INDUCIBLE NITRIC-OXIDE SYNTHASE IS PRESENT WITHIN HUMAN ATHEROSCLEROTIC LESIONS AND PROMOTES THE FORMATION AND ACTIVITY OF PEROXYNITRITE

Inflammatory cytokines associated with atherosclerosis may be capable of stimulating the synthesis and activity of inducible nitric oxide sy nthase (iNOS), which could further influence the pathologic features a ssociated with the disease. Although there is a certain amount of indi rect evidence to support the presence of iNOS in atherosclerosis, ther e has been no definitive study to confirm this. This study has assesse d the localization of iNOS within human normal and atherosclerotic ves sels by immunocytochemistry, Western blotting, and in situ hybridizati on. Further, activity of NO synthase has been assessed by detection of nitrotyrosine, which is a marker indicative of the formation and acti vity of the nitric oxide-derived oxidant, peroxynitrite. In Western bl ots of crude homogenates of atherosclerotic aorta, the iNOS antiserum reacted with a band of approximately 130 kd (the known molecular weigh t for iNOS), but no such band was seen in normal aorta. Immunostaining and in situ hybridization confirmed the presence of iNOS in atheroscl erotic vessels, in which it was specifically localized to (CD68-positi ve) macrophages, foam cells, and the vascular smooth muscle. The antis erum to nitrotyrosine reacted with a wide range of protein bands (appr oximately 180 to 30 kd) in Western blots of atherosclerotic aorta. The distribution of immunostaining for nitrotyrosine was virtually identi cal to that seen for iNOS and was present in macrophages, foam cells, and the vascular smooth muscle. In conclusion, these studies have demo nstrated that stimulated expression of iNOS is associated with atheros clerosis and that the activity of this enzyme under such conditions pr eferentially promotes the formation and activity of peroxynitrite. Thi s may be important in the pathology of atherosclerosis, which contribu tes to lipid peroxidation and to vascular damage.