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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)

Long-term cultures of retinal capillary endothelial cells (RCEC) and of pericytes were grown from collagenase-treated calf retinal vessels. By the use of mechanical separation and differential growth response in various media, pure cell lines were derived from cloned capillary cells as well as from multiple vessel fragments. RCEC and pericytes appeared different under phase contrast microscopy. In addition, RCEC produced factor VIII antigen and angiotensin-converting enzyme, and pericytes did not. RCEC preferred to grow in tumor-conditioned medium, whereas pericytes preferred a standard tissue culture medium supplemented with calf serum. Extracellular matrix, together with tubular structures, developed in postconfluent RCEC but not in pericytes. This study demonstrates that it is possible to grow long-term, pure cultures of RCEC and of pericytes that possess distinctive morphology, growth, and synthetic capabilities in vitro.
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PMID:Retinal vascular endothelial cells and pericytes. Differential growth characteristics in vitro. 629 92

We studied the extracellular localization of factor VIII-related antigen (VIIIR: Ag) in cultures of human endothelial cells. The cells deposited both VIIIR: Ag and fibronectin already during their initial adhesion phase and in immunofluorescence microscopy of spread cells extracellular VIIIR: Ag was localized to fibrils coaligning with pericellular fibronectin. When human fibroblasts, which do not synthesize VIIIR: Ag, were cultured in endothelial cell post-culture medium, a fibrillar matrix localization of VIIIR: Ag was seen, comparable to that of endothelial cell cultures. A fibrillar VIIIR: Ag-specific staining was also seen in cell-free pericellular matrices of endothelial cells, produced by deoxycholate treatment. In immunoelectron microscopy, VIIIR: Ag was seen in fibrillar extracellular material between and underneath the cells and in cell-free matrices of of endothelial cells as well. In immunofluorescence microscopy of cell-free matrices, VIIIR: Ag codistributed with both fibronectin and type III procollagen. Digestion of the matrices with purified bacterial collagenase abolished the type III procollagen-specific fluorescence, whereas the fibrillar VIIIR: Ag-specific staining, codistributing with fibronectin, remained unaffected. In electrophoresis of isolated, metabolically labelled endothelial cell matrices, major polypeptides with Mr 220-240; 180; 160 and 45 kD and some minor polypeptides were resolved. In addition, immunoblotting revealed fibronectin, VIIIR: Ag and type III procollagen as components of cell-free matrices of endothelial cells. Direct overlay of iodinated cellular fibronectin on electrophoretically separated polypeptides of cultured endothelial cells, transferred to nitrocellulose, suggested that fibronectin binds directly to VIIIR: Ag. Our results indicate that VIIIR: Ag produced by human endothelial cells is a component of the pericellular matrix and is not bound to collagen but may directly associate with fibronectin.
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PMID:Factor VIII-related antigen. A pericellular matrix component of cultured human endothelial cells. 631 62

Autogenous endothelial seeding (AES) of vascular prostheses (VP) using venous endothelial cells (EC) reduces platelet-VP interactions and improves patency rates in small caliber VP in dogs. To conserve patients' veins for use in coronary or limb bypass surgery, human trials of AES should require proof that adequate numbers of EC with the growth capacity to cover VP can be harvested from acceptably small pieces of peripheral vein. EC were isolated from excess saphenous vein segments remaining after coronary bypass surgery by filling veins with 0.1% CLS II collagenase at 37 degrees C for 15 min and removing EC by flushing the veins with culture medium. EC were cultured on fibronectin-coated dishes in medium 199 with 30% human serum and 300 micrograms/ml of endothelial cell growth factor. These cells grew to form confluent monolayers, and were identified as EC by tests for factor VIII antigen. Veins from 53 patients with a mean age of 55.8 +/- 9.8 (SD) years yielded vein segments with an average area of 1.9 +/- 0.6 cm2, from which an average of 5.3 +/- 2.8 X 10(4) cells were removed per cm2 of vein area. EC in culture underwent 14.3 +/- 1.4 population doublings with an average population doubling time of 1.8 +/- 0.3 days (N = 14 cultures), which allowed an 100-fold increase in cell number to occur in 11 to 12 days. These data suggest that the EC available from small vein segments in adult humans have the growth capacity to cover areas comparable in size to the luminal areas of VP commonly used in arterial surgery.
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PMID:Adult human saphenous vein endothelial cells: assessment of their reproductive capacity for use in endothelial seeding of vascular prostheses. 632 16

Colloidal gold is an electron-dense, lyophobic colloid that readily forms a stable electrostatic interaction with a variety of macromolecules. Monodispersed colloids ranging from 3-150 nm in diameter can be produced to provide the researcher with flexibility in selecting the optimally sized probe. Gold labeling of antibodies and lectins has been extensively used to study surface antigens and cell components. Recently, the use of gold labeling has been extended to study receptor-ligand binding, enzyme-substrate reactions, and transcellular pathways. Published applications include gold labeling of metabolites (low-density lipoproteins), enzymes (DNAase and RNAase, RNA polymerase, thrombin, collagenase, elastase), hormones (insulin, epidermal growth factor, glucagon), circulating plasma proteins (asialoglycoprotein, alpha 2-macroglobulin, factor VIII-von Willebrand factor), and endotoxins (tetanus toxin, cholera toxin). This broad spectrum of applications emphasizes the versatility and usefulness of colloidal gold as a probe in areas of cell biology related to receptors, endocytosis, transport, and functions of proteins.
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PMID:Colloidal gold: a pluripotent receptor probe. 635 33

Thirteen adult dogs underwent thoracoabdominal bypass operations with 6-mm, double-velour Dacron grafts 25 to 30 cm long. Experimental grafts were seeded with cultured autologous endothelial cells (n = 7). Unseeded grafts served as controls (n = 6). Endothelial cells were harvested from external jugular vein segments using 0.1% trypsin and 0.5% collagenase solutions. Grafts were studied at weeks 2 and 4. Endothelial cell coverage of experimental graft surfaces after two weeks was 60% to 70%, and approximately 80% after four weeks. Immunofluorescence using factor VIII-related antigen confirmed the graft's inner surface to be endothelium. Endothelial cell coverage in control grafts occurred as pannus ingrowth, and never exceeded more than 10% of the conduit surface. Generation of an early endothelial surface in prosthetic grafts is possible in a canine model using cultured autologous cells.
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PMID:Endothelial cell seeding of prosthetic vascular grafts: early experimental studies with cultured autologous canine endothelium. 644 93

The effect on platelet function of a monoclonal platelet antibody to platelet membrane glycoprotein I was tested. This antibody, AN51, inhibited ristocetin or bovine factor VIII-induced aggregation but did not modify ADP, collagen type I or type III, thrombin or arachidonic acid induced aggregations. Furthermore, the adhesion-aggregation of platelets induced by microfibrils was also inhibited by the antibody. Platelet adhesion to rabbit aorta subendothelium was impaired by the antibody. The persistent adhesion of platelets to collagenase-treated subendothelium was also inhibited. These findings strongly suggested that platelet membrane glycoprotein I could interact with a non-collagenic microfibrillar component of subendothelium. The binding of factor VIII/von Willebrand factor to platelet membrane in the presence of ristocetin was decreased in the binding site for factor VIII/von Willebrand factor to allow platelet adhesion to subendothelium.
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PMID:Monoclonal antibody to human platelet glycoprotein I. II. Effects on human platelet function. 679 59

A procedure for the isolation and long-term in vitro cultivation of endothelial cells from rat cerebral cortical microvasculature is described. Migrating cells emerged from collagenase-treated microvessel fragments as early as 1 to 2 days in culture. Migration continued and marked proliferation began 5 to 7 days after isolation and continued up to 12 to 14 days. Cell colonies developed and consisted of endothelial cells as determined by phase contrast microscopy and cell culture behavior. Proliferation of the endothelial cells was significantly enhanced (3- to 4-fold) with the addition of endothelial cell growth supplement at a concentration of 150 microgram. per ml. Cultures with endothelial cell growth supplement (ECGS) retained their characteristic endothelial morphology for 6 to 8 weeks, after which they exhibited a gradual deterioration and loss of their phenotypic appearance. The endothelial origin of these cells was demonstrated by positive immunofluorescent staining for factor VIII antigen and angiotensin-converting enzyme and lack of binding of rat smooth muscle myosin antibody. Ultrastructural examination of confluent endothelial cell cultures revealed intercellular junctional complexes consisting of gap junctions, punctate fusions, and tight junctions. Since long-term endothelial cell cultures derived from the cerebral microvasculature retain characteristic endothelial cell markers and in vivo markers and in vivo features of brain capillary endothelium, they can serve as a useful model system to characterize microvascular endothelium in a variety of disease states.
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PMID:Primary culture of rat cerebral microvascular endothelial cells. Isolation, growth, and characterization. 704 18

Epithelial cells, microvascular endothelial cells, and fibroblasts have been isolated in culture from normal urinary bladders of Fischer rats. Normal epithelial cells were cultured most efficiently when transitional epithelial sheets were plated on to collagen-coated roller flasks. The epithelial sheets were obtained by two micro-dissection techniques. In the first method, the epithelium was peeled as a large coherent sheet from the submucosal connective tissue following subepithelial injection of a collagenase solution, and after incubation of the bladders in the same enzyme solution. Epithelial sheets with intact basal cell layers were essential for culture success. On collagenous matrices, epithelial differentiation was similar to that in vivo. The in vitro transitional epithelium was composed of three cell layers, namely superficial, intermediate, and basal cells. Basal cells were attached to newly synthesized basal lamina by means of hemidesmosomes. Superficial cells were sealed at their apical lateral membranes by a junctional complex, i.e. a terminal bar. Asymmetric luminal membrane plaques were not apparent. In the second method, the epithelium was separated from the underlying connective tissue after collagenase--trypsin digestion of everted urinary bladders. Although the digest consisted mainly of epithelial cells, these rarely survived the first passage when plated on conventional plastic growth surfaces. After the third culture week, epithelial cells usually died and slowly growing colonies of fibroblasts or large flattened epitheloid cells became apparent. Epitheloid cells were identified by their typical ultrastructure as endothelial cells, showing Weibel--Palade bodies and pinocytotic caveolae. These cells were reactive with antiserum against factor VIII. The free surface of monolayer cultures was non-thrombogenic when incubated in the presence of platelets. Fibroblasts were isolated from heavily contaminated epithelial cell cultures after differential trypsinization. These three cells types represent the normal control cells of an in vitro tumor model for the study of invasiveness. All three cell types are involved in the formation and functional maintenance of the epithelial--stromal junction. The study of cell--cell and cell--matrix interactions may provide important clues for the understanding of tumor invasiveness, a process that starts at the epithelial--stromal junction and proceeds with its destruction.
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PMID:The isolation and characterization in vitro of normal epithelial cells, endothelial cells and fibroblasts from rat urinary bladder. 743 29

To examine potential mechanisms by which hematopoiesis may be regulated by endothelial cells within the bone marrow (BM) microenvironment, we have devised a technique for the in vitro study of the interaction of human BM microvascular endothelial cells (BMEC) with hematopoietic cells. Microvessels isolated by collagenase digestion of spicules obtained from filtered BM aspirate were plated on gelatin-coated plastic dishes, and colonies of endothelial cells grown from microvessel explants were further purified by Ulex europaeus lectin affinity separation. BMEC monolayers isolated by this technique grew in typical cobblestone fashion, stained positively with antibody to factor VIII/von Willebrand factor, and incorporated acetylated LDL. Immunohistochemical studies showed that BM microvessels and BMEC monolayers express CD34, PECAM, and thrombospondin. Incubation of resting BMEC with BM mononuclear hematopoietic cells resulted in the selective adhesion of relatively large numbers of CD34+ progenitor cells and megakaryocytes. The binding of purified BM-derived CD34+ progenitor cells to BMEC was dependent on divalent cations and was partially blocked by antibodies to CD34. IL-1 beta treatment of BMEC monolayers resulted in an increase of CD34+ progenitor cell adhesion by mechanisms independent of CD34 or divalent cations. BMEC exhibit specific affinity for CD34+ progenitor cells and megakaryocytes, suggesting that the BM microvasculature may play a role in regulating the trafficking, proliferation, and differentiation of lineage specific hematopoietic elements, and possibly of pluripotent stem cells within the CD34+ population.
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PMID:Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. 751 3

The influence of laminin on cell cultures derived from unilateral acoustic nerve schwannomas was investigated. Cell cultures were initiated from 12 schwannomas, removed via the enlarged middle cranial fossa approach. Tumor tissue was dispersed by collagenase treatment and cells seeded in uncoated or laminin-coated culture dishes. Confluent cultures were immunocytochemically characterized with antibodies against S-100, CD 68, factor VIII-related antigen and type IV collagen. Cell adhesion in response to different doses of laminin was evaluated with an electronic cell counter. The effect of laminin on cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxy-uridine (BRDU) into cellular DNA. Cells cultured on laminin as substrate appeared more differentiated with long, fusiform, cytoplasmic processes. Cultured cells stained positive for S-100, not for factor VIII-related antigen or CD 68. Only cells cultured on laminin deposited a dense extracellular network of type IV collagen. When laminin was added to the culture medium, cell attachment and proliferation was stimulated in a dose dependent manner. Maximal stimulation of both was observed with a laminin concentration of 50 micrograms/ml, which induced a nearly 2-fold increase in cell attachment and an approximately 66% increase in DNA content. Since laminin is a major component of the extracellular matrix in schwannomas, the possibility exists that laminin is also mitogenic for human neoplastic Schwann cells in situ.
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PMID:Laminin promotes differentiation, adhesion and proliferation of cell cultures derived from human acoustic nerve schwannoma. 757 28


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