Effects of homocysteine on proliferation, necrosis, and apoptosis of vascular smooth muscle cells in culture and influence of folic acid

Background: It is known that hyperhornocysteinemia is associated with an in creased risk of vascular disease, yet little is known about the pathogenic mechanisms underlying the action of homocysteine (Hcy) itself. Methods: We evaluated the effects of Hcy on cell proliferation, apoptosis, and necrosis in smooth muscle cells (SMCs) cultured for 24 h with different amounts of Hcy. The percentage of apoptotic and necrotic cells from the culture was ev aluated using two different techniques: annexin V-FITC and propidium iodide (PI) fluorescence and apoptosis TUNEL assay. Results: The addition of 10 m uM/l of Hcy to the medium was followed by a significant increase in cell pr oliferation and death, through apoptosis and necrosis, respectively. Notwit hstanding this apparent balance, a significant increase was found in the to tal number of cells present in Hcy-treated culture, thus demonstrating a po sitive dose-dependent correlation with Hcy concentrations in the culture me dium. The addition of folic acid to the culture medium significantly reduce d both Hcy concentrations in media and the effects of Hcy on the proliferat ion/apoptosis/necrosis balance of cells in culture. The percentages for apo ptotic cells and for cells with a necrotic morphology continued to increase as Hcy concentrations increased, although the absolute values were lower i n the culture treated than in that not treated with folic acid. Conclusions : In the presence of folic acid, at increasing concentrations of Hcy, the t otal number of cells in culture showed increases far less relevant with res pect to the control. Also the percentage of apoptotic cells to that of cell s with a necrotic morphology, although conserving the tendency to increase to growth of the concentrations of Hcy, have shown absolute values that wer e lower in the folic acid-treated cultures. (C) 2001 Elsevier Science Ltd. All rights reserved.