Xunde Wang

Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver

Nitrite represents a circulating and tissue storage form of NO whose bioactivation is mediated by the enzymatic action of xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin, myoglobin, and tissue heme proteins. Because the rate of NO generation from nitrite is linearly dependent on reductions in oxygen and pH levels, we hypothesized that nitrite would be reduced to NO in ischemic tissue and exert NO-dependent protective effects. Solutions of sodium nitrite were administered in the setting of hepatic and cardiac ischemia-reperfusion (I/R) injury in mice. In hepatic I/R, nitrite exerted profound dose-dependent protective effects on cellular necrosis and apoptosis, with highly significant protective effects observed at near-physiological nitrite concentrations. In myocardial I/R injury, nitrite reduced cardiac infarct size by 67%. Consistent with hypoxia-dependent nitrite bioactivation, nitrite was reduced to NO, S-nitrosothiols, N-nitros-amines, and iron-nitrosylated heme proteins within 1-30 minutes of reperfusion. Nitrite-mediated protection of both the liver and the heart was dependent on NO generation and independent of eNOS and heme oxygenase-1 enzyme activities. These results suggest that nitrite is a biological storage reserve of NO subserving a critical function in tissue protection from ischemic injury. These studies reveal an unexpected and novel therapy for diseases such as myocardial infarction, organ preservation and transplantation, and shock states.

Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer

Nitrite (NO2 −) is an intrinsic signaling molecule that is reduced to NO during ischemia and limits apoptosis and cytotoxicity at reperfusion in the mammalian heart, liver, and brain. Although the mechanism of nitrite-mediated cytoprotection is unknown, NO is a mediator of the ischemic preconditioning cell-survival program. Analogous to the temporally distinct acute and delayed ischemic preconditioning cytoprotective phenotypes, we report that both acute and delayed (24 h before ischemia) exposure to physiological concentrations of nitrite, given both systemically or orally, potently limits cardiac and hepatic reperfusion injury. This cytoprotection is associated with increases in mitochondrial oxidative phosphorylation. Remarkably, isolated mitochondria subjected to 30 min of anoxia followed by reoxygenation were directly protected by nitrite administered both in vitro during anoxia or in vivo 24 h before mitochondrial isolation. Mechanistically, nitrite dose-dependently modifies and inhibits complex I by posttranslational S-nitrosation; this dampens electron transfer and effectively reduces reperfusion reactive oxygen species generation and ameliorates oxidative inactivation of complexes II–IV and aconitase, thus preventing mitochondrial permeability transition pore opening and cytochrome c release. These data suggest that nitrite dynamically modulates mitochondrial resilience to reperfusion injury and may represent an effector of the cell-survival program of ischemic preconditioning and the Mediterranean diet.

Human Neuroglobin Functions as a Redox-regulated Nitrite Reductase

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ∼2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

S-Nitrosohemoglobin Is Unstable in the Reductive Erythrocyte Environment and Lacks O2/NO-linked Allosteric Function

Our previous results run counter to the hypothesis that S-nitrosohemoglobin (SNO-Hb) serves as anin vivo reservoir for NO from which NO release is allosterically linked to oxygen release. We show here that SNO-Hb undergoes reductive decomposition in erythrocytes, whereas it is stable in purified solutions and in erythrocyte lysates treated with an oxidant such as ferricyanide. Using an extensively validated methodology that eliminates background nitrite and stabilizes erythrocyte S-nitrosothiols, we find the levels of SNO-Hb in the basal human circulation, including red cell membrane fractions, were 46 ± 17 nm in human arterial erythrocytes and 69 ± 11 nm in venous erythrocytes, incompatible with the postulated reservoir function of SNO-Hb. Moreover, we performed experiments on human red blood cells in which we elevated the levels of SNO-Hb to 10,000 times the normal in vivo levels. The elevated levels of intra-erythrocytic SNO-Hb fell rapidly, independent of oxygen tension and hemoglobin saturation. Most of the NO released during this process was oxidized to nitrate. A fraction (25%) was exported as S-nitrosothiol, but this fraction was not increased at low oxygen tensions that favor the deoxy (T-state) conformation of Hb. Results of these studies show that, within the redox-active erythrocyte environment, the β-globin cysteine 93 is maintained in a reduced state, necessary for normal oxygen affinity, and incapable of oxygen-linked NO storage and delivery.

Effect of eculizumab on haemolysis-associated nitric oxide depletion, dyspnoea, and measures of pulmonary hypertension in patients with paroxysmal nocturnal haemoglobinuria.

Pulmonary hypertension (PH) is a common complication of haemolytic anaemia. Intravascular haemolysis leads to nitric oxide (NO) depletion, endothelial and smooth muscle dysregulation, and vasculopathy, characterized by progressive hypertension. PH has been reported in patients with paroxysmal nocturnal haemoglobinuria (PNH), a life‐threatening haemolytic disease. We explored the relationship between haemolysis, systemic NO, arginine catabolism and measures of PH in 73 PNH patients enrolled in the placebo‐controlled TRIUMPH (Transfusion Reduction Efficacy and Safety Clinical Investigation Using Eculizumab in Paroxysmal Nocturnal Haemoglobinuria) study. At baseline, intravascular haemolysis was associated with elevated NO consumption (P < 0·0001) and arginase‐1 release (P < 0·0001). Almost half of the patients in the trial had elevated levels (≥160 pg/ml) of N‐terminal pro‐brain natriuretic peptide (NT‐proBNP), a marker of pulmonary vascular resistance and right ventricular dysfunction previously shown to indicate PH. Eculizumab treatment significantly reduced haemolysis (P < 0·001), NO depletion (P < 0·001), vasomotor tone (P < 0·05), dyspnoea (P = 0·006) and resulted in a 50% reduction in the proportion of patients with elevated NT‐proBNP (P < 0·001) within 2 weeks of treatment. Importantly, the significant improvements in dyspnoea and NT‐proBNP levels occurred without significant changes in anaemia. These data demonstrated that intravascular haemolysis in PNH produces a state of NO catabolism leading to signs of PH, including elevated NT pro‐BNP and dyspnoea that are significantly improved by treatment with eculizumab.

Measurement of nitric oxide levels in the red cell: validation of tri-iodide-based chemiluminescence with acid-sulfanilamide pretreatment

The tri-iodide-based chemiluminescence assay is the most widely used methodology for the detection of S-nitrosothiols (RSNOs) in biological samples. Because of the low RSNO levels detected in a number of biological compartments using this assay, criticism has been raised that this method underestimates the true values in biological samples. This claim is based on the beliefs that (i) acidified sulfanilamide pretreatment, required to remove nitrite, leads to RSNO degradation and (ii) that there is auto-capture of released NO by heme in the reaction vessel. Because our laboratories have used this assay extensively without ever encountering evidence that corroborated these claims, we sought to experimentally address these issues using several independent techniques. We find that RSNOs of glutathione, cysteine, albumin, and hemoglobin are stable in acidified sulfanilamide as determined by the tri-iodide method, copper/cysteine assay, Griess-Saville assay and spectrophotometric analysis. Quantitatively there was no difference in S-nitroso-hemoglobin (SNOHb) or S-nitroso-albumin (SNOAlb) using the tri-iodide method and a recently described modified assay using a ferricyanide-enhanced reaction mix at biologically relevant NO:heme ratios. Levels of SNOHb detected in human blood ranged from 20–100 nm with no arterial-venous gradient. We further find that 90% of the total NO-related signal in blood is caused by erythrocytic nitrite, which may partly be bound to hemoglobin. We conclude that all claims made thus far that the tri-iodide assay underestimates RSNO levels are unsubstantiated and that this assay remains the “gold standard” for sensitive and specific measurement of RSNOs in biological matrices.

High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.

Pulmonary hypertension is associated with reduced nitric oxide bioavailability and early mortality in sickle cell disease (SCD). We previously demonstrated that placenta growth factor (PlGF), an angiogenic factor produced by erythroid cells, induces hypoxia-independent expression of the pulmonary vasoconstrictor endothelin-1 in pulmonary endothelial cells. Using a lentivirus vector, we simulated erythroid expression of PlGF in normal mice up to the levels seen in sickle mice. Consequently, endothelin-1 production increased, right ventricle pressures increased, and right ventricle hypertrophy and pulmonary changes occurred in the mice within 8 weeks. These findings were corroborated in 123 patients with SCD, in whom plasma PlGF levels were significantly associated with anemia, endothelin-1, and tricuspid regurgitant velocity; the latter is reflective of peak pulmonary artery pressure. These results illuminate a novel mechanistic pathway linking hemolysis and erythroid hyperplasia to increased PlGF, endothelin-1, and pulmonary hypertension in SCD, and suggest that strategies that block PlGF signaling may be therapeutically beneficial.

Circulating Blood Endothelial Nitric Oxide Synthase Contributes to the Regulation of Systemic Blood Pressure and Nitrite Homeostasis

Objective—Mice genetically deficient in endothelial nitric oxide synthase (eNOS−/−) are hypertensive with lower circulating nitrite levels, indicating the importance of constitutively produced nitric oxide (NO•) to blood pressure regulation and vascular homeostasis. Although the current paradigm holds that this bioactivity derives specifically from the expression of eNOS in endothelium, circulating blood cells also express eNOS protein. A functional red cell eNOS that modulates vascular NO• signaling has been proposed. Approach and Results—To test the hypothesis that blood cells contribute to mammalian blood pressure regulation via eNOS-dependent NO• generation, we cross-transplanted wild-type and eNOS−/− mice, producing chimeras competent or deficient for eNOS expression in circulating blood cells. Surprisingly, we observed a significant contribution of both endothelial and circulating blood cell eNOS to blood pressure and systemic nitrite levels, the latter being a major component of the circulating NO• reservoir. These effects were abolished by the NOS inhibitor L-NG-nitroarginine methyl ester and repristinated by the NOS substrate L-arginine and were independent of platelet or leukocyte depletion. Mouse erythrocytes were also found to carry an eNOS protein and convert 14C-arginine into 14C-citrulline in NOS-dependent fashion. Conclusions—These are the first studies to definitively establish a role for a blood-borne eNOS, using cross-transplant chimera models, that contributes to the regulation of blood pressure and nitrite homeostasis. This work provides evidence suggesting that erythrocyte eNOS may mediate this effect.

14-3-3 Binding and Phosphorylation of Neuroglobin during Hypoxia Modulate Six-to-Five Heme Pocket Coordination and Rate of Nitrite Reduction to Nitric Oxide

Background: Neuroglobin protects neurons from hypoxia; however, the underlying mechanisms for this effect remain poorly understood. Results: Hypoxia increases neuroglobin phosphorylation, binding to 14-3-3, and nitrite reduction to form nitric oxide. Conclusion: Hypoxia-dependent post-translational modifications to neuroglobin regulate the six-to-five heme pocket equilibrium and heme access to ligands. Significance: Hypoxia-regulated neuroglobin may contribute to the cellular adaptation to hypoxia. Neuroglobin protects neurons from hypoxia in vitro and in vivo; however, the underlying mechanisms for this effect remain poorly understood. Most of the neuroglobin is present in a hexacoordinate state with proximal and distal histidines in the heme pocket directly bound to the heme iron. At equilibrium, the concentration of the five-coordinate neuroglobin remains very low (0.1–5%). Recent studies have shown that post-translational redox regulation of neuroglobin surface thiol disulfide formation increases the open probability of the heme pocket and allows nitrite binding and reaction to form NO. We hypothesized that the equilibrium between the six- and five-coordinate states and secondary reactions with nitrite to form NO could be regulated by other hypoxia-dependent post-translational modification(s). Protein sequence models identified candidate sites for both 14-3-3 binding and phosphorylation. In both in vitro experiments and human SH-SY5Y neuronal cells exposed to hypoxia and glucose deprivation, we observed that 1) neuroglobin phosphorylation and protein-protein interactions with 14-3-3 increase during hypoxic and metabolic stress; 2) neuroglobin binding to 14-3-3 stabilizes and increases the half-life of phosphorylation; and 3) phosphorylation increases the open probability of the heme pocket, which increases ligand binding (CO and nitrite) and accelerates the rate of anaerobic nitrite reduction to form NO. These data reveal a series of hypoxia-dependent post-translational modifications to neuroglobin that regulate the six-to-five heme pocket equilibrium and heme access to ligands. Hypoxia-regulated reactions of nitrite and neuroglobin may contribute to the cellular adaptation to hypoxia.

Mixed haematopoietic chimerism for sickle cell disease prevents intravascular haemolysis.

Recent studies suggest that persistent intravascular haemolysis, a defining feature of sickle cell disease (SCD), is associated with severe long-term consequences, including pulmonary hypertension, which carries a 2-year mortality rate of up to 50% (Castro et al, 2003). Thus, the development of therapies that correct intravascular haemolysis and its complications are increasingly recognized as important for the long-term prognosis of the SCD patient. Haematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for SCD, and recent improvements in supportive care and the development of reduced-intensity conditioning regimens have substantially lessened the severity of the immediate toxicities of the transplant procedure (Locatelli, 2006). When lower intensity conditioning regimens are utilized, host haematopoiesis is incompletely eliminated, and mixed haematopoietic chimerism frequently results. The effects of persistent recipient erythro-poiesis on intravascular haemolysis are unknown, but would be anticipated to perpetuate intravascular haemolysis. Several serum biomarkers have been recently identified to strongly correlate with endothelial damage, pulmonary hypertension and prospective early mortality in SCD patients. These include increased soluble vascular cellular adhesion molecule 1 (sVCAM-1) levels, increased nitric oxide (NO) consumption, increased plasma free haemoglobin (Hb), and inverted argi-nine/ornithine ratio (Solovey et al, 1997; Reiter et al, 2002; Morris et al, 2005). Using these parameters, we examined the potential of partial donor engraftment to correct intravascular haemolysis in nine SCD patients, and hence to potentially avert the development of long-term SCD-associated complications. Of the 19 patients who underwent matched related donor HSCT for SCD across four transpalnt centres, each with a different conditioning regimen, nine developed mixed haematopoietic chimerism and were studied in detail. Heparinized blood was obtained from these nine patients upon enrolment onto Institutional Review Board-approved protocols. Overall mononuclear cell engraftment for all nine patients was measured by standard analysis of short tandem repeats from genomic DNA extracted from peripheral blood mononuclear cells. Indications for HSCT ranged from acute chest syndrome and strokes to intractable recurrent pain crises. As shown in Table I, patients 1–7 and 9 underwent nonmyeloablative transplant, while patient 8 underwent a fully myeloablative transplant. For patients 1–3, this period of partial donor engraftment, with levels ranging from 20% to 50%, lasted 5–11 months following HSCT. For patients 4–9, stable mixed chimerism was achieved with levels ranging from 42% to 90%. For patients 4–8, mixed chimerism was maintained even off immunosuppression. Patients 7 and 8 continue to stably demonstrate 55% and 42% peripheral blood donor engraftment, respectively, >4 years following transplant. Patient 9 has been continuously maintained on immunosuppression, and remains a mixed chimera. None of the subjects required red blood cell (RBC) transfusion beyond 1 month post-transplant unless they relapsed and none, while engrafted, experienced any SCD-related events. Table I Patient clinical characteristics. Despite differences in the conditioning regimens, all patients similarly demonstrated dramatic improvements in parameters of intravascular haemolysis while partially donor-engrafted. The median pretransplant values for indices of intravascular haemolysis of our cohort were elevated, similar to the general SCD population (Reiter et al, 2002). Following transplantation, significant normalization of these parameters was observed for each patient, to levels similar to normal donors. As shown in Fig 1A, lactate dehydrogenase (LDH) and free Hb concentration decreased from a median of 375 U/l (range: 255–1699) to 184 U/l (range: 122–260; P = 0.02), and from 19.7 μmol/l (range 5.5–75.9) to 1.6 μmol/l (range: 0.2–7.3; P = 0.02) respectively. Haptoglobin increased from a median value of 50 mg/l (range: 50–200) to 330 mg/l (range: 70–2420; P = 0.02). Fig 1 (A) Pre- and post-transplant plasma values for haptoglobin; LDH; free Hb concentration; nitric oxide (NO) consumption; sVCAM-1; and arginine/ornithine ratio. Median levels for each parameter are represented by the black filled diamonds. The dotted line ... Parameters of vascular function post-transplant similarly improved. Plasma NO consumption and sVCAM-1 levels significantly decreased from a median of 7.6 μmol/l (range: 1.9–49.7) to 1.3 μmol/l (range: 0.6–8.6; P = 0.02), and 952.1 ng/ml (range: 498.2–1067) to 543.1 ng/ml (range: 193.9–724; P = 0.02) respectively. The median arginine/ornithine ratio increased from 0.6 (range: 0.1–1.5) to 1.8 (range: 0.6–2.3; P = 0.06) following HSCT. The observed improvements in parameters of intravascular haemolysis and endothelial function were not related to presence of immunosuppression, which is routinely administered in the early post-transplant period as graft-vs.-host disease prophylaxis. Patients 4 and 6 maintained unchanged or improved levels of donor chimerism and extent of intravascular haemolysis and vascular function, both on and off immunosuppression (Fig 1B). We had sufficient samples from two of the three patients who experienced graft rejection (patients 1 and 3) to analyse in detail the impact of absence or presence of donor erythropoiesis on intravascular haemolysis. Temporary normalization of these parameters occurred with the presence of donor haematopoiesis, with reversion to abnormal baseline values following loss of the donor graft (Fig 1B). Direct indices of intravascular haemolysis (haptoglobin and total bilirubin) were the first to revert to abnormal, coincident with loss of donor engraftment. Free Hb concentration and NO consumption subsequently rose, consistent with the loss of the haemoglobin binding and clearance capacity of haptoglobin. Finally, a few months after graft rejection, a slight rise in LDH was observed. These findings directly demonstrate the dependence upon donor cells to achieve improvement in intravascular haemolysis and potentially on vascular function. To reconcile these results in the face of persistent recipient haematopoiesis, erythroid lineage-specific chimerism was analysed by RNA β-globin pyrosequencing. This method evaluates chimerism by quantitative sequencing of donor vs. host-derived erythroid lineage transcripts, based on detection of the sickle mutation (Wu et al, 2005). Peripheral blood RBCs were fully replaced by donor-derived erythrocytes for patients with at least 50% donor nucleated cell engraftment (Table I). For patients with <50% engraftment, 70–85% donor RBCs expression was observed. Post-transplant donor RBC enrichment relative to the degree of nucleated cell engraftment is probably related to factors such as decreased survival of SS erythrocytes and intrinsic SCD-associated ineffective erythropoiesis (Kean et al, 2003; Wu et al, 2005). Fig 1B shows that normalization of the parameters of haemolysis and endothelial dysfunction was coincidental with expression of donor RBCs, thus directly demonstrating the beneficial effect of donor erythrocytes. Although patients 1, 3 and 8 continued to produce recipient RBCs, which would be expected to perpetuate intravascular haemolysis, this degree of haemolysis is apparently within the buffering capacity of the system. Supporting these findings, RBC exchange and hyper-transfusion that reduce peripheral blood %HbS to 20–30% are clinically protective (Swerdlow, 2006). Taken together, our data mechanistically support mixed chimerism as a suitable endpoint of stem cell-based therapies for SCD.