Proliferation Of Human Umbilical Vein Research Articles

Adhesion, proliferation and viability of human umbilical vein endothelial cells cultured on the surface of biodegradable non-woven matrices modified with RGD peptides

Tissue engineering is a promising area for the production of small-diameter vascular grafts. In recent years, a number of strategies have been developed to make the polymer surfaces of vascular prostheses capable to selectively adhesion of endothelial cells. The arginine–glycine–aspartic acid (RGD) sequence (a cell adhesion site that is present on many extracellular matrix proteins) is the promising target for modification. The efficiency of attachment of endothelial cells can be influenced both by the structure of RGD peptide and the extent of linker group.Aim: to determine the optimal method for modification of non-woven matrices of polyhydroxybutyrate/ valerate and polycaprolactone (PHBV/PCL) by RGD-peptides leading to the increasing of adhesion, viability and proliferation of endothelial cells.Materials and methods. Electrospinning was used to produce 4 mm diameter tubular polymer matrices from PHBV/PCL. Modification of surface of polymer scaffolds was performed using 4,7,10-trioxa-1,13-tridekandiamin, hexamethylenediamine, glutaraldehyde, ninhydrin, ascorbic acid, a cyclic peptide c [RGDFK], RGDK, AhRGD. The quality of modification was assessed by ninhydrin test and determination of arginine-containing peptide. The structure of the surface of matrices before and after modification was studied by scanning electron microscopy. Adhesion, viability and proliferation of Human umbilical vein (HUVEC) endothelial cells cultured for 7 days on the surface of matrices in the presence of RGD and without one were examined using fluorescence and laser scanning microscopy after the cells were pre-stained with fluorescent nuclear dyes (ethidium bromide and Hoechst 33342), and also by special kits for proliferation assessment (Click-iTTM Plus EdU Alexa FluorTM 488 Imaging Kit).Results. RGD peptides bound to the matrix surface via a long linker (4,7,10-trioxa-1,13-tridecanediamine) were characterized by the increased bioavailability and activity. High level of cell adhesion, viability and proliferation were noted on the surface of RGDK and c[RGDFK] modified matrices, whereas their paired analogues with a short linker (hexamethylenediamine) showed low results of cellular viability even against satisfactory cell adhesion.Discussion. Non-woven matrices based on PHBV/PCL and modified using 4,7,10-trioxa-1,13-tridecanediamine showed better results in case of adhesion of HUVEC and subsequent preservation of cell viability and proliferation. RGD-containing peptides of RGDK and c [RGDFK] were more tropic to endothelial cell receptors.

Activation of Aryl Hydrocarbon Receptor Inhibits Proliferation of Human Umbilical Vein Endothelial Cells.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor which mediates a variety of biological processes including reproduction and in particular normal placentation. The AhR has been extensively studied as a receptor for environmental toxicants such as dioxin. Upon binding to these toxicants, the AhR is activated and could induce adverse developmental effects on many tissues including fetal cardiovasculature. Recently, normal physiological roles of AhR have begun to be recognized, particularly after the discovery of endogenous AhR ligands such as 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), which has a much lower toxicity as compared with the extreme deleterious effects of dioxin. However, little is known about AhR actions in endothelial cells and their underlying signaling mechanisms. Herein, we examined if ITE activated AhR and affected proliferation of human umbilical vein (HUV) endothelial (HUVE) cells in response to fetal bovine serum. The expression of AhR in HUV and HUVE cells was evaluated by immunohistochemistry and Western blot analysis, respectively. Cell proliferation was determined in vitro using the crystal violet cell proliferation assay. AhR was immunolocalized in endothelial cells of HUV and was expressed in HUVE cells in vitro. We observed that ITE dose-dependently (0.1 nM-5 μM) and time-dependently (2-6 days) inhibits (P < 0.05) HUVE cells proliferation. ITE also reduced (P < 0.05) AhR protein levels in a time-dependent manner, with an initial decrease seen at 6 hr and completely vanished after 48 hr, indicating that ITE induced robust AhR activation. AhR plays an important role in mediating HUVE cells growth and may account for the potential deleterious effects of environmental toxicants on embryonic/fetal vasculature and thus fetal development. (HL64703 to JZ, HD38843 to RRM and JZ, HL49210 to RRM) (poster)

High-yield expression of human vascular endothelial growth factor VEGF 165 in Escherichia coli and purification for therapeutic applications

Vascular endothelial growth factor (VEGF 165) is a potent mitogen that induces angiogenesis and vascular permeability in vivo and has demonstrated potential in therapeutic applications for accelerating wound healing. An industrial production method that provides high yield as well as high purity, quality, and potency is needed. The process described in this report involves a bacterial expression system capable of producing approximately 9 g of rhVEGF per liter of broth and a downstream purification process consisting of protein refolding and three chromatography steps prior to formulation of the drug substance. A high cell density (HCD) fed-batch fermentation process was used to produce rhVEGF in periplasmic inclusion bodies. The inclusion bodies are harvested from the cell lysate and subjected to a single-step protein solubilization and refolding operation to extract the rhVEGF for purification. Overall recovery yields observed during development, including refolding and chromatography, were 30 ± 6%. Host cell impurities are consistently cleared below target levels at both laboratory and large-scale demonstrating process robustness. The structure of the refolded and purified rhVEGF was confirmed by mass spectrometry, N-terminal sequencing, and tryptic peptide mapping while product variants were analyzed by multiple HPLC assays. Biological activity was verified by the proliferation of human umbilical vein derived endothelial cells.

Expression and characterization of human vascular endothelial growth factor (VEGF 165) in insect cells

Vascular endothelial growth factor (VEGF) is the best characterized multifunctional protein which plays a key role in normal and pathologic angiogenesis. The gene encoding the human VEGF 165 was cloned from the ovarian carcinoma cell line (OVCAR3) and expressed in insect cells using the baculovirus expression vector system. The recombinant human VEGF 165 (rhVEGF 165) protein produced by Sf21 ( Spodoptera frugiperda) cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide-linked dimer and secretion into the media. The rhVEGF 165 had a high affinity for heparin and this characteristic was used to purify this form to homogeneity by heparin affinity, Resource S and Resource RPC columns. The biological activity of the purified 42-kDa homodimer was shown by the induction of the proliferation of human umbilical vein derived endothelial cells. These results demonstrate that an angiogenic growth factor whose normal processing requires glycosylation and disulfide-bridge formation can be efficiently expressed in high concentration (up to 20 mg/L) in Sf21 cells.

Effects of C5a and FMLP on interleukin-8 production and proliferation of human umbilical vein endothelial cells.

Interleukin-8 (IL-8), C5a and N-formyl-methionyl-leucyl-phenylalanine (fMLP) are chemotactic peptides with predominant effects on leukocytes during inflammation. With emphasis on C5a we studied the regulation of the production of IL-8 by human umbilical vein endothelial cells (HUVEC) in vitro. Primary HUVEC cultures were incubated with IL-1alpha, TNFalpha, C5a and fMLP for 24 h and 48 h prior to measurement of IL-8 in supernatants of the cells by an enzyme immunoassay. Whereas IL-1alpha and TNFalpha significantly increased the levels of IL-8, C5a decreased the IL-8 production after 48 h. In addition, the ability of IL-1alpha, TNFalpha, C5a, fMLP and IL-8 to induce cell proliferation was compared by means of a 3H-thymidine incorporation assay. In contrast with IL-1alpha and TNFalpha, both C5a and fMLP increased cell proliferation of HUVEC. This increase occurred with increasing concentrations of C5a contrary to IL-8, which showed increased cell proliferation at low, but not high IL-8 concentrations.

Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5.

We previously reported that the binding of two-chain high molecular weight kininogen (HKa) to endothelial cells may occur through interactions with endothelial urokinase receptors. Since the binding of urokinase to urokinase receptors activates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding on endothelial cell proliferation. Unexpectedly, HKa inhibited proliferation in response to several growth factors, with 50% inhibition caused by approximately 10 nM HKa. This activity was Zn(2+) dependent and not shared by either single-chain high molecular weight kininogen (HK) or low molecular weight kininogen. HKa selectively inhibited the proliferation of human umbilical vein and dermal microvascular endothelial cells, but did not affect that of umbilical vein or human aortic smooth muscle cells, trophoblasts, fibroblasts, or carcinoma cells. Inhibition of endothelial proliferation by HKa was associated with endothelial cell apoptosis and unaffected by antibodies that block the binding of HK or HKa to any of their known endothelial receptors. Recombinant HK domain 5 displayed activity similar to that of HKa. In vivo, HKa inhibited neovascularization of subcutaneously implanted Matrigel plugs, as well as rat corneal angiogenesis. These results demonstrate that HKa is a novel inhibitor of angiogenesis, whose activity is dependent on the unique conformation of the two-chain molecule.

Vascular endothelial growth factor is efficiently synthesized in spite of low transfection efficiency of pSG5VEGF plasmids in vascular smooth muscle cells.

The limitation of lipotransfection with plasmid vectors is its low efficiency and the short-term expression of introduced genes. This is particularly important when the synthesis of high amounts of therapeutic products is required. However, growth factors with paracrine action overcome this problem. The aim of our study was to check whether the amounts of vascular endothelial growth factor (VEGF) generated after plasmid lipotransfection into vascular smooth muscle cells (VSMC) can be sufficient to stimulate endothelial cell proliferation. Two plasmids, pSG5-VEGF121 and pSG5-VEGF165, harboring human VEGF121 and VEGF165 isoforms were constructed and lipotransfected into COS-7 cells or to rat VSMC. The transfection efficiency, estimated by the expression of control, beta-galactosidase gene, was about 50% in COS-7 but rarely exceeded 5% in VSMC. However, despite this, the smooth muscle cells generated high amounts of VEGF protein, up to 3 ng/ml medium. The biological activity of this VEGF was confirmed by enhanced proliferation of human umbilical vein and coronary artery endothelial cells, stimulated with conditioned media of pSG5-VEGF transfected cells. Thus, the low transfection efficiency does not preclude the generation of high amounts of VEGF by VSMC. After reaching the maximum at about 48 h after transfection, the generation of VEGF decreased in the following days. Such a situation may be sufficient for the gene therapy of restenosis when the long-term expression of therapeutic gene(s) is not necessary. Thus, we suggest that the pSG5-VEGF121, and pSG5-VEGF165 plasmids can be used for therapeutic application.

High levels of biologically active vascular endothelial growth factor (VEGF) are produced by the baculovirus expression system.

Vascular endothelial growth factor (VEGF) is a recently discovered mitogen for endothelial cells in vitro, and a potent angiogenesis promoting factor in vivo. VEGF is secreted from producing cells as a homodimer, binds to specific receptors on the cell surface of endothelial cells, and is produced in four forms as a result of alternative splicing. We have expressed the cDNA encoding the 165 amino-acid long isoform of VEGF in insect cells using the baculovirus based expression vector. We show that infected insect cells secrete large amounts of VEGF. Antibodies directed against a synthetic peptide prepared from human VEGF identify the secreted factor. The baculovirus derived VEGF expressed in insect cells (inVEGF) binds directly to the VEGF receptors inVEGF competes with pure mammalian cells derived [125I]-VEGF for binding to the VEGF receptors that are present on the cell surface of endothelial cells. Furthermore, inVEGF is biologically active and induces the proliferation of human umbilical vein derived endothelial cells.