Fiammetta Monacelli

Advanced glycation end products and bone loss during aging.

Abstract: It is well known that bone mass density decreases with age. Age‐related bone mass loss is ascribed to several factors. Nonenzymatic glycation has been proposed as a new potential factor in the loss of bone during aging. In this study we evaluated the concentration of pentosidine, an advanced glycation end product, in cortical and trabecular bone and in the plasma of subjects undergoing orthopedic surgery. The relationship between these parameters and a clinical index of osteoporosis was also studied. Samples of bone and plasma of 104 nondiabetic subjects (74 women and 30 men), 72 ± 1 years old, were studied. Pentosidine was determined by HPLC after decalcification and hydrolysis. The radiologic Singh index was evaluated blindly by orthopedic surgeons to provide the degree of osteoporosis. Pentosidine concentration of cortical bone shows a significant exponential increase with age (r= 0.610, P < 0.001). This increase, however, is not seen in the trabecular bone, which is characterized by a large spread in the data. Interestingly the concentration of cortical pentosidine is also related to the Singh score (rs=−0.274, P < 0.01). Plasma pentosidine has a significant exponential correlation with age (r=+0.339, P < 0.001) and a linear correlation with the cortical bone pentosidine (r=+0.248, P < 0.05). This study demonstrates that pentosidine increases exponentially in cortical bone during aging, and is thus a good biomarker for the degree of bone mass density loss. The trabecular bone concentration of pentosidine is more variable, probably because of the turnover rate and the local environment; plasma pentosidine might provide information on the bone turnover rate.

Pentosidine Effects on Human Osteoblastsin Vitro

Osteoporosis, a multifactorial and progressive skeletal metabolic disease, is characterized by low‐mass density and structural deterioration of bone micro‐architecture that leads to enhanced bone fragility and increased susceptibility to fractures. Recently, it has been proposed that age‐related bone loss could be correlated with the glycoxidative process. The aim of the present study was to investigate the in vitro effects of pentosidine, a glycoxidative end product, on human osteoblasts (HOb). The mineralization rate, the specific bone markers (alkaline phosphatase [ALP], collagen Iα1 [COL Iα1], osteocalcin [BGP]), and the human receptor for advanced glycation end products (RAGE) gene expression have been evaluated. Pentosidine incubation of HOb caused a significant decrease in ALP, Col Iα1, and RAGE mRNA levels, but only the RAGE gene expression decreased with no dose dependency. Moreover, pentosidine incubation of osteoblasts hampered the formation of bone nodules. No effect was observed on BGP gene expression under all experimental conditions. Our data gives further support to a detrimental effect of AGEs on bone that leads to functional alterations of osteoblasts. This study addresses a crucial role of protein glycoxidation in the bone mineralization process. AGEs formation and accumulation in bone may be one of the first pathogenetic steps of bone remodeling in aging and in age‐related diseases, leading to enhanced bone mass loss.

Glycated fetal calf serum affects the viability of an insulin-secreting cell line in vitro.

The purpose of the present study was to evaluate the direct effects of advanced glycation end products (AGEs) on beta-cells by their exposure to a glycated serum to estimate the cellular viability and the related insulin secretion. Glycation of fetal calf serum was obtained by incubation with 50 mol/L ribose at 37 degrees C for 7 days; at the end of this incubation period, the pentosidine content ranged between 15 and 16 x 10(5) pmol/L. HIT-T15 cells, a pancreatic islet cell line, were grown and cultured for 5 days in Roswell Park Memorial Institute (RPMI) medium containing either not glycated (NGS) or glycated (GS) fetal calf serum. Cellular oxidative stress (ie, thiobarbituric acid-reactive substances) was assessed by high-performance liquid chromatography. Cellular viability was evaluated by detection of proliferation, cell necrosis, and cell apoptosis rate. The insulin secretion and the related intracellular content were evaluated by enzyme-linked immunosorbent assay. The present study reported, after 5 days of exposure to the glycation environment, a moderately reduced cellular proliferation (-20.44% +/- 2.92%) with a corresponding increase of cell necrosis (+67.7% +/- 1.56%) and cell apoptosis (+39.83% +/- 2.92%) rate in comparison with the untreated cells. Oxidative intracellular stress was higher in GS conditions compared with the NGS ones (+293.3% +/- 87.53%). Insulin release from GS-treated HIT-T15 cells was lower than that of NGS-treated cells both when cells were stimulated with low glucose concentration (2.8 mmol/L, -30.3% +/- 4.91%) or when they were challenged with high glucose concentration (16.7 mmol/L, -29.2% +/- 5.82%). Incubation of HIT-T15 cells with glycated serum also caused a significant decrease of insulin intracellular content (-44.47% +/- 9.98%). Thus, AGEs were shown to exert toxic effects on insulin-secreting cells. Chronically high intracellular oxidative stress, due to accumulation of AGEs, affects the insulin secretion machinery. The present data suggest a pivotal role of the non-enzymatic glycation process in the onset and progression of diabetes during aging and a direct adverse effect of a glycated environment on the pancreatic islet cells.

Reliability study in five languages of the translation of the pain behavioural scale Doloplus

Non‐verbal pain assessment scales are useful tools for pain evaluation in persons with communication disorders and moderate–severe dementia. The Doloplus® was one of the first scales to be developed and validated as a pain assessment tool in older adults with dementia. This study aims at evaluating the translation of the Doloplus® scale in five languages, as regards test–retest and inter‐rater reliability. Results show that both tests are good or excellent for the English, Italian, Portuguese and Spanish versions and moderate for the Dutch version. These results bring a unique opportunity to include the translated Doloplus® scale in daily assessment of elderly persons with communication disorders, and future studies should focus on enriching the validation of the scale in each language.

Nicotinamide phosphoribosyltransferase promotes epithelial-to-mesenchymal transition as a soluble factor independent of its enzymatic activity

Background: Nicotinamide phosphoribosyltransferase (NAMPT) acts both as an enzyme in the production of the coenzyme NAD+ and as a secreted cytokine. Results: In breast cancer cells, NAMPT induces the epithelial-to-mesenchymal transition, a process that underlies metastasis, as a secreted protein independent of its enzymatic activity. Conclusion: Secreted NAMPT promotes epithelial-to-mesenchymal transition. Significance: Extracellular NAMPT neutralization may be of therapeutic value. Boosting NAD+ biosynthesis with NAD+ intermediates has been proposed as a strategy for preventing and treating age-associated diseases, including cancer. However, concerns in this area were raised by observations that nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in mammalian NAD+ biosynthesis, is frequently up-regulated in human malignancies, including breast cancer, suggesting possible protumorigenic effects for this protein. We addressed this issue by studying NAMPT expression and function in human breast cancer in vivo and in vitro. Our data indicate that high NAMPT levels are associated with aggressive pathological and molecular features, such as estrogen receptor negativity as well as HER2-enriched and basal-like PAM50 phenotypes. Consistent with these findings, we found that NAMPT overexpression in mammary epithelial cells induced epithelial-to-mesenchymal transition, a morphological and functional switch that confers cancer cells an increased metastatic potential. However, importantly, NAMPT-induced epithelial-to-mesenchymal transition was found to be independent of NAMPT enzymatic activity and of the NAMPT product nicotinamide mononucleotide. Instead, it was mediated by secreted NAMPT through its ability to activate the TGFβ signaling pathway via increased TGFβ1 production. These findings have implications for the design of therapeutic strategies exploiting NAD+ biosynthesis via NAMPT in aging and cancer and also suggest the potential of anticancer agents designed to specifically neutralize extracellular NAMPT. Notably, because high levels of circulating NAMPT are found in obese and diabetic patients, our data could also explain the increased predisposition to cancer of these subjects.

Evidence for a role of the histone deacetylase SIRT6 in DNA damage response of multiple myeloma cells.

Multiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD(+)-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM.

Structural Alterations of Human Serum Albumin Caused by Glycative and Oxidative Stressors Revealed by Circular Dichroism Analysis

The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points.

Artificial Neural Networks Identify the Predictive Values of Risk Factors on the Conversion of Amnestic Mild Cognitive Impairment

The search for markers that are able to predict the conversion of amnestic mild cognitive impairment (aMCI) to Alzheimers disease (AD) is crucial for early mechanistic therapies. Using artificial neural networks (ANNs), 22 variables that are known risk factors of AD were analyzed in 80 patients with aMCI, for a period spanning at least 2 years. The cases were chosen from 195 aMCI subjects recruited by four Italian Alzheimers disease units. The parameters of glucose metabolism disorder, female gender, and apolipoprotein E epsilon3/epsilon4 genotype were found to be the biological variables with high relevance for predicting the conversion of aMCI. The scores of attention and short term memory tests also were predictors. Surprisingly, the plasma concentration of amyloid-beta (42) had a low predictive value. The results support the utility of ANN analysis as a new tool in the interpretation of data from heterogeneous and distinct sources.

Plasma levels of insulin and amyloid β42 are correlated in patients with amnestic Mild Cognitive Impairment

Epidemiological and experimental data suggest that type 2 diabetes (DM2) and sporadic late-onset Alzheimers disease (AD) share a common mechanism, that is able to produce accumulation of insulin and amyloid beta 42 (Abeta42), the major pathogenic events respectively of the two conditions. In 71 non diabetic patients with amnestic mild cognitive impairment we found a significant linear correlation between fasting plasma levels of insulin and Abeta42 (R = +0.25, P < 0.05). The levels of both peptides were elevated in comparison to 48 age-matched cognitively normal controls. The correlation of insulin and Abeta42 plasma levels suggests a pathogenic link between DM2 and sporadic AD.

Vitamin E-coated filter decreases levels of free 4-hydroxyl-2-nonenal during haemodialysis sessions

Uraemic subjects undergoing chronic haemodialysis show increased oxidative stress. The use of non-biocompatible filters and reduced antioxidative defences are important sources of reactive oxygen species (ROS) release. The highly oxidative environment accelerates the onset and progression of tissue damage and atherosclerotic cardiovascular disease. The aldehyde 4-hydroxyl-2-nonenal (HNE) is probably the best marker of oxidative stress. In this study, the concentration of plasma HNE was evaluated in eight uremic subjects during two sessions of haemodialysis: the first using a standard biocompatible filter and the second using a filter coated with vitamin E. Baseline plasma levels of HNE were elevated, and dropped during haemodialysis. At the end of the session, however, low levels were maintained only when the vitamin E-modified filter was used. By contrast, a marked increase in HNE was recorded at the end of the session in all subjects who underwent haemodialysis with the conventional filter. This study provides evidence that the vitamin E-coated filter plays a role in counteracting oxidative stress. The chronic use of vitamin E-modified filters in haemodialysed subjects might help to counterbalance oxidative attack and, consequently, contribute to preventing cardiovascular disease.