OBJECTIVE:Chronic hemolysis is a hallmark of sickle cell disease (SCD) and a driver of vasculopathy; however, the mechanisms contributing to hemolysis remain incompletely understood. Although XO ...(xanthine oxidase) activity has been shown to be elevated in SCD, its role remains unknown. XO binds endothelium and generates oxidants as a byproduct of hypoxanthine and xanthine catabolism. We hypothesized that XO inhibition decreases oxidant production leading to less hemolysis.
APPROACH AND RESULTS:Wild-type mice were bone marrow transplanted with control (AA) or sickle (SS) Townes bone marrow. After 12 weeks, mice were treated with 10 mg/kg per day of febuxostat (Uloric), Food and Drug Administration–approved XO inhibitor, for 10 weeks. Hematologic analysis demonstrated increased hematocrit, cellular hemoglobin, and red blood cells, with no change in reticulocyte percentage. Significant decreases in cell-free hemoglobin and increases in haptoglobin suggest XO inhibition decreased hemolysis. Myographic studies demonstrated improved pulmonary vascular dilation and blunted constriction, indicating improved pulmonary vasoreactivity, whereas pulmonary pressure and cardiac function were unaffected. The role of hepatic XO in SCD was evaluated by bone marrow transplanting hepatocyte-specific XO knockout mice with SS Townes bone marrow. However, hepatocyte-specific XO knockout, which results in >50% diminution in circulating XO, did not affect hemolysis levels or vascular function, suggesting hepatocyte-derived elevation of circulating XO is not the driver of hemolysis in SCD.
CONCLUSIONS:Ten weeks of febuxostat treatment significantly decreased hemolysis and improved pulmonary vasoreactivity in a mouse model of SCD. Although hepatic XO accounts for >50% of circulating XO, it is not the source of XO driving hemolysis in SCD.
Nitric oxide (NO) regulates blood pressure (BP) by binding the reduced heme iron (Fe2+) in soluble guanylyl cyclase (sGC) and relaxing vascular smooth muscle cells (SMC). We previously showed that ...sGC heme iron reduction (Fe3+ → Fe2+) is modulated by cytochrome b5 reductase 3 (CYB5R3). However, the in vivo role of SMC CYB5R3 in BP regulation remains elusive. Here, we generated conditional smooth muscle cell-specific Cyb5r3 knockout mice (SMC CYB5R3 KO) to test if SMC CYB5R3 loss impacts systemic BP in normotension and hypertension via regulation of sGC redox state. SMC CYB5R3 KO mice exhibited a 5.84 mmHg increase in BP and impaired acetylcholine-induced vasodilation in mesenteric arteries compared to controls. To drive sGC oxidation and elevate BP, we infused mice with angiotensin-II. We found SMC CYB5R3 KO mice exhibited a 14.75 mmHg BP increase and mesenteric arteries had diminished NO-dependent vasodilation, but increased responsiveness to sGC heme-independent activator BAY 58-2667 over controls. Furthermore, acute injection of BAY 58-2667 in angiotensin-II treated SMC CYB5R3 KO mice showed greater BP reduction compared to controls. Together, these data provide the first in vivo evidence that SMC CYB5R3 is a sGC heme reductase in resistance arteries and provides resilience against systemic hypertension development.
Xanthine oxidase (XO) catalyzes the catabolism of hypoxanthine to xanthine and xanthine to uric acid, generating oxidants as a byproduct. Importantly, XO activity is elevated in numerous hemolytic ...conditions including sickle cell disease (SCD); however, the role of XO in this context has not been elucidated. Whereas long-standing dogma suggests elevated levels of XO in the vascular compartment contribute to vascular pathology via increased oxidant production, herein, we demonstrate, for the first time, that XO has an unexpected protective role during hemolysis. Using an established hemolysis model, we found that intravascular hemin challenge (40 μmol/kg) resulted in a significant increase in hemolysis and an immense (20-fold) elevation in plasma XO activity in Townes sickle cell phenotype (SS) sickle mice compared to controls. Repeating the hemin challenge model in hepatocyte-specific XO knockout mice transplanted with SS bone marrow confirmed the liver as the source of enhanced circulating XO as these mice demonstrated 100% lethality compared to 40% survival in controls. In addition, studies in murine hepatocytes (AML12) revealed hemin mediates upregulation and release of XO to the medium in a toll like receptor 4 (TLR4)-dependent manner. Furthermore, we demonstrate that XO degrades oxyhemoglobin and releases free hemin and iron in a hydrogen peroxide-dependent manner. Additional biochemical studies revealed purified XO binds free hemin to diminish the potential for deleterious hemin-related redox reactions as well as prevents platelet aggregation. In the aggregate, data herein reveals that intravascular hemin challenge induces XO release by hepatocytes through hemin-TLR4 signaling, resulting in an immense elevation of circulating XO. This increased XO activity in the vascular compartment mediates protection from intravascular hemin crisis by binding and potentially degrading hemin at the apical surface of the endothelium where XO is known to be bound and sequestered by endothelial glycosaminoglycans (GAGs).
(Top) Canonical hemin scavenging pathway. (Bottom) Hemin overload releases XO via TLR-4 into the circulation leading to glycosaminoglycan binding on endothelium where XO generates H2O2 needed to “split” hemin and uric acid to chelate released Fe. Display omitted
•Intravascular heme crisis in mice is associated with a 20-fold elevation in circulating XO.•Pharmacologic inhibition or liver-specific knockout of XO results in diminished survival during heme crisis.•Free hemin induces a TLR4-dependent upregulation and export of XO in hepatocytes.•XO degrades oxyhemoglobin and free hemin in a H2O2-dependent manner.•Hemin-induced elevation of circulating XO may be a protective response.
Pulmonary and systemic vasculopathies are significant risk factors for early morbidity and death in patients with sickle cell disease (SCD). An underlying mechanism of SCD vasculopathy is vascular ...smooth muscle (VSM) nitric oxide (NO) resistance, which is mediated by NO scavenging reactions with plasma hemoglobin (Hb) and reactive oxygen species that can oxidize soluble guanylyl cyclase (sGC), the NO receptor. Prior studies show that cytochrome b5 reductase 3 (CYB5R3), known as methemoglobin reductase in erythrocytes, functions in VSM as an sGC heme iron reductase critical for reducing and sensitizing sGC to NO and generating cyclic guanosine monophosphate for vasodilation. Therefore, we hypothesized that VSM CYB5R3 deficiency accelerates development of pulmonary hypertension (PH) in SCD. Bone marrow transplant was used to create SCD chimeric mice with background smooth muscle cell (SMC)–specific tamoxifen-inducible Cyb5r3 knockout (SMC R3 KO) and wild-type (WT) control. Three weeks after completing tamoxifen treatment, we observed 60% knockdown of pulmonary arterial SMC CYB5R3, 5 to 6 mm Hg elevated right-ventricular (RV) maximum systolic pressure (RVmaxSP) and biventricular hypertrophy in SS chimeras with SMC R3 KO (SS/R3KD) relative to WT (SS/R3WT). RV contractility, heart rate, hematological parameters, and cell-free Hb were similar between groups. When identically generated SS/R3 chimeras were studied 12 weeks after completing tamoxifen treatment, RVmaxSP in SS/R3KD had not increased further, but RV hypertrophy relative to SS/R3WT persisted. These are the first studies to establish involvement of SMC CYB5R3 in SCD-associated development of PH, which can exist in mice by 5 weeks of SMC CYB5R3 protein deficiency.
•Vascular SMC CYB5R3 delays development of SCD-associated PH in mice.•PH in SCD can exist in mice by 5 weeks of age when SMC CYB5R3 protein is deficient.
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Phone-based unannounced pill counts to measure medication adherence are much more practical and less expensive than home-based unannounced pill counts, but their validity has not been widely ...assessed. We examined the validity of phone versus home-based pill counts using a simplified protocol streamlined for studies embedded in clinical care settings. A total of 100 paired counts were used to compare concordance between unannounced phone and home-based pill counts using interclass correlations. Discrepancy analyses using χ
2
tests compared demographic and clinical characteristics across patients who were concordant between phone and home-based pill counts and patients who were not concordant. Concordance was high for phone-based and home-based unannounced total pill counts, as well as individual medication counts and calculated adherence. This study demonstrates that a simplified phone-based pill count protocol can be implemented among patients from a routine clinical care setting and is a feasible means of monitoring medication adherence.
Heme crisis drives a number of hemolytic conditions including malaria, sepsis, blood transfusions, and cardiac bypass. Free heme, released from hemoglobin, causes endothelial damage via direct and ...iron (Fe)-mediated generation of reactive species, as well as activation of endothelial cells and macrophages leading to an inflammatory response. An enzyme that has been shown to have elevated activity in many hemolytic conditions is xanthine oxidase (XO). XO generates hydrogen peroxide (H2O2) as a byproduct of the oxidation of hypoxanthine and xanthine in the final steps of the purine degradation pathway. While XO activity is known to be increased in hemolytic diseases, its exact role has yet to be established.
In order to study the role of XO in hemolytic disease, we developed a novel model of heme crisis in which we injected mice with two identical doses of hemin one hour apart and monitored the mice for 24 hours in order to deplete heme scavenging mechanisms before inducing heme crisis. Heme crisis induced damage was evaluated by hemopexin ELISA, plasma XO activity by HPLC, H&E staining of liver, lung, and kidney tissue, qRT-PCR of inflammatory cytokines, and hematological analysis of circulating leukocytes, RBCs, and platelets. To isolate the role of XO, our heme crisis model was repeated; however, prior to hemin injection mice were pretreated with the FDA approved, XO inhibitor febuxostat (10 mg/kg/day) in drinking water. Liver, lung and kidney injury and inflammation was again evaluated with H&E staining, qRT-PCR of inflammatory cytokines, and hematological analysis. In addition, the interaction between heme and XO was explored in vitro using evaluation of hemin degradation via spectrophotometry and computational modeling.
We found that mice treated with two doses of 50 μmol/kg hemin had a 92.3% decrease in hemopexin, and a 20-fold increase in plasma XO activity compared to controls. H&E staining showed severe liver hemorrhaging, increased cell infiltration in the lung, and cellular disorganization in the kidney. The pro-inflammatory cytokines, IL-6, TNFα, and IL-1β, were all significantly increased in the liver, lung, and kidney, with IL-6 having the greatest fold change in all three organs. Systemic inflammation was also suggested via significant increases in circulating monocytes and granulocytes. Additionally, hematological analysis showed decreased RBCs and platelets, indicating additional hemolysis and platelet activation. While these markers of injury and inflammation were observed with 50 μmol/kg hemin, lower doses of hemin showed no effect. Together, these results indicate that our heme crisis model mimics the pro-inflammatory state, and organ damage observed in patients during severe hemolysis. Interestingly, when mice were pre-treated with febuxostat, organ damage was observed at lower doses of hemin (25 μmol/kg) compared to untreated mice, as observed by H&E staining. Inhibition of XO also had a significant impact on the inflammatory response. While circulating monocytes were decreased in mice pre-treated with febuxostat, the pro-inflammatory cytokines IL-6, TNFα, and IL-1β, were further exacerbated in the liver, lung, and kidney. This suggests that XO may play a role in mediating the inflammatory response induced by heme crisis. To explore how XO could mediate the inflammatory response we conducted in vitro enzymatic XO experiments with hemin. We found that XO was able to degrade hemin as observed by a decrease in absorbance at 618 nm. Additionally, based on a spectral shift observed when hemin and XO were incubated together, we hypothesized that XO may have the ability to bind hemin. This was further supported by computational modeling in which a potential heme binding site was discovered in the FAD domain of XO with a kd=128 nM. This suggests that XO may have the ability to bind hemin. Thus, during substrate oxidation, H2O2 is produced in the same XO domain of the potential heme binding site, allowing for increased chance of the H2O2 induced heme splitting reaction. We further hypothesize that the uric acid produced by xanthine oxidation may serve as an Fe chelator to scavenge free Fe released by a heme splitting reaction. By creating a microenvironment that can split heme and scavenge Fe, XO may be able to mediate the inflammatory response induced by heme crisis.
Straub:Bayer Pharmaceuticals: Research Funding.
Glucocorticoids might prevent bronchopulmonary dysplasia in extremely preterm infants but have adverse neurodevelopmental effects. In this trial involving preterm infants, there was little difference ...in survival without bronchopulmonary dysplasia or in the occurrence of neurodevelopmental impairment with hydrocortisone as compared with placebo.
Abstract only In cardiovascular disease, oxidative stress can drive soluble guanylyl cyclase (sGC) heme oxidation resulting in the loss of the sGC heme (apo-sGC), the impairment of nitric oxide (NO) ...binding and cGMP production, and vasoconstriction. Consequently, a new class of therapeutic compounds sGC activators have been developed which target oxidized and apo-sGC to cause irreversible, NO-independent reactivation of cGMP production and vasodilation. While sGC activators have had varied clinical success, surprisingly few studies have defined the impact of NO-independent sGC activation on vascular physiology in healthy conditions. We found mesenteric and pulmonary arteries are two log orders more sensitive to NO-independent sGC activator BAY 58-2667 induced vasodilation than aorta; no difference in NO-dependent sGC vasodilation between vessels was observed. These data indicate the presence of an activatable physiological pool of oxidized and/or apo-sGC in pulmonary and mesenteric arteries. We recently published that smooth muscle cell cytochrome b5 reductase 3 (CYB5R3) acts to reduce oxidized heme sGC back to its NO-sensitive reduced heme state during vascular disease. We found transgenic CYB5R3 overexpression (CYB5R3 OE) mice were more resistant to BAY 58-2667 mesenteric artery vasodilation and blood pressure lowering compared to wild-type controls (n=5-9) under physiologic conditions. Also, healthy CYB5R3 OE pulmonary arteries had a near complete loss of BAY 58-2667 vasodilation suggesting both mesenteric and pulmonary arteries contain a pool of oxidized sGC. We next asked if physiological H 2 O 2 production accounts for changes in BAY 58-2667 responsiveness. We found using mitochondrial-specific catalase overexpression mice, that BAY 58-2667 vasodilation did not differ from controls in any vascular bed (n=4-6). We next tested whether xanthine oxidase (XO), which can produce H 2 O 2 at the endothelial cell surface of vessels, can impact physiological BAY 58-2667 vasodilation. We found that Febuxostat, a XO inhibitor, led to a significant decrease in mesenteric artery BAY 58-2667 induced vasodilation from ~70% to ~30% (n=6). Combined, these data provide evidence for CYB5R3 and XO as regulators of physiological sGC resistance artery vasodilation.
The arterial resistance vasculature modulates blood pressure and flow to match oxygen delivery to tissue metabolic demand. As such, resistance arteries and arterioles have evolved a series of highly ...orchestrated cell-cell communication mechanisms between endothelial cells and vascular smooth muscle cells to regulate vascular tone. In response to neurohormonal agonists, release of several intracellular molecules, including nitric oxide, evokes changes in vascular tone. We and others have uncovered novel redox switches in the walls of resistance arteries that govern nitric oxide compartmentalization and diffusion. In this review, we discuss our current understanding of redox switches controlling nitric oxide signaling in endothelial and vascular smooth muscle cells, focusing on new mechanistic insights, physiological and pathophysiological implications, and advances in therapeutic strategies for hypertension and other diseases.
Abstract only Introduction: Stroke and silent infarcts are serious complications of sickle cell disease (SCD), occurring frequently in children. Decreased nitric oxide bioavailability and ...responsiveness contribute to neurovascular disease. Cytochrome b5 reductase 3 (Cyb5R3) is a heme iron reductase that reduces oxidized soluble guanylate cyclase heme iron (Fe 3+ --> Fe 2+ ) to preserve nitric oxide signaling. A loss-of-function Cyb5R3 missense variant (T117S) occurs with high frequency (0.23 minor allele) in persons of African ancestry. Hypothesis: We hypothesized that impaired reductase function of T117S Cyb5R3 exacerbates brain damage after ischemic stroke in SCD. Methods: Bone marrow transplant was used to create male SCD mice with wild type (SS/WT) or T117S (SS/T117S) Cyb5R3. Blood was sampled before and after middle cerebral artery occlusion (55 minutes occlusion, 48 hours reperfusion). Infarct volume (IV) was determined by 2,3,5-triphenyltetrazolium chloride. Intravascular hemolysis and correlation (Pearson’s R) of hematology changes with IV were determined. Baseline Walk-PHaSST (NCT00492531) data were analyzed for stroke occurrence. Results: Brain IV (63 vs 27 cm 3 , P=0.003) and mortality (3/6 vs 0/8) were greater in SS/T117S vs SS/WT. Red blood cells, hemoglobin and hematocrit declined as IV increased. Plasma oxyhemoglobin increased in parallel with IV (r = 0.74, P=0.09). There were different signatures to hematologic changes that occurred with IV in SCD. Relative to wild type, T117S contracted the erythroid compartment (red blood cell: -13% vs 13%, P=0.003; hematocrit: -20% vs 1%, P=0.008; hemoglobin: -18% vs 2%, P=0.007). Mean platelet volume correlated with IV in SS/T117S (r = 0.87, P=0.06), while the inverse occurred in SS/WT (r = -0.63, P=0.09) Monocytes increased in parallel with IV in SS/T117S (r = 0.73, P=0.16), but followed the opposite trajectory in SS/WT (r = -0.77, P=0.04). WalkPHaSST participants with T117S Cyb5R3 self-reported more ischemic stroke (7.4% vs 5.1%) relative to wild type. Conclusion: Cyb5R3 is an important modifier of the evolution and outcome of ischemic brain injury in SCD and its hematologic consequences. Our findings indicate a bidirectional relationship between stroke and anemia in SCD that may axially turn on Cyb5R3 activity.
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