on PloS one
by Grimaldi A, Balestrieri ML, D'Onofrio N, Di Domenico G, Nocera C, Lamberti M, Tonini G, Zoccoli A, Santini D, Caraglia M, Pantano F
Endothelial Progenitor Cells (EPCs), a minor subpopulation of the mononuclear cell fraction in peripheral blood, play a critical role in cancer development as they contribute to angiogenesis-mediated pathological neovascularization. In response to tumor cytokines, including VEGF, EPCs mobilize from the bone marrow into the peripheral circulation and move to the tumor bed where they incorporate into sprouting neovessels. In the present study, we evaluated the effects of everolimus (Afinitor, Novartis), a rapamycin analogue, alone or in combination with chloroquine, a 4-alkylamino substituted quinoline family member, one of the autophagy inhibitors, on EPCs biological functions. We found that either everolimus or chloroquine induce growth inhibition on EPCs in a dose-dependent manner after 72 h from the beginning of incubation. The combined administration of the two drugs to EPC was synergistic in inducing growth inhibition; in details, the maximal pharmacological synergism between everolimus and chloroquine in inducing growth inhibition on EPCs cells was recorded when chloroquine was administered 24 h before everolimus. Moreover, we have studied the mechanisms of cell death induced by the two agents alone or in combination on EPCs and we have found that the synergistic effect of combination on EPC growth inhibition was paralleled by increased apoptosis induction and reduced autophagy. These effects occurred together with biochemical features that are typical of reduced autophagic death such as increased co-immunoprecipitation between Beclin 1 and Bcl-2. Chloroquine antagonized the inhibition of the activity of Akt→4EBP1 axis mediated by everolimus and at the same time it blocked the feed-back activation of Erk-1/2 induced by RAD in EPCs. These data suggest a new strategy in order to block angiogenesis in tumours in which this process plays a key role in both the sustainment and spreading of cancer cells.
on Journal of cellular biochemistry
by Servillo L, D'Onofrio N, Longobardi L, Sirangelo I, Giovane A, Cautela D, Castaldo D, Giordano A, Balestrieri ML
Hyperglycaemia, a characteristic feature of diabetes mellitus, induces endothelial dysfunction and vascular complications by accelerating endothelial cell (EC) senescence and limiting the proliferative potential of these cells. Here we aimed to investigate the effect of stachydrine, a proline betaine present in considerable quantities in juices from fruits of the Citrus genus, on EC under high-glucose stimulation, and its underlying mechanism. The senescence model of EC was set up by treating cells with high-glucose (30 mM) for different times. Dose-dependent (0.001-1 mM) evaluation of cell viability revealed that stachydrine does not affect cell proliferation with a similar trend up to 72 h. Noticeable, stachydrine (0.1 mM) significantly attenuated the high-glucose induced EC growth arrest and senescence. Indeed, co-treatment with high-glucose and stachydrine for 48 h kept the percentage of EC in the G0 /G1 cell cycle phase near to control values and significantly reduced cell senescence. Western blot analysis and confocal-laser scanning microscopy revealed that stachydrine also blocked the high-glucose induced upregulation of p16(INK4A) and downregulation of SIRT1 expression and enzyme activity. Taken together, results here presented are the first evidence that stachydrine, a naturally occurring compound abundant in citrus fruit juices, inhibits the deleterious effect of high-glucose on EC and acts through the modulation of SIRT1 pathway. These results may open new prospective in the identification of stachydrine as an important component of healthier eating patterns in prevention of cardiovascular diseases.
on International journal of molecular sciences
by Servillo L, Giovane A, D'Onofrio N, Casale R, Cautela D, Castaldo D, Balestrieri ML
N(G),N(G)-dimethyl-L-arginine (ADMA) and N(G)-methyl-L-arginine (NMMA) are endogenous inhibitors of nitric oxide synthase (NOS). In contrast, N(G),N'(G)-dimethyl-L-arginine (SDMA) possesses only a weak inhibitory potency towards neuronal NOS and it is known to limit nitric oxide (NO) production by competing with L-arginine for cellular uptake. The inhibition of NOS is associated with endothelial dysfunction in cardiovascular diseases as well in chronic renal failure. L-homoarginine (HArg), a structural analog of L-arginine (Arg), is an alternative but less efficient substrate for NOS. Besides, it inhibits arginase, leading to an increased availability of L-arginine for NOS to produce NO. However, its relation with cardiovascular disease remains unclear. To date, several analytical methods for the quantitative determination of Arg, HArg, NMMA, AMDA, and SDMA in biological samples have been described. Here, we present a simple, fast, and accurate HPLC-ESI-MS/MS method which allows both the simultaneous determination and quantification of these compounds without needing derivatization, and the possibility to easily modulate the chromatographic separation between HArg and NMMA (or between SDMA and ADMA). Data on biological samples revealed the feasibility of the method, the minimal sample preparation, and the fast run time which make this method very suitable and accurate for analysis in the basic and clinical settings.
on International journal of cardiology
by Marfella R, Rizzo MR, Siniscalchi M, Paolisso P, Barbieri M, Sardu C, Savinelli A, Angelico N, Del Gaudio S, Esposito N, Rambaldi PF, D'Onofrio N, Mansi L, Mauro C, Paolisso G, Balestrieri ML
We examined the effects of peri-procedural intensive glycemic control during early percutaneous coronary intervention (PCI) on the number and differentiation of endothelial progenitor cells (EPCs) and myocardial salvage (MS) in hyperglycemic patients with first ST-elevation myocardial infarction (STEMI).
on Toxicology letters
by Pedata P, Bergamasco N, D'Anna A, Minutolo P, Servillo L, Sannolo N, Balestrieri ML
Air pollution exposure in industrialized cities is associated with an increased risk of morbidity and mortality attributed to cardiovascular diseases. Combustion exhausts emitted from motor vehicles and industries represent a major source of nanoparticles in the atmosphere. Flame-generated organic carbon nanoparticles (OC NPs) provide interesting model nanoparticles that simulate fresh combustion emissions near roadways or combustion sources. These model nanoparticles can be produced by controlling flame operating conditions and used to test possible toxicological mechanisms responsible for the observed health effects. OC NPs were used to investigate their possible effect on endothelial cells (EC) growth and production of proinflammatory lipid mediators. Results indicated a dose and time-dependent reduction in cell viability following incubation of EC with OC NPs for 24 and 48h. Fluorescence-activated cell sorting revealed that EC treated with OC NPs showed a cell proliferation index significantly lower than that of control cells and an increased apoptotic cell death. The annexin assay confirmed the increased apoptotic cell death. Moreover, OC NPs also induced a time-dependent increase of proinflammatory lysophospholipid production. These results, establishing that OC NPs induce EC proinflammatory lysophosholipid production and apoptotic cell death, provide the first evidence of the detrimental effect of OC NPs on EC.
on Journal of cellular physiology
by Vilasi A, Vilasi S, Romano R, Acernese F, Barone F, Balestrieri ML, Maritato R, Irace G, Sirangelo I
A range of debilitating human diseases is known to be associated with the formation of stable highly organized protein aggregates known as amyloid fibrils. The early prefibrillar aggregates behave as cytotoxic agents and their toxicity appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increase in free Ca(2+) that lead to apoptotic or necrotic cell death. However, specific signaling pathways that underlie amyloid pathogenicity remain still unclear. This work aimed to clarify cell impairment induced by amyloid aggregated. To this end, we used a combined proteomic and one-dimensional (1) H-NMR approach on NIH-3T3 cells exposed to prefibrillar aggregates from the amyloidogenic apomyoglobin mutant W7FW14F. The results indicated that cell exposure to prefibrillar aggregates induces changes of the expression level of proteins and metabolites involved in stress response. The majority of the proteins and metabolites detected are reported to be related to oxidative stress, perturbation of calcium homeostasis, apoptotic and survival pathways, and membrane damage. In conclusion, the combined proteomic and (1) H-NMR metabonomic approach, described in this study, contributes to unveil novel proteins and metabolites that could take part to the general framework of the toxicity induced by amyloid aggregates. These findings offer new insights in therapeutic and diagnostic opportunities.
on Journal of cellular physiology
by Sirangelo I, Irace G, Balestrieri ML
Amyloidosis is the accumulation of insoluble proteinaceous aggregates in vivo and is implicated in many neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's diseases. This article briefly reviews the current knowledge of amyloid aggregate toxicity and inflammatory signaling in the nervous system. In particular, we focus our attention on the role of platelet-activating factor (PAF) as mediator of amyloid cytotoxicity.
on Journal of agricultural and food chemistry
by Servillo L, Giovane A, Balestrieri ML, Casale R, Cautela D, Castaldo D
The occurrence and distribution in Citrus genus plants of N-methylated derivatives of tryptamine and their 5-hydroxylated forms are reported. Tryptamine, N-methyltryptamine, N,N-dimethyltryptamine, N,N,N-trimethyltryptamine, 5-hydroxytryptamine (serotonin), 5-hydroxy-N-methyltryptamine, 5-hydroxy-N,N-dimethyltryptamine (bufotenine), and 5-hydroxy-N,N,N-trimethyltryptamine (bufotenidine) were quantitated by LC-ESI-MS/MS. Leaves of all citrus plants examined contained N,N,N-trimethyltryptamine, a compound that we first discovered in the bergamot plant. Interestingly, we also found out that all plants examined contained 5-hydroxy-N,N-dimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine, compounds never described so far in the Citrus genus. As N,N,N-trimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine possess nicotine-like activity by exerting their action on acetylcholine receptors, it is conceivable that both represent the arrival point of a biosynthetic pathway aimed to provide Citrus plants with chemical defense against aggressors. This hypothesis is supported by our finding that leaves and seeds, which are more frequently attacked by biotic agents, are the parts of the plant where the highest levels of those compounds were found.
on Biology of reproduction
by Balestrieri ML, Gasparrini B, Neglia G, Vecchio D, Strazzullo M, Giovane A, Servillo L, Zicarelli L, D'Occhio MJ, Campanile G
The aim of this study was to compare the proteome profiles of the chorioamnion and corresponding caruncle for buffalo embryos that had either normal or retarded development on Day 25 after artificial insemination (AI). In experiment 1, embryos that were to subsequently undergo late embryonic mortality had a smaller width on Day 25 after AI than embryos associated with pregnancy on Day 45 after AI. In experiment 2, 25 Italian Mediterranean buffaloes underwent transrectal ultrasonography on Day 25 after AI, and pregnant animals were categorized as one of two groups based on embryonic width: normal embryos (embryonic width > 2.7 mm) and retarded embryos (embryonic width < 2.7 mm). Three buffaloes of each group were slaughtered on Day 27 after AI to collect chorioamnion and caruncle tissues for subsequent proteomic analyses. Two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometer analysis were used to ascertain the proteomic profiles. To confirm 2D-DIGE-results, three selected proteins were analyzed by Western blot. The proteomic profiles of the chorioamnion of retarded embryos and the corresponding caruncles showed differences in the expression of several proteins compared to normal embryos. In particular, a down-regulation was observed for proteins involved in protein folding (HSP 90-alpha, calreticulin), calcium binding (annexin A1, annexin A2), and coagulation (fibrinogen alpha-chain) (P < 0.05), whereas proteins involved in protease inhibition (alpha-1-antiproteinase, serpin H1, serpin A3-8), DNA and RNA binding (heterogeneous nuclear ribonucleoproteins A2/B1 and K), chromosome segregation (serine/threonine-protein phosphatase 2A), cytoskeletal organization (ezrin), cell redox homeostasis (amine oxidase-A), and hemoglobin binding (haptoglobin) were up-regulated (P < 0.05).
on Nitric oxide : biology and chemistry
by Servillo L, Giovane A, Cautela D, Castaldo D, Balestrieri ML
Endogenous methylarginines, N(G),N(G)-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), N(G)-N('G)-dimethyl-L-arginine (symmetric dimethylarginine; SDMA), and N(G)-monomethyl-L-arginine (monomethylarginine; NMMA) are supposed to be produced in human body through the methylation of protein arginine residues by protein arginine methyltransferases (PRMT) and released during proteolysis of the methylated proteins. Micromolar concentration of ADMA and NMMA can compete with arginine for nitric oxide synthase (NOS) reducing nitric oxide (NO) formation, whereas SDMA does not. Indeed, increased ADMA and SDMA plasma levels or a decreased arginine/ADMA ratio is related with risk factors for chronic kidney disease and cardiovascular disease. To the best of our knowledge the exogenous presence of methylarginines, like that in fruits and vegetables, has never been described so far. Here, we report the finding that methylarginines are ubiquitous in vegetables which represent an important part of human daily diet. Some of these vegetables contain discrete amounts of ADMA, SDMA, and NMMA. Specifically, among the vegetables examined, soybean, rye, sweet pepper, broad bean, and potato contain the highest ADMA and NMMA mean levels. Our results establish that the three methylarginines, in addition to being produced endogenously, can also be taken daily through the diet in conspicuous amounts. We propose that the contribution of the methylarginines contained in the vegetables of daily diet should be taken into account when the association between vegetable assumption and their levels is evaluated in clinical studies. Furthermore, a comprehensive understanding on the role of the digestive breakdown process and intestinal absorption grade of the methylarginines contained in vegetables is now needed.