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Human Umbilical Vein Endothelial Cells (human + umbilical_vein_endothelial_cell)
Selected AbstractsS -Allyl- L -Cysteine Sulfoxide Inhibits Tumor Necrosis Factor-Alpha Induced Monocyte Adhesion and Intercellular Cell Adhesion Molecule-1 Expression in Human Umbilical Vein Endothelial CellsTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 3 2010Chai Hui Abstract Garlic and its water-soluble allyl sulfur-containing compound, S -Allyl- L -cysteine Sulfoxide (ACSO), have shown antioxidant and anti-inflammatory activities, inhibiting the development of atherosclerosis. However, little is known about the mechanism(s) underlying the therapeutic effect of ACSO in inhibiting the formation of atherosclerostic lesion. This study aimed to investigate whether ACSO could modulate tumor necrosis factor-alpha (TNF-,)-induced expression of intercellular cell adhesion molecule-1, monocyte adhesion and TNF-,-mediated signaling in human umbilical vein endothelial cells. While TNF-, promoted the intercellular cell adhesion molecule-1 mRNA transcription in a dose- and time-dependent manner, ACSO treatment significantly reduced the levels of TNF-,-induced intercellular cell adhesion molecule-1 mRNA transcripts (P < 0.01). Furthermore, ACSO dramatically inhibited TNF-, triggered adhesion of THP-1 monocytes to endothelial cells and porcine coronary artery rings. Moreover, ACSO mitigated TNF-, induced depolarization of mitochondrial membrane potential and overproduction of superoxide anion, associated with the inhibition of NOX4, a subunit of nicotinamide adenine dinucleotide phosphate-oxidase, mRNA transcription. In addition, ACSO also inhibited TNF-,-induced phosphorylation of JNK, ERK1/2 and I,B, but not p38. Apparently, ACSO inhibited proinflammatory cytokine-induced adhesion of monocytes to endothelial cells by inhibiting the mitogen-activated protein kinase signaling and related intercellular cell adhesion molecule-1 expression, maintaining mitochondrial membrane potential, and suppressing the overproduction of superoxide anion in endothelial cells. Therefore, our findings may provide new insights into ACSO on controlling TNF-,-mediated inflammation and vascular disease. Anat Rec, 2010. © 2010 Wiley-Liss, Inc. [source] Chitosan Oligosaccharides Inhibit the Expression of Interleukin-6 in Lipopolysaccharide-induced Human Umbilical Vein Endothelial Cells Through p38 and ERK1/2 Protein KinasesBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 5 2010Hong-Tao Liu However, the potential roles of COS in the treatment of vascular inflammations remain unknown. In the present study, we examined the effects of COS on interleukin-6 (IL-6) production in human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS). Induction of HUVECs with LPS (100 ng/ml) increased the mRNA expression and protein secretion of IL-6 (versus the vehicle-treated group, p < 0.01), which were significantly reverted by the pre-treatment with COS (50,200 ,g/ml) for 24 hr before LPS exposure (versus the LPS-treated group, p < 0.05 or 0.01). Signal transduction studies showed that the pre-treatment of HUVECs with COS (50,200 ,g/ml) for 24 hr markedly inhibited the LPS-induced over-expression of phosphorylated p38 mitogen-activated protein kinase (MAPK), phosphorylated ERK1/2 and nuclear factor ,B (NF-,B). Moreover, the LPS-induced NF-,B activation was suppressed by the specific ERK1/2 inhibitor PD98059 (30 ,M) (versus the LPS-treated group, p < 0.01), but not by the specific p38 MAPK inhibitor SB203580 (25 ,M). Additionally, both MAPK inhibitors markedly suppressed LPS-induced IL-6 mRNA expression in HUVECs (versus the LPS-treated group, p < 0.01). In conclusion, our results suggest that COS inhibit LPS-induced up-regulation of IL-6 in HUVECs, and this can be regulated by at least two parallel signalling pathways: one via p38 MAPK pathway independent of NF-,B activation and one via ERK1/2 pathway dependent on NF-,B activation. [source] Growth Inhibition Activity of Thioacetal Artemisinin Derivatives Against Human Umbilical Vein Endothelial Cells.CHEMINFORM, Issue 8 2004Sangtae Oh Abstract For Abstract see ChemInform Abstract in Full Text. [source] Angiotensin II regulates endothelial cell migration through calcium influx via T-type calcium channel in human umbilical vein endothelial cellsACTA PHYSIOLOGICA, Issue 4 2010A. Martini Abstract Aim:, The T-type calcium channel is expressed in vascular endothelial cells, but its role in endothelial cell function is yet to be elucidated. We analysed the endothelial functional role of T-type calcium channel-dependent calcium under angiotensin II (Ang II) stimulation. Methods:, Human umbilical vein endothelial cells were co-incubated with hormone at 10,7 m and either Efonidipine 10,5 m or Verapamil 10,5 m or Mibefradil 10,5 m or Wortmannin 10,6 m. The contribution of Ang II receptors was evaluated using PD123319 10,7 m and ZD 7155 10,7 m. The calcium ion concentration was observed using Fluo-3 acetossimetil ester. The cells were observed after 3, 6, 9 and 12 h. Results:, The microfluorescence method points out that Ang II induces intracellular calcium modulation in time by distinct mechanisms. AT2 receptor blockade is necessary to observe significant increase in [Ca2+]i levels. Pre-treatment with Mibefradil abolishes Ang II -induced cell migration. Conclusions:, Our data show that Ang II, via AT1 receptor, modulates calcium concentration involving T-type calcium channel and L-type calcium channel but only the calcium influx via T-type calcium channels regulates endothelial cell migration which is essential for angiogenesis. [source] Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall componentsIMMUNOLOGY, Issue 2 2004Jaya Talreja Summary Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-,B translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components. [source] Human plasminogen kringle 1,5 reduces atherosclerosis and neointima formation in mice by suppressing the inflammatory signaling pathwayJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2010P. C. CHANG Summary.,Background:,Activation of vascular endothelial cells plays an important role in atherogenesis and plaque instability. Recent research has demonstrated that late-stage inhibition of plaque angiogenesis by angiostatin (kringle 1,4) reduces macrophage accumulation and slows the progression of advanced atherosclerosis. Kringle 1,5 (K1,5) is a variant of angiostatin that contains the first five kringle domains of plasminogen. Objective: To investigate whether K1,5 has an inhibitory effect on early-stage atherosclerosis, using the apolipoprotein E (ApoE)-deficient mouse model and a carotid artery ligation model. Methods: ApoE-deficient mice received K1,5 treatment for 4 weeks, and the severity of aortic atherosclerosis was measured. In the ligation model, the left common carotid arteries of C57BL/6 mice were ligated near the carotid bifurcation, and the mice received K1,5 for 4 weeks. Human umbilical vein endothelial cells were pretreated with K1,5 before tumor necrosis factor-, (TNF-,) treatment to explore the anti-inflammatory effect of K1,5. Results: The areas of the lesion in the aortas of ApoE-deficient mice that received K1,5 treatment were notably decreased, and the formation of carotid neointima in the C57BL/6 mice was decreased by treatment with K1,5. Expression of TNF-,-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 was inhibited by K1,5 treatment, possibly via downregulation of translocation of nuclear factor-,B and expression of reactive oxygen species. Conclusions: K1,5 reduced atherosclerosis and neointima formation in mice, possibly through inhibition of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in endothelial cells. [source] Zoledronate inhibits endothelial cell adhesion, migration and survival through the suppression of multiple, prenylation-dependent signaling pathwaysJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2007M. HASMIM Summary.,Background: Recent evidence indicates that zoledronate, a nitrogen-containing bisphosphonate used to treat conditions of increased bone resorption, may have anti-angiogenic activity. The endothelial cells signaling events modulated by zoledronate remain largely elusive. Objectives: The aim of this work was to identify signaling events suppressed by zoledronate in endothelial cells and responsible for some of its biological effects. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to zoledronate, isoprenoid analogs (i.e. farnesol and geranylgeraniol) and various inhibitors of signaling, and the effect on adhesion, survival, migration, actin cytoskeleton and signaling events characterized. Results: Zoledronate reduced Ras prenylation, Ras and RhoA translocation to the membrane, and sustained ERK1/2 phosphorylation and tumor necrosis factor (TNF) induced JNK phosphorylation. Isoprenoid analogs attenuated zoledronate effects on HUVEC adhesion, actin stress fibers and focal adhesions, migration and survival. Isoprenoid analogs also restored Ras prenylation, RhoA translocation to the membrane, sustained FAK and ERK1/2 phosphorylation and prevented suppression of protein kinase B (PKB) and JNK phosphorylation in HUVEC exposed to TNF in the presence of zoledronate. Pharmacological inhibition of Rock, a RhoA target mediating actin fiber formation, phosphatidylinositol 3-kinase, an activator of PKB, MEK1/2, an activator of ERK1/2, and JNK, recapitulated individual zoledronate effects, consistent with the involvement of these molecules and pathways and their inhibition in the zoledronate effects. Conclusions: This work has demonstrated that zoledronate inhibits HUVEC adhesion, survival, migration and actin stress fiber formation by interfering with protein prenylation and has identified ERK1/2, JNK, Rock, FAK and PKB as kinases affected by zoledronate in a prenylation-dependent manner. [source] Photochemically Modulated Endothelial Cell Thrombogenicity via the Thrombomodulin,Tissue Factor Pathway,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 5 2003Patrick Fungaloi ABSTRACT 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, ,= 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. [source] Design and validation of a pulsatile perfusion bioreactor for 3D high cell density culturesBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009Julie A. Chouinard Abstract This study presents the design and validation of a pulsatile flow perfusion bioreactor able to provide a suitable environment for 3D high cell density cultures for tissue engineering applications. Our bioreactor system is mobile, does not require the use of traditional cell culture incubators and is easy to sterilize. It provides real-time monitoring and stable control of pH, dissolved oxygen concentration, temperature, pressure, pulsation frequency, and flow rate. In this bioreactor system, cells are cultured in a gel within a chamber perfused by a culture medium fed by hollow fibers. Human umbilical vein endothelial cells (HUVEC) suspended in fibrin were found to be living, making connections and proliferating up to five to six times their initial seeding number after a 48-h culture period. Cells were uniformly dispersed within the 14.40,mm,× 17.46,mm,×,6.35,mm chamber. Cells suspended in 6.35-mm thick gels and cultured in a traditional CO2 incubator were found to be round and dead. In control experiments carried out in a traditional cell culture incubator, the scarcely found living cells were mostly on top of the gels, while cells cultured under perfusion bioreactor conditions were found to be alive and uniformly distributed across the gel. Biotechnol. Bioeng. 2009; 104: 1215,1223. © 2009 Wiley Periodicals, Inc. [source] The carbon monoxide releasing molecule (CORM-3) inhibits expression of vascular cell adhesion molecule-1 and E-selectin independently of haem oxygenase-1 expressionBRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2009H Song Background and purpose:, Although carbon monoxide (CO) can modulate inflammatory processes, the influence of CO on adhesion molecules is less clear. This might be due to the limited amount of CO generated by haem degradation. We therefore tested the ability of a CO releasing molecule (CORM-3), used in supra-physiological concentrations, to modulate the expression of vascular cell adhesion molecule (VCAM)-1 and E-selectin on endothelial cells and the mechanism(s) involved. Experimental approach:, Human umbilical vein endothelial cells (HUVECs) were stimulated with tumour necrosis factor (TNF)-, in the presence or absence of CORM-3. The influence of CORM-3 on VCAM-1 and E-selectin expression and the nuclear factor (NF)-,B pathway was assessed by flow cytometry, Western blotting and electrophoretic mobility shift assay. Key results:, CORM-3 inhibited the expression of VCAM-1 and E-selectin on TNF-,-stimulated HUVEC. VCAM-1 expression was also inhibited when CORM-3 was added 24 h after TNF-, stimulation or when TNF-, was removed. This was paralleled by deactivation of NF-,B and a reduction in VCAM-1 mRNA. Although TNF-, removal was more effective in this regard, VCAM-1 protein was down-regulated more rapidly when CORM-3 was added. CORM-3 induced haem oxygenase-1 (HO-1) in a dose- and time-dependent manner, mediated by the transcription factor, Nrf2. CORM-3 was still able to down-regulate VCAM-1 expression in HUVEC transfected with siRNA for HO-1 or Nrf2. Conclusions and implications:, Down-regulation of VCAM and E-selectin expression induced by CORM-3 was independent of HO-1 up-regulation and was predominantly due to inhibition of sustained NF-,B activation. [source] Influence of antiseptic agents on interleukin 8 release and transmigration of polymorphonuclear granulocytes in an in vitro model of peritonitisBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 7 2000W. Sendt Background The effect of antiseptic agents on peritoneal cells is ill defined. The influence of taurolidine (TAU) and polyhexamide (HEX) was investigated in an in vitro model. Methods Human umbilical vein endothelial cells (HUVECs) and human peritoneal mesothelial cells (HPMCs) were laid on collagen-coated filter inserts (HUVECs on the bottom, HPMCs on the top), thus representing a two-chamber peritoneal model. When confluence was reached, HPMCs were stimulated with 0·5 ml tumour necrosis factor (TNF) , 10 ,g ml,1 for 4 h. Afterwards 0·5 ml TAU (1 and 2 per cent) or 0·5 ml HEX (0·1 and 0·2 per cent) solutions were added to the upper compartment. After 1 h polymorphonuclear granulocytes (PMNs) (105 ml,1) were added to the lower compartment. After 2 and 6 h aliquots were taken from both compartments, transmigrated PMNs were counted and interleukin (IL) 8 concentrations were measured. Controls were either TNF-,-stimulated HPMCs or stimulated HPMCs where culture medium had been substituted for TNF-,. Significance of differences was assessed by analysis of variance with Bonferroni corrections. Correlations were calculated by linear regression analysis. Results Stimulation with TNF-, led to a time-dependent increase in PMN transmigration. IL-8 secretion into the apical compartment increased time dependently, resulting in a gradient between the two chambers. After substitution of the stimulus by culture medium, significantly less IL-8 was measured in both compartments. PMN transmigration was almost absent. Addition of HEX resulted in an initial increase in IL-8 levels comparable to TNF controls without further changes. A concentration-dependent decrease in IL-8 gradient was associated with reduced transmigration. The IL-8 gradient between the upper and lower chambers correlated significantly with PMN transmigration (r = 0·8205, P < 0·0001). Conclusion The decrease in IL-8 gradients by HEX and the diminished IL-8 response after addition of TAU may reflect either anti-inflammatory effects or cellular damage. Both antiseptic solutions reduced PMN migration, irrespective of continuous stimulation in this model. © 2000 British Journal of Surgery Society Ltd [source] Multiple effects of bevacizumab in angiogenesis: implications for its use in age-related macular degenerationACTA OPHTHALMOLOGICA, Issue 5 2009Angela Carneiro Abstract. Purpose:, This study aimed to elucidate the precise effects of bevacizumab in all steps in the neovascularization process in endothelial cells. Methods:, Human umbilical vein endothelial cells (HUVECs) were incubated with bevacizumab at concentrations within the clinically established range or with identical amounts of excipient. Cell cytotoxicity (evaluated by MTT assay), proliferation (by BrdU incorporation assay), apoptosis (by TUNEL assay), migration (by double-chamber assay) and vessel assembly in matrigel-coated plates were assessed in vitro. Mouse plug matrigel assays were performed to confirm in vitro results. Results:, Incubation of HUVECs with bevacizumab did not present cytotoxicity. Concentrations comparable with those after intravitreal doses of bevacizumab significantly reduced proliferation and migration capacity, and increased apoptotic rates in these cells. In addition, bevacizumab led to a significant decrease in the assembly of capillary-like structures on matrigel assay in comparison with excipient-treated cells. Further substantiating these in vitro findings, bevacizumab also inhibited angiogenesis in a mouse plug matrigel assay, as evaluated by haemoglobin content levels. Conclusions:, These results demonstrate that clinical doses of bevacizumab are able to prevent several steps of the angiogenic process. Bevacizumab is thus currently recommended for treating disorders that present augmented angiogenesis. [source] Levels of cysteinyl leukotriene receptor mRNA in human peripheral leucocytes: significantly higher expression of cysteinyl leukotriene receptor 2 mRNA in eosinophilsCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2001H. Mita Background Cysteinyl leukotrienes (CysLTs) have been implicated as important contributors in the pathophysiology of asthma and their biological effects are mediated by at least two distinct G-protein-coupled receptors. cDNA sequences of cysteinyl leukotriene receptor 1 (CysLTR1) and cysteinyl leukotriene receptor 2 (CysLTR2) have recently been elucidated. Objectives Our aim is to explore gene expression and the comparative expression of CysLTR1 mRNA and CysLTR2 mRNA in human peripheral blood leucocytes. Methods Gene expression of CysLTR1 and CysLTR2 mRNAs in human peripheral blood eosinophils, neutrophils, monocytes and T lymphocytes has been measured by competitive reverse transcription-polymerase chain reactions using RNA or DNA competitors. Results(a) When cellular levels of CysLTR1 mRNA were normalized to those of G3PDH mRNA, the relative concentration of CysLTR1 mRNA in eosinophils (43.8 ± 37.2, n = 29) was significantly higher than that in neutrophils (18.7 ± 23.3, n = 11), monocytes (0.93 ± 1.1, n = 10) and T lymphocytes (3.4 ± 2.4, n = 11). (b) When measured using each DNA competitor, mRNAs for both types of CysLTR coexisted in each type of leucocyte. The ratio of CysLTR1 mRNA to CysLTR2 mRNA was significantly lower in eosinophils (0.65 ± 0.42, n = 12) than in neutrophils (6.9 ± 4.9, n = 12), monocytes (1.8 ± 0.9, n = 10) and T lymphocytes (4.5 ± 5.7, n = 10). (c) Human umbilical vein endothelial cells expressed CysLTR2 mRNA, but not CysLTR1 mRNA. Conclusion These studies reveal that CysLTR1 mRNA and, in particular, CysLTR2 mRNA are abundantly expressed at high levels in eosinophils, raising the possibility that CysLTR2 may have an important physiological role in eosinophils and a CysLTR2 antagonist may be a good target for preventing signal transduction by CysLTs in eosinophils. [source] Triptolide functions as a potent angiogenesis inhibitorINTERNATIONAL JOURNAL OF CANCER, Issue 1 2010Ming-Fang He Abstract Triptolide is a key anti-inflammatory compound of the Chinese herbal medicine Tripterygium wilfordii Hook. f. (Celastraceae). It also possesses potent antitumor activity. In this study, we show that triptolide is an angiogenesis inhibitor based on various angiogenesis assays. The IC50 in in vitro assays was 45 nM, which was much lower than the plasma concentrations of triptolide in the rat or human administered with T. wilfordii extracts for treating inflammation. When dosed in vivo, triptolide potently inhibited angiogenesis at 100 nM in Matrigel plug assay. Triptolide at 0.75 mg/kg/day significantly blocked tumor angiogenesis and tumor progression in murine tumorigenesis assay. The underlying mechanism of triptolide correlated with downregulation of proangiogenic Tie2 and VEGFR-2 expression in human umbilical vein endothelial cell by semiquantitative RT-PCR and western blot analysis. Although Tie2 inhibition appeared to be a later event as compared with VEGFR-2, Tie2 overexpression significantly attenuated the inhibitory effect of triptolide on endothelial proliferation and network formation. By contrast, Tie2 knockdown mimicked the inhibitory effect of triptolide on endothelial network formation. Our findings suggest that antitumor action of triptolide is partly via inhibition of tumor angiogenesis by blocking 2 endothelial receptor-mediated signaling pathways, and triptolide can be a promising antiangiogenic agent. [source] Prostacyclin inhibits endothelial cell XIAP ubiquitination and degradationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Jun-Yang Liou To understand the role of prostacyclin (PGI2) in protecting endothelial cells from apoptosis, we evaluated the effects of carbaprostacyclin (cPGI2) on H2O2 -induced human umbilical vein endothelial cell (HUVEC) apoptosis. cPGI2 suppressed H2O2 -induced annexin V-positive cells in a concentration- and time-dependent manner. Pre-treatment of HUVEC with 50 µM cPGI2 for 4 h produced the maximal anti-apoptotic effect. Authentic PGI2 generated by adenoviral transfer of PGI2 synthetic genes exerted a similar protective effect. cPGI2 inhibited Smac/DIABLO release from mitochondria, caspase 3 activation, focal adhesion protein degradation, and cell detachment. cPGI2 selectively protected X-linked inhibitor of apoptosis protein (X-linked IAP, XIAP) from H2O2 -induced ubiquitination, and preserved XIAP protein levels. PD-98059 but not H-89 abrogated the protective action of cPGI2. cPGI2 increased ERK phosphorylation which was blocked by PD-98059. HUVEC stably transfected with dominant negative Ras abrogated XIAP preservation by cPGI2 while constitutive active Ras increased ERK phosphorylation and protected XIAP from degradation. Our results demonstrate for the first time that PGI2 inhibits XIAP ubiquitination and degradation via the Ras/MEK-1/ERK signaling pathway. Preservation of XIAP proteins represents a key mechanism by which PGI2 protects endothelial cells from oxidant-induced apoptosis. J. Cell. Physiol. 212:840,848, 2007. © 2007 Wiley-Liss, Inc. [source] Enamel matrix derivative exhibits angiogenic effect in vitro and in a murine modelJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 8 2003Kuo Yuan Abstract Objectives: Angiogenesis is one of the most critical events in the wound healing process. Any increase in angiogenesis could result in more rapid and complete healing. A recent study found that enamel matrix derivative (EMD) could accelerate early periodontal wound healing. We wanted to clarify whether EMD caused an angiogenic effect and, thus, possibly enhanced wound healing. Methods: We performed in vitro proliferation and chemotaxis assays on human umbilical vein endothelial cell (HUVEC) cultures, and a tissue culture assay using blood vessel fragments in fibrin gel. Collagen membranes soaked with EMD were implanted subcutaneously in mice to test the in vivo angiogenic effect. Results: While there were no significant differences between the negative control and EMD groups in the proliferation assay, EMD treatment did exhibit a significantly greater dose-dependent chemotactic effect on HUVEC than control group treatments. The tissue culture in fibrin gel showed new blood vessel outgrowths in the EMD groups, but none in the negative control group. In the animal studies, significantly more endothelial cells were detected in the EMD group of mice. Conclusions: Our findings show that EMD does exhibit some angiogenic effects. However, the underlying molecules and mechanisms are still unidentified. We discuss several possibilities. Zusammenfassung Ziele: Die Angiogenese gehört zu den kritischsten Ereignissen bei der Wundheilung. Eine Erhöhung der Angiogenese könnte zu einer rascheren und kompletteren Wundheilung führen. Kürzlich zeigte eine Studie, dass Schmelzmatrixderivate (EMD) die frühe parodontale Wundheilung beschleunigen könnte. Wir wollten klären, ob EMD einen angiogenetischen Effekt verursacht und dies möglicherweise die Wundheilung verbessert. Methoden: Wir führten in vitro Proliferations- und Chemotaxis-Assays an menschlichen Umbilicalvenen-Endothelzellen (HUVEC)Kulturen durch und studierten eine Gewebekultur unter Nutzung von Blutgefäßfragmenten in Fibringel. Kollagenmembranen mit EMD getränkt wurden subkutan in Mäuse implantiert, um den angiogenetischen Effekt in vivo zu testen. Ergebnisse: Während es keine signifikanten Differenzen zwischen den negativen Kontrollen und den EMD Gruppen in dem Proliferationsassay gab, zeigte die EMD Behandlung einen signifikant größeren, dosisabhängigen chemotaktischen Effekt auf HUVEC verglichen mit den Kontrollen. Die Gewebekultur im Fibringel zeigte neue Blutgefäßbildungen in den EMD-Gruppen, aber keine bei den Negativkontrollen. Bei den Tierstudien wurden signifikant mehr Endothelzellen in den EMD Mäusegruppen entdeckt. Schlussfolgerungen: Unsere Ergebnisse zeigen, dass EMD einige angiogenetische Effekte zeigt. Jedoch sind die zugrunde liegenden Moleküle und die Mechanismen noch nicht geklärt. Wir diskutieren verschiedene Möglichkeiten. Résumé Objectifs: L'Angiogenèse est un des plus critiques éléments lors du processus de cicatrisation. La moindre augmentation de l'angiogenèse peut entraîner une cicatrisation plus rapide et plus complète. Une récente étude a montré que les dérivés de la matrice amellaire (EMD) pouvait accélérer plus tôt la cicatrisation parodontale. Nous voulions clarifier la possible responsabilité de l'EMD dans l'angiogenèse et si oui, l'amélioration de la cicatrisation. Méthodes: Nous avons réalisé in vitro la prolifération et un essai de chimiotactisme sur des cultures de cellules endothéliales de la veine ombilicale humaine (HUVEC), et un essai de culture tissulaire en utilisant des fragments de vaisseaux sanguins dans un gel de fibrine. Des membranes de collagène gorgées d'EMD furent implantées en sous-cutanée chez des souris pour tester l'effet angiogénique in vivo. Résultats: Bien qu'il n'y eut pas de différences significatives entre le contrôle négatif et le groupe EMD pour le test de prolifération, le traitement par EMD présentait un effet chimiotactique dose- dépendant significativement plus élevé sur les HUVEC. La culture tissulaire sur gel de fibrine présentait une surcroissance de nouveaux vaisseaux sanguins pour le groupe EMD, mais pas dans le groupe contrôle. Plus de cellules endothéliales furent en outre détectées lors de l'étude animale, pour le groupe de souris traitées par EMD. Conclusions: Nos données montrent que l'EMD présente quelques effets angiogéniques. Cependant, les molécules et les mécanismes responsables ne sont toujours pas identifiés. Nous discutons quelques possibilités. [source] Epidermal growth factor released from platelet-rich plasma promotes endothelial cell proliferation in vitroJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2010M.-P. Bertrand-Duchesne Bertrand-Duchesne M-P, Grenier D, Gagnon G. Epidermal growth factor released from platelet-rich plasmapromotes endothelial cell proliferation in vitro. J Periodont Res 2009; doi: 10.1111/j.1600-0765.2009.01205.x. © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective:, The therapeutic benefits of platelet-rich plasma (PRP) for the promotion of healing and regeneration of periodontal tissues are thought to result from enrichment in growth factors released from platelets. The aim of this study was to evaluate the effects of specific growth factors released from PRP on endothelial cell proliferation. Material and Methods:, The levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in supernatants of calcium- and thrombin-activated PRP samples from five donors were quantified by enzyme-linked immunosorbent assay. Supernatants were treated with neutralizing antibodies specific to each growth factor, and the effects of these treatments on human umbilical vein endothelial cell (HUVEC) proliferation in vitro were determined. The effect of removing EGF from PRP supernatants with antibody-coated beads on HUVEC proliferation was also tested. Results:, Average concentrations of VEGF, PDGF-BB, bFGF and EGF in PRP supernatants were 189, 27,190, 39.5 and 513 pg/mL, respectively. The addition of EGF neutralizing antibodies to the PRP supernatants significantly reduced HUVEC proliferation (up to 40%), while such an inhibition was not observed following neutralization of the other growth factors. Removal of EGF from PRP supernatants by treatment with antibody-coated beads also resulted in a significant decrease in HUVEC proliferation. Recombinant EGF increased HUVEC proliferation in vitro in a dose-dependent manner. Conclusion:, This study showed that PRP supernatants are highly mitogenic for endothelial cells and provided evidence that this effect may be due, at least in part, to the presence of EGF. In vivo experiments are needed to confirm the roles of specific growth factors released from PRP in the healing of oral surgical and/or periodontal wounds. [source] Osteoblasts stimulated with pulsed electromagnetic fields increase HUVEC proliferation via a VEGF-A independent mechanism,BIOELECTROMAGNETICS, Issue 3 2009Richard A. Hopper Abstract The clinically beneficial effect of low frequency pulsed electromagnetic fields (ELF-PEMF) on bone healing has been described, but the exact mechanism of action remains unclear. A recent study suggests that there is a direct autocrine mitogenic effect of ELF-PEMF on angiogenesis. The hypothesis of this study is that ELF-PEMF also has an indirect effect on angiogenesis by manipulation of vascular endothelial growth factor (VEGF)-A-based paracrine intercellular communication with neighboring osteoblasts. Conditioned media experiments measured fetal rat calvarial cell (FRC) and human umbilical vein endothelial cell (HUVEC) proliferation using tritiated thymidine uptake. We demonstrate that ELF-PEMF (15 Hz, 1.8 mT, for 8 h) has an indirect effect on the proliferation rate of both endothelial cells and osteoblasts in vitro by altering paracrine mediators. Conditioned media from osteoblast cells stimulated with ELF-PEMF increased endothelial proliferation 54-fold, whereas media from endothelial cells stimulated with ELF-PEMF did not affect osteoblast proliferation. We examined the role of the pro-angiogenic mediator VEGF-A in the mitogenic effect of ELF-PEMF-stimulated osteoblast media on endothelial cells. The production of VEGF-A by FRC as measured by ELISA was not changed by exposure to PEMF, and blocking experiments demonstrated that the ELF-PEMF-induced osteoblast-derived endothelial mitogen observed in these studies was not VEGF-A, but some other soluble angiogenic mediator. Bioelectromagnetics 30:189,197, 2009. © 2008 Wiley-Liss, Inc. [source] Endothelial stimulation by small lymphocytic lymphoma correlates with secreted levels of basic fibroblastic growth factorBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2003Lisa Rimsza Summary. Lymph nodes (LN) involved with small lympho- cytic lymphoma (SLL) reportedly contain increased numbers of microvessels that may constitute a therapeutic target in this disease. We investigated the secretion of the angiogenic growth factor, basic fibroblastic growth factor (bFGF), from primary tissue cultures of 15 LN with SLL and 10 reactive LN. bFGF was detected from the resulting conditioned media (CM) in 13/15 SLL samples (mean 92 ± 30, range 5,420 pg/ml) but was undetectable in CM from all reactive lymph nodes. CM was also used in a 72-h human umbilical vein endothelial cell (HUVEC) proliferation assay. HUVEC proliferation increased in the presence of SLL CM (70 ± 17%, range ,4,194%), proportional to secreted levels of bFGF (R2 = 0·95), and was reversed by depleting bFGF from CM. Previous SLL studies have examined either patient serum samples or paraffin-embedded lymph node tissue sections. This is the first study to examine the secretion of an angiogenic growth factor from primary cultures of lymph node cells. Our results indicate that bFGF is probably the primary mediator responsible for increased angiogenesis in involved nodes. These findings may be pertinent to future investigation into the mechanisms of increased angiogenesis in SLL. [source] Antitumor activities of synthetic and natural stilbenes through antiangiogenic actionCANCER SCIENCE, Issue 10 2008Yoshiyuki Kimura We reported that the antitumor and antimetastatic actions of resveratrol might be due to the inhibition of tumor-induced angiogenesis. To search for anticancer agents with stronger activity than resveratrol, we examined the antiangiogenic effects of 21 synthetic and/or natural stilbenes. Among these 21 stilbenes, 2,3-, 3,4-, and 4,4,-dihydroxystilbene inhibited the pro-matrix metalloproteinase (pro-MMP),9 production in colon 26 cells at 5,25 µM, vascular endothelial growth factor (VEGF),induced human umbilical vein endothelial cell (HUVEC) migration at 10 and 25 µM, and VEGF-induced angiogenesis at 5,50 µM. Resvertarol inhibited the pro-MMP-9 production and VEGF-induced angiogenesis at 25 or 50 µM. Thus, the inhibition of pro-MMP-9 production in colon 26 cells and VEGF-induced angiogenesis by three dihydroxystilbenes were greater than those of resveratrol. The three dihydroxystilbenes (8 mg/kg, intraperitoneal injection) inhibited the tumor-induced neovascularization in colon 26,packed chamber-bearing mice and the tumor growth in colon 26,bearing mice. Furthermore, the three dihydroxystilbenes inhibited VEGF-induced VEGFR-2 phosphorylation. On the other hand, the three dihydroxystilbenes had no effect on VEGFR-1 and-2 expression, and VEGF-induced VEGFR-1 phosphorylation in HUVECs. These findings suggest that the inhibition of tumor-induced neovascularization by these three dihydroxystilbenes may be due to the inhibition of VEGF-induced endothelial cell migration and VEGF-induced angiogenesis through the inhibition of VEGF-induced VEGFR-2 phosphorylation in endothelial cells and pro-MMP-9 expression in colon 26 cells. (Cancer Sci 2008; 99: 2083,2096) [source] Sonic hedgehog derived from human pancreatic cancer cells augments angiogenic function of endothelial progenitor cellsCANCER SCIENCE, Issue 6 2008Madoka Yamazaki Hedgehog signaling is important in the pathogenesis of pancreatic cancer. Several recent observations suggest the involvement of sonic hedgehog (SHH) in postnatal neovascularization. We identified a novel role for SHH in tumor-associated angiogenesis in pancreatic cancer. Immunohistochemical analysis revealed that patched homolog 1 (PTCH1), both a receptor for and transcriptional target of hedgehog signaling, was expressed in a small fraction of endothelial cells within pancreatic cancer, but not in normal pancreatic tissue. When endothelial progenitor cells (EPC) isolated from human peripheral blood were cultured with supernatant from SHH-transfected 293 cells or pancreatic cancer cells, mRNA levels of vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 and angiopoietin-1 were significantly increased, whereas no such induction was observed in human umbilical vein endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMVEC). HUVEC tube formation was stimulated when cocultured with EPC, and preconditioning EPC with supernatant from KP-1 N pancreatic cancer cells highly expressing SHH significantly enhanced the effect. The effect was partially attenuated by specific inhibition of SHH with cyclopamine or a neutralizing antibody. These findings suggest that tumor-derived SHH can induce angiogenesis, and this is mediated by its effects on EPC specifically. Targeting SHH would be a novel therapeutic approach that can inhibit not only proliferation of cancer cells but also EPC-mediated angiogenesis. (Cancer Sci 2008; 99: 1131,1138) [source] Heterogeneity in lipopolysaccharide responsiveness of endothelial cells identified by gene expression profiling: role of transcription factorsCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2006G. C. Beck Summary Interindividual differences of endothelial cells in response to endotoxins might contribute to the diversity in clinical outcome among septic patients. The present study was conducted to test the hypothesis that endothelial cells (EC) with high and low proinflammatory potential exist and to dissect the molecular basis underlying this phenomenon. Thirty human umbilical vein endothelial cell (HUVEC) lines were stimulated for 24 h with lipopolysaccharide (LPS) and screened for interleukin (IL)-8 production. Based on IL-8 production five low and five high producers, tentatively called types I and II responders, respectively, were selected for genome-wide gene expression profiling. From the 74 genes that were modulated by LPS in all type II responders, 33 genes were not influenced in type I responders. Among the 41 genes that were increased in both responders, 17 were expressed significantly stronger in type II responders. Apart from IL-8, significant differences in the expression of proinflammatory related genes between types I and II responders were found for adhesion molecules [intercellular adhesion molecule (ICAM-1), E-selectin)], chemokines [monocyte chemoattractant protein (MCP-1), granulocyte chemotactic protein (GCP-2)], cytokines (IL-6) and the transcription factor CCAAT/enhancer binding protein-delta (C/EBP-,). Type I responders also displayed a low response towards tumour necrosis factor (TNF)-,. In general, maximal activation of nuclear factor (NF)-,B was achieved in type I responders at higher concentrations of LPS compared to type II responders. In the present study we demonstrate that LPS-mediated gene expression differs quantitatively and qualitatively in types I and II responders. Our results suggest a pivotal role for common transcription factors as a low inflammatory response was also observed after TNF-, stimulation. Further studies are required to elucidate the relevance of these findings in terms of clinical outcome in septic patients. [source] Angiotensin II regulates endothelial cell migration through calcium influx via T-type calcium channel in human umbilical vein endothelial cellsACTA PHYSIOLOGICA, Issue 4 2010A. Martini Abstract Aim:, The T-type calcium channel is expressed in vascular endothelial cells, but its role in endothelial cell function is yet to be elucidated. We analysed the endothelial functional role of T-type calcium channel-dependent calcium under angiotensin II (Ang II) stimulation. Methods:, Human umbilical vein endothelial cells were co-incubated with hormone at 10,7 m and either Efonidipine 10,5 m or Verapamil 10,5 m or Mibefradil 10,5 m or Wortmannin 10,6 m. The contribution of Ang II receptors was evaluated using PD123319 10,7 m and ZD 7155 10,7 m. The calcium ion concentration was observed using Fluo-3 acetossimetil ester. The cells were observed after 3, 6, 9 and 12 h. Results:, The microfluorescence method points out that Ang II induces intracellular calcium modulation in time by distinct mechanisms. AT2 receptor blockade is necessary to observe significant increase in [Ca2+]i levels. Pre-treatment with Mibefradil abolishes Ang II -induced cell migration. Conclusions:, Our data show that Ang II, via AT1 receptor, modulates calcium concentration involving T-type calcium channel and L-type calcium channel but only the calcium influx via T-type calcium channels regulates endothelial cell migration which is essential for angiogenesis. [source] Monocilia on chicken embryonic endocardium in low shear stress areasDEVELOPMENTAL DYNAMICS, Issue 1 2006Kim Van der Heiden Abstract During cardiovascular development, fluid shear stress patterns change dramatically due to extensive remodeling. This biomechanical force has been shown to drive gene expression in endothelial cells and, consequently, is considered to play a role in cardiovascular development. The mechanism by which endothelial cells sense shear stress is still unidentified. In this study, we postulate that primary cilia function as fluid shear stress sensors of endothelial cells. Such a function already has been attributed to primary cilia on epithelial cells of the adult kidney and of Hensen's node in the embryo where they transduce mechanical signals into an intracellular Ca2+ signaling response. Recently, primary cilia were observed on human umbilical vein endothelial cells. These primary cilia disassembled when subjected to high shear stress levels. Whereas endocardial,endothelial cells have been reported to be more shear responsive than endothelial cells, cilia are not detected, thus far, on endocardial cells. In the present study, we use field emission scanning electron microscopy to show shear stress-related regional differences in cell protrusions within the cardiovasculature of the developing chicken. Furthermore, we identify one of these cell protrusions as a monocilium with monoclonal antibodies against acetylated and detyrosinated alpha-tubulin. The distribution pattern of the monocilia was compared to the chicken embryonic expression pattern of the high shear stress marker Krüppel-like factor-2. We demonstrate the presence of monocilia on endocardial,endothelial cells in areas of low shear stress and postulate that they are immotile primary cilia, which function as fluid shear stress sensors. Developmental Dynamics 235:19,28, 2006. © 2005 Wiley-Liss, Inc. [source] Leptin and endothelin-1 mediated increased extracellular matrix protein production and cardiomyocyte hypertrophy in diabetic heart diseaseDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 5 2009Pijush Majumdar Abstract Background We investigated the role of leptin and its interaction with endothelin 1 (ET-1) in fibronectin (FN) synthesis and cardiomyocyte hypertrophy, two characteristic features of diabetic cardiomyopathy. Methods Endothelial cells [human umbilical vein endothelial cells (HUVECs)] were examined for FN production and neonatal rat cardiomyocytes for hypertrophy, following incubation with glucose, ET-1, leptin and specific blockers. FN, ET-1, leptin and leptin receptors mRNA expression and FN protein were measured. Myocytes were also morphometrically examined. Furthermore, hearts from streptozotocin-diabetic rats were analysed. Results Glucose caused increased FN mRNA and protein expression in HUVECs and cardiomyocytes hypertrophy along with upregulation of ET-1 mRNA, leptin mRNA and protein. Glucosemimetic effects were seen with leptin and ET-1. Leptin receptor antagonist (leptin quadruple mutant) and dual endothelin A endothelin B (ETA/ETB) receptor blocker bosentan normalized such abnormalities. Hearts from the diabetic animals showed hypertrophy and similar mRNA changes. Conclusion These data indicate that in diabetes increased FN production and cardiomyocyte hypertrophy may be mediated through leptin with its interaction with ET-1. Copyright © 2009 John Wiley & Sons, Ltd. [source] Development of Live Cell Chips to Monitor Cell Differentiation ProcessesENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 1 2008C. Maercker Abstract A big demand exists for high-throughput functional in vitro assays which can measure cellular phenotypes by molecular methods and therefore improve the resources of primary cells for cell therapy, tissue engineering and high-content screenings in drug development. This approach focuses on cellular adhesion which is an important differentiation process during homing of stem cells. Moreover, it is a promising method especially for adherent cells which are not accessible by classical cell sorting methods. The chip design includes a housing with electrodes to measure electric field densities and impedance, respectively. Moreover, specific coatings of the wells permit a perfect growth of the selected cell types. In parallel, protein biomarkers can be followed by light microscopy. So far, experiments have been started to discriminate between different cell densities and cell types. In addition, after stimulating human cardiac fibroblasts and human umbilical vein endothelial cells, concentrations of proteins involved in adhesion had been increased, and proteins were translocated within the cells. In ongoing experiments, different human cell lines and fibroblastoid mesenchymal stem cells isolated from fat tissue, umbilical cord, or bone marrow are tested in the chip. To optimize the adhesion conditions, the surfaces within the vials of the chip were specifically activated. Microscopy was adjusted to be able to measure cellular morphology in parallel. This concept allows to identify the behavior of mesenchymal stem cells, which cannot be described so far by standard biomarkers. In addition, simulation of the homing process of the cells within its stem cell niche in an in vitro assay is a promising setup for large-scale gain-of-function or loss-of-function screenings in functional genomics as well as for generating precursor cells relevant for the therapy of various diseases. [source] IL-10 inhibits endothelium-dependent T cell costimulation by up-regulation of ILT3/4 in human vascular endothelial cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2007Christian Abstract Effects of IL-10 on endothelium-dependent T cell activation have not been investigated in detail. We confirm expression of the IL-10 receptor and effective signaling via STAT-3 in human umbilical vein endothelial cells (HUVEC). In CD4 T cell cocultures with HUVEC, pretreatment of endothelial cells with IL-10 resulted in significant dose-dependent inhibition of CD4 T cell proliferation, which also occurred when IL-10 was removed after pretreatment before starting cocultures. Th1/Th2 polarization of proliferated T cells, endothelial nitric oxide (NO), or IL-12 production were unchanged. However, IL-10 stimulation resulted in up-regulation of SOCS-3, a negative regulator of cytokine secretion, and induction of the inhibitory surface molecules immunoglobulin-like transcript 3 and 4 (ILT3/ILT4) in EC, potentially involving glucocorticoid-induced leucine zipper (GILZ). Addition of blocking antibodies against ILT3/ILT4 to EC/T cell cocultures resulted in nearly complete reestablishment of T cell proliferation. In contrast, addition of soluble ILT3 or overexpression of ILT3 in cocultures significantly reduced T cell proliferation. No induction of foxp3+ regulatory T cells was seen. In conclusion, the T cell costimulatory potential of human EC is markedly suppressed by IL-10 due to up-regulation of ILT3/ILT4, obviously not involving generation of Treg. This identifies a novel action of IL-10 in EC and a potential therapeutical target for local immunomodulation. [source] Insulin/protein kinase B signalling pathway upregulates metastasis-related phenotypes and molecules in H7721 human hepatocarcinoma cell lineFEBS JOURNAL, Issue 18 2003Hui-Ling Qi The effect of insulin on cancer metastatic potential was studied in a human hepatocarcinoma cell line, H7721. Cell adhesion to human umbilical vein endothelial cells (HUVECs) and laminin as well as chemotactic cell migration and invasion were selected as the indices of metastasis-related phenotypes for assessment of metastatic potential ex vivo. The results indicated that insulin enhanced all of these metastasis-related phenotypes. After the cells were treated with specific inhibitor of PI3K (LY294002) or transfected with antisense cDNA of PKB (AS-PKB), all of the above phenotypes were attenuated, and they could not be significantly stimulated by insulin, indicating that the insulin effect on metastatic potential was mediated by PI3K and PKB. Only the monoclonal antibody to the sialyl Lewis X (SLex), but not antibodies to other Lewis antigens, significantly blocked the cell adhesion to HUVECs, cell migration and invasion, suggesting that SLex played a crucial role in the metastatic potential of H7721 cells. The upregulation of cell surface SLex and ,-1,3-fucosyltransferase-VII (,-1,3 Fuc T-VII, enzyme for SLex synthesis) was also mediated by PI3K and PKB, since LY294002 and AS-PKB also reduced the expressions of SLex and ,-1,3 FucT-VII, and attenuated the response to insulin. Furthermore, the alterations in the expressions of PKB protein and activity were correlated to the changes of metastatic phenotypes and SLex expression. Taken together, the insulin/PKB signalling pathway participated in the enhancement of metastatic potential of H7721 cells, which was mediated by the upregulation of the expression of SLex and ,-1,3 FucT-VII. [source] Transfection of the c- erbB2/neu gene upregulates the expression of sialyl Lewis X, ,1,3-fucosyltransferase VII, and metastatic potential in a human hepatocarcinoma cell lineFEBS JOURNAL, Issue 12 2001Fei Liu The pCMV4 plasmid containing the cancer-promoting gene, c- erbB2/neu, was cotransfected into the human hepatocarcinoma cell line 7721 with the pcDNA3 vector, which contains the ,neo' selectable marker. Several clones showing stable expression of c- erbB2/neu were established and characterized by determination of c- erbB2/neu mRNA and its encoded protein p185. Expression of Lewis antigens and ,1,3-fucosyltransferases and the biological behavior of 7721 cells after c- erbB2/neu transfection were studied using mock cells transfected with the vectors pCMV4 and pcDNA3 as controls. SLex expression on the surface of mock cells was high, whereas expression of SDLex, Lex and SLea was absent or negligible. This is compatible with the abundant expression of ,1,3-fucosyltransferase VII, very low expression of ,fucosyltransferase III/VI, and almost absent expression of ,1,3-fucosyltransferase IV in the mock cells. After transfection of c- erbB2/neu, expression of SLex and ,1,3-fucosyltransferase VII were simultaneously elevated, but that of ,fucosyltransferase III/VI was not altered. The expression of both SLex and ,1,3-fucosyltransferase VII correlated positively with the expression of c- erbB2/neu in different clones, being highest in clone 13, medium in clone 6, and lowest in clone 7. In addition, the adhesion of 7721 cells to human umbilical vein endothelial cells (HUVECs) or P-selectin, as well as cell migration and invasion, were increased in c- erbB2/neu -transfected cells. These increases also correlated positively with the expression intensities of c- erbB2/neu, SLex and ,1,3-fucosyltransferase VII in the different clones, whereas cell adhesion to fibronectin correlated negatively with these variables. mAbs to SLex (KM93) and SDLex (FH6) significantly and slightly, respectively, abolished cell adhesion to HUVECs or P-selectin and cell migration and invasion. mAbs to SDLex and SLea did not suppress cell adhesion to HUVECs nor inhibit cell migration and invasion. Transfection of ,1,3-fucosyltransferase VII cDNA into 7721 cells showed similar results to transfection of c- erbB2/neu, and the increased adhesion to HUVECs, cell migration, and invasion were also inhibited significantly by KM93 and slightly by FH6. These results indicate that expression of ,1,3-fucosyltransferase VII and its specific product, SLex, and their capacity for cell adhesion, migration and invasion are closely related. Therefore, the c- erbB2/neu gene is proposed to be a metastasis-promoting gene, and its effects are at least partially mediated by the increased expression of ,1,3-fucosyltransferase VII and SLex. [source] Functionalized, Swellable Hydrogel Layers as a Platform for Cell StudiesADVANCED FUNCTIONAL MATERIALS, Issue 8 2009Núria Marí-Buyé Abstract This paper reports the design, synthesis and characterization of thin films as a platform for studying the separate influences of physical and chemical cues of a matrix on the adhesion, growth and final phenotype of cells. Independent control of the physical and chemical properties of functionalized, swellable hydrogel thin films is achieved using initiated chemical vapor deposition (iCVD). The systematic variation in crosslink density is demonstrated to control the swelling ability of the iCVD hydrogel films based on 2-hydroxyethyl methacrylate (HEMA). At the same time, the incorporation of controllable concentrations of the active ester pentafluorophenyl methacrylate (PFM) allows easy immobilization of aminated bioactive motifs, such as bioactive peptides. Initial cell culture results with human umbilical vein endothelial cells (HUVEC) indicate that the strategy of using PFM to immobilize a cell-adhesion peptide motif onto the hydrogel layers promotes proper HUVEC growth and enhances their phenotype. [source] | ||||||||||||||||||||||||||||||||||||||||