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Regarding the importance of inhibiting VEGF and unique features of VHHs as a new generation of antibody-based therapeutics, the present study aimed to generate VHHs against the receptor binding domain of VEGF, thereby blocking of VEGF binding to its receptor. After preparing the gene repertoire of VHH fragments from an immunized camel, a VHH phage display library was constructed. We adopted a stringent successive biopanning to isolate the phages displaying VHH with high affinity to VEGF-RBD.A significant enrichment of phages that specifically bound to the target protein was obtained after six rounds of panning. Of the specific clones with high binding affinity screened by monoclonal phage ELISA, 52% shared the same VHH sequence, showing its high enrichment. Using molecular simulation of antigen-antibody interaction based on the crystallographic information of VEGF/VEGFR2, molecular dynamics simulations and MM/PBSA free energy calculations, we provide a reliable picture of the binding site of antibody on antigen. The key residues in the VEvhh1-VEGF interface were dissected and the energetics was analyzed by MM/PBSA. The results of studies revealed that VEvhh1 binds to the receptor binding site of VEGF with high binding energy and showed the highest affinity to the residues of VEGF which are responsible for VEGF binding to VEGFR2. Also the antibody potently covers these key functional residues of VEGF, thereby inhibiting VEGF binding to its receptor and probably abrogating its biological activity. This study may represent VEvhh1 as an anti-VEGF and anti-angiogenic candidate.

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در این مقاله اثر ضد رگ‌ زایی داروی آرتیمیزینین برای مقابله با رشد تومور سرطانی مورد بررسی قرار گرفته است.به این منظور از رویکرد مدلسازی استفاده شده است. ابتدا یکی از مدل‌ های موجود در ادبیات برای انطباق با نتایج تجربی، بهبود داده شده و سپس پارامتر های آن با استفاده از روش بیشترین شیب بر مبنای داده‌ های آزمایشگاهی جمع‌ آوری شده از موش‌ های آزمایشگاهی استخراج شده است. سپس بر مبنای مدل استخراج شده، روش پایداری لیاپانوف برای طراحی برنامه دارو دهی مد نظر قرار گرفته و یک برنامه دارو دهی برای درمان و کنترل رشد تومور سرطانی ارائه گردیده است. با استفاده از شبیه‌ سازی های رایانه ای نشان داده شده است که برنامه دارو دهی ارائه شده مناسب بوده و می‌ تواند منجر به ایجاد بهبود در روند درمان و کنترل رشد تومور سرطانی گردد. در طراحی برنامه دارو دهی مقدار سمیت داروی آرتیمیزینین نیز لحاظ شده است.

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Introduction: Breast cancer is one of the provalent cancers in the world. This cancer as well as other solid tumors, in the course of its development has phases in this order: Epithelial Dysplasia, Carcinoma Insitu, Invassiveness and metastasis. Breast cancer Diagnosis is generally made with pathology methods. In this survey, measuring Angiostatin (which is one of the most important and potent angiogensis inhibitor) in random urine as a noninvassive method was introduced to diagnose the disease. Materials and Methods: In this assay, random urine samples of 15 Breast cancer patient and 15 control urine samples were obtained, and assayed with improved sandwich direct ELISA. Results: Obtained result in statistical T-Test (Pvalue<0.03) showed significant correlation between urine angiostatin and breast cancer, that has coordinace with the result of patients sample pathology. Discussion: Angiostatin dosage in urine of patients of breast cancer is a good marker of non invassive diagnosis.

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Abnormal angiogenesis is associated with various diseases such as solid tumors and metastasis, retinopathies, and rheumatoid arthritis. VEGF-A is the most important mediator of angiogenesis among all growth factors. The bioactivity of VEGF is mediated by two tyrosine kinase receptors VEGFR-1 and VEGFR-2 present on endothelial cells. VEGF signaling through VEGFR-2 is the major angiogenic pathway that leads to stimulation of endothelial cell ingrowths into the tumor. It comprised of an extracellular portion, a cytoplasmic portion, and a short transmembrane domain. The extracellular portion consists of seven Ig-like domains (D1–D7), of which the 1st to 3rd domains function as ligand-binding sites. In the present work, a soluble recombinant extracellular domain 1-3 of VEGFR-2 was expressed in Pichia pastoris to inhibit the VEGF-induced angiogenesis. The 975 bp DNA fragment containing extracellular domain 1-3 kdr, was designed according to the nucleotide sequence at GenBank and protein sequence at Swiss-Prot. The recombinant secretory expression vector (pPinkαHC/KDR1-3) was constructed and transferred into yeast by electroporation. The high expression transformants were identified through complementation of adenine auxotrophy and cultured. KDR1-3 was expressed under the induction of %1 methanol and confirmed by using SDS-PAGE and western blot techniques. After being purified by affinity chromatography using Ni-NTA resins, binding of expressed product to hVEGF165 was proved by two direct ELISA and ELISA receptor binding assays. The data showed that human VEGFR-2 extracellular domain 1-3 with eukaryotic protein structure, that there is no reported paper about, was successfully expressed. 

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Integrin inhibitors may change conformational and dynamical properties of integrin, but its molecular properties in this process is not clearly understood. Tumstatin is an anti-angiogenesis protein derived from collagen XVIII, but little is known about how tumstatin applies its antiangiogenic and antitumor effects. It has been reported that 18 amino acids fragment of tumstatin has anti-tumor activities similar to tumstatin. We used molecular docking and molecular dynamic simulations to describe inhibitor activity of peptide in molecular level. We described the binding of this peptide to Hybrid/EGF-4 interface and that these interactions might contribute to improved hydrophobic interactions at these regions and also fixed the mobile domains of integrin. In the complex, we recognized a novel binding site on integrin for integrin inhibitors that may have critical role in integrin inhibition. These results support the idea that hydrophobic interactions between Hybrid/EGF-4 domain and peptide-anti tumor might contribute to stability of bended state and therefore inhibit integrin activation.  Our finding can be applied to understand the mechanism of out-in pathway integrin signaling and development of integrin targeted drug.

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Investigation of factors affecting endothelial cell proliferation is an essential part of angiogenesis studies. Given the importance of inhibiting angiogenesis in the treatment of cancers, and due to the side effects and high cost of anti-angiogenic drugs such as Avastin, the use of physical agents to aid in treatment and reduce the need for high doses of the drug is noteworthy. Magnetic fields are of interest due to their long-distance and non-invasive effects, and many studies have been conducted on their effects on biological phenomena, including angiogenesis, with inconsistent results. In the present study, the effect of a 2 mT alternating magnetic field with a frequency of 200 Hz and Austin on the proliferation of human umbilical vein endothelial cells (HUVEC) was investigated. Cells were treated for 48 hours under a mixture of 50 μg/ml solution of vascular endothelial growth factor (VEGEF) and Avastin at concentrations (zero (drug control), 50, 100, 200 and 400 μg/ml) as well as field treatment groups for They were exposed to magnetic fields for 3, 6, 12, 24 and 48 hours. Then, cell proliferation was assessed using Alamar Blue colorimetric test. Data were analyzed by three-way analysis of variance. According to the findings, the exposure times of 12, 24 and 48 hours showed a significant reduction in cell proliferation compared to the control group, but this difference was not significant in the 3 and 6 hour treatments. Also, the degree of interaction of these factors with each other on HUVEC proliferation was investigated.

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Tumor induced angiogenesis is the bridge between benign and malignant tumor growth stages. In this process, growth and migration of endothelial cells build capillaries to supply the tumor with blood for its further growth. Regarding the importance of capillary formation and blood flow in angiogenesis, simulation of this phenomenon plays important role in tumor growth and cancer development studies. In this work, considering intracellular, cellular, and extracellular scales a mathematical model of tumor-induced angiogenesis is used to consider mechanical effects of extracellular matrix on growth and migration of endothelial cells. These effects are matrix density and its fiber length. In this study, to model cellular dynamics, a discrete lattice based Monte Carlo method is used. Results show that migration of endothelial cells and development of capillaries are possible in a specified range of matrix density and matrix fiber length. Based on the results, medium matrix densities and low fiber length provide a suitable environment for capillaries growth and development. The model is a promising tool for modeling tumor induced angiogenesis and is a base for development of models for loop formation and blood flow in capillaries around tumor.

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Tissue engineering is a rapidly growing field of research for several decades, which is driven by the human urgent need for tissue substitutes and transplantable organs. Considering the advancements, the clinical applications in the field of tissue engineering have been limited until now. The major reason toward this limitation is the lack of sufficient blood supply for the tissue in the earliest phase after implantation. Time-consuming process of angiogenesis leads to inadequate vascularization and finally, death of cells and destruction of tissue. During recent years, by implementing a strategy called Inosculation, it has been tried to facilitate tissue vascularization by a preformed vasculature network within tissue structure. In the current research considering cellular nature of angiogenesis process, relying on a cell-based mathematical model, the effect of inosculation strategy is investigated through the dynamics of angiogenesis process with respect to extracellular, cellular and intracellular spatio-temporal scales. The results show the advantages of inosculation strategy over angiogenesis strategy in vascularization of tissue constructs. The model demonstrates the capability of inosculation strategy to improve the anastomosis probability, which is providing the crucial requisite for the blood to flow through capillary network. Furthermore, the cellular model was developed in a way that illustrates the effects of extracellular matrix on morphogenesis through branching phenomenon.

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Aims: Exosomes are considered as a protective and enriched source of shuttle microRNAs. However, the precise biological mechanism of exosomal microRNAs in recipient cells remains to be further clarified. The aim of this study waz to investigate Expression level of miR-9 in exosomes derived from ovarian epithelial carcinoma cells and the effects of exosome treatment on Vascular Endothelial Growth Factor )VEGF( expression in human umbilical vein endothelial cells.
Material and Methods: Exosomes were purified from the conditioned medium of ovarian epithelial carcinoma cells. Exosome size and morphology were examined by a scanning electron microscope. Purified exosomes were labeled with PKH26 red fluorescent labeling kit, then labeled exosomes were incubated with human umbilical vein endothelial cells (HUVECs) for 12h at 37°C, and the cellular uptake was monitored using an inverted fluorescence microscope. Expression levels of miR-9 and VEGF were measured by real-time PCR. Paired t-test was used for data analysis.
Findings: The purified MSCs-derived exosomes had a spherical shape with a diameter between 50 to 100nm. PKH26-labeled exosomes can be taken up by SKOV3 tumor cells with high efficiency. The expression levels of miR-9 in both ovarian tumor cells and their exosomes. Exosomes derived from ovarian tumor cells caused increased expression of VEGF in exosome-treated endothelial cells.
Conclusion: Exosomes derived from ovarian tumor cells led to increased expression of VEGF in endothelial cells. As miR-9 was enriched in both ovarian tumor cells and their exosomes, it seems that exosomal transfer of miR-9 may affect the expression of VEGF in endothelial cells during tumor angiogenesis.

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Aims: The aim of the present study was to evaluate the effects of lysophosphatidic acid (LPA) supplementation of human ovarian tissue culture media on the morphology of tissue and alteration in angiogenesis by expression of vascular endothelial growth factor (VEGF) after transplantation.
Materials & Methods: In the present experimental study, the human ovarian tissues (n=8) after collection from female-to-male transsexual people, were cut into small fragments (n=98). Then, vitrified-warmed and cultured 24 hours in two groups in the presence and absence of LPA, and finally they were transplanted to γ-irradiated mice (n=13). After two weeks the morphology of tissues was studied by hematoxylin and eosin staining and VEGF protein was detected by immunohistochemistry. The expression of VEGF gene was evaluated by real time RT-PCR.
Results: The morphology of both transplanted tissues was well preserved and follicles at different developmental stages were seen in all studied groups. Significantly a higher expression of VEGF gene was observed in the LPA-treated group compared to the non-treated once (p<0.05). Several blood vessels were shown positive reactions for VEGF antibody as green color in stroma of ovarian tissue sections in all studied groups.
Conclusion: Supplementation of human ovarian tissue culture media with LPA before transplantation could increase the expression of VEGF gene related to angiogenesis.
 

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A cardiac infarction is the leading cause of death worldwide. Although the common treatments, including medication and various grafts, are unable to return the patients to their normal life, a cardiac patch is a promising technique in the field of tissue engineering that can stimulate the natural regeneration process of the diseased tissue via a scaffold with appropriate mechanical properties, biocompatibility and electrical conductivity. In this study, the composite scaffolds based on alginate (ALG) were fabricated through freeze-drying and coated with different concentrations of graphene oxide (GO) to make ALG/xGO (x=0.01, 0.05 and 0.1 wt. %) scaffolds. The scaffolds were characterized in terms of morphology, physicochemical structure, tensile strength, electrical conductivity, and cell response and gene expression. The presence of GO provided interconnected pores in the composite scaffolds. Adding GO up to 0.1 wt.% significantly enhanced Young’s modulus up to 5.5 MPa and electrical conductivity up to 8.59 S.m^-1 (p≤0.05). Additionally, GO improved the vitality of human umbilical vein endothelial cells (HUVECs) compared to the scaffold without GO.  Investigating cell attachment of L929 fibroblasts indicated that the optimal content of GO at 0.05 wt.% can provide better places for cellular nesting due to the appropriate size of pores for cell/material interactions. The increase in the amount of GO up to 0.1 wt.% lead to a significant increase in gene expression of VEGFR-2 compared to the other scaffolds and tissue culture plate. We found that the prepared ALG/0.1GO composite scaffold could be appropriate for further experiments on cardiac tissue engineering applications.