Effect of endothelial‐cell‐conditioned medium on proteoglycan synthesis in cultured smooth muscle cells from pig aorta

Abstract

The effect of porcine endothelial-cell-conditioned medium on proteoglycan synthesis by pig aorta smooth muscle cells was studied under serum-free conditions. Maximal stimulation of [35S]-sulfate incorporation (50%) into medium-secreted and cell layer proteoglycans was observed after 20 min and 4 h incubation, respectively. This stimulation can be explained neither by increased secretion nor by oversulfation of medium-secreted [35S]-labeled proteoglycans. Those [35S]-proteoglycans secreted (for 24 h) in the presence of endothelial cell-conditioned medium were characterized by a higher hydrodynamic size than those secreted in the presence of control medium, without modification of glycosaminoglycan chain length. Agreement between the stimulation of incorporation of [35S]-sulfate into glycanic chains (50.1%) and [14C]-serine residues associated with glycosaminoglycans (49.9%) involved an increase in the number of glycanic chains linked to protein cores. The lesser stimulation of [14C]-serine incorporation into secreted proteins (18%) suggested that stimulation of glycosaminoglycan synthesis was not the direct consequence of enhanced protein synthesis. Proteoglycan synthesis was studied in the presence of para-nitrophenyl-beta-D-xyloside. Fractionation of medium-secreted [35S]-proteoglycans and xyloside-initiated glycosaminoglycans revealed that stimulation of [35S]-glycosaminoglycan protein core acceptor for glycanic chain initiation. Our results suggest that the factor(s) secreted by endothelial cells are able to modify smooth muscle cell proteoglycan synthesis by stimulating the first step of protein core glycosylation. This stimulation was accompanied by an increase in proteoglycan hydrodynamic size.

Platelet-derived Growth Factor Inhibits Smooth Muscle Cell Adhesion to Fibronectin by ERK-dependent and ERK-independent Pathways

“…Cell Culture-Primary cultures of SMC were established from media explants of pig aortas and were subsequently cultured up to seven passages in minimum essential medium with Earle's salts containing 2 mM glutamine, 50 units/ml penicillin, 50 g/ml streptomycin, and 10% fetal calf serum (22). Cultures were rendered quiescent by incubation in serum-free medium containing 0.2% (w/v) BSA for 96 h. The cells were placed into fresh medium 24 h before the experiments.…”

Biochemical and functional characterization of proteoglycans produced by SI/SId murine bone marrow stromal cell lines

“…Extracellular matrix growth factor binding studies For the preparation of cell-free ECMs, confluent cultures of bone marrow stromal cells in 25 cm2 tissue culture flasks were first washed for 10 min with sterile, endotoxin-free water, then with 0.125 mM NH,OH, followed by five washes with sterile, endotoxin-free water (Berrou et al, 1988). Preparations of cell-free ECM (Berrou et al, 1988) from Sld3 and GBU6 stromal cell cultures were incubated for 1 hr at 37°C with either 5 x concentrated WEHI-3B conditioned medium or blank culture medium, and then washed three times with culture medium. Blank flasks, in which the plastic substratum had been treated according to the protocol used for ECM preparation, were incubated with WEHI-3B conditioned medium or control medium in an identical manner.…”

Stimulation of large proteoglycan synthesis in cultured smooth muscle cells from pig aorta by endothelial cell‐conditioned medium

“…sulfate-labeled proteoglycans We have previously shown (Berrou et al, 1988) that, in serum-free conditions, ECCM stimulated proteogly- can synthesis by smooth muscle cells without modification of the structural features of their glycosaminoglycans. The increased synthesis of proteoglycan secreted into the medium can be detected as quickly as 20 min after the addition of ECCM to the cells.…”

“…After 20 min incubation, the medium was collected and the radioactivity incorporated into proteoglycans was determined by chromatography on Sephadex G-25 M, PD-10 columns (Berrou et al, 1988).…”

“…We have previously shown (Berrou et al, 1988) that endothelial cells secrete factor(s) that stimulate proteoglycan synthesis by quiescent smooth muscle cells. This stimulation requires a protein acceptor for glycosaminoglycan chain initiation and is accompanied by an increase in proteoglycan hydrodynamic size without modifications of the structural characteristics of their glycosaminoglycan chains.…”