No abstract

Plaque instability associated with acute coronary syndromes results in part from apoptosis and senescence of cells within the atherosclerotic (AS) lesion. Increased cellular oxidative stress has been proposed to contribute to plaque progression and changes in composition, leading to plaque instability. Our objective was to examine the role of NADPH oxidase in smooth muscle cell (SMC) phenotypes associated with an unstable plaque. Aortae were isolated from pre-lesion (8 weeks of age) and post-lesion (35 weeks of age) hypercholesterolemic mice (ApoE-/-/LDLR-/-, AS), and age-matched normal C57BL/6J mice. We observed an age-dependent increase in reactive oxygen species (ROS) in aorta from AS mice, with evidence for elevated ROS prior to lesion development. Whereas macrophage infiltration was restricted to the lesion, oxidized lipids extended beyond the plaque and into the vessel wall. Consistent with these findings, we observed dynamic changes in the expression of NADPH oxidases in AS vessels. Specifically, Nox1 expression was increased early and decreased with lesion progression, while induction of Nox4 was a late event. Nox2 and p22phox were elevated throughout lesion development. Similar to observations in aortae, SMCs isolated from the lesion of AS aortae had decreased Nox1 and increased Nox4 levels as compared to SMCs from normal mice. AS SMCs demonstrated increased generation of ROS, cell cycle arrest, evidence of senescence, and increased susceptibility to apoptosis. Overexpression of Nox4 in normal SMCs recapitulated the phenotypes of the AS SMCs. We conclude that increased expression of Nox4 in AS may drive SMC phenotypes that lead to the plaque instability and rupture responsible for myocardial infarction and stroke.

Objective: Vascular injury causes neointimal hypertrophy, which is characterized by redox-mediated matrix degradation and smooth muscle cell (SMC) migration and proliferation. We hypothesized that, as compared to the adjacent medial SMCs, neointimal SMCs produce increased superoxide via NADPH oxidase, which induces redox-sensitive intracellular signaling to activate matrix metalloproteinase-9 (MMP-9). Methods and Results: Two weeks after balloon injury, rat aorta developed a prominent neointima, containing increased expression of NADPH oxidase and reactive oxygen species (ROS) as compared to the medial layer. Next, SMCs were isolated from either the neointima or the media and studied in culture. Neointimal-derived SMCs exhibited increased Nox1 expression and ROS levels as compared to medial SMCs. Neointimal SMCs had higher cell growth rates than medial SMCs. ROS-dependent ERK1/2 phosphorylation was greater in neointimal SMCs. MMP-9 activity, as detected by gel zymography, was greater in neointimal SMCs under resting and stimulated conditions and was prevented by expression of an antisense to Nox1 or treatment with an ERK1/2 inhibitor. Conclusions: Following vascular injury, the increased expression of Nox1 in SMCs within the neointima initiates redox-dependent phosphorylation of ERK1/2 and subsequent MMP-9 activation.

Abstract-In intact vessels, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) act as an integrated system, possibly through reactive oxygen species (ROS). Using a coculture system we tested whether ECs modulate VSMC redox status by regulating activity of NAD(P)H oxidase and antioxidants. VSMC production of O 2 •Ϫ , H 2 O 2 , and NO was assessed using fluoroprobes and amplex-red. NAD(P)H oxidase subunit expression and oxidase activity were determined by Western blotting and chemiluminescence, respectively. Expression of thioredoxin, SOD, growth signaling pathways (PCNA, p21cip1, CDK4, ERK1/2, p38MAPK) was evaluated by immunoblotting. Thioredoxin activity was assessed by the insulin disulfide reduction assay. In cocultured conditions, VSMC ROS production was reduced by Ϸ50% without changes in NAD(P)H oxidase expression/activity versus monoculture (PϽ0.05). This was associated with decreased cell growth (PϽ0.05). Expression of Cu/Zn SOD and thioredoxin was increased in coculture versus monoculture VSMCs (PϽ0.01). Pretreatment of ECs with L-NAME (NOS inhibitor), NS-398 (Cox2 inhibitor), and HET0016 (20-HETE inhibitor) did not influence VSMC ROS formation, whereas CDNB, thioredoxin reductase inhibitor, abolished ROS modulating effects of ECs. These findings indicate that in a coculture system recapitulating intact vessels, ECs negatively regulate ROS production in VSMCs through thioredoxin upregulation. Functionally this is associated with growth inhibition. The modulatory actions of ECs are independent of NOS/NO, Cox2, and HETE and do not involve NAD(P)H oxidase. Our data identify novel mechanisms whereby ECs protect against VSMC oxidative stress, a process that may be important in maintaining vascular integrity.