[Phenotypic and functional characteristics of endothelial cells derived from human liver cancer].
OBJECTIVE
To analyze the phenotypic and functional characteristics of endothelial (T3A) cells derived from human hepatocellular cell carcinoma.


METHODS
Endothelial cells were isolated from human hepatocellular carcinoma specimens. The identification of T3A cells was performed by checking von Willebrand Factor (vWF), CD31, CD34 and Dil-Ac-LDL uptake. The cell surface fenestrations, a specific morphological feature of tumor derived EC, were investigated by scanning and transmission electron microscopy. The phenotypic characteristics of T3A cells were analyzed by fluorescence-activated cell sorter (FACS) and were further conformed by real-time PCR at transcription level. Furthermore, tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity was evaluated by 3-(4, 5-dimethythiazolyl) -2, -diphenyl-2H-tetrazolium-bromide (MTT) assay; Matrix metalloproteinase secretion was detected by zymography; Angiogenic ability in vitro was analyzed by culturing T3A cells in three-dimensional Matrigel plug. Coagulant and fibrinolytic activities were detected by enzyme-linked immunosorbent assay (ELISA).


RESULTS
The isolated T3A cells exhibited classic "spindle-shape" morphology and monolayer growth and contact inhibition properties. Immunofluorescent staining showed that T3A cells expressed vWF, CD31, CD34, and uptake of Dil-Ac-LDL at a high level. The cell surface fenestrations were observed on T3A cells by scanning and transmission electron microscopy. By FACS and real-time PCR, T3A cells were found to express alphav3, alphavbeta5 and TNF receptor p75 at high levels, and TNF receptor p55 and ICAM-1 at low levels, as compared with those in human liver sinusoidal endothelial cells (LSEC). In response to TNFalpha, LSEC exhibited a dose-dependent cytotoxicity, while T3A cells were resistant. Gelatin zymography showed that MMP-2 activity was higher in T3A cells than that in LSEC. In a three-dimensional plug of Matrigel, T3A cells exhibited stronger angiogenic ability as compared with LSEC. In addition, T3A cells released more tissue factor (TF), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1) and urine plasminogen activator (u-PA) than LSEC in response to TNFalpha.


CONCLUSIONS
Tumor-derived endothelial cells are phenotypically and functionally different from those derived from normal liver tissue.
Wu
Lian-Qiu
LQ
Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, China.
Zhang
Wen-Jian
WJ
Ye
Li-Ya
LY
Yang
Zhi-Hua
ZH
Lou
Jin-Ning
JN
D018952:Antigens, CD34 / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D015870:Gene Expression; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D016023:Integrins / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008168:Lung / Q000098:blood supply / Q000378:metabolism / Q000473:pathology*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D019030:Receptors, Vitronectin / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014407:Tumor Cells, Cultured; D053319:Tumor Necrosis Factor Decoy Receptors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014841:von Willebrand Factor / Q000378:metabolism
D018952:Antigens, CD34; D039302:Integrin alphaVbeta3; D016023:Integrins; D008077:Lipoproteins, LDL; D017395:Plasminogen Activator Inhibitor 1; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D047888:Receptors, Tumor Necrosis Factor, Type I; D019030:Receptors, Vitronectin; D053319:Tumor Necrosis Factor Decoy Receptors; D014409:Tumor Necrosis Factor-alpha; C032629:acetyl-LDL; C064660:integrin alphaVbeta5; D014841:von Willebrand Factor; D018799:Intercellular Adhesion Molecule-1; C069521:recombinant human tumor necrosis factor-binding protein-1; D010959:Tissue Plasminogen Activator; D020778:Matrix Metalloproteinase 2