Alessandro Mauriello

Localization of distinct F2-isoprostanes in human atherosclerotic lesions.

F2-Isoprostanes are prostaglandin (PG) isomers formed in situ in cell membranes by peroxidation of arachidonic acid. 8-epi PGF2alpha and IPF2alpha-I are F2-isoprostanes produced in humans which circulate in plasma and are excreted in urine. Measurement of F2-isoprostanes may offer a sensitive, specific, and noninvasive method for measuring oxidant stress in clinical settings where reactive oxygen species are putatively involved. We determined whether isoprostanes were present in human atherosclerotic lesions, where lipid peroxidation is thought to occur in vivo. 8-epi PGF2alpha ranged from 1.310-3.450 pmol/micromol phospholipid in atherectomy specimens compared with 0.045-0.115 pmol/micromol phospholipid (P < 0.001) in vascular tissue devoid of atherosclerosis. Corresponding values of IPF2alpha-I were 5.6-13.8 vs. 0.16-0.44 pmol/micromol phospholipid (P < 0.001). Levels of the two isoprostanes in vascular tissue were highly correlated (r = 0.80, P < 0.0001). Immunohistochemical studies confirmed that foam cells adjacent to the lipid necrotic core of the plaque were markedly positive for 8-epi PGF2alpha. These cells were also reactive with anti-CD68, an epitope specific for human monocyte/macrophages. 8-epi PGF2alpha immunoreactivity was also detected in cells positive for anti-alpha-smooth muscle actin antibody, which specifically recognizes vascular smooth muscle cells. Our results indicate that 8-epi PGF2alpha and IPF2alpha-I, two distinct F2-isoprostanes and markers of oxidative stress in vivo, are present in human atherosclerotic plaque. Quantitation of these chemically stable products of lipid peroxidation in target tissues, as well as in biological fluids, may aid in the rational development of antioxidant drugs in humans.

Insulin-Dependent Activation of Endothelial Nitric Oxide Synthase Is Impaired by O-Linked Glycosylation Modification of Signaling Proteins in Human Coronary Endothelial Cells

Background—Hyperglycemia impairs functional properties of cytosolic and nuclear proteins via O-linked glycosylation modification (O-GlcNAcylation). We studied the effects of O-GlcNAcylation on insulin signaling in human coronary artery endothelial cells. Methods and Results—O-GlcNAcylation impaired the metabolic branch of insulin signaling, ie, insulin receptor (IR) activation of the IR substrate (IRS)/phosphatidylinositol 3-kinase (PI3-K)/Akt, whereas it enhanced the mitogenic branch, ie, ERK-1/2 and p38 (mitogen-activated protein kinase). Both in vivo and in vitro phosphorylation of endothelial nitric oxide synthase (eNOS) by Akt were reduced by hyperglycemia and hexosamine activation. Insulin-induced eNOS activity in vivo was reduced by hyperglycemia and hexosamine activation, which was coupled to increased activation and expression of matrix metalloproteinase-2 and -9; these phenomena were reversed by inhibition of the hexosamine pathway. Finally, carotid plaques from type 2 diabetic patients showed increased endothelial O-GlcNAcylation with respect to nondiabetics. Conclusions—Our data show that hyperglycemia, through the hexosamine pathway, impairs activation of the IR/IRS/PI3-K/Akt pathway, resulting in deregulation of eNOS activity.

Contrast-Enhanced Ultrasound Imaging of Intraplaque Neovascularization in Carotid Arteries: Correlation With Histology and Plaque Echogenicity

OBJECTIVES This study was designed to evaluate contrast-enhanced ultrasound imaging of carotid atherosclerosis as a clinical tool to study intraplaque neovascularization. BACKGROUND Plaque neovascularization is associated with plaque vulnerability and symptomatic disease; therefore, imaging of neovascularization in carotid atherosclerosis may represent a useful tool for clinical risk stratification and monitoring the efficacy of antiatherosclerotic therapies. METHODS Thirty-two patients with 52 carotid plaques were studied by standard and contrast-enhanced ultrasound imaging. In 17 of these patients who underwent endarterectomy, the surgical specimen was available for histological determination of microvessel density by CD31/CD34 double staining. Plaque echogenicity and degree of stenosis at standard ultrasound imaging were evaluated for each lesion. Contrast-agent enhancement within the plaque was categorized as absent/peripheral (grade 1) and extensive/internal (grade 2). RESULTS In the surgical subgroup, plaques with higher contrast-agent enhancement showed a greater neovascularization at histology (grade 2 vs. grade 1 contrast-agent enhancement: median vasa vasorum density: 3.24/mm(2) vs. 1.82/mm(2), respectively, p = 0.005). In the whole series of 52 lesions, echolucent plaques showed a higher degree of contrast-agent enhancement (p < 0.001). Stenosis degree was not associated with neovascularization at histology or with the grade of contrast-agent enhancement. CONCLUSIONS Carotid plaque contrast-agent enhancement with sonographic agents correlates with histological density of neovessels and is associated with plaque echolucency, a well-accepted marker of high risk lesions, but it is unrelated to the degree of stenosis. Contrast-enhanced carotid ultrasound imaging may provide valuable information for plaque risk stratification and for assessing the response to antiatherosclerotic therapies, beyond that provided by standard ultrasound imaging.

Role of Inflammation in Atherosclerosis

Inflammation plays a major role in all phases of atherosclerosis. Stable plaques are characterized by a chronic inflammatory infiltrate, whereas vulnerable and ruptured plaques are characterized by an “active” inflammation involved in the thinning of the fibrous cap, predisposing the plaque to rupture. Although a single vulnerable atherosclerotic plaque rupture may cause the event, there are many other types of plaques, several of which are vulnerable. The existence of multiple types of vulnerable plaques suggests that atherosclerosis is a diffuse inflammatory process. A current challenge is to identify morphologic and molecular markers able to discriminate stable plaques from vulnerable ones, allowing the stratification of patients at high risk for acute cardiovascular and cerebrovascular events before clinical syndromes develop. With that aim in mind, this article summarizes the natural history of atherosclerotic plaques, focusing on molecular mechanisms affecting plaque progression and serum markers correlated with plaque inflammation.

Extracellular Proteases in Atherosclerosis and Restenosis

Extracellular proteolysis plays a key role in many pathophysiologic processes including cancer, inflammatory diseases, and cardiovascular conditions such as atherosclerosis and restenosis. Whereas matrix metalloproteinases are their best known member, many others are becoming better known. The extracellular proteases are a complex and heterogeneous superfamily of enzymes. They include metalloproteinases (matrix metalloproteinases, adamalysins, or pappalysins), serine proteases (elastase, coagulation factors, plasmin, tissue plasminogen activator, urokinase plasminogen activator), and the cysteine proteases (such cathepsins). In addition to their matrix degradation capabilities, they have other less well known biologic functions that include angiogenesis, growth factor bioavailability, cytokine modulation, receptor shedding, enhancing cell migration, proliferation, invasion, and apoptosis. This review discusses extracellular proteases relevant to the vasculature, their classification and function, and how protease disorders contribute to arterial plaque growth, including chronic atherosclerosis, acute coronary syndromes, restenosis, and vascular remodeling. These broad extracellular protease functions make them potentially interesting therapeutic targets.

Timp3 deficiency in insulin receptor–haploinsufficient mice promotes diabetes and vascular inflammation via increased TNF-α

Activation of inflammatory pathways may contribute to the beginning and the progression of both atherosclerosis and type 2 diabetes. Here we report a novel interaction between insulin action and control of inflammation, resulting in glucose intolerance and vascular inflammation and amenable to therapeutic modulation. In insulin receptor heterozygous (Insr+/-) mice, we identified the deficiency of tissue inhibitor of metalloproteinase 3 (Timp3, an inhibitor of both TNF-alpha-converting enzyme [TACE] and MMPs) as a common bond between glucose intolerance and vascular inflammation. Among Insr+/- mice, those that develop diabetes have reduced Timp3 and increased TACE activity. Unchecked TACE activity causes an increase in levels of soluble TNF-alpha, which subsequently promotes diabetes and vascular inflammation. Double heterozygous Insr+/-Timp3+/- mice develop mild hyperglycemia and hyperinsulinemia at 3 months and overt glucose intolerance and hyperinsulinemia at 6 months. A therapeutic role for Timp3/TACE modulation is supported by the observation that pharmacological inhibition of TACE led to marked reduction of hyperglycemia and vascular inflammation in Insr+/- diabetic mice, as well as by the observation of increased insulin sensitivity in Tace+/- mice compared with WT mice. Our results suggest that an interplay between reduced insulin action and unchecked TACE activity promotes diabetes and vascular inflammation.

DNA methylation silences miR-132 in prostate cancer

Silencing of microRNAs (miRNAs) by promoter CpG island methylation may be an important mechanism in prostate carcinogenesis. To screen for epigenetically silenced miRNAs in prostate cancer (PCa), we treated prostate normal epithelial and carcinoma cells with 5-aza-2′-deoxycytidine (AZA) and subsequently examined expression changes of 650 miRNAs by megaplex stemloop reverse transcription–quantitative PCR. After applying a selection strategy, we analyzed the methylation status of CpG islands upstream to a subset of miRNAs by methylation-specific PCR. The CpG islands of miR-18b, miR-132, miR-34b/c, miR-148a, miR-450a and miR-542-3p showed methylation patterns congruent with their expression modulations in response to AZA. Methylation analysis of these CpG islands in a panel of 50 human prostate carcinoma specimens and 24 normal controls revealed miR-132 to be methylated in 42% of human cancer cases in a manner positively correlated to total Gleason score and tumor stage. Expression analysis of miR-132 in our tissue panel confirmed its downregulation in methylated tumors. Re-expression of miR-132 in PC3 cells induced cell detachment followed by cell death (anoikis). Two pro-survival proteins—heparin-binding epidermal growth factor and TALIN2—were confirmed as direct targets of miR-132. The results of this study point to miR-132 as a methylation-silenced miRNA with an antimetastatic role in PCa controlling cellular adhesion.

Multicentric inflammation in epicardial coronary arteries of patients dying of acute myocardial infarction

OBJECTIVES We sought to test the hypothesis of whether inflammatory cell infiltration in patients dying of an acute myocardial infarction (MI) is a multifocal event involving multiple coronary branches. BACKGROUND Coronary instability is thought to reflect local disruption of a single vulnerable plaque. However, previous postmortem studies have not addressed the question of whether activation of inflammatory cells, particularly T lymphocytes, is limited to the culprit lesion only or rather diffuse in the coronary circulation. METHODS We performed a systematic flow cytometric study in three groups of autopsied patients (group 1 = acute MI; group 2 = old MI; group 3 = no ischemic heart disease). Cell suspensions of enzymatically digested coronary arteries were stained for flow cytometry with CD3, CD68, alpha-smooth muscle actin, and human leukocyte antigen (HLA)-DR antibodies. RESULTS The coronary plaques showed: 1) a higher proportion of inflammatory cells in groups 1 and 2 than in group 3; 2) a higher percentage of T lymphocytes in group 1 than in group 2 (11.67 +/- 0.70% vs. 5.67 +/- 0.74%, p = 0.001) and in group 2 than in group 3 (p = 0.008); and 3) diffuse cell activation in the whole coronary tree of group 1, but not of group 2 subjects. CONCLUSIONS Our study suggests that lymphocytes may play a key role in coronary instability by determining activation of various cellular types throughout the coronary circulation. Activated T lymphocytes and their products may well represent a new target in both the treatment and prevention of acute coronary syndromes.

TIMP3 Is Reduced in Atherosclerotic Plaques From Subjects With Type 2 Diabetes and Increased by SirT1

OBJECTIVE Atherosclerosis is accelerated in subjects with type 2 diabetes by unknown mechanisms. We identified tissue inhibitor of metalloproteinase 3 (TIMP3), the endogenous inhibitor of A disintegrin and metalloprotease domain 17 (ADAM17) and other matrix metalloproteinases (MMPs), as a gene modifier for insulin resistance and vascular inflammation in mice. We tested its association with atherosclerosis in subjects with type 2 diabetes and identified Sirtuin 1 (SirT1) as a major regulator of TIMP3 expression. RESEARCH DESIGN AND METHODS We investigated ADAM10, ADAM17, MMP9, TIMP1, TIMP2, TIMP3, and TIMP4 expression levels in human carotid atherosclerotic plaques (n = 60) from subjects with and without diabetes. Human vascular smooth muscle cells exposed to several metabolic stimuli were used to identify regulators of TIMP3 expression. SirT1 small interference RNA, cDNA, and TIMP3 promoter gene reporter were used to study SirT1-dependent regulation of TIMP3. RESULTS Here, we show that in human carotid atherosclerotic plaques, TIMP3 was significantly reduced in subjects with type 2 diabetes, leading to ADAM17 and MMP9 overactivity. Reduced expression of TIMP3 was associated in vivo with SirT1 levels. In smooth muscle cells, inhibition of SirT1 activity and levels reduced TIMP3 expression, whereas SirT1 overexpression increased TIMP3 promoter activity. CONCLUSIONS In atherosclerotic plaques from subjects with type 2 diabetes, the deregulation of ADAM17 and MMP9 activities is related to inadequate expression of TIMP3 via SirT1. Studies in vascular cells confirmed the role of SirT1 in tuning TIMP3 expression.

Aging and atherosclerosis in the rabbit: 1. Distribution, prevalence and morphology of atherosclerotic lesions

Aging is considered a risk factor in the pathogenesis of atherosclerosis. It is not clear, however, whether the relationship between aging and atherosclerosis is the result of increased susceptibility of the arterial wall related to intrinsic alterations or the expression of the increase in intensity or duration of exposure to risk factors. In this study, we used aged (median age 46 months) and young (4 months old) New Zealand white rabbits. Nine aged and 11 young rabbits received a hyperlipemic diet enriched with a low dose of cholesterol for 18 months. Eleven aged and 8 young rabbits, fed standard chow for the same period, were used as controls. Using morphologic and morphometric methods, we detected in aged hyperlipemic rabbits (a) a marked prevalence of fibroatheromatous plaques (as opposed to fatty streaks in young hyperlipemic rabbits); (b) aortic lesions more extensive and of greater dimensions than in young hyperlipemic rabbits; (c) fibroatheromatous plaques in carotids and raised fatty streaks in the large subepicardial coronary branches. Our results show an increased susceptibility of the aged arterial wall to hypercholesterolemia.