The Vascular Smooth Muscle Cell In Culture

Abstract

When smooth muscle from the aortic media of adult pigs, monkeys and rabbits are dispersed into single cells and seeded into primary culture they are in the contractile phenotype and do not undergo cell division when challenged with 5 or 10% whole blood serum. However, after 6-8 days the isolated cells undergo a spontaneous change in phenotype to a synthetic state whereby their contractility is lost and they become responsive to serum mitogens. This change in function is accompanied by distinct morphological changes and is reversible if the cells are seeded sufficiently dense (5 × 104 -1 × 105/ml) that a confluent monolayer of cells is achieved within 5-7 days of the original change in phenotype. If however the cells are seeded so sparsely (1 × 103 - 5 × 103/ml) that 2-3 weeks of proliferation is necessary for confluence to be achieved then the cells remain permanently in the synthetic phenotype and immediately responsive to serum mitogens upon subculture. While the cells are in the synthetic phenotype, whether proliferating or quiescent, they have a considerably decreased production of prostaglandins E2, F2α and I2. They also have a decreased ability to metabolize low density lipoprotein, a decrease in the specific activity of cholesteryl esterase and an increase in cholesterol ester deposits.The spontaneous change in phenotype from the contractile to synthetic state in culture can be prevented by seeding the contractile smooth muscle sufficiently dense (1 × 106/ ml) that a confluent monolayer is present from day 1. It can also be inhibited by a confluent monolayer of contractile smooth muscle or endothelial cells in co-culture with the sparsely-seeded smooth muscle such that the two cell layers are not in contact but bathed by the same nutrient medium. A factor which inhibits smooth muscle phenotypic change can also be extracted from pig and rabbit aortic tissue and its effect mimicked by sodium heparin at 50 units/ml.It is suggested that smooth muscle phenotype and its control are important but unrecognized factors in atherogenesis.

Hexamethylenebisacetamide selectively inhibits the proliferation of human and rat vascular smooth-muscle cells

“…Taken together, these data show that inhibition of rat VSMC proliferation by HMBA is correlated with retention of SM-I and SM-2 in primary cells and with re-expression of these proteins in passaged cells of altered morphology. This correlation between smooth-muscle-specific MHC and proliferation is consistent with the view that de-differentiation is an essential precursor to proliferation [9]. However, other work has shown that heparin causes retention of smooth-muscle-specific MHCs in primary rat VSMCs, but does not prevent the eventual proliferation of the cells, although the length of the cell cycle is increased (D. J. Grainger, C. M. Witchell, J.…”

“…When arterial VSMCs are dispersed into cell culture, they begin to lose smooth-muscle-specific isoforms of MHC and actin and accumulate their non-muscle equivalents before proliferating [5,8]. VSMCs in the de-differentiated state in vitro resemble the cells found in atheromatous plaques [3], and it has been proposed that de-differentiation is an essential precursor to proliferation [9]. If this hypothesis is correct, the de-differentiation process offers a potential target for the selective inhibition of VSMC proliferation, since a variety of other types of cells proliferate without apparently de-differentiating.…”

Effect of PUFAs on extracellular matrix production and remodeling in vascular smooth muscle cell cultures in an atherosclerotic model

“…All experiments were conducted when cell cultures were at the third or fourth passage Cells were seeded at approximately 2 × 10 4 cells per dish (60 mm) containing 4 mL fresh medium. Cells were identified as vascular SMCs by their hill‐and‐valley configuration at confluence and positive fluorescence staining for smooth muscle actin and myosin . Trypan blue (Sigma) was added to the cells by means of a hemocytometer and flasks were examined under inverted light microscope (Olympus) to count live and dead cells.…”

Terminally Alkylated Heparin. 2. Potent Antiproliferative Agent for Vascular Smooth Muscle Cells

“…SMCs, harvested from the media of rabbit aorta by a collagenase and elastase method as described previously, 10,11 were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco Laboratories Inc., Grand Island, NY) supplemented with 15% fetal bovine serum (FBS, Gibco Lab. Inc.), 50 IU/mL penicillin, 50 µg/mL streptomycin (Flow Lab., Irvine, Scotland), and 2.5 µg/mL amphotericin B (ICN Biomedicals Inc., Aurora, OH).…”