Fiorella Marcheselli

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.

MiR-146a as marker of senescence-associated pro-inflammatory status in cells involved in vascular remodelling

In order to identify new markers of vascular cell senescence with potential in vivo implications, primary cultured endothelial cells, including human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), human coronary artery endothelial cells (HCAECs) and ex vivo circulating angiogenic cells (CACs), were analysed for microRNA (miR) expression. Among the 367 profiled miRs in HUVECs, miR-146a, miR-9, miR-204 and miR-367 showed the highest up-regulation in senescent cells. Their predicted target genes belong to nine common pathways, including Toll-like receptor signalling (TLR) that plays a pivotal role in inflammatory response, a key feature of senescence (inflammaging). MiR-146a was the most up-regulated miR in the validation analysis (>10-fold). Mimic and antagomir transfection confirmed TLR’s IL-1 receptor-associated kinase (IRAK1) protein modulation in both young and senescent cells. Significant correlations were observed among miR-146a expression and β-galactosidase expression, telomere length and telomerase activity. MiR-146a hyper-expression was also validated in senescent HAECs (>4-fold) and HCAECs (>30-fold). We recently showed that CACs from patients with chronic heart failure (CHF) presented a distinguishing feature of senescence. Therefore, we also included miR-146a expression determination in CACs from 37 CHF patients and 35 healthy control subjects (CTR) for this study. Interestingly, a 1,000-fold increased expression of miR-146a was observed in CACs of CHF patients compared to CTR, along with decreased expression of IRAK1 protein. Moreover, significant correlations among miR-146a expression, telomere length and telomerase activity were observed. Overall, our findings indicate that miR-146a is a marker of a senescence-associated pro-inflammatory status in vascular remodelling cells.

Food restriction in female Wistar rats: V. Lipid peroxidation and antioxidant enzymes in the liver

The activities of antioxidant enzymes as well as the levels of basal and enzyme induced peroxidation have been investigated in liver of female Wistar undernourished rats. Food restriction was applied starting from the age of 3.5 months by feeding the animals on every-other-day schedule (EOD). Diet restriction prevented the age-dependent increase of basal and enzyme induced lipid peroxidation in both mitochondrial and microsomal liver membrane preparations. The activities of antioxidant enzyme, i.e. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) of liver decreased during aging in ad libitum fed rats. In the diet conditioned animals, a small increase of SOD and a complete recovery of CAT activities were observed. Present data support that food restriction improved the protection against peroxidation, and this may be in close relationship with the life prolonging effect of such a treatment.

Interrelationship among neutrophil efficiency, inflammation, antioxidant activity and zinc pool in very old age.

Neutrophils are the first barrier against infections. Aged neutrophils display impaired oxidative burst and phagocytosis with subsequent less capability to destroy bacteria. In successful ageing (nonagenarians), neutrophil efficiency (phagocytosis) increases. After ingested microbes, aged neutrophils are less prone to undergo apoptosis favouring chronic inflammation. Moreover, the superoxide dismutase (SOD) activity, which is necessary in avoiding ROS produced by oxidative burst, is limited in ageing. The mechanisms of age-related changes in neutrophil function are not fully understood, taking also into account that nonagenarians escape infections in comparison with elderly. Zinc pool may be involved because it is pivotal for neutrophil efficiency and SOD activity. Since zinc also controls the inflammation, via IL-6 and soluble factor of gp130 (sgp130), we have assessed the possible interrelationship among oxidative burst, apoptosis, inflammation, SOD, adhesion molecule Mac-1 and zinc pool in elderly and in nonagenarians. The oxidative burst and the capacity to increase Mac-1 after PMA stimulation decrease both in elderly and nonagenarians, but the latter display a slight increased neutrophil induced apoptosis, decreased sgp130, increased SOD, and more neutrophil zinc content, as it occurs in young-adults. Significant correlation exists between sgp130 and zinc pool in very old age. These findings suggest lower chronic inflammation in nonagenarians, via more zinc available, with subsequent long-life survival. Therefore, a more correct interrelationship among neutrophil efficiency, inflammation, antioxidant activity and zinc pool exists in successful ageing with subsequent more effectiveness to control the inflammatory response to pathogens.

Melatonin regulates the respiratory burst of human neutrophils and their depolarization

Pieri C, Recchioni R, Moroni F, Marcheselli F, Marra M, Marinoni Silvia, Di Primio R. Melatonin regulates the respiratory burst of human neutrophils and their depolarization. J. Pineal Res. 1998; 24:43–49.

MiR-21-5p and miR-126a-3p levels in plasma and circulating angiogenic cells: relationship with type 2 diabetes complications

Innovative biomarkers are required to manage type 2 diabetic patients (T2DM). We focused our study on miR-126-3p and miR-21-5p levels, as biomarkers of endothelial function and inflammation. MiRNAs levels were measured in plasma from 107 healthy subjects (CTR) and 193 diabetic patients (T2DM), 76 without (T2DM NC) and 117 with (T2DM C) complications. When diabetic complication were analysed as a whole, miR-126-3p and miR-21-5p levels declined significantly from CTR to T2DM NC and T2DM C patients. When miRNAs levels were related to specific complications, significantly higher miR-21-5p levels (0.46 ± 0.44 vs. 0.26±0.33, p < 0.001) and significant lower miR-126-3p levels (0.21±0.21 vs. 0.28±0.22, p = 0.032) were found in T2DM with previous major cardiovascular events (MACE) vs. all the others T2DM patients. To confirm these results we focused on circulating angiogenic cells (CACs) from a subgroup of 10 CTR, 15 T2DM NC and 15 T2DM patients with MACE. CACs from T2DM patients expressed higher miR-21-5p and lower miR-126-3p levels than CACs from CTR. Furthermore, CACs from T2DM + MACE showed the highest levels of miR-21-5p. Circulating miR-21-5p and miR-126-3p emerge as dynamic biomarkers of systemic inflammatory/angiogenic status. Their expression levels in CACs from T2DM with MACE suggest a shift from a proangiogenic to a proinflammatory profile.

Food restriction in female Wistar rats. I. Survival characteristics, membrane microviscosity and proliferative response in lymphocytes.

The effect of food restriction on the survival characteristics, membrane microviscosity and proliferative response in lymphocytes of female Wistar undernourished rats has been evaluated. Diet restriction was applied starting from the age of 3.5 months by feeding the animals on an every-other-day schedule (EOD). Diet restricted animals showed an increase of both mean, median and maximal life span as compared to the rats fed ad libitum (AL). Analyzing the survival curves by a parametric model, it emerged that undernutrition increased the individual resistance to environmental insults. In particular, it could be speculated that the positive influence was more pronounced in individuals with the lowest physiological capacities. The membrane microviscosity of lymphocytes was lower in EOD animals as compared to the AL ones even if one assumes a decrease in body temperature of 1-2 degrees C in EOD groups. The improvement of membrane microviscosity due to diet restriction may in part explain the improvement of proliferative response of lymphocytes from EOD groups.

Cellular senescence in cardiovascular diseases: Potential age-related mechanisms and implications for treatment

The aging process is associated with a loss of complexity in the dynamics of physiological systems that reduce the ability to adapt to stress, causing frailty and/or age-related diseases. At the cellular level, proliferative and/or oxidative-stress induced cell senescence associated with a pro-inflammatory state may greatly contribute to age-associated impaired tissue and organ functions. Senescence of endothelial and cardiac cells observed over normal aging, appear to be accelerated in age-related diseases and in particular, in cardiovascular disease (CVD). Although the molecular mechanisms of cellular senescence have been extensively studied, a complete understanding of their role in CVD is still limited. Cardiac, endothelial (EC), vascular smooth muscle (VSMC), leukocytic and stem cells (endothelial progenitor cells (EPC), embryonic stem cells (ESC) and haematopoietic stem cells (HSC)) may play a pivotal role on the maintenance and regeneration of cardiovascular tissue. Age-associated changes of such cells may enhance the risk of developing CVD. The purpose of this review is to illustrate how cellular senescence may affect tissue repair and maintenance toward CVD, focusing on the role played by telomere length and microRNA expression. Finally, interventions aimed at improving the age-related decline in vascular cells during aging and disease, as well as strategies to harness the regenerative capacity of stem cells in CVD will be discussed.

Conventional and novel diagnostic biomarkers of acute myocardial infarction: a promising role for circulating microRNAs.

Abstract Biomarkers play a critical role in the diagnosis of acute myocardial infarction (AMI), especially in patients with atypical clinical and/or electrocardiographic presentation or co-morbidities, like the elderly. High-sensitivity assays based on specific biomarkers (e.g. cardiac troponins) enabling earlier AMI diagnosis have recently become available in clinical practice. Although no single biomarker of myocardial necrosis is ever likely to afford AMI diagnosis, a combination including different biomarkers for necrosis and ischemia, like new circulating molecules (microRNAs), could enhance diagnostic specificity. We review the recent literature on conventional and novel AMI biomarkers, with special emphasis on circulating microRNAs.

Platelet as a physiological model to investigate apoptotic mechanisms in Alzheimer β-amyloid peptide production

Although neuronal apoptosis in Alzheimers disease is generally interpreted as the consequence of the toxicity of extracellular beta-amyloid (Abeta) peptide aggregates, some experimental results provide evidence that the Abeta overproduction can be the result of a primary neuronal degeneration. As platelets are considered a good model where to study proteolytic processing of the amyloid precursor protein (APP), we exposed platelets to the proapoptotic agent ionomycin and analyzed Abeta40 and Abeta42 levels in the intracellular and extracellular compartments. The activation of apoptotic pathways in platelets has been verified by mitochondrial membrane depolarization, exposure of phosphatidylserine, protease activation and morphological changes. A significative increase in intraplatelet Abeta40, but not Abeta42, was observed after 10 min treatment with ionomycin. Thus, the activation of apoptotic pathways in platelets determines an altered processing of APP leading to elevated levels of intracellular Abeta40. The specific intracellular production of Abeta40 represents a potential threat to the cells since very high local Abeta40 concentration increases the risk of its aggregation and toxicity. As a result, Abeta40 might be dangerous even before it becomes secreted rendering neurons highly vulnerable.