Heparan sulfate-degrading enzymes induce modulation of smooth muscle phenotype

Macrophages cocultured with rabbit aortic smooth muscle cells at a ratio of 1:3 degraded all the 35S-labeled heparan sulfate proteoglycan from the smooth muscle surface into free sulfate (Kav of 0.84 on Sepharose 6B). Concomitantly, the same macrophages induced a decrease in the volume fraction of myofilaments (Vvmyo) of the smooth muscle cells and a decrease in alpha-actin mRNA as a percentage of total actin mRNA. Both macrophage lysosomal lysate at neutral pH and heparinase degraded cell-free 35S-labeled matrix deposited by smooth muscle cells into fragments which eluted at a Kav of 0.63 and which were identified as heparan sulfate chains by their complete degradation in the presence of low pH nitrous acid. At acid pH the macrophage lysosomal lysate completely degraded the heparan sulfate to free sulfate (Kav 0.84). Both macrophage lysosomal lysate and commercial heparinase at neutral pH induced smooth muscle phenotypic change while other enzymes such as trypsin and chondroitin ABC lyase had no effect. It was therefore suggested that the active factor present in the macrophages is a lysosomal heparan sulfate-degrading endoglycosidase (heparinase). Only a small amount of heparan sulfate-degrading activity was released into the incubation medium by living macrophages, and there was no heparinase activity on their isolated plasma membranes, although proteolytic enzymes were evident in both instances. In pulse-chase studies, high Vvmyo smooth muscle cells were seen to constantly internalize and degrade 35S-labeled heparan sulfate proteoglycan from their own pericellular compartment, suggesting that this may be the mechanism by which smooth muscle phenotype is maintained under normal circumstances and that removal of heparan sulfate from the surface of smooth muscle cells and its degradation by macrophages temporarily interrupts this process, inducing smooth muscle phenotypic change.