Synthesis and Secretion of Thrombospondin and Fibronectin by Cultured Human Endothelial Cells

Mosher, Deane F.; Doyle, Mary Jean

doi:10.1016/b978-0-12-521980-8.50019-5

Cited by 17 publications

(20 citation statements)

References 44 publications

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“…Cultured human and bovine endothelial cells have recently been shown to synthesize and secrete thrombospondin (18)(19)(20). This finding raised the question of whether thrombospondin, like factor VIII/von Willebrand factor, is specific for platelets and endothelial cells or, like fibronectin, is synthesized by a wide variety of cell types.…”

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confidence: 96%

“…The radioactive postculture medium was removed, centrifuged at 8,000 x g for 2 min to remove cells and debris, and frozen at -35°C. The radioactive cell layers were washed twice, removed with a rubber policeman, pelleted by centrifugation, dissolved by boiling for 5 min in 2% NaDodSO4 containing protease inhibitors as described (20), and frozen until analyzed.In experiments in which the accumulation of thrombospondin antigen was measured by enzyme-linked immunosorbent assay (ELISA), fibroblasts were cultured in minimal essential medium containing 20% rabbit serum in 2-cm2 wells of multiwell plates. When the cells were confluent, the cells were washed, and the medium was replaced with 1 ml of fresh minimal essential medium containing 20% rabbit serum.…”

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confidence: 99%

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confidence: 99%

“…At various times after the medium change, the postculture medium was removed, centrifuged at 8,000 X g for 2 Preparation of Thrombospondin and Rabbit Polyclonal Anti-Thrombospondin Antibodies. Human platelet thrombospondin and rabbit polyclonal anti-thrombospondin were prepared as described (20).…”

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Cultured human fibroblasts synthesize and secrete thrombospondin and incorporate it into extracellular matrix.

Jaffe¹,

Ruggiero²,

Leung³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

254

125

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Thrombospondin, a major glycoprotein released from a granules of thrombin-stimulated platelets, is a disulfidebonded trimer of 160-kilodalton subunits. Cultured human foreskin and fetal lung fibroblasts secreted thrombospondin (determined by enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (15.7 and 5.8 Thrombospondin is a major platelet a granule glycoprotein that is secreted and then partially bound to platelet membranes when human platelets aggregate in response to thrombin (1)(2)(3)(4)(5)(6)(7)(8). Thrombospondin is a 450-kilodalton (kDal) protein and is composed of three large disulfide-linked subunits (9, 10). During platelet aggregation, thrombin-stimulated platelets develop a membrane-bound lectin-like activity (11-13), which originates from a granules and appears to play a role in mediating platelet aggregation by binding to a specific receptor on other platelets (14,15 3.7 X 10'°Bq). The radioactive postculture medium was removed, centrifuged at 8,000 x g for 2 min to remove cells and debris, and frozen at -35°C. The radioactive cell layers were washed twice, removed with a rubber policeman, pelleted by centrifugation, dissolved by boiling for 5 min in 2% NaDodSO4 containing protease inhibitors as described (20), and frozen until analyzed.In experiments in which the accumulation of thrombospondin antigen was measured by enzyme-linked immunosorbent assay (ELISA), fibroblasts were cultured in minimal essential medium containing 20% rabbit serum in 2-cm2 wells of multiwell plates. When the cells were confluent, the cells were washed, and the medium was replaced with 1 ml of fresh minimal essential medium containing 20% rabbit serum. At various times after the medium change, the postculture medium was removed, centrifuged at 8,000 X g for 2 min, and frozen until assayed. 998The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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confidence: 96%

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confidence: 99%

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confidence: 99%

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Cultured human fibroblasts synthesize and secrete thrombospondin and incorporate it into extracellular matrix.

Jaffe¹,

Ruggiero²,

Leung³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

254

125

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“…1. Lanes 1, thc whole digests; lanes 2 and 3, the fibronectin-binding fragments (2) and the fragments bound to ovalhumin -ATli-gel (3). Arrows point to the fibronectin-binding fragments their binding capacity for -fibronectin when incubated in the presence of Ca2+ (Fig.…”

Section: Fibvonectin-binding Domains Of Thrombospondinmentioning

confidence: 99%

Multiple domains are involved in the interaction of endothelial cell thrombospondin with fibronectin

Dardik¹,

Lahav²

1989

European Journal of Biochemistry

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Thrombospondin is a large multifunctional glycoprotein synthesized, secreted and incorporated into the extracellular matrix by several cell types in culture. It is also present in the blood platelet and is secreted following platelet activation. We have previously shown that thrombospondin co-distributes with fibronectin in the extracellular matrix and that it can bind directly to purified fibronectin. In order to elucidate the chemical aspects of thrombospondin incorporation into the extracellular matrix, we studied the interaction of endothelial cell thrombospondin and fibronectin. We find that endothelial cell thrombospondin has two distinct binding domains for fibronectin. One domain is on the 70-kDa core fragment, probably similar to that of platelet thrombospondin. The other domain is on the 27-kDa N-terminal fragment and is unique to endothelial cell thrombospondin. The dissociation constant of the intact endothelial-cell-derived molecule is 0.7 f 0.2 x M. Following fragmentation, the separate domains bind with somewhat lower affinity: the core domain binds with a Kd of 3.4 f 1.5 x lo-' M and the N-terminal domain binds with a Kd of 8.8 f 1.8 x lo-' M. Binding of the intact molecule is CaZ +-independent. By contrast, following tryptic fragmentation. binding of the 70-kDa fragment is practically lost. It can be restored, however, by removal of Ca2+, indicating that the binding site on this domain is either sequestered or becomes so following fragmentation. Heparin, which also binds to both fragments, competed with fibronectin binding to the 27-kDa fragment but not to the 70-kDa domain. The [act that heparin also competitively inhibits fibronectin binding of the intact molecule further supports sequestration of the fibronectin-binding domain on the 70-kDa core fragment. Our data suggest that endothelial-cell thrombospondin possesses two distinct binding sites for fibronectin, a low-affinity constitutively available one and a high-affinity one, possibly sequestered on the intact unbound molecule.

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Synthesis of extracellular matrix glycoproteins by cultured microvascular endothelial cells isolated from the dermis of neonatal and adult skin

Kramer

Fuh

Bensch

et al. 1985

Journal Cellular Physiology

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We examined the synthesis of extracellular matrix macromolecules by human microvascular endothelial cells isolated from the dermis of neonatal (foreskin) and adult (abdominal) skin. Electron microscopy showed that both cell types produced an extracellular matrix that was strictly localized to the subendothelial space. The subendothelial matrices were initially deposited as a single discontinuous layer of filamentous, electron-dense material that progressively became multilayered. Biosynthetic studies indicated that 2-4% of the newly synthesized protein was deposited in the subendothelial matrices by both cell types. Approximately 15-20% of the radiolabeled protein was secreted into the culture medium, and the remainder was confined to the cellular compartment. Biochemical and immunochemical analyses demonstrated the extracellular secretion of type IV collagen, laminin, fibronectin, and thrombospondin by the newborn and adult cells. Whereas type IV collagen was the predominant constituent of the matrix, fibronectin was secreted into the medium, with only small amounts being deposited in the matrix. Thrombospondin was a major constituent of the matrix produced by the newborn foreskin cells but was virtually absent in the matrix elaborated by the adult cells. However, both cell types did release comparable amounts of thrombospondin into their medium. Immunoperoxidase staining for type IV collagen revealed a fibrillar network in the subendothelial matrices produced by both adult and neonatal cells. In contrast, thrombospondin, which was detected only in the matrix of newborn cells, exhibited a spotty and granular staining pattern. The results indicate that the extracellular matrices synthesized by cultured human microvascular endothelial cells isolated from anatomically distinct sites and different stages of development and age are similar in ultrastructure but differ in their macromolecular composition.

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Synthesis and Secretion of Thrombospondin and Fibronectin by Cultured Human Endothelial Cells

Cited by 17 publications

References 44 publications

Cultured human fibroblasts synthesize and secrete thrombospondin and incorporate it into extracellular matrix.

Cultured human fibroblasts synthesize and secrete thrombospondin and incorporate it into extracellular matrix.

Multiple domains are involved in the interaction of endothelial cell thrombospondin with fibronectin

Synthesis of extracellular matrix glycoproteins by cultured microvascular endothelial cells isolated from the dermis of neonatal and adult skin

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