Enrico Ammirati

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.

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.)

Long-Term Heart Transplant Survival by Targeting the Ionotropic Purinergic Receptor P2X7

Background— Heart transplantation is a lifesaving procedure for patients with end-stage heart failure. Despite much effort and advances in the field, current immunosuppressive regimens are still associated with poor long-term cardiac allograft outcomes, and with the development of complications, including infections and malignancies, as well. The development of a novel, short-term, and effective immunomodulatory protocol will thus be an important achievement. The purine ATP, released during cell damage/activation, is sensed by the ionotropic purinergic receptor P2X7 (P2X7R) on lymphocytes and regulates T-cell activation. Novel clinical-grade P2X7R inhibitors are available, rendering the targeting of P2X7R a potential therapy in cardiac transplantation. Methods and Results— We analyzed P2X7R expression in patients and mice and P2X7R targeting in murine recipients in the context of cardiac transplantation. Our data demonstrate that P2X7R is specifically upregulated in graft-infiltrating lymphocytes in cardiac-transplanted humans and mice. Short-term P2X7R targeting with periodate-oxidized ATP promotes long-term cardiac transplant survival in 80% of murine recipients of a fully mismatched allograft. Long-term survival of cardiac transplants was associated with reduced T-cell activation, T-helper cell 1/T-helper cell 17 differentiation, and inhibition of STAT3 phosphorylation in T cells, thus leading to a reduced transplant infiltrate and coronaropathy. In vitro genetic upregulation of the P2X7R pathway was also shown to stimulate T-helper cell 1/T-helper cell 17 cell generation. Finally, P2X7R targeting halted the progression of coronaropathy in a murine model of chronic rejection as well. Conclusions— P2X7R targeting is a novel clinically relevant strategy to prolong cardiac transplant survival.

Selective up-regulation of the soluble pattern-recognition receptor pentraxin 3 and of vascular endothelial growth factor in giant cell arteritis: relevance for recent optic nerve ischemia.

OBJECTIVE To assess local expression and plasma levels of pentraxin 3 (PTX3) in patients with giant cell arteritis (GCA). METHODS Plasma and serum samples were obtained from 75 patients with GCA (20 of whom had experienced optic nerve ischemia in the previous 3 weeks and 24 of whom had experienced symptom onset in the previous 6 months and had no history of optic nerve ischemia) and 63 controls (35 age-matched healthy subjects, 15 patients with rheumatoid arthritis, and 13 patients with chronic stable angina). In 9 patients in whom GCA was recently diagnosed, circulating levels of interleukin-1β (IL-1β), IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12p70, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α (MIP-1α), CCL4/MIP-1β, CCL11/eotaxin, CXCL9/monokine induced by interferon-γ, CXCL10/interferon-γ-inducible 10-kd protein, tumor necrosis factor α (TNFα), interferon-γ, vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor, and FasL were measured via a multiplexed cytometric assay. PTX3 and VEGF concentrations were assessed by enzyme-linked immunosorbent assay. PTX3 and CD68 expression were determined by immunohistochemistry and immunofluorescence on temporal artery samples. RESULTS GCA patients with very recent optic nerve ischemia had significantly higher PTX3 and VEGF levels compared to other GCA patients and controls. GCA patients with a disease duration of <6 months had significantly higher PTX3 levels compared to other GCA patients and controls. Immunohistochemistry revealed selective PTX3 expression in the wall of inflamed arteries. CONCLUSION Our findings indicate that local expression of PTX3 is a feature of vascular inflammation in GCA; elevated circulating levels of PTX3 identify patients with very recent optic nerve ischemia or a recent diagnosis. Optic nerve ischemia is also associated with increased circulating VEGF levels.

Markers of Inflammation Associated with Plaque Progression and Instability in Patients with Carotid Atherosclerosis

Atherosclerosis is the focal expression of a systemic disease affecting medium- and large-sized arteries, in which traditional cardiovascular risk factor and immune factors play a key role. It is well accepted that circulating biomarkers, including C-reactive protein and interleukin-6, reliably predict major cardiovascular events, including myocardial infarction or death. However, the relevance of biomarkers of systemic inflammation to atherosclerosis progression in the carotid artery is less established. The large majority of clinical studies focused on the association between biomarkers and subclinical atherosclerosis, that is, carotid intima-media thickening (cIMT), which represents an earlier stage of the disease. The aim of this work is to review inflammatory biomarkers that were associated with a higher atherosclerotic burden, a faster disease progression, and features of plaque instability, such as inflammation or neovascularization, in patients with carotid atherosclerotic plaque, which represents an advanced stage of disease compared with cIMT. The association of biomarkers with the occurrence of cerebrovascular events, secondary to carotid plaque rupture, will also be presented. Currently, the degree of carotid artery stenosis is used to predict the risk of future cerebrovascular events in patients affected by carotid atherosclerosis. However, this strategy appears suboptimal. The identification of suitable biomarkers could provide a useful adjunctive criterion to ensure better risk stratification and optimize management.

High-Sensitivity C-Reactive Protein Is Within Normal Levels at the Very Onset of First ST-Segment Elevation Acute Myocardial Infarction in 41% of Cases: A Multiethnic Case-Control Study

OBJECTIVES This study sought to assess the prevalence of normal levels of high sensitivity C-reactive protein (hsCRP) at the very onset of ST-segment elevation myocardial infarction (STEMI). BACKGROUND Levels of hsCRP ≥2 mg/l identify individuals who benefit from lipid lowering and possibly anti-inflammatory agents, but how many patients develop infarction in spite of hsCRP levels <2 mg/l and thus would be ineligible for these treatments? METHODS We studied 887 patients with unequivocally documented STEMI as the first manifestation of coronary disease and 887 matched control subjects from urban areas of Italy, Scotland, and China. Blood samples were obtained before reperfusion strategies <6 h from symptoms onset in order to limit acute event-related increases. RESULTS hsCRP values were similar in samples obtained <2 h, 2 to 4 h, and 4 to 6 h from symptoms onset in all ethnic groups, consistent with the delayed hsCRP elevation after myocardial necrosis and thus indicative of pre-infarction levels. Median hsCRP values were significantly higher in patients than in control subjects: 2.49 (interquartile range [IQR]: 1.18 to 5.55) mg/l versus 1.32 (IQR: 0.58 to 3.10) mg/l (p < 0.0001), which is consistent with previous findings. However, 41% of patients had hsCRP levels <2 mg/l and conversely, 37% of control subjects had values ≥2 mg/l. CONCLUSIONS The measurement of hsCRP, with a 2 mg/l cutoff, would not have predicted 41% of unequivocally documented STEMIs in 3 ethnic groups without evidence of previous coronary disease, thus indicating both its limitations as an individual prognostic marker and as an indicator of a generalized inflammatory pathogenetic component of STEMI. New specific prognostic and therapeutic approaches should be found for such a large fraction of patients at risk.

Identification and Predictive Value of Interleukin-6+ Interleukin-10+ and Interleukin-6− Interleukin-10+ Cytokine Patterns in ST-Elevation Acute Myocardial Infarction

Rationale: At the onset of ST-elevation acute myocardial infarction (STEMI), patients can present with very high circulating interleukin-6 (IL-6+) levels or very low-IL-6– levels. Objective: We compared these 2 groups of patients to understand whether it is possible to define specific STEMI phenotypes associated with outcome based on the cytokine response. Methods and Results: We compared 109 patients with STEMI in the top IL-6 level (median, 15.6 pg/mL; IL-6+ STEMI) with 96 in the bottom IL-6 level (median, 1.7 pg/mL; IL-6− STEMI) and 103 matched controls extracted from the multiethnic First Acute Myocardial Infarction study. We found minimal clinical differences between IL-6+ STEMI and IL-6− STEMI. We assessed the inflammatory profiles of the 2 STEMI groups and the controls by measuring 18 cytokines in blood samples. We exploited clustering analysis algorithms to infer the functional modules of interacting cytokines. IL-6+ STEMI patients were characterized by the activation of 2 modules of interacting signals comprising IL-10, IL-8, macrophage inflammatory protein-1&agr;, and C-reactive protein, and monocyte chemoattractant protein-1, macrophage inflammatory protein-1&bgr;, and monokine induced by interferon-&ggr;. IL-10 was increased both in IL-6+ STEMI and IL-6− STEMI patients compared with controls. IL-6+IL-10+ STEMI patients had an increased risk of systolic dysfunction at discharge and an increased risk of death at 6 months in comparison with IL-6−IL-10+ STEMI patients. We combined IL-10 and monokine induced by interferon-&ggr; (derived from the 2 identified cytokine modules) with IL-6 in a formula yielding a risk index that outperformed any single cytokine in the prediction of systolic dysfunction and death. Conclusions: We have identified a characteristic circulating inflammatory cytokine pattern in STEMI patients, which is not related to the extent of myocardial damage. The simultaneous elevation of IL-6 and IL-10 levels distinguishes STEMI patients with worse clinical outcomes from other STEMI patients. These observations could have potential implications for risk-oriented patient stratification and immune-modulating therapies.

Dynamic assessment of ‘valvular reserve capacity’ in patients with rheumatic mitral stenosis

AIMS Mitral stenosis (MS) may exhibit a dynamic valvular reserve. When resting gradients and systolic pulmonary pressure (sPAP) do not reflect the real severity of the disease, a dynamic evaluation becomes necessary. The aim of the study was to assess the clinical utility of exercise echocardiography in symptomatic patients with apparently subcritical MS. METHODS AND RESULTS One hundred and thirty consecutive patients were referred for symptomatic MS. Patients with unimpressive resting MVA (>1-1.5 cm(2)) and mean PG (≥5-9 mmHg) underwent exercise echocardiography. Cardiac performance and mitral indices (MVA, peak/mean PG, sPAP) were measured. Exhaustion of valvular reserve capacity under exercise was defined as appearance of symptoms and sPAP > 60 mmHg. Forty-six patients (35%) (age: 53 ± 10 years; 74%, female) with resting MVA (1.2 ± 0.36 cm(2)), mean PG (6.8 ± 2.7 mmHg), and sPAP (38 ± 7 mmHg) inconsistent with symptoms underwent stress echocardiography. Exercise was stopped for dyspnoea (76%) or fatigue (24%). At peak workloads (57.2 ± 21.8 Watts), increased mean PG (17.2 ± 4.8 mmHg, P< 0.001) and sPAP (67.4 ± 11.4 mmHg; P< 0.0001) were observed, without change in MVA (1.25 ± 0.4 cm(2); P= n.s.). At univariate analysis, predictors of adaptation to exercise were age (-0.345; P = 0.024), mean PG (0.339; P= 0.023), and sPAP (0.354; P= 0.024); at multivariate analysis, best predictor was resting mean PG, although correlation was poor (-0.339; P= 0.015). CONCLUSION In MS with limiting symptoms despite unimpressive findings at rest, valvular capacity exhaustion should be tested on a dynamic background, as no single resting index can predict potential haemodynamic adaptation to exercise. In such context, the contribution of exercise echocardiography remains extremely valuable.

Questing for circadian dependence in ST-segment-elevation acute myocardial infarction: A multicentric and multiethnic study

Rationale: Four monocentric studies reported that circadian rhythms can affect left ventricular infarct size after ST-segment–elevation acute myocardial infarction (STEMI). Objective: To further validate the circadian dependence of infarct size after STEMI in a multicentric and multiethnic population. Methods and Results: We analyzed a prospective cohort of subjects with first STEMI from the First Acute Myocardial Infarction study that enrolled 1099 patients (ischemic time <6 hours) in Italy, Scotland, and China. We confirmed a circadian variation of STEMI incidence with an increased morning incidence (from 6:00 am till noon). We investigated the presence of circadian dependence of infarct size plotting the peak creatine kinase against time onset of ischemia. In addition, we studied the patients from the 3 countries separately, including 624 Italians; all patients were treated with percutaneous coronary intervention. We adopted several levels of analysis with different inclusion criteria consistent with previous studies. In all the analyses, we did not find a clear-cut circadian dependence of infarct size after STEMI. Conclusions: Although the circadian dependence of infarct size supported by previous studies poses an intriguing hypothesis, we were unable to converge toward their conclusions in a multicentric and multiethnic setting. Parameters that vary as a function of latitude could potentially obscure the circadian variations observed in monocentric studies. We believe that, to assess whether circadian rhythms can affect the infarct size, future study design should not only include larger samples but also aim to untangle the molecular time–dynamic mechanisms underlying such a relation.