Overexpression of Inducible Nitric Oxide Synthase by Neointimal Smooth Muscle Cells

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Abstract
—The formation of a neointima represents an important repair mechanism in response to vascular injury. It is associated with the expression of a specific set of genes by the intimal smooth muscle cells. Recently, expression of the inducible isoform of NO synthase (iNOS) has been identified 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 injury. 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 analyses likewise revealed that the high production of NO by intimal SMCs was due to overexpression of iNOS. Moreover, the same stimuli induced a higher transcriptional activity in intimal than in medial SMCs, as detected by transfection of a reporter gene under the iNOS promoter. Induction of iNOS led to a reduced proliferation in both medial and intimal SMCs. This inhibitory effect was, however, less pronounced in intimal than in medial SMCs. Similarly, intimal cells were less sensitive to NO-induced inhibition of mitochondrial respiration. When SMC clones were analyzed, there was no correlation between iNOS expression and growth pattern, suggesting that iNOS expression is independent of the morphological 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 resistant 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.