Isolation of vascular smooth muscle cell cultures with altered responsiveness to the antiproliferative effect of heparin.
Smooth muscle cell (SMC) hyperplasia in the arterial wall is an important component of both atherogenesis and post-vascular surgical restenosis. One naturally-occurring group of molecules which can suppress SMC proliferation in animal models and in cell culture systems are the complex carbohydrates of the heparan sulfate class, including heparin. In this communication, we have used retrovirus vectors to introduce several oncogenes into SMC: SV40 Large T antigen (SVLT), polyoma virus Large T antigen (PyLT), v-myc, and adenovirus E1a. We analyzed a total of 11 cultures. A combination of Western blot analysis, immunoprecipitation, and indirect immunofluorescence confirmed the expression of the infected oncogenic protein in each culture we isolated. All four oncogenes permitted the maintenance of a normal SMC phenotype, as assessed by the general morphology of cells in the light microscope and the presence of SMC-specific alpha-actin in an immunofluorescence assay. Doubling times in infected cells ranged from 20 to 33 hr, and final cell densities in infected cultures ranged from 4 x 10(4) to 5 x 10(5) cells per cm2. By comparison, the parent line had a doubling time of 30 hr and reached a final cell density of 1 x 10(5) cells per cm2. Despite the differences sometimes observed in these proliferation parameters, neither one was strongly correlated with heparin responsiveness. PyLT, v-myc, and E1a all produced SMC cultures or lines which retained sensitivity to the antiproliferative activity of heparin (ED50 = 50 micrograms/ml). In contrast, SVLT expression yielded SMC lines which were highly resistant to heparin (ED50 > 300 micrograms/ml). These results suggest that altered responsiveness to heparin is dependent upon which oncogenic protein is being expressed in the cells. The availability of cloned, immortal SMC lines with a wide range of heparin responsiveness should aid in the understanding of the cellular and molecular mechanism of action of this potentially important growth regulator and therapeutic agent.
Caleb
B L
BL
Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Hardenbrook
M
M
Cherington
V
V
Castellot
J J
JJ
D017500:Adenovirus E1A Proteins / Q000235:genetics; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D016259:Genes, myc / Q000502:physiology; D005822:Genetic Vectors / Q000502:physiology*; D006131:Growth Inhibitors / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley; D012190:Retroviridae / Q000235:genetics; D012680:Sensitivity and Specificity; D013539:Simian virus 40 / Q000235:genetics
D017500:Adenovirus E1A Proteins; D000952:Antigens, Polyomavirus Transforming; D005343:Fibrinolytic Agents; D006131:Growth Inhibitors; D006493:Heparin