Human endothelial cells synthesize, process, and secrete fibronectin molecules bearing an alternatively spliced type III homology (ED1)

Abstract

Cellular fibronectin (Fn) bearing an alternatively spliced extra type III structural repeat (ED1) is normally present at low concentrations in blood plasma. The source of this material remains uncertain. In this study, primary cultures of human umbilical vein endothelial cells (HUVEC) labeled with 35S-methionine were observed to synthesize Fn monomers both with and without this segment. Monomers isolated from cell lysates with antibodies to the ED1 sequence comigrated in nonreduced sodium dodecyl sulfate polyacrylamide gel electrophoresis with the slower (designated M1), but not the faster (designated M2), of two major monomeric populations that were recognized by antibodies raised to plasma-derived Fn. The differences between M1 and M2 were not due to glycosylation, since they were also observed between species of Fn monomer purified from cells grown in the presence of tunicamycin. M1 and M2 were both observed to incorporate with a similar rate into dimeric Fn, indicating that Fn monomers with and without the ED1 site can dimerize with similar efficiency. Analysis of reduced samples of Fn isolated from cells with anti-ED1 antibodies indicated the presence of both M1-M1 and M1-M2 dimers. In addition to being incorporated into extracellular matrix, ED1 + Fn was observed to be secreted in soluble form into the medium, potentially reflecting intravascular release of this protein by endothelial cells in vivo.

Preferential recognition of a fragment species of osteoarthritic synovial fluid fibronectin by antibodies to the alternatively spliced EIIIA segment

“…The anti‐human EIIIA mAb 3E2 (17, 20) and mAb IST‐9 (24) were obtained from Sigma and from Sera Lab (Crowley Down, UK), respectively. Anti‐EIIIA mAb 15/27 and mAb 52/54, derived from sister hybridomas, were purified on protein A–Sepharose (25, 26). The mAb 52/54 was coupled to preactivated Sepharose 4B according to the manufacturer's instructions (Amersham Pharmacia, Piscataway, NJ).…”

Endothelial Dysfunction in Preeclampsia

“…These findings include altered endothelial procoagulant expression, 36 increased concentration of the endothelial pressor, endothelin, [37][38][39][40][41][42][43][44][45][46] increased adhesion molecules [47][48][49] in the circulation, and direct markers of endothelial activation. [50][51][52][53][54][55][56][57][58][59][60][61] Most importantly, several of these markers are increased weeks to months before evident preeclampsia. 35,[62][63][64] On the basis of these findings, the concept that many of the pathophysiological changes of preeclampsia are initiated by altered endothelial function seems reasonable ( Fig.…”

Circulating Factors as Markers and Mediators of Endothelial Cell Dysfunction in Preeclampsia

“…In vitro studies have demonstrated that in addition to being incorporated into extracellular matrix, a soluble form of ED-A cFN is secreted into the medium, potentially reflecting intravascular release of this protein by human endothelial cells in vivo. 92 Cellular fibronectin plays a central role in the adhesion, morphology, and migration of endothelial cells. While it is a major component of the endothelial extracellular matrix, cFN is normally only a minor component of circulating fibronectin and thus is an accurate marker of endothelial cell injury.…”