Publications by authors named "Maria Donniacuo"

20 Publications

  • Page 1 of 1

An in vitro study to assess the effect of hyaluronan-based gels on muscle-derived cells: Highlighting a new perspective in regenerative medicine.

PLoS One 2020 6;15(8):e0236164. Epub 2020 Aug 6.

Department of Experimental Medicine, School of Medicine, University of Campania "Luigi Vanvitelli," Via L. De Crecchio, Naples, Italy.

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has been widely used for biomedical applications. Here, we have analyzed the effect of HA on the rescue of primary cells under stress as well as its potential to recover muscle atrophy and validated the developed model in vitro using primary muscle cells derived from rats. The potentials of different HAs were elucidated through comparative analyses using pharmaceutical grade a) high (HHA) and b) low molecular weight (LHA) hyaluronans, c) hybrid cooperative complexes (HCC) of HA in three experimental set-ups. The cells were characterized based on the expression of myogenin, a muscle-specific biomarker, and the proliferation was analyzed using Time-Lapse Video Microscopy (TLVM). Cell viability in response to H2O2 challenge was evaluated by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the expression of the superoxide dismutase enzyme (SOD-2) was assessed by western blotting. Additionally, in order to establish an in vitro model of atrophy, muscle cells were treated with tumor necrosis factor-alpha (TNF-α), along with hyaluronans. The expression of Atrogin, MuRF-1, nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB), and Forkhead-box-(Fox)-O-3 (FoxO3a) was evaluated by western blotting to elucidate the molecular mechanism of atrophy. The results showed that HCC and HHA increased cell proliferation by 1.15 and 2.3 folds in comparison to un-treated cells (control), respectively. Moreover, both pre- and post-treatments of HAs restored the cell viability, and the SOD-2 expression was found to be reduced by 1.5 fold in HA-treated cells as compared to the stressed condition. Specifically in atrophic stressed cells, HCC revealed a noteworthy beneficial effect on the myogenic biomarkers indicating that it could be used as a promising platform for tissue regeneration with specific attention to muscle cell protection against stressful agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236164PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410276PMC
October 2020

Protective effect of piceatannol and bioactive stilbene derivatives against hypoxia-induced toxicity in H9c2 cardiomyocytes and structural elucidation as 5-LOX inhibitors.

Eur J Med Chem 2019 Oct 10;180:637-647. Epub 2019 Jul 10.

Consorzio Sannio Tech-AMP Biotec, Appia Str. 7, Apollosa, BN, 82030, Italy; Institute of Food Sciences, National Research Council, Roma Str. 64, Avellino, 83100, Italy. Electronic address:

Stilbenes with well-known antioxidant and antiradical properties are beneficial in different pathologies including cardiovascular diseases. The present research was performed to investigate the potential protective effect of resveratrol (1) and piceatannol (2), against hypoxia-induced oxidative stress in the H9c2 cardiomyoblast cell line, and the underlying mechanisms. Compounds 1 and 2 significantly inhibited the release of peroxynitrite and thiobarbituric acid levels at na no- or submicromolar concentrations, and this effect was more evident in piceatannol-treated cells, that significantly increased MnSOD protein level in a concentration dependent manner. Furthermore, since piceatannol, which is far less abundant in natural sources, displayed a higher bioactivity than the parent compound, we hereby report on a very fast synthesis and detailed structure-based design of a focused stilbene library. Finally, taking into account that hypoxia-induced ROS accumulation also increases expression and activity of 5-lipoxygenase (5-LOX) with production of leukotrienes, we have disclosed structural key factors crucial for 5-LOX activity. Among the synthesized analogues ( 3-7), compound 7 was the most effective in improving cardiomyocytes viability and in 5-LOX inhibition. In conclusion, modeling and experimental studies provided the basis for further optimization of stilbene analogues as multi-target inhibitors of the inflammatory and oxidative pathway.
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http://dx.doi.org/10.1016/j.ejmech.2019.07.033DOI Listing
October 2019

Addition of the Aldose Reductase Inhibitor Benzofuroxane Derivative BF-5m to Prolonged and Moderate Exercise Training Enhanced Protection of the Rat Heart From Type-1 Diabetes.

Front Pharmacol 2019 16;10:392. Epub 2019 Apr 16.

Department of Experimental Medicine, Pharmacology Division, University of Campania "L. Vanvitelli", Naples, Italy.

Moderate exercise training may not be sufficient to exert beneficial effects on the cardiovascular system because of the long-term multifactorial etiology of diabetic complications. The addition of a proper pharmacological tool to the physical exercise should improve the outcomes of the diabetic damage. Here it is shown that 8 weeks exercise training of type 1 diabetic Sprague-Dawley (SD) rats resulted in a significantly increased heart rate, a 14% increase in the left ventricular ejection fraction (LVEF) increased plasma insulin levels and a 13% decrease in plasma glucose with respect to sedentary animals. The training also resulted in a 22% reduction in cardiac QT interval from a diabetic sedentary value of 185 ± 19 ms. Treatment of trained rats with the new antioxidant and NO-releasing aldose reductase 2 inhibitor 5(6)-(benzo[]thiazol-2-ylmethoxy) benzofuroxane BF-5m, 20 mg/kg/day, added a further and significant ( < 0.01 vs. sedentary) increase of the LVEF up to 38% at 8 week time point. The long QT interval recorded in trained rats was reduced to further 12% by addition to the training of pharmacological treatment with 20 mg/kg/day BF-5m. At this time, the association of the two treatments improved the expression into the cardiac tissue of sarcoplasmic reticulum Ca ATPase 2 (SERCA2) and manganese superoxide dismutase (MnSOD), and reduced the fibrosis.
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http://dx.doi.org/10.3389/fphar.2019.00392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6476970PMC
April 2019

Cardioprotective effect of a moderate and prolonged exercise training involves sirtuin pathway.

Life Sci 2019 Apr 5;222:140-147. Epub 2019 Mar 5.

Department of Experimental Medicine, Pharmacology Division, University of Campania "L. Vanvitelli", 80138, Naples, Italy. Electronic address:

Aim: To investigate the cardioprotective effects of prolonged and moderate exercise training on cellular and molecular events early after myocardial infarction.

Materials And Methods: Male Wistar rats were divided in sedentary or exercised group; both groups underwent to a myocardial infarction. All the molecular and immunohistochemical analyses on hearts of sedentary and exercised rats were performed 48 h after surgical procedure. SIRT1 and SIRT3 expression were measured and two of the pathways activated by sirtuins, p53-induced apoptosis and Forkhead boxO (FOXO)3a-induced oxidative stress, were investigated. All the experiments were performed also in presence of the SIRT inhibitor, EX527.

Key Findings: Fourty-eight hours post myocardial infarction, exercise training induced the activation of SIRT1 and SIRT3 pathway reducing cardiomyocytes apoptosis and oxidative damage. Molecular data were confirmed by immunohistochemical evaluations. These effects are more evident in border infarcted zone than in the remote myocardium.

Significance: Exercise training is a non-pharmacological prevention strategy in cardiovascular diseases and the sirtuins family seems to be as novel and attractive target in cardioprotection.
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http://dx.doi.org/10.1016/j.lfs.2019.03.001DOI Listing
April 2019

AT1-receptor blockade: Protective effects of irbesartan in cardiomyocytes under hypoxic stress.

PLoS One 2018 24;13(10):e0202297. Epub 2018 Oct 24.

Department of Experimental Medicine, Section of Pharmacology, Università degli Studi della Campania "Luigi Vanvitelli", Naples, Italy.

Hypoxia induces myocardial injury through the activation of inflammatory and oxidative processes. The pivotal role of the renin angiotensin system (RAS) in the pathogenesis of cardiovascular diseases has been firmly established in clinical trials and practice; in fact many experimental and clinical data have highlighted that its inhibition has a cardioprotective role. Activated RAS also stimulates inflammation directly inducing proinflammatory and oxidative gene expression. This study aimed to investigate the protective role of a pre-treatment (10 and 100 μM) with irbesartan on injury induced by 24 h of hypoxia in HL-1 cardiomyocytes; in particular, we have analyzed the natriuretic peptide (BNP) expression, a biomarker able to modulate inflammatory reaction to cardiac injury and some markers involved in oxidative stress and inflammation. Our results demonstrated that a pre-treatment with 100 μM irbesartan significantly increased SOD activity and catalase expression of 15 and 25%, respectively, compared to hypoxic cells (P<0.05). On the other hand, it was able to reduce the release of peroxynitrite and iNOS protein expression of 20 and 50% respectively (P<0.05). In addition irbesartan exerts an anti-inflammatory activity reducing Toll-like receptors (TLRs)-2 and -4 mRNA expression, TNF-alpha expression and activity (20%) and increasing the expression of the cytokine IL-17 (40%) (P<0.05 vs hypoxia). Our findings also showed that BNP induced by ischemia was significantly and in a concentration-dependent manner reduced by irbesartan. The findings of our study demonstrated that the AT1 receptor antagonist irbesartan exerts a protective role in an in vitro hypoxic condition reducing oxidative stress and inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202297PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200178PMC
March 2019

Role of statins and mevalonate pathway on impaired HDAC2 activity induced by oxidative stress in human airway epithelial cells.

Eur J Pharmacol 2018 Aug 18;832:114-119. Epub 2018 May 18.

Department of Experimental Medicine, University of Campania Luigi Vanvitelli, Naples, Italy.

In patients with chronic obstructive pulmonary disease (COPD) the inflammatory response is often steroid-resistant, likely since oxidative stress and cigarette smoking impair histone deacetylase 2 (HDAC2) activity. Since it has been demonstrated that statins may restore the HDAC2 activity in cultured human endothelial cells, the aim of this study was to investigate the effects of statins in reversing the steroid-resistance induced by oxidative stress. We evaluated the effects of simvastatin and dexamethasone on HDAC2 expression and activity, and the role of mevalonate and Rho/ROCK pathways in A549 cells, a human lung type II epithelial cell line stressed with HO. Our results documented that HO significantly reduced the HDAC2 expression and activity. In HO treated cells dexamethasone was unable to restore the activity of HDAC2, whereas simvastatin restored both the expression and the activity of this enzyme. Our data also showed that mevalonate reduced the activity of HDAC2 whereas Y27632, a Rho/ROCK inhibitor, had no effect on HDAC2 activity when co-administered with simvastatin. Our data suggest that statins could have the potential to restore corticosteroid sensitivity in A549 cells. The evidences of this study suggest that, although both mevalonate and Rho/ROCK pathways are involved in the detrimental effect elicited by oxidative stress, statins may restore the function and expression of depleted HDAC2 via modulating the mevalonate cascade, at least in A549 cells. In conclusion, the modulation of histone acetyltransferase/deacetylase activity may lead to the development of novel anti-inflammatory approaches to inflammatory lung diseases that are currently difficult to treat.
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http://dx.doi.org/10.1016/j.ejphar.2018.05.023DOI Listing
August 2018

Inhibition of p110δ PI3K prevents inflammatory response and restenosis after artery injury.

Biosci Rep 2017 Oct 27;37(5). Epub 2017 Sep 27.

Department of Biochemistry, Biophysics and General Pathology, University of Campania "L. Vanvitelli", Naples, Italy.

Inflammatory cells play key roles in restenosis upon vascular surgical procedures such as bypass grafts, angioplasty and stent deployment but the molecular mechanisms by which these cells affect restenosis remain unclear. The p110δ isoform of phosphoinositide 3-kinase (PI3K) is mainly expressed in white blood cells. Here, we have investigated whether p110δ PI3K is involved in the pathogenesis of restenosis in a mouse model of carotid injury, which mimics the damage following arterial grafts. We used mice in which p110δ kinase activity has been disabled by a knockin (KI) point mutation in its ATP-binding site (p110δ PI3K mice). Wild-type (WT) and p110δ mice were subjected to longitudinal carotid injury. At 14 and 30 days after carotid injury, mice with inactive p110δ showed strongly decreased infiltration of inflammatory cells (including T lymphocytes and macrophages) and vascular smooth muscle cells (VSMCs), compared with WT mice. Likewise, PI-3065, a p110δ-selective PI3K inhibitor, almost completely prevented restenosis after artery injury. Our data showed that p110δ PI3K plays a main role in promoting neointimal thickening and inflammatory processes during vascular stenosis, with its inhibition providing significant reduction in restenosis following carotid injury. p110δ-selective inhibitors, recently approved for the treatment of human B-cell malignancies, therefore, present a new therapeutic opportunity to prevent the restenosis upon artery injury.
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http://dx.doi.org/10.1042/BSR20171112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617917PMC
October 2017

Long-term administration of ranolazine attenuates diastolic dysfunction and adverse myocardial remodeling in a model of heart failure with preserved ejection fraction.

Int J Cardiol 2016 Aug 12;217:69-79. Epub 2016 May 12.

Department of Experimental Medicine, Section of Pharmacology, Second University of Naples, Via Costantinopoli 16, 80138 Naples, Italy.

Background: To investigate the effects of chronic administration of ranolazine (RAN) on experimental model of heart failure with preserved ejection fraction.

Methods: Seven-weeks old Dahl salt-sensitive rats were fed a high salt diet for 5weeks to induce hypertension. Afterwards, rats continued with a high salt diet and were administered either with vehicle or RAN (20mg/kg/die, ip) for the following 8weeks. Control rats were maintained on a low salt diet.

Results: While systolic parameters were not altered, diastolic parameters were changed in high salt animals. Hemodynamic analysis showed a decreased dP/dt min, increased LVEDP, longer time constant and steeper slope of the end-diastolic pressure-volume relationship. Treatment with RAN attenuated these alterations and determined a reduction in mortality. Additionally, the magnitude of myocardial hypertrophy and activation of PI3K/Akt pathway were reduced. Alteration in diastolic compliance as a consequence of elevated myocardial stiffness was confirmed by an increase of collagen deposition and activation of pro-fibrotic TGF-β/SMAD3/CTGF signaling. These effects were counteracted by RAN. High salt rats had a decrease in SERCA2 and an increase in Na(+)/Ca(2+) exchanger (NCX). Treatment with RAN reduced NCX expression and determined an increment of SERCA2. Moreover, the levels of nitrotyrosine and oxidized dyhydroethidium were higher in high salt rats. RAN induced a decrement of oxidative stress, supporting the concept that reduction in ROS may mediate beneficial effects.

Conclusions: Our findings support the possibility that diastolic dysfunction can be attenuated by RAN, indicating its ability to affect active relaxation and passive diastolic compliance.
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http://dx.doi.org/10.1016/j.ijcard.2016.04.168DOI Listing
August 2016

Effect of Prolonged Moderate Exercise on the Changes of Nonneuronal Cells in Early Myocardial Infarction.

Neural Plast 2015 22;2015:265967. Epub 2015 Jul 22.

Department of Experimental Medicine, Division of Pharmacology, The Second University of Naples, Via Costantinopoli 16, 80138 Naples, Italy.

Myocardial infarction (MI) is one of the leading causes of death in developed countries and it is characterized by several associated symptomatologies and poor quality of life. Recent data showed a possible interaction between infarction and brain inflammation and activity. Previous studies have demonstrated the beneficial effect of exercise training on deterioration in cardiac function after MI. In this study we analyzed in sedentary and trained rats the microglia and astrocytes 48 hours after MI in PVN, thalamus, prefrontal cortex, and hippocampus through immunofluorescence approach. We found significant changes in specific microglia phenotypes in the brain areas analyzed together with astrocytes activation. Prolonged exercise normalized these morphological changes of microglia and astrocytes in the prefrontal cortex, hippocampus, and thalamus but not in the PVN. Our data suggest that there is an early brain reaction to myocardial infarction induction, involving nonneuronal cells, that is attenuated by the prolonged exercise.
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http://dx.doi.org/10.1155/2015/265967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526216PMC
April 2016

G-CSF contributes at the healing of tunica media of arteriotomy-injured rat carotids by promoting differentiation of vascular smooth muscle cells.

J Cell Physiol 2016 01;231(1):215-23

Department of Experimental Medicine, Biotechnology, and Molecular Biology Section, Second University of Naples, Naples, Italy.

Restenosis is a complex pathophysiological disease whose causative mechanisms are not fully understood. Previous studies allowed us to demonstrate the efficacy of bone marrow mesenchymal stromal cells (MSCs) transplantation in limiting the pathophysiological remodeling in a model of arteriotomy-induced (re) stenosis. In the current research we studied the effectiveness of G-CSF treatment on male rate rats that were subjected carotid arteriotomy in order to evaluate a potentially effective non-invasive strategy that recapitulates the MSC-mediated recovery of injured vessels. WKY male rats were subjected carotid arteriotomy and given a nine day treatment (3 days pre- to 6 days post-arteriotomy) with G-CSF or saline. Carotids were harvested 7 and 30 days following arteriotomy (early- and late-phase, respectively). Although morphometrical analysis did not reveal differences in lumen narrowing between G-CSF- and PBS-carotids 30 days following arteriotomy, we detected a noticeable conservative effect of G-CSF treatment on vascular wall morphology. Histological and molecular analysis revealed an increase in cellularity within the tunica media with a concomitant increase of the VSMCs differentiation markers both at early- and late-phases of (re) stenotic response in G-CSF-treated carotids (Sm22-alpha, Myocd, and Smtn). These findings were accompanied by the downregulation of oxidative stress-related genes in G-CSF-injured rats. The effect exerted by G-CSF in our model of arteriotomy-induced (re) stenosis seemed support the recovery of the architecture of the tunica media of injured vessels by: (i) inducing VSMCs differentiation; and (ii) limiting the oxidative-stress response induced by arteriotomy.
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http://dx.doi.org/10.1002/jcp.25074DOI Listing
January 2016

Primary and Acquired Resistance of Colorectal Cancer to Anti-EGFR Monoclonal Antibody Can Be Overcome by Combined Treatment of Regorafenib with Cetuximab.

Clin Cancer Res 2015 Jul 2;21(13):2975-83. Epub 2015 Apr 2.

Oncologia Medica, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale "F. Magrassi e A. Lanzara," Seconda Università degli Studi di Napoli, Naples, Italy.

Purpose: In colorectal cancer, the activation of the intracellular RAS-RAF and PIK3CA-AKT pathways has been implicated in the resistance to anti-EGFR mAbs. We have investigated the role of regorafenib, an oral multikinase inhibitor, in combination with cetuximab, an anti-EGFR mAb, to overcome anti-EGFR resistance.

Experimental Design: We have tested, in vitro and in vivo, the effects of regorafenib in a panel of human colorectal cancer cell lines with a KRAS mutation (SW480, SW620, HCT116, LOVO, and HCT15) or with a BRAF mutation (HT29), as models of intrinsic resistance to cetuximab treatment, and in two human colorectal cancer cell lines (GEO and SW48) that are cetuximab-sensitive, as well as in their derived cells with acquired resistance to cetuximab (GEO-CR and SW48-CR).

Results: Treatment with regorafenib determined a dose-dependent growth inhibition in all colorectal cancer cell lines. The combined treatment with cetuximab and regorafenib induced synergistic antiproliferative and apoptotic effects in cetuximab-resistant cell lines by blocking MAPK and AKT pathways. Nude mice were injected s.c. with HCT116, HCT15, GEO-CR, and SW48-CR cells. The combined treatment caused significant tumor growth inhibition. Synergistic antitumor activity of regorafenib plus cetuximab was also observed in an orthotopic colorectal cancer model of HCT116 cells. In particular, the combined treatment induced a significant tumor growth inhibition in the primary tumor site (cecum) and completely prevented metastasis formation.

Conclusions: The combined treatment with cetuximab and regorafenib could be a strategy to overcome resistance to anti-EGFR therapies in metastatic colorectal cancer patients.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-0020DOI Listing
July 2015

Effects of chronic treatment with the new ultra-long-acting β2 -adrenoceptor agonist indacaterol alone or in combination with the β1 -adrenoceptor blocker metoprolol on cardiac remodelling.

Br J Pharmacol 2015 Jul 12;172(14):3627-37. Epub 2015 May 12.

Centre of Excellence for Cardiovascular Diseases, Second University of Naples, Naples, Italy.

Background And Purpose: The ability of a chronic treatment with indacaterol, a new ultra-long-acting β2 -adrenoceptor agonist, to reverse cardiac remodelling and its effects in combination with metoprolol, a selective β1 -adrenoceptor antagonist, were investigated on myocardial infarction in a rat model of heart failure (HF).

Experimental Approach: We investigated the effects of indacaterol and metoprolol, administered alone or in combination, on myocardial histology, β-adrenoceptor-mediated pathways, markers of remodelling and haemodynamic parameters in a rat model of HF. Five groups of rats were assessed: sham-operated rats; HF rats; HF + indacaterol 0.3 mg·kg(-1) ·day(-1) ; HF + metoprolol 100 mg·kg(-1) ·day(-1) ; HF + metoprolol + indacaterol. All pharmacological treatments continued for 15 weeks.

Key Results: Treatment with either indacaterol or metoprolol significantly reduced the infarct size in HF rats. However, the combination of indacaterol and metoprolol reduced the infarct size even further, reduced both BP and heart rate, reversed the decrease in ejection fraction, normalized left ventricular systolic and diastolic internal diameters, normalized the decreased β1 adrenoceptor mRNA expression as well as cardiac cAMP levels and reduced cardiac GPCR kinase 2 expression, compared with the untreated HF group.

Conclusion And Implications: The results of our study demonstrated an additive interaction between indacaterol and metoprolol in normalizing and reversing cardiac remodelling in our experimental model of HF. The translation of these findings to clinical practice might be of interest, as this combination of drugs could be safer and more effective in patients suffering from HF and COPD.
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http://dx.doi.org/10.1111/bph.13148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507164PMC
July 2015

Effects of sildenafil on the gastrocnemius and cardiac muscles of rats in a model of prolonged moderate exercise training.

PLoS One 2013 29;8(7):e69954. Epub 2013 Jul 29.

Department of Experimental Medicine, Section of Pharmacology L. Donatelli, Centre of Excellence for Cardiovascular Diseases, Second University of Naples, Naples, Italy.

Moderate exercise training improves energetic metabolism, tissue perfusion and induces cardiac and skeletal muscle remodeling. Sildenafil, a potent phosphodiesterase-5 inhibitor used to treat erectile dysfunction, reduces infarct size and increases tissue oxygenation in experimental models of cardiovascular disease. We have evaluated the effects of prolonged moderate exercise training and a repeat administration of sildenafil on the rat gastrocnemius and cardiac muscles. Animals were divided into two groups: sedentary and trained. Each group was subdivided into animals treated with vehicle or with two doses of sildenafil (10 or 15 mg/kg/day) during the last week of training. Physical exercise did not induce cardiac hypertrophy, whereas it increased mRNA levels of the PGC-1α, HIF-1α and VEGF genes, which are involved in mitochondrial biogenesis and angiogenesis, and reduced mRNA levels of FoxO3a, MuRF-1 and Atrogin-1. Sildenafil dose-dependently promoted both angiogenesis, as shown by increased capillary density, and muscle atrophy, as shown by muscle fibre size. These effects were more pronounced in trained animals. Our data confirm the beneficial effects of a moderate and prolonged training on cardiovascular and skeletal systems and document the positive and negative effects of sildenafil on these tissues at doses higher than those used in clinical practice. This report may impact on the use of sildenafil as a substance able to influence sports performance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069954PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726744PMC
March 2014

Local inhibition of ornithine decarboxylase reduces vascular stenosis in a murine model of carotid injury.

Int J Cardiol 2013 Oct 13;168(4):3370-80. Epub 2013 May 13.

Dept. of Experimental Medicine, Second University of Naples, Italy; Excellence Research Centre for Cardiovascular Diseases, Second University of Naples, Italy.

Objectives: Polyamines are organic polycations playing an essential role in cell proliferation and differentiation, as well as in cell contractility, migration and apoptosis. These processes are known to contribute to restenosis, a pathophysiological process often occurring in patients submitted to revascularization procedures. We aimed to test the effect of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, on vascular cell pathophysiology in vitro and in a rat model of carotid arteriotomy-induced (re)stenosis.

Methods: The effect of DFMO on primary rat smooth muscle cells (SMCs) and mouse microvascular bEnd.3 endothelial cells (ECs) was evaluated through the analysis of DNA synthesis, polyamine concentration, cell viability, cell cycle phase distribution and by RT-PCR targeting cyclins and genes belonging to the polyamine pathway. The effect of DFMO was then evaluated in arteriotomy-injured rat carotids through the analysis of cell proliferation and apoptosis, RT-PCR and immunohistochemical analysis of differential gene expression.

Results: DFMO showed a differential effect on SMCs and on ECs, with a marked, sustained anti-proliferative effect of DFMO at 3 and 8 days of treatment on SMCs and a less pronounced, late effect on bEnd.3 ECs at 8 days of DFMO treatment. DFMO applied perivascularly in pluronic gel at arteriotomy site reduced subsequent cell proliferation and preserved smooth muscle differentiation without affecting the endothelial coverage. Lumen area in DFMO-treated carotids was 49% greater than in control arteries 4 weeks after injury.

Conclusions: Our data support the key role of polyamines in restenosis and suggest a novel therapeutic approach for this pathophysiological process.
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http://dx.doi.org/10.1016/j.ijcard.2013.04.153DOI Listing
October 2013

Anti-inflammatory effect of simvastatin in an experimental model of spinal cord trauma: involvement of PPAR-α.

J Neuroinflammation 2012 Apr 26;9:81. Epub 2012 Apr 26.

Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, 98125 Messina, Italy.

Background: Statins such as simvastatin are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase used in the prevention of cardiovascular disease. In addition to their cholesterol-lowering activities, statins exert pleiotropic anti-inflammatory effects, which might contribute to their beneficial effects on lipid-unrelated inflammatory diseases. Recently it has been demonstrated that the peroxisome proliferator-activated receptor (PPAR)-α mediates anti-inflammatory effects of simvastatin in vivo models of acute inflammation. Moreover, previous results suggest that PPAR-α plays a role in control of secondary inflammatory process associated with spinal cord injury (SCI).

Methods: With the aim to characterize the role of PPAR-α in simvastatin activity, we tested the efficacy of simvastatin (10 mg/kg dissolved in saline i.p. 1 h and 6 h after the trauma) in an experimental model of SCI induced in mice by extradural compression of the spinal cord (T6-T7 level) using an aneurysm clip with a closing force of 24 g via a four-level T5-T8 laminectomy, and comparing mice lacking PPAR-α (PPAR-α KO) with wild type (WT) mice. In order to elucidate whether the effects of simvastatin are due to activation of the PPAR-α, we also investigated the effect of a PPAR-α antagonist, GW6471 (1 mg/kg administered i.p. 30 min prior treatment with simvastatin) on the protective effects of on simvastatin.

Results: Results indicate that simvastatin activity is weakened in PPAR-α KO mice, as compared to WT controls. In particular, simvastatin was less effective in PPAR-α KO, compared to WT mice, as evaluated by inhibition of the degree of spinal cord inflammation, neutrophil infiltration, nitrotyrosine formation, pro-inflammmatory cytokine expression, nuclear factor (NF)-κB activation, inducible nitric-oxide synthase (iNOS) expression, and apoptosis. In addition we demonstrated that GW6471 significantly antagonized the effect of the statin and thus abolished the protective effect.

Conclusions: This study indicates that PPAR-α can contribute to the anti-inflammatory activity of simvastatin in SCI.
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http://dx.doi.org/10.1186/1742-2094-9-81DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372420PMC
April 2012

Hyperbaric oxygen therapy reduces the toll-like receptor signaling pathway in multiple organ failures.

Intensive Care Med 2011 Jul 13;37(7):1110-9. Epub 2011 May 13.

Department of Experimental Medicine, Excellence Center for Cardiovascular Diseases, Second University of Naples, via De Crecchio 7, 80138, Naples, Italy.

Purpose: Zymosan-induced generalized inflammation is the only experimental model that reproduces characteristics of human multiple organ dysfunction syndrome (MODS). Toll-like receptors (TLRs) are key components in innate immune responses and their signaling pathway is known to activate target genes such as nuclear factor-κB (NF-κB) and cytokines that are involved in inflammation and immune responses. We previously reported that hyperbaric oxygen (HBO) therapy is effective in the treatment of severe zymosan-induced inflammation in MODS. The aim of this study was to investigate the effect of HBO exposure on TLR2 and TLR4 signal transduction and organ dysfunction during MODS induced by zymosan in the rat.

Methods: Male Wistar rats were randomized into four groups and treated as follows: (1) saline solution (control); (2) zymosan; (3) HBO 4 and 11 h after zymosan injection; (4) HBO 4 and 11 h after saline solution injection. Zymosan-induced damage of the lungs, liver, and small intestine was evaluated using histology and biochemistry. The activation of the TLR signaling pathway was measured with Western blot, reverse transcriptase polymerase chain reaction analysis (RT-PCR), and immunohistochemistry.

Results: Zymosan induced a severe inflammatory response characterized by the activation of the TLR signaling pathway and by an organ dysfunction. HBO exposure significantly reduced the development of lung, liver, and intestine injury in our experimental model. It also significantly reduced the zymosan-induced expression of TLR2 and TLR4, NF-κB activation, and cytokine production.

Conclusions: Taken together, these results suggest that, by interfering with the TLR pathway, HBO treatment may exert a protective effect against tissue injury caused by zymosan-induced generalized inflammation.
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http://dx.doi.org/10.1007/s00134-011-2241-1DOI Listing
July 2011

PPARα mediates the anti-inflammatory effect of simvastatin in an experimental model of zymosan-induced multiple organ failure.

Br J Pharmacol 2011 Jun;163(3):609-23

Department of Experimental Medicine, Section of Pharmacology 'L.Donatelli', Excellence Centre for Cardiovascular Diseases, Second University of Naples, Italy.

Background And Purpose: Zymosan-induced non-septic shock is a multi-factorial pathology that involves several organs including the kidneys, liver and lungs. Its complexity and diversity presents a continuing therapeutic challenge. Given their pleiotropic effect, statins could be beneficial in non-septic shock. One of the molecular mechanisms underlying the anti-inflammatory effect of statins involves the peroxisome proliferator-activated receptor (PPAR) α. We used a zymosan-induced non-septic shock experimental model to investigate the role of PPARα in the anti-inflammatory effects of simvastatin.

Experimental Approach: Effects of simvastatin (5 or 10 mg·kg(-1) i.p.) were analysed in PPARα knock-out (KO) and PPARα wild type (WT) mice after zymosan or vehicle administration. Organ injury in lung, liver, kidney and intestine was evaluated by immunohistology. PPARα mRNA expression and nuclear factor-κB activation were evaluated in all experimental groups, 18 h after study onset. Cytokine levels were measured in plasma, and nitrite/nitrate in plasma and peritoneal exudate. Nitric oxide synthase, nitrotyrosine and poly ADP-ribose were localized by immunohistochemical methods.

Key Results: Simvastatin significantly and dose-dependently increased the zymosan-induced expression of PPARα levels in all tissues analysed. It also dose-dependently reduced systemic inflammation and the organ injury induced by zymosan in lung, liver, intestine and kidney. These effects were observed in PPARαWT mice and in PPARαKO mice.

Conclusions And Implications: Simvastatin protected against the molecular and cellular damage caused by systemic inflammation in our experimental model. Our results also provide new information regarding the role of PPARα in the anti-inflammatory effects of statins.
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http://dx.doi.org/10.1111/j.1476-5381.2011.01248.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3101622PMC
June 2011

Rosiglitazone reduces the inflammatory response in a model of vascular injury in rats.

Shock 2009 Dec;32(6):638-44

Department of Experimental Medicine, Excellence Centre for Cardiovascular Diseases, Second University of Naples, Naples, Italy.

Thiazolidinediones are ligands that bind to and activate the nuclear peroxisome proliferator-activated receptor gamma. They are widely used as insulin sensitizers for the treatment of type 2 diabetes. Several studies have implicated the peroxisome proliferator-activated receptor gamma agonists rosiglitazone and pioglitazone in inflammatory events. To assess the anti-inflammatory properties of rosiglitazone, we investigated its effects on the molecular and cellular inflammatory response induced by a carotid injury in the rat. Male Wistar rats were randomized into a rosiglitazone-treated group (10 mg kg(-1) day(-1)) and a control group (0.9% w/v NaCl). The drug or vehicle was administered by gavage for 7 days before carotid injury and for up to 21 days after injury. The inflammatory markers p38 mitogen-activated protein kinase, cyclooxygenase 2, nuclear factor-kappaB, and heat shock protein 47 and the influx and activity of cells in response to injury were measured. Rosiglitazone treatment significantly reduced the expression of the inflammatory markers compared with control group. p38 mitogen-activated protein kinase and nuclear factor-kappaB started to decrease a few hours after injury, whereas cyclooxygenase 2 and heat shock protein 47 expression decreased 7 and 14 days, respectively, after injury. Rosiglitazone also reduced neointima formation and inflammatory cell infiltration. In conclusion, rosiglitazone negatively regulated the inflammatory events involved in tissue repair at molecular and cellular levels. These results suggest that rosiglitazone plays a protective role in inflammatory vascular diseases.
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http://dx.doi.org/10.1097/SHK.0b013e3181a5a377DOI Listing
December 2009

Myocardial adeno-associated virus serotype 6-betaARKct gene therapy improves cardiac function and normalizes the neurohormonal axis in chronic heart failure.

Circulation 2009 Jan 22;119(1):89-98. Epub 2008 Dec 22.

Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Background: The upregulation of G protein-coupled receptor kinase 2 in failing myocardium appears to contribute to dysfunctional beta-adrenergic receptor (betaAR) signaling and cardiac function. The peptide betaARKct, which can inhibit the activation of G protein-coupled receptor kinase 2 and improve betaAR signaling, has been shown in transgenic models and short-term gene transfer experiments to rescue heart failure (HF). This study was designed to evaluate long-term betaARKct expression in HF with the use of stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6).

Methods And Results: In HF rats, we delivered betaARKct or green fluorescent protein as a control via AAV6-mediated direct intramyocardial injection. We also treated groups with concurrent administration of the beta-blocker metoprolol. We found robust and long-term transgene expression in the left ventricle at least 12 weeks after delivery. betaARKct significantly improved cardiac contractility and reversed left ventricular remodeling, which was accompanied by a normalization of the neurohormonal (catecholamines and aldosterone) status of the chronic HF animals, including normalization of cardiac betaAR signaling. Addition of metoprolol neither enhanced nor decreased betaARKct-mediated beneficial effects, although metoprolol alone, despite not improving contractility, prevented further deterioration of the left ventricle.

Conclusions: Long-term cardiac AAV6-betaARKct gene therapy in HF results in sustained improvement of global cardiac function and reversal of remodeling at least in part as a result of a normalization of the neurohormonal signaling axis. In addition, betaARKct alone improves outcomes more than a beta-blocker alone, whereas both treatments are compatible. These findings show that betaARKct gene therapy can be of long-term therapeutic value in HF.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.108.803999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2647661PMC
January 2009

Inflammatory events in a vascular remodeling model induced by surgical injury to the rat carotid artery.

Br J Pharmacol 2006 Jan;147(2):175-82

Department of Experimental Medicine, Excellence Centre on Cardiovascular Diseases, Second University of Naples, Via Costantinopoli 16, 80138 Naples, Italy.

1.--The aim of our study was to gain insight into the molecular and cellular mechanisms of the inflammatory response to arterial injury in a rat experimental model. 2.--Rats (five for each experimental time) were subjected to brief clamping and longitudinal incision of a carotid artery and monitored for 30 days. Subsequently, Nuclear Factor-kappaB (NF-kappaB) expression was measured by electrophoretic mobility shift assay. Heat shock protein (HSP) 27, HSP47 and HSP70 were evaluated by Western blot. Morphological changes of the vessel wall were investigated by light and electron microscopy. 3.--In injured rat carotid artery NF-kappaB activity started immediately upon injury, and peaked between 2 and 3 weeks later. Western blot showed a significant increase of HSP47 and HSP70 7 days after injury. At 2 weeks postinjury, HSP27 expression peaked. Light microscopy showed a neointima formation, discontinuity of the media layer and a rich infiltrate. Among infiltrating cells electron microscopy identified dendritic-like cells in contact with lymphocytes. 4.--Our model of surgical injury induces a significant inflammatory process characterized by enhanced NF-kappaB activity and HSPs hyperexpression. Dendritic-like cells were for the first time identified as a novel component of tissue repair consequent to acute arterial injury.
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http://dx.doi.org/10.1038/sj.bjp.0706472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1615853PMC
January 2006