OVEREXPRESSION OF INDUCIBLE NITRIC-OXIDE SYNTHASE BY NEOINTIMAL SMOOTH-MUSCLE CELLS

The formation of a neointima represents an important repair mechanism in response to vascular injury. It is associated with the expression o f a specific set of genes by the intimal smooth muscle cells. Recently , expression oi the inducible isoform of NO synthase (iNOS) has been i dentified in injured arteries during neointimal formation, suggesting that intimal SMCs have a unique mechanism for regulating NO production . Therefore, we have analyzed the expression of iNOS in intimal SMCs. Although first expressed in the media within 1 day after injury, iNOS was confined to neointimal smooth muscle cells at 1 to 2 weeks after i njury. Isolated intimal SMCs were found to consistently reexpress iNOS in reaction to proinflammatory mediators. This was associated with a 5- to 8-fold higher output of NO in comparison with SMCs derived from the media of uninjured arteries. Western blot and Northern blot analys es likewise revealed that the high production of NO by intimal SMCs wa s due to overexpression of iNOS. Moreover, the same stimuli induced a higher transcriptional activity in intimal than in medial SMCs, as det ected by transfection of a reporter gene under the iNOS promoter. Indu ction of iNOS led to a reduced proliferation in both medial and intima l SMCs. This inhibitory effect was, however, less pronounced in intima l than in medial SMCs. Similarly, intimal cells were less sensitive to NO-induced inhibition of mitochondrial respiration. When SMC clones w ere analyzed, there was no correlation between iNOS expression and gro wth pattern, suggesting that iNOS expression is independent of the mor phological phenotype of SMCs. Together, our data show that the intimal SMC is the main iNOS-expressing cell type in the injured artery, that it responds more vividly to iNOS-inducing cytokines because of a more efficient activation of the iNOS promoter, and that it is more resist ant to the actions of NO compared with medial SMCs. Intimal production of NO via the inducible pathway may be important for the restoration of vascular homeostasis after injury.