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           Search results for: Recombinant Human Vascular Endothelial Growth Factor rh-VEGF165   

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Expression and characterization of recombinant human VEGF165 in the middle silk gland of transgenic silkworms.

Recombinant human vascular endothelial growth factor (rhVEGF) has important applications in therapeutic angiogenesis and inhibition of VEGF-mediated pathological angiogenesis. Previous studies have shown that rhVEGF can be produced in several expression systems, including Escherichia coli, yeasts, insect cells and mammalian cells. However, little is known regarding the effective production of this protein in organs of live organisms. Here, we report for the first time the expression and characterization of rhVEGF165 in the middle silk gland (MSG) of the transgenic silkworm line S1-V165. Our results confirmed that (1) rhVEGF165 was highly expressed in MSG cells and was secreted into the cocoon of S1-V165; (2) the dimeric form of rhVEGF165 could be easily dissolved from S1-V165 cocoons using an alkaline solution; (3) rhVEGF165 extracted from S1-V165 cocoons exhibited slightly better cell proliferative activity than the hVEGF165 standard in cultured human umbilical vein endothelial cells. This study provides an alternative strategy for the production of bioactive rhVEGF165 using the MSG of transgenic silkworms.

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Intraocular Penetration of a vNAR: In Vivo and In Vitro VEGF Neutralization.

Variable new antigen receptor domain (vNAR) antibodies are novel, naturally occurring antibodies that can be isolated from naïve, immune or synthetic shark libraries. These molecules are very interesting to the biotechnology and pharmaceutical industries because of their unique characteristics related to size and tissue penetrability. There have been some approved anti-angiogenic therapies for ophthalmic conditions, not related to vNAR. This includes biologics and chimeric proteins that neutralize vascular endothelial growth factor (VEGF), which are injected intravitreal, causing discomfort and increasing the possibility of infection. In this paper, we present a vNAR antibody against human recombinant VEGF (rhVEGF) that was isolated from an immunized shark. A vNAR called V13, neutralizes VEGF cytokine starting at 75 μg/mL in an in vitro assay based on co-culture of normal human dermal fibroblasts (NHDFs) and green fluorescence protein (GFP)-labeled human umbilical vein endothelial cells (HUVECs) cells. In the oxygen-induced retinopathy model in C57BL/6:Hsd mice, we demonstrate an endothelial cell count decrease. Further, we demonstrate the intraocular penetration after topical administration of 0.1 μg/mL of vNAR V13 by its detection in aqueous humor in New Zealand rabbits with healthy eyes after 3 h of application. These findings demonstrate the potential of topical application of vNAR V13 as a possible new drug candidate for vascular eye diseases.

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A Low Cost Implantation Model in the Rat That Allows a Spatial Assessment of Angiogenesis.

There is continual demand for animal models that allow a quantitative assessment of angiogenic properties of biomaterials, therapies, and pharmaceuticals. In its simplest form, this is done by subcutaneous material implantation and subsequent vessel counting which usually omits spatial data. We have refined an implantation model and paired it with a computational analytic routine which outputs not only vessel count but also vessel density, distribution, and vessel penetration depth, that relies on a centric vessel as a reference point. We have successfully validated our model by characterizing the angiogenic potential of a fibrin matrix in conjunction with recombinant human vascular endothelial growth factor (rhVEGF165). The inferior epigastric vascular pedicles of rats were sheathed with silicone tubes, which were subsequently filled with 0.2 ml of fibrin and different doses of rhVEGF165, centrically embedding the vessels. Over 4 weeks, tissue samples were harvested and subsequently immunohistologically stained and computationally analyzed. The model was able to detect variations over the angiogenic potentials of growth factor spiked fibrin matrices. Adding 20 ng of rhVEGF165 resulted in a significant increase in vasculature while 200 ng of rhVEGF165 did not improve vascular growth. Vascularized tissue volume increased during the first week and vascular density increased during the second week. Total vessel count increased significantly and exhibited a peak after 2 weeks which was followed by a resorption of vasculature by week 4. In summary, a simple implantation model to study vascularization with only a minimal workload attached was enhanced to include morphologic data of the emerging vascular tree.

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Lipoprotein Receptor-related Protein 6 Signaling is Necessary for Vasculogenic Differentiation of Human Dental Pulp Stem Cells.

The aim of this study was to evaluate the effects of Wnt signaling through lipoprotein receptor-related protein 6 (LRP6) and Frizzled6 on the endothelial differentiation of dental pulp stem cells (DPSCs). DPSCs were stably transduced with enhanced green fluorescent protein (EGFP)-tagged lentiviral vectors (short hairpin RNA-LRP6, short hairpin RNA-Frizzled6, or empty vector controls). We evaluated the effects of LRP6 and Frizzled6 on expression of endothelial markers and on capillary tube formation mediated by DPSCs induced with recombinant human Wnt1 (rhWnt1) and/or recombinant human vascular endothelial growth factor (rhVEGF). In vivo, tooth slices/scaffolds were seeded with LRP6-silenced, Frizzled6-silenced, or vector control DPSC cells and transplanted into immunodeficient mice. The density of blood vessels generated by DPSCs differentiated into vascular endothelial cells was analyzed by immunohistochemistry for EGFP. The rhWnt1 and rhVEGF induced expression of active β-catenin in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. Furthermore, VEGF and interleukin-8 were downregulated in LRP6-silenced DPSCs, but not in control DPSCs or in Frizzled6-silenced DPSCs (P < .05). Likewise, rhWnt1 and rhVEGF induced expression of the endothelial marker VEGF receptor-2 in control DPSCs and in Frizzled6-silenced DPSCs, but not in LRP6-silenced DPSCs. These data correlated with a trend for lower density of capillary sprouts generated by LRP6-silenced DPSCs when compared with control DPSCs in Matrigel. In vivo, tooth slice/scaffolds seeded with DPSC-short hairpinRNA-LRP6 cells showed lower density of human blood vessels (ie, EGFP-positive blood vessels), when compared with tooth slice/scaffolds seeded with vector control cells (P < .05). Collectively, these data demonstrated that LRP6 signaling is necessary for the vasculogenic differentiation of human DPSCs.

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Repair of large saddle defects of the mandibular ridge using dual growth factor release-An experimental pilot study in minipigs.

To test the hypothesis that the addition of small amounts of rhVEGF to rhBMP2 in a polymer carrier can accomplish equivalent repair effect as a reduced dosage of rhBMP2 compared to rhBMP2 alone.

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The Use of Sequential VEGF- and BMP2-Releasing Biodegradable Scaffolds in Rabbit Mandibular Defects.

Promising developments have materialized in reconstructive surgical procedures with the applications of tissue engineering. In our study, we used tissue scaffolds fabricated from polylactic acid-polyethylene glycol (PLLA-PEG) copolymers to ensure different release rates of selective growth factors recombinant human bone morphogenetic protein 2 [rhBMP-2] and vascular endothelial growth factor (rhVEGF165) in the repair of mandibular bone defects.

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Continuous delivery of rhBMP2 and rhVEGF165 at a certain ratio enhances bone formation in mandibular defects over the delivery of rhBMP2 alone--An experimental study in rats.

The aim of the present study was to test the hypothesis that different amounts of vascular endothelial growth factor and bone morphogenic protein differentially affect bone formation when applied for repair of non-healing defects in the rat mandible. Porous composite PDLLA/CaCO3 carriers were fabricated as slow release carriers and loaded with rhBMP2 and rhVEGF165 in 10 different dosage combinations using gas foaming with supercritical carbon dioxide. They were implanted in non-healing defects of the mandibles of 132 adult Wistar rats with additional lateral augmentation. Bone formation was assessed both radiographically (bone volume) and by histomorphometry (bone density). The use of carriers with a ratio of delivery of VEGF/BMP between 0.7 and 1.2 was significantly related to the occurrence of significant increases in radiographic bone volume and/or histologic bone density compared to the use of carriers with a ratio of delivery of ≤ 0.5 when all intervals and all outcome parameters were considered. Moreover, simultaneous delivery at this ratio helped to "save" rhBMP2 as both bone volume and bone density after 13 weeks were reached/surpassed using half the dosage required for rhBMP2 alone. It is concluded, that the combined delivery of rhVEGF165 and rhBMP2 for repair of critical size mandibular defects can significantly enhance volume and density of bone formation over delivery of rhBMP2 alone. It appears from the present results that continuous simultaneous delivery of rhVEGF165 and rhBMP2 at a ratio of approximately 1 is favourable for the enhancement of bone formation.

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Angiogenesis and bone regeneration of porous nano-hydroxyapatite/coralline blocks coated with rhVEGF165 in critical-size alveolar bone defects in vivo.

To improve the regenerative performance of nano-hydroxyapatite/coralline (nHA/coral) block grafting in a canine mandibular critical-size defect model, nHA/coral blocks were coated with recombinant human vascular endothelial growth factor(165) (rhVEGF) via physical adsorption (3 μg rhVEGF165 per nHA/coral block). After the nHA/coral blocks and VEGF/nHA/coral blocks were randomly implanted into the mandibular box-shaped defects in a split-mouth design, the healing process was evaluated by histological observation and histomorphometric and immunohistological analyses. The histological evaluations revealed the ingrowth of newly formed blood vessels and bone at the periphery and cores of the blocks in both groups at both 3 and 8 weeks postsurgery, respectively. In the histomorphometric analysis, the VEGF/nHA/coral group exhibited a larger quantity of new bone formation at 3 and 8 weeks postsurgery. The percentages of newly formed bone within the entire blocks in the VEGF/nHA/coral group were 27.3% ± 8.1% and 39.3% ± 12.8% at 3 weeks and 8 weeks, respectively, and these values were slightly greater than those of the nHA/coral group (21.7% ± 3.0% and 32.6% ± 10.3%, respectively), but the differences were not significant (P>0.05). The immunohistological evaluations revealed that the neovascular density in the VEGF/nHA/coral group (146 ± 32.9 vessel/mm(2)) was much greater than that in the nHA/coral group (105 ± 51.8 vessel/mm(2)) at the 3-week time point (P<0.05), but no significant difference was observed at the 8-week time point (341 ± 86.1 and 269 ± 50.7 vessel/mm(2), respectively, P>0.05). The present study indicated that nHA/coral blocks might be optimal scaffolds for block grafting in critical-size mandibular defects and that additional VEGF coating via physical adsorption can promote angiogenesis in the early stage of bone healing, which suggests that prevascularized nHA/coral blocks have significant potential as a bioactive material for bone regeneration in large-scale alveolar defects.

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Recombinant human vascular endothelial growth factor receptor 1 effectively inhibits angiogenesis in vivo.

Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. VEGF receptor‑1 (VEGFR‑1) acts as a decoy VEGF receptor that enables the regulation of VEGF on the vascular endothelium. In the present study, the recombinant human VEGFR1D1‑3/Fc (rhVEGFR‑1), which contains key domains for VEGF binding, was cloned and expressed in Chinese hamster ovary (CHO) cells. The rhVEGFR‑1 protein was purified using protein‑A affinity chromatography. The molecular weight of rhVEGFR‑1 was found to be ~162 and 81 kD in non‑reducing and reducing SDS‑PAGE, respectively. The majority of the final protein products were in the dimeric conformation. Western blot analysis revealed that rhVEGFR‑1 was only capable of binding to the full glycan form of rhVEGF‑165 and rhVEGF‑121. The dissociation constant for the binding of rhVEGFR‑1 to VEGF‑165, detected using Biacore, was 285 pM. In addition, rhVEGFR‑1 inhibited the proliferation and migration of human microvascular endothelial cells. In vivo experiments also demonstrated that rhVEGFR‑1 inhibited chicken chorioallantoic membrane neovascularization and angiogenesis in nude mice. In conclusion, an anti‑angiogenic recombinant soluble VEGFR was expressed (up to 5 mg/l) in CHO cells and was shown to be capable of inhibiting neovascularization in vivo and in vitro.

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Production of functional human vascular endothelial growth factor(165) in transgenic rice cell suspension cultures.

Vascular endothelial growth factors (VEGFs) are secreted by tumor cells and other cells exposed to hypoxia, and play a critical role in the development and differentiation of the vascular system. In this study, we investigated the production of functional recombinant human VEGF165 (rhVEGF165) in transgenic rice cell suspension culture. Complementary DNA was synthesized from human leukemia HL60 cells and cloned into expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with this recombinant vector by the Agrobacterium mediated method and the integration of the target gene into the plant genome was confirmed by genomic PCR. The expression of rhVEGF165 in the rice cells was determined by Northern blot and Western blot analyses. The accumulated rhVEGF165 protein in the culture medium was 19 mg/L after 18 days of culturing in a sugar-free medium. The rhVEGF165 was purified using a heparin HP column and its biological activity was tested on human umbilical vein endothelial cells (HUVECs). The purified rhVEGF165 significantly increased the proliferative activity of the HUVECs. Therefore, it was demonstrated that functional rhVEGF165 could be produced using transgenic rice suspension culture vector under the control of the RAmy3D promoter.

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