Chapter 2 - Biomedical applications of polymeric nitric oxide (NO) donors

N-Nitrosamines are well established motifs to release nitric oxide (NO) under photoirradiation. Herein, a series of amphiphilic N-nitrosamine-based block copolymers (BCPx-NO) are developed to attain controlled NO release under photoirradiation (365 nm, 3.71 mW/cm2). The water-soluble BCPx-NO forms micellar architecture in aqueous medium and exhibits a sustained NO release of 92-160 μM within 11.5 h, which is 36.8-64.0% of the calculated value. To understand the NO release mechanism, a small molecular NO donor (NOD) resembling the NO releasing functional motif of BCPx-NO is synthesized, which displays a burst NO release in DMSO within 2.5 h. The radical nature of the released NO is confirmed by electron paramagnetic resonance (EPR) spectroscopy. The gradual NO release from micellar BCPx-NO enhances antibacterial activity over NOD and exhibits a superior bactericidal effect on Gram-positive Staphylococcus aureus. In relation to biomedical applications, this work offers a comprehensive insight into tuning light-triggered NO release to improve antibacterial activity.

Nitric Oxide

Nitric oxide: physiology and pharmacology.

S-nitrosothiols have been implicated as intermediary transducers of nitric oxide bioactivity; however, the mechanisms by which these compounds affect cellular functions have not been fully established. In this study, we have examined the effect of S-nitrosothiol transport on intracellular thiol status and upon the activity of a target protein (caspase-3), in bovine aortic endothelial cells. We have previously demonstrated that the specific transport of amino acid-based S-nitrosothiols (S-nitroso-L-cysteine and S-nitrosohomocysteine) occurs via amino acid transport system L to generate high levels of intracellular protein S-nitrosothiols (Zhang, Y., and Hogg, N. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 7891-7896). In this study, we demonstrate that the transport of S-nitrosothiols is essential for these compounds to affect intracellular thiol levels and to modify intracellular protein activity. Importantly, the ability of these compounds to affect intracellular processes occurs independently of nitric oxide formation. These findings suggest that the major action of these compounds is not to liberate nitric oxide in the extracellular space but to be specifically transported into cells where they are able to modify cellular functions through nitric oxide-independent mechanisms.

The effect of aspirin and two nitric oxide donors on the infarcted heart in situ

RHAMM (Receptor for Hyaluronan‐Mediated Motility) is necessary for endothelial cell (EC) migration in vitro and angiogenesis in vivo, but the mechanisms are incompletely understood. Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) involves a signaling cascade through Src kinase and Akt, and results in eNOS phosphorylation. A number of diverse agonists, including VEGF, HDL, estradiol, acetylcholine and insulin, activate eNOS to produce NO. We hypothesized that RHAMM is necessary for the activation of Src kinase and eNOS to produce NO. To determine the molecular mechanism by which RHAMM regulates eNOS activation, we used cultured EC and RHAMM KO mice.RHAMM siRNA knockdown in HUVEC, BAEC or HPAEC inhibited the increased cell migration after single scratch wounding and eNOS activity observed with treatment with all EC agonists, and the exogenous NO donor NONOate rescued the migratory defect, suggesting that RHAMM is universally required for eNOS activation and NO production. RHAMM siRNA‐mediated decrease in cell migration and eNOS activity correlated with decreased Akt and eNOS phosphorylation but not Erk phosphorylation. In RHAMM KO mice, normal alveolarization and re‐endothelialization after arterial injury, processes that require NO, were delayed. RHAMM KO mice also did not respond to exogenous VEGF, but treatment with either inhaled NO (iNO) or Molsidomine normalized lung alveolarization and restored re‐endothelialization after arterial injury respectively.Since activation of Src is a proximal step in the signaling cascade, we examined the effect of RHAMM knockdown on Src activation. RHAMM siRNA treatment of EC was associated with hyperphosphorylation of the inhibitory site of Src (Y527) and an inhibition of the phosphorylation of the activation site of Src (Y416). In this process, the phosphatase Shp2 is critical in the dephosphorylation of Y527 and Src activation, and associates with VEGFR2. Interestingly, Shp2 and Src both co‐immunoprecipitated with RHAMM in vivo as well as in vitro. Compared to EC treated with scrambled siRNA, co‐immunoprecipitation of Shp2 and VEGFR2 after VEGF stimulation was significantly reduced in RHAMM siRNA‐treated EC. These data suggest that the RHAMM‐Shp2 complex facilitates Shp2 tethering to VEGFR2, which is necessary for the dephosphorylation of Y527 in Src and the downstream activation of the signaling pathway for NO production in endothelial cells. We speculate that RHAMM is a novel target for the regulation of the wide array of physiologic and pathological functions that endothelial NO regulates.Support or Funding InformationNIH: HL093535, HL079090, HL075930; Children's Medical Center Foundation; William Buchanan Chair in Pediatrics.

Four nitric oxide (NO) donors, S-nitrosoglutathione (GSNO), S-nitrosocysteine (CySNO), S-nitroso-N-acetylcysteine (SNAC), and 2-(2-S-nitroso propionamide) acetic acid (GAS) were prepared and their physicochemical characteristics were analyzed. Besides, the antibacterial properties of NO donors were investigated against Escherichia coli and Staphylococcus aureus. UV-visible absorption spectrum and Fourier transform infrared spectrum verified the successful preparation of RSNOs. All NO donors (10mmol l-1) could release NO continuously, and the amount of NO release was from 80.22 μmol l-1 to 706.63 μmol l-1, in which the release of NO from SNAC was the highest, and the release of NO from NaNO2 was the least. The inhibition zone indicated that all NO donors showed stronger antibacterial activity against E. coli and S. aureus, and the antibacterial ability was in the order of SNAC>GSNO>CySNO>GAS>NaNO2 for both E. coli and S. aureus (P<0.05). Scanning electron microscopy(SEM) showed that all NO donors could result in varying degrees of damage to cell wall and membrane of both E. coli and S. aureus and the damage of E. coli was more severe. Four alternative NO donors were successfully synthesized. All alternative NO donors showed better antibacterial properties against E. coli and S. aureus than NaNO2.

A Photo-triggered and photo-calibrated nitric oxide donor: Rational design, spectral characterizations, and biological applications

Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that beta-actin is associated with eNOS oxygenase domain and that association of eNOS with beta-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that beta-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326-333 of human eNOS, one of the putative ABSs, specifically bound to beta-actin and prevented eNOS association with beta-actin in vitro. Peptide 326 also prevented beta-actin-induced decrease in superoxide formation and increase in NO and L-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-beta-actin binding and to prevent beta-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-beta-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-beta-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that beta-actin binding to eNOS through residues 326-333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by beta-actin plays an important role in endothelial function.

Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi (T. cruzi), and the disease remains a major health problem in many Latin American countries. Several papers report that the killing of the parasite is dependent on the production of nitric oxide (NO). The endogenous free radical NO is an important cellular signalling molecule that plays a key role in the defense against pathogens, including T. cruzi. As T. cruzi is able to compromise host macrophages decreasing endogenous NO production, the administration of exogenous NO donors represents an interesting strategy to combat Chagas disease. Thus, the aims of this study were to prepare and evaluate the antimicrobial activity of NO-releasing polymeric nanoparticles against T. cruzi. Biocompatible polymeric nanoparticles composed of chitosan/sodium tripolyphosphate(TPP) were prepared and used to encapsulate mercaptosuccinic acid (MSA), which is a thiol-containing molecule. Nitrosation of free thiols (SH) groups of MSA were performed by the addition of equimolar amount of sodium nitrite (NaNO2), leading to the formation of S-nitroso-MSA-containing nanoparticles. These polymeric nanoparticles act as spontaneous NO donors, with free NO release. The results show the formation of nanoparticles with average hydrodynamic diameter ranging from 270 to 500 nm, average of polydispersity index of 0.35, and encapsulation efficiency in the range of 99%. The NO release kinetics from the S-nitroso-MSA-containing nanoparticles showed sustained and controlled NO release over several hours. The microbicidal activity of S-nitroso-MSA-containing nanoparticles was evaluated by incubating NO-releasing nanoparticles (200 - 600 μg/mL) with replicative and non-infective epimastigote, and non-replicative and infective trypomastigote forms of T. cruzi. In addition, a significant decrease in the percentage of macrophage-infected (with amastigotes) and NO-releasing nanoparticle-treated cells was observed. Taken together, our results reveal a potent toxic effect of NO-releasing polymeric nanoparticles against different life cycle forms of T. cruzi, indicating that the encapsulation of the NO donor S-nitroso-MSA represents an interesting approach to combat and to prevent Chagas disease.

The rapid decomposition of nitric oxide (NO) donors in aqueous environments remains a limitation for applications requiring extended NO release. Herein, we report the synthesis of dipalmitoylphosphatidylcholine-based liposomes capable of extended NO release using low molecular weight NO donors and a reverse-phase evaporation technique. The encapsulation of the NO donors within the liposomes enabled both prolonged NO release and enhanced storage compared to free NO donors alone. The NO-releasing liposomes also demonstrated enhanced efficacy against human pancreatic cancer cells. These NO-release vehicles represent attractive anticancer therapeutics due to their potential to store the majority of their NO payload until reaching cancerous tissue at which time the lower pH inherent to such environments will trigger an avalanche of NO.

In the vasculature it is well established that cGMP is involved in the relaxant response to nitric oxide (NO) and NO donors. However, there is an increasing evidence that alternative/additional pathways that are cGMP-independent may also exist. A key criterion for a response to NO or a NO donor drug to be classified as cGMP-independent is lack of (or incomplete) inhibition by the selective inhibitor of soluble guanylate cyclase, ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one). In many blood vessels cGMP-independent mechanisms contribute to the vasorelaxation, and in certain vascular beds cGMP-independent relaxation may be the predominant mechanism of action of NO and NO donors. NO donor drugs that generate NO "spontaneously", like authentic NO (i.e. solutions of NO gas), appear to exhibit a larger component of cGMP-independent vasorelaxation than do those drugs that require bioactivation in the tissue. The long lasting inhibition of responses to vasoconstrictors by S-nitrosothiols, persisting after removal of these NO donors, may be a cGMP-independent process, at least in some vessels. The mechanisms involved in the inhibition of vascular growth by NO and NO donors are predominantly cGMP-independent, as are the mechanisms responsible for the effects of NO donors on apoptosis in vascular smooth muscle and endothelial cells. The ability of NO and NO donors to inhibit platelet aggregation has a significant cGMP-independent component. cGMP-independent pathways are most often, though not exclusively, seen at high concentrations (microM - mM) of NO and NO donors. Hence, in relation to the actions of endogenous NO, these pathways may be particularly important in settings when the inducible isoform of NO-synthase is expressed. Furthermore, cGMP-independent pathways are enhanced in animal models of atherosclerosis and ischaemia. This suggests that it may be possible to target cGMP-independent mechanisms with selected NO donors in disease states.

A new oxidative pathway of nitric oxide production from oximes in plants

Previous studies in our laboratory have shown that mixed lineage kinase 3 (MLK3) can be activated following global ischemia. In addition, other laboratories have reported that the activation of MLK3 may be linked to the accumulation of free radicals. However, the mechanism of MLK3 activation remains incompletely understood. We report here that MLK3, overexpressed in HEK293 cells, is S-nitrosylated (forming SNO-MLK3) via a reaction with S-nitrosoglutathione, an exogenous nitric oxide (NO) donor, at one critical cysteine residue (Cys-688). We further show that the S-nitrosylation of MLK3 contributes to its dimerization and activation. We also investigated whether the activation of MLK3 is associated with S-nitrosylation following rat brain ischemia/reperfusion. Our results show that the administration of 7-nitroindazole, an inhibitor of neuronal NO synthase (nNOS), or nNOS antisense oligodeoxynucleotides diminished the S-nitrosylation of MLK3 and inhibited its activation induced by cerebral ischemia/reperfusion. In contrast, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (an inhibitor of inducible NO synthase) or nNOS missense oligodeoxynucleotides did not affect the S-nitrosylation of MLK3. In addition, treatment with sodium nitroprusside (an exogenous NO donor) and S-nitrosoglutathione or MK801, an antagonist of the N-methyl-D-aspartate receptor, also diminished the S-nitrosylation and activation of MLK3 induced by cerebral ischemia/reperfusion. The activation of MLK3 facilitated its downstream protein kinase kinase 4/7 (MKK4/7)-JNK signaling module and both nuclear and non-nuclear apoptosis pathways. These data suggest that the activation of MLK3 during the early stages of ischemia/reperfusion is modulated by S-nitrosylation and provides a potential new approach for stroke therapy whereby the post-translational modification machinery is targeted.

Nitric oxide releasing poly(vinylidene fluoride-co-hexafluoropropylene) films using a fluorinated nitric oxide donor to greatly decrease chemical leaching