The Cytoskeleton of Rat Aortic Smooth Muscle Cells Normal Conditions, Experimental Intimal Thickening, and Tissue Culture zyxw G. GABBIANI zyxwv Department zyxw of Pathology University of Geneva I21 I Geneva 4 Switzerland zyxw It is presently well accepted that smooth muscle cells (SMC) are the major contributor to the formation of atheromatous plaque.'-' However, little is known about morpho- logic and/or biochemical differences between SMC of the media and atheromatous SMC.'*2*S The cytoskeletal features of SMC in the arterial wall have been clarified only recently:"" practically all SMC contain intermediate filaments (IF) composed of vimentin and many (more than 50%) contain IF composed of desmin. However, vascular SMC are characterized by the prevalence of a special a-actin i~otype.~.~ It has been shown that the large majority of rat aortic SMC located in intimal thickening after endothelial injury (the most commonly used model for the atheromatous plaque) contain IF composed only of vimentin.' This suggests that changes in cytoskeletal elements may represent useful markers of SMC adaptation during experimental and possibly human atheromatosis. We have therefore compared the cytoskeletal features of rat aortic SMC during normal conditions, experimental intimal thickening, and in vitro Our results show that the modifications of cytoskeletal elements during intimal thickening or tissue culture furnish new useful information on SMC reaction to stimuli inducing their replication and movement. NORMAL CONDITIONS AND ISOLATED CELLS Total tissues were treated as previously described." Cells were isolated by means of enzymatic dige~ti0n.l~ Vimentin- and desmin-positive cells were identified by means of indirect immunofluorescent staining using affinity-purified polyclonal antibodies against vimentin purified from eye lens and desmin purified from chicken gizzard." For the biochemical evaluation of total actin we first determined the quantities of DNA and protein per mg of tissue in the different specimens;12 then, we evaluated actin as percentage of total protein by densitometric analysis of sodium dodecyl sulfate polyacrylamide gels. These data, correlated with the estimations of DNA and total proteins per mg of tissue, allowed us to calculate the quantities of actin per cell.12The proportion of actin isoforms was calculated in two-dimensional gels according to Quitschke and S~hechter.'~ The values were obtained as percentages of total actin in the same gel. Results using total tissues or isolated cells were similar and hence will be reported together. The percentage of vimentin-positive and vimentin plus desmin-positive cells zy 196