Publications by authors named "Paola Failli"

46 Publications

D-Tagatose Feeding Reduces the Risk of Sugar-Induced Exacerbation of Myocardial I/R Injury When Compared to Its Isomer Fructose.

Front Mol Biosci 2021 13;8:650962. Epub 2021 Apr 13.

Department of Drug Science and Technology, University of Turin, Turin, Italy.

It is known that fructose may contribute to myocardial vulnerability to ischemia/reperfusion (I/R) injury. D-tagatose is a fructose isomer with less caloric value and used as low-calorie sweetener. Here we compared the metabolic impact of fructose or D-tagatose enriched diets on potential exacerbation of myocardial I/R injury. Wistar rats were randomizedly allocated in the experimental groups and fed with one of the following diets: control (CTRL), 30% fructose-enriched (FRU 30%) or 30% D-tagatose-enriched (TAG 30%). After 24 weeks of dietary manipulation, rats underwent myocardial injury caused by 30 min ligature of the left anterior descending (LAD) coronary artery followed by 24 h' reperfusion. Fructose consumption resulted in body weight increase (49%) as well as altered glucose, insulin and lipid profiles. These effects were associated with increased I/R-induced myocardial damage, oxidative stress (36.5%) and inflammation marker expression. TAG 30%-fed rats showed lower oxidative stress (21%) and inflammation in comparison with FRU-fed rats. Besides, TAG diet significantly reduced plasmatic inflammatory cytokines and GDF8 expression (50%), while increased myocardial endothelial nitric oxide synthase (eNOS) expression (59%). Overall, we demonstrated that D-tagatose represents an interesting sugar alternative when compared to its isomer fructose with reduced deleterious impact not only on the metabolic profile but also on the related heart susceptibility to I/R injury.
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http://dx.doi.org/10.3389/fmolb.2021.650962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8076855PMC
April 2021

Acute visceral pain relief mediated by A3AR agonists in rats: involvement of N-type voltage-gated calcium channels.

Pain 2020 09;161(9):2179-2190

Department of Neuroscience, Psychology, Drug Research and Child Health-Neurofarba-Division of Pharmacology and Toxicology, Florence, Italy.

Abstract: Pharmacological tools for chronic visceral pain management are still limited and inadequate. A3 adenosine receptor (A3AR) agonists are effective in different models of persistent pain. Recently, their activity has been related to the block of N-type voltage-gated Ca2+ channels (Cav2.2) in dorsal root ganglia (DRG) neurons. The present work aimed to evaluate the efficacy of A3AR agonists in reducing postinflammatory visceral hypersensitivity in both male and female rats. Colitis was induced by the intracolonic instillation of 2,4-dinitrobenzenesulfonic acid (DNBS; 30 mg in 0.25 mL 50% EtOH). Visceral hypersensitivity was assessed by measuring the visceromotor response and the abdominal withdrawal reflex to colorectal distension. The effects of A3AR agonists (MRS5980 and Cl-IB-MECA) were evaluated over time after DNBS injection and compared to that of the selective Cav2.2 blocker PD173212, and the clinically used drug linaclotide. A3AR agonists significantly reduced DNBS-evoked visceral pain both in the postinflammatory (14 and 21 days after DNBS injection) and persistence (28 and 35 days after DNBS) phases. Efficacy was comparable to effects induced by linaclotide. PD173212 fully reduced abdominal hypersensitivity to control values, highlighting the role of Cav2.2. The effects of MRS5980 and Cl-IB-MECA were completely abolished by the selective A3AR antagonist MRS1523. Furthermore, patch-clamp recordings showed that A3AR agonists inhibited Cav2.2 in dorsal root ganglia neurons isolated from either control or DNBS-treated rats. The effect on Ca2+ current was PD173212-sensitive and prevented by MRS1523. A3AR agonists are effective in relieving visceral hypersensitivity induced by DNBS, suggesting a potential therapeutic role against abdominal pain.
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http://dx.doi.org/10.1097/j.pain.0000000000001905DOI Listing
September 2020

Adenosine A3 receptor activation inhibits pronociceptive N-type Ca2+ currents and cell excitability in dorsal root ganglion neurons.

Pain 2019 05;160(5):1103-1118

Division of Pharmacology and Toxicology, Department of NEUROFARBA, University of Florence, Italy.

Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A3AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A3AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A3AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca-activated K (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca influx via N-type voltage-dependent Ca channels, as Cl-IB-MECA-induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca currents, and its effect was inhibited by 56% in the presence of A3AR antagonist MRS1523, demonstrating that the majority of adenosine's effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca measurements confirmed the inhibitory role of A3AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A3AR activation could be mediated through N-type Ca channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3AR-based therapy as a viable approach to alleviate pain in different pathologies.
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http://dx.doi.org/10.1097/j.pain.0000000000001488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669900PMC
May 2019

Osteogenic transdifferentiation of vascular smooth muscle cells isolated from spontaneously hypertensive rats and potential menaquinone-4 inhibiting effect.

J Cell Physiol 2019 11 1;234(11):19761-19773. Epub 2019 Apr 1.

Department of Medical, Oral and Biotechnological Sciences, University "G. d'Annunzio" of Chieti-Pescara, Centro Scienze dell'Invecchiamento e Medicina Traslazionale (Ce.S.I.-Me.T.), StemTeCh Group, Chieti, Italy.

Vascular calcification (VC) is an active and cell-mediated process that shares many common features with osteogenesis. Knowledge demonstrates that in the presence of risk factors, such as hypertension, vascular smooth muscle cells (vSMCs) lose their contractile phenotype and transdifferentiate into osteoblastic-like cells, contributing to VC development. Recently, menaquinones (MKs), also known as Vitamin K2 family, has been revealed to play an important role in cardiovascular health by decreasing VC. However, the MKs' effects and mechanisms potentially involved in vSMCs osteoblastic transdifferentiation are still unknown. The aim of this study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of MKs family, in the modulation of the vSMCs phenotype. To achieve this, vascular cells from spontaneously hypertensive rats (SHR) were used as an in vitro model of cell vascular dysfunction. vSMCs from Wistar Kyoto normotensive rats were used as control condition. The results showed that MK-4 preserves the contractile phenotype both in control and SHR-vSMCs through a γ-glutamyl carboxylase-dependent pathway, highlighting its capability to inhibit one of the mechanisms underlying VC process. Therefore, MK-4 may have an important role in the prevention of vascular dysfunction and atherosclerosis, encouraging further in-depth studies to confirm its use as a natural food supplement.
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http://dx.doi.org/10.1002/jcp.28576DOI Listing
November 2019

Calcimimetic R-568 vasodilatory effect on mesenteric vascular beds from normotensive (WKY) and spontaneously hypertensive (SHR) rats. Potential involvement of vascular smooth muscle cells (vSMCs).

PLoS One 2018 9;13(8):e0202354. Epub 2018 Aug 9.

Aging and Translational Medicine Research Center (CeSI-MeT), University "G. d'Annunzio" of Chieti-Pescara, Chieti, Italy.

The potential role of calcimimetics as vasculotropic agents has been suggested since the discovery that calcium sensing receptors (CaSRs) are expressed in cardiovascular tissues. However, whether this effect is CaSR-dependent or -independent is still unclear. In the present study the vascular activity of calcimimetic R-568 was investigated in mesenteric vascular beds (MVBs) isolated from Spontaneously Hypertensive rats (SHR) and the relative age-matched Wistar-Kyoto (WKY) control rats. Pre-constricted MBVs were perfused with increasing concentrations of R-568 (10 nM- 30 μM) resulting in a rapid dose-dependent vasodilatation. However, in MVBs from SHR this was preceded by a small but significant vasoconstriction at lowest nanomolar concentrations used (10-300 nM). Pre-treatment with pharmacological inhibitors of nitric oxide (NO) synthase (NOS, L-NAME), KCa channels (CTX), cyclo-oxygenase (INDO) and CaSR (Calhex) or the endothelium removal suggest that NO, CaSR and the endothelium itself contribute to the R-568 vasodilatory/vasoconstrictor effects observed respectively in WKY/SHR MVBs. Conversely, the vasodilatory effects resulted by highest R-568 concentration were independent of these factors. Then, the ability of lower R-568 doses (0.1-1 μM) to activate endothelial-NOS (eNOS) pathway in MVBs homogenates was evaluated. The Akt and eNOS phosphorylation levels resulted increased in WKY homogenates and Calhex significantly blocked this effect. Notably, this did not occur in the SHR. Similarly, vascular smooth muscle cells (vSMCs) stimulation with lower R-568 doses resulted in Akt activation and increased NO production in WKY but not in SHR cells. Interestingly, in these cells this was associated with the absence of the biologically active dimeric form of the CaSR thus potentially contributing to explain the impaired vasorelaxant effect observed in response to R-568 in MVB from SHR compared to WKY. Overall, these findings provide new insight on the mechanisms of action of the calcimimetic R-568 in modulating vascular tone both in physiological and pathological conditions such as hypertension.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202354PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6084966PMC
February 2019

Adipose-derived stem cells decrease pain in a rat model of oxaliplatin-induced neuropathy: Role of VEGF-A modulation.

Neuropharmacology 2018 03 11;131:166-175. Epub 2017 Dec 11.

Department of Neuroscience, Psychology, Drug Research and Child Health - NEUROFARBA - Pharmacology and Toxicology Section, University of Florence, Florence, Italy.

Oxaliplatin therapy of colorectal cancer induces a dose-dependent neuropathic syndrome in 50% of patients. Pharmacological treatments may offer limited relief; scientific efforts are needed for new therapeutic approaches. Therefore we evaluated in a preclinical setting the pain relieving properties of mesenchymal stem cells and its secretome. Rat adipose stem cells (rASCs) were administered in a rat model of oxaliplatin-induced neuropathy. A single intravenous injection of rASCs reduced oxaliplatin-dependent mechanical hypersensitivity to noxious and non-noxious stimuli taking effect 1 h after administration, peaking 6 h thereafter and lasting 5 days. Cell-conditioned medium was ineffective. Repeated rASCs injections every 5 days relieved pain each time with a comparable effect. Labeled rASCs were detected in the bloodstream 1 and 3 h after administration and found in the liver 24 h thereafter. In oxaliplatin-treated rats, the plasma concentration of vascular endothelial growth factor (pan VEGF-A) was increased while the isoform VEGFb was upregulated in the spinal cord. Both alterations were reverted by rASCs. The anti-VEGF-A monoclonal antibody bevacizumab (intraperitoneally) reduced oxaliplatin-dependent pain. Studying the peripheral and central role of VEGFb in pain, we determined that the intraplantar and intrathecal injection of the growth factor induced a pro-algesic effect. In the oxaliplatin neuropathy model, the intrathecal infusion of bevacizumab, anti-rat VEGFb antibody and rASCs reduced pain. Adult adipose mesenchymal stem cells could represent a novel approach in the treatment of neuropathic pain. The regulation of VEGF-A is suggested as an effective mechanism in the complex response orchestrated by stem cells against neuropathy.
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http://dx.doi.org/10.1016/j.neuropharm.2017.12.020DOI Listing
March 2018

Role of Nitric Oxide, Nitric Oxide Synthase, Soluble Guanylyl Cyclase, and cGMP-Dependent Protein Kinase I in Mouse Stem Cell Cardiac Development.

Stem Cells Int 2016 20;2016:2868323. Epub 2016 Oct 20.

Department of Neurofarba, Pharmacology and Toxicology Unit, University of Florence, Florence, Italy.

. Nitric oxide (NO) can trigger cardiac differentiation of embryonic stem cells (ESCs), indicating a cardiogenic function of the NO synthetizing enzyme(s) (NOS). However, the involvement of the NO/NOS downstream effectors soluble guanylyl cyclase (sGC) and cGMP activated protein kinase I (PKG-I) is less defined. Therefore, we assess the involvement of the entire NO/NOS/sGC/PKG-I pathway during cardiac differentiation process. . Mouse ESCs were differentiated toward cardiac lineages by hanging drop methodology for 21 days. NOS/sGC/PKG-I pathway was studied quantifying genes, proteins, enzymatic activities, and effects of inhibition during differentiation. Percentages of beating embryoid bodies (mEBs) were evaluated as an index of cardiogenesis. . Genes and protein expression of enzymes were increased during differentiation with distinctive kinetics and proteins possessed their enzymatic functions. Exogenous administered NO accelerated whereas the blockade of PKG-I strongly slowed cardiogenesis. sGC inhibition was effective only at early stages and NOS blockade ineffective. Of NOS/sGC/PKG-I pathway, PKG-I seems to play the prominent role in cardiac maturation. . We concluded that exogenous administered NO and other pharmacological strategies able to increase the activity of PKG-I provide new tools to investigate and promote differentiation of cardiogenic precursors.
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http://dx.doi.org/10.1155/2016/2868323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5093303PMC
October 2016

Effect of the SOD mimetic MnL4 on in vitro and in vivo oxaliplatin toxicity: Possible aid in chemotherapy induced neuropathy.

Free Radic Biol Med 2016 Apr 28;93:67-76. Epub 2016 Jan 28.

Dipartimento di Neuroscienze, Psicologia, Scienze del Farmaco e Salute del Bambino (NEUROFARBA), Sezione di Farmacologia e Tossicologia, Università degli Studi di Firenze, Viale Pieraccini, 6, Firenze 50139, Italy. Electronic address:

Background: One of the most discomfortable dose-limiting adverse reactions of effective drugs for the treatment of solid tumors is a peripheral neuropathy which is the main reason for dose reduction and discontinuation of the therapy. We identified oxidative stress as one target of oxaliplatin toxicity in the search of possible adjuvant therapies to prevent neuropathy and alleviate pain. Therefore, we studied an effective SOD mimetic compound, MnL4, as a possible adjuvant treatment in in vitro cellular cultures and in vivo on a rat model of oxaliplatin-induced neuropathy.

Methods And Results: All rat manipulations were carried out according to the European Community guidelines for animal care. We performed experiments on SH-SY5Y, HT-29 and primary cortical rat astrocytes. Incubation with 100 µM oxaliplatin increased superoxide anion production and caspase 3/7 activity in the neuronal cell line SH-SY5Y and cortical astrocytes. MnL4 (10 µM) significantly reduced the increase in superoxide anion in both cell types, but prevented caspase 3/7 activity only in astrocytes. MnL4 reduced lipid peroxidation induced by oxaliplatin and normalized the intracellular calcium signal evoked by ATP and acetylcholine in astrocytes, preincubated with oxaliplatin. MnL4 did not interfere with the concentration- and time-dependent cytotoxic effects of oxaliplatin on the cancer cell lines HT-29 and LoVo. In vivo MnL4 reduced the response at mechanical noxious and mechanical and thermal non-noxious stimuli in oxaliplatin treated animals. Rat rota-rod performances were improved.

Conclusion: Since MnL4 exerts its beneficial effects without interfering with the anticancer activity of oxaliplatin, it could be proposed as adjuvant to prevent and reduce oxaliplatin induced neuropathy.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.01.023DOI Listing
April 2016

α7 Nicotinic Receptor Promotes the Neuroprotective Functions of Astrocytes against Oxaliplatin Neurotoxicity.

Neural Plast 2015 3;2015:396908. Epub 2015 Jun 3.

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Pharmacology and Toxicology Section, University of Florence, 50139 Florence, Italy.

Neuropathies are characterized by a complex response of the central nervous system to injuries. Glial cells are recruited to maintain neuronal homeostasis but dysregulated activation leads to pain signaling amplification and reduces the glial neuroprotective power. Recently, we highlighted the property of α7 nicotinic-acetylcholine-receptor (nAChR) agonists to relieve pain and induce neuroprotection simultaneously with a strong increase in astrocyte density. Aimed to study the role of α7 nAChR in the neuron-glia cross-talk, we treated primary rat neurons and astrocytes with the neurotoxic anticancer drug oxaliplatin evaluating the effect of the α7 nAChR agonist PNU-282987 (PNU). Oxaliplatin (1 μM, 48 h) reduced cell viability and increased caspase-3 activity of neuron monocultures without damaging astrocytes. In cocultures, astrocytes were not able to protect neurons by oxaliplatin even if glial cell metabolism was stimulated (pyruvate increase). On the contrary, the coculture incubation with 10 μM PNU improved neuron viability and inhibited apoptosis. In the absence of astrocytes, the protection disappeared. Furthermore, PNU promoted the release of the anti-inflammatory cytokine TGF-β1 and the expression of the glutamate-detoxifying enzyme glutamine synthetase. The α7 nAChR stimulation protects neurons from oxaliplatin toxicity through an astrocyte-mediated mechanism. α7 nAChR is suggested for recovering the homeostatic role of astrocytes.
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http://dx.doi.org/10.1155/2015/396908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4469839PMC
March 2016

Pretreatment with Relaxin Does Not Restore NO-Mediated Modulation of Calcium Signal in Coronary Endothelial Cells Isolated from Spontaneously Hypertensive Rats.

Molecules 2015 May 26;20(6):9524-35. Epub 2015 May 26.

NEUROFARBA, Section of Pharmacology & Toxicology, University of Florence, Viale G. Pieraccini 6, 50139 Florence, Italy.

We demonstrated that in coronary endothelial cells (RCEs) from normotensive Wistar Kyoto rats (WKY), the hormone relaxin (RLX) increases NO production and reduces calcium transients by a NO-related mechanism. Since an impairment of the NO pathway has been described in spontaneously hypertensive rats (SHR), the present study was aimed at exploring RLX effects on RCEs from SHR, hypothesizing that RLX could restore calcium responsiveness to NO. RCEs were isolated from WKY and SHR. Calcium transients were evaluated by image analysis after the administration of angiotensin II or α-thrombin. Angiotensin II (1 µM) caused a prompt rise of [Ca2+]i in WKY and SHR RCEs and a rapid decrease, being the decay time higher in SHR than in WKY. NOS inhibition increased calcium transient in WKY, but not in SHR RCEs. Whereas RLX pretreatment (24 h, 60 ng/mL) was ineffective in SHR, it strongly reduced calcium transient in WKY in a NO-dependent way. A similar behavior was measured using 30 U/mL α-thrombin. The current study offers evidence that RLX cannot restore NO responsiveness in SHR, suggesting an accurate selection of patients eligible for RLX treatment of cardiovascular diseases.
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http://dx.doi.org/10.3390/molecules20069524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272299PMC
May 2015

Different apoptotic pathways activated by oxaliplatin in primary astrocytes vs. colo-rectal cancer cells.

Int J Mol Sci 2015 Mar 9;16(3):5386-99. Epub 2015 Mar 9.

Department of Neuroscience, Psychology, Drug Research and Child Health - Neurofarba - Pharmacology and Toxicology Section, University of Florence, Florence 50139, Italy.

Oxaliplatin-based chemotherapy improves the outcomes of metastatic colorectal cancer patients. Its most significant and dose-limiting side effect is the development of a neuropathic syndrome. The mechanism of the neurotoxicity is unclear. The limited knowledge about differences existing between neurotoxic and antitumor effects hinders the discovery of effective and safe adjuvant therapies. In vitro, we suggested cell-specific activation apoptotic pathways in normal nervous cells (astrocytes) vs. colon-cancer cells (HT-29). In the present research we compared the apoptotic signals evoked by oxaliplatin in astrocytes and HT-29 analyzing the intrinsic and extrinsic apoptotic pathways. In astrocytes, oxaliplatin induced a mitochondrial derangement measured as cytosolic release of cytochrome C, increase in superoxide anion levels and decreased expression of the antiapoptotic protein Bcl-2. Caspase-8, a main initiator of the extrinsic process remained unaltered. On the contrary, in HT-29 oxaliplatin increased caspase-8 activity and Bid expression, thus activating the extrinsic apoptosis, while the Bcl-2 increased expression blocked the mitochondrial damage. Data suggest the preferred activation of the intrinsic apoptosis as oxaliplatin damage signaling in normal nervous cells. The extrinsic pathway prevails in tumor cells indicating a possible strategy for planning new molecules to treat oxaliplatin-dependent neurotoxicity without negatively influence chemotherapy.
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http://dx.doi.org/10.3390/ijms16035386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394482PMC
March 2015

Oxaliplatin neurotoxicity involves peroxisome alterations. PPARγ agonism as preventive pharmacological approach.

PLoS One 2014 18;9(7):e102758. Epub 2014 Jul 18.

Dept. of Neuroscience, Psychology, Drug Research and Child Health - Neurofarba - Pharmacology and Toxicology Section, University of Florence, Florence, Italy.

The development of neuropathic syndromes is an important, dose limiting side effect of anticancer agents like platinum derivates, taxanes and vinca alkaloids. The causes of neurotoxicity are still unclear but the impairment of the oxidative equilibrium is strictly related to pain. Two intracellular organelles, mitochondria and peroxisomes cooperate to the maintaining of the redox cellular state. Whereas a relationship between chemotherapy-dependent mitochondrial alteration and neuropathy has been established, the role of peroxisome is poor explored. In order to study the mechanisms of oxaliplatin-induced neurotoxicity, peroxisomal involvement was evaluated in vitro and in vivo. In primary rat astrocyte cell culture, oxaliplatin (10 µM for 48 h or 1 µM for 5 days) increased the number of peroxisomes, nevertheless expression and functionality of catalase, the most important antioxidant defense enzyme in mammalian peroxisomes, were significantly reduced. Five day incubation with the selective Peroxisome Proliferator Activated Receptor-γ (PPAR-γ) antagonist G3335 (30 µM) induced a similar peroxisomal impairment suggesting a relationship between PPARγ signaling and oxaliplatin neurotoxicity. The PPARγ agonist rosiglitazone (10 µM) reduced the harmful effects induced both by G3335 and oxaliplatin. In vivo, in a rat model of oxaliplatin induced neuropathy, a repeated treatment with rosiglitazone (3 and 10 mg kg(-1) per os) significantly reduced neuropathic pain evoked by noxious (Paw pressure test) and non-noxious (Cold plate test) stimuli. The behavioral effect paralleled with the prevention of catalase impairment induced by oxaliplatin in dorsal root ganglia. In the spinal cord, catalase protection was showed by the lower rosiglitazone dosage without effect on the astrocyte density increase induced by oxaliplatin. Rosiglitazone did not alter the oxaliplatin-induced mortality of the human colon cancer cell line HT-29. These results highlight the role of peroxisomes in oxaliplatin-dependent nervous damage and suggest PPARγ stimulation as a candidate to counteract oxaliplatin neurotoxicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102758PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103888PMC
November 2015

The polyphenol oleuropein aglycone protects TgCRND8 mice against Aß plaque pathology.

PLoS One 2013 8;8(8):e71702. Epub 2013 Aug 8.

Department of Neuroscience, Psychology, Drug Research and Child Health, Division of Pharmacology and Toxicology, University of Florence, Florence, Italy.

The claimed beneficial effects of the Mediterranean diet include prevention of several age-related dysfunctions including neurodegenerative diseases and Alzheimer-like pathology. These effects have been related to the protection against cognitive decline associated with aging and disease by a number of polyphenols found in red wine and extra virgin olive oil. The double transgenic TgCRND8 mice (overexpressing the Swedish and Indiana mutations in the human amyloid precursor protein), aged 1.5 and 4, and age-matched wild type control mice were used to examine in vivo the effects of 8 weeks dietary supplementation of oleuropein aglycone (50 mg/kg of diet), the main polyphenol found in extra virgin olive oil. We report here that dietary supplementation of oleuropein aglycone strongly improves the cognitive performance of young/middle-aged TgCRND8 mice, a model of amyloid-ß deposition, respect to age-matched littermates with un-supplemented diet. Immunofluorescence analysis of cerebral tissue in oleuropein aglycone-fed transgenic mice showed remarkably reduced ß-amyloid levels and plaque deposits, which appeared less compact and "fluffy"; moreover, microglia migration to the plaques for phagocytosis and a remarkable reduction of the astrocyte reaction were evident. Finally, oleuropein aglycone-fed mice brain displayed an astonishingly intense autophagic reaction, as shown by the increase of autophagic markers expression and of lysosomal activity. Data obtained with cultured cells confirmed the latter evidence, suggesting mTOR regulation by oleuropein aglycone. Our results support, and provide mechanistic insights into, the beneficial effects against Alzheimer-associated neurodegeneration of a polyphenol enriched in the extra virgin olive oil, a major component of the Mediterranean diet.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071702PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3738517PMC
April 2014

Therapeutic effects of the superoxide dismutase mimetic compound MnIIMe2DO2A on experimental articular pain in rats.

Mediators Inflamm 2013 6;2013:905360. Epub 2013 Jun 6.

Department of Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.

Superoxide anion (O(2) (•-)) is overproduced in joint inflammation, rheumatoid arthritis, and osteoarthritis. Increased O(2) (•-) production leads to tissue damage, articular degeneration, and pain. In these conditions, the physiological defense against O(2) (•-), superoxide dismutases (SOD) are decreased. The Mn(II) complex MnL4 is a potent SOD mimetic, and in this study it was tested in inflammatory and osteoarticular rat pain models. In vivo protocols were approved by the animal Ethical Committee of the University of Florence. Pain was measured by paw pressure and hind limb weight bearing alterations tests. MnL4 (15 mg kg(-1)) acutely administered, significantly reduced pain induced by carrageenan, complete Freund's adjuvant (CFA), and sodium monoiodoacetate (MIA). In CFA and MIA protocols, it ameliorated the alteration of postural equilibrium. When administered by osmotic pump in the MIA osteoarthritis, MnL4 reduced pain, articular derangement, plasma TNF alpha levels, and protein carbonylation. The scaffold ring was ineffective. MnL4 (10(-7) M) prevented the lipid peroxidation of isolated human chondrocytes when O(2) (•-) was produced by RAW 264.7. MnL4 behaves as a potent pain reliever in acute inflammatory and chronic articular pain, being its efficacy related to antioxidant property. Therefore MnL4 appears as a novel protective compound potentially suitable for the treatment of joint diseases.
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http://dx.doi.org/10.1155/2013/905360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3690261PMC
February 2014

UDP-glucose enhances outward K(+) currents necessary for cell differentiation and stimulates cell migration by activating the GPR17 receptor in oligodendrocyte precursors.

Glia 2013 Jul 2;61(7):1155-71. Epub 2013 May 2.

Divi Department of Neuroscience, Psychology, Drug Research and Child Health (NeuroFarBa), University of Florence, Viale Pieraccini, 6, 50139 Florence, Italy.

In the developing and mature central nervous system, NG2 expressing cells comprise a population of cycling oligodendrocyte progenitor cells (OPCs) that differentiate into mature, myelinating oligodendrocytes (OLGs). OPCs are also characterized by high motility and respond to injury by migrating into the lesioned area to support remyelination. K(+) currents in OPCs are developmentally regulated during differentiation. However, the mechanisms regulating these currents at different stages of oligodendrocyte lineage are poorly understood. Here we show that, in cultured primary OPCs, the purinergic G-protein coupled receptor GPR17, that has recently emerged as a key player in oligodendrogliogenesis, crucially regulates K(+) currents. Specifically, receptor stimulation by its agonist UDP-glucose enhances delayed rectifier K(+) currents without affecting transient K(+) conductances. This effect was observed in a subpopulation of OPCs and immature pre-OLGs whereas it was absent in mature OLGs, in line with GPR17 expression, that peaks at intermediate phases of oligodendrocyte differentiation and is thereafter downregulated to allow terminal maturation. The effect of UDP-glucose on K(+) currents is concentration-dependent, blocked by the GPR17 antagonists MRS2179 and cangrelor, and sensitive to the K(+) channel blocker tetraethyl-ammonium, which also inhibits oligodendrocyte maturation. We propose that stimulation of K(+) currents is responsible for GPR17-induced oligodendrocyte differentiation. Moreover, we demonstrate, for the first time, that GPR17 activation stimulates OPC migration, suggesting an important role for this receptor after brain injury. Our data indicate that modulation of GPR17 may represent a strategy to potentiate the post-traumatic response of OPCs under demyelinating conditions, such as multiple sclerosis, stroke, and brain trauma.
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http://dx.doi.org/10.1002/glia.22506DOI Listing
July 2013

Relaxant effect of a water soluble carbon monoxide-releasing molecule (CORM-3) on spontaneously hypertensive rat aortas.

Cardiovasc Drugs Ther 2012 Aug;26(4):285-92

Department of Preclinical and Clinical Pharmacology, University of Florence, Italy.

Purpose: Both carbon monoxide (CO) and nitric oxide (NO) are two gaseous molecules performing relevant functions in mammals. In order to better understand their actions in the cardiovascular system, we have investigated the effects of CORM-3, (tricarbonylchloro(glycinato)ruthenium(II), a water soluble CO-releasing molecule and SNAP (S-nitroso-N-acetyl-DL-penicillamine, a well known NO-releasing molecule) on aortas of normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR).

Methods: The isometric contraction of angiotensin II (AT-II) and endothelin-1 (ET-1) was evaluated in endothelium-denuded aortic strips.

Results: In control conditions, AT-II induced a similar concentration-dependent contraction in both WKY and SHR, while ET-1 was more effective in SHR aortic strips. CORM-3 or SNAP (10(-7)-3 × 10(-4) M) reduced the contraction induced by AT-II or ET-1 in a concentration-dependent way. Whereas the median inhibitory concentration of SNAP was significantly lower in WKY than in SHR, CORM-3 had a similar effect in both strains. The scaffold compound iCORM-3 was ineffective. Pretreatment with an inhibitor of soluble guanylyl cyclase (ODQ, 3 × 10(-6) M) marginally reduced CORM-3 relaxation in both strains, whereas it reduced relaxation induced by SNAP in WKY and, to a lesser extent, in SHR. The benzylindazole derivative YC-1 (10(-6) M), a sensitizer of soluble guanylate cyclase to the action of NO, significantly increased the relaxant effect of SNAP in AT-II precontracted aortic strips. The blocker of calcium-activated potassium channels, charybdotoxin (10(-8) M), reduced the relaxation induced by CORM-3 in both strains.

Conclusions: Different mechanisms seem to be implicated in CO- and NO-mediated vascular relaxation. Since the relaxant properties of CO are conserved in SHR aortas, CORM-3 could be a new potential agent for the treatment of hypertension, when NO donors show sub-optimal or absent responses.
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http://dx.doi.org/10.1007/s10557-012-6400-6DOI Listing
August 2012

Restoring nitric oxide cytosolic calcium regulation by cyclic guanosine monophosphate protein kinase I alpha transfection in coronary endothelial cells of spontaneously hypertensive rats.

J Vasc Res 2012 15;49(3):221-30. Epub 2012 Mar 15.

Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.

In microcoronary endothelial cells (RCEs) from spontaneously hypertensive rats (SHR), the nitric oxide (NO)/cyclic guanosine monophosphate (GMP)-dependent proteinkinase I (cGKI) pathway cannot regulate the cytosolic calcium ([Ca2+]i) dynamic as in RCEs from Wistar Kyoto rats (WKY). We investigated the altered downstream NO target in SHR cells and, since cGKI expression was low, whether the re-expression of cGKIα in SHR RCEs could restore NO calcium responsiveness. We measured [Ca2+]i dynamic by fura-2 imaging analysis and the cGKI level by RT-PCR and Western blot in SHR and WKY RCEs. Plasmids encoding for enhanced green fluorescence protein or cGKIα-enhanced green fluorescence protein were transiently transfected in SHR RCEs, and [Ca2+]i was evaluated. Angiotensin-II (AT-II) increased [Ca2+]i in a concentration-dependent way in both strains. Whereas in WKY, endogenously produced NO and cyclic GMP analog decreased the AT-II-induced [Ca2+]i transient, they were ineffective in SHR RCEs. The cGKI level was low in SHR cells. However, after cGKIα re-expression, endogenous NO decreased the AT-II-induced [Ca2+]i transient, while endothelial NO synthase and cGKI inhibition prevented it. The low expression of cGKI in SHR accounts for the absent regulation of the agonist-induced [Ca2+]i transient by the NO/cyclic GMP pathway. Studies on cGKI in humans could contribute to a better understanding of cardiovascular pathologies.
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http://dx.doi.org/10.1159/000332911DOI Listing
July 2012

Oxaliplatin-induced neuropathy: oxidative stress as pathological mechanism. Protective effect of silibinin.

J Pain 2012 Mar 9;13(3):276-84. Epub 2012 Feb 9.

Deparment of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy.

Unlabelled: Oxaliplatin is the standard treatment for advanced colorectal cancer. Its dose-limiting toxicity is the development of a painful neuropathic syndrome sustained by unclear mechanisms. Although the oxidative hypothesis is a matter of debate, direct data about oxidative damage induced in vivo by anticancer agents are lacking and the efficacy of the available antioxidant compounds are unsatisfactory. In a rat model of painful oxaliplatin-induced neuropathy (2.4 mgkg(-1) i.p., daily for 21 days), we described an important component of oxidative stress. In the plasma of oxaliplatin-treated rats, the increases in carbonylated protein and thiobarbituric acid reactive substances were the index of the resultant protein oxidation and lipoperoxidation, respectively. The same pattern of oxidation was revealed also in the sciatic nerve, and in the spinal cord where the damage reached the DNA level. The antioxidant compound silibinin (100 mgkg(-1) per os), administered once a day, starting from the first day of oxaliplatin injection until the 20th, prevented oxidative damage as did α-tocopherol. Repetitive administration of silibinin, as well as α-tocopherol, reduced oxaliplatin-dependent pain induced by mechanical and thermal stimuli. Antioxidants were also able to improve motor coordination. The antineuropathic effect of both molecules improved by about 50% oxaliplatin-induced behavioral alterations.

Perspective: This study characterizes oxidative stress parameters in a rat model of oxaliplatin-induced neuropathy. A relationship between the improvement of oxidative alterations and pain relief is established in rats treated with natural antioxidant compounds like α-tocopherol and silibinin. Silibinin could be a valid therapeutic option for chemotherapy-induced neuropathy.
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http://dx.doi.org/10.1016/j.jpain.2011.11.009DOI Listing
March 2012

Oxaliplatin elicits mechanical and cold allodynia in rodents via TRPA1 receptor stimulation.

Pain 2011 Jul 9;152(7):1621-1631. Epub 2011 Apr 9.

Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy Department of Molecular Cell Biology, Katholieke Universiteit, Leuven, Belgium Department of Pharmacology, Chiesi Farmaceutici, Parma, Italy Department of Pharmaceutical Chemistry, University of Ferrara, Ferrara, Italy Chemistry Department, University of Ferrara, Ferrara, Italy.

Platinum-based anticancer drugs cause neurotoxicity. In particular, oxaliplatin produces early-developing, painful, and cold-exacerbated paresthesias. However, the mechanism underlying these bothersome and dose-limiting adverse effects is unknown. We hypothesized that the transient receptor potential ankyrin 1 (TRPA1), a cation channel activated by oxidative stress and cold temperature, contributes to mechanical and cold hypersensitivity caused by oxaliplatin and cisplatin. Oxaliplatin and cisplatin evoked glutathione-sensitive relaxation, mediated by TRPA1 stimulation and the release of calcitonin gene-related peptide from sensory nerve terminals in isolated guinea pig pulmonary arteries. No calcium response was observed in cultured mouse dorsal root ganglion neurons or in naïve Chinese hamster ovary (CHO) cells exposed to oxaliplatin or cisplatin. However, oxaliplatin, and with lower potency, cisplatin, evoked a glutathione-sensitive calcium response in CHO cells expressing mouse TRPA1. One single administration of oxaliplatin produced mechanical and cold hyperalgesia in rats, an effect selectively abated by the TRPA1 antagonist HC-030031. Oxaliplatin administration caused mechanical and cold allodynia in mice. Both responses were absent in TRPA1-deficient mice. Administration of cisplatin evoked mechanical allodynia, an effect that was reduced in TRPA1-deficient mice. TRPA1 is therefore required for oxaliplatin-evoked mechanical and cold hypersensitivity, and contributes to cisplatin-evoked mechanical allodynia. Channel activation is most likely caused by glutathione-sensitive molecules, including reactive oxygen species and their byproducts, which are generated after tissue exposure to platinum-based drugs from cells surrounding nociceptive nerve terminals.
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http://dx.doi.org/10.1016/j.pain.2011.02.051DOI Listing
July 2011

Aldehyde dehydrogenase 7A1 (ALDH7A1) attenuates reactive aldehyde and oxidative stress induced cytotoxicity.

Chem Biol Interact 2011 May 19;191(1-3):269-77. Epub 2011 Feb 19.

Department of Pharmaceutical Sciences, University of Colorado Denver, Aurora, CO 80045, USA.

Mammalian aldehyde dehydrogenase 7A1 (ALDH7A1) is homologous to plant ALDH7B1 which protects against various forms of stress such as increased salinity, dehydration and treatment with oxidants or pesticides. Deleterious mutations in human ALDH7A1 are responsible for pyridoxine-dependent and folinic acid-responsive seizures. In previous studies, we have shown that human ALDH7A1 protects against hyperosmotic stress presumably through the generation of betaine, an important cellular osmolyte, formed from betaine aldehyde. Hyperosmotic stress is coupled to an increase in oxidative stress and lipid peroxidation (LPO). In this study, cell viability assays revealed that stable expression of mitochondrial ALDH7A1 in Chinese hamster ovary (CHO) cells provides significant protection against treatment with the LPO-derived aldehydes hexanal and 4-hydroxy-2-nonenal (4HNE) implicating a protective function for the enzyme during oxidative stress. A significant increase in cell survival was also observed in CHO cells expressing either mitochondrial or cytosolic ALDH7A1 treated with increasing concentrations of hydrogen peroxide (H(2)O(2)) or 4HNE, providing further evidence for anti-oxidant activity. In vitro enzyme activity assays indicate that human ALDH7A1 is sensitive to oxidation and that efficiency can be at least partially restored by incubating recombinant protein with the thiol reducing agent β-mercaptoethanol (BME). We also show that after reactivation with BME, recombinant ALDH7A1 is capable of metabolizing the reactive aldehyde 4HNE. In conclusion, ALDH7A1 mechanistically appears to provide cells protection through multiple pathways including the removal of toxic LPO-derived aldehydes in addition to osmolyte generation.
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http://dx.doi.org/10.1016/j.cbi.2011.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3387551PMC
May 2011

Low molecular weight compounds with transition metals as free radical scavengers and novel therapeutic agents.

Cardiovasc Hematol Agents Med Chem 2010 Jul;8(3):128-46

Department of Chemistry "Ugo Schiff", University of Florence, Florence, Italy.

Molecules able to modulate the levels of endogenous free radicals, such as reactive oxygen species (ROS) and nitric oxide (NO), are of pivotal interest for pharmacological and pharmaceutical sciences because of their potential therapeutic relevance. In fact, ROS and NO, which are normal products of cell metabolism, may play a dual beneficial/deleterious role, depending on local concentration and mode of generation. As such, they have been identified as key pathogenic factors for many inflammatory, vascular dysfunctional and degenerative disorders, including atherosclerosis, hypertension, cardiovascular and neurodegenerative diseases, cancer, diabetes mellitus, and ageing. Therefore, the identification and characterization of novel antioxidant/free radical scavenger molecules may expand the current therapeutic implements for the treatment and prevention of the above diseases. In this perspective, low molecular weight complexes of transition metals with organic scaffolds are viewed and investigated as promising pharmaceutical agents. These complexes take advantage of the known principles of inorganic chemistry, i.e. the ability of transition metals, Fe(II), Co(II), Mn(II) and Ru(II), to bind to and react with NO and/or ROS, to counterbalance excessive endogenous free radical generation in biological systems. Among NO scavengers, representative examples are iron complexes with dithiocarbamates or ruthenium compounds with polyamine-polycarboxylate scaffolds; on the other hand, manganese-based molecules appear effective as ROS scavengers. Of note, Mn(II)-containing molecules, currently under study as ROS scavengers, have major functional similarities to Mn-superoxide dismutase (SOD), a Mn-containing enzyme acting as potent endogenous anti-oxidant. In this article, we briefly summarize the state-of-the-art concerning the chemical and biological properties of transition metal ion complexes with low molecular weight synthetic ligands as ROS/NO scavengers provided with therapeutic effectiveness in animal models of free radical-induced diseases. A proper design of the organic scaffolds may yield metal complexes which are stable in aqueous solution in a wide range of physical and chemical conditions, thus preventing release of the metal and the related toxicity. These metal-based compounds may be viewed as a novel class of drugs helpful to reduce vascular dysfunction and oxidative tissue injury due to derangements of the endogenous generation/catabolism of NO and ROS.
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http://dx.doi.org/10.2174/187152510791698389DOI Listing
July 2010

Sex steroid receptors in male human bladder: expression and biological function.

J Sex Med 2010 Aug 20;7(8):2698-713. Epub 2010 Apr 20.

Sexual Medicine and Andrology Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy.

Introduction: In male, lower urinary tract symptoms (LUTS) have been associated, beside benign prostatic hyperplasia, to some unexpected comorbidities (hypogonadism, obesity, metabolic syndrome), which are essentially characterized by an unbalance between circulating androgens/estrogens. Within the bladder, LUTS are linked to RhoA/Rho-kinase (ROCK) pathway overactivity.

Aim: To investigate the effects of changing sex steroids on bladder smooth muscle.

Methods: ER α, ER β, GPR30/GPER1 and aromatase mRNA expression was analyzed in male genitourinary tract tissues, and cells isolated from bladder, prostate, and urethra. Estrogen and G1 effect on RhoA/ROCK signaling output like cell migration, gene expression, and cytoskeletal remodeling, and [Ca(2+) ](i) was also studied in hB cells. Contractile studies on bladder strips from castrated male rats supplemented with estradiol and testosterone was also performed.

Main Outcome Measures: The effects of classical (ER α, ER β) and nonclassical (GPR30/GPER1) estrogen receptor ligands (17 β-estradiol and G1, respectively) and androgens on RhoA/ROCK-.mediated cell functions were studied in hB cells. Contractility studies were also performed in bladder strips from castrated male rats supplemented with testosterone or estradiol.

Results: Aromatase and sex steroid receptors, including GPR30, were expressed in human bladder and mediates several biological functions. Both 17 β-estradiol and G1 activated calcium transients and induced RhoA/ROCK signaling (cell migration, cytoskeleton remodeling and smooth muscle gene expression). RhoA/ROCK inhibitors blunted these effects. Estrogen-, but not androgen-supplementation to castrated rats increased sensitivity to the ROCK inhibitor, Y-27632 in isolated bladder strips. In hB cells, testosterone elicited effects similar to estrogen, which were abrogated by blocking its aromatization through letrozole.

Conclusion: Our data indicate for the first time that estrogen-more than androgen-receptors up-regulate RhoA/ROCK signaling. Since an altered estrogen/androgen ratio characterizes conditions, such as aging, obesity and metabolic syndrome, often associated to LUTS, we speculate that a relative hyperestrogenism may induce bladder overactivity through the up-regulation of RhoA/ROCK pathway.
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http://dx.doi.org/10.1111/j.1743-6109.2010.01811.xDOI Listing
August 2010

A novel manganese complex effective as superoxide anion scavenger and therapeutic agent against cell and tissue oxidative injury.

J Med Chem 2009 Nov;52(22):7273-83

Department of Preclinical and Clinical Pharmacology, University of Florence, V. le G. Pieraccini 6, Florence, Italy.

Two cyclic polyamine-polycarboxylate ligands, 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (H(2)L3) and 4,10-dimethyl-1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (H(2)L4), and two noncyclic scaffolds, N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (H(3)L1) and ethylene-bisglycol-tetracetic acid (H(4)L2), form stable complexes with Mn(II) in aqueous solutions. Cyclic voltammograms show that the complexes with the most hydrophobic ligands, [MnL2](2-) and [MnL4], are oxidized at higher potential than [MnL1](-) and [MnL3]. The pharmacological properties of these molecules were evaluated as superoxide ion scavengers and anti-inflammatory compounds. Among the four complexes, [MnL4] was the most bioactive, being effective in the nanomolar/micromolar range. It abates the levels of key markers of oxidative injury on cultured cells and ameliorates the outcome parameters in animal models of acute and chronic inflammation. [MnL4] toxicity was very low on both cell cultures in vitro and mice in vivo. Hence, we propose [MnL4] as a novel stable oxygen radical scavenging molecule, active at low doses and with a low toxicity.
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http://dx.doi.org/10.1021/jm901298xDOI Listing
November 2009

Functional characterization of two isoforms of the P2Y-like receptor GPR17: [35S]GTPgammaS binding and electrophysiological studies in 1321N1 cells.

Am J Physiol Cell Physiol 2009 Oct 22;297(4):C1028-40. Epub 2009 Jul 22.

Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy.

The previously "orphan" G protein-coupled receptor GPR17 is structurally related to both P2Y nucleotide receptors and to receptors for cysteinyl leukotrienes. Genomic analysis revealed two putative open reading frames encoding for a "short" and a "long" receptor isoform of 339- and 367-amino acids, respectively, with the latter displaying a 28-amino acid longer NH(2) terminus. The short isoform has been recently "deorphanized," revealing dual responses to uracil nucleotides and cysteinyl leukotrienes. No information regarding the ligand specificity, tissue distribution, or pathophysiological roles of the long receptor isoform is available. In the present study, we cloned human long-GPR17, determined its tissue distribution, and characterized its pharmacological specificity in 1321N1 cells by [35S]GTPgammaS binding (which measures the ability of G protein-coupled receptor agonists to increase GTP binding to G proteins) and whole cell patch-clamp recording measuring receptor coupling to K+ channels. [35S]GTPgammaS binding in long-GPR17-expressing 1321N1 cells revealed concentration-dependent responses to uracil nucleotides (UDP-galactose = UDP > UDP-glucose) and cysteinyl leukotrienes (LTC4 > LTD4), which were counteracted by a purinergic (cangrelor) and a cysteinyl leukotriene antagonist (montelukast), respectively. The nonhydrolyzable ATP analog ATPgammaS also acted as an antagonist. GPR17 coupled to Gi and, to a lesser extent, Gq proteins. UDP-glucose and LTD(4) also induced increases in overall outward K+ currents, which were antagonized by the purinergic antagonists MRS2179 and cangrelor and by montelukast. We conclude that the previously uncharacterized long-GPR17 isoform is a functional receptor that is stimulated by both uracil nucleotides and cysteinyl leukotrienes. We also show that the signaling pathway of GPR17 involves the generation of outward K+ currents, an important protective mechanism that, in brain, is specifically aimed at reducing neuronal hyperexcitability and resultant neuronal injury.
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http://dx.doi.org/10.1152/ajpcell.00658.2008DOI Listing
October 2009

Losartan counteracts the hyper-reactivity to angiotensin II and ROCK1 over-activation in aortas isolated from streptozotocin-injected diabetic rats.

Cardiovasc Diabetol 2009 Jun 22;8:32. Epub 2009 Jun 22.

Dept. of Pharmacology, University of Florence, Viale G, Pieraccini 6, Florence, Italy.

Background: In streptozotocin-injected rats (STZ-rats), we previously demonstrated a role for angiotensin II (AT-II) in cardiac remodelling and insulin resistance partially counteracted by in vivo treatment with losartan, an AT-II receptor antagonist.We now aimed to investigate the effect of treating diabetic STZ-rats with losartan on diabetes vascular response to vasoconstrictors.

Methods: Male Wistar rats were randomly divided in four groups, two of them were assigned to receive losartan in the drinking water (20 mg/kg/day) until the experiment ending (3 weeks afterward). After 1 week, two groups, one of which receiving losartan, were injected in the tail vein with citrate buffer (normoglycemic, N and normoglycemic, losartan-treated, NL). The remaining received a single injection of streptozotocin (50 mg/kg in citrate i.v.) thus becoming diabetic (D) and diabetic losartan-treated (DL). Plasma glycaemia and blood pressure were measured in all animals before the sacrifice (15 days after diabetes induction).In aortic strips isolated from N, NL, D and DL rats we evaluated i) the isometric concentration-dependent contractile response to phenylephrine (Phe) and to AT-II; ii) the RhoA-kinase (ROCK1) activity and expression by enzyme-immunoassay and Western blot respectively.

Key Results: The concentration-dependent contractile effect of Phe was similar in aortas from all groups, whereas at all concentrations tested, AT-II contraction efficacy was 2 and half and 1 and half times higher in D and DL respectively in comparison with N and NL. AT-II contracture was similarly reduced in all groups by AT-II receptor antagonists, irbesartan or irbesartan plus PD123319. HA-1077 (10 microM), an inhibitor of ROCK1 activity, reduced AT-II efficacy (Deltamg/mg tissue w.w.) by -3.5 +/- 1.0, -4.6 +/- 1.9, -22.1 +/- 2.2 and -11.4 +/- 1.3 in N, NL, D and DL respectively). ROCK1 activity and expression were higher in D than in N/NL and DL aortas.

Conclusion And Implications: Aortas isolated from STZ-rats present hyper-contracture to AT-II mainly dependent on the up-regulation of ROCK1 expression/activity. In vivo losartan treatment partially corrects AT-II hyper-contracture, limiting the increase in ROCK1 expression/activity. These data offer a new molecular mechanism supporting the rationale for using losartan in the prevention of diabetic vascular complications.
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http://dx.doi.org/10.1186/1475-2840-8-32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711933PMC
June 2009

Silybin, a component of sylimarin, exerts anti-inflammatory and anti-fibrogenic effects on human hepatic stellate cells.

J Hepatol 2009 Jun 5;50(6):1102-11. Epub 2009 Apr 5.

Dipartimento di Medicina Interna, Università degli Studi di Firenze, Viale G.B. Morgagni, 85, 50134 Florence, Italy.

Background/aims: Hepatic fibrogenesis, a consequence of chronic liver tissue damage, is characterized by activation of the hepatic stellate cells (HSC). Silybin has been shown to exert anti-fibrogenic effects in animal models. However, scant information is available on the fine cellular and molecular events responsible for this effect. The aim of this study was to assess the mechanisms regulating the anti-fibrogenic and anti-inflammatory activity of Silybin.

Methods: Experiments were performed on HSC isolated from human liver and activated by culture on plastic.

Results: Silybin was able to inhibit dose-dependently (25-50 microM) growth factor-induced pro-fibrogenic actions of activated human HSC, including cell proliferation (P < 0.001), cell motility (P < 0.001), and de novo synthesis of extracellular matrix components (P < 0.05). Silybin (25-50 microM), inhibited the IL-1-induced synthesis of MCP-1 (P < 0.01) and IL-8 (P < 0.01) showing a potent anti-inflammatory activity. Silybin exerts its effects by directly inhibiting the ERK, MEK and Raf phosphorylation, reducing the activation of NHE1 (Na+/H+ exchanger, P < 0.05) and the IkBalpha phosphorylation. In addition, Silybin was confirmed to act as a potent anti-oxidant agent.

Conclusion: The results of the study provide molecular insights into the potential therapeutic action of Silybin in chronic liver disease. This action seems to be mostly related to a marked inhibition of the production of pro-inflammatory cytokines, a clear anti-oxidant effect and a reduction of the direct and indirect pro-fibrogenic potential of HSC.
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http://dx.doi.org/10.1016/j.jhep.2009.02.023DOI Listing
June 2009

Altered nitric oxide calcium responsiveness of aortic smooth muscle cells in spontaneously hypertensive rats depends on low expression of cyclic guanosine monophosphate-dependent protein kinase type I.

J Hypertens 2009 Jun;27(6):1258-67

Department of Pharmacology, Viale Pieraccini, 6-University of Florence, Florence, Italy.

Objectives: The nitric oxide/cyclic guanosine monophosphate (GMP)/cyclic GMP-dependent protein kinase type I (cGKI) pathway has been extensively investigated in the spontaneously hypertensive rat (SHR) as a possible pathogenetic factor. Therefore, we investigated the role of nitric oxide/cGKI on intracellular calcium dynamics ([Ca2+]i) of aortic smooth muscle cells isolated from control normotensive Wistar Kyoto rats (WKY) and SHR.

Methods: Rat aortic smooth muscle cells (RASMCs) were obtained from 12 to 16-week-old WKY and SHR. [Ca2+]i dynamics were monitored by imaging analysis of fura-2-loaded RASMCs. cGKI mRNA and cGKI protein expression were evaluated by reverse transcription-PCR and western blot. Plasmids codifying for enhanced green fluorescent protein (EGFP) or cGKIalpha-EGFP were transfected on SHR RASMCs.

Results: Angiotensin II similarly increased [Ca2+]i in WKY and SHR RASMCs. In WKY RASMCs, S-nitroso-N-acetyl-DL-penicillamine (SNAP, 1-100 micromol/l) reduced the decay time of angiotensin II-induced [Ca2+]i transient. On the contrary, in SHR cells, SNAP was ineffective. Dibutyryl cyclic GMP (1-100 nmol/l), a membrane-permeable cyclic GMP analogue, behaved similarly to SNAP. In naive SHR RASMCs, cGKI mRNA and cGKI protein were low or absent. After transfection of a plasmid encoding for cGKIalpha-EGFP, the [Ca2+]i dynamic of SHR-transfected cells regained sensitivity to the nitric oxide/cyclic GMP pathway.

Conclusion: The low expression of cGKI determines the lack of nitric oxide/cyclic GMP-dependent regulation on [Ca2+]i transient in SHR RASMCs. This alteration may contribute to the development of hypertension and explain suboptimal responses to nitroglycerin and other nitric oxide-releasing molecules in patients.
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http://dx.doi.org/10.1097/HJH.0b013e328329d18cDOI Listing
June 2009

Synthesis of new pyrazolo[5,1-c][1,2,4] benzotriazines, pyrazolo[5,1-c]pyrido[4,3-e][1,2,4] triazines and their open analogues as cytotoxic agents in normoxic and hypoxic conditions.

Bioorg Med Chem 2008 Nov 26;16(21):9409-19. Epub 2008 Sep 26.

Dipartimento di Scienze Farmaceutiche, Università degli Studi di Firenze, Via U. Schiff 6, 50019 Polo Scientifico, Sesto Fiorentino, Firenze, Italy.

The synthesis and antitumor activity in normoxic and hypoxic conditions of a series of pyrazolo[5,1-c][1,2,4]benzotriazine and its related analogues are reported. All compounds were tested on human colorectal adenocarcinoma cell line HCT-8 and for compounds 15 and 20, which show to have selective cytotoxicity in hypoxic and in normoxic conditions respectively, ROS production, cell cycle, and DNA fragmentation were measured. This preliminary study encouraged us to consider 15 and 20 as interesting leads for further optimization.
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http://dx.doi.org/10.1016/j.bmc.2008.09.055DOI Listing
November 2008

A carbon monoxide-releasing molecule (CORM-3) abrogates polymorphonuclear granulocyte-induced activation of endothelial cells and mast cells.

FASEB J 2008 Sep 12;22(9):3380-8. Epub 2008 Jun 12.

Department of Preclinical and Clinical Pharmacology, University of Florence, Viale G. Pieraccini 6, I-50139, Florence, Italy.

We hypothesized that circulating polymorphonuclear granulocytes (PMNs), vascular endothelial cells (ECs), and perivascular mast cells (MCs) may initiate and sustain the inflammatory response through the generation of the superoxide anion (O(2)(*-)) by PMNs primed by inflammatory stimuli, which in turn evoked the overexpression of adhesion molecules from ECs and release of histamine by MCs. To pin-point the role of carbon monoxide (CO) in curbing vascular inflammation, we studied the effect of a water-soluble CO-releasing molecule [tricarbonylchloro-glycinate-ruthenium (II); CORM-3] on an experimental model of vascular inflammation. The model consists of coincubating formyl-methionyl peptide (fMLP) -primed human PMNs with rat ECs or with rat MCs. The effects of CORM-3 were evaluated by measuring the generation of O(2)(*-) and the expression of CD11b in fMLP-primed PMNs; the expression of ICAM-1 and CD203c in ECs and MCs, respectively; and the release of histamine from MCs. Our results show that the chemotactic peptide fMLP primes PMNs to generate O(2)(*-) and overexpress CD11b, both events being central to the inflammatory process, while CORM-3 significantly decreases these events (IC(50)=1.66 microM for O(2)(*-) production; 1.20 microM for CD11b expression in human PMNs). The experiments also show that fMLP-primed PMNs increase the CD54 expression by coincubated ECs, and the expression of CD203c and the release of histamine by coincubated MCs. Once again, CORM-3 abolishes these events (IC(50)=6.78 microM for CD54 expression in ECs; 1.18 microM for CD203 expression; 1.15 microM for histamine release in MCs). Thus, CORM-3 exerts a powerful anti-inflammatory action by down-regulating the oxidative burst in PMNs, the overexpression of adhesion molecules in PMNs and ECs, the release of histamine, and the overexpression of an activation marker by MCs.
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http://dx.doi.org/10.1096/fj.08-107110DOI Listing
September 2008

Polyamine-polycarboxylate metal complexes with different biological effectiveness as nitric oxide scavengers. Clues for drug design.

J Med Chem 2008 Jun 17;51(11):3250-60. Epub 2008 May 17.

Department of Chemistry, University of Florence, Sesto Fiorentino, Florence, Italy.

The synthesis of the Fe(III), Co(II), Mn(II), and Ru(III) complexes with two polyamine-polycarboxylate ligands, N-(2-hydroxyethyl)ethylenediamine-N, N', N'-triacetic acid (H3L1) and ethylene bisglycol tetraacetic acid (H4L2) is reported. Potentiometric studies showed that these ligands form stable complexes in aqueous solution and no metal release occurs, thus accounting for their low toxicity in cultured RAW 264.7 macrophages. X-ray characterization of the [Co(L1)](-) complex showed that binding sites are available at the metal for NO binding. Efficiency of these compounds to bind NO was studied by UV-vis spectrophotometry. Then their NO-scavenging properties were assayed in a cell-free system under physiological conditions, using S-nitroso-N-acetyl-D,L-penicillamine (SNAP) as NO source. The L1 complexes caused the most effective reduction of free NO, [Mn(L1)](-) being the most efficient. Conversely, in NOS II induced RAW 264.7 macrophages, the Ru(III) and Co(II) complexes with L2 were the most effective compounds. [Ru(L2)](-) also afforded significant protection against lipopolysaccharide-induced endotoxic shock in the mouse in vivo.
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http://dx.doi.org/10.1021/jm701553uDOI Listing
June 2008