Maria Cristina Albertini

H2O2-induced block of glycolysis as an active ADP-ribosylation reaction protecting cells from apoptosis

ABSTRACT H2O2 treatment on U937 cells leads to the block of glycolytic flux and the inactivation of glyceraldehyde‐3‐phosphate‐dehydrogenase by a posttranslational modification (possibly ADP‐ribosylation). Glycolysis spontaneously reactivates after 2 h of recovery from oxidative stress; thereafter cells begin to undergo apoptosis. The specific ADP‐ribosylation inhibitor 3‐aminobenzamide inhibits the stress‐induced inactivation of glyceraldehyde‐3‐phosphate‐dehydrogenase and the block of glycolysis; concomitantly, it anticipates and increases apoptosis. Exogenous block of glycolysis (i.e., by culture in glucose‐free medium or with glucose analogs or after NAD depletion), turns the transient block into a stable one: this results in protection from apoptosis, even when downstream cell metabolism is kept active by the addition of pyruvate. All this evidence indicates that the stress‐induced block of glycolysis is not the result of a passive oxidative damage, but rather an active cell reaction programmed via ADP‐ribosylation for cell self‐defense.—Colussi, C., Albertini, M. C., Coppola, S., Rovidati, S., Galli, F., Ghibelli, L. H2O2‐induced block of glycolysis as an active ADP‐ribosylation reaction protecting cells from apoptosis. FASEB J. 14, 2266‐2276 (2000)

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.

Redox state, antioxidative activity and lipid peroxidation in erythrocytes and plasma of chronic ambulatory peritoneal dialysis patients

Red blood cells and plasma reduced and oxidized glutathione levels, glutathione peroxidase (GSH-Px) activity, thiobarbituric acid reactants (TBAR) of both chronic ambulatory peritoneal dialysis (CAPD) patients and a matched control group were investigated in this study. Oxidized and reduced pyridinic nucleotides in red blood cells (RBC), in which NADPH is a direct expression of hexose monophosphate shunt function, were also studied. The results obtained indicate that RBC and plasma are exposed to oxidative stress in CAPD. This condition is characterized by a decreased GSH/GSSG ratio, particularly evident in RBC as a consequence of the GSSG accumulation. Lipid peroxidation is increased, as indicated by raised TBAR levels, and reduced pyridinic nucleotides are decreased. Increased GSH-Px levels and unmodified or slightly increased GSH content were observed in the RBC but not in plasma, which showed decreased GSH and unmodified peroxidase activity. Peroxidase correlated positively with TBAR levels in the RBC lysates. In a subgroup of patients treated with erythropoietin (vs. untreated patients and controls) no differences were observed in the glutathione-related parameters studied. These data suggest that a mechanism for adaptation to oxidative conditions may be present in CAPD and its effects on RBC integrity are discussed in comparison with the hemodialysis conditions previously studied.

Erythrocyte Redox State in Uremic Anemia: Effects of Hemodialysis and Relevance of Glutathione Metabolism

Reduced and oxidized glutathione and pyridine coenzymes, glutathione-related enzymes and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) were investigated in the RBC of patients with chronic renal failure (CRF) and in age- and sex-matched controls. The effects of hemodialysis (HD) were also studied. A defective RBC redox state was shown in the CRF group based on a decreased GSH/GSSG ratio and NADPH levels. Increased activities of glutathione transferase (GSH-S-T) and Cu,Zn-SOD were observed before HD. Dialysis apparently restores the levels of antioxidant enzymes and at the same time strongly affects the redox state. Thus we can speculate that HD can generate severe redox impairment inducing damage in RBC and plasma antioxidant enzymes. Increased erythrocyte GSSG and GSM-S-T levels coupled with a reduced hexose monophosphate shunt (HMPS) function may be useful indexes of oxidative stress in uremic anemia.

Oxidative Damage during Hemodialysis Using a Vitamin-E-Modified Dialysis Membrane: A Preliminary Characterization

A comparison of the oxyradical exposure during hemodialysis (HD) carried out with vitamin-E-modified cellulose (CL-E) or conventional membranes, studying red blood cell (RBC) and plasma lipoperoxidation and RBC glutathione metabolism, was done. In this preliminary characterization of a new and original approach to the prevention of free radical damage in HD, the results obtained indicate that lipoperoxidation in plasma and RBC is decreased and therefore oxidative damage can be significantly decreased using CL-E dialysis membranes instead of conventional membranes.

Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells

Abstract:  Among the non‐neurological functions of melatonin, much attention is being directed to the ability of melatonin to modulate the immune system, whose cells possess melatonin‐specific receptors and biosynthetic enzymes. Melatonin controls cell behaviour by eliciting specific signal transduction actions after its interaction with plasma membrane receptors (MT1, MT2); additionally, melatonin potently neutralizes free radicals. Melatonin regulates immune cell loss by antagonizing apoptosis. A major unsolved question is whether this is due to receptor involvement, or to radical scavenging considering that apoptosis is often dependent on oxidative alterations. Here, we provide evidence that on U937 monocytic cells, apoptosis is antagonized by melatonin by receptor interaction rather than by radical scavenging. First, melatonin and a set of synthetic analogues prevented apoptosis in a manner that is proportional to their affinity for plasma membrane receptors but not to their antioxidant ability. Secondly, melatonins antiapoptotic effect required key signal transduction events including G protein, phospholipase C and Ca2+ influx and, more important, it is sensitive to the specific melatonin receptor antagonist luzindole.

Static magnetic fields enhance skeletal muscle differentiation in vitro by improving myoblast alignment

Static magnetic field (SMF) interacts with mammal skeletal muscle; however, SMF effects on skeletal muscle cells are poorly investigated. The myogenic cell line L6, an in vitro model of muscle development, was used to investigate the effect of a 80 ± mT SMF generated by a custom‐made magnet. SMF promoted myogenic cell differentiation and hypertrophy, i.e., increased accumulation of actin and myosin and formation of large multinucleated myotubes. The elevated number of nuclei per myotube was derived from increased cell fusion efficiency, with no changes in cell proliferation upon SMF exposure. No alterations in myogenin expression, a modulator of myogenesis, occurred upon SMF exposure. SMF induced cells to align in parallel bundles, an orientation conserved throughout differentiation. SMF stimulated formation of actin stress‐fiber like structures. SMF rescued muscle differentiation in the presence of TNF, a muscle differentiation inhibitor. We believe this is the first report showing that SMF promotes myogenic differentiation and cell alignment, in the absence of any invasive manipulation. SMF‐enhanced parallel orientation of myotubes is relevant to tissue engineering of a highly organized tissue such as skeletal muscle. SMF rescue of muscle differentiation in the presence of TNF may have important therapeutic implications.

Melatonin reduces endoplasmic reticulum stress and preserves sirtuin 1 expression in neuronal cells of newborn rats after hypoxia-ischemia

Conditions that interfere with the endoplasmic reticulum (ER) functions cause accumulation of unfolded proteins in the ER lumen, referred to as ER stress, and activate a homeostatic signaling network known as unfolded protein response (UPR). We have previously shown that in neonatal rats subjected to hypoxia–ischemia (HI), melatonin administration significantly reduces brain damage. This study assessed whether attenuation of ER stress is involved in the neuroprotective effect of melatonin after neonatal HI. We found that the UPR was strongly activated after HI. Melatonin significantly reduced the neuron splicing of XBP‐1 mRNA, the increased phosphorylation of eIF2α, and elevated expression of chaperone proteins GRP78 and Hsp70 observed after HI in the brain. CHOP, which plays a convergent role in the UPR, was reduced as well. Melatonin also completely prevented the depletion of SIRT‐1 induced by HI, and this effect was observed in the same neurons that over‐express CHOP. These results demonstrate that melatonin reduces ER stress induced by neonatal HI and preserves SIRT‐1 expression, suggesting that SIRT‐1, due to its action in the modulation of a wide variety of signaling pathways involved in neuroprotection, may play a key role in the reduction of ER stress and neuroprotection observed after melatonin.

Modulation of Caspase Activity Regulates Skeletal Muscle Regeneration and Function in Response to Vasopressin and Tumor Necrosis Factor

Muscle homeostasis involves de novo myogenesis, as observed in conditions of acute or chronic muscle damage. Tumor Necrosis Factor (TNF) triggers skeletal muscle wasting in several pathological conditions and inhibits muscle regeneration. We show that intramuscular treatment with the myogenic factor Arg8-vasopressin (AVP) enhanced skeletal muscle regeneration and rescued the inhibitory effects of TNF on muscle regeneration. The functional analysis of regenerating muscle performance following TNF or AVP treatments revealed that these factors exerted opposite effects on muscle function. Principal component analysis showed that TNF and AVP mainly affect muscle tetanic force and fatigue. Importantly, AVP counteracted the effects of TNF on muscle function when delivered in combination with the latter. Muscle regeneration is, at least in part, regulated by caspase activation, and AVP abrogated TNF-dependent caspase activation. The contrasting effects of AVP and TNF in vivo are recapitulated in myogenic cell cultures, which express both PW1, a caspase activator, and Hsp70, a caspase inhibitor. We identified PW1 as a potential Hsp70 partner by screening for proteins interacting with PW1. Hsp70 and PW1 co-immunoprecipitated and co-localized in muscle cells. In vivo Hsp70 protein level was upregulated by AVP, and Hsp70 overexpression counteracted the TNF block of muscle regeneration. Our results show that AVP counteracts the effects of TNF through cross-talk at the Hsp70 level. Therefore, muscle regeneration, both in the absence and in the presence of cytokines may be enhanced by increasing Hsp70 expression.