Sulfated proteoglycans synthesized by vascular endothelial cells in culture

Abstract

Metabolically labeled proteoglycans were isolated both from the culture medium and from the cell layer of cultured bovine aortic endothelial cells. Proteoglycans were fractionated by sequential gel filtration on Sepharose CL-4B, DEAE-cellulose column chromatography, and CsCl density gradient centrifugation. The culture medium contained two distinct proteoglycans: a large proteoglycan (Kav = 0.07) of low buoyant density containing heparan sulfate side chains (Mr congruent to 36,000) and a smaller proteoglycan (Kav = 0.45) of high density containing chondroitin sulfate chains (Mr congruent to 20,000). The chondroitin sulfate proteoglycan fraction contained a small amount (less than 10%) of dermatan sulfate. A very similar low density heparan sulfate proteoglycan was extracted from the cell layer with 2% sodium dodecyl sulfate in the presence of enzyme inhibitors. In addition, there was a high density proteoglycan of small size (Kav = 0.43) containing heparan sulfate side chains (Mr congruent to 20,000) in the cell layer. Analyses of proteoglycans synthesized by cultured human umbilical vein endothelial cells gave similar results, except that these cells produced more dermatan sulfate and unidentified oversulfated chondroitin sulfate chains. Morphologically atypical endothelial cells contained reduced levels of the large heparan sulfate proteoglycan. Both indirect immunofluorescence and direct immunoelectron microscopy revealed that the basement membrane-like matrix under monolayers of bovine endothelial cells reacted with antibodies against the basement membrane proteoglycan isolated from a basement membrane-producing tumor. By electron microscopy, this material was shown to consist of a fine filamentous meshwork containing discrete 10-20-nm diameter ruthenium red positive granules resembling those present in basement membranes of intact arteries.