Smooth muscle cells produce an inhibitor of endothelial cell growth.

Abstract

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...

Effects of porcine aortic smooth muscle cell conditioned medium on endothelial cell replication.

“…Our data demonstrating, in SMC conditioned medium, a nonfilterable inhibitory activity for EC proliferation are consistent with that of Schumacher et al 6 who described a 35 to 40 K dalton peptide in bovine aortic SMC conditioned medium that inhibits growth of proliferating EC. Other investigators have noted briefly that media of cultured SMC from calf pulmonary artery 12 and bovine aorta 1314 stimulate bovine aortic EC 1213 or bovine capillary EC 14 growth in vitro, but the nature of these activities has not been described further.…”

“…Other investigators have noted briefly that media of cultured SMC from calf pulmonary artery 12 and bovine aorta 1314 stimulate bovine aortic EC 1213 or bovine capillary EC 14 growth in vitro, but the nature of these activities has not been described further. Schumacher et al 6 described an EC growth stimulator in whole conditioned medium of bovine aortic SMC as heat labile, but we found that our EC growth-promoting substance, separated from the inhibitor by urtrafiltratjon, was retained after boiling even with visible loss of other materials as precipitates. This EC growth stimulator had a molecular weight less than 1000 daltons, was stable to extremes in pH, was not degraded by trypsin, and was completely stable to repeated freezing and thawing.…”

“…4 There is less information on the effect of SMC on EC. Eisenstein et al 5 found that normal aorta contains an EC growth inhibitor, and they subsequently demonstrated 6 that such an inhibitor is produced by SMC in culture. They noted that SMC conditioned medium also had EC growth-stimulatory activity but did not characterize it beyond establishing its heat lability.…”

Comparison of the phosphorylation events in membranes from proliferating vs. quiescent endothelial cells

“…Though several EC growth factors have been described including an acidic (Lemmon et al, 1982;Gordon et al, 1983;Maciag et al, 1984a) and a basic (Gospodarowicz et al, 1976(Gospodarowicz et al, , 1983 form of fibroblast growth factor, EC growth factor (Maciag et al, , 1984b, and several other acidic mitogens (Barritault et al, 1982;D'Amore and Klagsbrun, 19841, most laboratories successfully culture arterial EC in plasma-or serum-containing media without further supplementation. Several growth inhibitors specific for EC have been reported (Heimark and Schwartz, 1985;Schumacher et al, 1985), including one that is a membrane component of confluent but not subconfluent EC (Heimark and Schwartz, 1985). The mechanism of action of these growth inhibitors has not yet been determined.…”