3-nitrotyrosine and Carotid-Stenosis

Atherosclerosis is a chronic inflammatory vascular disease driven by the subendothelial accumulation of macrophages. The mechanism regulating the inflammatory response in macrophages during atherogenesis remains unclear. Because microRNAs (miRNAs) play a crucial role in cellular signaling by posttranscriptional regulation of gene expression, we studied the miRNA expression profiles during the progression of atherosclerosis.. Using an miRNA real-time polymerase chain reaction array, we found that macrophage-derived miR-342-5p and miR-155 are selectively upregulated in early atherosclerotic lesions in Apoe(-/-) mice. miR-342-5p directly targets Akt1 through its 3'-untranslated region. Akt1 suppression by miR-342-5p induces proinflammatory mediators such as Nos2 and II6 in macrophages via the upregulation of miR-155. The local application of an miR-342-5p antagomir inhibits the development of atherosclerosis in partially ligated carotid arteries. In atherosclerotic lesions, the miR-342-5p antagomir upregulated Akt1 expression and suppressed the expression of miR-155 and Nos2. This reduced Nos2 expression was associated with a diminished generation of nitrotyrosine in the plaques. Furthermore, systemic treatment with an inhibitor of miR-342-5p reduced the progression of atherosclerosis in the aorta of Apoe(-/-) mice.. Macrophage-derived miR-342-5p promotes atherosclerosis and enhances the inflammatory stimulation of macrophages by suppressing the Akt1-mediated inhibition of miR-155 expression. Therefore, targeting miR-342-5p may offer a promising strategy to treat atherosclerotic vascular disease.

Topics: Animals; Aortic Diseases; Apolipoproteins E; Atherosclerosis; Bone Morphogenetic Protein Receptors, Type II; Carotid Stenosis; Chemokine CCL2; DEAD-box RNA Helicases; Disease Progression; Gene Expression Regulation; Interleukin-6; Macrophage Activation; Macrophages; Mice; Mice, Knockout; MicroRNAs; Nitric Oxide Synthase Type II; Oligonucleotides; Proto-Oncogene Proteins c-akt; Ribonuclease III; RNA, Antisense; Signal Transduction; Tyrosine; Up-Regulation; Vasculitis

Oxidative stress is an important determinant in atherosclerosis development. Various markers of oxidative stress, such as oxidation of low-density lipoprotein (LDL), nitrosative stress, lipid peroxidation, and protein oxidation, have been implicated in the initiation and/or progression of atherosclerosis, but their association with plaque erosion and symptomatic carotid disease has not been fully defined. In addition, certain oxidative markers have been shown in various models to promote plaque remodeling through matrix metalloproteinase (MMP) activation.. To perform a global investigation of various oxidative stress markers and assess for potential relationships with destabilization and symptomatic development in human carotid plaques.. Thirty-six patients undergoing endarterectomy were evaluated and compared with 20 control specimens obtained at the time of autopsy. Differences between stable and unstable plaques, symptomatic and asymptomatic patients, and >or=90% and <90% stenosis were evaluated. Oxidized LDL (ox-LDL), nitrotyrosine (NT), malondialdehyde (MDA), and protein carbonyls (PCs) levels were determined in atheromatic plaques homogenates by corresponding biochemical assays. Immunohistochemical (IHC) analysis was also employed to determine the percentage and topological distribution of cells expressing NT and metalloproteinase-9 (MMP-9) in serial sections from corresponding atheromatic plaques. MMP-9 expression was further verified using Western blot analysis.. Ox-LDL was increased in symptomatic patients (P < .05). Also, ox-LDL and NT levels were significantly higher in unstable versus stable carotid plaques (P < .05, respectively). Furthermore, IHC serial section analysis, corroborated by statistical analysis, showed a topological and expressional correlation between NT and MMP-9 (P < .05). MDA and PCs levels, although increased in carotid plaques, did not distinguish stable from unstable carotid plaques as well as symptomatic from asymptomatic patients with various degrees of stenosis.. All types of investigated oxidative stress markers were significantly increased in human carotid plaques, but only ox-LDL levels were associated with clinical symptoms, while peroxynitrite products and MMP-9 were specifically related to plaque instability.

Topics: Aged; Aged, 80 and over; Biomarkers; Blotting, Western; Carotid Artery, Internal; Carotid Stenosis; Case-Control Studies; Disease Progression; Endarterectomy, Carotid; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunohistochemistry; Lipoproteins, LDL; Male; Malondialdehyde; Matrix Metalloproteinase 9; Middle Aged; Oxidative Stress; Prospective Studies; Protein Carbonylation; Rupture; Severity of Illness Index; Tyrosine; Up-Regulation

Topics: Actins; Aged; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Apoptosis; Blood Glucose; Carotid Stenosis; Caspase 3; Caspases; Collagen; Diabetes Mellitus, Type 2; Endarterectomy, Carotid; Female; Glycated Hemoglobin; Humans; Hyperglycemia; In Situ Nick-End Labeling; Interleukin-1; Macrophages; Male; Muscle, Smooth, Vascular; Oxidative Stress; Tumor Necrosis Factor-alpha; Tyrosine

Neointimal proliferation occurring after vascular or endovascular procedures is a major complication leading to end-organ or limb ischemia. In experimental models, balloon injury has been shown to induce NAD(P)H oxidase to produce vascular superoxide anion (O2*-) production, which has been implicated in cell proliferation, but a direct link is still unclear. We postulated that inhibition of arterial NAD(P)H oxidase, resulting in decreased O2*-, would lessen the neointimal hyperplasia caused by balloon injury to the common carotid artery (CCA). Sprague-Dawley rats were implanted with osmotic minipumps containing either vehicle, a cell-permeant peptide that inhibits NAD(P)H oxidase (gp91ds-tat, 10 mg/kg per day), or a scrambled peptide control (scrmb-tat). Two days after pump implantation, the left CCA was injured using an intravascular balloon embolectomy catheter (2F Fogarty). Systolic blood pressure was monitored by tail cuff. Fourteen days after injury, CCAs were harvested and analyzed by digital morphometry. Rats in both groups remained normotensive, with no significant differences in systolic blood pressure. Reactive oxygen species measurements after injury indicated a significant reduction in vascular O2*- in rats infused with gp91ds-tat, and the neointima/media area and thickness ratios were significantly lower in their arteries compared with control. On the contrary, no significant change in overall CCA diameter was observed in any group. Our data indicate that in response to balloon injury of the rat carotid artery, NAD(P)H oxidase activity contributes to neointimal hyperplasia and is involved in vascular cell proliferation and migration during restenosis.

Topics: Angioplasty; Animals; Carotid Arteries; Carotid Stenosis; Enzyme Inhibitors; Glycoproteins; Hyperplasia; Male; NADPH Oxidases; Rats; Rats, Sprague-Dawley; Superoxides; Tyrosine

Although the association between inflammation and atherosclerosis is well established, the biologic events that trigger the local inflammatory response within plaque are not fully understood. Cytotoxic free radicals and infectious agents, both of which are associated with an inflammatory response, have previously been implicated in the initiation and progression of atherosclerosis. In this study, we analyzed carotid plaque for evidence of oxidative vascular injury by determining the presence and distribution of inducible nitric oxide synthase (iNOS) expression and nitrotyrosine formation and for evidence of infection with cytomegalovirus.. Carotid plaque from 51 patients who underwent endarterectomy for either primary (n = 37) or recurrent (n = 14) stenosis were examined histologically (hematoxylin-eosin staining and Masson's trichrome staining) and with immunohistochemistry with specific antibodies to alpha-smooth muscle actin, macrophages (CD68), T-lymphocytes (CD3), and T-cell activation (human leukocyte antigen-DR). Twenty-eight specimens from patients with primary (n = 15) and recurrent (n = 13) stenosis were examined for the presence of iNOS and nitrotyrosine with immunohistochemistry and in situ hybridization (iNOS). Twenty-three additional specimens (22 primary, and 1 recurrent) were analyzed with antibodies to p53, cytomegalovirus, and the polymerase chain reaction (cytomegalovirus, n = 8).. Primary atherosclerotic lesions were either complex heterogenous cellular plaques (n = 29) or relatively acellular fibrous plaques (n = 8). Ten of 14 recurrent plaques were either complex or fibrous lesions, and the remaining four were typical of myointimal thickening. CD68-positive staining cells were detected in all specimens regardless of their structural morphology. CD3-positive cells were interspersed between macrophages in all heterogeneous cellular plaques and only infrequently noted in fibrous plaques. iNOS and nitrotyrosine immunoreactivity were detected in macrophages and smooth muscle cells in all complex and fibrous plaques and in two of four myointimal plaques. The presence of iNOS and nitrotyrosine in plaque correlated with the existence of symptoms in 80% of primary and 62% of recurrent lesions. Cytomegalovirus was detected in only two of 23 carotid specimens (9%).. The association between ischemic cerebrovascular symptoms and iNOS and nitrotyrosine immunoreactivity in complex primary and recurrent carotid plaque and the infrequent occurrence of cytomegalovirus in primary carotid lesions suggests that ongoing free radical oxidative damage rather than viral infection may contribute to plaque instability in patients with complex and fibrous carotid plaques.

Topics: Aged; Carotid Arteries; Carotid Stenosis; Cytomegalovirus Infections; Female; Humans; Immunohistochemistry; In Situ Hybridization; Intracranial Arteriosclerosis; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Polymerase Chain Reaction; Recurrence; Risk Factors; Tyrosine