Stimulation of human vascular endothelial cell growth by a platelet-derived growth factor and thrombin

Repair of a vascular wound is mediated by migration and subsequent replication of the endothelial cells that form the inner lining of blood vessels. We have measured the growth response of human umbilical vein endothelial cells (HuE) to two polypeptides that are transiently produced in high concentrations at the site of a wound; the platelet-derived growth factor (PDGF) and the protease thrombin. When 10(4) HuE cells are seeded as a dense island (2-mm diameter) in the center of a 16-mm tissue culture well in medium containing 20% human serum derived from platelet-poor plasma (PDS), no increase in cell number or colony size is observed. With the addition of 0.5 ng/ml partially purified PDGF, colony size increases and the number of cells after 8 days is 4.8 X 10(4). When human thrombin (1 microgram/ml) is added along with the PDGF, the cell number rises to 9.2 X 10(4). Thrombin alone stimulates no increase in cell number. Although partially purified PDGF stimulates endothelial cells maintained in PDS as well as those maintained in whole blood serum (WBS), pure PDGF is active only when assayed in medium that contains WBS and is supplemented with thrombin. These results suggest the existence of a second class of platelet-derived factors that enable HuE cells to respond to the mitogenic activity of the purified platelet mitogen and thrombin.     

Size effect in molecular imaging of vascular endothelial growth factor

Visualizing and quantifying vascular endothelial growth factor (VEGF) levels are crucial factors in understanding the tumor environment and monitoring antiangiogenic treatment. The small peptidic probes described by Fedorova et?al. in this issue appear to be superior to antibodies in reflecting the dynamics of VEGF as they facilitate image quantification.     

Total chemical synthesis of biologically active vascular endothelial growth factor

Efficient access: the 204-residue covalent-dimer vascular endothelial growth factor with full mitogenic activity was prepared from three unprotected peptide segments by one-pot native chemical ligations. The covalent structure of the synthetic VEGF was confirmed by precise mass measurement, and the three-dimensional structure of the synthetic protein was determined by high-resolution X-ray crystallography.     

Activity ranking of synthetic analogs targeting vascular endothelial growth factor receptor 2 by an integrated cell membrane chromatography system

Evaluating the biological activities of small molecules represents an important part of the drug discovery process. Cell membrane chromatography (CMC) is a well‐developed biological chromatographic technique. In this study, we have developed combined SMMC‐7721/CMC and HepG2/CMC with high‐performance liquid chromatography and time‐of‐flight mass spectrometry to establish an integrated screening platform. These systems was subsequently validated and used for evaluating the activity of quinazoline compounds, which were designed and synthesized to target vascular endothelial growth factor receptor 2. The inhibitory activities of these compounds towards this receptor were also tested using a classical caliper mobility shift assay. The results revealed a significant correlation between these two methods (R² = 0.9565 or 0.9420) for evaluating the activities of these compounds. Compared with traditional methods of evaluating the activities analogous compounds, this integrated cell membrane chromatography screening system took less time and was more cost effective, indicating that it could be used as a practical method in drug discovery.     

Effect of laser modified surface microtopochemistry on endothelial cell growth

The introduction of microelectronics technology in the area of biological sciences has brought forth previously unforeseeable applications such as DNA or protein biochips, miniaturized, multiparametric biosensors for high performance multianalyte assays, DNA sequencing, biocomputers, and substrates for controlled cell growth (i.e. tissue engineering). We developed and investigated a new method using “cold” excimer laser beam technology combined with microlithographical techniques to create surfaces with well defined 3D microdomains in order to delineate critical microscopic surface features governing cell–material interactions. Microfabricated surfaces with microgrooves 30–3 μm deep, 10–1 μm wide spaced 30 μm apart were obtained with micron resolution, by “microsculpturing” polymer model surfaces using a computer controlled laser KrF excimer beam coupled with a microlithographic projection technique. The laser beam after exiting a mask was focused onto the polymer target surface via an optical setup allowing for a 10-fold reduction of the mask pattern. Various 3D micropatterned features were obtained at the micron level. Reproducible submicron features could also be obtained using this method. Subsequently, model human umbilical endothelial cells (HUVEC) were cultured on the laser microfabricated surfaces in order to study the effects of specific microscopic surface features on cell deposition and orientation. Cell deposition patterns were found to be microstructure dependant, and showed cell orientation dependency for features in the cell range dimension, a behaviour significantly different from that of a previously studied cell model (osteoprogenitor cell). This model may be a promising in so far as it is very rapid (a time frame less than a second per square centimeter of micropatterned surface) and provides further insights into the effects of surface microtopography on cell response with possible applications in the field of biosensors, biomedical and/or pharmaceutical engineering sciences.     

Photochemically modulated endothelial cell thrombogenicity via the thrombomodulin-tissue factor pathways

Photodynamic therapy (PDT) is based on a photochemical reaction using a photosensitizer and light to produce reactive oxygen species that have biological effects. Although its application in some fields is largely based on thrombosis, in the vascular setting thrombosis must be prevented. In this study we examined the effects of PDT on the changes in activity of thrombomodulin (TM) and tissue factor (TF) as important regulators of the coagulation process of endothelial cells. Human umbilical vein endothelial cells were treated with PDT (chloro-aluminum-sulfonated phthalocyanine, lambda = 630 nm) at different light-energy doses, and TM and TF levels were measured using fluorescence spectroscopy. Microparticles (MP) were analyzed using flow cytometry analysis. PDT alters the thrombogenic state of endothelial cells by causing decreased expression of TM and increased expression of functional TF in a light-energy dose-dependent way. PDT-treated endothelial cells shed large numbers of MP containing high levels of TF. TF functionality of PDT-treated cells, measured by a Factor Xa-generating assay, was high. TF was located mostly intracellularly and in MP. The disturbed anticoagulant balance described in this study may explain the occurrence of thrombosis induced by PDT and, if not contained, dispute the suitability of PDT as an adjuvant modality to treat vascular restenosis.     

Effects of vascular endothelial growth factor (VEGF) and chondroitin sulfate A on human monocytic THP-1 cell migration

Angiogenesis serves as a crucial factor in disease development and progression, such as cancer metastasis, and monocyte migration is one of the key steps for angiogenesis. Therapeutic modulation of angiogenesis is a promising new therapeutic avenue under investigation. In this study, effects of vascular endothelial growth factor (VEGF) and chondroitin sulfate A on monocyte migration were investigated. Human monocytic THP-1 cells were from Riken Cell Bank (Tsukuba, Japan) and vascular endothelial cells (VECs) were obtained from swine thoracic aorta. The migration experimental system was adapted from Falcon™ Cell Culture Inserts with pore sizes of 3 and 8 μm cultured endothelial cells or not on the insert polyethylene terephthalate (PET) membranes. Four VEGF concentrations (0, 10, 50 and 100 ng/ml) and three concentrations of chondroitin sulfate A (0, 1.25 and 5.0 mg/ml) were used to investigate their effects on THP-1 cell migration ability through PET membranes and VECs monolayer. The THP-1 cell migration was evaluated by counting the number of migrated cells related to the total number of cells under a microscope. We counted the migration cells every 1 h on a Tatai-type hemocytometer using an inverted microscope for total 7 h. For inserts with pore sizes of 3 and 8 μm, the THP-1 cell migration increased with VEGF concentrations; however, cell migration decreased with the chondroitin sulfate A concentration. Our results demonstrated that VEGF accelerated monocyte migration through endothelial monolayer and chondroitin sulfate A is an effective inhibitor of monocyte migration for angiogenesis.     

Upconversion luminescence assay for the detection of the vascular endothelial growth factor,a biomarker for breast cancer

Upconversion nanoparticles (UCNPs) of the type α-NaYF₄:Yb³⁺, Er³⁺ and a typical diameter of 6–7 nm were synthesized by thermal decomposition of the respective rare-earth stearate. The oleic acid on the surface of the UCNPs was then replaced by aptamer DNA. The assay was performed in a microplate format with a capture probe immobilized in the wells. Following binding of the vascular endothelial growth factor (VEGF), an auxiliary probe DNA is added that is labeled with UCNPs and binds to the VEGF-loaded capture probe. The method enables highly sensitive and highly specific detection of the VEGF which is a marker for breast cancer. Under the optimum conditions, the intensity of the upconversion luminescence (at excitation/emission wavelengths of 980/541 nm) is linearly proportional to the VEGF concentration in the 50 pM to 2000 pM concentration range, with a 6 pM detection limit. The method was applied to the determination of VEGF in spiked serum, typically at a 500 pM level, and gave recoveries that ranged from 98 to 113 %, with RSDs between 2.9 and 3.6 %. This makes it a viable tool for early diagnosis of breast cancer.

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Graphical abstract An upconversion luminescence based assay is presented for trace analysis of vascular endothelial growth factor. It is based on a sequence fragment-linked technique of target-induced aptamer (AP) and upconversion nanoparticles (UCNPs) and has potential application in the early diagnosis of breast cancer.

     

Angiotensin II receptor blocker attenuates overexpression of vascular endothelial growth factor in diabetic podocytes

VEGF expressed in glomerular podocytes, is known to increase vascular permeability to macromolecules. Angiotensin II can stimulate the release of VEGF, and the protective effects of angiotensin II antagonist against diabetic glomerular injury suggest that the angiotensin II-induced VEGF is an important pathogenetic mechanism in the development of proteinuria during diabetic nephropathy although this mechanism is not fully understood. In this study, the changes of VEGF expression was examined in the experimental diabetic nephropathy to determine whether these changes were modified by renoprotective intervention by blockers of angiotensin II receptors. The streptozotocin- induced diabetic rats were treated with L-158,809, a blocker of angiotensin II receptors, for 12 weeks. Age-matched rats with L-158,809 served as controls. RT-PCR and immunohistochemistry were used to assess and quantify gene and protein expression of VEGF. A progressive increase in urinary protein excretion was observed in diabetic rats. Glomerular VEGF expression was significantly higher in diabetic rats than in the control groups, with a significant reduction in glomerular VEGF expression and proteinuria in L-158,809- treated diabetic rats. VEGF mRNA was also significantly higher in diabetic kidneys than in the control groups, with a significant reduction in VEGF mRNA in L-158,809-treated diabetic kidneys. These results demonstrates that VEGF expression is significantly increased in diabetic podocytes, and angiotensin II receptor antagonist attenuated these changes in VEGF expression and prevented the development of proteinuria in vivo. Attenuation of increased VEGF expression in podocytes could contribute to the renoprotective effects of angiotensin II receptor antagonists in diabetic nephropathy.     

The expression patterns of vascular endothelial growth factor and thrombospondin 2 after corneal alkali burn

The aim of the investigation was to explore the expression patterns of VEGF and TSP2 after corneal alkali burn in vivo. After the model of corneal alkali burn was established in mice, the expression levels of VEGF and TSP2 were determined by immunohistochemistry (IHC), RT-PCR, image analysis and statistical evaluation. Compared with control group, the expression level of VEGF increased significantly at 6h after alkali burn and reached its maximum at 12h. Then, it increased again till the second peak appeared at 96h and 192h. The VEGF-positive reaction mainly gathered in the stroma of cornea. On the other hand, the expression of TSP2 enhanced at 3h and attained two peaks at 6h and 96h, respectively, with the process of wound healing. TSP2 was expressed mainly in the base of epithelial layer. The expression patterns of VEGF and TSP2 reflect the complicated interaction with many factors including promoted and inhibited vascularization in vivo. Moreover, it might provide a novel method for controlling vascular hyperplasia in future clinical work according to the data of VEGF and TSP2.     

Determination of cell type‐specific proteome signatures of primary human leukocytes,endothelial cells,keratinocytes, hepatocytes,fibroblasts and melanocytes by comparative proteome profiling

Cells gain their functional specialization by different protein synthesis. A lot of knowledge with respect to cell type‐specific proteins has been collected during the last thirty years. This knowledge was built mainly by using antibodies. Nowadays, modern MS, which supports comprehensive proteome analyses of biological samples, may render possible the search for cell type‐specific proteins as well. However, a therefore necessary systematic MS study comprising many different cell types has not been performed until now. Here we present a proteome analysis strategy supporting the automated and meaningful comparison of any biological samples. We have presently applied this strategy to six different primary human cell types, namely leukocytes, endothelial cells, keratinocytes, hepatocytes, fibroblasts, and melanocytes. Comparative analysis of the resulting proteome profiles allowed us to select proteins specifically identified in one of the six cell types and not in any of the five others. Based on these results, we designated cell type‐specific proteome signatures consisting each of six such characteristic proteins. These signatures independently reproduced well‐known marker proteins already established for FACS analyses in addition to novel candidate marker proteins. We applied these signatures for the interpretation of proteome profiles obtained from the analyses of hepatocellular carcinoma‐associated tissue homogenates and normal liver tissue homogenates. The identification of members of the above described signatures gave us an indication of the presence of characteristic cells in the diseased tissues and thus supported the interpretation of the proteomics data of these complex biological samples.     

Biomedical and clinical applications of automated single cell electrophoresis

The automated single cell electrophoresis microscope Parmoquant has been used for the discrimination of lymphocytes and the study of the interaction of substances with cells and synthetic particles. Electrophoretic histograms allowed the determination of changes in the proportion of lymphocyte populations after kidney transplantation, during dialysis treatment, open heart surgery and during pregnancy. Discrimination of leukemic cells on the basis of electrophoresis was used as an additional parameter in diagnosis. In a mouse tumor model, histogram determination enabled the in vivo effect of the tumor necrosis factor on immune cells to be evaluated. Cell electrophoresis was shown to be suitable to detect the influence of antibodies, lectins and bacteria on the cell surface. Protein adsorption was studied on synthetic particles using cell electrophoresis. This method was applied to investigate the phenomena of blood interaction with biomaterials used in artificial organs and to determine differences in the protein composition of serum or other body fluids connected with diseases.     

Electrochemical control of growth factor presentation to steer neural stem cell differentiation

Let it grow: The conjugated polymer poly(3,4-ethylenedioxythiophene) (PEDOT) was synthesized with heparin as the counterion to form a cell culture substrate. The surface of PEDOT:heparin in the neutral state associated biologically active growth factors. Electrochemical in?situ oxidation of PEDOT during live cell culture decreased the bioavailability of the growth factor and created an exact onset of neural stem cell differentiation.     

Development of CE methods to analyze potential components of the angiogenic glycoprotein vascular endothelial growth factor 165

The vascular endothelial growth factor 165 (VEGF₁₆₅) is the predominant form of the complex VEGF family. This glycoprotein has, among others, an angiogenic effect in many physiological and pathological events. For this reason, its roles as a biomarker and as a therapeutic drug have been considered. However, very little is known about the existence of different forms of VEGF₁₆₅ arising from glycosylation and other potential PTMs. This aspect is crucial because it is known that for other glycoproteins the ratio between these isoforms actually acts as a biomarker for certain diseases and other physiological states. In addition, for therapeutic use of glycoproteins it is known that the biological activity may differ for the various isoforms. In this work CE methods to separate up to seven peaks without baseline resolution containing various forms of VEGF₁₆₅ are developed. Using a computer program previously developed in‐house peak assignment could be performed with accuracy close to 100%. In this way, comparison between recombinant human VEGF₁₆₅ expressed in insect cells, which is a glycosylating system, and in Escherichia coli cells, which are unable of performing glycosylation of proteins, has been possible. The methods developed, besides providing information about the existence of several forms of VEGF₁₆₅, mean a starting point that permits the study of the role of VEGF₁₆₅ as a potential biomarker of different diseases and physiological processes and to perform quality control of the recombinant drug during manufacturing. To the best of our knowledge this is the first time that CE methods for VEGF₁₆₅ have been developed.     

Low-level laser therapy promotes vascular endothelial growth factor receptor-1 expression in endothelial and nonendothelial cells of mice gastrocnemius exposed to snake venom

Crotalinae snake venoms cause severe local myonecrosis and microvasculature failure at the bite site. We evaluated whether low-level laser therapy (LLLT) could accelerate angiogenesis and myoregeneration in male Swiss mice injected with Bothrops moojeni venom through immunohistochemistry of the vascular endothelial growth factor receptor-1 (VEGFR-1). Envenomed gastrocnemius was either unirradiated (V) or irradiated with HeNe (VHN, 632.8 nm) or GaAs (VGA, 904 nm, 10000 Hz). Animals sacrificed at 3 and 12 h were irradiated once (4 J cm(-2)), at 24 h (twice) and at 3, 7, 21 days (4, 8, 22 times, respectively). At 3 days, LLLT increased angiogenesis (80%:HeNe vs 40%:GaAs), decreased neutrophils and increased proliferation of regenerating cells. However, after 21 days, myoregeneration observed in the VHN group appeared delayed compared with the V group. As LLLT improved revascularization, the suggestive delay in myoregeneration could be a dose-response inhibitory effect caused by multiple irradiations in myogenesis. The immunodetection of VEGFR-1 in neutrophils, macrophages, satellite cells, fibroblasts, Schwann cells and skeletal and smooth muscle fibers (not seen in saline-controls) at only the acute stages of envenoming suggests a mediator role for VEGFR-1 in local alterations. This is the first time that VEGFR-1 expression, and its modulation by photostimulation, has been demonstrated in endothelial and nonendothelial cells of snake envenomed skeletal muscle.     

Combination of integrin-binding peptide and growth factor promotes cell adhesion on electron-beam-fabricated patterns

Understanding and controlling cell adhesion on engineered scaffolds is important in biomaterials and tissue engineering. In this report we used an electron-beam (e-beam) lithography technique to fabricate patterns of a cell adhesive integrin ligand combined with a growth factor. Specifically, micron-sized poly(ethylene glycol) (PEG) hydrogels with aminooxy- and styrene sulfonate-functional groups were fabricated. Cell adhesion moieties were introduced using a ketone-functionalized arginine-glycine-aspartic acid (RGD) peptide to modify the O-hydroxylamines by oxime bond formation. Basic fibroblast growth factor (bFGF) was immobilized by electrostatic interaction with the sulfonate groups. Human umbilical vein endothelial cells (HUVECs) formed focal adhesion complexes on RGD- and RGD and bFGF-immobilized patterns as shown by immunostaining of vinculin and actin. In the presence of both bFGF and RGD, cell areas were larger. The data demonstrate confinement of cellular focal adhesions to chemically and physically well-controlled microenvironments created by a combination of e-beam lithography and "click" chemistry techniques. The results also suggest positive implications for addition of growth factors into adhesive patterns for cell-material interactions.