Proliferation and extracellular matrix production by human infragenicular smooth muscle cells in response to interleukin-1β

Purpose: Atherosclerotic peripheral vascular disease commonly involves the infragenicular arterial tree. Our study evaluated the effect of interleukin (IL)-1 beta on the proliferation of vascular smooth muscle cells (VSMCs) derived from atherosclerotic infragenicular arteries of human subjects who underwent below-knee amputation, as well as the role of IL-1 beta in VSMCs' production of extracellular matrix components, substances that are important in the transformation of VSMCs from the contractile to the synthetic phenotype. This transformation to the synthetic phenotype is an important step in the formation of the atherosclerotic lesion. Methods: Cultures were identified as being of smooth muscle origin through staining with the cytoskeletal marker, alpha-smooth muscle actin. Proliferation assays mere performed by seeding confluent cultures of passages 4 to 7 into six-well plates at 10,000 cells per well. After serum starvation, samples were incubated with IL-1 beta (1 ng/ml). Cell number was determined on a daily basis. To study extracellular matrix production, cells were propagated in tissue culture chamber slides in the absence or presence of growth media containing IL-1 beta. After fixation with 100% methanol, each sample was stained with a primary antibody specific for an extracellular matrix component. After staining with the fluorescein-tagged secondary antibody, each sample was examined using immunofluorescent microscopic examination. Results: The results of our proliferation assays showed that IL-1 beta caused a significant increase in the proliferation of VSMCs at 24, 48, 72, and 96 hours (p less than or equal to 0.003 when comparing IL-1 beta-treated samples with control specimens at each time period using unpaired t test). The number of IL-1 beta-treated cells at 96 hours was double the number present in the control samples (16,033 +/- 235 vs 8102 +/- 824). When compared with control samples, IL-1 beta was found to affect time production of extracellular matrix proteins by infragenicular VSMCs, IL-1 beta caused an increase in the production of fibronectin, a decrease in the production of laminin, and no change in the production of collagen type IV. Conclusions: These results suggest that interleukin-1 beta acts as a potent stimulant of the proliferation of human infragenicular VSMCs. IL-1 beta also acts to augment the production of fibronectin by these cells. Fibronectin has been implicated in the phenotypic transformation of VSMCs from the contractile to the synthetic state. Therefore, IL-IB may serve as an important regulatory factor in the development of atherosclerosis by stimulating the proliferation of VSMCs and their transformation to the synthetic state, two important steps in the formation of time atherosclerotic lesion.