Cottonseed-derived gossypol and ethanol extracts differentially regulate cell viability and VEGF gene expression in mouse macrophages.

Vascular endothelial growth factor (VEGF) plays an important role in chronic inflammation associated with several diseases. Many plant extracts have nutritional and healthy benefits by down-regulating VEGF expression, but there was no report on VEGF regulation by cottonseed extracts in any biological system. The objective was to investigate cell viability and VEGF expression regulated by gossypol and ethanol extracts using lipopolysaccharides (LPS) as a control. MTT, qPCR and immunoblotting techniques were used to monitor cell viability, VEGF mRNA and protein levels in mouse RAW264.7 macrophages. Gossypol dramatically reduced macrophage viability but cottonseed extracts and LPS exhibited minor effect on cell viability. VEGFb mRNA levels were approximately 40 fold of VEGFa in the macrophages. Gossypol increased VEGFa and VEGFb mRNA levels up to 27 and 4 fold, respectively, and increased VEGF protein. LPS increased VEGFa mRNA by sixfold but decreased VEGFb mRNA. LPS increased VEGF protein in 2-4 h but decreased in 8-24 h. Glanded seed extracts showed some stimulating effects on VEGF mRNA levels. Glandless seed coat extract showed increased VEGFb mRNA levels but its kernel extract reduced VEGF mRNA levels. This study demonstrated that gossypol and ethanol extracts differentially regulated cell viability and VEGF expression in mouse macrophages.

Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles.

Avascular transplantation of frozen-thawed testicular tissue fragments represents a potential future technique for fertility restoration in boys with cancer. A significant loss of spermatogonia was observed in xeno-transplants of human tissue most likely due to the hypoxic period before revascularization. To reduce the effect of hypoxia-reoxygenation injuries, several options have already been explored, like encapsulation in alginate hydrogel and supplementation with nanoparticles delivering a necrosis inhibitor (NECINH) or VEGF. While these approaches improved short-term (5 days) vascular surfaces in grafts, neovessels were not maintained up to 21 days; i.e., the time needed for achieving vessel stabilization. To better support tissue grafts, nanoparticles loaded with VEGF, PDGF and NECINH were developed. Testicular tissue fragments from 4-5-week-old mice were encapsulated in calcium-alginate hydrogels, either non-supplemented (control) or supplemented with drug-loaded nanoparticles (VEGF-nanoparticles; VEGF-nanoparticles + PDGF-nanoparticles; NECINH-nanoparticles; VEGF-nanoparticles + NECINH-nanoparticles; and VEGF-nanoparticles + PDGF-nanoparticles + NECINH-nanoparticles) before auto-transplantation. Grafts were recovered after 5 or 21 days for analyses of tissue integrity (hematoxylin-eosin staining), spermatogonial survival (immuno-histo-chemistry for promyelocytic leukemia zinc finger) and vascularization (immuno-histo-chemistry for α-smooth muscle actin and CD-31). Our results showed that a combination of VEGF and PDGF nanoparticles increased vascular maturity and induced a faster maturation of vascular structures in grafts.

Network pharmacology based virtual screening of active constituents of Prunella vulgaris L. and the molecular mechanism against breast cancer.

Prunella vulgaris L, a perennial herb widely used in Asia in the treatment of various diseases including cancer. In vitro studies have demonstrated the therapeutic effect of Prunella vulgaris L. against breast cancer through multiple pathways. However, the nature of the biological mechanisms remains unclear. In this study, a Network pharmacology based approach was used to explore active constituents and potential molecular mechanisms of Prunella vulgaris L. for the treatment of breast cancer. The methods adopted included active constituents prescreening, target prediction, GO and KEGG pathway enrichment analysis. Molecular docking experiments were used to further validate network pharmacology results. The predicted results showed that there were 19 active ingredients in Prunella vulgaris L. and 31 potential gene targets including AKT1, EGFR, MYC, and VEGFA. Further, analysis of the potential biological mechanisms of Prunella vulgaris L. against breast cancer was performed by investigating the relationship between the active constituents, target genes and pathways. Network analysis showed that Prunella vulgaris L. exerted a promising preventive effect on breast cancer by acting on tumor-associated signaling pathways. This provides a basis to understand the mechanism of the anti-breast cancer activity of Prunella vulgaris L.

A Network Pharmacology Approach to Estimate the Active Ingredients and Potential Targets of Cuscutae semen in the Treatment of Osteoporosis.

BACKGROUND Osteoporosis is a metabolic osteopathy characterized by abnormal bone mass and microstructure that has become a public health problem worldwide. Cuscutae semen (CS) is a traditional Chinese medicine (TCM) that has a positive effect on the prevention and treatment of osteoporosis. However, the mechanism of CS is unclear. Therefore, this study aimed to reveal the possible molecular mechanism involved in the effects of CS on osteoporosis based on a network pharmacology approach. MATERIAL AND METHODS The inactive and active ingredients of CS were identified by searching the pharmacology analysis platform of the Chinese medicine system (TCMSP), and the targets of osteoporosis were screened in the relevant databases, such as GeneCards, PubMed, and the Comparative Toxicogenomics Database (CTD). The network of "medicine-ingredients-disease-targets (M-I-D-T)" was established by means of network pharmacology, and the key targets and core pathways were determined by R analysis. Molecular docking methods were used to evaluate the binding activity between the target and the active ingredients of CS. RESULTS Eleven active ingredients were identified in CS, and 175 potential targets of the active ingredients were also identified from the TCMSP. Moreover, we revealed 22 539 targets related to osteoporosis in the 3 well-established databases, and we determined the intersection of the disease targets and the potential targets of the active ingredients; 107 common targets were identified and used in further analysis. Additionally, biological processes and signaling pathways involved in target action, such as fluid shear stress, atherosclerosis, cancer pathways, and the TNF signaling pathway, were determined. Finally, we chose the top 5 common targets, CCND1, EGFR, IL6, MAPK8, and VEGFA, for molecular docking with the 11 active ingredients of CS. CONCLUSIONS This study suggested that CS has multiple ingredients and multiple targets relevant to the treatment of osteoporosis. We determined that the active ingredient, sesamin, may be the most crucial ingredient of CS for the treatment of osteoporosis. Additionally, the network pharmacology method provided a novel research approach to analyze the function of complex ingredients.

Andrographolide binds to ATP-binding pocket of VEGFR2 to impede VEGFA-mediated tumor-angiogenesis.

Vasculogenesis and angiogenesis are process of formation of blood vessels. Blood vessels are evolved to distribute nutrients and oxygen to distant organs. These vessels are crucial for growth and repair of wounded tissue. During tumor condition there occurs imbalance in the growth of blood vessels which leads to neo-angiogenesis. Neo-angiogenesis is major perpetrator behind the establishment of tumor. Tumor cells secrete pro-angiogenic factor VEGFA which binds to VEGFR2 present over surface of endothelial cells and triggers formation of new blood vessels. To inhibit tumor-angiogenesis, a physiologically-safe small molecule inhibitor was screened which can potentially interact with kinase domain of VEGFR2 and inhibit its activity. Molecular-docking module and biochemical analysis identified andrographolide as one of the best docking molecules that binds to ATP-binding pocket of VEGFR2 and inhibits its kinase activity. Thus, for a more radical approach towards safe VEGFR2 inhibitor, andrographolide was repurposed to inhibit tumor-angiogenesis and reduce tumor burden.

A bioactive nano-calcium phosphate paste for in-situ transfection of BMP-7 and VEGF-A in a rabbit critical-size bone defect: results of an in vivo study.

The aim of this study was to prepare an injectable DNA-loaded nano-calcium phosphate paste that is suitable as bioactive bone substitution material. For this we used the well-known potential of calcium phosphate in bone contact and supplemented it with DNA for the in-situ transfection of BMP-7 and VEGF-A in a critical-size bone defect. 24 New Zealand white rabbits were randomly divided into two groups: One group with BMP-7- and VEGF-A-encoding DNA on calcium phosphate nanoparticles and a control group with calcium phosphate nanoparticles only. The bone defect was created at the proximal medial tibia and filled with the DNA-loaded calcium phosphate paste. As control, a bone defect was filled with the calcium phosphate paste without DNA. The proximal tibia was investigated 2, 4 and 12 weeks after the operation. A histomorphological analysis of the dynamic bone parameters was carried out with the Osteomeasure system. The animals treated with the DNA-loaded calcium phosphate showed a statistically significantly increased bone volume per total volume after 4 weeks in comparison to the control group. Additionally, a statistically significant increase of the trabecular number and the number of osteoblasts per tissue area were observed. These results were confirmed by radiological analysis. The DNA-loaded bone paste led to a significantly faster healing of the critical-size bone defect in the rabbit model after 4 weeks. After 12 weeks, all defects had equally healed in both groups. No difference in the quality of the new bone was found. The injectable DNA-loaded calcium phosphate paste led to a faster and more sustained bone healing and induced an accelerated bone formation after 4 weeks. The material was well integrated into the bone defect and new bone was formed on its surface. The calcium phosphate paste without DNA led to a regular healing of the critical-size bone defect, but the healing was slower than the DNA-loaded paste. Thus, the in-situ transfection with BMP-7 and VEGF-A significantly improved the potential of calcium phosphate as pasty bone substitution material.

Binding of Resveratrol to Vascular Endothelial Growth Factor Suppresses Angiogenesis by Inhibiting the Receptor Signaling.

Resveratrol is a polyphenol commonly found in plants and food health products, such as grape and red wine, and was identified for its binding to vascular endothelial growth factor (VEGF) by using HerboChips screening. The binding, therefore, resulted in alterations of VEGF binding to its receptor and revealed the roles of VEGF in angiogenesis. Several lines of evidence gave support to the inhibitory activities of resveratrol in VEGF-triggered angiogenesis. In human umbilical vein endothelial cells (HUVECs), compared with a VEGF-induced group, resveratrol, at a high concentration, suppressed VEGF-mediated endothelial cell proliferation, cell migration, cell invasion, and tube formation by 80 ± 9.01%, 140 ± 3.78%, 110 ± 7.51%, and 120 ± 10.26%, respectively. Moreover, resveratrol inhibited the subintestinal vessel formation in zebrafish embryo. In signaling cascades, application of resveratrol in HUVECs reduced the VEGF-triggered VEGF receptor 2 phosphorylation and c-Jun N-terminal kinase phosphorylation. Moreover, the VEGF-mediated phosphorylations of endothelial nitric oxide synthase, protein kinase B, and extracellular signal-regulated kinase were obviously decreased by (3 ± 0.37)-, (2 ± 0.27)- and (6 ± 0.23)-fold, respectively, in the presence of resveratrol at high concentration. Parallelly, the VEGF-induced reactive oxygen species formation was significantly decreased by 50 ± 7.88% to 120 ± 14.82% under resveratrol treatment. Thus, our results provided support to the antiangiogenic roles of resveratrol, as well as its related signaling mechanisms, in attenuating the VEGF-mediated responses. The present results supported possible development of resveratrol, which should be considered as a therapeutic agent in terms of prevention and clinical treatment of diseases related to angiogenesis.

Anti-angiogenic Activity of Major Phenolics in Tamarind Assessed with Molecular Docking Study on VEGF Kinase Proteins.

BACKGROUND AND OBJECTIVE: The waste products of the tamarind canning industry have been discarded; however, it has potential health benefits. Herein, the study was carried out HPLC profiling of phenolic constituents of Tamarindis indica pericarp and seeds. Furthermore, the cytotoxic activity against HUH-7 cells was evaluated and assessed with molecular docking study on angiogenesis-related VEGF kinase proteins in addition to evaluating the level of released VEGF in treated HUH-7 cells by ELISA. MATERIALS AND METHODS: Folin-ciocalteu and AlCl3 assays were used for quantification of total phenolics (TPC) and total flavonoids (TFC) contents, respectively. Molecular docking study was done on VEGF kinase proteins. RESULTS: TPC and TFC of pericarp and seeds were 0.35±0.02 g GAE g-1 DE and 0.12±0.009 g CE g-1 DE, 0.39±0.01 g GAE g-1 DE and 0.03±0.006 g CE g-1 DE, respectively. In pericarp, 8 phenolics were tentatively identified, where (+)-catechin was the major (27,386.04 µg g-1 DE) followed by gallic acid and naringenin (931.47, 500.42 µg g-1 DE) respectively. While in seeds, 11 phenolics were tentatively identified, where naringenin was the major (95,305.47 µg g-1 DE) followed by (+)-catechin and rutin (54,930.29, 15,361.66 µg g-1 DE) respectively. Aqueous and methanol seeds extracts exhibit cytotoxic effect with IC50 27.4±1.81 and 13.4±0.94 µg mL-1, respectively, it was more potent than aqueous and methanol pericarp extracts which had IC50 132±5.82 and 61.6±3.16 µg mL-1. The tested phenolics were fit on the active sites of VEGF kinase targets with varied degree of interactions. The cytotoxic and anti-angiogenic activities were confirmed in light of phenolics docking interactions. CONCLUSION: Results demonstrate for the first time that phenolics could inhibit angiogenesis via inhibiting kinase proteins, which could therefore be developed as antiangiogenic drugs.

Surface functionalization superparamagnetic nanoparticles conjugated with thermoresponsive poly(epsilon-lysine) dendrons tethered with carboxybetaine for the mild hyperthermia-controlled delivery of VEGF.

UNLABELLED: Vascular endothelial growth factor (VEGF) is the growth factor responsible for the triggering of angiogenesis, the process of blood vessel formation supporting the long-term viability of any repaired or regenerated tissue. As the growth factor is effective only when concentration gradients are generated, new shuttles need to be developed that ensure both the control of gradients at the site of tissue repair and the release of VEGF at physiological levels. Magnetic hyperthermia is the production of heat induced by magnetic materials through their exposure to an external oscillating magnetic field. In this paper, magnetic nanoparticles capable of generating controllable hyperthermia were functionalised with hyperbranched poly(epsilon-lysine) peptides integrating in their core parallel thermoresponsive elastin-like peptide sequences and presenting an uppermost branching generation tethered by the zwitterionic amino acid carboxybetaine. The results show that these functionalised magnetic nanoparticles avidly bind VEGF and release it only upon generation of mild-hyperthermic pulses generated by oscillating magnetic filed. The VEGF release occurred in a temperature range at which the elastin-like peptides collapse. It is proposed that, through the application of an external magnetic field, these magnetic carriers could generated gradients of VEGF in vivo and allow its tuned delivery in a number of clinical applications. STATEMENT OF SIGNIFICANCE: The present paper for the first time reveals the possibility to control the delivery of VEGF through mild hyperthermia stimuli generated by a oscillating magnetic field. To this purpose, magnetic nanoparticles of high size homogeneity and coated with a thin coating of poly(acrylic acid) were functionalised with a novel class of poly(epsilon lysine) dendrimers integrating in their structure a thermoresponsive amino acid sequence mimicking elastin and exposing at high density a zwitterionic modified amino acid, the carboxybetaine, known to be able to bind macromolecules. Physicochemical and biochemical characterisation elegantly show the link between the thermal properties of the nanoparticles and of the dendrimer change of conformation and how this enable the release of VEGF at temperature values compatible with the growth factor stability.

Scopoletin, an active principle of tree tobacco (Nicotiana glauca) inhibits human tumor vascularization in xenograft models and modulates ERK1, VEGF-A, and FGF-2 in computer model.

We recently reported the antineovascularization effect of scopoletin on rat aorta and identified its potential anti-angiogenic activity. Scopoletin could be useful as a systemic chemotherapeutic agent against angiogenesis-dependent malignancies if its antitumorigenic activity is investigated and scientifically proven using a suitable human tumor xenograft model. In the present study, bioassay-guided (anti-angiogenesis) phytochemical investigation was conducted on Nicotiana glauca extract which led to the isolation of scopoletin. Further, anti-angiogenic activity of scopoletin was characterized using ex vivo, in vivo and in silico angiogenesis models. Finally, the antitumorigenic efficacy of scopoletin was studied in human colorectal tumor xenograft model using athymic nude mice. For the first time, an in vivo anticancer activity of scopoletin was reported and characterized using xenograft models. Scopoletin caused significant suppression of sprouting of microvessels in rat aortic explants with IC50 (median inhibitory concentration) 0.06µM. Scopoletin (100 and 200mg/kg) strongly inhibited (59.72 and 89.4%, respectively) vascularization in matrigel plugs implanted in nude mice. In the tumor xenograft model, scopoletin showed remarkable inhibition on tumor growth (34.2 and 94.7% at 100 and 200mg/kg, respectively). Tumor histology revealed drastic reduction of the extent of vascularization. Further, immunostaining of CD31 and NG2 receptors in the histological sections confirmed the antivascular effect of scopoletin in tumor vasculature. In computer modeling, scopoletin showed strong ligand affinity and binding energies toward the following angiogenic factors: protein kinase (ERK1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2). These results suggest that the antitumor activity of scopoletin may be due to its strong anti-angiogenic effect, which may be mediated by its effective inhibition of ERK1, VEGF-A, and FGF-2.

Combined Use of Oligopeptides, Fragment Libraries, and Natural Compounds: A Comprehensive Approach To Sample the Druggability of Vascular Endothelial Growth Factor.

The modulation of protein-protein interactions (PPIs) is emerging as a highly promising tool to fight diseases. However, whereas an increasing number of compounds are able to disrupt peptide-mediated PPIs efficiently, the inhibition of domain-domain PPIs appears to be much more challenging. Herein, we report our results related to the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR). The VEGF-VEGFR interaction is a typical domain-domain PPI that is highly relevant for the treatment of cancer and some retinopathies. Our final goal was to identify ligands able to bind VEGF at the region used by the growth factor to interact with its receptor. We undertook an extensive study, combining a variety of experimental approaches, including NMR-spectroscopy-based screening of small organic fragments, peptide libraries, and medicinal plant extracts. The key feature of the successful ligands that emerged from this study was their capacity to expose hydrophobic functional groups able to interact with the hydrophobic hot spots at the interacting VEGF surface patch.

Functionalization of titanium implants using a modular system for binding and release of VEGF enhances bone-implant contact in a rodent model.

AIMS: To test the immobilization of vascular endothelial growth factor (VEGF165 ) on the surface of titanium implants using DNA oligonucleotide (ODN) anchor strands for the ability to enhance periimplant bone formation. MATERIALS AND METHODS: DNA oligonucleotides were anchored to the surface of sandblasted acid-etched (SAE) titanium screw implants and were hybridized with complementary strands of ODN conjugated to rhVEGF165 . The implants were tested against blank SAE implants and SAE implants with nano-anchored ODN. The implants were inserted into the tibiae of 36 Sprague-Dawley rats. Primary outcome parameters were bone-implant contact (BIC), amount of new bone formation and periimplant bone density (BD). density after 1, 4 and 13 weeks. Unit of analysis has been the individual implant. RESULTS: Implants with rhVEGF165 hybridized to ODN anchor strands exhibited significantly increased average BIC after 1 month compared to blank implants and implants with anchored ODN strands. CONCLUSIONS: It is concluded that rhVEGF165 immobilized on the surface of titanium implants through nano-anchored oligonucleotide strands can accelerate BIC of sandblasted and etched titanium implants to a certain extent. The radius of effect of the growth factor appears to be limited to tissue immediately adjacent to the implant surface.

Potent inhibition of VEGFR-2 activation by tight binding of green tea epigallocatechin gallate and apple procyanidins to VEGF: relevance to angiogenesis.

SCOPE: Excessive concentrations of vascular endothelial growth factor (VEGF) drive angiogenesis and cause complications such as increased growth of tumours and atherosclerotic plaques. The aim of this study was to determine the molecular mechanism underlying the potent inhibition of VEGF signalling by polyphenols. METHODS AND RESULTS: We show that the polyphenols epigallocatechin gallate from green tea and procyanidin oligomers from apples potently inhibit VEGF-induced VEGF receptor-2 (VEGFR-2) signalling in human umbilical vein endothelial cells by directly interacting with VEGF. The polyphenol-induced inhibition of VEGF-induced VEGFR-2 activation occurred at nanomolar polyphenol concentrations and followed bi-phasic inhibition kinetics. VEGF activity could not be recovered by dialysing VEGF-polyphenol complexes. Exposure of VEGF to epigallocatechin gallate or procyanidin oligomers strongly inhibited subsequent binding of VEGF to human umbilical vein endothelial cells expressing VEGFR-2. Remarkably, even though VEGFR-2 signalling was completely inhibited at 1 µM concentrations of polyphenols, endothelial nitric oxide synthase was shown to still be activated via the PI3K/Akt signalling pathway which is downstream of VEGFR-2. CONCLUSION: These data demonstrate for the first time that VEGF is a key molecular target for specific polyphenols found in tea, apples and cocoa which potently inhibit VEGF signalling and angiogenesis at physiological concentrations. These data provide a plausible mechanism which links bioactive compounds in food with their beneficial effects.

The use of pH-sensitive functional selenium nanoparticles shows enhanced in vivo VEGF-siRNA silencing and fluorescence imaging.

The utility of small interfering RNAs (siRNAs) has shown great promise in treating a variety of diseases including many types of cancer. While their ability to silence a wide range of target genes underlies their effectiveness, the application of therapies remains hindered by a lack of an effective delivery system. In this study, we sought to develop an siRNA-delivery system for VEGF, a known signaling molecule involved in cancer, that consists of two selenium nanoparticles SeNPs and G2/PAH-Cit/SeNPs. A G2/PAH-Cit/SeNP is a pH-sensitive delivery system that is capable of enhancing siRNA loading, thus increasing siRNA release efficiency and subsequent target gene silencing both in vitro and in vivo. In vivo experiments using G2/PAH-Cit/SeNPs@siRNA led to significantly higher accumulation of siRNA within the tumor itself, VEGF gene silencing, and reduced angiogenesis in the tumor. Furthermore, the G2/PAH-Cit/SeNP delivery system not only enhanced anti-tumor effects on tumor-bearing nude mice as compared to SeNPs@siRNA, but also resulted in weak occurrence of lesions in major target organs. In sum, this study provides a new class of siRNA delivery system, thereby providing an alternative therapeutic route for cancer treatment.

Discovering small molecule ligands of vascular endothelial growth factor that block VEGF-KDR binding using label-free microarray-based assays.

We present here a label-free microarray-based assay platform that we used to identify inhibitors of vascular endothelial growth factor (VEGF)-kinase-insertion domain receptor (KDR) binding. Supported by a combination of special ellipsometry-based optical detection and small molecule microarrays (SMM), this platform consists of three assays: (1) the first assay detects binding of a target protein with SMM and identifies ligands to the protein as inhibitor candidates; (2) the second assay detects binding of a receptor protein with identical SMM and subsequent binding of the target protein (a sandwich assay) to identify the ligands to the receptor protein that do not interfere with the target-receptor binding; (3) the third assay detects binding of the target protein to the receptor protein in the presence of the ligands of the target protein identified from the first assay, with the receptor protein immobilized to a solid surface through the ligands identified in the second assay, to yield dose-response curves. Using this platform, we screened 7,961 compounds from the National Cancer Institute and found 12 inhibitors to VEGF-KDR (VEGFR2) interactions with IC50 ranging from 0.3 to 60 µM. The inhibitory potency of these inhibitors found in the microarray-based assay was confirmed by their inhibition of VEGF-induced VEGFR2 phosphorylation in a cell-based assay.

Cotinine-conjugated aptamer/anti-cotinine antibody complexes as a novel affinity unit for use in biological assays.

Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/ anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/ anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.

Vascular-targeted photothermal therapy of an orthotopic murine glioma model.

AIM: To develop nanoshells for vascular-targeted photothermal therapy of glioma. MATERIALS & METHODS: The ability of nanoshells conjugated to VEGF and/or poly(ethylene glycol) (PEG) to thermally ablate VEGF receptor-2-positive endothelial cells upon near-infrared laser irradiation was evaluated in vitro. Subsequent in vivo studies evaluated therapy in mice bearing intracerebral glioma tumors by exposing tumors to near-infrared light after systemically delivering saline, PEG-coated nanoshells, or VEGF-coated nanoshells. The treatment effect was monitored with intravital microscopy and histology. RESULTS: VEGF-coated but not PEG-coated nanoshells bound VEGF receptor-2-positive cells in vitro to enable targeted photothermal ablation. In vivo, VEGF targeting doubled the proportion of nanoshells bound to tumor vessels and vasculature was disrupted following laser exposure. Vessels were not disrupted in mice that received saline. The normal brain was unharmed in all treatment and control mice. CONCLUSION: Nanoshell therapy can induce vascular disruption in glioma.

Cotinine-conjugated aptamer/anti-cotinine antibody complexes as a novel affinity unit for use in biological assays

Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.

Proangiogenic activity of plant extracts in accelerating wound healing - a new face of old phytomedicines.

Angiogenesis, the formation of new capillaries from pre-existing vascular network, plays an important role in physiological and pathological processes such as embryonic development, wound healing, and development of atherosclerosis. Extension of the circulatory network is also considered to be one the most important factors during cancerogenesis. Inhibition of angiogenesis may lead to inhibition of tumor growth whereas stimulation may improve wound healing. Research achievements suggest the use of plants and their extracts as potential therapeutic agents with pro- or antiangiogenic activity. Since the anticancer and antiangiogenic properties of many phytomedicines have been amply reviewed elsewhere this paper will focus on the treatment of vascular insufficiency in wound healing. Globally accepted herbal drugs are thought to be safe and effective, however, there is a need for more evidence-based confirmation in controlled and validated trials. Among the most frequently studied proangiogenic phytochemicals are ginsenosides from Panax ginseng, beta-sitosterol from Aloe vera, calycosin from Radix Astragali, and extracts from Hippophae rhamnoides L. and Angelica sinensis.

Development and characterization of rhVEGF-loaded poly(HEMA-MOEP) coatings electrosynthesized on titanium to enhance bone mineralization and angiogenesis.

Osteointegration of titanium implants could be significantly improved by coatings capable of promoting both mineralization and angiogenesis. In the present study, a copolymeric hydrogel coating, poly-2-hydroxyethyl methacrylate-2-methacryloyloxyethyl phosphate (P(HEMA-MOEP)), devised to enhance calcification in body fluids and to entrap and release growth factors, was electrosynthesized for the first time on titanium substrates and compared to poly-2-hydroxyethyl methacrylate (PHEMA), used as a blank reference. Polymers exhibiting negatively charged groups, such as P(HEMA-MOEP), help to enhance implant calcification. The electrosynthesized coatings were characterized by X-ray photoelectron spectroscopy and atomic force microscopy. MG-63 human osteoblast-like cell behaviour on the coated specimens was investigated by scanning electron microscopy, MTT viability test and osteocalcin mRNA detection. The ability of negatively charged phosphate groups to promote hydroxyapatite-like calcium phosphate deposition on the implants was explored by immersing them in simulated body fluid. Similar biological responses were observed in both coated specimens, while calcium-phosphorus globules were detected only on P(HEMA-MOEP) surfaces pretreated with alkaline solution. Testing of the ability of P(HEMA-MOEP) hydrogels to entrap and release human recombinant vascular endothelial growth factor, to tackle the problem of insufficient oxygen and nutrient delivery, suggested that P(HEMA-MOEP)-coated titanium prostheses could represent a multifunctional material suitable for bone restoration applications.