A stimulatory effect of Artemisia leaf extract on the proliferation of cultured endothelial cells

To investigate the effect of the hot water extract from Artemisia leaf (Artemisia princeps Panpanini) (AFE) on the proliferation of endothelial cells, the cells from bovine aorta were cultured for up to 96 h in the presence of 1, 5, 10 or 50 micrograms/ml AFE in RPMI1640 medium supplemented with 10% fetal bovine serum. After a 72 h culture, the cell number was significantly increased by AFE at 1, 5 and 10 micrograms/ml. An increase in the cell number by 5 micrograms/ml AFE observed after a 72 or 96 h treatment. The incorporations of both [3H]thymidine and [14C]leucine by the growing cells were significantly increased by 5 micrograms/ml AFE after a 72 h treatment. In addition, the incorporation of [3H]thymidine by either growing or confluent cells was significantly increased by 50 micrograms/ml AFE after a 72 h treatment. The stimulatory activity of AFE was recognized in the low-molecular-weight fraction (molecular weight less than or equal to 10000 dalton). These results clearly indicated that AFE contained some low-molecular-weight component(s) which stimulates the proliferation of vascular endothelial cells in vitro.     

Gardenia fruit extract does not stimulate the proliferation of cultured vascular smooth muscle cells, A10

We investigated the effect of a hot water extract from Gardenia fruit (Gardenia jasminoides Ellis) (GFE), which has a stimulatory effect on endothelial cell proliferation, on the proliferation of A10 cells, an established cell line of vascular smooth muscle cell from murine aorta in a culture system. GFE did not change the number of A10 cells after a 48 h culture. GFE significantly increased the incorporation of [3H]thymidine and [14C]leucine into the acid-soluble fraction of bovine aortic endothelial cell layers, but significantly decreased that of A10 cells. These results suggested that GFE stimulates the proliferation of endothelial cells but not of A10 cells. In the endothelial cell culture, GFE significantly increased the accumulation of basic fibroblast growth factor, which is an autocrine for endothelial cell proliferation in medium and low-affinity (glycosaminoglycans-binding) fractions, while A10 cells did not produce a significant amount of the factor. Since it is postulated that a selective stimulation of endothelial cell proliferation by increasing the production of basic fibroblast growth factor is appropriate for prevention of arteriosclerosis and thrombosis, GFE may contain a beneficial component as a useful drug.     

1,3-dimethylisoguaninium,an antiangiogenic purine analog from the sponge Amphimedon paraviridis

An antiangiogenic purine analog, 1,3-dimethylisoguaninium (1), was isolated from the ethanol (EtOH) extract of the Okinawan sponge Amphimedon paraviridis. The structure was elucidated on the basis of its spectral properties and X-ray crystallographic analysis. Compound 1 exhibited specific inhibition of the basic fibroblast growth factor (bFGF)-induced proliferation of bovine aorta endothelial cells (BAECs). Moreover, compound 1 reduced the tube formation of BAECs in a time-dependent manner.     

Stimulants from gardeniae fructus for cultured endothelial cell proliferation.

Bioassay-guided fractionation of Gardeniae Fructus extract (GFE), which stimulates the proliferation of cultured endothelial cells, led to the isolation of glycerol and D-mannitol. Both compounds significantly increased the incorporation of [3H]thymidine and [14C]leucine into the acid-insoluble fraction of bovine aortic endothelial cell layers in culture. This clearly indicated that glycerol and D-mannitol are active components of GFE on endothelial cell proliferation. On the other hand, they did not change the number of cultured vascular smooth muscle cells from bovine aorta. Glycerol and D-mannitol may be beneficial drugs for vascular disorders.     

Inhibition of corneal neovascularization by rapamycin

The purpose of this study was to determine whether rapamycin could inhibit corneal angiogenesis induced by basic fibroblast growth factor (bFGF). Using human dermal microvascular endothelial cells (HDMECs), we examined the effect of rapamycin on cell proliferation and migration, and the expression of vascular endothelial growth factor (VEGF). The rabbit's eye was implanted intrastromally into the superior cornea with pellet containing bFGF for the control group and pellet containing bFGF and rapamycin for the rapamycin group. Biomicrographically, corneal angiogenesis was evaluated for 10 days after pellet implantation. The neovascularized cornea also was examined histologically. bFGF induced corneal neovascularization was significantly reduced by treatment with rapamycin. Using in vitro model, rapamycin strongly inhibited bFGF induced proliferation, migration, and VEGF secretion of HDMECs. We could observe that the bFGF induced corneal angiogenesis was inhibited by rapamycin in a micropocket rabbit model. The score of neovascularization was significantly decreased in the rapamycin group than in the control group at 10 days after pellet implantation. Histologically, the cornea of rapamycin group also showed much less new vessels than that of control group. Collectively, rapamycin appears to inhibit bFGF induced angiogenesis in a rabbit corneal micropocket assay and may have therapeutic potential as an antiangiogenic agent.     

Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.     

Application of heparinized cellulose matrices for substrate-mediated bFGF peptide and transgene delivery to stimulate cellular proliferation

We investigated the use of heparinized cellulose matrices (H-CM) as affinity substrates for binding of basic fibroblast growth factor (bFGF), a heparin-binding peptide, to facilitate cellular proliferation and substrate-mediated transgene delivery. Using human HT-1080 fibroblasts and Saos-2 osteoblasts as cellular models, we showed that H-CM was a friendly substrate for cellular adhesion. Once adhered, cells received stimulation from the bound bFGF, leading to enhanced proliferation. Furthermore, taking advantage of the negative zeta potential of H-CM, we applied electrostatic adsorption to immobilize cationic poly-ethylenimine/DNA polyplexes onto the surface for transgene delivery upon cellular adhesion. Because bFGF stimulated cellular proliferation, we observed a significant increase in transfection efficiency in comparison to transfection on H-CM without the bFGF binding. We showed that H-CM was capable of mediating both bFGF peptide and bFGF transgene delivery to induce a synergistic stimulation of cellular proliferation, thus offering a useful device for fabrication of tissue scaffolds.     

Bee (Apis mellifera) venom produced toxic effects of higher amplitude in rat thoracic aorta than in skeletal muscle--an ultrastructural study

In this study, changes produced in aorta and triceps surae muscle of Wistar rats as response to bee venom (BV) envenomation were analyzed by transmission electron microscopy and morphometry. A subchronic treatment of 30 days with daily doses of 700 μg BV/kg and an acute-lethal treatment with a single dose of 62 mg BV/kg were performed. The subchronic treatment resulted in endothelial cell retraction, a thicker subendothelial layer, and thinner elastic laminae and musculoelastic layers in aorta, and thicker endothelium and basal laminae in skeletal muscle. In both tissues polymorphous, swollen mitochondria with disrupted cristae were observed. The acute treatment produced extensive endothelial lesions, breakdown of the collagen layer and migration of muscle cells toward the intima in the aorta, and dilatation of endoplasmic reticulum in the skeletal muscle cells. Mitochondria were almost devoid of cristae or with few circular cristae in the smooth muscle cells while most of the mitochondria presented abnormal circular cristae in the skeletal muscle cells. Degenerative alterations in the aorta were of higher intensity in our experiments-both the intima and media strongly responded to BV, in contrast to those found at the level of the skeletal muscle cells where a moderate degenerative myopathy was recorded.     

The identification of proliferation and tumour-induced proteins in human endothelial cells: a possible target for tumour therapy

The continued growth and spread of tumours is dependent on the proliferation of the endothelial cells of their vasculature. The presence of proliferation- or tumour-induced surface proteins on these endothelial cells would offer a suitable epitope for monoclonal antibody therapy of tumours. Using cultured human umbilical and capillary endothelial cells, we have stimulated them with simple mitogens and tumour conditioned media and examined the proteins induced by [35S]methionine incorporation and 125I-surface-labelling. Two-dimensional polyacrylamide gel electrophoresis revealed the induction of proliferation and tumour-related antigens on the surface of the endothelial cells. Subsequent monoclonal antibody studies suggest that tumour specific surface proteins are present on most tumour endothelium.     

THE EFFECT OF LASER IRRADIATION ON THE RELEASE OF bFGF FROM 3T3 FIBROBLASTS

Studies have shown that low-level laser irradiation increases the proliferation of fibroblasts in cell culture. The mechanism of action is unknown. Basic fibroblast growth factor (bFGF) is a multifunctional polypeptide that has been detected in most tissues and which supports cell proliferation and differentiation. The purpose of this study was to determine whether laser irradiation (660 nm) can stimulate production of bFGF from fibroblast cells in cell culture. Our study showed that fibroblasts irradiated with laser energy at 2.16 J/cm² demonstrated increased cell proliferation and enhanced production of bFGF, whereas fibroblasts irradiated with laser energy at 3.24 J/cm² neither demonstrated increased cell proliferation or an enhanced release of bFGF as compared to the control group. These results provide direct evidence that the proliferation of fibroblasts as a result of stimulation by low level laser irradiation may be associated with the autocrine production of bFGF from fibroblasts.     

Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.     

The role of adiponectin in the production of IL-6, IL-8, VEGF and MMPs in human endothelial cells and osteoblasts: implications for arthritic joints

This study was performed to evaluate the contribution of adiponectin to the production of interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and MMP-13 in human endothelial cells and osteoblasts in arthritic joints. Cultured human umbilical vascular endothelial cells (HUVECs) and osteoblasts were stimulated with adiponectin (1 or 10 μg ml⁻¹) or IL-1β (0.1 ng ml⁻¹) in the presence or absence of hypoxia for 24 h. The protein expression patterns were examined by analyzing culture supernatants using the enzyme-linked immunosorbent assay (ELISA). Adiponectin significantly stimulated the production of VEGF, MMP-1 and MMP-13 in osteoblasts but not in endothelial cells, whereas it significantly stimulated the production of IL-6 and IL-8 in both endothelial cells and osteoblasts. The increase in VEGF production induced by adiponectin was significantly greater than that induced by IL-1β. The production of IL-6 and IL-8 in adiponectin-stimulated endothelial cells was approximately 10-fold higher than that in IL-1β-stimulated endothelial cells; in osteoblasts, adiponectin-induced IL-6 and IL-8 secretion was approximately twofold higher than that induced by IL-1β. In addition, IL-8 production in endothelial cells was approximately sevenfold higher than in osteoblasts. However, IL-6 levels were similar between the two cell types, suggesting that adiponectin may be involved in the production of IL-8 in endothelial cells, which may have an important role in neutrophil recruitment to arthritic joints. Furthermore, the increases in protein expression induced by adiponectin were differentially regulated by hypoxia. In conclusion, adiponectin has a more important role than does IL-1β in the production of mediators that drive synovitis and joint destruction in endothelial cells and osteoblasts at physiological concentrations.     

Salvianolic acid A inhibits PDGF-BB induced vascular smooth muscle cell migration and proliferation while does not constrain endothelial cell proliferation and nitric oxide biosynthesis

Proliferation and migration of vascular smooth muscle cells (VSMCs) are critical events in the initiation and development of restenosis upon percutaneous transluminal coronary angioplasty (PTCA). Polyphenols have been suggested to ameliorate post-angioplasty restenosis. Salvianolic A (SalA) is one of the most abundant polyphenols extracted from salvia. In this study, we investigated the effect of salvianolic A (SalA) on the migration and proliferation of VSMCs. We found a preferential interaction of SalA with cellular systems that rely on the PDGF signal, but not on the EGF and bFGF signal. SalA inhibits PDGF-BB induced VSMC proliferation and migration in the concentration range from 0.01 to 0.1 μM. The inhibition of SalA on VSMC proliferation is associated with cell cycle arrest. We also found that SalA inhibits the PDGFRβ-ERK1/2 signaling cascade activated by PDGF-BB in VSMCs. In addition, SalA does not influence the proliferation of endothelial cells, the synthesis of NO and eNOS protein expression. Our results suggest that SalA inhibits migration and proliferation of VSMCs induced by PDGF-BB via the inhibition of the PDGFRβ-ERK1/2 cascade, but that it does not constrain endothelial cell proliferation and nitric oxide biosynthesis. Thus, the present study suggests a novel adjunct pharmacological strategy to prevent angioplasty-related restenosis.     

The basic research on physiological property of functionalized hyaluronan—I. Effect of hyaluronan and sulfated hyaluronan on cell proliferation of human epidermal keratinocytes

Hyaluronan (HA) is one of the polysaccharides that is found widely in connective tissue of mammals, and it has no sulfate group and high molecular weight in comparison with other glycosaminoglycans. Glycosaminoglycans are deeply concerned with the manifestation of biofunctions not only by their physical properties but also by physiological ones. In this study, sulfated HA (S‐HA) with various degrees of sulfate substitution and high molecular weight will be synthesized in order to give HA new biological functions. Moreover, the effect of HA and S‐HA on cell proliferation of human epidermal keratinocytes in vitro will be discussed. HA did not affect lag phase, but growth rate (metabolic turnover) of the cell in a logarithmic growth phase which was controlled by the molecular weight of HA. S‐HA stimulated the cell proliferation in the low concentration region under 1 μg/ml. While it inhibited the cell proliferation in the high concentration region over 10 μg/ml. It strongly suppressed the cell proliferation in the logarithmic growth metaphase. These facts were considered to be caused by the change of the cell‐matrix and/or cell–cell interactions. Copyright © 2004 John Wiley & Sons, Ltd.     

Soluble mediators from mesenchymal stem cells suppress T cell proliferation by inducing IL-10

Mesenchymal stem cells (MSCs) can inhibit T cell proliferation; however, the underlying mechanisms are not clear. In this study, we investigated the mechanisms of the immunoregulatory activity of MSCs on T cells. Irradiated MSCs co-cultured with either naïve or pre-activated T cells in a mixed lymphocyte reaction (MLR) significantly suppressed T cell proliferation in a dose-dependent manner, irrespective of allogeneic disparity between responders and MSCs. Transwell assays revealed that the suppressive effect was primarily mediated by soluble factors that induced apoptosis. Splenocytes stimulated with alloantigen in the presence of the MSC culture supernatant (CS) produced a significant amount of IL-10, which was attributed to an increase in the number of IL-10 secreting cells, confirmed by an ELISPOT assay. The blockade of IL-10 and IL-10 receptor interaction by anti-IL-10 or anti-IL-10-receptor antibodies abrogated the suppressive capacity of MSC CS, indicating that IL-10 plays a major role in the suppression of T cell proliferation. The addition of 1-methyl-DL-tryptophan (1-MT), an indoleamine 2,3-dioxygenase (IDO) inhibitor, also restored the proliferative capacity of T cells. In conclusion, we demonstrated that soluble mediators from culture supernatant of MSCs could suppress the proliferation of both naïve and pre-activated T cells in which IL-10 and IDO play important roles.     

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.     

Culture and Differentiation of Rat Neural Stem/Progenitor Cells in a Three-Dimensional Collagen Scaffold

A stable and fast method for constructing a neural-like tissue from rat neural stem/progenitor cells (rNS/PCs) based on three-dimensional (3D) collagen gel is described. First step, the collagen-embedded rNS/PCs expanded with the medium consisting of DMEM/F12/RPMI1640 (1:1:1) supplemented with EGF and bFGF was used to expand the cells in gel in 96-well plates until the average diameter of cell clusters was about 50–100 μm with the cell density higher than 10⁷ cells/mL. In the second step, the initial medium was replaced with NB/B-27 supplemented with bFGF and BDNF. The results show that cells in collagen presented neural-like morphology and maintained live cell rate around 82 % in neural network pattern at least for 42 days under static conditions. The cell–collagen constructs were detected by immunofluorescence and immunohistochemistry test after 42 days of culture, part of cells still maintained the character of rNS/PCs, and others differentiated into neurons, astrocytes, and oligodendrocytes. Our 3D neural-like tissue construct was similar to the neural tissue in morphology and cell compositions. They thus have a potential to be used for drug screening, detection of environment toxins, and replacement therapy.     

人工细胞外基质对血管内皮细胞生存的影响

通过物理吸附方法, 利用胶原、 聚赖氨酸和融合蛋白VEGF-Fc对聚苯乙烯培养板表面进行改性, 以研究细胞外基质材料对血管内皮细胞的影响. 结果表明, 3种蛋白显著提高了聚苯乙烯表面的亲水性. 内皮细胞的黏附、 增殖、 细胞骨架蛋白染色和血管性血友病因子(vWF)免疫染色实验结果表明, 胶原、 聚赖氨酸和VEGF-Fc基质均能有效提高血管内皮细胞的黏附, 其中胶原可与VEGF协同作用促进内皮细胞分化表型的表达; VEGF-Fc基质兼具了VEGF的生物学活性, 可促进内皮细胞的黏附和增殖以及vWF功能性蛋白的表达. 本研究为诱导材料表面内皮化和血管新生的生物活性材料的设计开发提供了新思路.     

Cytotoxic fraction from Artemisia sacrorum Ledeb. against three human cancer cell lines and separation and identification of its compounds

Artemisia sacrorum Ledeb. was extracted by 95% ethanol and water, respectively. By partitioning the 95% ethanol extract successively with different solvents and separating the water extract by macroporous resin, nine separate parts were obtained. According to the results of in vitro experiments, the CH?Cl? (dichloromethane) fraction showed the most pronounced cytotoxic activity against HepG2, HT-29 and MCF-7 cells, with EC?? values 122.35, 49.76 and 28.51?μg?mL?1, respectively, at 48?h. Following this, the compounds of the CH?Cl? fraction were separated and identified. Ten compounds were isolated from A. sacrorum Ledeb. and identified by spectral analysis. Four compounds, including acacetin, were isolated for the first time from A. sacrorum Ledeb.