Heparan sulfate expression is affected by inflammatory stimuli in primary human endothelial cells

Abstract

In diabetes the endothelium is either chronically or transiently exposed to hyperglycemic conditions. In addition, endothelial dysfunction in diabetes is related to changes in the inflammatory response and the turnover of extracellular matrix. This study was undertaken to study the effects of inflammatory stimuli on one particular matrix component, the heparan sulfate (HS) proteoglycans (PGs) synthesized by primary human umbilical cord vein endothelial cells (HUVEC). Such cells were cultured in vitro in 5 mM and 25 mM glucose. The latter concentration was used to mimic hyperglycemic conditions in short-term experiments. HUVEC were also cultured in the presence of the inflammatory agents tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), interleukin 1β (IL-1β) and transforming growth factor β (TGF-β). The cells were labeled with (35)S-sulfate and (35)S-PGs were recovered for further analyses. The major part of the (35)S-PGs was secreted to the medium, irrespective of type of stimuli. Secreted (35)S-PGs were therefore isolated and subjected to further analyses. TNF-α and IL-1α slightly increased the release of (35)S-PGs to the culture medium, whereas IL-1β treatment gave a significant increase. The different treatments neither changed the ratio of (35)S-HS and (35)S-chondroitin sulfate (CS) nor the macromolecular properties of the (35)S-PGs. However, the (35)S-HS chains were slightly increased in size after TNF-α treatment, and slightly decreased after TGF-β treatment, but not affected by the other treatments. Compositional analysis of labeled disaccharides showed changes in the amount of 6-O-sulfated glucosamine residues after treatment with TNF-α, IL-1α and IL-1β. Western immunoblotting showed that major HSPGs recovered from these cells were collagen XVIII, perlecan and agrin, and that secretion of these distinct PGs was increased after IL-1β stimulation. Hence, short term inflammatory stimuli increased the release of HSPGs in HUVEC and affected both the size and sulfation pattern of HS, depending on type of stimuli.