Giuseppe Danilo Norata

Deficiency of the Long Pentraxin PTX3 Promotes Vascular Inflammation and Atherosclerosis

Background— Immune responses participate in several phases of atherosclerosis; there is, in fact, increasing evidence that both adaptive immunity and innate immunity tightly regulate atherogenesis. Pentraxins are a superfamily of acute-phase proteins that includes short pentraxins such as C-reactive protein or long pentraxins such as PTX3, a molecule acting as the humoral arm of innate immunity. To address the potential role of PTX3 in atherogenesis, we first investigated the expression of PTX3 during atherogenesis, generated double-knockout mice lacking PTX3 and apolipoprotein E, and then studied the effect of murine PTX3 deficiency on plasma lipids, atherosclerosis development, and gene expression pattern in the vascular wall. Methods and Results— PTX3 expression increases in the vascular wall of apolipoprotein E–knockout mice from 3 up to 18 months of age. Double-knockout mice lacking PTX3 and apolipoprotein E were fed an atherogenic diet for 16 weeks. Aortic lesions were significantly increased in double-knockout mice and mice heterozygous for PTX3 compared with apolipoprotein E–knockout mice. Mice lacking PTX3 showed a more pronounced inflammatory profile in the vascular wall as detected by cDNA microarray and quantitative polymerase chain reaction analysis and an increased macrophage accumulation within the plaque. Finally, lesion size correlated with the number of bone marrow monocytes. Conclusion— PTX3 has atheroprotective effects in mice, which, in light of the cardioprotective effects recently reported, suggests a cardiovascular protective function of the long pentraxin 3 through the modulation of the immunoinflammatory balance in the cardiovascular system.

LOX-1, OxLDL, and Atherosclerosis

Oxidized low-density lipoprotein (OxLDL) contributes to the atherosclerotic plaque formation and progression by several mechanisms, including the induction of endothelial cell activation and dysfunction, macrophage foam cell formation, and smooth muscle cell migration and proliferation. Vascular wall cells express on their surface several scavenger receptors that mediate the cellular effects of OxLDL. The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the main OxLDL receptor of endothelial cells, and it is expressed also in macrophages and smooth muscle cells. LOX-1 is almost undetectable under physiological conditions, but it is upregulated following the exposure to several proinflammatory and proatherogenic stimuli and can be detected in animal and human atherosclerotic lesions. The key contribution of LOX-1 to the atherogenic process has been confirmed in animal models; LOX-1 knockout mice exhibit reduced intima thickness and inflammation and increased expression of protective factors; on the contrary, LOX-1 overexpressing mice present an accelerated atherosclerotic lesion formation which is associated with increased inflammation. In humans, LOX-1 gene polymorphisms were associated with increased susceptibility to myocardial infarction. Inhibition of the LOX-1 receptor with chemicals or antisense nucleotides is currently being investigated and represents an emerging approach for controlling OxLDL-LOX-1 mediated proatherogenic effects.

Leptin:Adiponectin Ratio Is an Independent Predictor of Intima Media Thickness of the Common Carotid Artery

Background and Purpose— The evaluation of the leptin:adiponectin ratio (L:A) has been suggested as an atherosclerotic index in patients with type 2 diabetes and a useful parameter to assess insulin resistance in patients with and without diabetes. Methods— We investigated, therefore, the relationship between L:A ratio and intima media thickness (IMT), an independent predictor of cardiovascular disease, in 110 healthy males. Results— L:A ratio was significantly correlated to body mass index, waist, hip, waist-to-hip ratio, systolic blood pressure, IMT, high-density lipoprotein, apolipoprotein A-I, glucose, and the homeostasis model of insulin resistance–revised. No significant correlation was observed with age, diastolic blood pressure, low-density lipoprotein, triglycerides, apolipoprotein B, ApoB/ApoA-I ratio, insulin, alanine transaminase, &ggr;-glutamyl-transferase, and resistin. In addition, when the relationship between IMT and adiponectin or leptin alone was analyzed, only leptin plasma levels significantly associated with IMT (r=0.301, P<0.01). In a multiple regression analysis including in the statistical model the risk factors known to affect IMT (age, systolic blood pressure, diastolic blood pressure, high-density lipoprotein cholesterol, triglycerides, total cholesterol, body mass index, glucose, and L:A ratio), we observed that only age, L:A, and glucose were independent predictors of IMT. As expected, obese subjects (body mass index >30 kg/m2) showed a significantly higher L:A ratio compared with nonobese subjects (1.20 versus 0.42, respectively, P<0.001); in addition, subjects with the metabolic syndrome showed a significantly higher L:A ratio level (0.79) compared with subjects without (0.52) (P<0.01). Conclusions— We show here that the L:A ratio is a powerful independent predictor of IMT in healthy subjects and correlates with several anthropometric, metabolic, and clinical parameters better than each single adipokine.

The Long Pentraxin PTX3: A Modulator of the Immunoinflammatory Response in Atherosclerosis and Cardiovascular Diseases

Innate and adaptive immune responses participate in atherosclerosis. Pentraxins, an essential component of the humoral arm of innate immunity, are a superfamily of acute phase proteins highly conserved during evolution and can be classified as short pentraxins such as C-reactive protein (CRP) and long pentraxins such as PTX3. The latter has an unrelated, long N-terminal domain coupled to the C-terminal pentraxin domain and differs from CRP in gene organization, cellular source, and recognized ligands. PTX3 in humans, like CRP, is a marker of atherosclerosis and correlates with the risk of developing vascular events. Although CRP sequence and regulation have not been conserved during evolution between mouse and man, the conservation of sequence, gene organization, and regulation of PTX3 in evolution enables one to address the question regarding its pathophysiologic roles in genetically modified mice. Deficiency of PTX3 is associated with increased heart damage with a greater no-reflow area and increased inflammatory response in a model of acute myocardial infarction (MI) caused by coronary artery ligation. More recently, deficiency of PTX3 on an apolipoprotein E knockout background was associated with increased atherosclerosis, macrophage accumulation within the plaque, and a more pronounced inflammatory profile in the vascular wall. Although these observations point to a cardiovascular protective effect of PTX3, they also suggest the possibility that the increased levels of PTX3 in subjects with cardiovascular disease (CVD) may reflect a protective physiologic response that correlates with the severity of the disease. In summary, data that are accumulating suggest that the increase of pentraxins in atherosclerosis could not be regarded as a harmful response but rather a further attempt to protection of our body.

Emerging role of high density lipoproteins as a player in the immune system

High density lipoproteins (HDL) possess a number of physiological activities. The most studied and, perhaps, better understood is the ability of HDL to promote excess cholesterol efflux from peripheral tissues and transport to the liver for excretion, a mechanism believed to confer protection against atherosclerotic cardiovascular disease. The ability of HDL to modulate cholesterol bioavailability in the lipid rafts, membrane microdomains enriched in glycosphingolipids and cholesterol, is evolutionary conserved and affects the properties of cells involved in the innate and adaptive immune response, tuning inflammatory response and antigen presentation functions in macrophages as well as B and T cell activation. Also sphingosine-1 phosphate (S1P), a major active sphingolipid carried by HDL, is of relevance in the pathogenesis of several immuno-inflammatory disorders through the modulation of macrophage and lymphocyte functions. Furthermore, HDL influence the humoral innate immunity by modulating the activation of the complement system and the expression of pentraxin 3 (PTX3). Finally, in humans, HDL levels and functions are altered in several immune-mediated disorders, such as rheumatoid arthritis, systemic lupus eritematosus, Crohns disease and multiple sclerosis as well as during inflammatory responses. Altogether these observations suggest that the effects of HDL in immunity could be related, to either the ability of HDL to modulate cholesterol content in immune cell lipid rafts and to their role as reservoir for several biologically active substances that may impact the immune system.

Long Pentraxin 3, a Key Component of Innate Immunity, Is Modulated by High-Density Lipoproteins in Endothelial Cells

Objective—High-density lipoproteins (HDL) are endowed with cardiovascular protective activities. In addition to their role in reverse cholesterol transport, HDL exert several beneficial effects on endothelial cells, including the induction of endothelial nitric oxide synthase and prostacyclin release, and the control of the immune and inflammatory response. Methods and Results—To identify possible mechanisms involved in these effects we investigated the modulation of the expression of acute phase proteins of the pentraxin superfamily, such as C-reactive protein (CRP), serum amyloid P component protein (SAP), and the long pentraxin 3 (PTX3) by HDL in human endothelial cells. HDL induced PTX3 mRNA expression and protein release, whereas no effect was observed on CRP and SAP expression. This effect was mainly dependent on the activation of the lysosphingolipids receptors-PI3K/Akt axis and was mimicked by sphingosine 1 phosphate and other S1P mimetics. This observation was confirmed in vivo; indeed an increased expression of PTX3 mRNA was detected in the aorta of transgenic mice overexpressing human apoA-I, compared to apoA-I knock-out mice. Furthermore, plasma levels of PTX3 significantly increased in C57BL/6 mice injected with HDL. Conclusions—These data suggest that part of the atheroprotective effects of HDL could result from the modulation of molecules that act as sensors of the immunoinflammatory balance in the vascular wall.

Circulating CD4+CD25hiCD127lo Regulatory T-Cell Levels Do Not Reflect the Extent or Severity of Carotid and Coronary Atherosclerosis

Objective—Regulatory T (Treg) cells play a protective role in experimental atherosclerosis. In the present study, we investigated whether the levels of circulating Treg cells relate to the degree of atherosclerosis in carotid and coronary arteries. Methods and Results—We studied 2 distinct populations: (1) 113 subjects, selected from a free-living population (carotid study), in which we measured the intima-media thickness of the common carotid artery, as a surrogate marker of initial atherosclerosis; and (2) 75 controls and 125 patients with coronary artery disease (coronary study): 36 with chronic stable angina, 50 with non-ST-elevation acute coronary syndrome, 39 with ST-elevation acute myocardial infarction. Treg-cell levels were evaluated by flow cytometry (Treg cells identified as CD3+CD4+CD25highCD127low) and by mRNA expression of forkhead box P3 or of Treg-associated cytokine interleukin 10. In the carotid study, no correlation was observed between Treg-cell levels and intima-media thickness. No differences in Treg-cell levels were observed comparing rapid versus slow intima-media thickness progressors from a subgroup of patients (n=65), in which prospective data on 6-year intima-media thickness progression were available. In the coronary group, Treg-cell levels were not altered in chronic stable angina patients. In contrast, nonunivocal variations were observed in patients suffering an acute coronary syndrome (with a Treg-cell increase in ST-elevation acute myocardial infarction and a Treg-cell decrease in non-ST-elevation acute coronary syndrome patients). Conclusion—The results suggest that determination of circulating Treg-cell levels based on flow cytometry or mRNA assessment is not a useful indicator of the extent or severity of atherosclerosis.

Effector Memory T cells Are Associated With Atherosclerosis in Humans and Animal Models

Background— Adaptive T-cell response is promoted during atherogenesis and results in the differentiation of naïve CD4+T cells to effector and/or memory cells of specialized T-cell subsets. Aim of this work was to investigate the relationship between circulating CD4+T-cell subsets and atherosclerosis. Methods and Results— We analyzed 57 subsets of circulating CD4+T cells by 10-parameter/8-color polychromatic flow cytometry (markers: CD3/CD4/CD45RO/CD45RA/CCR7/CCR5/CXCR3/HLA-DR) in peripheral blood from 313 subjects derived from 2 independent cohorts. In the first cohort of subjects from a free-living population (n=183), effector memory T cells (TEM: CD3+CD4+CD45RA−CD45RO+CCR7− cells) were strongly related with intima-media thickness of the common carotid artery, even after adjustment for age (r=0.27; P<0.001). Of note, a significant correlation between TEM and low-density lipoproteins was observed. In the second cohort (n=130), TEM levels were significantly increased in patients with chronic stable angina or acute myocardial infarction compared with controls. HLA-DR+TEM were the TEM subpopulation with the strongest association with the atherosclerotic process (r=0.37; P<0.01). Finally, in animal models of atherosclerosis, TEM (identified as CD4+CD44+CD62L−) were significantly increased in low-density lipoprotein receptor and apolipoprotein E deficient mice compared with controls and were correlated with the extent of atherosclerotic lesions in the aortic root (r=0.56; P<0.01). Conclusions— Circulating TEM cells are associated with increased atherosclerosis and coronary artery disease in humans and in animal models and could represent a key CD4+T-cell subset related to the atherosclerotic process. (J Am Heart Assoc. 2012;1:27-41.)

Circulating soluble receptor for advanced glycation end products is inversely associated with body mass index and waist/hip ratio in the general population

Advanced glycation end products, AGEs, and its specific receptor, RAGE, are involved in vascular complications. A role for the soluble form of RAGE (sRAGE), which acts as a decoy for AGE, has been documented in patients with diabetes but no information is available in non-diabetic subjects. The aim of this study was to investigate the association of plasma levels of sRAGE with cardiometabolic risk factors in the general population. In addition we evaluated the relation of the common -374A/T polymorphism of RAGE with plasma levels of sRAGE. One hundred and seventy-six healthy subjects free of diabetes or coronary artery disease untreated for hypertension, dyslipidemia or cardiometabolic related diseases were randomly selected for this study from the general population. Plasma sRAGE were negatively and significantly correlated with BMI, waist/hip circumference ratio and fasting glycemia, while a positive correlation was observed with apolipoprotein A-I. These correlations were observed mainly in women who showed significantly higher sRAGE levels (1744+/-660 pg/mL vs 1414+/-649 pg/mL; P<0.05). In a stepwise regression analysis waist circumference was independently associated with sRAGE and, when waist circumference was excluded, BMI was independently associated with sRAGE. Finally in overweight subjects (BMI>25 kg/m(2)) plasma sRAGE was significantly lower compared to lean subjects (1460+/-640 pg/mL vs 1710+/-693 pg/mL; P<0.05). In healthy subjects plasma levels of sRAGE were negatively correlated with BMI and waist/hip ratio supporting a possible protective role for these proteins before any evidence of diabetic or vascular complications.

Proprotein convertase subtilisin/kexin type 9 (PCSK9): From structure–function relation to therapeutic inhibition

AIMS This short review aims at summarizing the current information on Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) structure and function focusing also on the therapeutic possibilities based on the inhibition of this protein. DATA SYNTHESIS PCSK9 has been recently discovered as the third gene involved in autosomal dominant hypercholesterolemia. PCSK9 binds and favors degradation of the low-density lipoprotein receptor (LDLR) and thereby modulates the plasma levels of LDL-cholesterol (LDL-C). Some of the natural occurring PCSK9 mutations increase the protein function (gain of function) and cause hypercholesterolemia, whereas loss of function mutations associate with hypocholesterolemia. Since the loss of a functional PCSK9 in humans is not associated with apparent deleterious effects, this protease is an attractive target for the development of lowering plasma LDL-C agents, either alone or in combination with statins. CONCLUSION Inhibition of PCSK9 is emerging as a novel strategy for the treatment of hypercholesterolemia and data obtained from pre-clinical studies show that use of monoclonal antibodies, antisense oligonucleotides and short interfering RNA are effective in reducing LDL-C, clinical studies, accompanied by a better understanding of PCSK9 biology, are now necessary to address whether these new compounds will have a future in clinical practice.