Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method for the isolation and long-term culture of human microvessel endothelial cells from mammary adipose tissue (HuMMEC) obtained at breast reduction surgery has been developed. Pure cultures of HuMMEC were isolated by sequential digestion of the fat with collagenase and trypsin followed by specific selection of microvessel fragments with Ulex europaeus agglutinin-1 coated magnetic beads (Dynabeads). The resulting cells formed contact-inhibited monolayers on gelatin and fibronectin substrates and capillary-like "tubes" on Matrigel; they also expressed von Willebrand factor, angiotensin-converting enzyme, and accumulated acetylated low density lipoprotein. Further immunofluorescence characterization revealed the presence of antigens for the endothelial cell specific monoclonal antibodies EN4 and H4-7/33. In addition, the origin of these cells was confirmed by the demonstration of the cell adhesion molecules, platelet endothelial cell adhesion molecule-1 (CD31), and endothelial leukocyte adhesion molecule-1 (ELAM-1/E-selectin) upon stimulation with tumor necrosis factor (TNF) alpha. HuMMEC were found to express-1 ELAM-1 at lower levels of TNF alpha (< 10 ng/ml) than required by human umbilical vein endothelial cells. These cells should provide a useful in vitro model for studying various aspects of microvascular biology and pathology.
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PMID:Isolation and characterization of microvessel endothelial cells from human mammary adipose tissue. 768 48

The pulmonary vasculature is of great physiological/pathological significance. We have isolated and cultured microvessel endothelial cells (HuLEC) from lung tissue obtained from lung transplant recipients by modification of published methods. Pure cultures of HuLEC were isolated by mechanical disaggregation of the tissue prior to sequential dispase and trypsin digestion to obtain microvessel fragments. Magnetic beads (Dynabeads) coated with Ulex europaeus agglutinin-1 were then used to enhance the purity of cultures at the first passage. HuLEC formed contact-inhibited "cobblestone" monolayers on gelatin and fibronectin substrates and capillary-like "tubes" on Matrigel and accumulated acetylated low-density lipoprotein. Immunofluorescent characterization of these cells revealed the presence of von Willebrand Factor, angiotensin-converting enzyme, and thrombomodulin and the expression of antigens for the endothelial cell-specific monoclonal antibodies EN4, PAL-E, and H4-7/33. The endothelial origin of these cells was confirmed by the demonstration of the cell adhesion molecules, platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), and E-selectin (endothelial leukocyte adhesion molecule-1/ELAM-1) upon stimulation with TNF alpha. These cells should provide a useful tool for studying various aspects of pathology and biology of the pulmonary microvasculature in vitro.
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PMID:Human lung microvessel endothelial cells: isolation, culture, and characterization. 841 55

Tumor growth beyond a certain size requires angiogenesis. Experimental evidence shows that once tumors leave the pre-angiogenic phenotype to become angiogenic, metastases often start to evolve. The aim of this study was to develop a reproducible immunohistochemical technique and method to characterize the neovascularization in archival prostate cancer tissue by quantifying the microvessel density (MVD). Archival tumor specimens from 64 consecutively diagnosed prostate cancer patients were immunostained for von Willebrand Factor (vWF), endothelial antigen and for CD31 combined with the use of different digestive enzymes (trypsin and pronase) and heating in a microwave oven. Both the mean and the maximal MVD, and the reproducibility of the method were estimated. Finally, the mean MVD, the maximal MVD, and clinical characteristics were correlated with the crude survival of the patient population. The immunohistochemical staining for vWF to measure the maximal MVD was found to be a reproducible method of characterizing the individual tumor. Both a univariate and a multivariate analysis demonstrated that the maximal MVD, in contrast to the mean MVD, was significantly associated with survival in prostate cancer patients. We conclude that evaluation of angiogenesis by immunostaining the endothelial cells for vWF measured by the MVD in the most vascularized areas of the tumor is a reproducible method of characterizing the individual prostate tumor. Maximal MVD proved to be an independent prognostic parameter useful in conjunction with other known prognostic markers in human prostate cancer.
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PMID:Immunohistochemical determination of tumor angiogenesis measured by the maximal microvessel density in human prostate cancer. 963 68

Microvascular endothelial cells (MVEC), which differ from large vessel endothelial cells, have been isolated successfully from lungs of various species, including man. However, contamination by nonendothelial cells remains a major problem in spite of several technical improvements. In view of the organ specificity of MVEC, endothelial cells should be derived from the tissue involved in the diseases one wishes to study. Therefore, to investigate some of the immunopathological mechanisms leading to acute respiratory distress syndrome (ARDS), we have attempted to isolate lung MVEC from patients undergoing thoracic surgery for lung carcinoma and patients dying of ARDS. The method described here includes four main steps: (1) full digestion of pulmonary tissue with trypsin and collagenase, (2) aggregation of MVEC induced by human plasma, (3) Percoll density centrifugation, and (4) selection and transfer of MVEC after local digestion with trypsin/EDTA under light microscopy. Normal and ARDS-derived lung MVEC purified by this technique presented contact inhibition (i.e., grew in monolayer), and expressed classical endothelial markers, including von Willebrand factor (vWF), platelet endothelial cell adhesion molecule 1(PECAM-1, CD31), and transcripts for the angiotensin converting enzyme (ACE). The cells also formed capillarylike structures, took up high levels of acetylated low-density lipoprotein (Ac-LDL), and exhibited ELAM-1 inducibility in response to TNF. Contaminant cells, such as fibroblasts, smooth muscle cells, or pericytes, were easily recognized on the basis of morphology and were eliminated by selection of plasma-aggregated cells under light microscopy. The technique presented here allows one to study the specific involvement and contribution of pulmonary endothelium in various lung diseases.
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PMID:An improved method for isolation of microvascular endothelial cells from normal and inflamed human lung. 971 12

Angiogenesis, defined as the growth of new vessels from pre-existing vessels, involves microvascular rather than large vessel endothelial cells. Accordingly, microvascular endothelial cell (MEC) proliferation assays are an appropriate in-vitro model of angiogenesis. We have developed a method for the isolation and long-term culture of large numbers of MEC from the human myometrium, tissue readily available from hysterectomy specimens. Human myometrial MEC were positively selected from tissue dissociated sequentially with collagenase and trypsin using Ulex europeaus antigen-1 (UEA)-coated dynabeads. Cultured myometrial MEC displayed characteristic endothelial phenotype and function for up to 14 passages: cobblestone morphology, formed capillary-like tubes on Matrigel, expressed CD31, Factor VIII-related antigen, bound UEA lectin, incorporated 1,1'-dioctadecyl-1,3,3,3', 3'-tetramethylindocarbocyanine perchlorate-labelled acetylated low density lipoprotein, migrated and proliferated in response to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), but not epidermal growth factor. Optimal growth of human myometrial MEC occurred in a simple medium comprising M199, 5 ng/ml bFGF, 15% human serum, 5% fetal calf serum (FCS) and heparin. Human serum was essential for growth, although there was a synergistic effect when FCS was included. Almost identical dose-response curves were obtained for bFGF- and VEGF-induced myometrial MEC proliferation in early and late passage cells. Therefore myometrial MEC are a good model for in-vitro studies of uterine angiogenesis, since they have a stable phenotype and proliferative responsiveness to VEGF and bFGF for up to 14 passages.
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PMID:Isolation, characterization and long-term culture of human myometrial microvascular endothelial cells. 1065 98

The isolation and long-term culture of murine endothelial cells (ECs) has often proven a difficult task. In this paper we describe a quick, efficient protocol for the isolation of microvascular endothelial cells from murine tissues. Murine lung or heart are mechanically minced and enzymatically digested with collagenase and trypsin. The single cell suspension obtained is then incubated with an anti-CD31 antibody, anti-CD105 antibody and with biotinylated isolectin B-4. Pure EC populations are finally obtained by magnetic bead separation using rat anti-mouse Ig- and streptavidin-conjugated microbeads. EC cultures are subsequently expanded and characterised. The surface molecule expression by the primary cultures of murine EC obtained from lung and heart tissue is analysed and compared to that of a murine endothelioma and of primary cultures of murine renal tubular epithelial cells. The phenotype and morphology of these cultures remain stable over 10-15 passages in culture, and no overgrowth of contaminating cells of non-endothelial origin is observed at any stage.
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PMID:Isolation of endothelial cells from murine tissue. 1103 33

Pericytes cover the abluminal surface of capillaries and venules and are thought to play an important role in microvascular regulation and pathology. The purpose of this study was to isolate and characterize human dermal microvascular pericytes (HDMPC), a minor cell type in the skin but a relatively easily obtainable human source of tissue. We developed and compared two procedures that differed in the preselection method. Isolation of dermal microvessel fragments from neonatal foreskins by trypsin digestion was followed by mechanical release of subepidermal tissue, collagenase treatment, and sieving through 100- and 30-microm meshes. After subcultivation, pericytes were preselected either by isolation of outgrowing capillary fragments or by 3G5-coupled magnetic beads. Pericytes were selected finally by cultivation of single cells in endothelial cell-conditioned media. Cultured HDMPC were seen to be large and well spread with irregular edges and prominent stress fibers. They lack contact inhibition, are positive for 3G5 antigen, alpha-smooth muscle actin, and vimentin, and are negative for the endothelial cell marker CD31, diI-acetylated low-density lipoprotein uptake, cytokeratin 5, 6, and 18, and S100 protein. Using both preselection methods, we could establish purified cell cultures of HDMPC. The results of these studies represent the first report of HDMPC isolation.
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PMID:Isolation and in vitro characterization of human dermal microvascular pericytes. 1125 95

In an attempt to identify novel diagnostic markers for mast cell (MC)-proliferative disorders, serial bone marrow (bm) sections of 22 patients with mastocytosis (systemic indolent mastocytosis, n = 19; mast cell leukemia [MCL], n = 1; isolated bm mastocytosis, n = 2) were analyzed by immunohistochemistry using antibodies against CD2, CD15, CD29, CD30, CD31, CD34, CD45, CD51, CD56, CD68R, CD117, HLA-DR, bcl-2, bcl-x(L), myeloperoxidase (MPO), and tryptase. Staining results revealed expression of bcl-x(L), CD68R, and tryptase in neoplastic MCs (focal dense infiltrates) in all patients. Mastocytosis infiltrates were also immunoreactive for CD45, CD117 (Kit), and HLA-DR. In most cases, the CD2 antibody produced reactivity with bm MCs in mastocytosis, whereas in control cases (reactive bm, immunocytoma, myelodysplastic syndrome), MCs were consistently CD2 negative. Expression of bcl-2 was detectable in a subset of MCs in the patient with MCL, whereas no reactivity was seen in patients with SIM or bm mastocytosis. Mastocytosis infiltrates did not react with antibodies against CD15, CD30, CD31, CD34, or MPO. In summary, our data confirm the diagnostic value of staining for tryptase, Kit, and CD68R in mastocytosis. Apart from these, CD2 may be a novel useful marker because MCs in mastocytosis frequently express this antigen, whereas MCs in other pathologic conditions are CD2 negative.
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PMID:Immunohistochemical properties of bone marrow mast cells in systemic mastocytosis: evidence for expression of CD2, CD117/Kit, and bcl-x(L). 1138 74

Coronary microvascular endothelial cells exert (patho)physiological effects on the function of cardiac myocytes, which may be studied experimentally using pure cell populations. As an essential pre-requisite to the investigation of cells from gene-modified mice, we studied the phenotypic properties of coronary microvascular endothelial cells isolated from normal mice, and biochemically characterized the superoxide production by these cells. Microvascular endothelial cells were isolated from devitalized mouse ventricular tissue after sequential digestion with collagenase, trypsin and DNase. Coronary microvascular endothelial cells were separated from cardiac myocytes and other cells by differential centrifugation, plating and culture. Mouse coronary microvascular endothelial cells showed an irregular "cobblestone" morphology at confluence, were >98% positive for CD31 by FACS analysis, and were also positive for VE-cadherin and endothelial-type nitric oxide synthase (eNOS) by confocal microscopy. The cells took up fluorescently labelled, acetylated low-density lipoprotein, but were negative for a alpha -smooth muscle actin, desmin and cytokeratin. Unlike human endothelial cells, mouse coronary microvascular endothelial cells only weakly expressed von Willebrand factor. Immunoblotting showed that the mouse cells expressed components of a phagocyte-type NADPH oxidase. They exhibited NADPH-dependent O(2)(-)-generating activity, which was increased by angiotensin II but completely inhibited by diphenyleneiodonium. Thus, mouse coronary microvascular endothelial cells express both eNOS and NADPH oxidase, interactions between which may play a role in endothelial cell pathophysiology.
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PMID:Phenotypic properties and characteristics of superoxide production by mouse coronary microvascular endothelial cells. 1144 17

The topography and phenotype of mast cells in the human area postrema, together with correlation between mast-cell density and microvessel density (MVD), were analysed in 16 brains. Transverse serial sections of formalin-fixed, paraffin-embedded brainstems were stained with toluidine blue and alcian blue/safranin stainings, and with anti-tryptase and anti-CD31 monoclonal antibodies. The mean (+/- SD) numbers of mast cells per section were 1.3 +/- 0.8 and 1.2 +/- 0.7 with toluidine blue and alcian blue/safranin, respectively, whereas anti-tryptase monoclonal antibody showed a mean of 5.1 +/- 2.4 cells. Mast cells were alcian blue- and safranin-positive in 56%, because of the coexistence of low-sulphated (blue-staining) and high-sulphated (red-staining) granules. No significant linear correlation between mast-cell density (4.9 mm(-2)) and MVD (114.5 mm(-2)) was found (r(2) = 0.19, P = 0.09). Mast cells were frequently located close to blood vessels (55%) (33% to venules, 22% to arterioles), indicating that their products play a role in the regulation of blood flow and in vessel permeability in the area postrema. Mast cells were located subependymally in 44% and close to the dorsal aspect of the nucleus of the tractus solitarius in 31%, suggesting a subregional distribution.
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PMID:The distribution of mast cells in the human area postrema. 1503 21


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