Helene Sage

Evidence from molecular cloning that SPARC, a major product of mouse embryo parietal endoderm, is related to an endothelial cell 'culture shock' glycoprotein of Mr 43,000.

We describe the molecular cloning and characterization of a secreted, acidic, cysteine‐rich glycoprotein (SPARC) of apparent Mr 43,000 which is a major product of mouse embryo parietal endoderm. These cells are specialized for the synthesis of a rapidly expanding basement membrane, but SPARC is not itself an integral matrix component. We show that SPARC is related structurally and antigenically to an Mr 43,000 glycoprotein secreted in large amounts by bovine aortic endothelial cells as part of a ‘culture shock’ response to in vitro conditions promoting their proliferation and migration.

Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice.

SPARC (Secreted Protein that is Acidic and Rich in Cysteine), a Ca++-binding glycoprotein also known as osteonectin, is produced in significant amounts by injured or proliferating cells in vitro. To elucidate the possible function of SPARC in growth and remodeling, we examined its distribution in embryonic and adult murine tissues. Immunohistochemistry on adult mouse tissues revealed a preferential association of SPARC protein with epithelia exhibiting high rates of turnover (gut, skin, and glandular tissue). Fetal tissues containing high levels of SPARC included heart, thymus, lung, and gut. In the 14-18-day developing fetus, SPARC expression was particularly enhanced in areas undergoing chondrogenesis, osteogenesis, and somitogenesis, whereas 10-day embryos exhibited selective staining for this protein in Reicherts membrane, maternal sinuses, and trophoblastic giant cells. SPARC displayed a Ca++-dependent affinity for hydrophobic surfaces and was not incorporated into the extracellular matrix produced by cells in vitro. We propose that in some tissues SPARC associates with cell surfaces to facilitate proliferation during embryonic morphogenesis and normal cell turnover in the adult.

Regulation of Collagen Gene Expression

Publisher Summary This chapter discusses the regulation of collagen protein synthesis of the cell, tissue, or whole animal level. Although in many cases these earlier studies pointed the way to the cellular and molecular biological experiments currently in progress. The collagens are a family of related proteins characterized by the repeating tripeptide sequence Gly-X-Y, in which X is often proline and Y is often hydroxyproline, and a triple-helical structure. This minimal definition of a collagen encompasses the twelve commonly recognized collagen types, a number of different collagens not yet accorded a type number, and several proteins whose structures and functions are sufficiently different from the collagens to warrant their exclusion from the collagen gene family. In addition, the effects of growth and differentiation factors on collagen expression during embryogenesis, tissue remodeling, wound repair, and normal morphogenesis are currently subjects of active study. Control of collagen synthesis in cells is complex and reflects both the translational and pre-translational mechanisms that are dependent on the cellular proliferative state. For most of the mediators discussed in this section, the net effect on collagen mRNA abundance has been shown, but the specific mechanisms remain to be established.

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, 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 the culture medium; the major products were flbronectln, a glycoprotein similar or identical to platelet thrombospondln, 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 culture medium was collagenous and consisted of Types I and III collagen In a ratio 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. (Arteriosclerosis 1:427-442, November/December 1981)

Endothelial cells from umbilical vein and a hemangioendothelioma secrete basement membrane largely to the exclusion of interstitial procollagens.

The biosynthesis of extracellular matrix proteins by primary cultures of endothelial cells from human umbilical vein, and by clones from a murlne hemangioendothelioma, was studied and compared to that reported for endothellum cultured from other sources. Umbilical vein endothelial cells secreted two glycoproteins – flbronectln and thrombospondin – which comprise the major proportion of the protein In the culture media of bovine aortic, venous, and corneal endothelial cells. These blosynthetlc products were absent from hemangioendothelioma cultures. However, In contrast to bovine endothellum from large vessels and cornea, which secrete primarily Type III procollagen Into the culture medium, both the umbilical vein and hemangioendothelioma cultures secrete Type IV (basement membrane) procollagen. In addition, EC, a novel endothelial collagen type that has been characterized in bovine endothelial cell supernates, was not present In the umbilical vein or tumor-derived endothellum. The production of basement membrane procollagen as the major collagen type In the medium of these cultures probably reflects the nature of the vascular bed from which the endothelial cells originated, rather than differences In species or In cellular Isolation and subculture. We suggest that endothelial cells from different vascular environments could display variations In growth, migration, morphology, and response to exogenous blood-borne factors as a result of their relationship to an extracellular matrix/subendothellum composed of diverse structural glycoproteins.

[4] Preparation and characterization of procollagens and procollagen-collagen intermediates

Publisher Summary This chapter describes specific methodology for the purification of the five different procollagen types both from tissues and from sources that synthesize these proteins in vitro. Smooth muscle cell cultures are chosen to isolate procollagen because the cells are easily propagated in tissue culture, they produce relatively large amounts of types I and III procollagen, and they alsosynthesize small but detectable amounts of types IV and V procollagen. Types I and III procollagen purification involves: isolation of smooth muscle cells; metabolic labeling; and ion-exchange chromatography. Because fractionation on DEAE-cellulose is performed under conditions in which procollagen retains its native conformation, it is critical, after removal of the labeled culture medium from the cells, to perform all procedures at 0–4 ° to prevent denaturation of the protein. Type II pro T collagen is prepared from chick sternal cartilages or from matrix-free cartilage cells. After removal of sterna the cells were filtered through lens paper and centrifuged. The culture medium was processed and chromatographed on DEAE-cellulose. Type II procollagen is migrated on SDS-PAGE similarly to type I procollagen before reduction and to the pros α(I) chain after reduction. Type IV procollagen can be prepared and characterized predominantly from three biosynthetic systems: the EHS sarcoma, human amniotic fluid cells, and rat parietal yolk sac. Procedures for isolation of type IV procollagen are the same as those just described for types I and III. In type V(AB) rocollagen purification are excised from fifty 19-day-old chick embryos and incubated in DMEM. Labeling is performed using 50 μCi of [ 3 H]proline. To remove any precipitated material and was chromatographed on a DEAE-cellulose column.

Type VIII collagen in murine development. Association with capillary formation in vitro.

Bovine endothelial and human astrocytoma cells, and a limited number of other normal and malignant cells, synthesize three chains that have been identified as type VIII collagen (180 kDa, 125 kDa, and 100 kDa). Digestion with pepsin converts these forms to major fragments of 65 kD (based on globular protein standards). In this study we have examined the structure and distribution of type VIII collagen in developing mice by immunohistological and immunoblotting techniques. Temporal and tissue-specific expression was observed in embryonic heart, cranial mesenchyme, and placental capillaries. Western blotting of embryonic and neonatal tissues showed major species of 125 and 65 kDa in the brain, placenta, heart, lung, and thymus. The predominant band in pepsin-treated tissues was 60-70 kDa, with additional forms of 250 and 150 kDa in neonatal heart and lung. Type VIII collagen was also synthesized by endothelial cells, forming capillary tubes in vitro. We suggest that type VIII collagen functions in cellular organization and differentiation, and that its various forms reflect not only tissue-specific processing but the presence of several related chains.

Interaction of platelets and purified collagens in a laminar flow model

Damage to the endothelial surface of the vessel wall can result in exposure of circulating blood components to collagen and other subendothelial structures. Collagen types I, III, IV, and V have been demonstrated in the vessel wall by chemical and immunohistological methods; type V is thrombin-sensitive, and is present on the endothelial cell surface. In an earlier study using a rocking model, both unstimulated and ADP-induced platelet adherence was reduced on wells coated with type V collagen in comparison to uncoated wells; and increased on plastic surfaces coated with types III and IV collagen in comparison to those coated with type V collagen. The present study was designed to determine the effect of erythrocytes and shear rate on platelet adherence to these purified collagen types in a laminar flow system. With platelet-rich plasma, adherence of labeled platelets was much lower in the laminar flow system compared with the rocking model. Erythrocytes significantly enhanced platelet adherence to surfaces that were untreated or absorbed with collagen types I, III, and IV. However, this enhancement was not seen in the presence of type V collagen. These studies provide additional evidence for the selectively nonthrombogenic nature of type V collagen.

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.

Diminished platelet adherence to type V collagen.

Different types of collagen vary in their influence on platelet reactivity. Collagen Types III, IV, and V were obtained from human placental tissue, and Type I collagen was prepared from rat skin. Each collagen type was coated onto a plastic surface. Each collagen-coated surface or appropriate plastic surface control was studied using citrated human 51 Cr-labeled platelet-rich plasma in both the presence and absence of 10 μM adenosine 5-diphosphate (ADP). Both unstimulated and ADP-induced platelet adherence were: 1) reduced by Type V collagen coating in comparison to uncoated wells; and 2) increased by Types III and IV collagen coating in comparison to Type V coated or plastic surfaces. Addition of the fast-acting thrombin inhibitor dansylarginine (DAPA) had no significant effect on unstimulated and ADP-induced platelet adherence to Type III, IV or V collagen-coated surfaces. Type I collagen-coated surfaces, studied only in the presence of DAPA, caused greater platelet adherence than those coated with Types III, IV, or V collagen. We conclude that Type V collagen may be less thrombogenic than Types, I, III, or IV.