic, reverse-phase C18 HPLC system to measure genistein concentration in blood and retinas of streptozotocin (65 mg/kg IV)-diabetic and non-diabetic rats receiving two types of genistein-rich diet (150 and 300 mg/kg) for ten days. Genistein's decay exhibited a two-compartmental open model. Half-lives of distribution and elimination were 2.09 and 71.79 min, with no difference between groups. Genistein steady-state concentration in blood for 150 and 300 mg/kg diet did not differ between diabetic (0.259 ± 0.07 and 0.26 ± 0.06 μg/ml) and non-diabetic rats (0.192 ± 0.05 and 0.183 ± 0.09 μg/ml). In retina, genistein concentration was significantly higher in diabetic rats (1.05 ± 0.47 and 0.997 ± 0.47 μg/gm wt. vs. 0.087 ± 0.11 and 0.314 ± 0.18 μg/gm wt., p < 0.05). The study determined that increasing genistein dose did not change its bioavailability, perhaps due to the poor aqueous solubility. The retina's increased genistein could be due to increased permeability of blood-retinal barrier that occurs early in diabetes., Competing Interests: Declaration of competing interest None., (Copyright © 2021 The Japanese Society for the Study of Xenobiotics. All rights reserved.)

Bevacizumab is a chimeric monoclonal human-murine antibody originated from murine monoclonal antibody (muMAb A4.6.1) with the human immunoglobulin IgG1. BVZ binds the extracellular portion of vascular endothelial growth factor receptors (VEGFR), which have tyrosine kinase activity. The mechanism of action of BVZ involves binding to VEGFR, Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), inducing homodimerization of two receptor subunits, and, consequently, autophosphorylation of their tyrosine kinase domains located inside the cytoplasm. With the advent of nanostructured systems it is increasingly necessary to look for safe analytical methods, ensuring the reliability of the results obtained by them, becoming essential to ensure the quality of medicines. In this work, the incorporation of bevacizumab in to different drug delivery systems was presented. Moreover, detailed investigation was performed about methods for qualitative and quantitative analyses of bevacizumab, including, biological fluids, and drug delivery systems, were investigated. Most recently high performance liquid chromatography coupled with various detectors, liquid chromatography, mass spectrometry and ELISA were used for this purpose. Thus, this review was performed to evaluate the benefits of bevacizumab carried by nanostructured systems and the analytical methods available for detection and quantification of these drugs.

Bevacizumab is a full-length human monoclonal antibody used to treat various neovascular diseases such as wet age-related macular degeneration (AMD), diabetic eye disease and other problems of the retina. Monthly intravitreal injections of bevacizumab (Avastin ® ) are effective in the treatment of wet AMD. However, there is a growing demand in the development of sustained release ophthalmic formulations. Therefore, this study aims, for the first time, to develop a rapid, simple, and sensitive method using size exclusion chromatography coupled with fluorescence detection for routine quantification of bevacizumab in ophthalmic formulations and during in vitro release studies. The selected chromatographic conditions included an aqueous mobile phase composed of 35 mM sodium phosphate buffer and 300 mM sodium chloride (pH 6.8), a flow rate of 0.5 mL/min, and the fluorescence detector was operated at excitation and emission wavelengths of 280 and 340 nm, respectively. The peak area-concentration relationship maintained its linearity over concentration range of 0.1-20 μg/mL (R 2 = 0.9993), and the quantitation limit was 100 ng/mL. The method was validated for specificity, accuracy, precision, and robustness. The developed method had a run time of 6 min at temperature 25 °C, making it a unique validated method for rapid and cost-effective quantification of bevacizumab., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Diet-based chemoprevention of cancer has emerged as an interesting approach to evade the disease or even target its early phases, reducing its incidence or slowing down tumor progression. In its basis in the essential role of angiogenesis for tumor growth and metastasis, angioprevention proposes the use of inhibitors of angiogenesis in cancer prevention. The anti-angiogenic potential exhibited by many natural compounds contained in many Mediterranean diet constituents makes this dietary pattern especially interesting as a source of chemopreventive agents, defined within the angioprevention strategy. In this review, we focus on natural bioactive compounds derived from the main foods included in the Mediterranean diet that display anti-angiogenic activity, as well as their possible use as angiopreventive agents.

Background: Angiogenesis inhibition research is a cutting edge area in angiogenesis-dependent disease therapy, especially in cancer therapy. Recently, studies on anti-angiogenic peptides have provided promising results in the field of cancer treatment., Methods: A non-redundant dataset of 135 anti-angiogenic peptides (positive instances) and 135 non anti-angiogenic peptides (negative instances) was used in this study. Also, 20% of each class were selected to construct an independent test dataset (see Additional files 1, 2). We proposed an effective machine learning based R package (AntAngioCOOL) to predict anti-angiogenic peptides. We have examined more than 200 different classifiers to build an efficient predictor. Also, more than 17,000 features were extracted to encode the peptides., Results: Finally, more than 2000 informative features were selected to train the classifiers for detecting anti-angiogenic peptides. AntAngioCOOL includes three different models that can be selected by the user for different purposes; it is the most sensitive, most specific and most accurate. According to the obtained results AntAngioCOOL can effectively suggest anti-angiogenic peptides; this tool achieved sensitivity of 88%, specificity of 77% and accuracy of 75% on the independent test set. AntAngioCOOL can be accessed at https://cran.r-project.org/ ., Conclusions: Only 2% of the extracted descriptors were used to build the predictor models. The results revealed that physico-chemical profile is the most important feature type in predicting anti-angiogenic peptides. Also, atomic profile and PseAAC are the other important features.

Ranolazine (RZ) is an anti-anginal drug with a distinct mechanism of action and widely employed in patients with chronic angina. Its measurement is essential in clinical environment to ensure adequate drug level and understand the redox mechanism which gives an idea of in-vivo fate of the drug. In view of this, an exemplary voltammetric approach is proposed here for determination of RZ utilizing glassy carbon electrode (GCE) fabricated with WO 3 decorated graphene nanocomposite. The structural and morphological characterizations of modifier were made by employing XRD, FESEM, EDAX, HRTEM, XPS, Raman and FT-IR spectroscopy which revealed successful formation of the nanocomposite. As a result of high electrical conductivity and large effective surface area of WO 3 nanoparticles and graphene nanosheets, the developed sensor WO 3 /Graphene/GCE displayed effectual and unrelenting electron interceding behavior exhibiting higher peak currents at lower potentials for RZ oxidation. Using square wave voltammetry, the drug showcased well-defined voltammetric response in Britton-Robinson buffer at pH 4.5 in concentration range from 0.2-1.4 μM and 1.4-14 μM with the low detection limit of 0.13 μM. The developed protocol was then implemented successfully to quantify RZ in commercially accessible pharmaceutical tablets with satisfactory recovery (99.8%-100.2%). The experimental results illustrated the applicability of the fabricated sensor for drug quality control and clinical analysis along with pharmacokinetic studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Angiogenesis is one of the key processes in the growth and development of tumors. Class-3 semaphorins (Sema3) are characterized as axon guidance factors involved in tumor angiogenesis by interacting with the vascular endothelial growth factor signaling pathway. Sema3 proteins convey their regulatory signals by binding to neuropilins and plexins receptors, which are located on the effector cell. These processes are regulated by furin endoproteinases that cleave RXRR motifs within the Sema, plexin-semaphorins-integrin, and C-terminal basic domains of Sema3 protein. Several studies have shown that the furin-mediated processing of the basic domain of Sema3F and Sema3A is critical for association with receptors. It is unclear, however, if this mechanism can also be applied to other Sema3 proteins, including the main subject of this study, Sema3C. To address this question, we generated a variant of the full-length human Sema3C carrying point mutation R745A at the basic domain at the hypothetical furin recognition site 742RNRR745, which would disable the processing of Sema3C at this specific location. The effects produced by this mutation were tested in an in vitro angiogenesis assay together with the wild-type Sema3C, Sema3A, and Sema3F proteins. Our results showed that the inhibitory effect of Sema3C on microcapillary formation by human umbilical vein endothelial cells could be abrogated upon mutation at the Sema3C basic domain within putative furin cleavage site 742RNRR745, indicating that this site was essential for the Sema3 biological activity.

Introduction: Proximity Extension Assay (PEA) is a direct one-step protein quantification method using a pair of DNA oligonucleotides linked to antibodies against the target molecule. It requires polyclonal or two monoclonal antibodies (mAbs) that bind to target epitopes close enough to form a DNA duplex which is quantified by real-time PCR. Bevacizumab, an anti-cancer drug, is a mAb against vascular endothelial growth factor with common cardiovascular adverse effects. It is widely used off-label to treat neovascular eye disorders by intravitreal application of small doses. Even then, certain amount reaches systemic circulation which is considered relevant regarding safety. We aimed to set-up a PEA-based assay for bevacizumab in human plasma and to preliminary evaluate it in patients treated intravitreally., Methods: We tested (PEA, quantitative PCR) several combinations of commercial mAbs and a Fab fragment against bevacizumab. The best combination was used to quantify bevacizumab in three patients donating plasma before and 24 h after the first intravitreal injection., Results: A combination of a mAb and a Fab fragment (HCA184 and HCA182, Bio-Rad Laboratories, Inc.) performed best: standard curve R 2 0.98, linear dynamic range 1-1000 pM, lower limit of quantification 1 pM (149 pg/mL) and a satisfactory precision (coefficient of variation 12%). All pre-dose patient concentrations were zero, while post-dose concentrations were 10.94, 13.73 and 55.49 ng/mL, in line with previous reports., Discussion: This is the first set-up of a PEA-based assay for quantification of bevacizumab in human plasma. Its good performance and high sensitivity support further evaluation for potential uses particularly when the expected concentrations are low., (Copyright © 2018. Published by Elsevier Inc.)

Metastasis is the major hindrance in the treatment of all cancers, including laryngeal squamous cell carcinoma. Intensive researches are under way to identify the effective natural polyphenols with anti-metastatic ability for cancer treatment. Wheatgrass, an herbal plant has been reported to show anticancer effects. Hence, in this study, we aimed to analyze the anti-metastatic effect of methanol extract of wheatgrass (MEWG). The levels of metastatic marker proteins were determined by western blot. PI3K and AKT levels were determined by real time (RT)-PCR analysis. In silico molecular docking was done to check the interaction of the 14 components (identified by HPLC/GCMS) of MEWG with PI3K and AKT. MEWG effectively decreased the metastatic protein expressions, namely VEGF, MMP-9 and COX-2 and increased TIMP-2. RT-PCR results showed reduced m-RNA levels of both PI3K and AKT when compared to control. Molecular docking studies revealed interaction of most of the identified compounds of the extract with the important residues of PI3K and AKT. These findings indicate that MEWG inhibits metastasis and angiogenesis in Hep-2 cells possibly via PI3K/AKT due to the cumulative effect of polyphenols and other constituent present in extract. The compounds of the extract were also found to be directly involved in inhibition of AKT/PI3K, thus could help to restrain metastasis.

Black carrot (Daucus carota L. ssp. sativus var. atrorubens Alef.) is a valuable source of carbohydrates, minerals and vitamins and contains also high amounts of anthocyanins giving the characteristic deep-purple color. These latter compounds are known as natural dyes used in the food and pharmaceutical industry that have recently attracted much attention for their healthful properties. The aim of this work was to investigate for the first time the polyphenolic profile and biological properties of a black carrot crude extract (BCCE) through an in-depth analysis of the main polyphenolic classes evaluating its antioxidant, cytoprotective and anti-angiogenic properties. Twenty five polyphenols were quantified by LC-DAD-FLD-MS/MS analysis (anthocyanins 78.06%, phenolic acids 17.89% and other flavonoids 4.06%) with polyglycosylated cyanidins as major components. In addition, BCCE showed a strong antioxidant and free radical scavenging activity particularly in the hydrogen transfer-based assays (ORAC and β-carotene bleaching) and a significant increase in the cell viability. Furthermore, BCCE exhibited a strong anti-angiogenic activity at the highest concentration assayed on the chick chorioallantoic membrane (50μg/egg). In conclusion, the obtained results demonstrated the antioxidant, cytoprotective and anti-angiogenic properties of BCCE, which highlight that the higher biological activity of BCCE is probably due to the synergic effects exerted by various polyphenolic classes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Kudingcha is a particularly bitter tasting tea that has been widely used in China to eliminate fever and itching eyes, and to clear blood toxins. Kudingcha is considered of value for its potential anticancer effects that are attributed to the presence of characteristic bioactive ingredients. The chlorogenic acid (CGA) derivatives 3-0-caffeoylquinic acid, 5-0-caffeoylquinic acid, 3,5-0-dicaffeoylquinic acid, and 4,5-0-dicaffeoylquinic acid were separated from Ilex kudingcha C.J. Tseng extract by high-performance liquid chromatography (HPLC)-photodiode array detector (PDA) and HPLC-nuclear magnetic resonance (NMR). In Tg(flk1:EGFP) zebrafish embryos at 52 hours postfertilization (hpf), angiogenesis was significantly inhibited by kudingcha extract (KDCE) at concentrations of 400 and 500 μg/mL and CGA also showed significant inhibition in embryos treated with 80, 100, and 130 μg/mL. Endothelial cell apoptosis showed a dose-dependent increase in response to KDCE and CGA. CGA derivatives from KDCE could have potential as anticancer agents against tumor angiogenesis.

Hard ticks (Acari: Ixodidae) are blood-sucking ectoparasites characterized by the extended period of their attachment to their host. To access their bloodmeal, ticks secrete saliva containing a range of molecules that target the host's inflammation, immune system, and hemostatic components. Some of these molecules reportedly possess antiangiogenic and antitumor properties. The present study describes our investigation, the first of its kind, of the antiangiogenic and antitumoral effects of the Hyalomma dromedarii Koch, 1844 (Acari: Ixodidae), salivary gland extract (SGE), which inhibited the adhesion and migration of Human Umbilical Vein Endothelial Cells (HUVECs) in a dose-dependent manner, as well as angiogenesis in the Chick Chorioallantoic Membrane model. Interestingly, H. dromedarii SGE exerted an antiproliferative effect on U87 glioblastoma cells and inhibited their adhesion and migration to fibrinogen. These results open up new possibilities for characterizing and developing new molecules involved in the key steps of tumor progression., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Prevention of postoperative endophthalmitis (POE) is a goal of every ophthalmic procedure and also in intravitreal therapy (IVT). This comparative retrospective study targets whether systemic and topical preoperative antibiotic prophylaxis (PAP) regimen does prevent POE after IVT in a higher rate compared with topical PAP alone. Out of 6111 IVT performed over a period of 103.5 months, in the study group A patients were treated with systemic and topical PAP (2881 IVT), and in the study group B patients were treated only with topical PAP (3230 IVT). Intravitreal drugs were anti-VEGF, triamcinolone and dexamethasone implants. The incidence of POE in group A (1/2881 or 0.035 % per injection) was not significantly different (P = 0.4) from that in suite B (1/3230 or 0.031 % per injection). Our study states that systemic PAP does not modify the risk of postoperative endophthalmitis in patients treated with IVT.

The capabilities of red wine against lipid oxidation and angiogenesis were evaluated by using a fish oil emulsion system and an in vivo zebrafish embryos model, respectively. The red wine contained 12 different antioxidant phenolics which levels were led by anthocyanins (140.46 mg/L), catechin (55.08 mg/L), and gallic acid (46.76 mg/L). The diversity of the phenolics in red wine was greater than the tea, coffee, or white wine selected as a peer control in this study. The total phenolics concentration of red wine was 305.53 mg/L, although the levels of tea, coffee, and white wine were 85.59, 76.85, and 26.57 mg/L, respectively. The activity of red wine in scavenging DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals was approximately 4 times higher than the tea and 8 times than the coffee or white wine. The red wine showed the highest capability in preventing long chain PUFA oxidation in the fish oil emulsion. Because of the outstanding antioxidant activity of red wine, the red wine dried extract was used to monitor its inhibitory effect against angiogenesis by using transgenic zebrafish embryos (Tg[fli1:egfp] y1 ) with fluorescent blood vessels. After incubated in 100 μg/mL of the extract solution for 26 h pf, each of the embryos had a lower number of intersegmental vessel than the control embryo. The inhibition rate of red wine extract against growing of angiogenic blood vessel reached 100%., (© 2017 Institute of Food Technologists®.)

Purpose:: Avastin® (bevacizumab) is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody given as an off-label drug by intravitreal administration for treatment of ocular diseases. The drug's clinical application and its cost-benefit profile has generated demand for its division into single-use vials to meet the low volume and low-cost doses necessary for intraocular administration. However, the safety of compounding the drug in single-use vials is still under discussion. In this study, the stability and efficacy of Avastin® repacked in individual single-use glass vials and glass ampoules by external compounding pharmacies were evaluated., Methods:: Polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), dynamic light scattering (DLS), and turbidimetry were selected to detect the formation of aggregates of various sizes. Changes in bevacizumab biological efficacy were investigated by using an enzyme-linked immunosorbent assay (ELISA)., Results:: Repacked and reference bevacizumab showed similar results when analyzed by PAGE. By SEC, a slight increase in high molecular weight aggregates and a reduction in bevacizumab monomers were observed in the products of the three compounding pharmacies relative to those in the reference bevacizumab. A comparison of repacked and reference SEC chromatograms showed that the mean monomer loss was ≤1% for all compounding pharmacies. Protein aggregates in the nanometer- and micrometer-size ranges were not detected by DLS and turbidimetry. In the efficacy assay, the biological function of repacked bevacizumab was preserved, with <3% loss of VEGF binding capacity relative to that of the reference., Conclusion:: The results showed that bevacizumab remained stable after compounding in ampoules and single-use glass vials; no significant aggregation, fragmentation, or loss of biological activity was observed.

Background: Bevacizumab is widely used for ophthalmic purposes. Recently, counterfeit bevacizumab has become a matter of concern. We analysed samples of suspected counterfeit formulations of bevacizumab and assessed the possibility of using simple tests in the clinic by ophthalmologists to prevent the use of counterfeit preparations in patients., Methods: We did a protein analysis using Bradford assay and SDS-PAGE to confirm the presence of bevacizumab in 16 samples - 6 suspected and 10 others. The samples were also subjected to physicochemical analysis such as osmolarity, chloride content and pH. The samples tested negative for protein were analysed by mass spectrometry to detect drugs used in place of bevacizumab. We standardized the method of frothing and precipitation analysis for identifying authentic samples of bevacizumab before their clinical use., Results: Five of the 16 samples tested were negative for the presence of bevacizumab. The physicochemical parameters also supported the protein analysis test. However, no ionizable organic compound (other drug[s]) was detected by mass spectrometry., Conclusion: Ophthalmic use of counterfeit bevacizumab can be prevented by simple methods such as the frothing and precipitation tests. These can identify the absence of an active drug.

Purpose: A simple suspension method has been developed for tube administration, in which tablets (and capsules) are disintegrated in hot water (55℃) without grinding (or opening) them. In the present study, we evaluated the feasibility of this simple suspension method for the preparation of lenalidomide (Celgene, Summit, New Jersey and USA) suspension by testing the stability of this drug at 55℃ and its adsorbability on the tube., Methods: We examined, by high-performance liquid chromatography, the time-dependent changes in the concentration of lenalidomide in suspensions of the drug prepared by the simple suspension method. The high-performance liquid chromatography analyses of lenalidomide were performed on Prominence LC-20AB/SPD-20 A (Shimadzu, Kyoto, Japan) with a ZORBAX SB-C18 RR analytical column (Agilent Technologies, Santa Clara, California, USA; particle size: 2.1 × 100 mm, 3.5 µm) at a flow rate of 0.4 mL/min. A solvent system consisting of 10 mM ammonium acetate (pH 7.0)/acetonitrile was used as the eluent and the eluate was detected by UV at 254 nm., Results: Lenalidomide was confirmed to remain stable in hot water at 55℃ for 24 h in the prepared suspension by the simple suspension method, and more than 99% of the drug could be recovered from the suspension. In addition, 94.5-98.0% of the drug amount could pass through a percutaneous endoscopic gastrostomy tube. Lenalidomide was scarcely adsorbed on to the percutaneous endoscopic gastrostomy tube made of polyurethane or polyvinyl chloride., Conclusion: Lenalidomide was found to be stable even in hot water and was not adsorbed on to the percutaneous endoscopic gastrostomy tube., (© The Author(s) 2015.)

Purpose: To measure the hydrodynamic radii of intravitreal anti-VEGF drugs ranibizumab, aflibercept and bevacizumab with μs time-resolved phosphorescence anisotropy., Methods: Ruthenium-based dye Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2, whose lifetime of several hundred nanoseconds is comparable to the rotational correlation time of these drugs in buffer, was used as a label. The hydrodynamic radii were calculated from the rotational correlation times of the Ru(bpy)2(mcbpy - O - Su - ester)(PF6)2-labelled drugs obtained with time-resolved phosphorescence anisotropy measurements in buffer/glycerol solutions of varying viscosity., Results: The measured radii of 2.76±0.04 nm for ranibizumab, 3.70±0.03 nm for aflibercept and 4.58±0.01 nm for bevacizumab agree with calculations based on molecular weight and other experimental measurements., Conclusions: Time-resolved phosphorescence anisotropy is a relatively simple and straightforward method that allows experimental measurement of the hydrodynamic radius of individual proteins, and is superior to theoretical calculations which cannot give the required accuracy for a particular protein.

Zebrafish have been traditionally used in ecotoxicology and developmental biology. However, due to the advances in available methodologies and the similitude with mammals, it has been increasingly used in other fields. One of the most recent fields using zebrafish is food research, being the focus of this review. Most relevant and recent publications including food component toxicity and key metabolic effects together with effectiveness on some zebrafish disease models have been reviewed. This model is a good intermediate tool between in vitro and rodent models, because it provides information from a complete organism in a fast and cost-effective manner. Definitely, in the near future, we will see this model being used by the ingredient suppliers and scientists in order to show the potential impact on health of several compounds.

Antivascular endothelial growth factor (Anti-VEGF) agents have been widely used for a variety of ocular disorders. The etiology of sustained ocular hypertension following intravitreal administration of anti-VEGF agents is yet to be unraveled. Our study investigates and characterizes the presence of intravitreally injected bevacizumab in the aqueous outflow channels of a rat model. Choroidal neovascularization (CNV) was induced by diode laser photocoagulation to the right eye of twelve Brown Norway rats. Bevacizumab (25 mg/ml) was injected intravitreally after 3 days. Immediately after bevacizumab injection, and 3, 6, 24 and 48 h later, animals were euthanized for immunofluorescence staining. Donkey anti-human IgG labeled with Alexa Fluor(®) 488 was used for bevacizumab immunoreactivity detection. Anti-CD31 antibody was used as a marker for Schlemm's canal endothelial cells. Untreated eyes were used as negative controls. The intensity of the immunostaining was analyzed qualitatively. Bevacizumab immunoreactivity was found in the aqueous outflow channels including the trabecular meshwork and Schlemm's canal immediately after injection, and declined incrementally within the following hours. Forty-eight hours after the injection, no bevacizumab staining was detected in the aqueous outflow channel structures. Our manuscript demonstrates the presence of bevacizumab in the trabecular meshwork and Schlemm's canal structures after intravitreal injection in a CNV induced rat model. Bevacizumab molecules passed through the aqueous outflow channels within 48 h after intravitreal bevacizumab injection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Centipeda minima is a Chinese herbal medicine used in the treatment of various diseases including cancer. An ethanol extract of the herb, its four fractions with different polarities, and two volatile oils prepared by steam distillation (SD) and supercritical fluid extraction (SFE) were investigated for their anti-angiogenic activity in a wild-type zebrafish model using a quantitative endogenous alkaline phosphatase (EAP) assay. The SFE oil displayed potent anti-angiogenic activity. Fifteen sesquiterpene lactones (SLs; compounds 1-15) isolated from the SFE oil were evaluated for their anti-angiogenic effect. Results revealed that pseudoguaianolide type SLs (1-8) inhibited vessel formation in the zebrafish embryos while guaianolide type SLs (9-15) showed little effect. Among the active ones, 6-O-angeloylenolin (1), a major component of SFE oil, possessed the strongest effect by reducing vessel formation in zebrafish embryos to 40% of the control value at 29.7 µM. Further study using the Tg (fli1a:EGFP) y1-type zebrafish model revealed that it blocked both intersegmental blood vessels (ISVs) and subintestinal vessels plexus (SIVs) formation in zebrafish embryos. Real-time polymerase chain reaction assay on the wild-type zebrafish embryos suggested that 6-O-angeloylenolin affected multiple molecular targets related to angiogenesis including VEGF receptor, angiopoietin, and its receptors. Taken together, our findings demonstrate that C. minima possesses anti-angiogenic activity, and 6-O-angeloylenolin is a promising candidate for the development of an anti-angiogenic agent.

Crowberry, Empetrum nigrum var. japonicum, is widely used in folk medicine and grows naturally in Korea. Although some constituents and biological activity of Korean crowberry have been examined, there is little detailed information available. In this study, we investigated the effects of ethanol extracts of crowberry (EECB) on the inhibition of angiogenesis, both in vitro and in vivo. The effects of EECB were tested on in vitro models of angiogenesis, that is, tube formation and proliferation of human umbilical vein endothelial cells (HUVECs). EECB exhibited significant inhibitory effects on tube formation of HUVECs in a concentration-dependent manner. In addition, crowberry significantly suppressed the proliferation of HUVECs in a concentration-dependent manner. Furthermore, strong antiangiogenic activity of EECB samples was observed in the in vivo assay using chick embryo chorioallantoic membrane (CAM). These results indicate that crowberry may have potential applications in the prevention and treatment of angiogenesis-dependent human diseases.

Artemisia arborescens, also known as tree wormwood, is a typical species of the Mediterranean flora. It has been used in folk medicine for its antispasmodic, anti-pyretic, anti-inflammatory, and abortifacient properties. In the current study, the application of multidimensional comprehensive gas chromatography (GC×GC), allowed to obtain a detailed fingerprint of the essential oil from A. arborescens aerial parts, highlighting an abundant presence of chamazulene followed by camphor, β-thujone, myrcene, and α-pinene. Moreover, flavonoids in the dichloromethane extract were analyzed by means of liquid chromatography with photodiode array and atmospheric pressure chemical ionization-mass spectrometry detections (HPLC-PDA and HPLC-APCI-MS). Six polymethoxyflavones were identified and three of them, including chrysosplenetin, eupatin, and cirsilineol, were described in this species for the first time. The anti-angiogenic activity was investigated in the dichloromethane extract by two in vivo models, chick chorioallantoic membrane (CAM) and zebrafish embryos. Results showed that this extract produced a strong reduction on vessel formation, both on zebrafish (57% of inhibition, 0.1 mg/mL) and chick chorioallantoic membrane (58% of inhibition, 0.8 mg/mL). The high separation power and sensitivity of the analytical methodology applied confirmed the safety of A. arborescens essential oil for human consumption, due to the very low level of the psychotrope α-thujone determined. Moreover, the knowledge of the flavonoidic profile holds a great significance for the use of A. arborescens as a valuable source of anti-angiogenic compounds that might contribute to the valorization of the phytotherapeutic potential of this plant., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Eight-armed PEG was functionalized with furyl and maleimide groups (8armPEG20k-Fur and 8armPEG20k-Mal); degradable hydrogels were obtained by cross-linking via Diels-Alder chemistry. To increase the stability to degradation, the macromonomers were modified by introducing a hydrophobic 6-aminohexanoic acid spacer between PEG and the reactive end-groups (8armPEG20k-Ahx-Fur and 8armPEG20k-Ahx-Mal). In an alternative approach, the number of reactive groups per macromonomer was increased by branching the terminal ends of eight-armed PEG with lysine (Lys) and Ahx residues (8armPEG20k-Lys-Ahx-Fur2 and 8armPEG20k-Lys-Ahx-Mal2). The hydrolytic resistance of the synthesized macromonomers was determined by UV spectroscopy; the obtained hydrogels were characterized by rheology and degradation studies. The degradation time of 5% (w/v) 8armPEG20k-Ahx hydrogels (28days) was twice as long as the degradation time of 5% (w/v) 8armPEG20k hydrogels (14days); this is explained by increased hydrolytic resistance of the maleimide group. Using dendritic 8armPEG20k-Lys-Ahx macromonomers substantially increased the stability of the resulting hydrogels; degradation of 5% (w/v) 8armPEG20k-Lys-Ahx hydrogels occurred after 34 weeks. 8armPEG20k hydrogels had the largest mesh size of all tested hydrogels, while hydrogels made from dendritic 8armPEG20k-Lys-Ahx macromonomers showed the smallest value. To evaluate their potential for the controlled release of therapeutic antibodies, the hydrogels were loaded with bevacizumab. The incorporated bevacizumab was released over 10 days (8armPEG20k) and 42days (8armPEG20k-Ahx), respectively; release from 8armPEG20k-Lys-Ahx hydrogels was not completed after 105 days. In summary, we believe that 8armPEG20k-Ahx or 8armPEG20k-Lys-Ahx hydrogels could serve as controlled release system for therapeutic antibodies such as bevacizumab., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Lately, drug-coated balloons have been introduced in interventional cardiology as an approach to treat occluded blood vessel. They were developed for the rapid transfer of antiproliferative drugs during the angioplasty procedure in stenosed vessels with the intent to reduce the risk of restenosis. In this study five different paclitaxel (PTX) balloon coatings were tested in vitro in order to examine how solvents and additives influence coating stability and drug transfer rates. PTX-coated balloons were advanced through a guiding catheter and a simulated coronary artery pathway under perfusion and were then inflated in a hydrogel acceptor compartment. The fractions transferred to the gel, remaining on the balloon and the PTX lost in the simulated coronary pathway were then analysed. The results obtained suggest that the solvent used for the coating process strongly influences the surface structure and the stability of the coating. Ethanol/water and acetone based PTX coatings showed the lowest drug transfer rates to the simulated vessel wall (both <1%) due to their high drug losses during the prior passage through the coronary artery model (more than 95%). Balloons coated with PTX from ethyl acetate-solutions showed smaller drug loss (83%±9%), but most of the remaining PTX was not transferred (mean balloon residue approximately 15%). Beside the solvent, the use of additives seemed to have a great impact on transfer properties. The balloon pre-treatment with a crosslinked polyvinylpyrrolidone (PVP) film was able to increase the PTX transfer rate from less than 1% (without PVP) to approximately 6%. The best results in this study were obtained for balloon coatings with commercially available SeQuent© Please balloons containing the contrast agent iopromide. For this formulation drug transfer rates of approximately 17% were determined. Fluorescence microscopic imaging could visualize the particulate transfer of labelled PTX from the balloon surface during dilatation. The findings of this study underline the importance of drug adhesion and coating stability for the efficiency of PTX transfer., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Purpose: To compare and examine the storage stability of compounded bevacizumab in polycarbonate (PC) and polypropylene (PP) syringes over a 6-month period. PC syringes have been used in a recent clinical study and bevacizumab stability has not been reported for this type of syringe., Methods: Repackaged bevacizumab was obtained from Moorfields Pharmaceuticals in PC and PP syringes. Bevacizumab from the stored syringes was analysed at monthly time points for a 6-month period and compared with bevacizumab from a freshly opened vial at each time point. SDS-PAGE electrophoresis and size-exclusion chromatography (SEC) was used to observe aggregation and degradation. Dynamic light scattering (DLS) provided information about the hydrodynamic size and particle size distribution of bevacizumab in solution. VEGF binding and the active concentration of bevacizumab was determined by surface plasmon resonance (SPR) using Biacore., Results: SDS-PAGE and SEC analysis did not show any changes in the presence of higher molecular weight species (HMWS) or degradation products in PC and PP syringes from T0 to T6 compared with bevacizumab sampled from a freshly opened vial. The hydrodynamic diameter of bevacizumab in the PC syringe after 6 months of storage was not significantly different to bevacizumab taken from a freshly opened vial. Using SPR, the VEGF binding activity of bevacizumab in the PC syringe was comparable to bevacizumab taken from a freshly opened vial., Conclusion: No significant difference over a 6-month period was observed in the quality of bevacizumab repackaged into prefilled polycarbonate and polypropylene syringes when compared with bevacizumab that is supplied from the vial.

Background: Consumption of medicinal plants to overcome diseases is traditionally belongs to the characteristics of most cultures on this earth. Sudan has been a host and cradle to various ancient civilizations and developed a vast knowledge on traditional medicinal plants. The present study was undertaken to evaluate the antioxidant, antiangiogenic and cytotoxic activities of six Sudanese medicinal plants which have been traditionally used to treat neoplasia. Further the biological activities were correlated with phytochemical contents of the plant extracts., Methods: Different parts of the plants were subjected to sequential extraction method. Cytotoxicity of the extracts was determined by dimethylthiazol-2-yl)- 2,5diphenyl tetrazolium bromide (MTT) assay on 2 human cancer (colon and breast) and normal (endothelial and colon fibroblast) cells. Anti-angiogenic potential was tested using ex vivo rat aortic ring assay. DPPH (1,1-diphenyl-2-picrylhydrazyl) assay was conducted to screen the antioxidant capabilities of the extracts. Finally, total phenolic and flavonoid contents were estimated in the extracts using colorimetric assays., Results: The results indicated that out of 6 plants tested, 4 plants (Nicotiana glauca, Tephrosia apollinea, Combretum hartmannianum and Tamarix nilotica) exhibited remarkable anti-angiogenic activity by inhibiting the sprouting of microvessels more than 60%. However, the most potent antiangiogenic effect was recorded by ethanol extract of T. apollinea (94.62%). In addition, the plants exhibited significant antiproliferative effects against human breast (MCF-7) and colon (HCT 116) cancer cells while being non-cytotoxic to the tested normal cells. The IC50 values determined for C. hartmannianum, N. gluaca and T. apollinea against MCF-7 cells were 8.48, 10.78 and 29.36 μg/ml, respectively. Whereas, the IC50 values estimated for N. gluaca, T. apollinea and C. hartmannianum against HCT 116 cells were 5.4, 20.2 and 27.2 μg/ml, respectively. These results were more or less equal to the standard reference drugs, tamoxifen (IC50 = 6.67 μg/ml) and 5-fluorouracil (IC50 = 3.9 μg/ml) tested against MCF-7 and HCT 116, respectively. Extracts of C. hartmannianum bark and N. glauca leaves demonstrated potent antioxidant effect with IC50s range from 9.4-22.4 and 13.4-30 μg/ml, respectively. Extracts of N. glauca leaves and T apollinea aerial parts demonstrated high amount of flavonoids range from 57.6-88.1 and 10.7-78 mg quercetin equivalent/g, respectively., Conclusions: These results are in good agreement with the ethnobotanical uses of the plants (N. glauca, T. apollinea, C. hartmannianum and T. nilotica) to cure the oxidative stress and paraneoplastic symptoms caused by the cancer. These findings endorse further investigations on these plants to determine the active principles and their mode of action.

Introduction: Bevacizumab is recommended in first line chemotherapy for advanced non-squamous non-small cell lung cancer (NSCLC). Paclitaxel is a doublet drug also widely used in cisplatin-based regimens. Paclitaxel plus bevacizumab is a standard regimen as first line treatment of metastatic breast cancer. Since there is no guideline for third line treatment of NSCLC, some oncology units use paclitaxel-bevacizumab in some NSCLC patient after relapse of the second line. The aim of this study was to assess retrospectively the feasibility and efficacy of this treatment option in stage IV NSCLC., Methods: Patients who had received the combination of paclitaxel (80 to 90 mg/m(2) in a weekly schedule) and bevacizumab (7.5 to 15 mg/kg every 21 days) in the thoracic oncology departments of two university hospitals in the Rhone-Alpes Department were retrospectively reviewed., Results: Twelve patients underwent this treatment. Their mean age was 56 years and their performance status was less or equal to 1 in 50 % of the cases. The chemotherapy was given as fifth line in 67 % of the patients and 67 % were antiangiogenic naïve. They received a mean of 6 courses. The overall response rate was 33 % and the disease control rate 66 %. Median progression-free survival was 5.1 months (95 %CI 1.0-9.1). Ten patients (82 %) experienced toxicity, the majority grade 1 to 2 events, and only one a grade 3 event (febrile neutropenia). There were no severe bleeding episodes., Conclusion: Combined paclitaxel-bevacizumab chemotherapy seems feasible in patients with NSCLC. The toxicity profile is acceptable. This regimen should be evaluated in further prospective studies., (Copyright © 2013 SPLF. Published by Elsevier Masson SAS. All rights reserved.)

Background: In our previous studies conducted on Ardisia crispa roots, it was shown that Ardisia crispa root inhibited inflammation-induced angiogenesis in vivo. The present study was conducted to identify whether the anti-angiogenic properties of Ardisia crispa roots was partly due to either cyclooxygenase (COX) or/and lipoxygenase (LOX) activity inhibition in separate in vitro studies., Methods: Benzoquinonoid fraction (BQ) was isolated from hexane extract by column chromatography, and later analyzed by using gas chromatography-mass spectrometry (GC-MS). Anti-angiogenic effect was studied on mouse sponge implantation assay. Ardisia crispa ethanolic rich fraction (ACRH), quinone-rich fraction (QRF) and BQ were screened for COX assay to evaluate their selectivity towards two isoforms (COX-1 and COX-2), The experiment on soy lipoxygenase (LOX) inhibitory assay was also performed to determine the inhibitory effect of ACRH, QRF and BQ on soy LOX., Results: BQ was confirmed to consist of 2-methoxy-6-undecyl-1,4-benzoquinone, when compared with previous data. Antiangiogenesis study exhibited a reduction of mean vascular density (MVD) in both ACRH and QRF, compared to control. In vitro study showed that both ACRH and QRF inhibited both COX-1 and COX-2, despite COX-2 inhibition being slightly higher than COX-1 in BQ. On the other hand, both ACRH and QRF were shown to have poor LOX inhibitory activity, but not BQ., Conclusions: In conclusion, ACRH and QRF might possibly exhibit its anti-angiogenic effect by inhibiting cyclooxygenase. However, both of them were shown to possess poor LOX inhibitory activity. On the other hand, BQ displayed selectivity to COX-2 inhibitory property as well as LOX inhibitory effect.

Hypertensive disorders of pregnancy represent the second commonest cause of direct maternal death and complicate an estimated 5-10 % of pregnancies. Classification systems aim to separate hypertension similar to that seen outside pregnancy (chronic and gestational hypertension) from the potentially fatal pregnancy-specific conditions. Preeclampsia, HELLP syndrome, and eclampsia represent increasing severities of this disease spectrum. The American College of Obstetricians and Gynecologists' 2013 guidelines no longer require proteinuria as a diagnostic criterion, because of its variable appearance in the disease spectrum. The cause involves inadequate cytotrophoblastic invasion of the myometrium, resulting in placental hypoperfusion and diffuse maternal endothelial dysfunction. Changes in angiogenic and antiangiogentic peptide profiles precede the onset of clinical preeclampsia. Women with preeclampsia should be closely monitored and receive magnesium sulfate intravenously if severe features, HELLP syndrome, or eclampsia occur. Definitive therapy is delivery of the fetus. Hypertension in pregnancy increases future maternal risk of hypertension and cardiovascular disorders.

In this work, the stability of Bevacizumab (Avastin(®)) repackaged in individual 1 mL single-use syringes and stored at different conditions was assessed. Bevacizumab repackaged in single-use syringes results from the off-label use of the drug as an intravitreal agent in the treatment of retinal diseases. Bevacizumab stability was assessed by assaying the anti-VEGF activity using an indirect ELISA method and a Dynamic Light Scattering study. The thermal stability of the drug was also studied by calorimetric analysis, aimed to evaluate thermodynamic parameters associated to the thermal unfolding process. Furthermore, microbiological and fungal tests on the Bevacizumab syringes were performed. As a result, a significant decrease of the anti-VEGF activity was detected when syringes were exposed to UV light at a temperature of 37°C. Under these conditions, the Dynamic Light Scattering study showed an increase of the average size of Bevacizumab; probably due to aggregation. In conclusion, Bevacizumab stability, when stored under different conditions, was assessed considering three different aspects: anti-VEGF activity, microbial contamination and physico-chemical properties. Bevacizumab was found to be stable, under sterile conditions, for 3 months at 4°C and for 7 days at room temperature, exposed to indirect light sources, while a brief exposure of the drug to direct UV radiation proved detrimental to drug stability.

Angiogenesis exhibits a significant effect on tumor progression. Inhibiting angiogenesis may provide significant advantages over currently available therapeutics for cancer therapies thus, the development of a system of screening angiogenesis is essential. In the present study, a novel available system of screening angiogenesis inhibitors by four steps was developed. The chorioallantoic membrane (CAM), yolk sac membrane and early chick embryo blood island assay were initially performed to obtain possible antitumor compounds. The MMTV‑PyMT transgenic breast cancer mouse model was used for final screening and to confirm potential antitumor effects. Four angiogenesis inhibitors were isolated from 480 compounds, which were obtained from ICCB known bioactives library, by a combination of the CAM, yolk sac membrane and early chick embryo blood island assay. The MMTV‑PyMT mouse was treated with one of four agents and it was demonstrated that the tumor volume was significantly inhibited. These results demonstrate that the four‑step screening system is feasible.

Background: Regeneration of periodontal tissues is one of the most important goals for the treatment of periodontal disease. The technology of plasma rich in growth factors provides a biologic approach for the stimulation and acceleration of tissue healing. The purpose of this study is to evaluate the biologic effects of this technology on primary human periodontal ligament fibroblasts., Methods: The authors studied the response of periodontal ligament cells to this pool of growth factors on cell proliferation, cell migration, secretion of several biomolecules, cell adhesion, and expression of α2 integrin. Cell proliferation and adhesion were evaluated by means of a fluorescence-based method. Cell migration was performed on culture inserts. The release of different biomolecules by periodontal ligament fibroblasts was quantified through enzyme-linked immunosorbent assay. The α2 integrin expression was assessed through Western blot., Results: This autologous technology significantly stimulated cell proliferation, migration, adhesion, and synthesis of many growth factors from cells including vascular endothelial growth factor, thrombospondin 1, connective tissue growth factor, hepatocyte growth factor, and procollagen type I. The α2 integrin expression was lower in plasma rich in growth factor-treated cells compared to non-stimulated cells, although no statistically significant differences were observed., Conclusion: This plasma rich in growth factors exerts positive effects on periodontal ligament fibroblasts, which could be positive for periodontal regeneration.

As with other protein therapeutics, trebananib (AMG 386), an investigational peptide Fc-fusion protein ("peptibody") that inhibits angiogenesis by neutralizing the interaction of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) with the Tie2 receptor, has the potential to trigger an immune response in cancer patients treated with the therapeutic. An electrochemiluminescence bridging anti-drug antibody (ADA) assay that was utilized to support early-phase clinical trials in the development of trebananib was found to lack adequate sensitivity and drug tolerance in later-phase clinical studies when higher doses of trebananib were administered. Therefore, we developed a surface plasmon resonance (SPR) immunoassay method utilizing a secondary confirmatory detector antibody (goat anti-human IgG F[ab']2) known to cross-react with human IgG and IgM to better assess the potential impact of immunogenicity on the pharmacokinetics, pharmacodynamics, and toxicity of trebananib. The SPR method was more sensitive than the electrochemiluminescence bridging assay because of signal amplification from the confirmatory binding of the detector antibody; drug tolerance was improved since antibody binding avidity does not affect detection on this platform. Despite the inability of the confirmatory detector antibody to bind angiopoietins in protein-free buffer, false-positive ADA results were generated from patient serum samples containing Ang1 and Ang2 through an apparently specific binding between the angiopoietins and the confirmatory detector antibody, likely mediated by the interaction of the angiopoietins with serum immunoglobulins. Addition to the sample diluent of a human antibody that specifically binds to Ang1 and Ang2 with high affinity resulted in a complete block of angiopoietin interference without affecting ADA detection. This biosensor-based assay provides a reliable method for assessing immunogenicity in phase 3 clinical trials., (© 2013.)

To address the inadequacy of current assays, we developed a directed in vivo lymphangiogenesis assay (DIVLA) by modifying an established directed in vivo angiogenesis assay. Silicon tubes (angioreactors) were implanted in the dorsal flanks of nude mice. Tubes contained either growth factor-reduced basement membrane extract (BME)-alone (negative control) or BME-containing vascular endothelial growth factor (VEGF)-D (positive control for lymphangiogenesis) or FGF-2/VEGF-A (positive control for angiogenesis) or a high VEGF-D-expressing breast cancer cell line MDA-MD-468LN (468-LN), or VEGF-D-silenced 468LN. Lymphangiogenesis was detected superficially with Evans Blue dye tracing and measured in the cellular contents of angioreactors by multiple approaches: lymphatic vessel endothelial hyaluronan receptor-1 (Lyve1) protein (immunofluorescence) and mRNA (qPCR) expression and a visual scoring of lymphatic vs blood capillaries with dual Lyve1 (or PROX-11 or Podoplanin)/Cd31 immunostaining in cryosections. Lymphangiogenesis was absent with BME, high with VEGF-D or VEGF-D-producing 468LN cells and low with VEGF-D-silenced 468LN. Angiogenesis was absent with BME, high with FGF-2/VEGF-A, moderate with 468LN or VEGF-D and low with VEGF-D-silenced 468LN. The method was reproduced in a syngeneic murine C3L5 tumor model in C3H/HeJ mice with dual Lyve1/Cd31 immunostaining. Thus, DIVLA presents a practical and sensitive assay of lymphangiogenesis, validated with multiple approaches and markers. It is highly suited to identifying pro- and anti-lymphangiogenic agents, as well as shared or distinct mechanisms regulating lymphangiogenesis vs angiogenesis, and is widely applicable to research in vascular/tumor biology.

Background: Angiogenesis plays a critical role in embryonic development and various physiological processes. However, excessive angiogenesis is associated with several pathological conditions including cancer. Pithecellobium jiringa (Jack) Prain is a traditional medicinal plant from the family Leguminosae. It is native to the Southeast Asia, where it has been used traditionally for treatment of various ailments such as hypertension and diabetes. The present work is aimed to study antioxidant and antiangiogenesis activities of P. jiringa ethanol extracts., Methods: P. jiringa fruit rinds were extracted with ethanol and 50% ethanol. The antioxidant property was analysed using, 1,1-diphenyl-2-picryl-hydrazyl free radical scavenging assay. Phytochemical analysis was performed using thin layer chromatography and colorimetric methods. Then, cell growth inhibition was studied against a panel of human cell lines by MTT test. In vitro inhibition of angiogenesis was studied by the following assays: isolated rat aortic rings cell viability, colony formation, endothelial cell migration, endothelial tube formation on matrigel, and expression of vascular endothelial growth factor by endothelial cells. In vivo antiangiogenesis effect was studied by utilising fertilised chick embryos assay. The results were statistically analysed by analysis of variance., Results: Ethanolic and 50% hydro-ethanolic extracts showed relatively high concentration of total phenolics associated with potent antioxidant activity. The rat aortic rings study conducted showed potent inhibition of the microvessels outgrowth with IC50s 5.27 ± 0.81 μg/ml (ethanolic) and 4.45 ± 0.63 μg/ml (50% hydro-ethanolic). Both extracts arrested the growth of human endothelial cells via down-regulation of VEGF expression, leading to inhibition of other angiogenesis cascades including migration of endothelial cells, and formation of capillary network on matrigel matrix. The extracts also inhibited the neovascularisation of chick embryo chorioallantoic membrane., Conclusions: P. jiringa extracts inhibit angiogenesis by blocking the VEGF expression thus inhibiting endothelial cells proliferation, migration and differentiation most likely due to presence of the antioxidant phenolics.

Objective: To test the hypothesis that high circulating concentrations of maternal anti-angiogenic factors are associated with increased risk of respiratory distress syndrome (RDS)., Study Design: This is a nested case-control study of nulliparous women who delivered less than 37 weeks of gestation within the Calcium for Preeclampsia Prevention (CPEP) trial. The study included 116 women with preeclampsia or gestational hypertension and 323 normotensive controls. Soluble fms-like tyrosine kinase 1 (sFlt1), placental growth factor (PlGF) and soluble endoglin (sEng) in maternal serum were measured at 21-32 weeks of gestation., Results: Preterm infants born to hypertensive mothers were more likely to develop RDS (22.5% vs. 20.9%, p = 0.03). After adjustment for gestational age at delivery, the odds ratio for the relationship between hypertension in pregnancy and RDS was 2.18 (95% CI 1.08-4.39). In hypertensive pregnancies women whose infants developed RDS had significantly higher circulating mean sFlt1 levels during midpregnancy (21-32 weeks of gestation) even after adjustment for gestational age at delivery (21,516 pg/mL vs. 7,000 pg/mL, p = 0.01)., Conclusions: Preterm preeclampsia and gestational hypertension, characterized by high circulating levels of sFlt1, are associated with a twofold increased risk of RDS in infants delivered before 37 weeks. Among women with these hypertensive pregnancies circulating sFlt1 concentrations during midpregnancy were substantially higher in women whose infants developed RDS.

Mirror syndrome is a rare disorder in which fetal hydrops is associated with maternal oedema, proteinuria and hypertension. The aetiology of the maternal condition is unknown, but it is thought to be related to preeclampsia. Few descriptions exist of placental morphology in mirror syndrome, but placentomegaly is consistently observed. In this case placental morphology showed villous oedema and syncytial nuclear aggregates where villi were in direct contact. Immunoperoxidase staining for VEGFR1 and Endoglin was more intense in mirror syndrome compared to gestational-age matched controls,and at a similar level to a case of preeclampsia. Placentally-derived anti-angiogenic factors may be involved in the pathogenesis of mirror syndrome., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: The anti-angiogenic monoclonal antibody, bevacizumab, is currently used by intravitreal administration as off-label drug to treat age-related macular degeneration or other ophthalmologic diseases. For this purpose, commercial bevacizumab is repackaged in 1mL polypropylene syringes under sterile conditions. However, no complete study on the stability of this hospital-based preparation is available., Methods: Commercial bevacizumab (25mg/mL; Avastin(®)) was aseptically repackaged in 1mL polypropylene syringes, stored at 4°C, and analyzed within the preparation day (D0), after 30 days (D30) and 90 days (D90). Some syringes were kept for up to 8 months to observe possible instability. Several complementary and stability-indicating analytical methods were used to assess in details the primary, secondary and tertiary structure of the antibody during its conservation: ionic chromatography, size-exclusion chromatography, peptide mapping, 2nd derivative UV and IR spectroscopy, turbidimetry, diffraction laser spectroscopy, thermal denaturation curves, microscopic examination and image analysis., Results: We clearly demonstrate that the commercial solution of bevacizumab can be safely repackaged in polypropylene syringes and stored up to 3 months at 4°C without alteration of its primary, secondary and tertiary structure. The only difference observed is the contamination of the syringe content by silicone oil microdroplets, which is quite immediate and does not change significantly during the storage in terms of number and size., Conclusion: Our results support the off-label use of repackaged bevacizumab by intravitreal administration, at least from a pharmaceutical point of view, with a validated stability of 3 months. This stability period is largely enough to practical situations and support current practices, such as in advance or batch preparations, which present major advantages in terms of GMP respect, workload optimization and financial savings., (Copyright © 2012. Published by Elsevier Masson SAS.)

Chromatographic fingerprinting has been widely accepted as a crucial method for qualitative and quantitative analyses of bioactives within traditional Chinese medicine. A fingerprint provides detailed information, specific for any given herb, thus facilitating the quality control measures of a given traditional Chinese medicine. In this article, quality assessment of Radix Scrophulariae was achieved by using high performance liquid chromatography combining diode-array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS). Eight batches of sample obtained from different origins in China were used to establish the fingerprint and quantitative analyses. By comparing the retention times, UV and MS spectral data with reference standards, four characteristic peaks in the chromatograms were confirmed as corresponding to acetoside, angoroside C, cinnamic acid, and harpagoside. In addition, other two characteristic peaks were tentatively identified, following the literature interpretation of HPLC-ESI-MS and LC-MS/MS (affording structural information) to be sibirioside A and scrophuloside B(4), respectively. The results indicated that the newly developed HPLC-DAD-MS fingerprint method would be suitable for quality control of Radix Scrophulariae., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The use of intra-oral soft-tissue-engineered devices has demonstrated potential for oral mucosa regeneration. The aim of this study was to investigate the temporal expression of angiogenic biomarkers during wound healing of soft tissue reconstructive procedures comparing living cellular constructs (LCC) with autogenous free gingival grafts. Forty-four human participants bilaterally lacking sufficient zones of attached keratinized gingiva were randomly assigned to soft tissue surgery plus either LCC or autograft. Wound fluid samples were collected at baseline and weeks 1, 2, 3, and 4 post-operatively and analyzed for a panel of angiogenic biomarkers: angiogenin (ANG), angiostatin (ANT), PDGF-BB, VEGF, FGF-2, IL-8, TIMP-1, TIMP-2, GM-CSF, and IP-10. Results demonstrated a significant increase in expression of ANT, PDGF-BB, VEGF, FGF-2, and IL-8 for the LCC group over the autograft group at the early stages of wound repair. Although angiogenic biomarkers were modestly elevated for the LCC group, no clinical correlation with wound healing was found. This human investigation demonstrates that, during early wound-healing events, expression of angiogenic-related biomarkers is up-regulated in sites treated with LCC compared with autogenous free gingival grafts, which may provide a safe and effective alternative for regenerating intra-oral soft tissues (ClinicalTrials.gov number, NCT01134081).

Ethnopharmacological Relevance: Gynura segetum is a popular medicinal plant in Indonesia and Malaysia, known to possess various medicinal properties especially for treatment of cancer, diabetes and hypertension., Aim of the Study: This study was carried out to evaluate the anti-angiogenic effect of Gynura segetum leaves extracts and its fractions. The chemical compositions of the active extracts were also determined., Materials and Methods: The anti-angiogenic activity of Gynura segetum leaves extracts and its fractions was evaluated in vivo using the chick embryo chorioallantoic membrane (CAM) assay. Gas chromatography-mass spectrometry (GC-MS) analysis was carried out to identify the chemical compositions of the active extracts., Results: The CAM treated with Gynura segetum leaves extracts and its fractions (100μg/disc) showed a significantly greater anti-angiogenic effect compared to the positive control suramin (50μg/disc). Chemical analysis of the active extracts from the leaves of Gynura segetum yielded nine known compounds: undecane (1), neophytadine (2), hexadecanoic acid, methyl ester (3), 9,12-octadecadienoic acid, methyl ester (4), 9,12,15-octadecatrienoic acid, methyl ester (5), phytol (6), tetradecanal (7), octadecanoic acid, methyl ester (8) and γ-sitosterol (9)., Conclusions: These results suggested that Gynura segetum has anti-angiogenic activity. The plant may be used as a potential source for protection against cancer., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: The temporomandibular joint (TMJ) cartilage consists of condylar cartilage and disc and undergoes continuous remodeling throughout post-natal life. To maintain the integrity of the TMJ cartilage, anti-angiogenic factors play an important role during the remodeling process. In this study, we investigated the expression of the anti-angiogenic factor, chondromodulin-1 (ChM-1), in TMJ cartilage and evaluate its potential role in TMJ remodeling., Methods: Eight TMJ specimens were collected from six 4-month-old Japanese white rabbits. Safranin-O staining was performed to determine proteoglycan content. ChM-1 expression in TMJ condylar cartilage and disc was determined by immunohistochemistry. Three human perforated disc tissue samples were collected for investigation of ChM-1 and vascular endothelial growth factor (VEGF) distribution in perforated TMJ disc., Results: Safranin-O stained weakly in TMJ compared with tibial articular and epiphyseal cartilage. In TMJ, ChM-1 was expressed in the proliferative and hypertrophic zone of condylar cartilage and chondrocyte-like cells in the disc. No expression of ChM-1 was observed in osteoblasts and subchondral bone. ChM-1 and VEGF were both similarly expressed in perforated disc tissues., Conclusions: ChM-1 may play a role in the regulation of TMJ remodeling by preventing blood vessel invasion of the cartilage, thereby maintaining condylar cartilage and disc integrity.

Neoplastic neovascularization is regulated not only by stimulators, but also by inhibitors of angiogenesis and might be the result of a net balance between the positive and negative regulators. Endostatin (ES) is a potent inhibitor of angiogenesis. The expression of ES has not been investigated in patients with osteosarcomas (OSAs). The aim of this study was to determine whether there is a correlation between the expression of ES and clinicopathologic parameters and/or outcomes in patients with OSAs. We made tissue microarrays from 46 cases of OSA and analyzed the expression of ES using immunohistochemistry. Staining was assessed in a semi-quantitative manner by scoring the proportion of positive tumor cells over the total number of tumor cells. A sample was defined as ES-positive when 10% or more of the tumor cells were stained positively throughout the tumor core. ES was localized to the cytoplasm of the tumor cells. 32.6% (15/46) of the patients were ES-positive. The expression of ES was positively correlated with tumor size (p = 0.011), histologic grade (p = 0.034), stage (p = 0.025), and distant metastasis (p = 0.036). Our results suggest that the expression of ES is increased in OSA, and ES may be used as a prognostic marker in patients with OSAs.

Vascular-disrupting strategies impair a tumor's blood vessel network, which is essential for tumor progression and metastasis. Vascular-disrupting agents (VDAs) cause a rapid and selective vascular shutdown in tumors to produce extensive secondary neoplastic cell death due to ischemia. A lead agent in this therapeutic strategy is the tubulin depolymerizing agent combretastatin-A4 phosphate (CA4P). Used alone, CA4P induces extensive necrosis in a wide variety of preclinical cancer models and significant blood flow reductions in the patient tumors. Preclinical and clinical data further indicate that CA4P can effectively be combined with chemotherapy or radiotherapy. Finally, the potential of combining VDAs with antiangiogenic therapies has shown considerable promise in preclinical models and such combinations are now beginning to be evaluated in patients.

We evaluated the chemopreventive potential of Azadirachta indica (neem) leaf fractions based on in vitro antioxidant assays, and in vivo inhibitory effects on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. In addition we also identified the major constituents in neem leaf fractions by HPLC. Analysis of the free radical scavenging activities and reducing potential of crude ethanolic extract (CEE), ethyl acetate fraction (EAF) and methanolic fraction (MF) of neem leaf revealed a concentration-dependent increase in antioxidant potential that was in the order EAF>MF>CEE. Administration of neem leaf fractions reduced the incidence of DMBA-induced HBP carcinomas at a lower concentration compared to the crude extract. Chemoprevention by neem leaf fractions was associated with modulation of phase I and phase II xenobiotic-metabolising enzymes, lipid and protein oxidation, upregulation of antioxidant defences, inhibition of cell proliferation and angiogenesis, and induction of apoptosis. However, EAF was more effective than MF in terms of antiproliferative and antiangiogenic effects, and expression of CYP isoforms. The greater efficacy of EAF may be due to higher content of constituent phytochemicals as revealed by HPLC analysis. The results of the present study suggest that the antioxidant properties of neem leaf fractions may be responsible for modulating key hallmark capabilities of cancer cells such as cell proliferation, angiogenesis and apoptosis in the HBP carcinogenesis model.

Malignant melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Angiogenesis - the growth of new vessels from preexisting vasculature - is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. We previously described a class of anti-angiogenic isoforms of VEGF, VEGF(xxx)b, that inhibit tumour growth in animal models, and are downregulated in some cancers, but have not been investigated in melanoma. To determine whether VEGF(xxx)b expression was altered in melanoma, PCR and immunohistochemistry of archived human tumour samples were used. In normal epidermis and in a proportion of melanoma samples, VEGF(xxx)b staining was seen. Some melanomas had much weaker staining. Subsequent examination revealed that expression was significantly reduced in primary melanoma samples (both horizontal and vertical growth phases) from patients who subsequently developed tumour metastasis compared with those who did not (analysis of variance (ANOVA) P<0.001 metastatic vs nonmetastatic), irrespective of tumour thickness, while the surrounding epidermis showed no difference in expression. Staining for total VEGF expression showed staining in metastatic and nonmetastatic melanomas, and normal epidermis. An absence of VEGF(xxx)b expression appears to predict metastatic spread in patients with primary melanoma. These results suggest that there is a switch in splicing as part of the metastatic process, from anti-angiogenic to pro-angiogenic VEGF isoforms. This may form part of a wider metastatic splicing phenotype.