Emanuela Masini

Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P.

We evaluated the effects of nitric oxide (NO) generators and endogenous production of NO elicited by substance P (SP) in the angiogenesis process. Angiogenesis was monitored in the rabbit cornea in vivo and in vitro by measuring the growth and migration of endothelial cells isolated from coronary postcapillary venules. The angiogenesis promoted in the rabbit cornea by [Sar9]-SP-sulfone, a stable and selective agonist for the tachykinin NK1 receptor, and by prostaglandin E1 (PGE1), was potentiated by sodium nitroprusside (SNP). Conversely, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), given systemically, inhibited angiogenesis elicited by [Sar9]-SP-sulfone and by PGE1. Endothelial cells exposed to SNP exhibited an increase in thymidine incorporation and in total cell number. Exposure of the cells to NO generating drugs, such as SNP, isosorbide dinitrate, and glyceryl trinitrate, produced a dose-dependent increase in endothelial cell migration. Capillary endothelial cell proliferation and migration produced by SP were abolished by pretreatment with the NO synthase inhibitors N omega-mono-methyl-L-arginine (L-NMMA), N omega-nitro-L-arginine (L-NNA), and L-NAME. Exposure of the cells to SP activated the calcium-dependent NO synthase. Angiogenesis and endothelial cell growth and migration induced by basic fibroblast growth factor were not affected by NO synthase inhibitors. These data indicate that NO production induced by vasoactive agents, such as SP, functions as an autocrine regulator of the microvascular events necessary for neovascularization and mediates angiogenesis.

Modulation of Prostaglandin Biosynthesis by Nitric Oxide and Nitric Oxide Donors

The biosynthesis and release of nitric oxide (NO) and prostaglandins (PGs) share a number of similarities. Two major forms of nitric-oxide synthase (NOS) and cyclooxygenase (COX) enzymes have been identified to date. Under normal circumstances, the constitutive isoforms of these enzymes (constitutive NOS and COX-1) are found in virtually all organs. Their presence accounts for the regulation of several important physiological effects (e.g. antiplatelet activity, vasodilation, and cytoprotection). On the other hand, in inflammatory setting, the inducible isoforms of these enzymes (inducible NOS and COX-2) are detected in a variety of cells, resulting in the production of large amounts of proinflammatory and cytotoxic NO and PGs. The release of NO and PGs by the inducible isoforms of NOS and COX has been associated with the pathological roles of these mediators in disease states as evidenced by the use of selective inhibitors. An important link between the NOS and COX pathways was made in 1993 by Salvemini and coworkers when they demonstrated that the enhanced release of PGs, which follows inflammatory mechanisms, was nearly entirely driven by NO. Such studies raised the possibility that COX enzymes represent important endogenous “receptor” targets for modulating the multifaceted roles of NO. Since then, numerous papers have been published extending the observation across various cellular systems and animal models of disease. Furthermore, other studies have highlighted the importance of such interaction in physiology as well as in the mechanism of action of drugs such as organic nitrates. More importantly, mechanisticstudies of how NO switches on/off the PG/COX pathway have been undertaken and additional pathways through which NO modulates prostaglandin production unraveled. On the other hand, NO donors conjugated with COX inhibitors have recently found new interest in the understanding of NO/COX reciprocal interaction and potential clinical use. The purpose of this article is to cover the advances which have occurred over the years, and in particular, to summarize experimental data that outline how the discovery that NO modulates prostaglandin production has impacted and extended our understanding of these two systems in physiopathological events.

A role for superoxide in gentamicin-mediated nephropathy in rats

Gentamicin is an antibiotic effective against Gram-negative infection, whose clinical use is limited by its nephrotoxicity. Oxygen free radicals are considered to be important mediators of gentamicin-mediated nephrotoxicity, but the exact nature of the radical in question is not known with certainty. We have investigated the potential role of superoxide in gentamicin-induced renal toxicity by using M40403, a low molecular weight synthetic manganese containing superoxide dismutase mimetic, which selectively removes superoxide. Administration of gentamicin at 100 mg/kg, s.c. for 5 days to rats induced a marked renal failure, characterised by a significant decrease in creatinine clearance and increased plasma creatinine levels, fractional excretion of sodium, lithium, urine gamma glutamyl transferase (gamma GT) and daily urine volume. A significant increase in kidney myeloperoxidase activity and lipid peroxidation was also observed in gentamicin-treated rats. M40403 (10 mg/kg, i.p. for 5 days) attenuated all these parameters of damage. Immunohistochemical localisation demonstrated nitrotyrosine formation and poly(ADP-ribose) synthetase (PARS) activation in the proximal tubule of gentamicin-treated rats. Renal histology examination confirmed tubular necrosis. M40403 significantly prevented gentamicin-induced nitrotyrosine formation, poly(ADP-ribose) synthetase activation and tubular necrosis. These results confirm our hypothesis that superoxide anions play an important role in gentamicin-mediated nephropathy and support the possible clinical use of low molecular weight synthetic superoxide dismutase mimetics in those conditions that are associated with over production of superoxide.

Platelets and inflammation: role of platelet-derived growth factor, adhesion molecules and histamine.

Abstract. Despite the fact that the relationship between platelets and the inflammatory and immune responses has been reviewed previously, the allocation of platelets among the inflammatory cells is still at issue. Recent developments in our understanding of platelet-associated signalling events have offered new potential insights into platelet functions in inflammatory and immune-related diseases. In recent years, it has been established that a range of molecules, mainly associated with the platelet surface and/or the platelet granules, regulate the capacity of platelets to cross-talk with other inflammatory cells during the process of inflammation, and of vascular inflammation in particular. This is the case with platelet-derived growth factor (PDGF), secreted from platelet alpha-granules, with P-selectin, expressed on the platelet surface, and with platelet histamine, which is secreted from platelets in response to aggregatory and inflammatory stimuli. The nature and mechanism of action of these r egulatory molecules, physiologically present in platelets and mobilised upon platelet activation and aggregation, is the subject of this review. The participation of platelets, through PDGF, P-selectin and histamine, is also discussed in overtly inflammatory disorders, such as acute respiratory distress syndrome, mesangial glomerulonephritis, chronic inflammatory bowel disease, disseminated intravascular inflammation, and allergic vasculitis, focusing on possible pharmacological interventions specifically active against growth factors, adhesion molecules and platelet histamine.

Oxidative Stress by Monoamine Oxidase Mediates Receptor-Independent Cardiomyocyte Apoptosis by Serotonin and Postischemic Myocardial Injury

Background— Serotonin (5-hydroxytryptamine [5-HT]), released by activated platelets during cardiac ischemia, is metabolized by the mitochondrial enzyme monoamine oxidase A (MAO-A). Because hydrogen peroxide is one of the byproducts of 5-HT degradation by MAO-A, we investigated the potential role of reactive oxygen species generated by MAOs in 5-HT-dependent cardiomyocyte death and post-ischemia-reperfusion cardiac damage. Methods and Results— Treatment of isolated adult rat cardiomyocytes with 5-HT induced intracellular oxidative stress and cell apoptosis. The apoptotic cascade triggered by 5-HT involves release of cytochrome c, upregulation of proapoptotic Bax protein, and downregulation of antiapoptotic Bcl-2 protein. These effects were prevented by inhibition of amine transporter or MAO, antioxidants, or iron chelation. In contrast, cardiomyocyte apoptosis was only slightly affected by the 5-HT2B receptor antagonist SB 206553. In vivo, inhibition of MAO-A largely reduced myocardial ultrastructural damage induced by 30 minutes of ischemia followed by 60 minutes of reperfusion in the rat heart. Cardioprotective effects of MAO inhibitors were associated with the prevention of postischemic oxidative stress, neutrophil accumulation, and mitochondrial-dependent cell death and were not reverted by SB 206553. Administration of MAO-A inhibitors during ischemia was still effective in preventing cardiac damage. Conclusions— Our results supply the first direct evidence that oxidative stress induced by MAO is responsible for receptor-independent apoptotic effects of 5-HT in cardiomyocytes and postischemic myocardial injury. These findings provide new insight into the mechanisms of 5-HT action in the heart and may constitute the basis for novel therapies.

Antiglaucoma carbonic anhydrase inhibitors: a patent review

Introduction: Glaucoma is one of the major causes of blindness, affecting together with age-related macular degeneration > 70 million people worldwide. One of the therapeutic options for its management is based on the inhibition of the metalloenyme carbonic anhydrase (CA, EC 4.2.1.1). CA inhibitors (CAIs) diminish intraocular pressure (IOP) by reducing the rate of bicarbonate formation and thus secretion of the aqueous humor. Areas covered: The main classes of clinically used antiglaucoma CAIs are the sulfonamides with systemic (acetazolamide, methazolamide, ethoxzolamide and dichlorophenamide) and topical (dorzolamide and brinzolamide) action. A patent literature review covering the period 2007 – 2013 is presented. Expert opinion: This review presents an overview of the patent literature in the CAI antiglaucoma drug design field during the past 6 years. Most of the patents deal with sulfonamide/sulfamide/sulfamate CAIs, sulfonamides incorporating NO-donating moieties, as well as hybrids incorporating sulfonamide and prostaglandin (PG) analogs, were also reported. There is an urgent need for new antiglaucoma CAIs/approaches to treat and diagnose this disease in the very near future, as the last drug which has been discovered in the field (latanoprost) dates back > 10 years ago.

Relaxin‐induced increased coronary flow through stimulation of nitric oxide production

1 Relaxin (RLX) is a multifunctional hormone which, besides its role in pregnancy and parturition, has also been shown to influence the cardiovascular system. In this study, we investigated the effect of RLX on coronary flow of rat and guinea‐pig hearts, isolated and perfused in a Langendorff apparatus. RLX was either added to the perfusion fluid at a concentration of 5 × 10−9 M for a 20‐min perfusion, or given as a bolus into the aortic cannula at concentrations of 10−9 M, 5 × 10−9 M and 10−8 M dissolved in 1 ml of perfusion fluid. 2 RLX, given either for a 20‐min perfusion or as a bolus in the aortic cannula to guinea‐pig and rat isolated hearts, increased the coronary flow and the amount of nitrite, a stable end‐product of nitric oxide (NO) metabolism, that appeared in the perfusates in a concentration‐dependent fashion. 3 The increase in coronary flow and in nitrite in the perfusates induced by RLX was significantly reduced by pretreatment with the nitric oxide synthase (NOS) inhibitor, NG‐monomethyl‐L‐arginine (L‐NMMA, 10−4 M). 4 The effects of RLX on coronary flow and nitrite amounts in the perfusates were compared with those induced by the endothelium‐dependent vasodilator agent, acetylcholine (ACh, 10−8 − 10−7 M), and by the endothelium‐independent vasodilator agent, sodium nitroprusside (SNP, 10−7‐10−6M). The results obtained show that RLX is more effective than ACh and SNP in increasing coronary flow. 5 The results of this study show that RLX increases coronary flow through stimulation of NO production; hence this hormone should be regarded as a novel agent capable of improving myocardial perfusion.

Dithiocarbamates strongly inhibit carbonic anhydrases and show antiglaucoma action in vivo

A series of dithiocarbamates were prepared by reaction of primary/secondary amines with carbon disulfide in the presence of bases. These compounds were tested for the inhibition of four human (h) isoforms of the zinc enzyme carbonic anhydrase, CA (EC 4.2.1.1), hCA I, II, IX, and XII, involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX). Several low nanomolar inhibitors targeting these CAs were detected. The X-ray crystal structure of the hCA II adduct with morpholine dithiocarbamate evidenced the inhibition mechanism of these compounds, which coordinate to the metal ion through a sulfur atom from the dithiocarbamate zinc-binding function. Some dithiocarbamates showed an effective intraocular pressure lowering activity in an animal model of glucoma.

Expression of matrix metalloproteinase 1, matrix metalloproteinase 2, and matrix metalloproteinase 9 in carcinoma of the head and neck

Numerous reports have documented a direct involvement of matrix metalloproteinase (MMP) overexpression in the development and progression of head and neck squamous cell carcinoma (HNSCC). In this study, the authors examined whether the expression of MMPs in HNSCC is correlated with other steps involved in tumor growth and metastasis, like angiogenesis, activation the nitric oxide (NO) pathway, and alteration of the p53 tumor suppressor gene.

Cyclooxygenase-2 Activation Mediates the Proangiogenic Effect of Nitric Oxide in Colorectal Cancer

Purpose: Up-regulation of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes has been reported in colorectal cancer. We aimed at evaluating the possible interaction between the nitric oxide and COX-2 pathways, and its effect on promoting tumor angiogenesis. Experimental Design: Expression of iNOS, COX-2, vascular endothelial growth factor (VEGF), and CD31 was analyzed in tumor samples and corresponding normal mucosa obtained from 46 surgical specimens. We also evaluated iNOS activity, prostaglandin E2 (PGE2), cyclic GMP and cyclic AMP production in the same specimens. Nitrite/nitrate levels, and PGE2 and VEGF production were assessed in HCT116 and HT29 colon cancer cell lines after induction and selective inhibition of the two enzyme pathways. Results: A significant correlation was found between iNOS and COX-2 immunohistochemical expression. PGE2 production significantly correlated with iNOS activity and cGMP levels. A significant correlation was also found among PGE2 production, microvessel density, and VEGF expression. Coinduction of both iNOS and COX-2 activities occurred after lipopolysaccharide (LPS) and epidermal growth factor (EGF) treatment in HCT116 and HT29 cells. Inhibition of iNOS by 1400W significantly reduced both LPS- and EGF-induced PGE2 production. Treatment with LPS, EGF, and arachidonic acid significantly increased VEGF production in the iNOS-negative/COX-2-positive HT29 cells. This effect was completely reversed by treatment with the selective COX-2 inhibitor celecoxib. Conclusions: Our data showed a prominent role of nitric oxide in stimulating COX-2 activity in colorectal cancer. This interaction is likely to produce a cooperative effect in promoting angiogenesis through PGE2-mediated increase in VEGF production.