Aldo Dobrina

Anakinra, a Recombinant Human Interleukin-1 Receptor Antagonist, Inhibits Apoptosis in Experimental Acute Myocardial Infarction

Background— Experimental interleukin-1 receptor antagonist gene overexpression has shown that interleukin-1 receptor antagonist is cardioprotective during global cardiac ischemia. The aim of the present study was to test the impact of an exogenous recombinant human interleukin-1 receptor antagonist (anakinra) in experimental acute myocardial infarction. Methods and Results— Two animal studies were conducted: one of immediate anakinra administration during ischemia in the mouse and one of delayed anakinra administration 24 hours after ischemia in the rat. Seventy-eight Institute of Cancer Research mice and 20 Wistar rats underwent surgical coronary artery ligation (or sham operation) and were treated with either anakinra 1 mg/kg or NaCl 0.9% (saline). Treatment was administered during surgery and then daily for 6 doses in the mice and starting on day 2 daily for 5 doses in the rats. Twenty-eight mice underwent infarct size assessment 24 hours after surgery, 6 saline-treated mice and 22 mice treated with increasing doses of anakinra (1 mg/kg [n=6], 10 mg/kg [n=6], and 100 mg/kg [n=10]); 6 mice were euthanized at 7 days for protein expression analysis. The remaining animals underwent transthoracic echocardiography before surgery and 7 days later just before death. Cardiomyocyte apoptosis was measured in the peri-infarct regions. The antiapoptotic effect of anakinra was tested in a primary rat cardiomyocyte culture during simulated ischemia and in vitro on caspase-1 and -9 activities. At 7 days, 15 of the 16 mice (94%) treated with anakinra were alive versus 11 of the 20 mice (55%) treated with saline (P=0.013). No differences in infarct size at 24 hours compared with saline were observed with the 1- and 10-mg/kg doses, whereas a 13% reduction in infarct size was found with the 100-mg/kg dose (P=0.015). Treatment with anakinra was associated with a significant reduction in cardiomyocyte apoptosis in both the immediate and delayed treatment groups (3.1±0.2% versus 0.5±0.3% [P<0.001] and 4.2±0.4% versus 1.1±0.2% [P<0.001], respectively). Compared with saline-treated animals, anakinra-treated mice and rats showed signs of more favorable ventricular remodeling. In vitro, anakinra significantly prevented apoptosis induced by simulated ischemia and inhibited caspase-1 and -9 activities. Conclusions— Administration of anakinra within 24 hours of acute myocardial infarction significantly ameliorates the remodeling process by inhibiting cardiomyocyte apoptosis in 2 different experimental animal models of AMI. This may open the door for using anakinra to prevent postischemic cardiac remodeling and heart failure.

Increased myocardial apoptosis in patients with unfavorable left ventricular remodeling and early symptomatic post-infarction heart failure

OBJECTIVES The purpose of this study was to evaluate a potential correlation between apoptotic rate (AR), post-infarction left ventricular (LV) remodeling, and clinical characteristics in subjects who died late (>or=10 days) after an acute myocardial infarction (AMI) with evidence of persistent occlusion of the infarct-related artery at autopsy. BACKGROUND Apoptosis contributes to myocardiocyte loss in cardiac disease and may have a pathophysiologic role in post-infarction LV remodeling. METHODS The AR was calculated at the site of infarction and in remote unaffected LV regions, using co-localization of in situ end labeling for deoxyribonucleic acid fragmentation and immunohistochemistry for caspase-3, in 14 subjects who died within two months after AMI. Correlation between AR and clinical characteristics such as age, site of AMI, transmural extension, multivessel coronary disease, and signs and/or symptoms of heart failure (HF), at the time of initial hospitalization for AMI or subsequently before death, was assessed using non-parametric statistical tests. Parameters of LV remodeling including diameters, free wall thickness, diameter-to-wall-thickness ratio, and mass were measured at gross examination at autopsy. Values are expressed as median (interquartile range). RESULTS Among clinical variables, early symptomatic post-infarction HF (9 cases, 64%) was associated with nearly fourfold increased AR at the site of infarction (26.2% [24.5% to 28.8%] vs. 6.4% [1.9% to 13.3%], p = 0.001). Moreover, AR both at the site of infarction and in unaffected regions was significantly correlated with parameters of progressive LV remodeling (p < 0.05). CONCLUSIONS Our data show that in patients dying >or=10 days after AMI, myocardial apoptosis is strongly associated with and may be a major determinant of unfavorable LV remodeling and early symptomatic post-infarction HF.

Atherosclerosis and inflammation. Patterns of cytokine regulation in patients with peripheral arterial disease

Inflammatory phenomena at sites of atherosclerotic plaques are increasingly thought to be major determinants of the progression and clinical outcome of atherosclerotic disease. Therefore, attention is being paid to systemic markers/mediators which may reflect the inflammatory activity in the plaques. This study evaluates the pattern of the main proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6), their soluble receptors/antagonist, and a variety of inflammatory markers, in patients with peripheral arterial disease (PAD). Eight patients with PAD suffering from claudicatio intermittens (CI), eight with critical limb ischemia (CLI) and eight controls (C) were studied. Blood samples were collected at baseline in all groups and. for C and CI, immediately after and 4 h after a 30-min treadmill test. Baseline: no differences in cytokine plasma levels were detected among the three groups. In contrast, soluble receptors of TNF (type I and II) and of IL-6, and IL-1beta receptor antagonist (IL-1ra) were increased in CI and CLI patients, as compared to C. Of note, IL-Ira correlated with the occurrence and stage of the disease in a highly significant proportion of the patients, reaching a predictive value for the disease of P < 0.0001. The opposite trend was observed for the soluble receptor of IL-1beta. Notably, in the patients no alterations could be found in white blood cell counts, expression of CD11c adherence molecule by circulating monocytes or, in vitro. O2- release from zymosan-activated neutrophils. Moreover, plasma levels of platelet activating factor (PAF), of neutrophil elastase and of the acute phase reactants C-reactive protein (CRP) and alpha1-acid glycoprotein were not found to be significantly altered. In contrast, the acute-phase proteins alpha1-antitrypsin (alpha1AT) and haptoglobin (HG) were found to be increased. Effect of treadmill: IL-1beta and TNFalpha remained at baseline levels following exercise, and IL-6 dropped to undetectable levels. Among cytokine antagonists, again the most relevant changes concerned the IL-1ra, which was significantly increased immediately after the treadmill test, both in CI and C, and returned to baseline levels after 4 h. In contrast, soluble TNFalpha, IL-1beta and IL-6 receptors, PAF, and the other markers of leukocyte activation were not found to be altered. Soluble TNFalpha and IL-6 receptors were shown to inhibit the biological effects of their ligands. Similarly, IL-1ra and the acute phase proteins alpha1AT and HG have been reported to exert anti-inflammatory functions. The increased plasma levels of these agents, together with low levels of inflammatory cytokines and other pro-inflammatory mediators such as PAF and alpha1-acid glycoprotein, appear to draw an undescribed picture, so far, of upregulation of a composite systemic anti-inflammatory mechanism in atherosclerotic patients. IL-1ra appears to be a reliable marker of the state of activation of this mechanism. These results may provide a basis for developing new insights into the pathogenesis of the atherosclerotic disease.

Widespread Myocardial Inflammation and Infarct-Related Artery Patency

Background—Diffuse coronary vascular inflammation is associated with acute coronary syndromes. However, it is unknown whether inflammation also occurs within the myocardium. Therefore, this study was aimed at assessing the presence of activated cells in unaffected remote myocardium of patients with acute myocardial infarction (AMI), in comparison to the peri-infarct region from the same cases, and in comparison to myocardial specimens from control hearts. Methods and Results—Sixteen patients dying 1 to 12 weeks after AMI and 16 control subjects were selected at autopsy. Myocardial specimens were taken at remote unaffected viable regions and at peri-infarct regions in cases with AMI. Confocal microscopy was performed to measure the number of activated cells (DR+), T-lymphocytes (CD3+), and activated T-lymphocytes (CD3+/DR+). Activated cells and activated T-lymphocytes were found in remote unaffected regions in 11 of 16 cases (69%), in peri-infarct zone in all cases (100%), and in none of the control hearts (0%, P <0.001 versus others). A greater myocardial inflammatory burden in remote regions but not in peri-infarct regions was associated with persistent infarct-related artery occlusion (P <0.05). Conclusions—This study for the first time shows the presence of activated T-lymphocytes in remote unaffected myocardial regions in approximately two thirds of patients with recent AMI. Because these cells are associated with persistent infarct-related artery occlusion, our data may suggest that an antigenic stimulus present also in the myocardium triggers an immune response that may be critical to precipitate artery occlusion.

Complement-endothelial cell interactions: pathophysiological implications.

The function of the endothelial cells can be modulated by humoral factors present in the circulation and in the extravascular fluid, including proteins of the complement system. This review examines the multiple interactions between the complement system and the endothelial cells and their functional consequences on inflammation, coagulation and regulation of vascular tone. The implications of these interactions in the induction and progression of the vascular lesions occurring in atherosclerosis, ischemia/reperfusion and xenotransplantation and the possible therapeutic approaches in terms of complement regulation are also discussed.

Evidence of CXC, CC and C chemokine production by lymphatic endothelial cells

Although production of chemokines by vascular endothelial cells has been documented, there is only limited information regarding the expression of chemokines by the lymphatic endothelium. Here we used lymphatic endothelial cells (LEC) derived from experimentally induced murine lymphangiomas to investigate the pattern of chemokine expression by these cells. Histological analysis of the lymphatic hyperplasia revealed the presence of leucocytes in the tissues surrounding the lesions, suggesting the presence of chemoattractant activity. A functional chemotactic assay on human polymorphonuclear cells and on purified subpopulations of murine leucocytes using culture supernatants from LEC primary cultures confirmed the presence of chemoattractant activity. The identity of different cytokines of the CXC, CC and C subfamilies was investigated by reverse trancriptase–polymerase chain reaction on total endothelial cell RNA. Amplified fragments corresponding to KC, IP10, Mig‐1, BCL, MIP‐2, SLC, RANTES, MCP‐1, C10, and Lptn were obtained, and confirmed by Southern blot and sequencing. In contrast, MIP‐1α, MIP‐1β, and MIP‐1γ were not detected. Higher levels of expression were revealed by Northern blot analysis for Mig‐1, MCP‐1 and C10. The lymphatic endothelium‐restricted production of these chemokines was also confirmed by in situ hybridization. The presence of high C10 mRNA expression levels in LEC was particularly unexpected, because the production of this molecule has been previously identified only in cells of the haematopoietic lineage. These observations represent the first detailed analysis of chemokine production by lymphatic endothelial cells and may account, in part, for the mechanism of leucocyte recruitment into the lymphatics, and of lymphocyte recirculation within the lymphatic system.

Prognostic value of interleukin-1 receptor antagonist in patients undergoing percutaneous coronary intervention.

Elevated plasma levels of inflammatory markers, such as C-reactive protein (CRP), have been associated with adverse outcome in selected patients with coronary artery disease (CAD) treated with coronary angioplasty or stenting. The aim of this study was to evaluate the predictive value of preprocedural interleukin-1 receptor antagonist (IL-1Ra) plasma levels for long-term major adverse cardiac events (MACE) in a series of unselected patients with symptomatic CAD treated with percutaneous coronary intervention (PCI). Seventy-three consecutive patients (62 men, aged 62 +/- 9 years) undergoing PCI were enrolled in a prospective follow-up study. IL-1Ra and CRP plasma levels were measured before the procedure; 36 patients (49%) had unstable angina pectoris on admission, 37 (51%) had chronic stable angina pectoris, and 30 (41%) had multivessel CAD, 15 of whom underwent multivessel PCI. Success was achieved in all 73 patients, with coronary stenting performed in 63 (86%). Follow-up clinical assessment included occurrence of MACE at 3, 6, 12, and 18 months. Logistic regression analysis, performed to determine independent predictors of MACE, identified IL-1Ra levels in the upper quartile as the only independent predictive factor of MACE at 18 months (19% in the fourth quartile vs 0% in the first quartile; p = 0.032). Patients with high preprocedural CRP levels (fourth quartile) had a nonsignificant increased risk of MACE (p = 0.09). Thus, preprocedural IL-1Ra plasma levels appear to be a valuable independent predictive factor of MACE in unselected patients undergoing PCI.

Genotypes and haplotypes in the IL-1 gene cluster: Analysis of two genetically and diagnostically distinct groups of Alzheimer patients

Increased risk of Alzheimers disease (AD) has been associated with polymorphisms in the IL-1 gene cluster, and in particular with the IL-1alpha-889 T/T genotype. However, this association is still unclear, and needs further investigation. In order to clarify the role of these polymorphisms in the complex pathogenesis of AD we examined genotype and haplotype frequencies of the two C-to-T SNPs at position -889 and -551 in the IL-1alpha and IL-1beta genes, respectively, and of the 86 bp VNTR intron-2 polymorphisms in the IL-1Ra gene. The analysis was performed in two genetically and diagnostically distinct groups of sporadic AD from Italy and the USA. In the Italian group a significant association between the IL-1alpha-889 T/T genotype and AD (OR=3.022, 95% CI: 1.001-9.119) was found, whereas no difference was found in the group from the USA. Results were also compared with previously published studies that analyzed the same IL-1 polymorphisms in AD. In both groups, the analysis of the estimated haplotypes shows that AD patients and controls who carry the IL-1beta-511 C allele, were also more frequently carriers of the IL-1Ra 1 allele (haplotypes -C-1). The total frequency of the two -C-1 haplotypes (C-C-1 plus T-C-1) was about one half of the total frequency of the eight estimated haplotypes. This was confirmed by significant linkage disequilibrium between these two loci in both the Italian and USA groups. In the Italian group a weak association of the T-C-2 haplotype with the disease (OR=1.648, 95% CI: 1.519-1.788) was also found, whereas in the USA group no difference was found. Although ours and other published data on different samples of Caucasian and non-Caucasian AD show a great heterogeneity in the frequencies of the IL-1alpha-889, the IL-1beta-511 and the IL-1Ra VNTR gene polymorphisms, we confirm the role of the IL-1alpha-889 T/T genotype as a risk factor for sporadic AD, and show the presence of an allelic association between IL-1beta C and IL-1Ra 1 alleles in both the Italian and the USA groups, confirmed by the presence of significant levels of linkage disequilibrium between these two loci.

Endothelin-1 induces connective tissue growth factor expression in cardiomyocytes.

Endothelin (ET)-1 is a vasoconstrictor involved in cardiovascular diseases. Connective tissue growth factor/CCN2 (CTGF) is a fibrotic mediator overexpressed in human atherosclerotic lesions, myocardial infarction, and hypertension. In different cell types CTGF regulates cell proliferation/apoptosis, migration, and extracellular matrix (ECM) accumulation and plays important roles in angiogenesis, chondrogenesis, osteogenesis, tissue repair, cancer and fibrosis. In the present study, we investigated the ET-1 signaling which triggers CTGF expression in cultured adult mouse atrial-muscle HL-1 cells used as a model system. ET-1 activated the CTGF promoter and induced CTGF expression at both mRNA and protein levels. Real-time PCR analysis revealed CTGF induction also in isolated rat heart preparations perfused with ET-1. Several intracellular signals elicited by ET-1 via ET receptors and even Epidermal Growth Factor Receptor (EGFR) contributed to the up-regulation of CTGF, including ERK activation and induction of the AP-1 components c-fos and c-jun, as also evaluated by ChIP analysis. Moreover, in cells treated with ET-1 the expression of ECM component decorin was abolished by CTGF silencing, indicating that CTGF is involved in ET-1 induced ECM accumulation not only in a direct manner but also through downstream effectors. Collectively, our data indicate that CTGF could be a mediator of the profibrotic effects of ET-1 in cardiomyocytes. CTGF inhibitors should be considered in setting a comprehensive pharmacological approach towards ET-1 induced cardiovascular diseases.

Portosystemic shunts in a large cohort of patients with liver cirrhosis: detection rate and clinical relevance

BackgroundThis study aimed to determine the detection rate and clinical relevance of portosystemic collaterals.MethodsWe studied 326 cirrhotics. Portosystemic collaterals, portal vein diameter, and splenic area were evaluated by color Doppler sonography; esophageal varices were detected by endoscopy.ResultsOf the cirrhotics, 130 had portosystemic collaterals (39.9% total, left gastric vein 11%, paraumbilical vein 7.4%, splenorenal shunts 13.8%, and combined shunts 7.7%). Cirrhotics without portosystemic collaterals or with a paraumbilical vein had a significantly narrower portal vein diameter than cirrhotics with a left gastric vein (P < 0.001). Cirrhotics with a paraumbilical vein had a significantly smaller splenic area than cirrhotics with a left gastric vein (P < 0.001), splenorenal shunts (P < 0.001), combined shunts (P < 0.001), or without portosystemic collaterals (P < 0.05). A significant association between portosystemic collaterals and Child’s classes or presence and type of esophageal varices was found (P < 0.0001 and P = 0.0004, respectively). The highest prevalence of Child’s class C and large (F-3) esophageal varices was found in cirrhotics with a left gastric vein (41.7% and 36.1%, respectively), whereas esophageal varices were absent in 47.4% of cirrhotics without portosystemic collaterals and in 58.3% of cirrhotics with a paraumbilical vein.ConclusionsThe left gastric vein is associated with some sonographic and clinical markers of disease severity, whereas the absence of portosystemic collaterals or the presence of paraumbilical veins seems to identify cirrhotics with markers predictive of a more favorable clinical course.