NIR-activated electrospun nanodetonator dressing enhances infected diabetic wound healing with combined photothermal and nitric oxide-based gas therapy.

Diabetic wounds pose a challenge to healing due to increased bacterial susceptibility and poor vascularization. Effective healing requires simultaneous bacterial and biofilm elimination and angiogenesis stimulation. In this study, we incorporated polyaniline (PANI) and S-Nitrosoglutathione (GSNO) into a polyvinyl alcohol, chitosan, and hydroxypropyltrimethyl ammonium chloride chitosan (PVA/CS/HTCC) matrix, creating a versatile wound dressing membrane through electrospinning. The dressing combines the advantages of photothermal antibacterial therapy and nitric oxide gas therapy, exhibiting enduring and effective bactericidal activity and biofilm disruption against methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Escherichia coli. Furthermore, the membrane's PTT effect and NO release exhibit significant synergistic activation, enabling a nanodetonator-like burst release of NO through NIR irradiation to disintegrate biofilms. Importantly, the nanofiber sustained a uniform release of nitric oxide, thereby catalyzing angiogenesis and advancing cellular migration. Ultimately, the employment of this membrane dressing culminated in the efficacious amelioration of diabetic-infected wounds in Sprague-Dawley rats, achieving wound closure within a concise duration of 14 days. Upon applying NIR irradiation to the PVA-CS-HTCC-PANI-GSNO nanofiber membrane, it swiftly eradicates bacteria and biofilm within 5 min, enhancing its inherent antibacterial and anti-biofilm properties through the powerful synergistic action of PTT and NO therapy. It also promotes angiogenesis, exhibits excellent biocompatibility, and is easy to use, highlighting its potential in treating diabetic wounds.

Nitrosative stress under microaerobic conditions triggers inositol metabolism in Pseudomonas extremaustralis.

Bacteria are exposed to reactive oxygen and nitrogen species that provoke oxidative and nitrosative stress which can lead to macromolecule damage. Coping with stress conditions involves the adjustment of cellular responses, which helps to address metabolic challenges. In this study, we performed a global transcriptomic analysis of the response of Pseudomonas extremaustralis to nitrosative stress, induced by S-nitrosoglutathione (GSNO), a nitric oxide donor, under microaerobic conditions. The analysis revealed the upregulation of genes associated with inositol catabolism; a compound widely distributed in nature whose metabolism in bacteria has aroused interest. The RNAseq data also showed heightened expression of genes involved in essential cellular processes like transcription, translation, amino acid transport and biosynthesis, as well as in stress resistance including iron-dependent superoxide dismutase, alkyl hydroperoxide reductase, thioredoxin, and glutathione S-transferase in response to GSNO. Furthermore, GSNO exposure differentially affected the expression of genes encoding nitrosylation target proteins, encompassing metalloproteins and proteins with free cysteine and /or tyrosine residues. Notably, genes associated with iron metabolism, such as pyoverdine synthesis and iron transporter genes, showed activation in the presence of GSNO, likely as response to enhanced protein turnover. Physiological assays demonstrated that P. extremaustralis can utilize inositol proficiently under both aerobic and microaerobic conditions, achieving growth comparable to glucose-supplemented cultures. Moreover, supplementing the culture medium with inositol enhances the stress tolerance of P. extremaustralis against combined oxidative-nitrosative stress. Concordant with the heightened expression of pyoverdine genes under nitrosative stress, elevated pyoverdine production was observed when myo-inositol was added to the culture medium. These findings highlight the influence of nitrosative stress on proteins susceptible to nitrosylation and iron metabolism. Furthermore, the activation of myo-inositol catabolism emerges as a protective mechanism against nitrosative stress, shedding light on this pathway in bacterial systems, and holding significance in the adaptation to unfavorable conditions.

Targeted delivery of NO donor and ROS scavenger for synergistic treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by high level of reactive oxygen species (ROS) and proinflammatory cytokines, which facilitate the activation of the inflammatory signaling such as NF-κB pathway and exacerbate the development of inflammation. Herein, we designed a nanodrug by encapsulating the NO donor S-nitrosoglutathione (GSNO) into an emulsion and coating the surface with a polydopamine (PDA) layer to yield GSNO@PDA, which simultaneously scavenged the extra ROS and suppressed NF-κB signaling for potent RA treatment. To enhance the cellular uptake and NO generation efficiency, dextran sulfate (DS) and Cu2+ were anchored on the surface of GSNO@PDA to obtain the final formulation GSNO@PDA@DS. Our results demonstrated that GSNO@PDA@DS were successfully prepared and the modification of DS effectively boosted the cellular uptake of GSNO@PDA@DS. Moreover, GSNO@PDA@DS lowered cellular ROS and elevated intracellular NO, resulting in a decrease of M1 phenotype, inhibition of NF-κB pathway and down-regulation of proinflammatory cytokine tumor necrosis factor-α (TNF-α). Further in vivo studies confirmed that GSNO@PDA@DS significantly relieved symptoms and bone erosion by regulating the microenvironment of RA, highlighting the potential of GSNO@PDA@DS for RA therapy through ROS scavenging and NO-mediated suppression of inflammatory signaling.

Moderate modulation by S-nitrosoglutathione of photorespiratory enzymes in pea (Pisum sativum) leaves, compared to the strong effects of high light.

When plants are exposed to water stress, photosynthesis is downregulated due to enhanced reactive oxygen species (ROS) and nitric oxide (NO). In contrast, photorespiratory metabolism protected photosynthesis and sustained yield. Modulation of photorespiration by ROS was established, but the effect of NO on photorespiratory metabolism was unclear. We, therefore, examined the impact of externally added NO by using S-nitrosoglutathione (GSNO), a natural NO donor, in leaf discs of pea (Pisum sativum) under dark or light: moderate or high light (HL). Maximum NO accumulation with GSNO was under high light. The presence of 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, prevented the increase in NO, confirming the release of NO in leaves. The increase in S-nitrosothiols and tyrosine-nitrated proteins on exposure to GSNO confirmed the nitrosative stress in leaves. However, the changes by GSNO in the activities and transcripts of five photorespiratory enzymes: glycolate oxidase, hydroxypyruvate reductase, catalase, glycerate kinase, and phosphoglycolate phosphatase activities were marginal. The changes in photorespiratory enzymes caused by GSNO were much less than those with HL. Since GSNO caused only mild oxidative stress, we felt that the key modulator of photorespiration might be ROS, but not NO.

Candidate molecules as alternative nitric oxide donors with better antibacterial property against Escherichia coli and Staphylococcus aureus.

AIMS: 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. METHODS AND RESULTS: UV-visible absorption spectrum and Fourier transform infrared spectrum verified the successful preparation of RSNOs. All NO donors (10 mmol 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. CONCLUSION: Four alternative NO donors were successfully synthesized. All alternative NO donors showed better antibacterial properties against E. coli and S. aureus than NaNO2.

Synergistic wound repair effects of a composite hydrogel for delivering tumor-derived vesicles and S-nitrosoglutathione.

Treating chronic wounds requires transition from proinflammatory M1 to anti-inflammatory M2 dominant macrophages. Based on the role of tumor extracellular vesicles (tEVs) in regulating the phenotypic switching from M1 to M2 macrophages, we propose that tEVs may have a beneficial impact on alleviating the overactive inflammatory microenvironment associated with refractory wounds. On the other hand, as a nitric oxide donor, S-nitrosoglutathione (GSNO) can regulate inflammation, promote angiogenesis, enhance matrix deposition, and facilitate wound healing. In this study, a guar gum-based hydrogel with tEVs and GSNO was designed for the treatment of diabetic refractory wounds. This hybrid hydrogel was formed through the phenyl borate bonds, which can automatically disintegrate in response to the high reactive oxygen species (ROS) level at the site of refractory diabetic wounds, releasing tEVs and GSNO. We conducted a comprehensive evaluation of this hydrogel in vitro, which demonstrated excellent performance. Meanwhile, using a full-thickness excision model in diabetic mice, the wounds exposed to the therapeutic hydrogel healed completely within 21 days. The increased closure rate was associated with macrophage polarization and collagen deposition, accelerated fibroblast proliferation, and increased angiogenesis in the regenerating tissues. Therefore, this multifunctional hybrid hydrogel appears to be promising for clinical applications.

Nitric oxide alleviates salt stress through protein S-nitrosylation and transcriptional regulation in tomato seedlings.

MAIN CONCLUSION: NO enhances the resistance of tomato seedlings to salt stress through protein S-nitrosylation and transcriptional regulation, which involves the regulation of MAPK signaling and carbohydrate metabolism. Nitric oxide (NO) regulates various physiological and biochemical processes and stress responses in plants. We found that S-nitrosoglutathione (GSNO) treatment significantly promoted the growth of tomato seedling under NaCl stress, indicating that NO plays a positive role in salt stress resistance. Moreover, GSNO pretreatment resulted in an increase of endogenous NO level, S-nitrosothiol (SNO) content, S-nitrosoglutathione reductase (GSNOR) activity and GSNOR expression under salt stress, implicating that S-nitrosylation might be involved in NO-alleviating salt stress. To further explore whether S-nitrosylation is a key molecular mechanism of NO-alleviating salt stress, the biotin-switch technique and liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) were conducted. A total of 1054 putative S-nitrosylated proteins have been identified, which were mainly enriched in chloroplast, cytoplasm and mitochondrion. Among them, 15 and 22 S-nitrosylated proteins were involved in mitogen-activated protein kinase (MAPK) signal transduction and carbohydrate metabolism, respectively. In MAPK signaling, various S-nitrosylated proteins, SAM1, SAM3, SAM, PP2C and SnRK, were down-regulated and MAPK, MAPKK and MAPKK5 were up-regulated at the transcriptional level by GSNO treatment under salt stress compared to NaCl treatment alone. The GSNO pretreatment could reduce ethylene production and ABA content under NaCl stress. In addition, the activities of enzyme identified in carbohydrate metabolism, their expression at the transcriptional level and the metabolite content were up-regulated by GSNO supplication under salt stress, resulting in the activation of glycolysis and tricarboxylic acid cycle (TCA) cycles. Thus, these results demonstrated that NO might beneficially regulate MAPK signaling at transcriptional levels and activate carbohydrate metabolism at the post-translational and transcriptional level, protecting seedlings from energy deficiency and salinity, thereby alleviating salt stress-induced damage in tomato seedlings. It provides initial insights into the regulatory mechanisms of NO in response to salt stress.

Effect of Nitric Oxide on the Functioning of the P-Glycoprotein Transporter.

We studied the effect of nitric oxide (NO) on the functioning of P-glycoprotein transporter (Pgp) in Caco-2 cells. NO donor S-nitrosoglutathione (GSNO) was used in concentrations of 1, 10, 50, 100, and 500 µM; the duration of exposure was 24 h. The content of Pgp was analyzed by the Western blotting, activity of the transport protein was analyzed by the transport of its substrate fexofenadine. It was shown that GSNO in concentrations of 10 and 50 µM increased the content and activity of Pgp. Increasing the GSNO concentration to 500 µM led to the development of nitrosative stress and a decrease in the content and activity of the transporter protein.

Mechanisms of Regulation of the P-Glycoprotein Transporter Protein Functioning under the Action of Nitric Oxide.

Mechanisms of regulation of the P-glycoprotein (Pgp) transporter under the action of nitric oxide (NO) were studied in Caco-2 cells. S-Nitrosoglutathione (GSNO) was used as a NO donor, which was added to the cells at concentrations 1, 10, 50, 100, and 500 µM and incubated for 3, 24, or 72 h. The amount of Pgp was analyzed using Western blotting, activity was determined by monitoring transport of its substrate, fexofenadine. The study showed that a short-term exposure to GSNO for 3 h at 500 µM concentration caused increase in the concentration of peroxynitrite in Caco-2 cells, which reduced the activity, but not the amount of Pgp. Increase in the duration of exposure to 24 h increased the amount and activity of Pgp at GSNO concentrations of 10 and 50 µM, increased the amount without increasing activity at 100 µM concentration, and decreased the amount of the transporter protein at 500 µM. Duration of exposure to GSNO of 72 h at concentration of 10 µM resulted in the increase of the amount and activity of Pgp, while at concentration of 100 and 500 µM it decreased the amount of the transport protein. At the same time, it was shown using specific inhibitors that the increase in the amount of Pgp under the influence of low concentrations of GSNO was realized through the NO-cGMP signaling pathway, and the effect of the higher concentration of GSNO and the respective development of nitrosative stress was realized through Nrf2 and the constitutive androstane receptor.

Enhancing S-nitrosoglutathione reductase decreases S-nitrosylation of Drp1 and reduces neuronal apoptosis in experimental subarachnoid hemorrhage both in vivo and in vitro.

Subarachnoid hemorrhage (SAH) is a hemorrhagic stroke with a high mortality and disability rate. Nitric oxide (NO) can promote blood supply through vasodilation, leading to protein S-nitrosylation. However, the function of S-nitrosylation in neurons after SAH remains unclear. Excessive NO in the pathological state is converted into S-nitrosoglutathione (GSNO) and stored in cells, which leads to high S-nitrosylation of intracellular proteins and causes nitrosative stress. S-nitrosoglutathione reductase (GSNOR) promotes GSNO degradation and protects cells from excessive S-nitrosylation. We conducted an in vivo rat carotid puncture model and an in vitro neuron hemoglobin intervention. The results showed that SAH induction increased NO, GSNO, neuron protein S-nitrosylation, and neuronal apoptosis, while decreasing the level and activity of GSNOR. GSNOR overexpression by lentivirus decreased GSNO but had little effect on NO. GSNOR overexpression also improved short- and long-term neurobehavioral outcomes in rats and alleviated nitrosative stress. Furthermore, GSNOR reduced neuronal apoptosis and played a neuroprotective role by alleviating Drp1 S-nitrosylation, reducing mitochondrial division. Thus, the regulation of GSNOR in early brain injury and neuronal denitrosylation may play an important role in neuroprotection.

S-nitrosoglutathione alleviates hyperglycemia-induced neurobehavioral deficits involving nitro-oxidative stress and aberrant monaminergic system.

The present study evaluated the protective role of S-nitrosoglutathione (GSNO) in preventing hyperglycemia-induced nitro-oxidative stress and alterations in monoaminergic system associated with neurobehavioral deficits in mice. Mice were subjected to diabetes by intraperitoneal injection of streptozotocin (40 mg/kg body weight) for 5 days, whereas GSNO (100 µg/kg body weight) was administered daily via oral route for 8 weeks. Diabetic mice showed deficits in neurobehavioral functions associated with memory, learning, anxiety and motor coordination. These neurobehavioral deficits observed in diabetic mice may be attributed to decrease in norepinephrine (NE), dopamine (DA), serotonin (5-HT) and increased monoamine oxidase (MAO) activity in cortex and hippocampus. Further, a significant increase in reactive oxygen species (ROS), protein carbonyls, nitrotyrosine (NT) and lipid peroxidation were observed in brain regions of diabetic animals suggesting increased nitro-oxidative stress. Hyperglycemia induced nitro-oxidative stress appears to involve reduction in redox ratio (GSH/GSSG) and enzymatic antioxidants; catalase (CAT) and superoxide dismutase (SOD) in cortex and hippocampus. However, GSNO supplementation was able to ameliorate alterations in monoaminergic system and nitro-oxidative stress in the brain regions thereby restoring neurobehavioural functions. These findings suggest GSNO as potential therapeutic molecule to prevent diabetic encephalopathy.

Improved Polymer Hemocompatibility for Blood-Contacting Applications via S-Nitrosoglutathione Impregnation.

Blood-contacting medical devices (BCMDs) are inevitably challenged by thrombi formation, leading to occlusion of flow and device failure. Ideal BCMDs seek to mimic the intrinsic antithrombotic properties of the human vasculature to locally prevent thrombotic complications, negating the need for systemic anticoagulation. An emerging category of BCMD technology utilizes nitric oxide (NO) as a hemocompatible agent, as the vasculature's endothelial layer naturally releases NO to inhibit platelet activation and consumption. In this paper, we report for the first time the novel impregnation of S-nitrosoglutathione (GSNO) into polymeric poly(vinyl chloride) (PVC) tubing via an optimized solvent-swelling method. Material testing revealed an optimized GSNO-PVC material that had adequate GSNO loading to achieve NO flux values within the physiological endothelial NO flux range for a 4 h period. Through in vitro hemocompatibility testing, the optimized material was deemed nonhemolytic (hemolytic index <2%) and capable of reducing platelet activation, suggesting that the material is suitable for contact with whole blood. Furthermore, an in vivo 4 h extracorporeal circulation (ECC) rabbit thrombogenicity model confirmed the blood biocompatibility of the optimized GSNO-PVC. Platelet count remained near 100% for the novel GSNO-impregnated PVC loops (1 h, 91.08 ± 6.27%; 2 h, 95.68 ± 0.61%; 3 h, 97.56 ± 8.59%; 4 h, 95.11 ± 8.30%). In contrast, unmodified PVC ECC loops occluded shortly after the 2 h time point and viable platelet counts quickly diminished (1 h, 85.67 ± 12.62%; 2 h, 54.46 ± 10.53%; 3 h, n/a; 4 h, n/a). The blood clots for GSNO-PVC loops (190.73 ± 72.46 mg) compared to those of unmodified PVC loops (866.50 ± 197.98 mg) were significantly smaller (p < 0.01). The results presented in this paper recommend further investigation in long-term animal models and suggest that GSNO-PVC has the potential to serve as an alternative to systemic anticoagulation in BCMD applications.

S-Nitrosoglutathione Limits Apoptosis and Reduces Pulmonary Vascular Dysfunction After Bypass.

BACKGROUND: During hypoxia or acidosis, S-nitrosoglutathione (GSNO) has been shown to protect the cardiomyocyte from ischemia-reperfusion injury. In a randomized double-blinded control study of a porcine model of paediatric cardiopulmonary bypass (CPB), we aimed to evaluate the effects of 2 different doses (low and high) of GSNO. METHODS: Pigs weighing 15-20 kg were exposed to CPB with 1 hour of aortic cross-clamp. Prior to and during CPB, animals were randomized to receive low-dose (up to 20 nmol/kg/min) GSNO (n = 8), high-dose (up to 60 nmol/kg/min) GSNO (n = 6), or normal saline (n = 7). Standard cardiac intensive care management was continued for 4 hours post-bypass. RESULTS: There was a reduction in myocyte apoptosis after administration of GSNO (P = .04) with no difference between low- and high-dose GSNO. The low-dose GSNO group had lower pulmonary vascular resistance post-CPB (P = .007). Mitochondrial complex I activity normalized to citrate synthase activity was higher after GSNO compared with control (P = .02), with no difference between low- and high-dose GSNO. CONCLUSIONS: In a porcine model of CPB, intravenous administration of GSNO limits myocardial apoptosis through preservation of mitochondrial complex I activity, and improves pulmonary vascular resistance. There appears to be a dose-dependent effect to this protection.

Nitric oxide-targeted protein phosphorylation during human sperm capacitation.

Among many other molecules, nitric oxide insures the correct progress of sperm capacitation by mediating phosphorylation events. For a more comprehensive understanding of how this happens, we capacitated human spermatozoa from healthy men in the presence/absence of S-Nitrosoglutathione, a nitric oxide donor, two nitric oxide synthase inhibitors, NG-Nitro-L-arginine Methyl Ester Hydrochloride and Aminoguanidine Hemisulfate salt and, finally, with/without L-Arginine, the substrate for nitric oxide synthesis, and/or human follicular fluid. When analyzing the phosphorylation of protein kinase A substrates and tyrosine residues, we particularly observed how the inhibition of nitric oxide synthesis affects certain protein bands (~ 110, ~ 87, ~ 75 and ~ 62 kD) by lowering their phosphorylation degree, even when spermatozoa were incubated with L-Arginine and/or follicular fluid. Mass spectrometry analysis identified 29 proteins in these species, related to: spermatogenesis, binding to the zona pellucida, energy and metabolism, stress response, motility and structural organization, signaling and protein turnover. Significant changes in the phosphorylation degree of specific proteins could impair their biological activity and result in severe fertility-related phenotypes. These findings provide a deeper understanding of nitric oxide's role in the capacitation process, and consequently, future studies in infertile patients should determine how nitric oxide mediates phosphorylation events in the species here described.

Pretreatment with S-Nitrosoglutathione Attenuates Septic Acute Kidney Injury in Rats by Inhibiting Inflammation, Oxidation, and Apoptosis.

OBJECTIVE: We aimed to investigate the protective effect of s-nitrosoglutathione (SNG) pretreatment on acute kidney injury (AKI) in septic rats. METHODS: We constructed a rat model of sepsis by cecal ligation and puncture and observed the survival of the rats. We obtained kidney and blood samples from rats, observed the pathological damage to the kidney tissues, and evaluated kidney function and the expression levels of inflammatory factors. We also detected the expression of induced nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the kidneys by immunohistochemistry and evaluated the apoptosis of kidney tubular epithelial cells (KTEC) by TUNEL. RESULTS: Pretreatment with SNG significantly reduced the mortality of septic rats, attenuated kidney pathological damage, and decreased the levels of serum creatinine, plasma neutrophil gelatinase-associated lipocalin, and plasma kidney injury molecule-1. Moreover, SNG pretreatment decreased the levels of TNF-α and IL-1ß in serum and kidney and reduced the expressions of NO, iNOS, PGE2, and COX-2 in the kidneys. Furthermore, pretreatment with SNG significantly reduced the apoptotic rate of KTEC and decreased the levels of caspase-3 and Bax mRNA, but increased the level of Bcl-2 mRNA. CONCLUSION: Pretreatment with SNG has a protective effect on AKI in septic rats, and the specific mechanisms are related to inhibition of inflammation, oxidation, and apoptosis.

S-nitrosoglutathione reductase maintains mitochondrial homeostasis by promoting clearance of damaged mitochondria in porcine preimplantation embryos.

OBJECTIVES: S-nitrosoglutathione reductase (GSNOR), a protein denitrosylase, protects the mitochondria from mitochondrial nitrosative stress. Mammalian preimplantation embryos are mitochondria-rich, but the effects of GSNOR on mitochondrial function in preimplantation embryos are not well-studied. In the present study, we investigate whether GSNOR plays a role in mitochondrial regulation during porcine preimplantation embryo development. MATERIALS AND METHODS: GSNOR dsRNA was employed to knock down the expression of GSNOR, and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), a pan-NOS inhibitor, was used to prevent protein S-nitrosylation. Mitochondrial amount and function in embryo development were assessed by performing immunofluorescence staining, Western blot, fluorescent probe and real-time reverse transcription PCR. RESULTS: GSNOR knock-down significantly impaired blastocyst formation and quality and markedly induced the increase in protein S-nitrosylation. Notably, GSNOR knock-down-induced overproduction of S-nitrosylation caused mitochondrial dysfunction, including mitochondrial membrane potential depolarization, mitochondria-derived reactive oxygen species (ROS) increase and ATP deficiency. Interestingly, GSNOR knock-down-induced total mitochondrial amount increase, but the ratio of active mitochondria reduction, suggesting that the damaged mitochondria were accumulated and mitochondrial clearance was inhibited. In addition, damaged mitochondria produced more ROS, and caused DNA damage and apoptosis. Importantly, supplementation with L-NAME reverses the increase in S-nitrosylation, accumulation of damaged mitochondria, and oxidative stress-induced cell death. Interestingly, autophagy was downregulated after GSNOR knock-down, but reversed by L-NAME treatment. Thus, GSNOR maintains mitochondrial homeostasis by promoting autophagy and the clearing of damaged mitochondria in porcine preimplantation embryos.

S-Nitrosoglutathione-Based Nitric Oxide-Releasing Nanofibers Exhibit Dual Antimicrobial and Antithrombotic Activity for Biomedical Applications.

The novel use of nanofibers as a physical barrier between blood and medical devices has allowed for modifiable, innovative surface coatings on devices ordinarily plagued by thrombosis, delayed healing, and chronic infection. In this study, the nitric oxide (NO) donor S-nitrosoglutathione (GSNO) is blended with the biodegradable polymers polyhydroxybutyrate (PHB) and polylactic acid (PLA) for the fabrication of hemocompatible, antibacterial nanofibers tailored for blood-contacting applications. Stress/strain behavior of different concentrations of PHB and PLA is recorded to optimize the mechanical properties of the nanofibers. Nanofibers incorporated with different concentrations of GSNO (10, 15, 20 wt%) are evaluated based on their NO-releasing kinetics. PLA/PHB + 20 wt% GSNO nanofibers display the greatest NO release over 72 h (0.4-1.5 × 10-10  mol mg-1 min-1 ). NO-releasing fibers successfully reduce viable adhered bacterial counts by ≈80% after 24 h of exposure to Staphylococcus aureus. NO-releasing nanofibers exposed to porcine plasma reduce platelet adhesion by 64.6% compared to control nanofibers. The nanofibers are found noncytotoxic (>95% viability) toward NIH/3T3 mouse fibroblasts, and 4',6-diamidino-2-phenylindole and phalloidin staining shows that fibroblasts cultured on NO-releasing fibers have improved cellular adhesion and functionality. Therefore, these novel NO-releasing nanofibers provide a safe antimicrobial and hemocompatible coating for blood-contacting medical devices.

Characterization of Rat Cardiovascular System by Anacrotic/Dicrotic Notches in the Condition of Increase/Decrease of NO Bioavailability.

We characterized modes of action of NO-donor S-nitrosoglutathione (GSNO) and NO-synthase inhibitor l-NAME derived from dicrotic (DiN) and anacrotic (AnN) notches of rat arterial pulse waveform (APW) in the condition of increased/decreased NO bioavailability. The cross-relationship patterns of DiN and AnN with 34 hemodynamic parameters (HPs) induced by GSNO and l-NAME are presented. After GSNO bolus administration, approximate non-hysteresis relationships were observed in the difference between DiN-AnN (mmHg) blood pressure (BP) and other 19 HPs, suggesting that these HPs, i.e., their signaling pathways, responding to NO concentration, are directly connected. Hysteresis relationships were observed between DiN-AnN (mmHg) and other 14 HPs, suggesting that signaling pathways of these HPs are indirectly connected. The hysteresis relationships were only observed between the time interval DiN-AnN (ms) and other 34 HPs, indicating no direct connection of signaling pathways. The cross-relationship patterns of DiN-AnN (mmHg), but not DiN-AnN (ms), induced by l-NAME were in accordance to the increased NO bioavailability induced by GSNO. In conclusion, we found the non-hysteresis/hysteresis cross-relationship "patterns" of DiN-AnN intervals to other HPs in the presence of GSNO that revealed their direct or indirect signaling pathways connections. This may contribute to our understanding of biological effects of natural substances that modulate NO production and/or NO signaling pathways.

Challenging development of storable particles for oral delivery of a physiological nitric oxide donor.

Nitric oxide (NO) deficiency is often associated with several acute and chronic diseases. NO donors and especially S-nitrosothiols such as S-nitrosoglutathione (GSNO) have been identified as promising therapeutic agents. Although their permeability through the intestinal barrier have recently be proved, suitable drug delivery systems have to be designed for their oral administration. This is especially challenging due to the physico-chemical features of these drugs: high hydrophilicity and high lability. In this paper, three types of particles were prepared with an Eudragit® polymer: nanoparticles and microparticles obtained with a water-in-oil-in-water emulsion/evaporation process versus microparticles obtained with a solid-in-oil-in-water emulsion/evaporation process. They had a similar encapsulation efficiency (around 30%), and could be freeze-dried then be stored at least one month without modification of their critical attributes (size and GSNO content). However, microparticles had a slightly slower in vitro release of GSNO than nanoparticles, and were able to boost by a factor of two the drug intestinal permeability (Caco-2 model). Altogether, this study brings new data about GSNO intestinal permeability and three ready-to-use formulations suitable for further preclinical studies with oral administration.

Wound healing action of nitric oxide-releasing self-expandable collagen sponge.

Mounting evidence showing that local nitric oxide (NO) delivery may significantly improve the wound healing process has stimulated the development of wound dressings capable of releasing NO topically. Herein, we describe the preparation of a self-expandable NO-releasing hydrolyzed collagen sponge (CS), charged with the endogenously found NO donor, S-nitrosoglutathione (GSNO). We show that cold pressed and GSNO-charged CS (CS/GSNO) undergo self-expansion to its original 3D shape upon water absorption to a swelling degree of 2,300 wt%, triggering the release of free NO. Topical application of compressed CS/GSNO on wounds in an animal model showed that exudate absorption by CS/GSNO leads to the release of higher NO doses during the inflammatory phase and progressively lower NO doses at later stages of the healing process. Moreover, treated animals showed significant increase in the mRNA expression levels of monocyte chemoattractant protein-1 (MCP-1), murine macrophage marker (F4/80), transforming growth factor beta (TGF-ß), stromal cell-derived factor 1 (SDF-1), insulin-like growth factor-1 (IGF-1), nitric oxide synthase(iNOS), and matrix metalloproteinase(MMP-9). Cluster differentiation 31 (CD31), vascular endothelial growth factor (VEGF), and F4/80 were measured on Days 7 and 12 by immunohistochemistry in the cicatricial tissue. These results indicate that the topical delivery of NO enhances the migration and infiltration of leucocytes, macrophages, and keratinocytes to the wounded tissue, as well as the neovascularization and collagen deposition, which are correlated with an accelerated wound closure. Thus, self-expandable CS/GSNO may represent a novel biocompatible and active wound dress for the topical delivery of NO on wounds.