Roberta Galeazzi

Age-related differences in the expression of circulating microRNAs: miR-21 as a new circulating marker of inflammaging

Circulating microRNAs (miRs) have been investigated as diagnostic/prognostic biomarkers in human diseases. However, little is known about their expression throughout the aging process. Eleven healthy individuals aged 20, 80 and 100 years underwent miR plasma profiling. The validation cohort consisted of 111 healthy adults (CTR) aged 20-105 years and included 30 centenarians. In addition, 34 patients with cardiovascular disease (CVD) and 15 healthy centenarian offspring (CO) were enrolled. An exploratory factorial analysis grouped the miRs into three main factors: factor 1 primarily higher in 20-year-old subjects, but these differences did not reach statistical significance, factor 2 primarily higher in octogenarians and factor 3 primarily higher in centenarians. MiR-21, the most highly expressed miR of factors 2 and 3, was further validated, confirming the differences in the age groups. MiR-21 expression was higher in the CVD patients and lower in the CO compared to the age-matched CTR. MiR-21 was correlated with C-reactive protein and fibrinogen levels. TGF-β signaling was the predicted common pathway targeted by miRs of factors 2 and 3. TGF-βR2 mRNA, a validated miR-21 target, showed the highest expression in the leukocytes from a subset of the octogenarians. Our findings suggest that miR-21 may be a new biomarker of inflammation.

Tumor necrosis factor-alpha gene -308G>A polymorphism is associated with ST-elevation myocardial infarction and with high plasma levels of biochemical ischemia markers.

ObjectivesAs is well known, acute myocardial infarction presents two electrocardiogram (EKG) patterns, ST-elevation (STEMI) and no ST-elevation (NSTEMI), characterized by different coronary artery thrombotic occlusion. Growing evidence shows that inflammation plays a central role in the pathogenesis of acute myocardial infarction. Among the factors that promote inflammation and arterial thrombosis, one of the most important is the proinflammatory cytokine tumor necrosis factor-&agr;. The expression of this cytokine is modulated by a polymorphism located at nucleotide −308 of tumor necrosis factor-&agr; promoter gene. The objective of our study is to verify whether tumor necrosis factor-&agr; −308 polymorphism is associated with risk of acute myocardial infarction (STEMI and NSTEMI) or with biochemical myocardial ischemia markers, such as troponin I, creatine kinase-MB, lactate dehydrogenase and myoglobin. MethodsWe analyzed tumor necrosis factor-&agr; −308 polymorphism in a total of 603 study participants: 293 elderly patients affected by acute myocardial infarction (STEMI and NSTEMI) and 310 healthy controls. ResultsWe found that individuals carrying the tumor necrosis factor-&agr; −308 AG+AA genotypes are significantly more represented among acute myocardial infarction patients affected by STEMI than among NSTEMI patients (OR=1.86, 95% CI 1.08–3.21, p=0.027) and healthy controls (OR=1.64, 95% CI 1.03–2.64, p=0.046). Furthermore, the patients carrying tumor necrosis factor-&agr; −308 AG+AA genotypes displayed significant increased levels of biochemical myocardial ischemia markers. ConclusionsOur study shows a significant association between the tumor necrosis factor-&agr; −308 polymorphism and the occurrence of STEMI, and suggests that the tumor necrosis factor-&agr; −308 polymorphism could play a role in the pathogenesis of cardiac ischemic damage, AA+AG genotype carrier individuals being likely to be affected by more severe ischemic damage than the rest of the population.

Remodelling of biological parameters during human ageing: evidence for complex regulation in longevity and in type 2 diabetes.

Factor structure analyses have revealed the presence of specific biological system markers in healthy humans and diseases. However, this type of approach in very old persons and in type 2 diabetes (T2DM) is lacking. A total sample of 2,137 Italians consisted of two groups: 1,604 healthy and 533 with T2DM. Age (years) was categorized as adults (≤65), old (66–85), oldest old (>85–98) and centenarians (≥99). Specific biomarkers of routine haematological and biochemical testing were tested across each age group. Exploratory factorial analysis (EFA) by principal component method with Varimax rotation was used to identify factors including related variables. Structural equation modelling (SEM) was applied to confirm factor solutions for each age group. EFA and SEM identified specific factor structures according to age in both groups. An age-associated reduction of factor structure was observed from adults to oldest old in the healthy group (explained variance 60.4% vs 50.3%) and from adults to old in the T2DM group (explained variance 57.4% vs 44.2%). Centenarians showed three-factor structure similar to those of adults (explained variance 58.4%). The inflammatory component became the major factor in old group and was the first one in T2DM. SEM analysis in healthy subjects suggested that the glucose levels had an important role in the oldest old. Factorial structure change during healthy ageing was associated with a decrease in complexity but showed an increase in variability and inflammation. Structural relationship changes observed in healthy subjects appeared earlier in diabetic patients and later in centenarians.

Albumin protects human red blood cells against Aβ25-35-induced lysis more effectively than ApoE

Inhibition of the lysis of human red blood cells (RBCs) exposed to amyloid peptide A&bgr;25–35 is an in vitro model for screening natural and synthetic substances potentially protective against amyloid damage. In this system, human serum and a component, namely apolipoprotein E (apoE), completely prevent RBC lysis. This report demonstrates that albumin, another serum component, is 8-fold more protective: a concentration of 12.5 μg/ml protects RBCs against 20 μM-A&bgr;25–35, and prevents the formation of fibrillar A&bgr;25–35 aggregates stainable by Congo Red. The biological relevance of these findings is suggested by the following: (1) a large fraction (∼90%) of circulating A&bgr;1–42 is bound to albumin; (2) albumin immunoreactivity is present in brain amyloid plaques; and (3) incubation of A&bgr; with albumin rapidly decreases detectable levels of free A&bgr; suggesting epitope masking. The results add new and important functional consequences to the amyloid-albumin relationship and imply that experimental systems investigating A&bgr; cytotoxicity should consider the protective interaction of albumin.

Association of MT1A haplotype with cardiovascular disease and antioxidant enzyme defense in elderly Greek population: comparison with an Italian cohort.

Metallothioneins (MT), the antioxidant zinc-binding proteins, seem to mediate cardioprotection. It has been postulated that zinc homeostasis and MT function may be altered, as a consequence of oxidative stress, in cardiovascular disease (CVD), with a potential implication of MT genetic polymorphisms. The present study explores the role of +647A/C and +1245A/G MT1A polymorphisms on the susceptibility to CVD, zinc status and enzyme antioxidant activity, in the Greek and Italian populations. The country selection was based on the lower zinc status and the reduced zinc dietary intake in Greece than in Italy despite the similar Mediterranean dietary pattern. A total of 464 old, healthy control subjects and 369 old CVD patients more than 70 years of age were studied. Logistic regression model indicated that +1245 MT1A G+ genotype significantly increased the risk of CVD in Greece (34.4% vs. 23.2%; odds ratio=1.88, 95% confidence interval=1.14-3.08; P=.013) but not in Italy. Haplotype analysis showed an increment of CG haplotype frequency in CVD Greek patients (17.4% vs. 10.6%, P<.05). Differential country-related frequency distribution was also recorded. Applying a multivariate regression model, +647/+1245 MT1A haplotype was associated with a modulation of enzyme antioxidant activities in both countries. Decreased plasma zinc and reduced intracellular Zn release, as well as increased enzyme antioxidant activity, were more apparent in Greek healthy donors than in Italy. In conclusion, +1245 MT1A polymorphism and +647/+1245 MT1A haplotype are implicated in CVD in Greece but not in Italy, suggesting a role of gene-diet interaction in the disease predisposition.

Helicobacter pylori masks differences in homocysteine plasma levels between controls and type 2 diabetic patients

Background Data in the literature have not clarified whether type 2 diabetes mellitus affects homocysteine plasma levels. Different variables able to influence homocysteine could be the cause of these controversial findings. An important but neglected confounding factor is Helicobacter pylori, which has been demonstrated to be a cause of elevated levels of homocysteine and which is prevalent in the Caucasian population, ranging from 30 to 40% incidence. Starting from these findings we wanted to verify whether differences in homocysteine levels exist between a type 2 diabetic population and a control group, taking into account the presence/absence of Helicobacter pylori.

Telomere/telomerase system impairment in circulating angiogenic cells of geriatric patients with heart failure

BACKGROUND The functional characteristics of circulating angiogenic cells (CACs) are impaired in congestive heart failure (CHF) patients, suggesting that CAC dysfunction could contribute to CHF pathogenesis. However, the underlying mechanisms are only partly unraveled. No data are currently available regarding telomere/telomerase system in CACs of CHF patients. METHODS CACs were obtained from 80 subjects: 40 healthy control subjects (CTR) [median age (IQR), 80 (76-85 yrs)] and 40 patients affected by post-ischemic cardiomyopathy CHF [median age (IQR), 82 (77-89)]. CAC and leukocyte telomere length, assessed as T/S ratio, and telomerase (TERT) activity were determined in all the enrolled subjects. Specificity and sensitivity of CAC and leukocyte T/S in discriminating between CHF and CTR were evaluated using Receiver Operator Characteristic (ROC) curve analysis and reported as AUC values. CD34+/VEGFR2+ number and pro-inflammatory cytokines plasma levels, such as IL-6 and TNF-α, were also measured. RESULTS CAC T/S and TERT activity were significantly reduced in CHF patients compared to CTR subjects. In leukocytes, only a significant T/S reduction was observed. AUC values were higher for CAC T/S with respect to leukocyte T/S (AUC=0.89, and AUC=0.73, P<0.01, respectively). In multivariate analysis, leukocyte T/S, CAC T/S, CAC TERT activity and NT-proBNP levels were confirmed as parameters significantly associated with CHF. CD34+/VEGFR2+ number, IL-6 and TNF-α plasma levels were significantly increased in CHF patients. CONCLUSIONS CACs from CHF patients are characterized by telomere/telomerase system impairment, providing new insight into the clinical relevance of CACs in CHF pathogenesis.

Association among 1267 A/G HSP70-2, -308 G/A TNF-α polymorphisms and pro-inflammatory plasma mediators in old ZincAge population.

Proinflammatory cytokines and heat shock proteins play relevant roles in the pathogenesis of inflammatory diseases. We investigated whether Hsp70 1267 A/G and TNF-α −308 G/A polymorphisms are associated with proinflammatory mediators, zinc status and laboratory parameters in 1,078 healthy elderly from ZincAge study. Hsp70 1267 A/G genotype and allele distribution were similar among various European countries, while a TNF-α genetic heterogeneity was observed between the Northern and the Southern European populations, with a major frequency of the −308 A variant in France, Germany and Poland. We used linear regression models to test additive, dominant or recessive associations of each SNP with proinflammatory mediators, laboratory parameters, metallothioneins and zinc status. Hsp70 1267 A/G SNP, but not TNF-α −308 G/A SNP, influences TNF-α and IL-6 plasma levels under additive, dominant and recessive models (for TNF-α only). An association between Hsp70 1267 A/G SNP and zinc plasma levels was observed in the dominant model. In particular, G allele carriers showed increased circulating pro-inflammatory cytokines and zinc. Moreover, both these SNPs affect creatinine levels suggesting a possible influence on renal function. In conclusion, Hsp70 1267 A/G SNP is associated with pro-inflammatory cytokine production in healthy elderly and might represent a possible determinant of individual susceptibility to inflammatory diseases.

Combination of biomarkers to predict mortality in elderly patients with myocardial infarction

OBJECTIVE The elderly subjects affected by Acute Myocardial Infarction (AMI) have the highest risk of mortality. Our study was designed to improve the capability of mortality risk stratification in elderly AMI patients through the concurrent evaluations of different biomarkers, including genetic markers. METHODS AND RESULTS One-year follow-up study was performed in 250 elderly AMI patients. The combination of high total Homocysteine (tHcy), low folate and vitamin B12 plasma levels (18.0+/-9.0 micromol/l; 4.4+/-1.2 ng/ml; 404.2+/-287.5 pg/ml, respectively) and elevated CRP plasma levels (> or =6 mg/dl) identify the highest-risk pathway of heart mortality (RR=4.20, IC 95% 1.62-10.89, P<0.002) with respect to the combination of low total tHcy, high folate and vitamin B12 plasma levels (12.4+/-5.2 micromol/l; 8.9+/-2.5 ng/ml; 546.9+/-379.8 pg/ml, respectively) and low CRP plasma levels (<6 mg/dl). CONCLUSION In elderly AMI patients the concomitant elevation of CRP and tHcy, associated with folate and vitamin B12 low levels, could be considered a significant predictive heart mortality risk factor.

Differential course of HIV-1 infection and apolipoprotein E polymorphism

We studied the course of infection with human immunodeficiency virus type 1 (HIV-1) in relation to apolipoprotein E (APOE) polymorphism found for 209 Italians treated at Infectious Disease Clinics in Rome and Modena. Clinically, patients were classified into four groups according to the yearly rate of decline in CD4+ cell count (LTNP: long-term non-progression; SLOW, ’NORMAL’ or RAPID). Patients at both extremes of the clinical spectrum, i.e. those who rapidly progressed to AIDS and those with stable high CD4 cell counts, had few APOE ɛ4 and ɛ2 alleles (P = 0.04). Detailed clinical information was then used to construct four model-based clinical profiles using grade-of-membership analysis (GoM), predictive of APOE genotypic frequencies: 1. The clinical profile associated with good long-term prognosis lacked ɛ2 (P=0.01); 2. Disease progression to AIDS was associated with ɛ4 and ɛ2, most evident for zidovudine-lamivudine regimens without a protease inhibitor (P = 0.03); and, 3. AIDS patients had low ɛ4 and ɛ2 frequencies, consistent with a high mortality rate among ɛ4+ and ɛ2+ AIDS patients. These findings suggest allele-specific immunomodulatory effects involving inherited APOE isoform important enough to alter the clinical course of HIV infection and, possibly, drug efficacy. They imply a connection between lipid metabolism and immunity potentially relevant to common disorders.