Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rapid method for the isolation of endothelial cells from canine adrenal glands is described. The technique is based on collagenase digestion of adrenal glands following mechanical tissue disruption. The endothelial cells attach and spread onto plastic petri dishes coated with a commercial attachment factor approximately one hour after seeding. Duration to first passage was approximately one week. Doubling times were 2-3 days. Cells were positively identified with immunoperoxidase staining for von Willebrand factor antigen.
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PMID:Isolation and short term culture of canine adrenal microvascular endothelial cells. 991 26

Experimental evidence has directly implicated matrix metalloproteinases (MMPs) in the remodeling of the stromal tissue surrounding tumors. Thus, MMP inhibitors could limit the expansion of both neoplastic cell compartment and endothelial cell compartment of a tumor. Much of the work on the role of MMP inhibitors has concentrated on their inhibitory effects on tumor cell invasion. We have examined the effects of a new MMP inhibitor, KB-R7785 (acting on MMP-1, MMP-3, and MMP-9), on tumor angiogenesis and metastasis of murine colon adenocarcinoma (C-26) in two tumor models in BALB/c mice (transparent chamber model and lung colonization model). KB-R7785 has not shown inhibitory effects on in vitro growth of either C-26 or KOP2.16 murine endothelial cells. In vivo, KB-R7785 administrated twice daily for 15 days (100 mg/kg, i.p.), starting the day of tumor inoculation (5 x 10(5) C26 cells) in transparent chamber, has resulted in 88.2% suppression of tumor growth, compared with that in vehicle-administered mice (controls). Tumors grown in controls have doubled their area in 3.3 days, whereas those treated by KB-R7785 progressed almost four times slower (tumor area doubling time, 12 days). KB-R7785 rendered centrally avascular tumors with only a rim of peripheral neovasculature, which had significant lower functional vascular density and vascular area than the corresponding parameters in control tumors 10 days after inoculation [79.9+/-6.7 cm/cm2 versus 164.1+/-10.1 cm/cm2 (P < 0.01) and 19.8+/-1.5% versus 42.6+/-2.7% (P < 0.01), respectively]. In the lung colonization model (tail vein inoculation of 5 x 10(5) C-26 cells), administration of KB-R7785 (100 mg/kg, i.p.) twice daily for 20 days has reduced the number of surface metastasis by 85.8% and abolished the tumor burden, as compared with controls. The few metastatic colonies found in the lungs of KB-R7785 treated mice appeared to be dormant (i.e., staining with von Willebrand factor antibody revealed few, if any, positive cells within the metastatic foci from MMP inhibitor-treated lungs, whereas terminal deoxynucleotidyl transferase-mediated nick end labeling showed a 4-fold increase in the rate of tumor cell apoptosis compared with controls. The fact that KB-R7785 interferes with early steps of angiogenesis and cancer spread suggests that MMP inhibitors may control both primary and secondary tumor growths by limiting the expansion of endothelial cells, as well as cancer cells, composing the tumors.
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PMID:Controlling tumor angiogenesis and metastasis of C26 murine colon adenocarcinoma by a new matrix metalloproteinase inhibitor, KB-R7785, in two tumor models. 1009 56

The avian and mammalian tectorial membranes both contain two non-collagenous glycoproteins, alpha and beta-tectorin. To determine whether variations in the primary sequences of the chick and mouse alpha-tectorins account for differences in subunit composition and matrix structure of the tectorial membranes in these two species, cDNAs spanning the entire open reading frame of chick alpha-tectorin were cloned and the derived amino acid sequence was compared with that of mouse alpha-tectorin. Chick alpha-tectorin shares 73% amino acid sequence identity with mouse alpha-tectorin and, like mouse alpha-tectorin, is composed of three distinct modules: an N-terminal region similar to the G1 domain of entactin, a central region that shares identity with zonadhesin and contains three full and two partial von Willebrand factor type D repeats, and a C-terminal region containing a zona pellucida domain. The central region of chick alpha-tectorin contains fewer potential N-glycosylation sites than that of mouse alpha-tectorin and is cleaved at two additional sites. Differences in the glycosylation and proteolytic processing of chick and mouse alpha-tectorin may therefore account for the variation observed in the composition and structure of the collagenase-insensitive matrices of the avian and mammalian tectorial membranes. In situ hybridisation and Northern blot analysis of chick inner ear tissue indicate that the spatial and temporal patterns of alpha and beta-tectorin mRNA expression in the developing chick inner ear are different, suggesting the two tectorins may each form homomeric filaments.
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PMID:Chick alpha-tectorin: molecular cloning and expression during embryogenesis. 1032 99

Solid tumors depend on angiogenesis for growth and metastasis. It has been shown that blood vessel density, as determined by counting the number of capillaries in clustered bursts, is a significant prognostic factor in carcinomas. It is unclear, however, whether vessel density is a prognostic factor in sarcomas. In this study, we examined angiogenesis in sarcomas of various grades and compared their vascular patterns to those of carcinomas. Microvessels were identified by von Willebrand factor staining. The matrix of multiple sarcoma and breast carcinoma specimens were extracted and subjected to Western analysis of various angiogenic factors and inhibitors. Metalloproteinase inhibitor presence was also determined by in situ hybridization. In breast carcinomas, capillaries were clustered in bursts within the stroma of the tumor, whereas the sarcoma capillaries were homogeneously distributed in the tumor stroma. Random blood vessel density per high power field in sarcomas did not correlate with patient prognosis. The matrix of sarcomas and carcinomas contained both angiogenic stimulators and inhibitors. Tissue inhibitor of metalloproteinase-1 was found predominantly in fibroblasts and myofibroblasts in the matrix of carcinoma specimens. The difference in the pattern of angiogenesis in sarcomas and carcinomas may be attributable to the presence of fibroblasts and myofibroblasts in carcinomas, resulting in the compartmentalization of bursts of angiogenic factors. The homogeneous appearance of vessel density in sarcomas observed in the present study would be the consequence of the influence of a single compartment.
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PMID:Different patterns of angiogenesis in sarcomas and carcinomas. 1058 66

Neovascularization may be necessary for better and longer function of transplanted islets. Vascular endothelial growth factor (VEGF) is known to be one of the most important factors of angiogenesis. Recently, VEGF was reported to be expressed in islets of normal pancreas. We studied the expression of VEGF and neovascularization related peptides in transplanted islets. To determine the angiogenic microcapillary, immunochemical staining was performed for Factor VIII-related antigen (von Willebrand factor [vWF]) and platelet endothelial cell adhesion molecule-1/CD31 (PECAM-1), both of which are known as markers of the angiogenic microvessel. Transplantable islets were isolated from Lewis rats (8-10 weeks of age) by discontinuous dextran gradient after collagenase digestion. Seven to twelve hundred islets were injected into the portal vein (IPV group, n = 7) or transplanted into subnephrocapsular cavity (SNC, n = 12) of the same descent rats. In the IPV group, the liver was resected 1 hour, 1 week, or 4 weeks after transplantation (Tx). In the SNC group, the kidney was resected 1, 3, 7, or 28 days after Tx. Each tissue was fixed in formaldehyde and embedded in paraffin. Serial 4-microm slices were immunostained for insulin, VEGF, PECAM-1, or vWF using specific antibodies. In IPV group, insulin-positive cells were VEGF positive as were in the normal pancreas at all time points. Islets of 1 hour after Tx were barely PECAM-1 positive as were in normal pancreas, but islets became weakly stained at 7 and 28 days after Tx. In vWF staining, transplanted islets showed stronger staining than those in the normal pancreas. In SNC group, VEGF was also stained in insulin-positive cells at 1, 3, 7, and 28 days. In PECAM-1 staining, islets of 1 day after Tx were barely stained as were in normal pancreas. However, the staining was increasingly enhanced from 3 to 7 days and then appeared weakened at 28 days after Tx. In vWF staining, islets were always vWF positive, as was seen in IPV group. This study revealed that PECAM-1 appeared in islets after islet Tx, suggesting that neovascularization occurs within the islet grafts. On the other hand, VEGF of transplanted islet did not obviously vary with time. Enhancement of the neovascularization may lead to better results of islet Tx.
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PMID:Immunohistochemical studies on vascular endothelial growth factor and platelet endothelial cell adhesion molecule-1/CD-31 in islet transplantation. 1097 11

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

Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary.
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PMID:Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue. 1157 14

Coronary microvascular endothelial cells (CMECs) play an important role in many physiological processes. Porcine CMECs from large breed pigs have been isolated and successfully cultured. However, because micropigs offer research advantages over large breed pigs, micropig CMEC (MPCMEC) cultures may be useful as an alternative in vitro porcine model for cardiovascular studies. We isolated MPCMECs from six Panepinto micropigs using a simplified technique and developed a system for their successful culture. MPCMECs were isolated by collagenase digestion of left ventricular samples obtained using sterile techniques. Primary isolates of MPCMECs grew steadily in complete DMEM supplemented with 20% FBS, 4 mM MgSO(4), and 500 microM dibutyryl cAMP and reached confluence in 7-10 days. Endothelial origin was demonstrated by rapid (4-h) uptake of acetylated low-density lipoprotein, immunostaining for the presence of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), von Willebrand factor (vWf)-related antigen, vascular endothelial cadherin (VE-cadherin), endothelial nitric oxide synthase (eNOS), and by positive staining using two fluorescein isothiocyanate-labeled endothelial-specific lectins, Dolichos biflorus agglutinin and Ulex europaeus agglutinin-1. MPCMECs also exhibited immunostaining for alpha-smooth muscle actin. MPCMECs were successfully subcultured in the absence of dibutyryl cAMP and continued to express PECAM-1 and vWf, but not eNOS, to passage six. The typical morphology of subconfluent MPCMECs consisted of elongated cells that grew in a swirling, herringbone pattern.
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PMID:Cultivation and characterization of coronary microvascular endothelial cells: a novel porcine model using micropigs. 1220 52

We examined the role of matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs), and plasminogen activator (PA) in transmyocardial laser revascularization (TMLR)-induced angiogenesis. TMLR was accomplished with a carbon dioxide laser in seven dogs whose left anterior descending coronary artery (LAD) was ligated. Seven control dogs underwent only LAD ligation, and four dogs underwent a sham operation, consisting only of a left thoracotomy. Two weeks later, transmural myocardial samples were harvested from the distributions of the LAD and the left circumflex artery for substrate zymography, immunohistochemical staining, and in situ zymography. MMP-1, MMP-2, TIMP-1, TIMP-2, and urokinase-type PA levels in the distribution of the LAD were higher in the laser group than in the control or sham group. Counts of von Willebrand factor-positive microvessels and smooth muscle alpha-actin-positive arterioles demonstrated that the angiogenesis and ateriogenesis was promoted in the laser group and correlated directly with the number of MMP-stained microvessels. We conclude that TMLR induces the expression of MMPs, TIMPs, and urokinase-type PA and that these proteinases play an important role in angiogenesis after TMLR.
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PMID:Role of MMPs and plasminogen activators in angiogenesis after transmyocardial laser revascularization in dogs. 1238 87

Liver sinusoidal endothelial cells (LSEC) have been reported to express MHC class II, CD80, CD86, and CD11c and effectively stimulate naive T cells. Because dendritic cells (DC) are known to possess these characteristics, we sought to directly compare the phenotype and function of murine LSEC and DC. Nonparenchymal cells from C57BL/6 mice were obtained by collagenase digestion of the liver followed by density gradient centrifugation. From the enriched nonparenchymal cell fraction, LSEC (CD45(-)) were then isolated to 99% purity using immunomagnetic beads. Flow cytometric analysis of LSEC demonstrated high expression of CD31, von Willebrand factor, and FcgammaRs. However, unlike DC, LSEC had low or absent expression of MHC class II, CD86, and CD11c. LSEC demonstrated a high capacity for Ag uptake in vitro and in vivo. Although acetylated low-density lipoprotein uptake has been purported to be a specific function of LSEC, we found DC captured acetylated low-density lipoprotein to a similar extent in vivo. Consistent with their phenotype, LSEC were poor stimulators of allogeneic T cells. Furthermore, in the absence of exogenous costimulation, LSEC induced negligible proliferation of CD4(+) or CD8(+) TCR-transgenic T cells. Thus, contrary to previous reports, our data indicate that LSEC alone are insufficient to activate naive T cells.
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PMID:Liver sinusoidal endothelial cells are insufficient to activate T cells. 1521 Jul 79


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