Two Distinct Phenotypes of Rat Vascular Smooth Muscle Cells: Growth Rate and Production of Tumor Necrosis Factor-α

Abstract

The monoclonal theory of atherosclerosis postulates that a subpopulation of vascular smooth muscle cells (VSMCs) is selectively expanded in response to pathologic stimuli and accumulates in vascular intima. The purpose of this research was to clone VSMC, determine growth rates of the clones and their ability to release the mitogenic cytokine tumor necrosis factor-α (TNF-α). With approval of the institutional animal care and use committee, VSMCs were isolated and cultured from the thoracic aortas of three rats. To confirm that the cells in primary culture were of smooth muscle origin, they were immunostained with anti-α-smooth muscle-actin antibodies. Single cell-derived individual colonies with uniform appearance were surrounded by cloning rings, released with trypsin, and expanded. Growth rates of the clones were assessed by the mitochondrial dependent reduction of methyltetrazolium (MTT) to formazan after 24-hour stimulation with 10 per cent serum. Additionally, cloned cells were stimulated with 0.1, 1,10, and 20 μg/mL lipopolysaccharide (LPS) for 24 hours, and TNF-α was determined in the culture medium. Data were analyzed by ANOVA. Two clones were isolated that could be divided into categories based on distinctly different morphology: 1) spindle-shaped (SP) or 2) epithelioid-shaped (EP) VSMCs. The SP clone had a growth rate that was 25 per cent higher than the EP clone ( P < 0.05). Also, the SP clone had significantly higher release of TNF-α in response to LPS. For instance, TNF-α released in response to 0.1 μg/mL of LPS in the SP clone was 157 ± 45 pg/mL versus 21 ± 8.5 pg/mL in the EP clone ( P < 0.05). Primary cultures of rat VSMCs are heterogeneous and consist of at least two morphologically distinct cell types. These clones are different in growth rate and cytokine production. It is possible that selective expansion of one of these clones contributes to formation of stenotic vascular lesions.