A potent inhibitor of endothelial cell growth and neovascularization has previously been isolated from both bovine and human aorta. In an attempt to determine if the major cellular component of that tissue, smooth muscle cells, synthesized molecules with this activity, we studied the effects of serum-free conditioned medium from bovine aortic smooth muscle on the growth of cultured endothelial cells from bovine aorta. The smooth muscle conditioned medium was found to contain both growth inhibitory and stimulating activities that could be distinguished by their heat stability. The inhibitor is precipitable by ammonium sulfate, heat stable (80° C), and inactivated by dithiothreitol, trypsin, and 2-mercaptoethanol. It has an estimated molecular weight of approximately 35,000 to 40,000 daltons (by column chromatography). It therefore appears that smooth muscle cells produce an inhibitor of endothelial cell growth that belongs to a class of natural endothelial cell growth inhibitors derived from avascular tissues we tentatively term "avasculins." Proof of its identity with the inhibitor isolated from intact aorta (or other tissues) must await purification. heparan sulfate fragments, 78 and others) are known to affect the growth of one or both of these cell types. Inhibitors of EC growth have been identified in a number of avascular tissues, including cartilage, aorta, and ocular tissues.9 -11 One potent factor found in aortic extracts, which is as yet only partially purified, passes through filter membranes with a nominal cutoff of 10,000 daltons and is effective in inhibiting neovascular growth in vivo 12 -14 and EC proliferation in culture.10 ' 12 - 14 In an attempt to determine whether this activity was synthesized by SMC, the major cell type found in the aortic wall, we designed tissue culture experiments to determine the effects of smooth Received May 14, 1984; revision accepted September 20, 1984. muscle conditioned medium (SMCM) on the growth of EC in culture. The data indicate that cultured SMC produced both inhibitory and stimulatory effects on EC growth. The inhibitory factor has properties in common with the EC growth inhibitor extracted from intact aorta.
MethodsWe cultured second to tenth passage EC and SMC from bovine aorta as previously described 10 with slight modifications. Briefly, EC were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with either 10% heat-inactivated calf serum (CS) or 10% heat-inactivated plasma-derived serum (PDS, Hyclone). A standard amount of antibiotics was also added to the growth medium.SMC were cultured in RPMI-1640 supplemented with 10% heat-inactivated CS and with the same antibiotics used for EC. Balb/c 3T3 cells were cultured in DMEM and B16 F10 mouse melanoma cells (from Isaiah Fidler) in Waymouth's medium, both with 10% heat-inactivated CS. All cells were cultured in a humidified atmosphere of 5% CCyair at 37° C.Preliminary growth inhibition assays were done in Linbro 96 well trays (Flow Laboratories, Inc., McLean, Virginia). Stock cultures of E...
