At the cardiovascular level, nitric oxide (NO) controls smooth muscle functions, maintains vascular integrity, and exerts an antihypertensive effect. Metal-nonoates are a recently discovered class of NO donors, with NO release modulated through the complexation of the N-aminoethylpiperazine N-diazeniumdiolate ligand to metal ions, and thus representing a significant innovation with respect to the drugs traditionally used. In this study, we characterized the vascular protective effects of the most effective compound of this class, Ni(PipNONO)Cl, compared with the commercial N-diazeniumdiolate group derivate, diethylenetriamine/nitric oxide (DETA/NO). Ni(PipNONO)Cl induced a concentration-dependent relaxation of precontracted rat aortic rings. The ED50 was 0.67 µM, compared with 4.3 µM obtained with DETA/NO. When tested on cultured microvascular endothelial cells, Ni(PipNONO)Cl exerted a protective effect on the endothelium, promoting cell proliferation and survival in the picomolar range. The administration of Ni(PipNONO)Cl to vascular smooth muscle cells reduced the cell number, promoting their apoptosis at a high concentration (10 µM). Inhibition of smooth muscle cell migration, a hallmark of atherosclerosis, was accompanied by cytoskeletal rearrangement and loss of lamellipodia. When added to isolated platelets, Ni(PipNONO)Cl significantly reduced ADP-induced aggregation. Since atherosclerosis is accompanied by an inflammatory environment, cultured endothelial cells were exposed to interleukin (IL)-1β. In the presence of IL-1β, Ni(PipNONO)Cl inhibited cyclooxygenase-2 and inducible nitric oxide synthase upregulation, and reduced endothelial permeability and the platelet and monocyte adhesion markers CD31 and CD40 at the plasma membrane. Overall, these data indicate that Ni(PipNONO)Cl exerts vascular protective effects relevant for vascular dysfunction and prevention of atherosclerosis and thrombosis.
Nitric oxide (NO), a free radical gaseous molecule, is a multifunctional messenger with diverse physiologic functions, such as dilation of blood vessels, inhibition of platelet aggregation, and suppression of smooth muscle cell proliferation (Moncada and Higgs, 1993; Higashi et al., 2003). It is continuously produced by the endothelium through the action of endothelial nitric oxide synthase (eNOS), and rapidly diffuses in the vessel wall and inside the lumen, where it exerts protective effects. When endothelial functionality is significantly reduced, the risk of endothelial dysfunction–related pathologies, such as atherosclerosis, thrombosis, and ischemia, increases (Cannon, 1998). Indeed, from a pharmacologic point of view, drugs inducing the recovery of endothelial survival and function exert beneficial effects as preventive agents or as therapeutic interventions. Statins, angiotensin-converting enzyme inhibitors, and NO donors/generators can control endothelial dysfunction and prevent the evolution of atherosclerosis, hypertension, and other cardiovascular diseases by promoting eNOS expression/activity or inducing NO release (Förstermann and Sessa, 2012; Majumder et al., 2014).
NO is also recognized to inhibit vascular smooth muscle cell proliferation in a cGMP-dependent manner (Jeremy et al., 1999), and has an impact on circulating platelets, in which both cGMP-dependent and -independent mechanisms potently inhibit their adhesion and aggregation (Crane et al., 2005). Similarly, NO is a potent inflammatory inhibitor of endothelial cell and monocyte activation (Bath, 1993).
In a previous article, we reported the synthesis, characterization, and NO-releasing properties of compounds named metal-nonoates, which are derived from N-aminoethylpiperazine N-diazeniumdiolate (HPipNONO). Simple metal-nonoates, such as [Cu(PipNONO)Cl] and [Ni(PipNONO)Cl], release NO at a slower (Cu) or comparable (Ni) rate to HPipNONO (half-life of approximately 5 minutes at 25°C) in aqueous buffer at physiologic pH (Ziche et al., 2008). Metal-nonoates promoted proliferation of bovine coronary endothelial cells in a cGMP-dependent manner (Ziche et al., 2008). This study aimed to corroborate the protective effects of metal-nonoates on human endothelial cells of various origins, such as human umbilical vein endothelial cells (HUVECs) and human cardiac microvascular endothelial cells (HCMEC), and the molecular mechanisms implicated, focusing on the activity of Ni(PipNONO)Cl, which follows a simple exponential curve for the release of NO and was not previously characterized for its biologic activity. In this study, the biologic and pharmacologic properties of Ni(PipNONO)Cl are compared with those of the parent free ligand, HPipNONO, and the commercially available N-diazeniumdiolate group (NONOate), diethylenetriamine/nitric oxide (DETA/NO), which is extensively used in pharmacologic assays and is easier to handle than diethylamine/NO. The compound was able to induce vasorelaxation in precontracted rat aortic rings. Additional protective actions by Ni(PipNONO)Cl on vascular components were demonstrated as inhibition of smooth muscle cell migration and proliferation, and platelet aggregation. Reversion of an inflammatory phenotype [induced by interleukin (IL)-1β] was obtained in the presence of the metal-nonoate.
Materials and Methods
The NONOate derivatives HPipNONO and Ni(PipNONO)Cl, and the control and decomposition compound Ni(Pip)Cl2 (Fig. 1, A–C, respectively) were synthesized in high purity and were spectrally and analytically characterized by Noxamet Ltd. (Milan, Italy) as previously reported (Ziche et al., 2008). DETA/NO, releasing 2 mol of NO for each mole of the compound, was from Sigma-Aldrich (St. Louis, MO). DETA/NO was chosen from preliminary biologic experiments because of its ease in handling and reproducibility compared with diethylamine/NO. IL-1β was from ReliaTech (Wolfenbüttel, Germany). For the biologic assays, the compounds were dissolved in dimethylsulfoxide and then solubilized in culture medium or buffer-maintained in ice until the administration to the cells/aortic rings.
HUVECs were purchased from Promocell (Heidelberg, Germany) and HCMECs were from ScienCell (M-Medical, Milan, Italy). Both cell lines were grown in complete endothelial growth medium (Lonza, Basel, Switzerland), supplemented with 10% fetal bovine serum. Human umbilical artery smooth muscle cells (HUASMs) were from Clonetics (Lonza) and were grown in SmBM Basal Medium (Lonza). Cells were cultured at 37°C in 5% CO2. Cells were split 1:3 twice a week, and used until passage 10.
Survival and Proliferation Assay.
Endothelial and smooth muscle cell proliferation was evaluated following the same protocol. One thousand cells/well (of a 96-well multiplate) were allowed to adhere in 10% serum for 3 to 4 hours and then test substances were added in medium with 0.1% serum. All experimental points were run in triplicate. After 2 days, cells were fixed, stained, and randomly counted at 20× original magnification in five fields (Monti et al., 2013). Data are reported as the number of counted cells/well.
Chemotaxis experiments were performed by the Boyden chamber technique (Neuroprobe 48-well microchemotaxis chamber; Biomap Snc, Agrate Brianza, Italy), with the filter coated with collagen and fibronectin (Sigma-Aldrich) (Solito et al., 2013). HUASMs (1.25 × 104) previously treated with Ni(PipNONO)Cl (1 µM) were added to the upper wells of the chamber. Lower wells contained 0.1% fetal bovine serum. All experimental points were run in triplicate. After 4 hours of incubation, cells were fixed and stained with the Diff-Quik kit (Biomap Snc). Migrated cells present in five fields/well were counted at 40× original magnification. Data are reported as the number of counted cells/well.
Measurement of Endothelial Permeability.
Cells were seeded on collagen-coated insert membranes (Corning/Sigma-Aldrich) containing a high density of 0.4-µm diameter pores, and the inserts were placed into a 12-multiwell plate. Cells were seeded at 8 × 104 cells/insert and cultured with complete medium for 72 hours. Confluent monolayers were pretreated with Ni(PipNONO)Cl (0.1 nM, 30 minutes) and then with IL-1β (100 ng/ml, 6 hours). Then 40 kDa fluorescein isothiocyanate-dextran (10 μM) was added on top of the cells, allowing the fluorescent molecules to pass through the endothelial cell monolayer in the lower compartment. The extent of permeability was determined after 15 minutes by measuring the fluorescence in a plate reader (Infinite 200 Pro SpectraFluor; Tecan, Mannedorf, Switzerland) at 485 and 535 nm excitation and emission, respectively (Solito et al., 2013).
Subconfluent endothelial and smooth muscle cells were seeded in 6-cm diameter petri dishes. After adherence, HUASMs were treated for the indicated times with Ni(PipNONO)Cl (0.1 nM or 1 µM, respectively), whereas HUVECs were serum-starved or treated with IL-1β before treatment (100 ng/ml, 6 or 18 hours). Serum starvation was used as a condition to induce reactive oxygen species–mediated dysfunction (Monti et al., 2010).
Protein extraction and Western blot were performed as previously described (Monti et al., 2013). Electrophoresis (50 μg of protein/sample) was carried out in 4–12% Bis-Tris Gels (Life Technologies, Carlsbad, CA). Proteins were then blotted onto nitrocellulose membranes, incubated overnight with antibodies [anti–phospho-extracellular signal-regulated kinase (ERK)1/2, anti–caspase-3, anti-p53, anti–phospho-Akt (each at 1:1000, nos. 9106S, 9662S, 9282S, and 9271S, respectively; Cell Signaling Technology, Danvers, MA), anti–cyclooxygenase-2 (COX-2) (at 1:200, no. 160112; Cayman, Ann Arbor, MI), anti–inducible nitric oxide synthase (iNOS) and anti-CD40 (each at 1:500, nos. sc-651 and sc-975, respectively; Santa Cruz Biotechnology, Dallas, TX), anti–phospho-eNOS (Ser1177) and anti–phospho-eNOS (Thr495) (each at 1:1000, nos. 07-428 and 04-811, respectively; Upstate Biotechnology/Millipore, Billerica, MA)], and then detected by enhanced chemiluminescence system (Bio-Rad, Hercules, CA). Results were normalized to those obtained by using an antibody against β-actin (no. A5441; Sigma-Aldrich), eNOS (no. 610297; BD Transduction, Franklin Lakes, NJ), and Akt, ERK, and total caspase-3 (nos. 9272, 9102, and 9662S, respectively; Cell Signaling Technology), when appropriate.
The cytoskeletal proteins tubulin, desmin, and β-actin were visualized in HUASM cultures by confocal microscopy. CD31 and CD40 expression was evaluated in HUVEC monolayers. Cells (2 × 104) were seeded on 1-cm-round glass coverslips. After 24 hours, cells were washed and treated with the indicated stimuli. Cells were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS). Unspecific binding sites were blocked in 3% bovine serum albumin (BSA) and then cells were incubated for 2 hours with antibodies [anti-CD31 (no. MAB2148; Chemicon/Millipore), anti-CD40 (no. sc-975; Santa Cruz Biotechnology), and anti–β-actin, anti-desmin, and anti–β-tubulin (nos. A5441, D8281, and T3526, respectively; Sigma-Aldrich)] diluted 1:50 in PBS containing 0.5% BSA. Cells were then washed, incubated for 1 hour with a rhodamine- or fluorescein-conjugated secondary antibody (#T6778 and F5262, respectively; Sigma-Aldrich) diluted 1:50 in PBS with 0.5% BSA. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich). Coverslips were mounted in Mowiol 4-88 (Calbiochem, La Jolla, CA) and pictures of stained cells were taken through a confocal microscope (Zeiss LSM500; Carl Zeiss, Oberkochen, Germany).
Rat Aorta Vessel Ring Preparations.
Effects of NONOates were tested on isolated thoracic aortic rings of male normotensive Sprague-Dawley rats (250–350 g; Charles River Italia, Calco, Italy), which were kept in temperature- and humidity-controlled rooms (22°C, 50%) with lights on from 7:00 AM to 7:00 PM, with water and food available ad libitum. All procedures, carried out in accordance with the Italian law (n.116, January 27, 1992), which reflects the European Directive 2010/63/UE, were approved by the University of Siena ethical board and the Italian Ministry of Health. All efforts were made to minimize the number of animals used and their suffering. Animals were anesthetized (intraperitoneally) with a mixture of Ketavet (30 mg/kg ketamine; Intervet, Aprilia, Italy) and Xilor (8 mg/kg xylazine; Bio 98, San Lazzaro, Italy), and were decapitated and exsanguinated. The aorta was immediately excised, cut into rings of 3-mm width (4–6 rings for each aorta), and bathed in Krebs’ solution as previously described (Ziche et al., 2008). Changes in tension were recorded by means of an isometric transducer connected to a computerized system (Biopac, Goleta, CA). After an equilibration period of 60 minutes, the presence of endothelium was confirmed by the responsiveness to acetylcholine (10 µM; Sigma-Aldrich). The value of tension developed after phenylephrine (0.3 μM; Sigma-Aldrich) administration was taken as 100% and the effects of the agents were referred to this value.
Platelet-Rich Plasma Preparation and Platelet Aggregation.
The following experiments were carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. After a detailed informed consent form was signed, blood (9 ml) was collected in the morning from resting and fasting male healthy subjects by a 19-G needle without stasis. The blood was then stored in acid/citrate/dextrose (1:9) for platelet studies (Puccetti et al., 2003). In particular, donors were not treated in the previous 2 weeks with corticosteroids, nonsteroidal anti-inflammatory drugs (except acetaminophen), and antibacterial drugs, and they did not suffer from recurring or chronic pathologies.
For platelet isolation, citrated blood was centrifuged, within 15 minutes from collection, at 100g for 20 minutes at 18°C to obtain platelet-rich plasma (PRP). This was separated from the top two-thirds of the supernatant to avoid contamination by other cells. The remaining blood was centrifuged at 1500g for 10 minutes to obtain platelet-poor plasma.
Platelet aggregation studies were performed in a four-channel PAP-4 aggregometer (Bio/Data Corporation, Horsham, PA) and results are expressed as the change in percentage of transmitted light. Platelet aggregation in PRP (0.5-ml aliquots) was induced by 5 μM ADP. All measurements were performed in duplicate and read 8 minutes after stimulation. With 5 μM ADP, the platelet aggregation curve has a biphasic appearance. The first curve (shape change) was considered as platelet activity reduction if maximal aggregation was <30% at 3 minutes. The second curve (release reaction) was considered as platelet activity reduction if maximal aggregation was <40% at 8 minutes. In the latter event, platelet activity was considered blocked if maximal aggregation was <20% (no irreversible aggregation obtained). Data are reported as the slope or rate of aggregation, the percentage of maximal aggregation of the first curve, and the percentage of maximal aggregation of the second curve, considering 100% the effect given by ADP alone.
Measurement of cGMP Levels.
cGMP levels were measured on cell extracts from confluent cell monolayers by an enzyme-immunoassay kit (Cayman Chemical, Ann Arbor, MI) as previously described (Monti et al., 2010). Cell monolayers were treated with 1 mM 3-isobutyl-5-methyl-xanthine (Sigma-Aldrich) for 15 minutes before stimulation. cGMP levels were assayed in cell lysates according to the manufacturer’s instructions, whereas proteins were measured in the pellet by Bradford’s procedure. Data are expressed as the fold of increase versus control.
Data represent means ± S.E.M. of n determinations. Statistical analysis was performed by means of the t test for unpaired data or by analysis of variance followed by the Bonferroni test for comparison among groups of data. P < 0.05 was considered statistically significant.
Protective Effects of Metal-Nonoates on Endothelial Cells.
Reduction of vascular tone is a typical feature of NO donors. The NONOate derivatives DETA/NO, HPipNONO, and Ni(PipNONO)Cl, and the control compound Ni(Pip)Cl2 were screened in rat aortic ex vivo preparations containing intact endothelium. Ni(PipNONO)Cl was more effective and potent than either DETA/NO or HPipNONO in inducing relaxation in precontracted aortic rings. The EC50 values (concentration achieving 50% of relaxation) were 0.67 µM for Ni(PipNONO)Cl and 4.3 µM for DETA/NO (Fig. 1D).
After the preliminary characterization, the new NONOates were evaluated in functional responses of cultured vascular cells. The integrity of the endothelial cell layer is a prerequisite for maintenance of vascular tone and blood fluidity. Thus, the potential pro-proliferative/prosurvival effects of metal-nonoates were evaluated in human endothelial cells, specifically the widely used HUVEC and the cardiac microvascular endothelium HCMEC. Endothelial cells were exposed to increasing concentrations (0.0001–100 nM) of the NONOate derivatives for 48 hours and the cell number was microscopically counted. Ni(PipNONO)Cl induced endothelial cell proliferation, but neither HPipNONO nor Ni(Pip)Cl2 gave any effect. Ni(PipNONO)Cl was the most effective on both HUVECs and HCMECs compared with DETA/NO (Fig. 2A and Table 1, respectively). Maximal proliferation was obtained in the presence 0.1 and 1 nM of Ni(PipNONO)Cl in HUVECs and HCMECs, respectively. By increasing Ni(PipNONO)Cl concentrations, a reduction in cell number could be found without any evidence of cytotoxicity.
The activation of signaling pathways correlated with cell proliferation and survival (ERK1/2 and Akt phosphorylation) and the inhibition of hallmarks of apoptosis (caspase-3 cleavage and p53 expression) were evaluated in HUVECs exposed to Ni(PipNONO)Cl (0.1 nM). The parameters were evaluated at increasing time points (5–60 minutes for pERK1/2 and pAkt, and 1–9 hours for cleaved caspase-3 and p53 expression). To better appreciate the ability of Ni(PipNONO)Cl to reduce cell death signal activation, serum starvation was used to mimic endothelial dysfunction (Monti et al., 2010). The results, reported in Fig. 2B, demonstrated that Ni(PipNONO)Cl induced Akt and ERK1/2 phosphorylation as early as at 5 minutes. pAKT was maximal at 15 minutes and persisted at 60 minutes. Death markers, such as cleaved caspase-3 and p53, were both inhibited after 3 hours (Fig. 2C). Moreover, eNOS activity, which is necessary to prevent endothelial dysfunction, an early event in the development of arteriosclerotic lesions (Schmidt et al., 2008), was evaluated. Western blot analysis for eNOS Ser1177 and Thr495 phosphorylation pattern was run to document eNOS activation (Monti et al., 2010). Ni(PipNONO)Cl (0.1 nM) promoted eNOS Ser1177 phosphorylation (maximal effect at 5 minutes) and, at the same time, eNOS Thr495 dephosphorylation (Fig. 2D), suggesting enzyme functioning.
Thus, these biochemical data corroborate the functional responses of endothelium, which maintains its integrity by stimulation of cell proliferation and survival, through inhibition of apoptosis, and by reducing endothelial dysfunction.
Antiatherogenic Effect of Metal-Nonoates on Vascular Smooth Muscle Cells.
Inhibition of smooth muscle cells growth and migration is a goal of antiatherogenic therapy. Ni(PipNONO)Cl, but not HPipNONO or Ni(Pip)Cl2, dose-dependently reduced the number of smooth muscle cells after 48 hours (Fig. 3A). DETA/NO was less effective than Ni(PipNONO)Cl in reducing the cell number. The inhibitory effect on cell proliferation, induced by Ni(PipNONO)Cl, correlated with an inhibition of Akt and ERK1/2 signaling and induction of apoptosis, evaluated as caspase-3 cleavage and p53 expression (Fig. 3, B and C, respectively).
Cell migration was also analyzed to evaluate the antiatherogenic effect of Ni(PipNONO)Cl. Cell chemotaxis of nonadherent smooth muscle cells was evaluated in HUASMs exposed to Ni(PipNONO)Cl. The metal-nonoate significantly reduced cell migration (Fig. 4A). To confirm that the antimigratory activity was not due to the cytotoxic effect exerted by the compound, a trypan blue exclusion test was performed. After 30 minutes of treatment with Ni(PipNONO)Cl (10 µM), cell death was 3.9% ± 0.08%, a value that was maintained up to 1 hour (4.5% ± 0.07%), demonstrating that the inhibition of migration was not due to cytotoxicity.
To support the antimigratory activity of the compound, immunofluorescence analysis of cytoskeletal components (tubulin, desmin, and β-actin) was performed. Our data revealed a rearrangement of all of the cytoskeletal components analyzed, and the loss of lamellipodia structures associated with cell movement (Fig. 4, B–D, respectively).
To verify the release of NO in HUASMs, the levels of cGMP were measured as the outcome of NO targeting on soluble guanylate cyclase. Indeed, Ni(PipNONO)Cl was able to increase 2-fold the cGMP levels after 15 minutes of incubation (214% ± 9% of basal; P < 0.001).
In Vitro Antithrombotic Effect of Metal-Nonoates.
NONOate compounds were then evaluated for their capacity to inhibit platelet aggregation. Human PRP was stimulated with ADP in the absence/presence of increasing concentrations of the NONOates. The data, reported in Table 2, demonstrate that platelet aggregation by 5 µM ADP was significantly reduced both in terms of shape change (first curve) and release reaction (second curve) by 30 μM HPipNONO, 10 μM Ni(PipNONO)Cl, and 30 μM DETA/NO, thus inducing a complete blocking of irreversible aggregation. Ni(PipNONO)Cl was clearly the most effective among the NO donors tested (P < 0.001).
Ni(PipNONO)Cl Prevents IL-1β–Induced Progression of Molecular and Cellular Events in Atherosclerosis.
Proinflammatory processes initiated in the endothelium represent a crucial step in atherosclerosis progression throughout all stages of the pathology. IL-1β, one of the major cytokines involved in vascular inflammation (Dinarello, 2005), was used as an experimental model to investigate the anti-inflammatory effect of Ni(PipNONO)Cl during atherosclerosis.
IL-1β (100 ng/ml) impaired endothelial cell proliferation by activating the apoptotic cascade, as documented by increased caspase-3 cleavage (Fig. 5). This effect was reverted by the pretreatment (30 minutes) of HUVECs with NO donors. In particular, Ni(PipNONO)Cl (0.1 nM) was more effective to recover cell proliferation than DETA/NO (0.1 nM, 30 minutes) (Fig. 5A), inhibiting caspase-3 activation (Fig. 5B).
The anti-inflammatory effect of Ni(PipNONO)Cl was also confirmed analyzing the expression of proinflammatory biochemical effectors, such as COX-2 and iNOS (Fig. 6, A and B). Ni(PipNONO)Cl (0.1 nM) inhibited the expression of both COX-2 and iNOS when cells were stimulated with IL-1β (100 ng/ml, 6 or 18 hours, respectively).
Cell permeability was studied as a functional parameter of the inflammatory process. IL-1β–induced cell permeability was inhibited by Ni(PipNONO)Cl (Fig. 6C).
Endothelial CD40 expression is upregulated in areas of inflammation and is considered as a marker of inflammation and mononuclear cell recruitment during atherosclerosis (Zirlik et al., 2007; Urban et al., 2011). IL-1β (100 ng/ml, 6 hours) promoted CD40 expression in HUVECs, whereas the pretreatment with Ni(PipNONO)Cl (0.1 nM, 30 minutes) protected cells from the upregulation of this inflammatory marker (Fig. 7). Moreover, the metal-nonoate inhibited the expression of CD31, a platelet adhesion marker, in HUVECs stimulated with IL-1β (Fig. 7B).
Taken together, these data confirm the anti-inflammatory and antiatherosclerotic effects of Ni(PipNONO)Cl, documenting its pharmacologic activity in the relevant stages of the pathologic process.
Endothelial cell dysfunction is now recognized as a characteristic hallmark of different cardiovascular pathologies (Bonetti et al., 2003). The reduced production of the gaseous transmitter NO accounts for vasoconstriction, induction of smooth muscle cell proliferation and migration, platelet aggregation, and in general the acquisition of an inflammatory phenotype that controls monocyte adhesion to and transmigration through the intima (Libby, 2002). Thus, from a therapeutic point of view, the restoration of adequate NO levels has been shown to revert endothelial dysfunction, improving cardiovascular wellness. However, the advances in NO donor drug development have been limited due to tolerance, appearance of side effects, and limited therapeutic improvements with respect to other classes of cardiovascular protective agents (Daiber et al., 2008; Iachini Bellisarii et al., 2012). Thus, there is a need for new generation of NO donor drugs to be used in an extended range of pathologies related to the cardiovascular system.
Metal-nonoates are effective NO donors developed and characterized a few years ago (Ziche et al., 2008). The presence of a metal center, and its likely interaction with the NONOate group, allows the molecules to display characteristic kinetics of NO release, but their efficacy is not simply related to their rate of NO release. For instance, the slow releasing compound Cu(PipNONO)Cl (half-life of approximately 1.5 hours) (Ziche et al., 2008) proved to be less effective in preliminary assays than the Ni derivative and was not further considered in the present cellular studies (data not shown). Ni(PipNONO)Cl proves to be more effective in inducing vasorelaxation compared with the metal free NONOate compound and a commercially available NONOate, DETA/NO, which is one of the most extensively used in the pharmacological assays. When tested on cultured cells, Ni(PipNONO)Cl is able to promote endothelial cell survival and proliferation through the activation of Akt and ERK1/2 signaling pathways. Notably, the maintenance of endothelial functions is achieved at very low concentration (picomolar range) and the effect is preserved for 48 hours with a single dose, showing that the efficacy of the compound extends for times much beyond the rate of in vitro NO release (Ziche et al., 2008). Moreover, endothelial cell functionality is also improved, as demonstrated by increased eNOS phosphorylation at Ser1177 residue and Thr495 dephosphorylation, the signature of eNOS enzyme activation. eNOS has an important role in the maintenance of cardiovascular functions (Förstermann and Sessa, 2012) and its activation strengthens the protective effects induced in the vasculature by Ni(PipNONO)Cl, thus finely controlling the events at the basis of several pathologies such as atherogenesis (Fig. 8).
Among the various cell players involved in the pathogenesis of atherogenesis or postangioplasty restenosis, vascular muscle cell activation is a key event in the formation of the fibrous cap and the neointima (Ross, 1993). Proliferative and migratory properties of smooth muscle cells are regulated by the presence of NO and in particular by its second messenger cGMP (Förstermann and Sessa, 2012). Ni(PipNONO)Cl inhibits smooth muscle cell proliferation and migration, being the two functional responses correlated with increased apoptosis markers (p53 upregulation and cleavage of caspase-3) and inhibition of the phosphorylation of ERK1/2 and Akt, linked to proliferation and migration. Cytoskeletal rearrangements and lamellipodia disappearance are also promoted by treatment with Ni(PipNONO)Cl. As mentioned above, the increase of cGMP is responsible for the inhibition of the smooth muscle cell activation. As expected, Ni(PipNONO)Cl doubled the levels of cGMP, confirming the involvement of the soluble guanylate cyclase pathway as previously demonstrated (Ziche et al., 2008).
With regard to platelet activity, specific concentrations of the metal-nonoate induce a significant block of irreversible aggregation due to stimulation with ADP. Such data are relevant in terms of antithrombotic effects because the reduction of ADP-induced platelet aggregation is now proven to be relevant in clinical studies evaluating the effects of ADP-receptor blockade drugs (i.e., clopidogrel) (Aradi et al., 2013). Indeed, several studies indicate a close relation between cardiovascular outcome and the magnitude of reduction for ADP-induced platelet aggregation during antiplatelet treatment (Parodi et al., 2011). As above, Ni(PipNONO)Cl demonstrates to be the most effective compound, reinforcing its vascular protective property.
Proinflammatory cytokines, such as IL-1β, commonly found during vascular dysfunction, may induce the activation of chemotactic factors, other cytokines, and cell adhesion molecules, all of which contribute to the inflammatory process (Ross, 1993). Interestingly, the protective effects of Ni(PipNONO)Cl extend to reducing endothelial cell expression of inflammatory hallmarks activated by IL-1β, such as the inducible enzymes COX-2 and iNOS, and monocyte and platelet adhesion markers, such as CD40 and CD31. Ni(PipNONO)Cl indeed inhibits the expression and plasma membrane localization of these indicators upregulated in cultured endothelium by IL-1β exposure (Fig. 8).
Collectively, from this ex vivo and in vitro characterization, we can propose the metal-nonoates as a promising approach to develop new drugs active against atherosclerosis and other cardiovascular pathologies characterized by endothelial dysfunction as a pathogenic mechanism, and Ni(PipNONO)Cl in particular as a lead compound for further chemical/pharmaceutical developments. The in vivo confirmation of safety and therapeutic potential is currently under investigation.
The authors thank F. Casella for administrative support.
Participated in research design: Monti, Morbidelli.
Conducted experiments: Monti, Solito, Puccetti, Pasotti.
Performed data analysis: Monti, Solito, Puccetti, Pasotti.
Wrote or contributed to the writing of the manuscript: Roggeri, Monzani, Casella, Morbidelli.
- Received July 28, 2014.
- Accepted September 17, 2014.
This research was supported by the Ministry of Education, University, and Research [Grant DM 593-2000, according to article 11] and Agenzia Provinciale per lo Sviluppo Locale (APLSO, Siena) [(to Noxamet Ltd)]. M.M., L.Pa., and R.R. were Noxamet Ltd fellows.
- bovine serum albumin
- diethylenetriamine/nitric oxide
- endothelial nitric oxide synthase
- extracellular signal-regulated kinase
- human cardiac microvascular endothelial cell
- N-aminoethylpiperazine N-diazeniumdiolate
- human umbilical artery smooth muscle cell
- human umbilical vein endothelial cell
- inducible nitric oxide synthase
- metal complexes containing the N-aminoethylpiperazine N-diazeniumdiolate ligand
- nitric oxide
- N-diazeniumdiolate group
- phosphate-buffered saline
- platelet-rich plasma
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics