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H Sage

Publications and source records attributed to H Sage.

At least 55 records · Page 3Linked to original sources
PubMed1982

Endothelial cells secrete a novel collagen type in vitro independently of prolyl hydroxylation.

Endothelial cells from bovine aorta, vena cava, and cornea secrete a novel collagen in vitro (Sage et al., 1980). Endothelial collagen (EC), which is sensitive to pepsin and to several neutral proteases, exhibited an additional unusual property in its mode of secretion. In the absence of added sodium ascorbate, EC was secreted by both aortic and corneal endothelial cells at levels which were very similar to those observed in cultures supplemented with this vitamin. In contrast, the secretion of type III procollagen, which normally constitutes 75-80% of collagenous protein in the culture medium, was significantly decreased in ascorbate-deficient cultures. Incubation of aortic endothelial cells with alpha, alpha'-dipyridyl, an inhibitor of prolyl and lysyl hydroxylases, reduced the extent of prolyl hydroxylation in total culture medium protein by 98% but also did not affect the secretion of EC. The secretion of EC by endothelial cells appears to be independent of a requirement for prolyl hydroxylation. This property differs markedly from the secretory characteristics of the interstitial procollagens and more closely resembles that described for type IV (basement membrane) procollagen.

2,2'-Dipyridyl↗
PubMed1982

Collagens of basement membranes.

Recent biochemical and immunohistochemical studies have described several components of basement membranes including heparan sulfate proteoglycan, 2 high molecular weight glycoproteins (fibronectin and laminin), and 2 collagen types (IV and V). These collagens have several properties which distinguish them from other types that are located in the interstitium: (a) type IV forms an amorphous, felt-like matrix, and neither IV nor V is found in large, cross-banded fibrils, (b) both have an increased content of hydrophobic amino acids, (c) the precursor (pro) forms are larger than those of interstitial collagens, (d) type IV contains interruptions within the triple helix, and e) both IV and V are resistant to human skin collagenase but are substrates for selected neutral proteases derived from mast cells, macrophages, and granulocytes. By immunofluorescence staining, type IV collagen has been localized to basement membranes at the dermal-epidermal junction, in capillaries, and beneath endothelial cells in larger vessels. Ultrastructurally it has been shown to be a specific component of the lamina densa. Type V collagen has been localized to the pericellular matrices of several cells types and may be specific for extramembranous structures which are closely associated with basal laminae. Other collagenous proteins have been described which may be associated with the extracellular matrix. One of these is secreted by endothelial cells in culture and by peptide mapping represents a novel collagen type. It is secreted under ascorbate-free conditions and is highly sensitive to proteolytic degradation. It has been proposed that a dynamic reciprocity exists between cells and their extracellular matrix which partially determines cell shape, biosynthesis, migration, and attachment. Examples of phenotypic modulation in several of these phenomena have been shown with endothelial cells grown on different substrates and isolated from different vascular environments.

Basement Membrane↗
PubMed1982

Structure-function relationship in the evolution of elastin.

The evolution of the structure of the rubber-like protein elastin, found in connective tissues which are subjected to periodic physiological stress, was studied with respect to its phylogenetic distribution, fiber morphology and arrangement, response to deformation, and amino acid composition. Aortae and other tissues from several vertebrates and invertebrates were examined for the presence of elastin, which was defined on the basis of a characteristic amino acid composition, the presence of the unique crosslinks desmosine and isodesmosine, and by histologic criteria. The protein was present in all vertebrates except the primitive jawless fishes and was absent from all invertebrates which were examined. In addition, the morphology of aortic elastin fibers differed markedly among the vertebrate families. Biochemical analysis revealed increases in both the degree of crosslinking and hydrophobicity in elastins from higher vertebrates (mammals, birds) as compared to those from bony fish. Mammalian elastin displayed an increased tendency toward coacervation (polymerization into aggregated structures) at 37 degrees C and behaved differently from a conventional elastomer when stretched in a microcalorimeter. Selection for an increasingly hydrophobic elastin appears to have paralleled the development of a highly-pressurized, closed circulatory system in homeothermic animals. The data do not support a common genetic origin for elastin and other connective tissue proteins. Significant variations in amino acid composition among aortic elastins from different species, however, indicate that genetically distinct elastin types could have arisen by divergence from a common ancestral gene.

Amino Acids↗
PubMed1982

Interactions of vascular wall cells with collagen gels.

Culture properties of bovine aortic endothelial cells and bovine aortic smooth muscle cells have been examined in relation to collagen gels. Endothelial cells grown on collagen maintain typical monolayer morphology not only in relationship to the overlying medium but also with respect to the collagen substrate. Endothelial cells placed within a collagen matrix assume a mycelial pattern resembling that of the microvasculature. Overlayment of confluent endothelial cells with collagen induces separation of the cells, changes in morphology, and reinitiation of growth. These shape changes do not require protein synthesis, appear to be independent of fibronectin, are not inhibited by cytochalasin D, but are inhibited by colchicine. Endothelial cell growth can be reinitiated by collagen. This phenomenon appears to be related to a change in cell shape and perhaps to separation of cells at the intercellular junction. In contrast, smooth muscle cells plated on collagen infiltrate the gels and assume a spindle-like, elongated morphologic appearance. Overlayment with collagen does not alter smooth muscle cell shape. Migration into the collagen gels is significantly enhanced when cells are cultured in medium containing platelet-released products, independent of growth stimulation itself. Migration is accompanied by collagen gel degradation. Release of labeled collagen into the medium by smooth muscle cells and appearance of TCA fragments in collagenase assay suggest secretion of collagenase. In summary, endothelial cells, but not smooth muscle cells, are restricted to the surface of collagen gels. The ability of the smooth muscle cells to invade is stimulated by platelet products and may be related to the synthesis of a smooth muscle collagenase.

Animals↗
PubMed1981

Isolation and characterization of a glycoprotein secreted by aortic endothelial cells in culture. Apparent identity with platelet thrombospondin.

A high molecular weight glycoprotein, reported to be secreted by endothelial cells (Sage, H., Crouch, E., and Bornstein, P. (1979) Biochemistry 18, 5433-5442), has been purified to apparent homogeneity from culture medium of adult bovine aortic endothelial cells. Purification was achieved by ammonium sulfate fractionation and successive chromatography on gelatin-Sepharose, Sephacryl S-300, and hydroxylapatite. The glycoprotein was found to be a disulfide-linked oligomer with a subunit molecular weight of 190,000, as judged by its mobility on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels. The endothelial cell-derived protein is distinct from high molecular weight serum glycoproteins such as fibronectin and alpha 2-macroglobulin. However, immunological and structural studies indicate that the Mr = 190,000 glycoprotein is either identical with or closely related to thrombospondin, a glycoprotein contained in platelet granules and released in response to thrombin-induced aggregation.

Amino Acids↗
PubMed1981

Susceptibility of type V collagen to neutral proteases: evidence that the major molecular species is a thrombin-sensitive heteropolymer, [alpha 1(V)]2 alpha 2(V).

The susceptibility of human type V collagen to several neutral proteases was examined. Thrombin cleaved both the alpha 1(V) and alpha 2(V) chains of this protein at 34 degrees C, producing two pairs of fragments with apparent molecular weights of 95000 and 10000 on sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Two-dimensional 125I-labeled peptide mapping of the larger fragments demonstrated that the upper band [which comigrated with alpha 1(I)] was derived from both the alpha 1(V) and alpha 2(V) chains, while the other component [which comigrated with alpha 2(I) was a product of alpha 1(V) alone. Cleavage of type V collagen, containing alpha 3(V) chains, with thrombin produced an analogous pattern with three high molecular weight bands. Chymotrypsin and trypsin cleaved type V collagen at 37 degrees C but not at lower temperatures. Digestion of type V collagen with elastase at 37 degrees C resulted in selective proteolysis of alpha 2(V), leaving alpha 1(V) essentially intact. Pepsin treatment of type V collagen from which alpha 2(V) had been removed by elastase treatment resulted in nearly complete degradation of alpha 1(V). These data support the hypothesis that a major fraction of native type V collagen is a heteropolymer with the chain composition [alpha 1(V)]2 alpha 2(V). Cleavage of type V collagen by thrombin may have physiologic significance in that breakdown of pericellular matrix may be an important step in the response of a tissue to injury.

Chymotrypsin↗
PubMed1981

Characterization of cell matrix associated collagens synthesized by aortic endothelial cells in culture.

Several collagen types have been isolated and characterized from bovine aortic endothelial cells and their associated extracellular matrix. Two collagens, which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the alpha 1(III), alpha 1(V), and alpha 2(V) collagen chains, were isolated by salt precipitation from pepsin digests of cell layer proteins. Two of these chains were further purified by molecular-sieve and ion-exchange chromatography and were identified as alpha 1(III) and alpha 1(V) by one-dimensional peptide maps generated with mast cell protease and cyanogen bromide. In contrast to type III collagen, which was found in both the culture medium and cell layer, type V collagen appeared to be restricted to the cell layer. In addition to their occurrence as cell layer constituents, both types III and V collagens were localized to an extracellular matrix after the cells had been removed from the culture dishes by detergent. Preliminary studies based on peptide maps comparing type III collagen from the cell layer and culture medium provide evidence for structural heterogeneity within this collagen type.

Animals↗
PubMed1981

A new mapping technique for collagen chains.

A new, highly sensitive method for peptide mapping of collagen chains has been developed which utilizes a modification of the radioiodination technique in polyacrylamide gels described by Elder et al. (1977b). Optimal conditions include the use of the Bolton-Hunter reagent to produce 125I-labeled collagen with the enzyme proteinase K, prior to resolution of the cleavage products by two-dimensional electrophoresis and chromatography. Unambiguous results were obtained by restricting comparison among collagens to a given set which was radioiodinated using the same procedure, i.e., in solution, or in a dried or hydrated gel. Iodination of collagens in solution, followed by proteinase K digestion, resulted in highly reproducible maps which were free of background contamination and which permitted characterization of chains with a defined mobility of SDS-PAGE after the iodination procedure. This technique has provided additional evidence that the alpha 1, alpha 2, and alpha 3 chains of type V collagen are structurally unique. In addition, relationships among several fragments from pepsin-treated type IV collagen, which consists of two distinct chains, could be further elucidated.

Collagen↗
PubMed1980

A unique, pepsin-sensitive collagen synthesized by aortic endothelial cells in culture.

A unique collagen, designated EC, has been isolated from the culture medium of adult bovine aortic endothelial cells. After diethylaminoethylcellulose chromatography of [3H]proline-labeled culture medium, three non-disulfide-bonded bacterial collagenase-sensitive components with apparent Mr of 177000 (EC 1), 125000 (EC 2), and 100000 (EC 3) were demonstrated. Molecular sieve chromatography, cyanogen bromide cleavage, and two-dimensional peptide mapping of radioiodinated EC fragments produced by protease digestion suggest that the lower molecular weight components originate from EC 1. Both EC 1 and EC 2 were digested by pepsin within 10 min to products of less than 60000 molecular weight, under conditions which supported only limited proteolysis of other native collagens. A pepsin-resistant fragment of Mr 50000, derived from a digest of EC 2, contained equal amounts of hydroxyproline and proline, suggesting that at least a portion of the endothelial collagen contains a stable, collagen-like triple helix. Comparative mapping using mast cell protease and cyanogen bromide cleavage, followed by polyacrylamide gel electrophoresis, indicates that the primary structure of this collagen differs from that of other known collagen types.

Animals↗
PubMed1980

Studies of morphologically atypical ("sprouting") cultures of bovine aortic endothelial cells. Growth characteristics and connective tissue protein synthesis.

Morphologic and biochemical studies were performed on cultures of bovine aortic endothelial cells which had developed a second growth pattern that has been referred to as "sprouting" (Gospodarowicz and Mecher, '78; Schwartz '78). These morphologically atypical cells undergrew the intact endothelial cell monolayer and appeared only after the cells had reached confluence. They were ultrastructurally very similar to endothelial cells, but synthesized reduced amounts of fibronectin and a predominance of type I procollagen, rather than the types III and IV procollagens synthesized by monolayer endothelial cells. It is suggested that these cells represent phenotypically altered endothelial cells that differ in biosynthesis of secreted proteins and display a reduced contact-inhibition.

Animals↗
PubMed1980

Structural basis for apparent heterogeneity of collagens in human basement membranes: type IV procollagen contains two distinct chains.

Fetal cells isolated from human amniotic fluid synthesize type IV procollagen when grown in monolayer culture. The procollagen, which contains two biochemically distinct chains, was found to be structurally and immunologically related to type IV collagen chains and collagenous fragments isolated from human placenta. Limited pepsin digestion of the intact procollagen that was deposited in the cell layer during culture produced a heterogeneous population of collagenous peptides comparable to that obtained during isolation of type IV collagens from human tissues. These studies support the hypothesis that basement membranes contain at least two genetically distinct type IV procollagen chains and suggest that the heterogeneity of collagenous components obtained after pepsin digestion of tissues and isolated basement membranes can result from degradative cleavage of the procollagen at a limited number of protease-sensitive sites.

Amniotic Fluid↗
PubMed1979

Collagen synthesis by bovine aortic endothelial cells in culture.

Endothelial cells isolated from bovine aorta synthesize and secrete type III procollagen in culture. The procollagen, which represents the major collagenous protein in culture medium, was specifically precipitated by antibodies to bovine type III procollagen and was purified by diethyl-aminoethylcellulose chromatography. Unequivocal identification of the pepsin-treated collagen was made by direct comparison with type III collagen isolated by pepsin digestion of bovine skin, utilizing peptide cleavage patterns generated by vertebrate collagenase, CNBr, and mast cell protease. The type III collagen was hydroxylated to a high degree, having a hydroxyproline/proline ratio of 1.5:1.0. Pulse-chase studies indicated that the procollagen was not processed to procollagen intermediates or to collagen. Pepsin treatment of cell layers, followed by salt fractionation at acidic and neutral pH, produced several components which were sensitive to bacterial collagenase and which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with alpha A, alpha B, and type IV collagen chains purified from human placenta by similar techniques. Bovine aortic endothelial cells also secreted fibronectin and a bacterial collagenase-insensitive glycoprotein which, after reduction, had a molecular weight of 135,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (using procollagen molecular weight standards) and which was not precipitable by antibodies to cold-insoluble globulin or to alpha 2-macroglobulin. Collagen biosynthesis by these cells provides an interesting model system for studying the polarity of protein secretion and the attachment of cells to an extracellular matrix. The presence of type III collagen in the subendothelium and the specific interaction of this protein with fibronectin and platelets suggest the involvement of this collagen in thrombus formation following endothelial cell injury.

Animals↗
PubMed1979

Characterization of a novel collagen chain in human placenta and its relation to AB collagen.

A novel collagen chain, termed alpha C, has been isolated from human placenta by limited pepsin digestion. The collagen containing the alpha C chain copurifies with placental AB collagen during selective salt precipitation but is virtually absent from fetal birth membranes, which contain relatively larger amounts of AB. Both native AB and alpha C-containing collagens are resistant to human skin collagenase under conditions that support cleavage of type I by greater than 90%. The alpha C chain was separated from alpha B by phosphocellulose chromatography and subsequently from alpha P by chromatography on CM-cellulose. Its amino acid composition is distinct from alpha A and alha B although all three chains posses compositional features in common; the carbohydrate content of the alpha C chain was intermediate between those of alpha A and alpha B. Analysis by NaDodSO4-polyacrylamide gel electrophoresis of peptides produced by CNBr cleavage and by limited digestion with the enzyme mast cell protease indicated different and unique products for the alpha A, alpha B, and alpha C chains. The data support the existence of another collagen chain which is related to the alpha A and alpha B chains but which is structurally unique. The proteins containing these chains may in turn comprise a subfamily of collagen isotypes which represents a divergence from and/or specialization of the type IV basement membrane collagens.

Amnion↗
PubMed1979

Studies on the evolution of elastin--I. Phylogenetic distribution.

1. Aortae and other tissues from numerous animals were examined for the presence of the rubbery protein elastin by (a) chemical purification and amino acid analysis, (b) presence of the cross-linking amino acids desmosine and isodesmosine and (c) histological staining. 2. Elastin was found in all vertebrates examined (42 species) with the exception of cyclostomes (3 species). It was absent from all invertebrates tested (14 species). 3. The amino acid compositions of vertebrate elastins showed marked and interesting interspecies variations.

Amino Acids↗
PubMedDate not supplied

Secretory phenotypes of endothelial cells in culture: comparison of aortic, venous, capillary, and corneal endothelium.

Endothelial cells from different tissues display variations in morphology, intercellular junctions, cell surface and growth properties, and in production of basal lamina components, both in vivo and in vitro. We have investigated the spectra of extracellular proteins secreted by bovine endothelial cells cultured from large vessels, cornea, and capillaries. Aortic, venous, and corneal endothelial cells displayed highly similar patterns of protein synthesis as judged by analysis of the culture medium; the major products were fibronectin, a glycoprotein similar or identical to platelet thrombospondin, and Type III procollagen. Ion-exchange chromatography, followed by peptide mapping, confirmed the presence of EC, a novel endothelial collagen previously described in bovine aortic endothelial cell cultures. Minor variations were found in the collagens of the cell layers: Type III, the predominant interstitial collagen, was associated with the basement membrane Types IV and V and, in the case of corneal endothelium, with Type I. In contrast, capillary endothelial cells secreted significantly more collagen than did the aortic, venous, and corneal cells. Approximately 50% of the protein in the culture medium was collagenous and consisted of Types I and III collagen in a ratio of 2:3. These interstitial collagens were the only types detected in capillary cell layers as well. The pattern and overall rate of collagen synthesis by capillary endothelial cells in vitro contrasted significantly with that of the other endothelial cell types and closely resembled that described for cultures of sprouting endothelium. These alterations in secretory phenotype may reflect: 1) a true difference in cell type between capillary and other types of endothelium, 2) differences resulting from cell isolation and initial culture conditions, or 3) a correlation between growth regulation and protein synthesis.

Animals↗
PubMedDate not supplied

Collagen type III induced ex vivo thrombogenesis in humans. Role of platelets and leukocytes in deposition of fibrin.

Exposure of type III collagen coats on plastic cover slips in parallel-plate perfusion chambers to flowing nonanticoagulated human blood resulted in deposition of platelets and fibrin. Blood was drawn directly from an antecubital vein by an occlusive roller pump over the collagen coats in chambers having flow slits of different dimensions, so that wall shear rates of 100, 650, and 2600 s-1 were obtained at 10 ml/min. Coagulation was minimally activated during the passage of blood from the vein to the chamber as shown by fibrinopeptide A levels of 3.7 ng/ml after 5-minute perfusions. The surface coverage with platelets increased from 18% at 100 s-1 to 59% at 2600 s-1, and the corresponding thrombus volumes increased from 2 to 22 microns 3/microns 2, respectively. This contrasted with the coverage with fibrin on collagen, which decreased from 28% at 100 s-1 to 9% at 2600 s-1. Fibrin deposits on the thrombi covered 6% of the surface irrespective of the shear rate, indicating that some of the deposited platelets accelerated the deposition of fibrin. The type III collagen preparation did not activate factor XII and did not possess tissue factor activity, indicating that the surface itself was not procoagulant. However, a correlation between deposited leukocytes and surface coverage with fibrin was observed (r = 0.78, p less than 0.01), suggesting a role for these cells in the deposition of fibrin. The data demonstrate that thrombogenesis is triggered by pure type III collagen, although the deposition of fibrin is not initiated by the collagen itself but presumably by deposited leukocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Coagulation↗