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.

Tracheomalacia in bronchopulmonary dysplasia: Trachealis hyper-relaxant responses to S-nitrosoglutathione in a hyperoxic murine model.

BACKGROUND: Bronchopulmonary dysplasia (BPD) with airway hyperreactivity is a long-term pulmonary complication of prematurity. The endogenous nonadrenergic, noncholinergic signaling molecule, S-nitrosoglutathione (GSNO) and its catabolism by GSNO reductase (GSNOR) modulate airway reactivity. Tracheomalacia is a major, underinvestigated complication of BPD. We studied trachealis, left main bronchus (LB), and intrapulmonary bronchiolar (IPB) relaxant responses to GSNO in a murine hyperoxic BPD model. METHODS: Wild-type (WT) or GSNOR knockout (KO) newborn mice were raised in 60% (BPD) or 21% (control) oxygen during the first 3 weeks of life. After room air recovery, adult trachealis, LB, and IPB smooth muscle relaxant responses to GSNO (after methacholine preconstriction) were studied using wire myographs. Studies were repeated after GSNOR inhibitor (GSNORi) pretreatment and in KO mice. RESULTS: GSNO relaxed all airway preparations. GSNO relaxed WT BPD trachealis substantially more than WT controls (P < .05). Pharmacologic or genetic ablation of GSNOR abolished the exaggerated BPD tracheal relaxation to GSNO and also augmented BPD IPB relaxation to GSNO. LB ring contractility was not significantly different between groups or conditions. Additionally, GSNORi treatment induced relaxation of WT IPBs but not trachealis or LB. CONCLUSION: GSNO dramatically relaxed the trachealis in our BPD model, an effect paradoxically reversed by loss of GSNOR. Conversely, GSNOR inhibition augmented IBP relaxation. These data suggest that GSNOR inhibition could benefit both the BPD trachealis and distal airways, restoring relaxant responses to those of room air controls. Because therapeutic options are limited in this high-risk population, future studies of GSNOR inhibition are needed.

Nitric oxide-generating compound GSNO suppresses porcine circovirus type 2 infection in vitro and in vivo.

BACKGROUND: Nitric oxide (NO), an important signaling molecule with biological functions, has antimicrobial activity against a variety of pathogens including viruses. To our knowledge, little information is available about the regulatory effect of NO on porcine circovirus type 2 (PCV2) infection. This study was conducted to investigate the antiviral activity of NO generated from S-nitrosoglutathione (GSNO), during PCV2 infection of PK-15 cells and BALB/c mice. RESULTS: GSNO released considerable NO in the culture medium of PK-15 cells, and NO was scavenged by its scavenger hemoglobin (Hb) in a dose-dependent manner. NO strongly inhibited PCV2 replication in PK-15 cells, and the antiviral effect was reversed by Hb. An in vivo assay indicated that GSNO treatment reduced the progression of PCV2 infection in mice, evident as reductions in the percentages of PCV2-positive sera and tissue samples and in the viral DNA copies in serum samples. GSNO also improved the growth performance and immune organs (spleens and thymuses) of the PCV2-infected mice to some degree. CONCLUSIONS: Our data demonstrate that the NO-generating compound GSNO suppresses PCV2 infection in PK-15 cells and BALB/c mice, indicating that NO and its donor, GSNO, have potential value as antiviral drugs against PCV2 infection.

S-Nitrosoglutathione Attenuates Airway Hyperresponsiveness in Murine Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is characterized by lifelong obstructive lung disease and profound, refractory bronchospasm. It is observed among survivors of premature birth who have been treated with prolonged supplemental oxygen. Therapeutic options are limited. Using a neonatal mouse model of BPD, we show that hyperoxia increases activity and expression of a mediator of endogenous bronchoconstriction, S-nitrosoglutathione (GSNO) reductase. MicroRNA-342-3p, predicted in silico and shown in this study in vitro to suppress expression of GSNO reductase, was decreased in hyperoxia-exposed pups. Both pretreatment with aerosolized GSNO and inhibition of GSNO reductase attenuated airway hyperresponsiveness in vivo among juvenile and adult mice exposed to neonatal hyperoxia. Our data suggest that neonatal hyperoxia exposure causes detrimental effects on airway hyperreactivity through microRNA-342-3p-mediated upregulation of GSNO reductase expression. Furthermore, our data demonstrate that this adverse effect can be overcome by supplementing its substrate, GSNO, or by inhibiting the enzyme itself. Rates of BPD have not improved over the past two decades; nor have new therapies been developed. GSNO-based therapies are a novel treatment of the respiratory problems that patients with BPD experience.

Inhibition on the S-nitrosylation of MKK4 can protect hippocampal CA1 neurons in rat cerebral ischemia/reperfusion.

S-nitrosylation, the nitric oxide-derived post-translational modification of proteins, plays critical roles in various physiological and pathological functions. In this present study, a rat model of cerebral ischemia and reperfusion by four-vessel occlusion was generated to assess MKK4 S-nitrosylation. Immunoprecipitation and immunoblotting were performed to evaluate MKK4 S-nitrosylation and phosphorylation. Neuronal loss was observed using histological detection. These results indicated that endogenous NO promoted the S-nitrosylation of MKK4. However, application of the exogenous NO donor S-nitrosoglutathione (GNSO), an inhibitor of the neuronal nitric oxide synthase 7-nitroindazole (7-NI), and the N-methyl-d-aspartate receptor (NMDAR) antagonist MK801 diminished I/R-induced S-nitrosylation and phosphorylation. These compounds also markedly decreased cerebral I/R-induced degeneration and death of neurons in hippocampal CA1 region in rats. Taken together, we demonstrated for the first time, that cerebral ischemia/reperfusion can induce S-nitrosylation of MKK4. We also found that inhibiting S-nitrosylation and activation of MKK4 resulted in marked decreases in neuronal degeneration and apoptosis, potentially via NMDAR-mediated mechanisms. These findings may lead to a new field of inquiry to investigate the underlying pathogenesis of stoke and the development of novel treatment strategies.

In Situ Microparticles Loaded with S-Nitrosoglutathione Protect from Stroke.

Treatment of stroke, especially during the first hours or days, is still lacking. S-nitrosoglutathione (GSNO), a cerebroprotective agent with short life time, may help if administered early with a sustain delivery while avoiding intensive reduction in blood pressure. We developed in situ forming implants (biocompatible biodegradable copolymer) and microparticles (same polymer and solvent emulsified with an external oily phase) of GSNO to lengthen its effects and allow cerebroprotection after a single subcutaneous administration to Wistar rats. Arterial pressure was recorded for 3 days (telemetry, n = 14), whole-blood platelet aggregation up to 13 days (aggregometry, n = 58), and neurological score, cerebral infarct size and edema volume for 2 days after obstruction of the middle cerebral artery by autologous blood clots (n = 30). GSNO-loaded formulations (30 mg/kg) induced a slighter and longer hypotension (-10 vs. -56 ± 6 mmHg mean arterial pressure, 18 h vs. 40 min) than free GSNO at the same dose. The change in pulse pressure (-50%) lasted even up to 42 h for microparticles. GSNO-loaded formulations (30 mg/kg) prevented the transient 24 h hyper-aggregability observed with free GSNO and 7.5 mg/kg-loaded formulations. When injected 2 h after stroke, GSNO-loaded microparticles (30 mg/kg) reduced neurological score at 24 (-62%) and 48 h (-75%) vs. empty microparticles and free GSNO 7.5 mg/kg and, compared to free GSNO, divided infarct size by 10 and edema volume by 8 at 48 h. Corresponding implants reduced infarct size and edema volume by 2.5 to 3 times. The longer (at least 2 days) but slight effects on arterial pressures show sustained delivery of GSNO-loaded formulations (30 mg/kg), which prevent transient platelet hyper-responsiveness and afford cerebroprotection against the consequences of stroke. In conclusion, in situ GSNO-loaded formulations are promising candidates for the treatment of stroke.

S-nitrosoglutathione reduces tau hyper-phosphorylation and provides neuroprotection in rat model of chronic cerebral hypoperfusion.

We have previously reported that treatment of rats subjected to permanent bilateral common carotid artery occlusion (pBCCAO), a model of chronic cerebral hypoperfusion (CCH), with S-nitrosoglutathione (GSNO), an endogenous nitric oxide carrier, improved cognitive functions and decreased amyloid-ß accumulation in the brains. Since CCH has been implicated in tau hyperphosphorylation induced neurodegeneration, we investigated the role of GSNO in regulation of tau hyperphosphorylation in rat pBCCAO model. The rats subjected to pBCCAO had a significant increase in tau hyperphosphorylation with increased neuronal loss in hippocampal/cortical areas. GSNO treatment attenuated not only the tau hyperphosphorylation, but also the neurodegeneration in pBCCAO rat brains. The pBCCAO rat brains also showed increased activities of GSK-3ß and Cdk5 (major tau kinases) and GSNO treatment significantly attenuated their activities. GSNO attenuated the increased calpain activities and calpain-mediated cleavage of p35 leading to production of p25 and aberrant Cdk5 activation. In in vitro studies using purified calpain protein, GSNO treatment inhibited calpain activities while 3-morpholinosydnonimine (a donor of peroxynitrite) treatment increased its activities, suggesting the opposing role of GSNO vs. peroxynitrite in regulation of calpain activities. In pBCCAO rat brains, GSNO treatment attenuated the expression of inducible nitric oxide synthase (iNOS) expression and also reduced the brain levels of nitro-tyrosine formation, thereby indicating the protective role of GSNO in iNOS/nitrosative-stress mediated calpain/tau pathologies under CCH conditions. Taken together with our previous report, these data support the therapeutic potential of GSNO, a biological NO carrier, as a neuro- and cognitive-protective agent under conditions of CCH.

Treatment of bowel in experimental gastroschisis with a nitric oxide donor.

OBJECTIVE: To reduce the harmful effect of bowel exposure to amniotic fluid in gastroschisis, we used the nitric oxide (NO) donor S-nitrosoglutathione (GSNO) in an animal model of gastroschisis and assessed the ideal concentration for treatment of changes in bowel. STUDY DESIGN: Gastroschisis was surgically induced in rat fetuses on day 18.5 of gestation. The fetuses were divided into 5 groups (n = 12 animals/group): control (C), gastroschisis (G), gastroschisis + GSNO 5 µmol/L (GNO1), gastroschisis + GSNO 0.5 µmol/L (GNO2), and gastroschisis + GSNO 0.05 µmol/L (GNO3). On day 21.5 of gestation, fetuses were collected by cesarean delivery. Body and intestinal weight were measured and the bowels were either fixed for histometric and immunohistochemical study or frozen for Western blotting. We analyzed bowel morphometry on histological sections and expression of the NO synthase (NOS) enzymes by Western blotting and immunohistochemistry. Data were analyzed by analysis of variance or Kruskal-Wallis test when appropriate. RESULTS: Morphological and histometric measurements of weight, diameter, and thickness of the layers of the intestinal wall decreased with GSNO treatment, especially in the GNO3 group, when compared with the G group (P < .05). The expression of neuronal NOS, endothelial NOS, and inducible NOS decreased mainly in GNO3 group compared to the G group (P < .05), with no difference compared to C group (P > .05). CONCLUSION: Fetal treatment with 0.05 µmol/L GSNO resulted in significant improvement of bowel morphology in gastroschisis.

S-nitrosoglutathione efflux in the erythrocyte.

Glutathione is an abundant molecule inside erythrocyte, originating S-nitrosoglutathione (GSNO) by reacting with nitric oxide (NO). GSNO has been regarded as a store and transporter of NO, with significant interest as a potential therapeutic agent, acting as an NO donor.NO metabolism inside the erythrocyte generates several derivatives, which can be altered by external and internal stimuli such as acetylcholine (ACh), a natural substrate of acetylcholinesterase (AChE). In spite of the knowledge gained in the last decades concerning NO efflux in erythrocytes little is known regarding erythrocyte GSNO efflux, which has also a significant role in microcirculation. Hence, the objective of this research was to evaluate the efflux of GSNO, concomitant with the efflux of NO, after stimulation with AChE effectors. To achieve these goals, the in vitro effect of AChE modulators - ACh and timolol - in erythrocyte NO and GSNO were studied. Timolol is an erythrocyte AChE inhibitor. Venous blood samples were collected from 18 healthy Caucasian men. For each blood sample, erythrocyte suspensions were obtained and incubated in the absence (controls) and presence of ACh and timolol maleate (10 µM final concentration of each modulator). Both timolol and ACh induced significant GSNO efflux in the erythrocyte when compared to the control; however the efflux was lower in the presence of timolol compared to ACh. Although erythrocyte NO efflux in presence of timolol is similar to the control, the efflux decreased when compared to the ACh treatment. The presence of timolol induces significant decrease of intra-erythrocyte GSNO levels, relative to control and ACh treatment. In conclusion, when erythrocytes were stimulated with ACh or timolol, GSNO efflux occurred associated with NO efflux. These new results bring new insight into the metabolism of erythrocyte NO and new possible therapeutic applications for GSNO.

S-Nitrosoglutathione protects the spinal bladder: novel therapeutic approach to post-spinal cord injury bladder remodeling.

AIMS: Bladder and renal dysfunction are secondary events of the inflammatory processes induced by spinal cord injury (SCI). S-Nitrosoglutathione (GSNO), an endogenous nitrosylating agent is pleiotropic and has anti-inflammatory property. Hence, GSNO ameliorates inflammatory sequelae observed in bladder and renal tissues after SCI. Thus, we postulate that GSNO will improve the recovery of micturition dysfunction by quenching the bladder tissue inflammation associated with SCI. METHODS: Contusion-based mild SCI was induced in female Sprague-Dawley rats. Sham operated rats served as the controls. SCI rats were gavaged daily with GSNO (50 µg/kg) or vehicle. Bladder function was assessed by urodynamics at 2 and 14 days following SCI. Urine protein concentration and osmolality were measured. Bladder and kidney tissues were analyzed by histology and immunofluorescence for a variety of endpoints related to inflammation. RESULTS: Two days after SCI, urodynamics demonstrated a hyperreflexive bladder with overflow and no clear micturition events. By Day 14, vehicle animals regained a semblance of a voiding cycle but with no definite intercontraction intervals. GSNO-treated SCI-rats showed nearly normal cystometrograms. Vehicle-treated SCI rats had increased bladder wet weight, proteinuria, and urine osmolality at Day 14, which was reversed by GSNO treatment. In addition, the SCI-induced increase in immune cell infiltration, collagen deposition, iNOS, and ICAM-1 expression and apoptosis were attenuated by GSNO. CONCLUSIONS: These results indicate that oral administration of GSNO hastens the recovery of bladder function after mild contusion-induced SCI through dampening the inflammation sequelae. These findings also suggest that GSNO-mediated redox modulation may be a novel therapeutic target for the treatment of mild SCI-induced renal and bladder dysfunction.

Preclinical therapeutic potential of a nitrosylating agent in the treatment of ovarian cancer.

This study examines the role of s-nitrosylation in the growth of ovarian cancer using cell culture based and in vivo approaches. Using the nitrosylating agent, S-nitrosoglutathione (GSNO), a physiological nitric oxide molecule, we show that GSNO treatment inhibited proliferation of chemoresponsive and chemoresistant ovarian cancer cell lines (A2780, C200, SKVO3, ID8, OVCAR3, OVCAR4, OVCAR5, OVCAR7, OVCAR8, OVCAR10, PE01 and PE04) in a dose dependent manner. GSNO treatment abrogated growth factor (HB-EGF) induced signal transduction including phosphorylation of Akt, p42/44 and STAT3, which are known to play critical roles in ovarian cancer growth and progression. To examine the therapeutic potential of GSNO in vivo, nude mice bearing intra-peritoneal xenografts of human A2780 ovarian carcinoma cell line (2 × 10(6)) were orally administered GSNO at the dose of 1 mg/kg body weight. Daily oral administration of GSNO significantly attenuated tumor mass (p<0.001) in the peritoneal cavity compared to vehicle (phosphate buffered saline) treated group at 4 weeks. GSNO also potentiated cisplatin mediated tumor toxicity in an A2780 ovarian carcinoma nude mouse model. GSNO's nitrosylating ability was reflected in the induced nitrosylation of various known proteins including NFκB p65, Akt and EGFR. As a novel finding, we observed that GSNO also induced nitrosylation with inverse relationship at tyrosine 705 phosphorylation of STAT3, an established player in chemoresistance and cell proliferation in ovarian cancer and in cancer in general. Overall, our study underlines the significance of S-nitrosylation of key cancer promoting proteins in modulating ovarian cancer and proposes the therapeutic potential of nitrosylating agents (like GSNO) for the treatment of ovarian cancer alone or in combination with chemotherapeutic drugs.

Pharmacological treatment of the basic defect in cystic fibrosis.

Cystic fibrosis (CF) is a genetic disease due to a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel in epithelial cells. There are about 1900 mutations, divided in several groups, for example, stop mutations, mutations affecting the permeability of the channel, and mutations in which the mutated CFTR is recognized as abnormal and destroyed. Pharmacological treatment has become possible for stop mutations (about 10% of the patients), and for a rare mutation affecting channel permeability. For the majority of patients, however, that have a mutation in which the mutated CFTR is destroyed on its way to the cell membrane, research is still in progress, although a number of compounds have been identified that (at least partly) corrects the error in chloride transport.

S-nitrosoglutathione inhibits inducible nitric oxide synthase upregulation by redox posttranslational modification in experimental diabetic retinopathy.

PURPOSE: Diabetic retinopathy (DR) is associated with nitrosative stress. The purpose of this study was to evaluate the beneficial effects of S-nitrosoglutathione (GSNO) eye drop treatment on an experimental model of DR. METHODS: Diabetes (DM) was induced in spontaneously hypertensive rats (SHR). Treated animals received GSNO eye drop (900 nM or 10 µM) twice daily in both eyes for 20 days. The mechanisms of GSNO effects were evaluated in human RPE cell line (ARPE-19). RESULTS: In animals with DM, GSNO decreased inducible nitric oxide synthase (iNOS) expression and prevented tyrosine nitration formation, ameliorating glial dysfunction measured with glial fibrillary acidic protein, resulting in improved retinal function. In contrast, in nondiabetic animals, GSNO induced oxidative/nitrosative stress in tissue resulting in impaired retinal function. Nitrosative stress was present markedly in the RPE layer accompanied by c-wave dysfunction. In vitro study showed that treatment with GSNO under high glucose condition counteracted nitrosative stress due to iNOS downregulation by S-glutathionylation, and not by prevention of decreased GSNO and reduced glutathione levels. This posttranslational modification probably was promoted by the release of oxidized glutathione through GSNO denitrosylation via GSNO-R. In contrast, in the normal glucose condition, GSNO treatment promoted nitrosative stress by NO formation. CONCLUSIONS: In this study, a new therapeutic modality (GSNO eye drop) targeting nitrosative stress by redox posttranslational modification of iNOS was efficient against early damage in the retina due to experimental DR. The present work showed the potential clinical implications of balancing the S-nitrosoglutathione/glutathione system in treating DR.

S-Nitrosoglutathione improves haemodynamics in early-onset pre-eclampsia.

AIMS: To determine the effects of in vivo S-nitrosoglutathione (GSNO) infusion on cardiovascular function, platelet function, proteinuria and biomarker parameters in early-onset pre-eclampsia. METHODS: We performed an open-label dose-ranging study of GSNO in early-onset pre-eclampsia. Six women underwent GSNO infusion whilst receiving standard therapy. The dose of GSNO was increased incrementally to 100 µg min(-1) whilst maintaining blood pressure of >140/80 mmHg. Aortic augmentation index, aortic pulse wave velocity, blood pressure and maternal-fetal Doppler parameters were measured at each dose. Platelet P-selectin, protein-to-creatinine ratio and soluble anti-angiogenic factors were measured pre- and postinfusion. RESULTS: Augmentation index fell at 30 µg min(-1) S-nitrosoglutathione (-6%, 95% confidence interval 0.6 to 13%), a dose that did not affect blood pressure. Platelet P-selectin expression was reduced [mean (interquartile range), 6.3 (4.9-7.6) vs. 4.1 (3.1-5.7)% positive, P = 0.03]. Soluble endoglin levels showed borderline reduction (P = 0.06). There was a borderline significant change in pre-to-postinfusion protein-to-creatinine ratio [mean (interquartile range), 0.37 (0.09-0.82) vs. 0.23 (0.07-0.49) g mmol(-1) , P = 0.06]. Maternal uterine and fetal Doppler pulsatility indices were unchanged. CONCLUSIONS: In early-onset pre-eclampsia, GSNO reduces augmentation index, a biomarker of small vessel tone and pulse wave reflection, prior to affecting blood pressure. Proteinuria and platelet activation are improved at doses that affect blood pressure minimally. These effects of GSNO may be of therapeutic potential in pre-eclampsia, a condition for which no specific treatment exists. Clinical studies of GSNO in early-onset pre-eclampsia will determine whether these findings translate to improvement in maternal and/or fetal outcome.

Protective role of S-nitrosoglutathione (GSNO) against cognitive impairment in rat model of chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion (CCH), featuring in most of the Alzheimer's disease spectrum, plays a detrimental role in brain amyloid-ß (Aß) homeostasis, cerebrovascular morbidity, and cognitive decline; therefore, early management of cerebrovascular pathology is considered to be important for intervention in the impending cognitive decline. S-nitrosoglutathione (GSNO) is an endogenous nitric oxide carrier modulating endothelial function, inflammation, and neurotransmission. Therefore, the effect of GSNO treatment on CCH-associated neurocognitive pathologies was determined in vivo by using rats with permanent bilateral common carotid artery occlusion (BCCAO), a rat model of chronic cerebral hypoperfusion. We observed that rats subjected to permanent BCCAO showed a significant decrease in learning/memory performance and increases in brain levels of Aß and vascular inflammatory markers. GSNO treatment (50 µg/kg/day for 2 months) significantly improved learning and memory performance of BCCAO rats and reduced the Aß levels and ICAM-1/VCAM-1 expression in the brain. Further, in in vitro cell culture studies, GSNO treatment also decreased the cytokine-induced proinflammatory responses, such as activations of NFκB and STAT3 and expression of ICAM-1 and VCAM-1 in endothelial cells. In addition, GSNO treatment increased the endothelial and microglial Aß uptake. Additionally, GSNO treatment inhibited the ß-secretase activity in primary rat neuron cell culture, thus reducing secretion of Aß, suggesting GSNO mediated mechanisms in anti-inflammatory and anti-amyloidogenic activities. Taken together, these data document that systemic GSNO treatment is beneficial for improvement of cognitive decline under the conditions of chronic cerebral hypoperfusion and suggests a potential therapeutic use of GSNO for cerebral hypoperfusion associated mild cognitive impairment in Alzheimer's disease.

Stimulation of functional recovery via the mechanisms of neurorepair by S-nitrosoglutathione and motor exercise in a rat model of transient cerebral ischemia and reperfusion.

PURPOSE: Stroke disability stems from insufficient neurorepair mechanisms. Improvement of functions has been achieved through rehabilitation or therapeutic agents. Therefore, we combined exercise with a neurovascular protective agent, S-nitrosoglutathione (GSNO), to accelerate functional recovery. METHODS: Stroke was induced by middle cerebral artery occlusion for 60 min followed by reperfusion in adult male rats. Animals were treated with vehicle (IR group), GSNO (0.25 mg/kg, GSNO group), rotarod exercise (EX group) and GSNO plus exercise (GSNO+EX group). The groups were studied for 14 days to determine neurorepair mechanisms and functional recovery. RESULTS: Treated groups showed reduced infarction, decreased neuronal cell death, enhanced neurotrophic factors, and improved neurobehavioral functions. However, the GSNO+EX showed greater functional recovery (p < 0.05) than the GSNO or the EX group. A GSNO sub group, treated 24 hours after IR, still showed motor function recovery (p < 0.001). The protective effect of GSNO or exercise was blocked by the inhibition of Akt activity. CONCLUSIONS: GSNO and exercise aid functional recovery by stimulating neurorepair mechanisms. The improvements by GSNO and exercise depend mechanistically on the Akt pathway. A combination of exercise and GSNO shows greater functional recovery. Improved recovery with GSNO, even administered 24 hours post-IR, demonstrates its clinical relevance.

Drug development in preeclampsia: a 'no go' area?

Drug development in pregnancy and particularly in preeclampsia has been long neglected. Preeclampsia is a leading cause of maternal mortality, and early-onset preeclampsia can result in serious long-lasting consequences to the neonate. Many treatments have been trialed with varying success including vitamin supplementation, low-molecular-weight heparins, and aspirin. In this commentary, we particularly focus on the current status of drugs in development specifically aimed at preeclampsia. We outline the current understanding of the causes of the endothelial dysfunction seen in preeclampsia and, as such, potential therapeutic targets. With treatment of preeclampsia being largely unchanged in decades, there is an urgent need for novel therapies particularly those directed at the underlying causes that may allow for extremely preterm delivery, and its myriad consequences, to be avoided.

S-nitrosoglutathione decreases inflammation and bone resorption in experimental periodontitis in rats.

BACKGROUND: S-nitrosoglutathione (GSNO) is a nitric oxide donor that may exert antioxidant, anti-inflammatory, and microbicidal actions and is thus a potential drug for the topical treatment of periodontitis. In this study, the effect of intragingival injections of GSNO-containing polyvinylpyrrolidone (PVP) formulations is evaluated in a rat model of periodontitis. METHODS: Periodontal disease was induced by placing a sterilized nylon (000) thread ligature around the cervix of the second left upper molar of the animals, which received intragingival injections of PVP; saline; or PVP/GSNO solutions which corresponded to GSNO doses of 25, 100, and 500 nmol; 1 hour before periodontitis induction, and thereafter, daily for 11 days. RESULTS: PVP/GSNO formulations at doses of 25 and/or 100, but not 500 nmol caused significant inhibition of alveolar bone loss, increase of bone alkaline phosphatase, decrease of myeloperoxidase activity, as well as significant reduction of inflammatory and oxidative stress markers when compared to saline and PVP groups. These effects were also associated with a decrease of matrix metalloproteinases 1 and 8, inducible nitric oxide synthase, and nuclear factor-κB immunostaining in the periodontium. CONCLUSION: Local intragingival injections of GSNO reduces inflammation and bone loss in experimental periodontal disease.

Spinal cord injury induced arrest in estrous cycle of rats is ameliorated by S-nitrosoglutathione: novel therapeutic agent to treat amenorrhea.

INTRODUCTION: Amenorrhea following spinal cord injury (SCI) has been well documented. There has been little research on the underlying molecular mechanisms and therapeutics. AIM: The purpose of the present study was to investigate the effect of GSNO in ameliorating SCI-induced amenorrhea through affecting the expression of CX43, NFkB, and ERß protein. METHODS: SCI was induced in female SD rats at the T9-T10 level. Estrous stage was determined by vaginal smear. GSNO (50 µg/kg body weight) was gavage fed daily. Animals were sacrificed on day 7 and 14 post SCI. Ovaries were fixed for histological and biochemical studies. Expression levels of ERß, CX-43, and NFkB were analyzed by Western blot and immunofluorescence. MAIN OUTCOME MEASURES: GSNO hastens resumption of the estrous cycle following SCI-induced transient arrest. RESULTS: Resumption of estrous cycle was hastened by GSNO. Atretic and degenerating follicles seen in the ovary of SCI rats on day 14 post-SCI were decreased in GSNO treated animals. The increased CX43 expression observed with SCI ovary was decreased by GSNO. ERß expression decreased significantly on day 7 and 14 post-SCI and was restored with GSNO treatment. Following SCI, NFkB expression was increased in the ovarian follicles and the expression was reduced with GSNO administration. The number of terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate (UTP) nick end labeling positive follicular and luteal cells was increased after SCI. GSNO-treated animals had significantly fewer apoptotic cells in the ovary. CONCLUSION: SCI-induced amenorrhea is accompanied by an increase in CX43 expression and a decrease in ERß expression. SCI animals treated with GSNO resumed the estrous cycle significantly earlier. These results indicate a potential therapeutic value for GSNO in treating amenorrhea among SCI patients.