Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 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

Mucinous (colloid) carcinoma and well- to moderately-differentiated adenocarcinoma of the colon differ in the pattern and the amount of mucin secretion and perhaps in their behaviour and clinical outcome. To ascertain why these differences exist and to elucidate the mechanisms of tumour progression, we examined two model human cell lines derived from colorectal mucinous carcinoma (C1a) and moderately differentiated adenocarcinoma (HM3) which show typical pathological and mucin staining patterns of the respective type of carcinomas to nude mouse tumour xenografts. Specifically, we sought to determine if there were quantitative and qualitative differences in mucin synthesis, in mucin gene expression and in biological properties between the two model cell lines. Northern blot analysis showed that MUC2 mRNA levels were significantly higher in C1a cells compared with HM3 cells, while those of MUC3, -5 and -6 mRNA were lower. C1a cells secreted approximately five times more radiolabelled apomucin and 1.5 times more glycosylated apomucin than HM3 cells. When the carbohydrate side-chain length of secreted mucins by these cell lines were examined by beta-elimination followed by P4 column chromatography, C1a mucins had mostly short carbohydrate side-chains, while HM3 cells had predominantly longer side-chains. Western blot analysis of the cell homogenate showed higher expression of MUC2 apomucin and mucin-associated carbohydrate antigens, such as T, Tn and sialyl Tn, with decreased sialyl Le(x) expression in C1a cells compared with HM3. Immunohistochemical analysis of 35 colorectal adenocarcinoma and 25 mucinous colorectal carcinoma tissues also demonstrated increased MUC2 apomucin, T, Tn and sialyl Tn antigens in the mucinous cancer specimens. Examination of the biological properties of these cell lines showed that C1a cells had significantly higher in vitro invasive activity in assays of invasion and collagenase activity and significantly lower E-selectin binding and liver colonisation activities in nude mice. These results indicate that colorectal mucinous carcinoma cells differ considerably from colorectal adenocarcinoma cells, both qualitatively and quantitatively, in the pattern of mucin gene expression and in the synthesis and secretion of mucin. In addition, biological studies showed that mucinous carcinoma cells have a greater degree of invasiveness, but less liver colonising activity. These results suggest that the biological and mucin characteristics of mucinous carcinoma cells contribute to extensive local invasion through tissue stroma as the predominant mechanism of tumour progression, while the biological and mucin characteristics of well- to moderately-differentiated colorectal adenocarcinoma contribute to progression via distant metastasis formation.
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PMID:Mucins secreted by cell lines derived from colorectal mucinous carcinoma and adenocarcinoma. 929 18

A rapid, reproducible method for the isolation of murine endothelial cells (ECs) has been developed. Murine ECs were highly enriched by collagenase digestion of mechanically minced lung and subcutaneous sponge implants followed by specific selection with rat anti-mouse CD31 (i.e., PECAM-1) monoclonal antibody-coated magnetic beads (Dynabeads). Pure EC populations were isolated from primary cultures by a second cycle of immunomagnetic selection. The cells from the lung were then cloned by a limiting-dilution method to exclude the possibility of nonendothelial cell contamination. Of the 300 cells plated, 29 clones (approximately 10%) were obtained. The clones were positive for CD31 as measured by flow cytometry, and one clone from the lungs (1G11) and the cells from sponge implants (designated as SIECs) were then subjected to subsequent culture in vitro for 40 and 30 passages (up to 5 months), respectively. Characterization was performed on cells between passage 3 and 10. Both cell types formed contact-inhibited monolayers on gelatin and capillary-like "tubes" on Matrigel. However, 1G11 cells exhibited a "cobblestone" morphology, whereas SIECs had a fibroblast-like appearance at confluence. By flow cytometry and enzyme-linked immunosorbent assay, these cells constitutively expressed CD31, VE-cadherin (cadherin-5), CD34, ICAM-1, VCAM-1, and P-selectin. After stimulation with 30 ng/mL of tumor necrosis factor-alpha, the cells became positive for E-selectin (at 4 hours poststimulation) and the expression of ICAM-1, VCAM-1, and P-selectin was upregulated (after 24 hours of stimulation). The presence of VE-cadherin in 1G11 cells and SIECs was confirmed by fluorescence microscopy and Northern blot analysis. The phenotype and morphology of both cell types were stable during 5 months of culture, and there was no evidence of overgrowth by contaminating cells. Taken together, the approach outlined herein may provide a general strategy for the isolation and culture of ECs from a variety of murine tissues. The general strategy outlined here is simple, effective, and flexible, allowing the inclusion of further positive or negative selection steps.
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PMID:A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants. 930 41

Certain regions of coronary and other arteries undergo cyclic flexure due to attachment to the heart or crossing of joints. Such motion gives rise to fluctuations in transmural stress and luminal shear stress. It is well known that cyclic variation of these biomechanical forces influences many aspects of vascular cell biology including gene expression. The purpose of this work was to investigate the hypothesis that cyclic flexure of arterial segments influences their gene expression. Bilateral porcine femoral arteries were obtained fresh from the abattoir. One vessel was mounted in an ex vivo perfusion system and subjected to an intraluminal pressure of 60 mmHg and flow of 50 ml/min to serve as a control. The other vessel was mounted in a second perfusion system with similar hemodynamic conditions, but also subjected to controlled cyclic bending consistent with that found in coronary arteries in vivo. Reverse transcriptase-polymerase chain reaction analysis demonstrated that E-selectin and matrix metalloproteinase-1 (MMP-1) were consistently and significantly downregulated in the specimens subjected to 4 h of cyclic bending as compared to the control (n = 8, p < 0.05). Our results show that cyclic flexure of arterial segments in vitro may influence their gene expression. Further investigation should follow this novel observation and focus on other known mediators to more carefully elucidate the consequence of cyclic flexure on arterial pathobiology.
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PMID:Gene expression is altered in perfused arterial segments exposed to cyclic flexure ex vivo. 1037 28

The extravasation of metastatic cells is regulated by molecular events involving the initial adhesion of tumor cells to the endothelium and subsequently the migration of cells in the host connective tissue. E-selectin on endothelial cells and sialyl Lewis X carbohydrate component on tumor cells are mainly involved in the adhesion of colon carcinoma cells to the endothelium of target organ. Interaction of T84 colon cancer cells to purified E-selectin in vitro caused an increase in the tyrosine phosphorylation of a number of proteins as well as the modulation of cellular properties correlated to the metastatic phenotype. Specifically, E-selectin-stimulated actin reorganization, increased collagenase secretion, and induced cell migration. Treatment of T84 cells with herbimycin A inhibited cell adhesion as well as selectin-induced increase of cell migration, and cytoskeleton assembly. Our data demonstrate that binding of cancer cells to E-selectin starts signal transduction pathways which may affect the tumor metastatic abilities.
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PMID:E-selectin modulates the malignant properties of T84 colon carcinoma cells. 1205 73

Intrasplenic administration of a colon adenocarcinoma cell line, colon 26, induced tumor necrosis factor (TNF) alpha protein expression around the central and portal veins of the liver at 3 days, and liver metastases by 24 days after the tumor injection, in 90% of wild-type (WT) mice. To explore the roles of TNF-alpha in the process, we administered colon 26 cells into tumor necrosis factor receptor p55 (TNF-Rp55) knockout (KO) mice. Less than 50% of TNF-Rp55 KO mice developed liver metastasis with significantly lower liver weights and the volumes of metastatic foci. These observations suggest the critical roles of TNF-Rp55-mediated signals in this liver metastasis model. The intrasplenic tumor injection induced mRNA expressions of vascular endothelial growth factor, heparin-binding epidermal growth factor, matrix metalloproteinase-9, and tissue inhibitor of matrix metalloproteinase-1 at similar levels in the livers of both WT and TNF-Rp55 KO mice. Immunohistochemical analyses of the livers of WT mice after tumor injection demonstrated the enhanced expression of vascular cell adhesion molecule (VCAM)-1 and E-selectin on sinusoidal endothelial cells. Enhanced E-selectin expression was similarly observed in the liver of TNF-Rp55 KO mice after tumor injection. However, the enhancement in VCAM-1 mRNA expression and its protein production was significantly attenuated in the liver of TNF-Rp55 KO mice when compared with WT mice. Collectively, these observations suggest that TNF-Rp55-mediated signals can up-regulate both VCAM-1 expression in the liver and subsequent liver metastasis after intrasplenic tumor injection.
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PMID:Essential roles of tumor necrosis factor receptor p55 in liver metastasis of intrasplenic administration of colon 26 cells. 1243 67

Tibolone is a synthetic progestin with estrogenic and progestogenic properties, used to alleviate menopausal syndromes and for osteoporosis prophylaxis in postmenopausal women. However, only little data are available on tibolone and breast cancer risk and on the effects of tibolone on the cardiovascular system. Therefore, in the present in vitro study, we investigated the effect of tibolone on the growth of the human breast cancer cell line, MCF-7, and on the direct effects of tibolone on the vasculature, i.e. human female coronary endothelial and smooth muscle cells. In the breast cancer cell experiments, tibolone was examined alone and in the presence of 0.1 nM estradiol in the concentration range from 0.001 microM to 1 microM. Tibolone lead to significant cell growth in the concentration range of 0.01 micro M to 1 microM and was not able to inhibit estradiol-induced proliferation at the concentrations of 0.01 microM and 0.1 microM. In the vascular endothelial cell culture experiments, tibolone was tested at the concentrations 0.1 microM, 1 microM and 10 microM. Tibolone reduced the synthesis of endothelin as well as the concentrations of E-selectin, PAI-1 and pro-MMP-1. The magnitude of the effects on these markers varied and was in the range of 11%-42%. Concerning smooth muscle cells, tibolone elicited no changes in the proliferation compared to control values. These data suggest that tibolone does have tumour cell-growth promoting effects in vitro. However, tibolone can positively influence the synthesis of markers in cell cultures of human female coronary artery, which modulate vascular tone and which play a decisive role in the various stages of atherosclerosis. Drawing a clinical consequence from our experiments would result in not recommending the use of tibolone in postmenopausal women at high risk for breast cancer development until long-term controlled clinical studies have been performed on the effect of tibolone administration and breast cancer risk. Experimental studies, such as the present one, are useful to explore mechanisms, but clearly cannot replace clinical studies.
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PMID:Effects of tibolone on human breast cancer cells and human vascular coronary cells. 1255 24

Gastrointestinal tract associated lymphoid tissue is considered to be the main replication site for enteroviruses. In order to invade tissues to reach pancreatic islets, cardiac muscles, and other secondary replication sites, the virus has to survive circulation in the blood and find a way to get through endothelial cells. In the present study, the susceptibility of human endothelial cells to infections caused by human parechovirus 1 and several prototype strains of enteroviruses, representing different species (human poliovirus, human enterovirus B and C), and acting through different receptor families was examined. Primary endothelial cells isolated from human umbilical vein by collagenase perfusion and also an established human endothelial cell line, HUVEC, were used. Primary endothelial cells were highly susceptible to several serotypes of enteroviruses (coxsackievirus A13, echoviruses 6, 7, 11, 30, and poliovirus 1). However, coxsackievirus A 9 and echovirus 1 infected only a few individual cells while human parechovirus 1 and coxsackie B viruses did not show evidence of replication in primary endothelial cells. In general, primary endothelial cells were more sensitive to infection-induced cytolytic effect than HUVEC. Activation of endothelial cells by interleukin-1beta did not change the pattern of enterovirus infection. Immunofluorescence stainings of infected primary endothelial cells showed that expression of activation markers, E-selectin, and intercellular adhesion molecule-1, was clearly increased by several virus infections and the former molecule also by exposing cells to UV-light inactivated coxsackieviruses. In contrast, human leukocyte antigen-DR expression was not increased by virus infection.
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PMID:Enterovirus infection and activation of human umbilical vein endothelial cells. 1276 7

Several genes are regulated by tocopherols which can be categorized, based on their function, into five groups: genes that are involved in the uptake and degradation of tocopherols (Group 1) include alpha-tocopherol transfer protein (alpha-TTP) and cytochrome P450 (CYP3A); genes that are associated with lipid uptake and atherosclerosis (Group 2) include CD36, SR-BI and SR-AI/II. Genes that modulate the expression of extracellular proteins (Group 3) include tropomyosin, collagen(alpha1), MMP-1, MMP-19 and connective tissue growth factor (CTGF). Genes that are related to inflammation, cell adhesion and platelet aggregation (Group 4) include E-selectin, ICAM-1, integrins, glycoprotein IIb, II-2, IL-4 and IL-beta. Group 5 comprises genes coding for proteins involved in cell signaling and cell cycle regulation and consists of PPAR-gamma, cyclin D1, cyclin E, Bcl2-L1, p27 and CD95 (Apo-1/Fas ligand). The expression of P27, Bcl2, alpha-TTP, CYP3A, tropomyosin, II-2, PPAR-gamma, and CTGF appears to be up-regulated by one or more tocopherols whereas all other listed genes are down-regulated. Several mechanisms may underlie tocopherol-dependent gene regulation. In some cases protein kinase C has been implicated due to its deactivation by alpha-tocopherol and its participation in the regulation of a number of transcription factors (NF-kappaB, AP-1). In other cases a direct involvement of PXR/RXR has been documented. The antioxidant responsive element (ARE) appears in some cases to be involved as well as the transforming growth factor beta responsive element (TGF-beta-RE). This heterogeneity of mediators of tocopherol action suggests the need of a common element that could be a receptor or a co-receptor, able to interact with tocopherol and with transcription factors directed toward specific regions of promoter sequences of sensitive genes. Here we review recent results of the search for molecular mechanisms underpinning the central signaling mechanism.
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PMID:Regulation of gene expression by alpha-tocopherol. 1531 6

Interactions between members of the TNF ligand superfamily with their cognate TNF receptors play a crucial role in maintaining immune homeostasis in normal individuals, while dysregulation of certain TNF-ligands and receptors contributes to the pathogenesis of autoimmunity. Identification of novel members of the TNF ligand and receptor families will promote our understanding of the pathogenesis of systemic autoimmune diseases, thus facilitating the development of novel therapeutic approaches. TNF-like weak inducer of apoptosis (TWEAK), a recently identified member of the TNF ligand family, induces PGE2, MMP-1, IL-6, IL-8, RANTES, and IP-10 in fibroblasts and synoviocytes, and upregulates ICAM-1, E-selectin, IL-8, and MCP-1 in endothelial cells. The receptor for TWEAK, Fn14, is expressed in various organs including the kidney; it is intriguing that some of these chemokines induced by TWEAK are crucial in the pathogenesis of lupus nephritis. Furthermore, others have described upregulated TWEAK expression on the surface of T cells in human lupus. In this paper we review the possible roles of TWEAK/TWEAK receptor interactions in the pathogenesis of inflammatory and systemic autoimmune diseases, with particular focus on systemic lupus erythematosus. TWEAK blockade may be helpful therapeutically in restoration of tolerance, but is more likely to modify inflammatory damage in target organs.
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PMID:The role of TWEAK/Fn14 in the pathogenesis of inflammation and systemic autoimmunity. 1535 86


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