OBJECTIVE Diabetes mellitus is a known risk factor for atherosclerosis. Because initiation and/or progression of the atherosclerotic process is associated with alterations in vascular smooth muscle cell growth and differentiation, the present studies were conducted to evaluate the effect of diabetes mellitus on the proliferative behaviour of cultured aortic smooth muscle cells. METHODS Male New Zealand White rabbits were made diabetic with a single intravenous injection of alloxan monohydrate (100 mg.kg-1) in saline. Primary cultures of smooth muscle cells were established from thoracic aortic segments of control and diabetic rabbits and used to develop multiple cell strains. The proliferative capability of secondary cultures was determined by measurements of [3H]-thymidine incorporation into DNA, cell counts, and protein content in control and diabetic cultures. The serum dependence of cellular growth was evaluated by incubation of cultured cells in growth medium supplemented with various fetal calf serum concentrations. RESULTS Cultures of diabetic origin incorporated thymidine to a greater extent than control cultures. Although the efficiency of cell attachment was not different between control and diabetic cells, diabetic cells had a shorter population doubling time than control cells [41.08(SEM 4.15) h v 58.08(6.79) h] and achieved higher final densities than control cultures. The serum dependence of smooth muscle cell cultures for viability and growth was different between the two groups. CONCLUSIONS These findings support the hypothesis that diabetes induces changes in vascular smooth muscle cell proliferation which may be associated with the onset or progression of the atherogenic process observed in diabetes.

Technical grade dinitrotoluene (DNT) is a mixture of approximately 76% 2,4DNT, 19% 2,6-DNT and 5% other isomers. Because DNT is commonly used in the manufacture of explosives and several commercial products, concerns have been raised that this chemical might represent a significant occupational hazard (Levine, 1987). Toxicity studies in rodents have shown that prolonged administration of DNT causes cancers of the liver, gall bladder, and kidney and benign tumors of connective tissues. Bond et al. (1981) have proposed that the hepatocarcinogenic effect of DNT is mediated by a toxic metabolite formed by intestinal flora upon reduction of nitrobenzylalcohol, an oxidative metabolite of dinitrotoluene.

Atherosclerotic vascular disease is the most common complication of diabetes mellitus. Enhanced vascular smooth muscle cell proliferation plays a central role in atherosclerotic lesion formation. Studies using explant cultures have demonstrated that aortic smooth muscle cells from rats with experimental or genetic diabetes have enhanced rates of proliferation when compared to controls. However, this method of culture may select for cells with enhanced migratory potential. In the present studies, aortic smooth muscle cells were successfully cultured from control and diabetic rabbits after enzymatic and mechanical dispersion from thoracic aortic segments. The proliferative patterns of control cells were characterized and growth rates of diabetic cells were compared to controls. Primary cultures from control rabbits grew after an initial 5-day lag period to achieve threefold increases in cell number by 9 days. Subcultures of aortic smooth muscle cells entered the logarithmic phase of growth after 2 days, reaching the plateau phase of growth in 5-7 days and achieving three to fourfold increases in cell number. The final density to which cultures grew was not affected by the number of cells attached on day 1 for the range studied. Cells from diabetic rabbits displayed shorter doubling times and reached greater densities at confluence than did cells from controls. These data support the hypothesis that diabetes induces an atherogenic response. The dissociated rabbit aortic smooth muscle cell culture provides a model in which to study diabetes-induced modulation of cell proliferation that is amenable to pharmacological manipulation to investigate agonist and growth factor-induced responses.