The chick embryo chorioallantoic membrane patient-derived xenograft (PDX) model.

The chick embryo chorioallantoic membrane (CAM) CAM is an extraembryonic membrane generated by the fusion of the chorion with the vascularized allantoic membrane. It performs multiple functions during embryonic development, including respiration, calcium transport from the eggshell, acid-base homeostasis, and ion/water reabsorption from the allantoic fluid. The CAM is a widely used model for the study of angiogenesis, anti-angiogenesis, tumor growth, and metastasis as well as drug efficacy. Ethical approval is omitted if experiments are terminated at embryonic day 14 in most countries, facilitating screenings of pharmacological or physics-based therapies with high reproducibility at large scales supporting the 3Rs principle. Being naturally immunodeficient, the chick embryo accepts transplantation from various tissues and species without immune response. This review article is focused on the analysis of the literature and personal data concerning the effects of patient-derived xenografts (PDX) on the CAM.

Improving germline transmission efficiency in chimeric chickens using a multi-stage injection approach.

Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. Compared to the other routes of viral vector administration, the embryo's bloodstream at Hamburger-Hamilton (HH) stages 14-15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14-15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.

Novel Peptide NT/K-CFY Derived from Kringle Structure of Neurotrypsin Inhibits Neovascularization.

Purpose: To assess the anti-neovascularization effect of a novel peptide NT/K-CFY derived from the kringle domain of neurotrypsin.Materials and Methods: Cell migration, lumen formation and cell proliferation assays were performed to determine the anti-neovascularization effect of NT/K-CFY in primary human umbilical vein endothelial cells (HUVECs). Chick chorioallantoic membrane (CAM) and oxygen-induced retinopathy (OIR) models were established to assess the anti-angiogenic role of NT/K-CFY in vivo. The retinal expression of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) was examined by western blot and real-time PCR in OIR model.Results: The in vitro results showed that NT/K-CFY effectively and safely decreased VEGF-induced cell migration, cell proliferation and tube formation in HUVECs. In addition, NT/K-CFY showed certain efficacy in angiogenesis inhibition in chicken embryos and oxygen-treated mouse pups. Moreover, the CFY peptide also improved retinal blood perfusion and reversed the abnormal expression of VEGF and PEDF in OIR mouse model.Conclusion: NT/K-CFY peptide strongly inhibits neovascularization in vitro and vivo. This novel peptide may become a promising therapeutic agent for ocular angiogenesis-related diseases.

Spatial immobilization of endogenous growth factors to control vascularization in bone tissue engineering.

The intimate crosstalk between endothelial and bony cells is essential for the reconstruction of bone tissue defects. Indeed, a successful bone repair is greatly dependent on the formation of new blood vessels, to ensure the supply of nutrients and gases, as well as the removal of metabolites. Bone morphogenetic proteins (BMPs) and vascular endothelial growth factor (VEGF) are involved on cells differentiation and bone vascularization aiming to develop viable bone tissue. Herein it is hypothesized that endogenous BMP-2 and VEGF bound in a parallel arrangement over a single nanofibrous substrate (NFM) can lead to a successful osteogenic and angiogenic differentiation of mesenchymal stem cells. For that, an engineered biofunctional system was developed comprising anti-BMP-2 and anti-VEGF antibodies, immobilized over an electrospun NFMs in a parallel pattern design, with the attempt to recreate the vasculature of bone tissue. The osteogenic and angiogenic potential of this engineered biofunctional system was demonstrated by culturing human bone marrow-derived mesenchymal stem cells (hBM-MSCs) during 21 days without exogenous induction. A chick chorioallantoic membrane (CAM) assay showed that the engineered biofunctional system, comprising bound endogenous BMP-2 and VEGF, is able to induce an increased angiogenic response. The angiogenic ability of this system, together with the osteogenic inductor BMP-2, enable obtaining an effective vascularized bone tissue engineering approach.

Angiogenesis and follicular development in ovarian tissue of cattle following vitrification and post-warming culture on chicken chorioallantoic membrane.

The purpose of this study was to assess the viability and growth of follicles in ovarian tissues of cattle vitrified using two non-permeating cryoprotectants (sucrose and trehalose) and two cryodevices (cryovial and cryotop). Cortical slices (1-2 mm3) from cattle ovaries (n = 5) were assigned to one of the 14 treatment groups. Cortical slices were vitrified in a TCM199 medium supplemented with ethylene glycol, DMSO, calf serum and either 0.5 M sucrose or trehalose, in cryovials or on cryotops. After warming, cortical slices were either fixed immediately for histology or grafted on a chorioallantoic membrane (CAM) of 10-day old chick embryos. Angiogenesis in ovarian tissues was determined. Viable and atretic preantral (primordial, primary and secondary) follicle densities were examined histologically. There was angiogenesis (chicken) in cortical slices grafted on the CAM by day 5 of culture, however, there was no difference for blood vessel densities when there was use of non-permeating cryoprotectants or cryodevices. Total, viable and atretic follicle densities did not differ (P > 0.05) with use of non-permeating cryoprotectants or cryodevices. The proportion of viable follicles was greater (P < 0.001) in fresh-control than CAM culture-control or vitrification groups. The inclusion of sucrose in the vitrification solution resulted in a larger number of atretic follicles than in the fresh-control group (P < 0.05). In summary, sucrose and trehalose, and cryotop and cryovial were equally suitable for vitrification of ovarian tissues of cattle. Vitrification of ovarian tissues of cattle with subsequent use of CAM culture adversely affected follicular development.

WIN55,212-2 induces caspase-independent apoptosis on human glioblastoma cells by regulating HSP70, p53 and Cathepsin D.

Despite the standard approaches to treat the highly aggressive and invasive glioblastoma (GBM), it remains incurable. In this sense, cannabinoids highlight as a promising tool, because this tumor overexpresses CB1 and/or CB2 receptors and being, therefore, can be susceptible to cannabinoids treatment. Thus, this work investigated the action of the cannabinoid agonist WIN55-212-2 on GBM cell lines and non-malignant cell lines, in vitro and in vivo. WIN was selectively cytotoxic to GBM cells. These presented blebbing and nuclear alterations in addition to cell shrinkage and chromatin condensation. WIN also significantly inhibited the migration of GAMG and U251 cells. Finally, the data also showed that the antitumor effects of WIN are exerted, at least to some extent, by the expression of p53 and increased cathepsin D in addition to the decreased expression of HSP70.This data can indicate caspase-independent cell death mechanism. In addition, WIN decreased tumoral perimeter as well as caused a reduction the blood vessels in this area, without causing lysis, hemorrhage or blood clotting. So, the findings herein presented reinforce the usefulness of cannabinoids as a candidate for further evaluation in treatment in glioblastoma treatment.

Preclinical alternative model for analysis of porous scaffold biocompatibility in bone tissue engineering.

Using scaffolds with appropriate porosity represents a potential approach for repair of critical-size bone defects. Vascularization is essential for bone formation and healing. This study investigates methods for monitoring angiogenesis within porous biopolymer scaffolds on the basis of polyhydroxybutyrate (PHB)/chitosan. We use the chick and quail chorioallantoic membrane (CAM) assay as an in vivo model focused on the formation of new blood vessels inside the implant structure. Chemical properties of the surface in biopolymer scaffold matrix were characterized as well as the tissue reaction of the CAM. Implantation of a piece of polymer scaffold results in vascular reaction, documented visually and by ultrasound biomicroscopy. Histological analysis shows myofibroblast reaction (smooth muscle actin-positive cells) without excessive collagen deposition. Cell invasion is observed inside the implant, and QH1 marker, detecting hemangioblasts and endothelial cells of quail origin, confirms the presence of vascular network. The CAM assay is a rapid and easy way to test biocompatibility and vasculogenic potential of new candidate scaffolds for bone tissue bioengineering with respect to the 3R´ s.

Hyaluronic acid-endostatin2-alft1 (HA-ES2-AF) nanoparticle-like conjugate for the target treatment of diseases.

Anti-flt1 peptide (GNQWFI, AF) specifically binds to Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), thereby inhibiting the interaction of VEGFR1 with a series of ligands. ES2 (IVRRADRAAVP) can effectively inhibit the proliferation and invasion of endothelial cells and play a key role in anti-angiogenesis. AF and ES2 peptides differ in their activity. To better exploit the advantages of both, we designed a new peptide called ES2-AF (IVRRADRAAVPGGGGGGNQWFI). Hyaluronic acid (HA) is widely used in the pharmaceutical industry because of its biodegradable and high load performance. The HA-specific cell surface receptor CD44 was highly expressed in the tumour site during the anti-tumour study. Therefore, we used HA as a modifier to chemically modify ES2-AF; it was expected that the modified compound would have preferable solubility, stronger targeting, longer half-life, and better anti-angiogenesis effects in vivo. In this study, the anti-proliferative, anti-migration and targeting activities of HA-ES2-AF in vitro were studied by MTT, ELISA, transwell and SPR assays. Meanwhile, the anti-neovascularization activity of HA-ES2-AF in vivo was studied by CAM assay, and the targeting of HA-ES2-AF to tumour tissue was studied by bioimaging techniques. Finally, we also studied the half-life of HA-ES2-AF in vivo. In short, the bioactivity of the new peptide ES2-AF was enhanced to a certain extent, and ES2-AF modified by HA had higher anti-neovascularization activity in vitro and in vivo, had stronger targeting to tumour tissue, and had a significantly prolonged half-life in vivo. These results laid the foundation for its further development into targeting anti-tumour drugs.

Injectable and tunable hyaluronic acid hydrogels releasing chemotactic and angiogenic growth factors for endodontic regeneration.

Bioengineered soft tissues on any meaningful scale or complexity must incorporate aspects of the functional tissue, namely a vasculature, providing cells oxygen and nutrients critical for their survival. However, the ability of tissue engineering strategies to promote a fast revascularization is critically limited. Particularly in endodontic regenerative therapies, the complicated anatomy of the root canal system, and the narrow apical access limit the supply of new blood vessels and pulp tissue ingrowth. Here we characterize the viscoelastic and microstructural properties of a class of injectable hyaluronic acid (HA) hydrogels formed in situ, reinforced with cellulose nanocrystals (CNCs) and enriched with platelet lysate (PL), and test its ability to promote cells recruitment and proangiogenic activity in vitro. The incorporation of CNCs enhanced the stability of the materials against hydrolytic and enzymatic degradation. Moreover, the release of the chemotactic and pro-angiogenic growth factors (GFs) (PDGF and VEGF) from the PL-laden hydrogels showed an improved sustained profile proportional to the amount of incorporated CNCs. The PL-laden hydrogels exhibited preferential supportive properties of encapsulated human dental pulp cells (hDPCs) in in vitro culture conditions. Finally, PL-laden hydrogels stimulated chemotactic and pro-angiogenic activity by promoting hDPCs recruitment and cell sprouting in hDPCs/human umbilical vein endothelial cell co-cultures in vitro, and in an ex vivo model. These results support the use of the combined system as a scaffold for GFs delivery and cells recruitment, thereby exhibiting great clinical potential in treating injuries in vascularized tissues. STATEMENT OF SIGNIFICANCE: Innovative strategies for improved chemotactic and pro-angiogenic features of TE constructs are needed. In this study, we developed an injectable HA/CNC/PL hydrogel with improved structural and biologic properties, that not only provide a sustained release of chemotactic and proangiogenic GFs from PL but also enhance the cells' viability and angiogenic activity. As a result of their unique traits, the developed hydrogels are ideally suited to simultaneously act as a GFs controlled delivery system and as a supportive matrix for cell culture, recruitment, and revascularization induction, holding great potential for the regeneration of vascularized soft tissues, such as the dentin-pulp complex.

Antifungal mechanism of action of Schinus lentiscifolius Marchand essential oil and its synergistic effect in vitro with terbinafine and ciclopirox against dermatophytes.

OBJECTIVES: The aim of this study was to evaluate the antifungal, antichemotactic and antioxidant activities of Schinus lentiscifolius essential oil, as well as its combined effect with terbinafine and ciclopirox, against dermatophytes. METHODS: Essential oil was analysed by GC-MS. The antifungal activity and the mechanism of action were determined by broth microdilution, sorbitol and ergosterol assays, as well as scanning electron microscopy. The checkerboard method was used for evaluating the interactions with commercial antifungal agents. The antioxidant and antichemotactic activities were measured using the DPPH and the modified Boyden chamber methods, respectively. KEY FINDINGS: Chemical analysis revealed the presence of 33 compounds, the primary ones being γ-eudesmol (12.8%) and elemol (10.5%). The oil exhibited 97.4% of antichemotactic activity and 37.9% of antioxidant activity. Antifungal screening showed effect against dermatophytes with minimum inhibitory concentration values of 125 and 250 µg/ml. Regarding the mechanisms of action, the assays showed that the oil can act on the fungal cell wall and membrane. Synergistic interactions were observed using the combination with antifungals, primarily terbinafine. CONCLUSIONS: Schinus lentiscifolius essential oil acted as a chemosensitizer of the fungal cell to the drug, resulting in an improvement in the antifungal effect. Therefore, this combination can be considered as an alternative for the topical treatment of dermatophytosis.

Strategic design of cardiac mimetic core-shell nanofibrous scaffold impregnated with Salvianolic acid B and Magnesium l-ascorbic acid 2 phosphate for myoblast differentiation.

The major loss of myocardial tissue extracellular matrix after infarction is a serious complication that leads to heart failure. Regeneration and integration of damaged cardiac tissue is challenging since the functional restoration of the injured myocardium is an incredible task. The injured micro environment of myocardium fails to regenerate spontaneously. The emergence of nano-biomaterials would be a promising approach to regenerate such a damaged cardiomyocytes tissue. Here, we have fabricated a dual bioactive embedded nanofibrous cardiac patch via coaxial electrospinning technique, to mimic the topographical and chemical cues of the natural cardiac tissue. The proportion and the concentration of the polymers were optimized for tailored delivery of bioactives from a spatio-temporally designed scaffold. The functionalization of polymeric core shell nanofibrous scaffold with dual bioactives enhanced the physico-chemical and bio-mechanical properties of the scaffolds that has resulted in a 3-dimensional topography mimicking the natural cardiac like extracellular matrix. The sustained delivery of bioactive signals, improved cell adhesion, proliferation, migration and differentiation could be attributed to its highly interconnected nanofibrous matrix with good extended morphology. Further, the expression of cardiac specific markers were found to increase on investigation of mRNA by real time PCR studies and proteins by immunofluorescence and western blotting techniques, confirming cell - biomaterial interactions. Flow cytometry analysis authenticated a potent mitochondrial membrane potential of cells treated with nanocomposite. In addition, in ovo studies in chicken chorioallantoic membrane assay confirm the efficacy of the developed scaffold in inducing angiogenesis required for maintaining its viability after transplantation onto the infarcted zone. These promising results demonstrate the potential of the composite nanofibrous scaffold as an effective biomaterial substrate for cardiac regeneration providing cues for development of novel cardiac therapeutics.

Short-term culture of adult bovine ovarian tissues: chorioallantoic membrane (CAM) vs. traditional in vitro culture systems.

BACKGROUND: A suitable culture system is important for follicle growth in adult bovine ovarian tissue. This study aimed to assess the avian chorioallantoic membrane (CAM) for short-term culture of adult bovine ovarian tissues compared with a traditional in vitro culture system. METHODS: Ovarian cortical tissues (1-2 mm3), collected from slaughtered adult cows, were randomly assigned to control, CAM or in vitro culture groups. In the control group, ovarian tissues were fixed with paraformaldehyde without culture. In CAM and in vitro culture groups, the ovarian tissues were cultured for up to 5 days and then fixed. Ovarian tissues were examined on culture days 0, 1, 3 and 5 for angiogenesis, follicle morphology and growth. In all groups, primordial and growing (healthy and atretic) follicle densities were determined. RESULTS: In the CAM culture, the avian blood vessel density increased (p < 0.01) over time with a decline (p < 0.001) in the bovine blood vessel density. Healthy primordial, atretic primordial and healthy growing follicle densities were higher (p < 0.05) in CAM-cultured ovarian tissues than in vitro-cultured tissues. Regardless of the culture system, the density of healthy primordial follicles decreased (p < 0.001) over time with an increase in healthy growing follicles on day 3 (p < 0.01) and an increase in atretic (primordial and growing) follicles during the 5-day culture period (p < 0.001). The proportions of healthy primordial and atretic growing follicles were also affected by culture day (p < 0.001). CONCLUSIONS: The CAM culture in chick embryos supported the bovine ovarian tissue grafts for 3 days demonstrating that CAM can be used as a satisfactory short-term culture system to assess ovarian tissue health, and to study follicle activation and development.

Identification of Reference Genes for Analysis of microRNA Expression Patterns in Equine Chorioallantoic Membrane and Serum.

MicroRNAs (miRNAs) have important posttranscriptional regulatory abilities, and there is considerable interest in evaluating their expression patterns in different pathophysiological states. The most common method of quantifying miRNA expression is quantitative reverse transcription PCR; however, the identification of tissue-specific and species-specific reference miRNA is a prerequisite for miRNA expression analysis. Currently, no reference genes have been described for evaluating miRNA expression in equine serum and chorioallantoic membrane (CAM) during pregnancy. The aim of the present study was to characterize reference genes for normalization of miRNA expression in CAM and serum in the pregnant equine. To identify the most stable miRNAs in serum, expression of potential candidates was evaluated in serum samples from diestrous mares, pregnant mares and geldings. To identify the most stable miRNAs in CAM, expression of potential candidates was evaluated in CAM, collected from mares at 4, 6 and 10 months of pregnancy and immediately postpartum. From a previously generated miRNA sequencing dataset, two separate lists of potential reference miRNAs were identified (serum and CAM) using the NormFinder program, in addition to the commonly used small RNA normalizers, 5S rRNA and U6 snRNA. The putative reference miRNAs were selected using geNorm and NormFinder. In case of a nonsignificant correlation between the results of ranking and stability value between these two programs, ranking from BestKeeper was also included. NormFinder and geNorm consistently identified eca-miR-21-5p, eca-let-7a-5p and eca-miR-10a-5p as the three most stable reference genes for the normalization of serum miRNAs. Within CAM samples, the average ranking obtained from the ranking of NormFinder, geNorm and BestKeeper identified eca-miR-8908a-1-5p, eca-miR-369-5p and eca-miR-106a-5p as the three most stable miRNAs. These observations provide information about equine-specific reference genes that can be used for normalizing miRNAs expression patterns in CAM and serum during the equine pregnancy.

Anti-angiogenic activity and mechanism of a chemically sulfated natural glucan from Phellinus ribis.

A sulfated polysaccharide named PRP-S16 was obtained by sulfation of a glucan from Phellinus ribis using the chlorosulfonic acid method. PRP-S16 could significantly block the formation of new vessels in chicken chorioallantoic membrane (CAM). It could also inhibit the proliferation, migration, and tube formation and significantly reduced the mRNA expression of vascular endothelial growth factor (VEGF) in EA.hy926 endothelial cells. Western blotting indicated that PRP-S16 down regulated the protein expression of VEGF and VEGF receptor-1 (VEGFR-1), and inhibited the phosphorylation of VEGFR-2, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK1/2) in EA.hy926 cells. These findings suggest that the mechanism of action of the anti-angiogenesis activity of PRP-S16 is related to the inhibition of VEGF-induced signaling pathway. PRP-S16 might be utilized as an effective antagonist in combating diseases associated with angiogenesis.

Sema3A drastically suppresses tumor growth in oral cancer Xenograft model of mice.

BACKGROUND: Multiple studies suggest anti-angiogenesis to be a promising and rational option in cancer treatment. Interestingly, the axonal sprouting inhibitor semaphorin 3A (Sema3A), a potent suppressor of tumor angiogenesis in various cancer models, is lowly expressed in human oral cancer. Thus, we hypothesized that overexpression of Sema3A in human oral cancer cells may have potential therapeutic effects. METHODS: The LentiSema3A-EGFP was first constructed and transduced to the tongue squamous cell carcinoma cell line SSC-9. Angiogenesis assay was performed with endothelial cell tube formation assay and chorioallantoic membrane (CAM) assay. Tumor xenografts model was used to evaluate the effect of Sema3a on the tumor growth. Finally, western blot was performed to study the mechanisms of inhibiting angiogenesis by Sema3A. RESULTS: In vitro and in vivo approaches revealed that Sema3A significantly inhibited tube formation of endothelial cells and reduced angiogenesis in CAM assay. In addition, overexpression of Sema3A in the tongue squamous cell carcinoma cell line SSC-9 resulted in significantly reduced angiogenesis and drastically suppressed tumor growth in mice. Mechanistically, Sema3A inhibited the phosphorylation of VEGFR2, as well as Src and FAK, downstream of the VEGF/VEGFR2 pathway. CONCLUSION: Our results demonstrated that overexpression of Sema3A in oral cancer cells drastically suppressed tumor growth by inhibiting angiogenesis. Our findings provide a basis for the development of novel therapeutics in the management of oral cancer.

Experimental tumor growth of canine osteosarcoma cell line on chick embryo chorioallantoic membrane (in vivo studies).

The chick embryo chorioallantoic membrane (CAM) model is extensively used in human medicine in preclinical oncological studies. The CAM model has several advantages: low cost, simple experimental approach, time saving and following "3R principles". Research has shown that the human osteosarcoma cell lines U2OS, MMNG-HOS, and SAOS can form tumors on the CAM. In veterinary medicine, this has been described only for feline fibrosarcomas, feline mammary carcinomas and canine osteosarcomas. However, in case of canine osteosarcomas, it has been shown that only non-adherent osteosarcoma stem cells isolated from KTOSA5 and CSKOS cell lines have the ability to form microtumors on the CAM after an incubation period of 5 days, in contrast to adherent KTOSA5 and CSKOS cells. In the presented study, we have proven that the commercial adherent canine osteosarcoma cell line (D-17) can form vascularized tumors on the CAM after the incubation period of 10 days.

An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane.

The chorioallantoic membrane (CAM) containing tiny blood vessels is an alternative to large animals for studies involving angiogenesis and tissue engineering. However, there is no technique to design the direction of growing blood vessels on the CAM at the microscale level for tissue engineering experiments. Here, a methodology is provided to direct blood vessel formation on the surface of a three-dimensional egg yolk using a cubic artificial eggshell with six functionalized membranes. A structure on the lateral side of the eggshell containing a straight channel and an interlinked chamber was designed, and the direction and formation area of blood vessels with blood flow was artfully defined by channels with widths of 70-2000 µm, without sharply reducing embryo viability. The relationship between the size of interlinked chamber and the induction of blood vessels was investigated to establish a theory of design. Role of negative and positive pressure in the induction of CAM with blood vessels was investigated, and air pressure change in the culture chamber was measured to demonstrate the mechanism for blood vessel induction. Histological evaluation showed that components of CAM including chorionic membrane and blood vessels were induced into the channels. Based on our design theory, blood vessels were induced into arrayed channels, and channel-specific injection and screening were realized, which demonstrated proposed applications. The platform with position- and space-controlled blood vessels is therefore a powerful tool for biomedical research, which may afford exciting applications in studies involved in local stimulation of blood vessel networks and those necessary to establish a living system with blood flow from a beating heart.

Combination of ruthenium(II)-arene complex [Ru(η6-p-cymene)Cl2(pta)] (RAPTA-C) and the epidermal growth factor receptor inhibitor erlotinib results in efficient angiostatic and antitumor activity.

Ruthenium-based compounds show strong potential as anti-cancer drugs and are being investigated as alternatives to other well-established metal-based chemotherapeutics. The organometallic compound [Ru(η6-p-cymene)Cl2(pta)], where pta = 1,3,5-triaza-7-phosphaadamantane (RAPTA-C) exhibits broad acting anti-tumor efficacy with intrinsic angiostatic activity. In the search for an optimal anti-angiogenesis drug combination, we identified synergistic potential between RAPTA-C and the epidermal growth factor receptor (EGFR) inhibitor, erlotinib. This drug combination results in strong synergistic inhibition of cell viability in human endothelial (ECRF24 and HUVEC) and human ovarian carcinoma (A2780 and A2780cisR) cells. Additionally, erlotinib significantly enhances the cellular uptake of RAPTA-C relative to treatment with RAPTA-C alone in human ovarian carcinoma cells, but not endothelial cells. Drug combinations induce the formation of chromosome bridges that persist after mitotic exit and delay abscission in A2780 and A2780cisR, therefore suggesting initiation of cellular senescence. The therapeutic potential of these compounds and their combination is further validated in vivo on A2780 tumors grown on the chicken chorioallantoic membrane (CAM) model, and in a preclinical model in nude mice. Immunohistochemical analysis confirms effective anti-angiogenic and anti-proliferative activity in vivo, based on a significant reduction of microvascular density and a decrease in proliferating cells.

Davallia bilabiata exhibits anti-angiogenic effect with modified MMP-2/TIMP-2 secretion and inhibited VEGF ligand/receptors expression in vascular endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Davallia bilabiata Hosokawa (D. bilabiata), also called GuSuiBu, is popularly used as a substitute for Drynaria fortunei J. Sm for rheumatoid and degenerative arthritis in traditional Chinese medicine. Little is known about the underlying mechanisms of anti-angiogenesis responsible for arthritis in D. bilabiata which needs to be elucidated. AIM OF THE STUDY: The present study is intended to investigate the anti-angiogenic effect of D. bilabiata associated with the modulation of matrix metalloproteinases (MMPs) and down regulation of vascular endothelial growth factor (VEGF) ligand/receptors both in vivo and in vitro. MATERIALS AND METHODS: We investigated the potential anti-angiogenic effect of D. bilabiata by the in vivo neovascularization of chick chorioallantoic membranes (CAM) assay, and the in vitro migration and matrix-induced tube formation assay using human umbilical vascular endothelial cells (HUVECs). The expressions of MMP-2, TIMP-2, RECK and VEGF/VEGFR were analyzed by real-time RT-PCR or Western blot method. RESULTS: One major compound from water extract of D. bilabiata was identified as Epicatechin 3-O-ß-D-allopyranoside. D. bilabiata was confirmed to inhibit in vivo angiogenesis by CAM assay. D. bilabiata also exhibited in vitro anti-angiogenic and anti-regrowth effects as demonstrated by tube formation assay, transwell migration assay and wound healing assay. The mRNA expressions of MMP-2, and MMP-14 were decreased. On the contrary, tissue inhibitor of metalloproteinase-2 (TIMP-2), reversion-inducing cysteine-rich protein with kazal motifs (RECK) were increased by D. bilabiata. The extracellular MMP-2 activity was found to be reduced both in vitro and in vivo by D. bilabiata as determined by gelatin zymography. Results from western blot analysis and ELISA further demonstrated the decrease of MMP-2 and increase of TIMP-2 secretion after D. bilabiata treatment. The gene expressions of VEGF-A, -B, -C, -D and VEGFR-1, -2, -3 were all inhibited by D. bilabiata. CONCLUSION: We concluded that the anti-angiogenic effect of D. bilabiata was associated with the decreased MMP-2 activity mediated by the upregulation of TIMP-2 and RECK, and the suppression of VEGF/VEGFRs expression.

The chick embryo chorioallantoic membrane (CAM) assay.

During avian development the mesodermal layers of the allantois and chorion fuse to form the chorioallantoic membrane (CAM). This structure rapidly expands generating a rich vascular network that provides an interface for gas and waste exchange. The CAM allows to study tissue grafts, tumor growth and metastasis, drugs delivery and toxicologic analysis, and angiogenic and anti-angiogenic molecules. The CAM is relatively simple, quick, and low-cost model that allows screening of a large number of pharmacological samples in a short time; does not require administrative procedures for obtaining ethics committee approval for animal experimentation. Moreover, being naturally immunodeficient, the chick embryo may receive transplantations from different tissues and species, without immune responses.