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

Abstract

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.

Disruption of the subendothelial basement membrane during neutrophil diapedesis in an in vitro construct of a blood vessel wall.

“…In vivo, emigrating neutrophils invade the endothelial cell layer and pause atop the basement membrane 3. Although a similar pattern has been reported in extracts ofsubendothelial matrices synthesized by human microvascular endothelial cells, laminin, an additional basement membrane component, cannot be readily detected on SDS gels in these or other systems (40,41,48 until pseudopodia begin to extend through the matrix (1,(14)(15)(16)(17)(18). To determine whether the basement membrane synthesized by cultured endothelial cells retarded neutrophil movement in a similar fashion, the ability of emigrating neutrophils to transmigrate the vessel wall construct was monitored.…”

Exogenous leukocyte and endogenous elastases can mediate mitogenic activity in pulmonary artery smooth muscle cells by release of extracellular matrix-bound basic fibroblast growth factor

“…This first extract was pooled with a second extract using 0.5% NP40 in hypotonic buffer (2 min, 37°C). The first extract caused the cells to become swollen and partially detach from the ECM and the second extraction was used to dissolve the cellular membranes and remove intact nuclei (Kramer et al, 1985;Boudreau and Rabinovitch, 1991). The ECM remained intact, free of cellular debris, and firmly attached to the entire area of the tissue culture well.…”

Chinese hamster ovary cell adhesion to human platelet thrombospondin is dependent on cell surface heparan sulfate proteoglycan.

“…thrombospondin is found in the extracellular matrix of several types of cells such as endothelial cells (3)(4)(5)(6), aortic smooth muscle cells (6), and fibroblasts (7). A major function of platelet thrombospQndin appears to be stabilization of the fibrinogen bridges between platelets formed during platelet aggregation (8)(9)(10).…”