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.     

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.     

Possible mechanism of the stimulatory effect of Artemisia leaf extract on the proliferation of cultured endothelial cells: involvement of basic fibroblast growth factor

To investigate the possible mechanism of the stimulatory effect of a hot water extract from Artemisia leaf (Artemisia princeps PANPANINI) (AFE) on the proliferation of endothelial cells, cells from bovine aorta were cultured for 72 h in RPMI1640 medium supplemented with 10% fetal calf serum in the presence of 5 micrograms/ml AFE. The AFE treatment significantly increased the cell number after culture, while in the presence of 10 micrograms/ml unfractionated heparin, AFE conversely decreased it. This implied that AFE enhanced the cell growth promotion by basic fibroblast growth factor (bFGF). The accumulation of bFGF was significantly increased in the culture medium, in the low-affinity (glycosaminoglycans-binding) fraction, and in the cell extract fraction, but was unchanged in the high-affinity (receptor-binding) fraction. The contents of [35S]sulfate-labeled glycosaminoglycans in both cell layer and the medium were not increased by AFE treatment. The proliferation of A10 cells, an established cell line of smooth muscle cells from murine aorta, was not stimulated by AFE. A10 cells did not produce a significant amount of bFGF in the presence or absence of AFE. Thus, the production of bFGF was considered to be involved in AFE stimulation of cell proliferation. In conclusion, it was suggested that AFE stimulated endothelial cell proliferation by increasing the production of bFGF rather than by an increase in the number of bFGF receptors and the content of glycosaminoglycans in the cell layer. The enhanced reserve of bFGF in the low-affinity fraction of cell layer and in the medium would cause the AFE-stimulated proliferation of endothelial cells.     

Proliferation of endothelial cells on the plasma-treated segmented-polyurethane surface: attempt of construction of a small caliber hybrid vascular graft and antithrombogenicity

To prepare a porous segmented-polyurethane (SPU) tube, a solution of SPU containing different concentrations of NaCl was coated on a glass rod and the coated SPU was immediately immersed in water. When the surface of the porous SPU, where bovine aortic endothelial cells are not normally capable of adhering and proliferating, was modified by plasma treatment, the proliferation of endothelial cells could be drastically improved. The cells proliferated confluently on the porous SPU surface prepared at low concentrations of NaCl below 10 g per 100 ml, but poorly on the porous surface prepared at high concentrations of NaCl. The construction of a hybrid vascular graft consisting of a porous SPU tube (2 mm in inner diameter, 5 cm in length) and endothelial cells was attempted. The cells cultured on the inner surface of the tube proliferated to confluency everywhere. From an in vitro antithrombogenic evaluation test, which involved the use of human blood, the present hybrid graft can be considered to provide an inert surface against thrombus formation and blood coagulation. Negligible changes in shape of human leukocytes in contact with bovine aortic endothelial cell surface occurred, suggesting that the bovine aortic endothelial cells used are immunologically less active against human blood.     

Vascular endothelial and smooth muscle cell culture on NaOH-treated poly(epsilon-caprolactone) films: a preliminary study for vascular graft development

Tissue engineering offers the potential of providing vessels that can be used to replace diseased and damaged native blood vessels. The endothelization of a synthetic vascular graft minimizes the failures associated with blood clotting and platelet activation. The aim of this study was to culture vascular-derived endothelial and smooth muscle cells on both untreated and NaOH-treated poly(epsilon-caprolactone) (PCL) films, a biocompatible and bio-resorbable polymer, and to evaluate the behavior of both cell types as a preliminary study for vascular graft development. PCL films were prepared by hot pressing; characterized by DSC, IR, SEM, and scanning force microscopy; and treated with NaOH to increase the surface hydrophilicity before cell culture. Endothelial and smooth muscle cells, isolated from pig cava vein, were characterized by immunofluorescence and confocal microscopy studies of endothelial nitric oxide synthase and alpha-smooth muscle actin. Good adhesion, growth, viability and morphology of both the endothelial and smooth muscle cells on PCL films were obtained, but a light stimulation of mitochondrial activity was observed during short culture times. NaOH treatment improved the adhesion and enhanced the proliferation in both cell types. This verified the possible use of this modified polymer as a support in the preparation of a synthetic vascular graft. [Diagram: see text] SEM micrograph of smooth muscle cells cultured on NaOH-treated PCL film. (Original magnification: 1000x).     

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.     

Synthesis of novel 1,3,4-trisubstituted pyrazole derivatives and their evaluation as antitumor and antiangiogenic agents

Several 1,3,4-trisubstituted pyrazole derivatives were synthesized and screened for their cytotoxic effect in a primary 3 tumor cell line test at 10(-4) M drug concentration. Compounds 19 and 20 reduced the growth of one or more of these cell lines to less than 32% and escalated up to evaluation in the full panel of 60 human tumor cell lines at a minimum of 5 concentrations at 10 fold dilutions. Compound N'-(1-[1-[4-nitrophenyl]-3-phenyl-1H-pyrazol-4-yl]methylene)-2-chlorobenzohydrazide 19 proved to be the most active of these derivatives with full panel median growth inhibition (GI50), total growth concentration (TGI) and median lethal concentration (LC50) mean graph mid-point (MG-MID) of 3.79, 12.5 and 51.5 microM, respectively. In addition, compounds 19, 39, 40, 41, 43, 45, 47 were tested for their antiangiogenic properties by testing their ability to inhibit human umbilical vein endothelial cells (HUVECs) proliferation, cord formation and migration in response to chemoattractant. 3-Acetyl-2-(1-(4-nitrophenyl)-3-phenylpyrazol-4-yl)-5-(4-pyridyl)-1,3,4-oxadiazoline 39 showed significant antiangiogenic profile at non-cytotoxic doses, with HUVEC proliferation inhibition IC50 of 7.60 microM, chemotaxis IC50 of 0.86 microM and was superior to the reference celecoxib 2 in both tests. Furthermore, in contrary to the references TNP-470 and celecoxib, all the tested compounds interfered with the migratory function of HUVECs in response to vascular endothelium growth factor (VEGF) rather than the endothelial cells proliferation.     

Controlling the Poly(ε‐caprolactone) Degradation to Maintain the Stemness and Function of Adipose‐Derived Mesenchymal Stem Cells in Vascular Regeneration Application

Biodegradable poly(ε‐caprolactone) (PCL) scaffolds with adipose‐derived mesenchymal stem cells (ADSCs) have been used in vascular regeneration studies. An evaluation method of the effect of PCL degradation products (DP) on the viability, stemness, and differentiation capacities of ADSCs is established. ADSCs are cultured in medium containing different concentrations of PCL DP before evaluating the effect of PCL DP on the cell apoptosis and proliferation, cell surface antigens, adipogenic and osteogenic differentiation capacities, and capacities to differentiate into endothelial cells and smooth muscle cells. The results demonstrate that PCL DP exceed 0.05 mg mL^?1 may change the stemness and differentiation capacities of ADSCs. Therefore, to control the proper concentration of PCL DP is essential for ADSCs in vascular regeneration application.     

Synthesis of 1,4-diphenylbutadiene derivatives: novel inducer of tissue-type plasminogen activator (t-PA) in cultured bovine endothelial cells

(E,E)-1,4-Diphenylbutadiene derivatives were synthesized by utilizing the Stobbe reaction of dimethyl succinate as a key step. Their stereoisomers were also synthesized stereoselectively by means of the cross-coupling reaction of the vinylstannanes and the vinylbromides, which were obtained from the propiolic acid esters by stereoselective hydrostannation, as a key step. To discover novel stimulators of fibrinolysis in vascular endothelial cells, the synthesized compounds were added to cultured bovine endothelial cells to determine the activity of the plasminogen activator in the conditioned medium. Of the synthesized compounds, three compounds were found to stimulate the activity of the plasminogen activator in endothelial cells. In addtition, these compounds inhibited thrombus formation in a rat model of venous thrombosis.     

Characterization of Poly(vinyl alcohol)/Chitosan Hydrogels as Vascular Tissue Engineering Scaffolds

In this study, PVA/Chitosan hydrogels were fabricated by freeze/thaw cycles and further crosslinking with a KOH/Na₂SO₄ coagulation bath and the effect of freeze/thaw cycle number on cell behaviour was evaluated. The surface of the hydrogels were further modified with Collagen type I adsorption and seeded with bovine aortic vascular smooth muscle and endothelial cells. Increasing the number of freeze/thaw cycles resulted in a marked change in surface morphology, hydrophilicity and protein adsorption. Collagen coating caused an increase in initial attachment and proliferation. We concluded that hydrogels that have undergone 3 freeze/thaw cycles were optimum for cell attachment both in the presence and the absence of coating.     

Heterotypic cell pair co-culturing on patterned microarrays

We present a pair-wise co-culturing technique that creates large numbers of heterotypic cell pairs in patterned arrays. Lithographic patterning produces arrays with thousands of traps, each designed to accommodate only two cells and confine them at these sites for co-culturing. Two variants are introduced: a random seeding method that sediments a mixture of two cell types onto the array, and an approach that incorporates ferromagnetic thin films into the arrays and attracts cells that have been attached to ferromagnetic nanowires to the array sites through dipole interactions. The array technique includes the utilization of custom image analysis software that extracts data from multi-channel fluorescence images and records information about the cells in every trap, enabling the acquisition of accurate, high-statistics data. The applicability of the technique was demonstrated in experiments examining proliferation rates in pairs of bovine pulmonary artery endothelial and smooth muscle cells. Results demonstrated that heterotypic interactions favored smooth muscle cell proliferation while disfavoring endothelial cell proliferation. This is one example of a variety of cell-cell interactions that could be probed with this method.     

Effects of flavonoid derivatives on human microvascular endothelial cells

Some natural compounds, including flavonoids, are active in vasculature re-growth during hair follicle disruption, but their effects have not been yet evaluated directly on microvascular endothelial cells. Skin vascularisation regulates the physiological blood supply required for hair growth and its dysregulation is the basis of several human diseases. Follicle-derived vascular endothelial growth factor (VEGF) release from follicular keratinocytes promotes perifollicular vascularisation and increases follicle and hair size, while blockade of VEGF-mediated angiogenesis leads to impaired hair growth. Here, we tested three flavonoids, namely visnadin (VSD), hesperidin (HSP) and baicalin (BC), on cultured human microvascular endothelial cells (HMEC), comparing their effects with minoxidil (MXD), a synthetic drug broadly used in the treatment of androgenetic alopecia. The response to these compounds was assayed in terms of endothelial survival, proliferation, tubulogenesis and proangiogenic signalling. We show that BC promotes HMEC proliferation, while both VSD and MXD enhance tubulogenesis. Interestingly, only HSP increases VEGFR-2 phosphorylation.     

UO22+对某些细胞毒性的初探

建立了由多种金属离子和小分子配体组成的多相细胞液热力学平衡模型,模拟研究了UO22+在组织液和细胞液的形态。体外培养了SD大鼠成骨细胞和人肾小管上皮细胞,通过体外细胞生长抑制实验探索了UO22+对成骨细胞及肾小管上皮细胞的毒性。研究表明,在细胞液中,当各形态UO22+物质总浓度[U]=8.4×10-6mol/L时,当pH为6.0～6.5时,UO22+主要以固相(UO2)3(PO4)2存在,当pH为6.8～7.5时,UO22+主要以水溶性[UO2(CO3)3]4-存在;当[U]=1.3×10-3mol/L时,在整个细胞液pH范围内,固相(UO2)3(PO4)2占主导地位。体外细胞生长抑制实验表明,UO22+对成骨细胞的生长具有抑制作用,能显著降低肾小管上皮细胞的存活率,具有明显的细胞毒性。     

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.     

EFFECTS OF PHOTODYNAMIC TREATMENT OF PLATELETS OR ENDOTHELIAL CELLS in vitro ON PLATELET AGGREGATION

The purpose of this work was to gain insight into the role played by platelets and endothelial cells in the development of thrombogenic vascular events, observed after in vivo photodynamic therapy (PDT), by studying the in vitro effects of PDT on isolated human platelets and cultured human and bovine endothelial cells. Exposure to Photofrin II (PII) and light caused platelets to rapidly lose their ability to aggregate. Photofrin II alone at high concentrations also exerted inhibitory effects on aggregation. Endothelial cells exposed to PII- and phthalocyanine (GaCl-PcS2,3 or Zn-PCS1,2)-mediated PDT released potent platelet anti- and disaggregating activity which could be identified as prostacyclin by the following criteria: a close correlation between the time and dose dependent anti-aggregating effects and released 6-keto-PGF1 alpha (the spontaneous hydrolysis product of PGI2, determined by radioimmunoassay), the inhibition of these effects by indomethacin, accumulation of 6-keto-PGF1 alpha metabolite in the media of cells treated with PDT (as determined by HPLC analysis), and the absence of evidence for significant nitric oxide production. This prostacyclin release occurred following plasma membrane damage. Although no pro-aggregating activity was observed, endothelial cells were found to release considerable amounts of arachidonic acid and prostaglandin F2 alpha in response to PDT. These data, which indicate powerful anti-thrombogenic effects in vitro, are in sharp contrast to the vascular effects of PDT in vivo which are characterized by severe platelet aggregation, and imply that the in vivo effects involve additional components of the vascular system.     

Stimulation of Microvascular Networks on Sulfonated Polyrotaxane Surfaces with Immobilized Vascular Endothelial Growth Factor

Modulation of material properties and growth factor application are critical in constructing suitable cell culture environments to induce desired cellular functions. Sulfonated polyrotaxane (PRX) surfaces with immobilized vascular endothelial growth factors (VEGFs) are prepared to improve network formation in vascular endothelial cells. Sulfonated PRXs, whereby sulfonated α‐cyclodextrins (α‐CDs) are threaded onto a linear poly(ethylene glycol) chain capped with bulky groups at both terminals, are coated onto surfaces. The molecular mobility of sulfonated PRX surfaces is modulated by tuning the number of threading α‐CDs. VEGF is immobilized onto surfaces with varying mobility. Low mobility and VEGF‐immobilization reinforce cell proliferation, yes‐associated protein activity, and rhoA, pdgf, ang‐1, and pecam‐1 gene expression. Highly mobile surfaces and soluble VEGF weakly affect these cell responses. Network formation is strongly stimulated in vascular endothelial cells only on low‐mobility VEGF‐immobilized surfaces, suggesting that molecular mobility and VEGF immobilization synergistically control cell function.     

Antithrombogenicity of cultured endothelial cell-detached surface

To examine the antithrombogenicity of cultured endothelial cell-detached surface, a simple hybrid vascular model tube consisting of a glass tube and endothelial cells was constructed. To detach the endothelial cells from the inner surface of the model tube, a steady shear stress of 2 or 8 N m(-2) was imposed onto the surface of endothelial cell monolayer by means of a coaxial double cylinder rotational-type apparatus. Coagulation of blood in contact with the endothelial cell-detached surface was examined using a damped oscillation rheometer. Coagulation of whole blood in the cell-detached tube occurred at about 40 min, which was almost the same as that in the endothelial cell-coated tube. A few platelets without shape change adhered to the endothelial cell-detached surface. These data suggest that the endothelial cell-detached surface may exhibit antithrombogenic and anticoagulant surfaces. Biochemical analyses showed that the glass surface, where endothelial cell was detached, was covered with components such as collagen type IV that is considered to be produced from the endothelial cells on the glass surface.     

Selective inhibitory effect of epigallocatechin-3-gallate on migration of vascular smooth muscle cells

In order to prevent restenosis after angioplasty or stenting, one of the most popular targets is suppression of the abnormal growth and excess migration of vascular smooth muscle cells (VSMCs) with drugs. However, the drugs also adversely affect vascular endothelial cells (VECs), leading to the induction of late thrombosis. We have investigated the effect of epigallocatechin-3-gallate (EGCG) on the proliferation and migration of VECs and VSMCs. Both cells showed dose-dependent decrease of viability in response to EGCG while they have different IC(50) values of EGCG (VECs, 150 mM and VSMCs, 1050 mM). Incubating both cells with EGCG resulted in significant reduction in cell proliferation irrespective of cell type. The proliferation of VECs were greater affected than that of VSMCs at the same concentrations of EGCG. EGCG exerted differential migration-inhibitory activity in VECs vs. VSMCs. The migration of VECs was not attenuated by 200 mM EGCG, but that of VSMCs was significantly inhibited at the same concentration of EGCG. It is suggested that that EGCG can be effectively used as an efficient drug for vascular diseases or stents due to its selective activity, completely suppressing the proliferation and migration of VSMCs, but not adversely affecting VECs migration in blood vessels.     

Enhancing endothelial differentiation of human mesenchymal stem cells by culture on a nanofibrous polycaprolactone/(poly-glycerol sebacate)/gelatin scaffold

Cardiovascular diseases have always been one of the main causes of death worldwide and eventually one of the major medical concerns. Tissue engineering is promising strategies of treating cardiovascular, which can be an effective approach with the design of appropriate scaffold. In this study, to develop engineering basement membrane for endothelial differentiation with good cell attachment, we produced polycaprolactone (PCL)/poly (glycerol sebacate) (PGS)/gelatin nanofibrous scaffold via electrospinning. Attenuated total reflectance-Fourier transform infrared and the proton nuclear magnetic resonance results confirmed the chemical structure of synthesized PGS. Scanning electron microscope images of the electrospun scaffold revealed that the nanofibers are smooth, continues and uniform. Moreover, due to the presence of hydrophilic functional groups in the scaffold, the contact angle is in the appropriate range for cell adhesion especially endothelial cells. The elastic modulus and ultimate tensile stress of electrospun scaffold were calculated 1.32 ± 0.27 MPa and 1.23 ± 0.18 MPa respectively. Quantitative polymerase chain reaction was performed for evaluation of endothelial differentiation of mesenchymal stem cells cultured on standard plate and fibrous scaffold under chemical stimulation with growth factor. Specific endothelial gene expression results postulated that our modified scaffold could support and significantly promote endothelial differentiation of MSCs.     

Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.