Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture.

Abstract

Low density vascular smooth muscle (VSM) cell cultures maintained on extracelIular-matrix (ECM)-coated dishes and plated in the presence of either plasma or serum will proliferate actively when serum-containing medium is replaced by a synthetic medium supplemented with three factors: high density lipoprotein (HDL, 250,ug protein/ml) ; insulin (2 .5 pg/ ml) or somatomedin C (10 ng/ml) ; and fibroblast growth factor (FGF, 100 ng/ml) or epidermal growth factor (EGF, 50 ng/ml) . The omission of any of these three factors from the synthetic medium results in a lower growth rate of the cultures, as well as in a lower final cell density once cultures reach confluence . When cells are plated in the total absence of serum, transferrin (10ILg/ml) is also required to induce optimal cell growth .The effects of the substrate and medium supplements on the life span of VSM cultures have also been analyzed . Cultures maintained on plastic and exposed to medium supplemented with 5% bovine serum underwent 15 generations . However, when maintained on ECM-coated dishes the serum-fed cultures had a life span of at least 88 generations. Likewise, when cultures were maintained in a synthetic medium supplemented with HDL and either FGF or EGF, an effect on the tissue culture life span by the substrate was observed . Cultures maintained on plastic underwent 24 generations, whereas those maintained on ECM-coated dishes could be passaged repeatedly for 58 generations . These experiments demonstrate the influence of the ECM-substrate not only in promoting cell growth but also in increasing the longevity of the cultures .Previous studies have shown that bovine vascular smooth muscle (VSM) cells maintained on dishes coated with extracellular matrix (ECM) proliferate equally well when exposed to either plasma or serum (13). Among the plasma components that could affect the proliferation of VSM cells are the high (HDL) and low density lipoproteins (LDL), both of which have been reported to be mitogenic for that cell type when cultures are exposed to lipoprotein-deficient serum (LPDS ; 3, 5, 28) or to serum from abetalipoproteinemic subjects (24). Insulin, epidermal growth factor (EGF), and fibroblast growth factor (FGF) might also affect VSM cell proliferation, inasmuch as these agents have been shown to be mitogenic for a wide variety of cell types maintained under serum-free conditions (1, 2, 31) as well as for VSM cell cultures exposed to plasma (16,18) . Transferrin could also be required (1, 2, 31) to make iron available to the cells.To define which of these factors are important for proliferation of VSM cells, we have studied the proliferative response of low density VSM cell cultures that were plated in the presence of serum or plasma, and then exposed to a synthetic medium supplemented with LDL, HDL, insulin, and FGF, and have compared the effects of the substrate (either ECM or plastic) on the proliferative response to these agents. Our results indicate that VSM cells maintained on ECM-coated dishes proliferate actively when e...

Stimulation of vascular smooth muscle cell prostacyclin and prostaglandin E2 synthesis by plasma high and low density lipoproteins.

“…Gospodarowicz et al and other investigators have reported that plasma factors, including the lipoproteins, may influence the growth characteristics and morphology of vascular smooth muscle cells grown in tissue culture (Gospodarowicz et al, 1981;Tauber et al, 1980Tauber et al, , 1981a. Figure 5 shows phase-contrast photomicrographs illustrating the effects of serumfree M-199, and serum-free M-199 containing HDL 3 , LDL, or lipoprotein-deficient serum (LPDS), on the morphology of the cultured vascular smooth muscle cells over a 24-hour incubation period.…”

Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats.

“…Confluent vascular smooth muscle cells were rendered quiescent and inositol lipids prelabeled to equilibrium by incubation in serum-free medium that contained myo- [2][3] H]inositol (5 /iCi/mmol) for 48 hours. Thereafter, cells were washed three times with phosphatebuffered saline solution, 1 ml of isotonic phosphatebuffered saline solution that contained 50 mM LiCl was added, and preincubation was carried out for 30 minutes at 37° C. After preincubation, cells were exposed to EGF (2 minutes at 37° C) at levels and times shown in the relevant figures (see Results).…”

High-density lipoproteins induce a rapid and transient release of Ca2+ in cultured fibroblasts

“…The proliferation rate has been shown to increase linearly with increasing amounts of HDL present in the culture medium [6,7], but the mechanism underlying the mitogenic effect of HDL is unknown. To verify that HDL3 had a mitogenic effect on the cultured cells in the present system, we measured the incorporation of [3H]thymidine in the presence of different concentrations of HDL3, which was isolated by ultracentrifugation.…”

“…First, they deplete peripheral cells of cholesterol by two different mechanisms: (a) an unspecific exchange of cholesterol molecules between HDL particles and the plasma membrane of cells [1], and (b) binding of apoproteins to specific binding proteins on the cell surface, which induces cholesterol transport from intracellular stores to the plasma membrane, but does not directly promote efflux to the HDL particle [2][3][4][5]. Secondly, HDLs have been reported to be mitogenic for several cell types [6,7].…”