Tissue factor (TF)-producing cells were identified in normal human vessels and atherosclerotic plaques by in situ hybridization and immunohistochemistry using a specific riboprobe for TF mRNA and a polyclonal antibody directed against human TF protein. TF mRNA and protein were absent from endothelial cells lining normal internal mammary artery and saphenous vein samples. In normal vessels TF was found to be synthesized in scattered cells present in the tunica media as well as fibroblast-like adventitial cells surrounding vessels. Atherosclerotic plaques contained many cells synthesizing TF mRNA and protein. Macrophages present as foam cells and monocytes adjacent to the cholesterol clefts contained TF mRNA and protein, as did mesenchymalappearing intimal cells. Significant TF protein staining was found deposited in the extracellular matrix surrounding mRNA-positive cells adjacent to the cholesterol clefts and within the necrotic cores. These results suggest that deposition of TF protein in the matrix of the necrotic core of the atherosclerotic plaque may contribute to the hyperthrombotic state of human atherosclerotic vessels.

Monocytes appear to be central to atherogenesis both as the progenitors of foam cells and as a potential source of growth factors mediating intimal hyperplasia, but the chemical messages which stimulate the influx of monocytes into human atheroma remain unknown. Monocyte chemoattractant protein-i (MCP-1) is a recently described molecule with powerful monocyte chemotactic activity expressed by monocytes, vascular endothelial cells, and smooth muscle cells in culture. To begin to address the role of MCP-1 in vivo, we examined 10 normal arteries and 14 diseased human arteries for MCP-1 expression by in situ hybridization. MCP-1 mRNA was detected in 16% of 10,768 cells counted in human carotid endarterectomy specimens with highest expression seen in organizing thrombi (33%) and in macrophage rich areas bordering the necrotic lipid core (24%) as compared to the fibrous cap (8%) and the necrotic lipid core itself (5%). Based on immunohistochemical staining of serial sections and on cell morphology, MCP-1 mRNA appeared to be expressed by vascular smooth muscle cells (VSMC), mesenchymal appearing intimal cells (MICs), and macrophages. By contrast, few cells expressing MCP-1 mRNA were found in normal arteries (< 0.1%). These data suggest a potential role for MCP-1 in mediating monocytic infiltration of the artery wall. (J.

Recent studies suggest that superoxide production by the NADPH/NADH oxidase may be involved in smooth muscle cell growth and the pathogenesis of hypertension. We previously showed that angiotensin II (Ang II) activates a p22phoxbased NADPH/NADH oxidase in cultured rat vascular smooth muscle cells and in animals made hypertensive by infusion of Ang II. To investigate the mechanism responsible for this increased oxidase activity, we examined p22phox mRNA expression in rats made hypertensive by implanting an osmotic minipump that delivered Ang II (0.7 mg/kg per day). Blood pressure began to increase 3 days after the start of Ang II infusion and remained elevated for up to 14 days. Expression of p22phox mRNA in aorta was also increased after 3 days and reached a maximum increase of 338 +/- 41% by 5 days after pump implantation compared with the value after sham operation. This increase in mRNA expression was accompanied by an increase in the content of the corresponding cytochrome (twofold) and NADPH oxidase activity (179 +/- 11% of that in sham-operated rats 5 days after pump implantation). Treatment with the antihypertensive agents losartan (25 mg/kg per day) or hydralazine (15 mg/kg per day) inhibited this upregulation of mRNA levels and activity. Furthermore, infusion of recombinant heparin-binding superoxide dismutase decreased both blood pressure and p22phox mRNA expression. In situ hybridization of aortic tissue showed that p22phox mRNA was expressed in medial smooth muscle as well as in the adventitia. These findings suggest that Ang II-induced hypertension activates the NADPH/NADH oxidase system by upregulating mRNA levels of one or several components of this oxidase system, including the p22phox, and that the NADPH/NADH oxidase system is associated with the pathology of hypertension in vivo.

Abstract-Restenosis, a frequent complication of coronary angioplasty, is associated with increased superoxide (O 2 · Ϫ ) production. Although the molecular identity of the responsible oxidase is unclear, an NAD(P)H oxidase appears to be involved. In smooth muscle, p22phox and 2 homologues of gp91phox, nox1 and nox4, are expressed, whereas fibroblasts contain gp91phox. To begin investigating the possibility that these oxidase components might contribute to the increased O 2 · Ϫ that accompanies neointimal formation, we measured their expression after balloon injury of the rat carotid artery. The increase in O 2 · Ϫ production 3 to 15 days after surgery was not due to inflammatory cell infiltration but appeared to be derived from medial and neointimal smooth muscle cells and adventitial fibroblasts. Nox1 and p22phox mRNAs were increased 2.7-and 3.6-fold, respectively, at day 3 after injury and remained elevated for 15 days. gp91Phox was increased 7 to 15 days after injury, and nox4 expression was increased 2-fold, but only at day 15 after surgery. These results confirm and extend our previous in vitro data and suggest that in the vasculature, the nox-based NAD(P)H oxidases serve different functions. This dynamic regulation of oxidase components may be critical to smooth muscle phenotypic modulation in restenosis and atherosclerosis. Key Words: neointimal formation Ⅲ superoxide Ⅲ NAD(P)H oxidase Ⅲ balloon injury Ⅲ nox R estenosis is a frequent complication of percutaneous transluminal coronary angioplasty. This pathophysiological process is characterized by arterial wall remodeling and intimal hyperplasia, resulting in luminal narrowing at the site of balloon dilation. Immediately after endothelial denudation, extensive death of medial smooth muscle cells (SMCs) occurs, which is then followed by significant proliferation of the remaining SMCs. 1 These cells, together with activated adventitial cells (myofibroblasts), migrate through the internal elastic lamina to form the neointimal layer. The mechanisms responsible for the cellular events resulting in restenosis are incompletely understood.

Atherosclerosis is associated with reduced endothelium-derived relaxing factor bioactivity. To determine whether this is due to decreased synthesis of nitric oxide synthase (NOS), we examined normal and atherosclerotic human vessels by in situ hybridization and immunocytochemistry by using probes specific for endothelial (ecNOS), inducible (iNOS), and neuronal (nNOS) NOS isoforms, ecNOS was detected in endothelial cells overlying normal human aortas, fatty streaks, and advanced atherosclerotic lesions. A comparison of the relative expression of ecNOS to von Willebrand factor on serial sections of normal and atherosclerotic vessels indicated that there was a decrease in the number of endothelial cells expressing ecNOS in advanced lesions. iNOS and nNOS were not detected in normal vessels, but widespread production of these isoforms was found in early and advanced lesions associated with macrophages, endothelial cells, and mesenchymal-appearing intimal cells. These data suggest that there is (1) a loss of ecNOS expression by endothelial cells over advanced atherosclerotic lesions and (2) a significant increase in overall NOS synthesis by other cell types in advanced lesions composed of the ecNOS, nNOS, and iNOS isoforms. We hypothesize that the increased expression of NOS and presumably NO in atherosclerotic plaques may be related to cell death and necrosis in these tissues.

These data suggest that adventitial myofibroblasts contribute to the process of vascular lesion formation by proliferating, synthesizing growth factors, and possibly migrating into the neointima. Increased synthesis of alpha-smooth muscle actin observed in the adventitial cells after arterial injury may constrict the injured vessel and contribute to the process of arterial remodeling and late lumen loss after angioplasty.

Background— In the arterial endothelium, laminar flow and cyclical stretch induce expression of NO synthase (NOS). We hypothesized that atrial fibrillation (AF) causes a downregulation of atrial endocardial NOS expression and NO · production. Because NO · has antithrombotic properties, this may contribute to thromboembolism in AF. Methods and Results— In pigs, AF was produced with rapid atrial pacing at 600 bpm for 1 week, whereas controls had atrial pacing at 100 bpm. All animals had catheter ablation of the AV junction and ventricular pacing at 100 bpm. NO · production from freshly isolated tissue was measured by a NO · -specific microelectrode. Left atrial basal and stimulated NO · production was decreased in AF by 73% and 71% ( P <0.01). Endocardial NOS expression, determined by Western analysis, was also significantly decreased by 46%. Expression of the prothombotic protein plasminogen activator inhibitor 1 (PAI-1) is known to be regulated by NO · and was increased in the left atrium by 1.8-fold in AF ( P <0.05). NO · concentration was decreased in the left atrial appendage, although NOS expression was not affected. Neither NOS concentration, NO · levels, nor PAI-1 expression were altered in the aortas or right atria of animals with AF. Conclusions— AF is associated with a marked decrease in endocardial NOS expression and NO · bioavailability and an increase in PAI-1 expression in the left atrium. These data suggest that organized atrial contraction is needed to maintain normal endocardial expression of NOS. It is likely that loss of this antithrombotic enzyme contributes to the thromboembolic phenomena commonly observed in AF.

Abstract. Smooth muscle cells (SMC) in rat carotid artery leave the quiescent state and proliferate after balloon catheter injury, but the signals for mitogenesis are not known. In this study, the possibility that cells within damaged arteries produce a growth factor that could act locally to stimulate SMC replication and repair was examined. We found that the genes for PDGF-A and -B (ligand) and PDGF receptor (oe and/~ subunits) were expressed in normal and injured carotid arteries and were independently regulated during repair of carotid injury. Two phases of PDGF ligand and receptor gene expression were observed: (a) In the early stage, a large decrease in PDGF ~-receptor mRNA levels preceded 10-to 12-fold increases in PDGF-A transcript abundance in the first 6 h after wounding. No change in PDGF c~-receptor or PDGF-B gene expression was found at these times. (b) In the chronic phase, 2 wk after injury, neointimal tissue had lower levels of PDGF u-receptor mRNA (threefold) and higher levels of PDGF ~-receptor mRNA (three-to fivefold) than did restored media. Moreover, in situ hybridization studies identified a subpopulation of neointimal SMC localized at or near the luminal surface with a different pattern of gene expression than the underlying carotid SMC. Luminal SMC were strongly positive for PDGF-A and PDGF/3-receptor transcripts, while showing little or no hybridization for PDGF-B or PDGF or-receptor. Immunohistochemical studies showed strongly positive staining for PDGF-A in SMC along the luminal surface. These data show that changes in PDGF ligand and receptor expression occur at specific times and locations in injured carotid artery and suggest that these changes may play a role in regulating arterial wound repair.