Evaluation of Angiogenesis in an Acellular Porous Biomaterial Based on Polyhydroxybutyrate and Chitosan Using the Chicken Ex Ovo Chorioallantoic Membrane Model.

The chorioallantoic membrane (CAM) is a highly vascularized avian extraembryonic membrane widely used as an in vivo model to study angiogenesis and its inhibition in response to tissues, cells, or soluble factors. In recent years, the use of CAM has become an integral part of the biocompatibility testing process for developing biomaterials intended for regenerative strategies and tissue engineering applications. In this study, we used the chicken ex ovo CAM assay to investigate the angiogenic potential of innovative acellular biopolymer polyhydroxybutyrate/chitosan (PHB/CHIT) scaffold, which is intended for the treatment of hard tissue defects, depending on treatment with pro- and anti-angiogenic substances. On embryonic day (ED) 7, the experimental biomaterials were placed on the CAM alone or soaked in vascular endothelial growth factor (VEGF-A), saline solution (PHY), or tyrosine kinase inhibitor (SU5402). After 72 h, the formation of vessels was analyzed in the surrounding area of the scaffold and inside the pores of the implants, using markers of embryonic endothelium (WGA, SNA), myofibroblasts (α-SMA), and macrophages (KUL-01). The morphological and histochemical analysis showed strong angiogenic potential of untreated scaffolds without additional effect of the angiogenic factor, VEGF-A. The lowest angiogenic potential was observed in scaffolds soaked with SU5402. Gene expression of pro-angiogenic growth factors, i.e., VEGF-A, ANG-2, and VE-CAD, was upregulated in untreated scaffolds after 72 h, indicating a pro-angiogenic environment. We concluded that the PHB/CHIT has a strong endogenous angiogenic potential and could be promising biomaterial for the treatment of hard tissue defects.

Usnic Acid Isolated from Usnea antarctica (Du Rietz) Reduced In Vitro Angiogenesis in VEGF- and bFGF-Stimulated HUVECs and Ex Ovo in Quail Chorioallantoic Membrane (CAM) Assay.

Natural products include a diverse set of compounds of drug discovery that are currently being actively used to target tumor angiogenesis. In the present study, we evaluated the anti-angiogenic activities of secondary metabolite usnic acid isolated from Usena antarctica. We investigated the in vitro effects on proliferation, migration, and tube formation of VEGF- and bFGF-stimulated HUVECs. Ex ovo anti-angiogenic activity was evaluated using the CAM assay. Our findings demonstrated that usnic acid in the concentration of 33.57 µM inhibited VEGF (25 ng/mL) and bFGF (30 ng/mL)-induced HUVECs proliferation, migration, and tube formation. The ex ovo CAM model was used to confirm the results obtained from in vitro studies. VEGF- and bFGF-induced vessel formation was inhibited by usnic acid after 72 h in over 2-fold higher concentrations compared to in vitro. Subsequently, histological sections of affected chorioallantoic membranes were stained with hematoxylin-eosin and alcian blue to determine the number and diameter of vessels as well as the thickness of the individual CAM layers (ectoderm, mesoderm, endoderm). Usnic acid was able to suppress the formation of VEGF- and bFGF-induced vessels with a diameter of less than 100 µm, which was demonstrated by the reduction of mesoderm thickness as well.

Antiproliferative Effect of Phellodendron amurense Rupr. Based on Angiogenesis.

Phellodendron amurense Rupr. is medicinal plant used for supplemental therapy of various diseases based on their positive biological activities. The aim of this study was evaluated the main metabolite, safety of application and anticancer potential. Berberine was determined by HPLC as main alkaloid. Harmful character was determined by irritation test in ovo. The potential cancerogenic effect was studied in vitro on a cellular level, in ovo by CAM assay and in vivo on whole organism Artemia franciscana. Extract from the bark of Phellodendron amurense showed antiproliferative and antiangiogenic effects. The results of our work showed promising anticancer effects based also on the inhibition of angiogenesis with minimum negative effects.

In Vivo Study of Osteochondral Defect Regeneration Using Innovative Composite Calcium Phosphate Biocement in a Sheep Model.

This study aimed to clarify the therapeutic effect and regenerative potential of the novel, amino acids-enriched acellular biocement (CAL) based on calcium phosphate on osteochondral defects in sheep. Eighteen sheep were divided into three groups, the treated group (osteochondral defects filled with a CAL biomaterial), the treated group with a biocement without amino acids (C cement), and the untreated group (spontaneous healing). Cartilages of all three groups were compared with natural cartilage (negative control). After six months, sheep were evaluated by gross appearance, histological staining, immunohistochemical staining, histological scores, X-ray, micro-CT, and MRI. Treatment of osteochondral defects by CAL resulted in efficient articular cartilage regeneration, with a predominant structural and histological characteristic of hyaline cartilage, contrary to fibrocartilage, fibrous tissue or disordered mixed tissue on untreated defect (p < 0.001, modified O'Driscoll score). MRI results of treated defects showed well-integrated and regenerated cartilage with similar signal intensity, regularity of the articular surface, and cartilage thickness with respect to adjacent native cartilage. We have demonstrated that the use of new biocement represents an effective solution for the successful treatment of osteochondral defects in a sheep animal model, can induce an endogenous regeneration of cartilage in situ, and provides several benefits for the design of future therapies supporting osteochondral defect healing.

A Comprehensive Study Monitoring the Venom Composition and the Effects of the Venom of the Rare Ethiopian Endemic Snake Species Bitis parviocula.

The Ethiopian endemic snake of the species Bitis parviocula, recognized for its colorful patterns, might be more interesting as we look deeper into the venom activity. We assayed the effects of venoms from the most widespread venomous African Bitis arietens and closely related species Bitis parviocula using The Hen's Egg Test-Chorioallantoic membrane test (HET-CAM) and Chicken embryotoxicity screening test (CHEST), acetylcholinesterase (AChE) analysis, cytotoxicity assay performed on cell lines and protein analysis of selected venoms. Our results indicated that B. parviocula venom contains vasoactive compounds that have a direct effect on blood vessels. The AChE analysis showed significant ability inhibiting AChE activity in embryonic tissue. Cytotoxicity observed on A549 ATCC® CCL-185™ cells indicates the possible presence of cytotoxic agents in B. parviocula venom. We proved previously described differences in the composition of venom obtained from B. arietans and B. parviocula by using electrophoresis and total protein concentration. Based on similarities in vasoactive effects observed after administration of venoms onto a chicken chorioallantoic membrane, we suggest that venom from B. arietans and B. parviocula might share certain venom proteins responsible for haemotoxicity. The main active components of B. parviocula venom are unknown. Our results suggest that it might be worth performing proteomic analysis of B. parviocula venom as it might contain medically valuable compounds.

PHB/CHIT Scaffold as a Promising Biopolymer in the Treatment of Osteochondral Defects-An Experimental Animal Study.

Biopolymer composites allow the creation of an optimal environment for the regeneration of chondral and osteochondral defects of articular cartilage, where natural regeneration potential is limited. In this experimental study, we used the sheep animal model for the creation of knee cartilage defects. In the medial part of the trochlea and on the medial condyle of the femur, we created artificial defects (6 × 3 mm2) with microfractures. In four experimental sheep, both defects were subsequently filled with the porous acellular polyhydroxybutyrate/chitosan (PHB/CHIT)-based implant. Two sheep had untreated defects. We evaluated the quality of the newly formed tissue in the femoral trochlea defect site using imaging (X-ray, Computer Tomography (CT), Magnetic Resonance Imaging (MRI)), macroscopic, and histological methods. Macroscopically, the surface of the treated regenerate corresponded to the niveau of the surrounding cartilage. X-ray examination 6 months after the implantation confirmed the restoration of the contour in the subchondral calcified layer and the advanced rate of bone tissue integration. The CT scan revealed a low regenerative potential in the bone zone of the defect compared to the cartilage zone. The percentage change in cartilage density at the defect site was not significantly different to the reference area (0.06-6.4%). MRI examination revealed that the healing osteochondral defect was comparable to the intact cartilage signal on the surface of the defect. Hyaline-like cartilage was observed in most of the treated animals, except for one, where the defect was repaired with fibrocartilage. Thus, the acellular, chitosan-based biomaterial is a promising biopolymer composite for the treatment of chondral and osteochondral defects of traumatic character. It has potential for further clinical testing in the orthopedic field, primarily with the combination of supporting factors.

Potential Effect of Pseudevernia furfuracea (L.) Zopf Extract and Metabolite Physodic Acid on Tumour Microenvironment Modulation in MCF-10A Cells.

Lichens comprise a number of unique secondary metabolites with remarkable biological activities and have become an interesting research topic for cancer therapy. However, only a few of these metabolites have been assessed for their effectiveness against various in vitro models. Therefore, the aim of the present study was to assess the effect of extract Pseudevernia furfuracea (L.) Zopf (PSE) and its metabolite physodic acid (Phy) on tumour microenvironment (TME) modulation, focusing on epithelial-mesenchymal transition (EMT), cancer-associated fibroblasts (CAFs) transformation and angiogenesis. Here, we demonstrate, by using flow cytometry, Western blot and immunofluorescence microscopy, that tested compounds inhibited the EMT process in MCF-10A breast cells through decreasing the level of different mesenchymal markers in a time- and dose-dependent manner. By the same mechanisms, PSE and Phy suppressed the function of Transforming growth factor beta (TGF-ß)-stimulated fibroblasts. Moreover, PSE and Phy resulted in a decreasing level of the TGF-ß canonical pathway Smad2/3, which is essential for tumour growth. Furthermore, PSE and Phy inhibited angiogenesis ex ovo in a quail embryo chorioallantoic model, which indicates their potential anti-angiogenic activity. These results also provided the first evidence of the modulation of TME by these substances.

Dimethyl Sulfoxide: Morphological, Histological, and Molecular View on Developing Chicken Liver.

Dimethyl sulfoxide (DMSO) is widely used as a solvent for small hydrophobic drug molecules. However, the safe volume allowing to avoid its embryotoxic effect has been poorly studied. In this study, we documented the effects of dimethyl sulfoxide (DMSO) in the developing chicken embryo at morphological, histological, and molecular levels. We focused on the developing chicken liver as the main organ involved in the process of detoxification. In our study, 100% DMSO was administered subgerminally onto the eggshell membrane (membrana papyracea) at various volumes (5, 10, 15, 20, 25, 30, 35, and 50 µL) on 4th embryonic day (ED). We focused on histopathological alterations of the liver structure, and noticed the overall impact of DMSO on developing chicken embryos (embryotoxicity, malformation). At the molecular level, we studied cytochrome P450 complex (CYP) isoform's activities in relation to changes of CYP1A5, CYP3A37, and CYP3A80 gene expression. Total embryotoxicity after application of different doses of DMSO on ED 4, and the embryo lethality increased with increasing DMSO amounts. Overall mortality after DMSO administration ranged below 33%. Mortality was increased with higher amounts of DMSO, mainly from 20 µL. The highest mortality was observed for the highest dose of DMSO over 35 µL. The results also showed a decrease in body weight with increased application volumes of DMSO. At the histological level, we observed mainly the presence of lipid droplets and dilated bile canaliculi and sinusoids in samples over the administration of 25 µL of DMSO. While these findings were not statistically significant, DMSO treatment caused a significant different up-regulation of mRNA expression in all studied genes. For CYP1A5, CYP3A37, and CYP3A80 DMSO volumes needed were 15 µL, 10 µL, and 20 µL, respectively. A significant down-regulation of all studied CYP isoform was detected after application of a DMSO dose of 5 µL. Regarding the morphological results, we can assume that the highest safe dose of DMSO without affecting chicken embryo development and its liver is up to 10 µL. This conclusion is corroborated with the presence of number of malformations and body weight reduction, which correlates with histological findings. Moreover, the gene expression results showed that even the lowest administered DMSO volume could affect hepatocytes at the molecular level causing down-regulation of cytochrome P450 complex (CYP1A5, CYP3A37, CYP3A80).

Characterization of Properties, In Vitro and In Vivo Evaluation of Calcium Phosphate/Amino Acid Cements for Treatment of Osteochondral Defects.

Novel calcium phosphate cements containing a mixture of four amino acids, glycine, proline, hydroxyproline and either lysine or arginine (CAL, CAK) were characterized and used for treatment of artificial osteochondral defects in knee. It was hypothesized that an enhanced concentration of extracellular collagen amino acids (in complex mixture), in connection with bone cement in defect sites, would support the healing of osteochondral defects with successful formation of hyaline cartilage and subchondral bone. Calcium phosphate cement mixtures were prepared by in situ reaction in a planetary ball mill at aseptic conditions and characterized. It was verified that about 30-60% of amino acids remained adsorbed on hydroxyapatite particles in cements and the addition of amino acids caused around 60% reduction in compressive strength and refinement of hydroxyapatite particles in their microstructure. The significant over-expression of osteogenic genes after the culture of osteoblasts was demonstrated in the cement extracts containing lysine and compared with other cements. The cement pastes were inserted into artificial osteochondral defects in the medial femoral condyle of pigs and, after 3 months post-surgery, tissues were analyzed macroscopically, histologically, immunohistochemically using MRI and X-ray methods. Analysis clearly showed the excellent healing process of artificial osteochondral defects in pigs after treatment with CAL and CAK cements without any inflammation, as well as formation of subchondral bone and hyaline cartilage morphologically and structurally identical to the original tissues. Good integration of the hyaline neocartilage with the surrounding tissue, as well as perfect interconnection between the neocartilage and new subchondral bone tissue, was demonstrated. Tissues were stable after 12 months' healing.

Galectin-8 Favors VEGF-Induced Angiogenesis: In Vitro Study in Human Umbilical Vein Endothelial Cells and In Vivo Study in Chick Chorioallantoic Membrane.

BACKGROUND/AIM: Although it has been accepted that the tandem repeat galectin-8 (Gal-8) is linked to angiogenesis, the underlying mechanisms in endothelial cells has remained poorly understood. In this study we aimed to investigate the effect of Gal-8 on selected biological processes linked to angiogenesis in in vitro and in vivo models. MATERIALS AND METHODS: In detail, we assessed how exogenously added human recombinant Gal-8 (with or without vascular endothelial growth factor - VEGF) affects selected steps involved in vessel formation in human umbilical vein endothelial cells (HUVECs) as well as using the chick chorioallantoic membrane (CAM) assay. Gene expression profiling of HUVECs was performed to extend the scope of our investigation. RESULTS: Our findings demonstrate that Gal-8 in combination with VEGF enhanced cell proliferation and migration, two cellular events linked to angiogenesis. However, Gal-8 alone did not exhibit any significant effects on cell proliferation or on cell migration. The molecular analysis revealed that Gal-8 in the presence of VEGF influenced cytokine-cytokine receptor interactions, HIF-1 and PI3K/AKT signaling pathways. Gal-8 alone also targeted cytokine-cytokine receptor interactions, but with a different expression profile as well as a modulated focal adhesion and TNF signaling. CONCLUSION: Gal-8 promotes a pro-angiogenic phenotype possibly in a synergistic manner with VEGF.

Changes in the Acute-Phase Protein Concentrations and Activities of Some Enzymes in Pigs Following the Repair of Experimentally Induced Articular Cartilage Defects Using Two Types of Biocement Powder.

The objective of the study was to assess the usefulness of acute-phase proteins (APPs) and serum enzymes in the evaluation of post-operative state after cartilage reconstruction in an animal model (Sus scrofa domesticus). Fifteen clinically healthy female pigs were evaluated during the first 30 days after the repair of experimentally induced articular cartilage defects using two types of biocement powders. Animals were divided into groups according to the type of biocement powder used: CAK-with amino acids (n = 6), C-without amino acids (n = 6) and the control group (Ctr) was without biocement (n = 3). The concentrations of selected APPs-serum amyloid A (SAA), haptoglobin (Hp) and C-reactive protein (CRP), and the activities of some serum enzymes-creatine kinase (CK), alkaline phosphatase (AP), and lactate dehydrogenase (LD) were measured one day before the surgery and on days 7, 14, and 30 after the surgical intervention. The most significant changes during the evaluated period were observed in the concentrations of SAA (p < 0.001) and Hp (p < 0.001), with marked increase of values 7 days after surgery. There was a numerical, but not statistically significant, difference between CAK, C and Ctr groups (p > 0.05). Marked variations were observed also in the activities of the evaluated enzymes, with the most significant changes in the activity of AP in the CAK group (p < 0.001). Presented results suggest possible usefulness of some APPs and serum enzymes in the evaluation of post-operative inflammatory state after the reconstruction of articular cartilage defects.

Canine Bone Marrow-derived Mesenchymal Stem Cells: Genomics, Proteomics and Functional Analyses of Paracrine Factors.

Adult stem cells have become prominent candidates for treating various diseases in veterinary practice. The main goal of our study was therefore to provide a comprehensive study of canine bone marrow-derived mesenchymal stem cells (BMMSC) and conditioned media, isolated from healthy adult dogs of different breeds. Under well-defined standardized isolation protocols, the multipotent differentiation and specific surface markers of BMMSC were supplemented with their gene expression, proteomic profile, and their biological function. The presented data confirm that canine BMMSC express important genes for differentiation toward osteo-, chondro-, and tendo-genic directions, but also genes associated with angiogenic, neurotrophic, and immunomodulatory properties. Furthermore, using proteome profiling, we identify for the first time the dynamic release of various bioactive molecules, such as transcription and translation factors and osteogenic, growth, angiogenic, and neurotrophic factors from canine BMMSC conditioned medium. Importantly, the relevant genes were linked to their proteins as detected in the conditioned medium and further associated with angiogenic activity in chorioallantoic membrane (CAM) assay. In this way, we show that the canine BMMSC release a variety of bioactive molecules, revealing a strong paracrine component that may possess therapeutic potential in various pathologies. However, extensive experimental or preclinical trials testing canine sources need to be performed in order to better understand their paracrine action, which may lead to novel therapeutic strategies in veterinary medicine.

Polyhydroxybutyrate/Chitosan 3D Scaffolds Promote In Vitro and In Vivo Chondrogenesis.

The articular cartilage is an avascular and aneural tissue and its injuries result mostly in osteoarthritic changes and formation of fibrous tissue. Efforts of scientists worldwide are focused on restoration of cartilage with increase in life quality of patients. Novel polymeric polyhydroxybutyrate/chitosan (PCH) porous 3D scaffolds were developed and characterized. The rat mesenchymal stem cells (MSCs) were seeded in vitro on PCH scaffolds by a simple filtration of MSCs suspension over scaffolds using syringe. The chondrogenesis of cell-scaffold constructs was carried out in supplemented chondrogenic cultivation medium. After 2 and 4 weeks of in vitro culturing cell-scaffold constructs in chondrogenic differentiation medium, the cartilage extracellular matrix components like glycosaminoglycans and collagens were identified in scaffolds by biochemical assays and histological and immunohistochemical staining. Preliminary in vivo experiments with acellular scaffolds, which filled the artificially created cartilage defect in sheep knee were done and evaluated. Cells released from the bone marrow cavity have penetrated into acellular PCH scaffold in cartilage defect and induced tissue formation similar to hyaline cartilage. The results demonstrated that PCH scaffolds supported chondrogenic differentiation of MSCs in vitro. Acellular PCH scaffolds were successfully utilized in vivo for reparation of artificially created knee cartilage defects in sheep and supported wound healing and formation of hyaline cartilage-like tissue.

3D Reconstruction and Evaluation of Accessory Hepatic Veins in Right Hemilivers in Laboratory Animals by Metrotomography: Implications for Surgery.

BACKGROUND The current study investigated the detection of accessory hepatic veins and their vascular territories in the right hemiliver in rats, guinea pigs, and rabbits, which has become a prerequisite for newly developed clinical procedures. We compared the anatomical continuity of accessory hepatic veins with accessory hepatic veins existing in human livers. MATERIAL AND METHODS The analysis of accessory hepatic veins was performed using a corrosion cast method in combination with computer tomography (CT). RESULTS In normal livers, accessory hepatic veins were regularly found. The length of these veins was 0.88±0.29 (cm ±SD) in rats, 1.10±0.39 in guinea pigs, and 1.28±0.48 in rabbits. Accessory hepatic veins became a part of the draining vessel draining into segment VI and VII; represented by interpolating and following Chouinard's segmental concept. CONCLUSIONS The importance of detecting accessory hepatic veins lies in the identification of structures requiring special attention during surgery, in reduction of surgical complications, and in choosing the best approach to maintain the vitality of a drainage segment. The vascular reconstruction should be done during surgical interventions.

The Serum Protein Profile and Acute Phase Proteins in the Postoperative Period in Sheep after Induced Articular Cartilage Defect.

Although several new implants have been developed using animal studies for the treatment of osteochondral and cartilage defects, there is a lack of information on the possible metabolic and biochemical reactions of the body to the implantation of biomaterials and cartilage reconstruction. Therefore, this study was aimed at evaluating the serum protein pattern and the alterations in the concentrations of selected acute phase proteins in five clinically healthy female sheep before and after the reconstruction of experimentally induced articular cartilage defects using polyhydroxybutyrate/chitosan based biopolymer material. The concentrations of total serum proteins (TSP), protein fractions, and selected acute phase proteins-serum amyloid A (SAA), haptoglobin (Hp), and C-reactive protein (CRP)-were measured before and on days seven, 14, and 30 after the surgical intervention. The TSP concentrations showed no marked differences during the evaluated period. Albumin values decreased on day seven and day 14 after surgery. In the concentrations of α1-, α2-, ß-, and γ2-globulins, a gradual significant increase was observed during the postoperative period (p < 0.05). The γ1-globulins decreased slightly seven days after surgery. The concentrations of SAA, Hp, and CRP increased significantly after the surgical intervention with a subsequent decrease on day 30. Presented results suggest marked alterations in the serum protein pattern after surgical intervention.

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.

Dynamics of Evans blue clearance from cerebrospinal fluid into meningeal lymphatic vessels and deep cervical lymph nodes.

Objectives Recently, it has been confirmed, that excess fluid and waste products from the brain are drained into the cerebrospinal fluid (CSF) and afterwards cleared via the olfactory route and/or lymphatic vessels in the brain dura and corresponding extracranial lymphatic structures. Therefore, the aim of present study was to monitor time-dependent uptake of Evans blue (EB) tracer from subarachnoid space into the meningeal lymphatic vessels and extracranial lymph nodes in rats during 3 hours-12 days. Methods EB was injected into the cisterna magna of anesthetized rats and after required survival, plasma, brain dura matter and corresponding lymph nodes (cervical, thoracic and lumbar) were dissected and processed for lymphatic vessels analyses using immunofluorescence and immunohistochemistry. Furthermore, we have used sensitive ultra-high-performance liquid chromatography (UHPLC) method for the determination of EB concentrations in selected samples. Results Using a combination of imaging methods, we have detected two different types of the vascular structures in the brain dura and in deep cervical lymph nodes. The blood vessels, which were RECA-1 + positive and the lymphatic-like vessels, expressing bright intense red fluorescence of EB tracer. Subsequently, using UHPLC with UV detection, we have quantified the EB concentration in positive structures by 3 hours up to 12 days after tracer delivery. A significant increase of EB concentration was detected in deep cervical lymph nodes already at 3 hours with a peak at 1 day that decreased to about one-tenth of its peak value by 12 days. Similar pattern was detected in brain dura. On the contrary, the brain tissue and plasma were almost negative for EB tracer during all tested time periods. Conclusion Our results demonstrate the dynamic changes of EB in meningeal lymphatic vessels and in deep cervical lymph nodes, thus recapitulating the downstream outflow of intracisternally injected tracer during 3 hours-12 days via dura mater lymphatic vessels towards corresponding extracranial draining system, particularly the deep cervical lymph nodes.

ß-Escin Effectively Modulates HUVECS Proliferation and Tube Formation.

In the present study we evaluated the anti-angiogenic activities of ß-escin (the major active compound of Aesculus hippocastanum L. seeds). Human umbilical-vein endothelial cells (HUVECs) were used as an in vitro model for studying the molecular mechanism underlying the anti-angiogenic effect of ß-escin. We investigated the in vitro effects on proliferation, migration, and tube formation of HUVECs and in vivo anti-angiogenic activity was evaluated in a chick chorioallantoic membrane (CAM) angiogenesis assay. Moreover, the effect on gene expressions was determined by the RT2 ProfilerTM human angiogenesis PCR Array. It was found that ß-escin exerts inhibitory effect on the basic fibroblast growth factor (bFGF)-induced proliferation, migration and tube formation, as well as CAM angiogenesis in vivo. The inhibition of critical steps of angiogenic process observed with ß-escin could be partially explained by suppression of Akt activation in response to bFGF. Moreover, the anti-angiogenic effects of ß-escin could also be mediated via inhibition of EFNB2 and FGF-1 gene expressions in endothelial cells. In conclusion, ß-escin affects endothelial cells as a negative mediator of angiogenesis in vitro and in vivo and may therefore be considered as a promising candidate for further research elucidating its underlying mechanism of action.

Brain-Cortex Microglia-Derived Exosomes: Nanoparticles for Glioma Therapy.

The function and integrity of the nervous system require interactive exchanges among neurons and glial cells. Exosomes and other extracellular vesicles (EVs) are emerging as a key mediator of intercellular communication, capable of transferring nucleic acids, proteins and lipids influencing numerous functional and pathological aspects of both donor and recipient cells. The immune response mediated by microglia-derived exosomes is most prominently involved in the spread of neuroinflammation, neurodegenerative disorders, and brain cancer. Therefore, in the present study we describe a reproducible and highly efficient method for yielding purified primary microglia cells, followed by exosome isolation and their characterization. An in vitro biological assay demonstrates that microglia-derived exosomes tested on a 3D spheroid glioma culture were able to inhibit tumor invasion in time course. These results evidence that brain microglia-derived exosomes could be used as nanotherapeutic agents against glioma cells.

Comparative Genomics of Bacillus amyloliquefaciens Strains Reveals a Core Genome with Traits for Habitat Adaptation and a Secondary Metabolites Rich Accessory Genome.

The Gram positive, non-pathogenic endospore-forming soil inhabiting prokaryote Bacillus amyloliquefaciens is a plant growth-promoting rhizobacterium. Bacillus amyloliquefaciens processes wide biocontrol abilities and numerous strains have been reported to suppress diverse bacterial, fungal and fungal-like pathogens. Knowledge about strain level biocontrol abilities is warranted to translate this knowledge into developing more efficient biocontrol agents and bio-fertilizers. Ever-expanding genome studies of B. amyloliquefaciens are showing tremendous increase in strain-specific new secondary metabolite clusters which play key roles in the suppression of pathogens and plant growth promotion. In this report, we have used genome mining of all sequenced B. amyloliquefaciens genomes to highlight species boundaries, the diverse strategies used by different strains to promote plant growth and the diversity of their secondary metabolites. Genome composition of the targeted strains suggest regions of genomic plasticity that shape the structure and function of these genomes and govern strain adaptation to different niches. Our results indicated that B. amyloliquefaciens: (i) suffer taxonomic imprecision that blurs the debate over inter-strain genome diversity and dynamics, (ii) have diverse strategies to promote plant growth and development, (iii) have an unlocked, yet to be delimited impressive arsenal of secondary metabolites and products, (iv) have large number of so-called orphan gene clusters, i.e., biosynthetic clusters for which the corresponding metabolites are yet unknown, and (v) have a dynamic pan genome with a secondary metabolite rich accessory genome.