Syndecan-4 regulates the bFGF-induced chemotactic migration of endothelial cells
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Chemotaxis assay
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Chemotaxis assay
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Purpose To explore the role of green tea on basic fibroblast growth factor (bFGF) in human breast cancer cells and human umbilical vein endothelial cells (HUVECs) and its mechanisms. Methods bFGF concentrations were determined by ELISA. Northern blot hybridization was performed to detect the expression of mRNA. Results 40 μg/mL GTE or EGCG could decrease the levels of bFGF peptide secreted into conditioned media as well as the bFGF peptide levels in both HUVECs and human breast cancer cells; this effect was dose dependent. 40 μg/mL GTE and EGCG decreased the mRNA levels of bFGF in MDA-MB231 cells. Conclusions Green tea can inhibit bFGF expression in human breast cancer cells and human umbilical vein endothelial cells through multiple levels. This may be the partial mechanism for green tea to inhibit angiogenesis in cancers.
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Matrigel matrix is widely used for supporting tumour implantation. However, some tumour cells are unable to degrade matrigel matrix resulting in residual matrigel at the time point of the biodistribution study or therapy experiment. In vitro cell uptake of tumour affine compounds into tumour cells embedded in matrigel was compared with matrigel free tumour cells. Matrigel accumulation exceeded cellular uptake of the tumour affine peptides. This suggests that matrigel might have an influence on the acquired biodistribution data when it is still present at the time point of the study. A quantitation of residual matrigel in tumour explants fourteen days after tumour implantation with a matrigel-tumour cell mixture showed that the overall matrigel content in the case of MCF-7 and AR42J tumours was about 23%. In order to evaluate the extent of accumulation of compounds in matrigel, nude mice bearing either tumours, tumour-matrigel-mixtures or matrigel alone received intravenous injections of fluorophor tagged tumour specific peptides. Fluorescence microscopy of cryosectioned matrigel, matrigel-tumour mixtures and tumour explants showed that the labelled compounds were matrigel associated and tumour cell associated with a higher fluorescence intensity in matrigel. In summary, matrigel matrix can influence biodistribution studies. It leads to believe in a higher tumour accumulation. Therefore, either a number of control experiments or the separation of matrigel from the tumour is necessary in order to obtain correct biodistribution data.
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Infrared imaging was applied to investigate a reconstituted basement membrane, known as Matrigel, in three-dimensional cell cultures. Matrigel, in the vicinity of the colonies, was examined for four breast cancer cell lines presenting different 3D colony morphologies. MCF-7 and T-47D present mass colonies, SKBR-3 grape-like colonies and MDA-MB-231 stellate colonies associated with a more invasive phenotype. The edge of the cell colonies was found to be significantly depleted in Matrigel. Except in a limited number of cases, Matrigel appeared to be thinner at the edges of the colonies but not completely destroyed or torn off as it would be for a purely mechanical effect. When a PCA was run on the spectra of one or several colonies, the score images on PC#3 and PC#4 presented structures in the Matrigel areas which appeared as fringes, lines, dots or regular patterns. This effect represents a very small fraction of the total variance but is reproducible for all the 4 cell lines. PC#4 presents systematically a maximum near 1624 cm(-1) and a minimum around 1700 cm(-1). When spectra are normalized, the effect is less marked but does not disappear. The nature of the variations that exist in the Matrigel layer is therefore not solely related to thickness but also to the chemical composition. At this stage, the weakness of the effect prevents a thorough investigation.
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Different angiogenic assays in vitro have helped to define various events underlying angiogenesis. In this report we have compared the phenotypic modifications of human umbilical vein endothelial cells (HUVE cells) and human dermal fibroblasts using Matrigel and collagen gels. Both HUVE cells and human dermal fibroblasts form a network of anastomosing cords that apparently resemble blood capillaries when grown on Matrigel. The whole network was formed by several cellular aggregates joined to each other by cellular cords. Lumen formation was not observed in this angiogenic system. In opposite, considerable differences between HUVE cells and human dermal fibroblasts were observed in the three-dimensional angiogenic assay on collagen gels described by Montesano et al [14]. These results indicate that data obtained with angiogenic systems using Matrigel must be interpreted with caution and that the assay described by Montesano et al [14], is more reliable to describe angiogenesis.
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AIM: To investigate the effect of basic fibroblast growth factor (bFGF) on C-type natriuretic peptide (CNP) production, release and mRNA expression. METHODS: Human endothelial cell cultured; CNP was measured by radioimmunoassay method;CNP mRNA expression was determined by RT-PCR technique. RESULTS: bFGF could augment CNP synthesis in human endothelial cells. Compared with control group,25 ng, 50 ng, 100 ng bFGF increased CNP contents in endothelial cells by 88% (P0.05), 95% (P0.05), 187% (P0.01), respectively.100 ng bFGF also stimulated CNP release from cultured human endothelial cell. In addition, 25 ng, 50 ng and 100 ng bFGF stimulated CNP mRNA expression of cultured human endothelial cells in a dose-dependent manner. CONCLUSION: bFGF might regulate CNP synthesis,release and mRNA expression in cultured umbilical human endothelial cells.
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Senescent cells can spread the senescent phenotype to other cells by secreting senescence-associated secretory phenotype factors. The resulting paracrine senescent cells make a significant contribution to the burden of senescent cell accumulation with age. Previous efforts made to characterize paracrine senescence are unreliable due to analyses being based on mixed populations of senescent and non-senescent cells. Here, we use dipeptidyl peptidase-4 (DPP4) as a surface maker to isolate senescent cells from mixed populations. Using this technique, we enrich the percentage of paracrine senescence from 40% to 85%. We then use this enriched culture to characterize DPP4+ primary and paracrine senescent cells. We observe ferroptosis dysregulation and ferrous iron accumulation as a common phenomenon in both primary and paracrine senescent cells. Finally, we identify ferroptosis induction and ferrous iron-activatable prodrug as a broad-spectrum senolytic approach to ablate multiple types of primary and paracrine senescent cells.
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Vascular endothelial growth factor (VEGF) has an ability to induce the migration of human umbilical vein endothelial cells (HUVEC). The objective of this study is to prepare several patterns of gelatin hydrogels for VEGF release and evaluate the 3-dimensional pattern of HUVEC migration in Matrigel by VEGF release. VEGF was incorporated into the gelatin hydrogel sheet to achieve the sustained release and generate the concentration gradient of VEGF. When Matrigel was put on the gelatin hydrogel sheet incorporating VEGF, the VEGF was released into the Matrigel to form a gradient pattern of VEGF concentration in the Matrigel with time and the area of VEGF released by the Matrigel depended upon the position of gelatin hydrogel sheet put on. In addition, HUVEC were seeded on the surface of Matrigel to evaluate the ability of VEGF released to enhance the cell migration into the Matrigel. HUVEC were migrated with time into the Matrigel to the direction and the position of VEGF released. It is concluded that the VEGF release induces the migration of HUVEC in Matrigel based on the concentration gradient and the position of VEGF formed in Matrigel.
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De novo adipogenesis at the implanted site of a basement membrane extract (Matrigel) was induced through controlled release of basic fibroblast growth factor (bFGF). bFGF was incorporated into biodegradable gelatin microspheres for its controlled release. When the mixture of Matrigel and bFGF-incorporated gelatin microspheres was implanted subcutaneously into the back of mice, a clearly visible fat pad was formed at the implanted site 6 weeks later. Histologic examination revealed that the de novo formation of adipose tissue accompanied with angiogenesis was observed in the implanted Matrigel at bFGF doses of 0.01, 0.1, and 1 microg/site, the lower and higher doses being less effective. The de novo formation induced by the bFGF-incorporated microspheres was significantly higher than that induced by free bFGF of the same dose. The mRNA of a lipogenesis marker protein, glycerophosphate dehydrogenase, was detected in the formed adipose tissues, biochemically indicating de novo adipogenesis. Free bFGF, the bFGF-incorporated gelatin microspheres, or Marigel alone and bFGF-free gelatin microspheres with or without Matrigel did not induce formation of adipose tissue. This de novo adipogenesis by mixture of Matrigel and the bFGF-incorporated gelatin microspheres will provide a new idea for tissue engineering of adipose tissue.
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