Cold exposure induces vaso-occlusion and pain in sickle mice that depend on complement activation.

Vaso-occlusive pain episodes (VOE) cause severe pain in patients with sickle cell disease (SCD). Vaso-occlusive events promote ischemia/reperfusion pathobiology that activates complement. We hypothesized that complement activation is linked to VOE. We used cold to induce VOE in the Townes sickle homozygous for hemoglobin S (HbSS) mouse model and complement inhibitors to determine whether anaphylatoxin C5a mediates VOE. We used a dorsal skinfold chamber to measure microvascular stasis (vaso-occlusion) and von Frey filaments applied to the plantar surface of the hind paw to assess mechanical hyperalgesia in HbSS and control Townes mice homozygous for hemoglobin A (HbAA) mice after cold exposure at 10°C/50°F for 1 hour. Cold exposure induced more vaso-occlusion in nonhyperalgesic HbSS mice (33%) than in HbAA mice (11%) or HbSS mice left at room temperature (1%). Cold exposure also produced mechanical hyperalgesia as measured by paw withdrawal threshold in HbSS mice compared with that in HbAA mice or HbSS mice left at room temperature. Vaso-occlusion and hyperalgesia were associated with an increase in complement activation fragments Bb and C5a in plasma of HbSS mice after cold exposure. This was accompanied by an increase in proinflammatory NF-κB activation and VCAM-1 and ICAM-1 expression in the liver. Pretreatment of nonhyperalgesic HbSS mice before cold exposure with anti-C5 or anti-C5aR monoclonal antibodies (mAbs) decreased vaso-occlusion, mechanical hyperalgesia, complement activation, and liver inflammatory markers compared with pretreatment with control mAb. Anti-C5 or -C5aR mAb infusion also abrogated mechanical hyperalgesia in HbSS mice with ongoing hyperalgesia at baseline. These findings suggest that C5a promotes vaso-occlusion, pain, and inflammation during VOE and may play a role in chronic pain.

The BACH1 inhibitor ASP8731 inhibits inflammation and vaso-occlusion and induces fetal hemoglobin in sickle cell disease.

In sickle cell disease (SCD), heme released during intravascular hemolysis promotes oxidative stress, inflammation, and vaso-occlusion. Conversely, free heme can also activate expression of antioxidant and globin genes. Heme binds to the transcription factor BACH1, which represses NRF2-mediated gene transcription. ASP8731, is a selective small molecule inhibitor of BACH1. We investigated the ability of ASP8731 to modulate pathways involved in SCD pathophysiology. In HepG2 liver cells, ASP8731 increased HMOX1 and FTH1 mRNA. In pulmonary endothelial cells, ASP8731 decreased VCAM1 mRNA in response to TNF-α and blocked a decrease in glutathione in response to hemin. Townes-SS mice were gavaged once per day for 4 weeks with ASP8731, hydroxyurea (HU) or vehicle. Both ASP8731 and HU inhibited heme-mediated microvascular stasis and in combination, ASP8731 significantly reduced microvascular stasis compared to HU alone. In Townes-SS mice, ASP8731 and HU markedly increased heme oxygenase-1 and decreased hepatic ICAM-1, NF-kB phospho-p65 protein expression in the liver, and white blood cell counts. In addition, ASP8731 increased gamma-globin expression and HbF+ cells (F-cells) as compared to vehicle-treated mice. In human erythroid differentiated CD34+ cells, ASP8731 increased HGB mRNA and increased the percentage of F-cells 2-fold in manner similar to HU. ASP8731 and HU when given together induced more HbF+ cells compared to either drug alone. In CD34+ cells from one donor that was non-responsive to HU, ASP8731 induced HbF+ cells ~2-fold. ASP8731 and HU also increased HBG and HBA, but not HBB mRNA in erythroid differentiated CD34+ cells derived from SCD patients. These data indicate that BACH1 may offer a new therapeutic target to treat SCD.

Factor XII contributes to thrombotic complications and vaso-occlusion in sickle cell disease.

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMß2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.

Sickle red blood cell-derived extracellular vesicles activate endothelial cells and enhance sickle red cell adhesion mediated by von Willebrand factor.

Endothelial activation and sickle red blood cell (RBC) adhesion are central to the pathogenesis of sickle cell disease (SCD). Quantitatively, RBC-derived extracellular vesicles (REVs) are more abundant from SS RBCs compared with healthy RBCs (AA RBCs). Sickle RBC-derived REVs (SS REVs) are known to promote endothelial cell (EC) activation through cell signalling and transcriptional regulation at longer terms. However, the SS REV-mediated short-term non-transcriptional response of EC is unclear. Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 h. Compared with AA REVs, SS REVs promoted human pulmonary microvascular ECs (HPMEC) activation indicated by increased von Willebrand factor (VWF) expression. Under microfluidic conditions, we found abnormal SS RBC adhesion to HPMECs exposed to SS REVs. This enhanced SS RBC adhesion was reduced by haeme binding protein haemopexin or VWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs. Consistent with these observations, haemin- or SS REV-induced microvascular stasis in SS mice with implanted dorsal skin-fold chambers that was inhibited by ADAMTS13. The adhesion induced by SS REVs was variable and was higher with SS RBCs from patients with increased markers of haemolysis (lactate dehydrogenase and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Our results emphasise the critical contribution made by REVs to the pathophysiology of SCD by triggering acute microvascular EC activation and abnormal RBC adhesion. These findings may help to better understand acute pathophysiological mechanism of SCD and thereby the development of new treatment strategies using VWF as a potential target.

MASP-2 and MASP-3 inhibitors block complement activation, inflammation, and microvascular stasis in a murine model of vaso-occlusion in sickle cell disease.

Patients with sickle cell disease (SCD) have ongoing hemolysis that promotes endothelial injury, complement activation, inflammation, vaso-occlusion, ischemia-reperfusion pathophysiology, and pain. Complement activation markers are increased in SCD in steady-state and further increased during vaso-occlusive crisis (VOC). However, the mechanisms driving complement activation in SCD have not been completely elucidated. Ischemia-reperfusion and heme released from hemoglobin during hemolysis, events that characterize SCD pathophysiology, can activate the lectin pathway (LP) and alternative pathway (AP), respectively. Here we evaluated the role of LP and AP in Townes sickle (SS) mice using inhibitory monoclonal antibodies (mAb) to mannose binding lectin (MBL)-associated serine protease (MASP)-2 or MASP-3, respectively. Townes SS mice were pretreated with MASP-2 mAb, MASP-3 mAb, isotype control mAb, or PBS before they were challenged with hypoxia-reoxygenation or hemoglobin. Pretreatment of SS mice with MASP-2 or MASP-3 mAb, markedly reduced Bb fragments, C4d and C5a in plasma and complement deposition in the liver, kidneys, and lungs collected 4 hours after challenge compared to control mAb-treated mice. Consistent with complement inhibition, hepatic inflammation markers NF-ĸB phospho-p65, VCAM-1, ICAM-1, and E-selectin were significantly reduced in SS mice pretreated with MASP-2 or MASP-3 mAb. Importantly, MASP-2 or MASP-3 mAb pretreatment significantly inhibited microvascular stasis (vaso-occlusion) induced by hypoxia-reoxygenation or hemoglobin. These studies suggest that the LP and the AP are both playing a role in promoting inflammation and vaso-occlusion in SCD. Inhibiting complement activation via the LP or the AP might inhibit inflammation and prevent VOC in SCD patients.

Plasma-Derived Hemopexin as a Candidate Therapeutic Agent for Acute Vaso-Occlusion in Sickle Cell Disease: Preclinical Evidence.

People living with sickle cell disease (SCD) face intermittent acute pain episodes due to vaso-occlusion primarily treated palliatively with opioids. Hemolysis of sickle erythrocytes promotes release of heme, which activates inflammatory cell adhesion proteins on endothelial cells and circulating cells, promoting vaso-occlusion. In this study, plasma-derived hemopexin inhibited heme-mediated cellular externalization of P-selectin and von Willebrand factor, and expression of IL-8, VCAM-1, and heme oxygenase-1 in cultured endothelial cells in a dose-responsive manner. In the Townes SCD mouse model, intravenous injection of free hemoglobin induced vascular stasis (vaso-occlusion) in nearly 40% of subcutaneous blood vessels visualized in a dorsal skin-fold chamber. Hemopexin administered intravenously prevented or relieved stasis in a dose-dependent manner. Hemopexin showed parallel activity in relieving vascular stasis induced by hypoxia-reoxygenation. Repeated IV administration of hemopexin was well tolerated in rats and non-human primates with no adverse findings that could be attributed to human hemopexin. Hemopexin had a half-life in wild-type mice, rats, and non-human primates of 80-102 h, whereas a reduced half-life of hemopexin in Townes SCD mice was observed due to ongoing hemolysis. These data have led to a Phase 1 clinical trial of hemopexin in adults with SCD, which is currently ongoing.

Soluble MD-2 and Heme in Sickle Cell Disease Plasma Promote Pro-Inflammatory Signaling in Endothelial Cells.

Recent evidence indicates that hemolysis in sickle cell disease (SCD) promotes inflammation via innate immune signaling through toll-like receptor 4 (TLR4). Free heme released by hemolyzed red blood cells can bind to myeloid differentiation factor-2 (MD-2) and activate TLR4 pro-inflammatory signaling on endothelium to promote vaso-occlusion and acute chest syndrome in murine models of SCD. MD-2 is co-expressed with TLR4 on cell membranes, but in inflammatory conditions, soluble MD-2 (sMD-2) is elevated in plasma. sMD-2 levels were significantly increased in human and murine sickle (SS) plasma as compared to normal (AA) plasma. Human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells incubated with human SS plasma had significant increases in pro-inflammatory IL-8, IL-6, and soluble VCAM-1 secretion compared to endothelial cells incubated with AA plasma. The increase in HUVEC IL-8 secretion was blocked by depletion of sMD-2 from SS plasma and enhanced by the addition of sMD-2 to AA plasma. The TLR4 signaling inhibitor, TAK-242, inhibited HUVEC IL-8 secretion in response to SS plasma by 85%. Heme-agarose pull-down assays and UV/Vis spectroscopy demonstrated that heme binds to sMD-2. Hemopexin, a high affinity heme-binding protein, inhibited HUVEC IL-8 secretion induced by SS plasma or SS and AA plasma supplemented with sMD-2. These data suggest that sMD-2 bound to heme might play an important role in pro-inflammatory signaling by endothelium in SCD.

Noncanonical Roles of Caspase-4 and Caspase-5 in Heme-Driven IL-1ß Release and Cell Death.

Excessive release of heme from RBCs is a key pathophysiological feature of several disease states, including bacterial sepsis, malaria, and sickle cell disease. This hemolysis results in an increased level of free heme that has been implicated in the inflammatory activation of monocytes, macrophages, and the endothelium. In this study, we show that extracellular heme engages the human inflammatory caspases, caspase-1, caspase-4, and caspase-5, resulting in the release of IL-1ß. Heme-induced IL-1ß release was further increased in macrophages from patients with sickle cell disease. In human primary macrophages, heme activated caspase-1 in an inflammasome-dependent manner, but heme-induced activation of caspase-4 and caspase-5 was independent of canonical inflammasomes. Furthermore, we show that both caspase-4 and caspase-5 are essential for heme-induced IL-1ß release, whereas caspase-4 is the primary contributor to heme-induced cell death. Together, we have identified that extracellular heme is a damage-associated molecular pattern that can engage canonical and noncanonical inflammasome activation as a key mediator of inflammation in macrophages.

Vasculo-toxic and pro-inflammatory action of unbound haemoglobin, haem and iron in transfusion-dependent patients with haemolytic anaemias.

Increasing evidence suggests that free haem and iron exert vasculo-toxic and pro-inflammatory effects by activating endothelial and immune cells. In the present retrospective study, we compared serum samples from transfusion-dependent patients with ß-thalassaemia major and intermedia, hereditary spherocytosis and sickle cell disease (SCD). Haemolysis, transfusions and ineffective erythropoiesis contribute to haem and iron overload in haemolytic patients. In all cohorts we observed increased systemic haem and iron levels associated with scavenger depletion and toxic 'free' species formation. Endothelial dysfunction, oxidative stress and inflammation markers were significantly increased compared to healthy donors. In multivariable logistic regression analysis, oxidative stress markers remained significantly associated with both haem- and iron-related parameters, while soluble vascular cell adhesion molecule 1 (sVCAM-1), soluble endothelial selectin (sE-selectin) and tumour necrosis factor α (TNFα) showed the strongest association with haem-related parameters and soluble intercellular adhesion molecule 1 (sICAM-1), sVCAM-1, interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) with iron-related parameters. While hereditary spherocytosis was associated with the highest IL-6 and TNFα levels, ß-thalassaemia major showed limited inflammation compared to SCD. The sVCAM1 increase was significantly lower in patients with SCD receiving exchange compared to simple transfusions. The present results support the involvement of free haem/iron species in the pathogenesis of vascular dysfunction and sterile inflammation in haemolytic diseases, irrespective of the underlying haemolytic mechanism, and highlight the potential therapeutic benefit of iron/haem scavenging therapies in these conditions.

Identification of a Heme Activation Site on the MD-2/TLR4 Complex.

Myeloid differentiation factor-2 (MD-2) binds lipopolysaccharide (LPS) and initiates toll-like receptor-4 (TLR4) pro-inflammatory signaling. Heme also activates TLR4 signaling, but it is unknown if heme interacts with MD-2. Therefore, we examined MD-2 for a potential heme activation site. Heme-agarose and biotin-heme/streptavidin-agarose pulled down recombinant MD-2, which was inhibited by excess free heme. UV/visible spectroscopy confirmed MD-2-heme binding. To determine whether MD-2 was required for heme-mediated TLR4 signaling, HEK293 cells were transfected with MD-2, TLR4, CD14, and an NF-κB luciferase reporter, and then stimulated with heme or LPS. Heme or LPS treatment elicited robust reporter activity. Absence of MD-2, TLR4 or CD14 plasmid abolished NF-κB reporter responses to heme or LPS. In silico analysis identified two potential heme docking sites on MD-2 near conserved amino acids W23/S33/Y34 and Y36/C37/I44. Heme-induced NF-κB activity was reduced by 39 and 78% in HEK293 cells transfected with MD-2 mutants W23A and Y34A, respectively, compared to WT-MD-2. NF-κB activation by LPS was not affected by the same mutants. Biotinyl-heme/streptavidin-agarose pulled down 68% less W23A and 80% less W23A/S33A/Y34A mutant MD-2 than WT-MD-2. In contrast, at the Y36/C37/I44 MD-2 site, heme-induced NF-κB activity was significantly increased by mutants Y36A (191% of WT-MD-2) and unchanged by mutants C37A and I44A (95 and 92%, respectively, of WT-MD-2). In conclusion, these data suggest that heme binds and activates TLR4 signaling at amino acids W23 and Y34 on MD-2.

The multifaceted role of ischemia/reperfusion in sickle cell anemia.

Sickle cell anemia is a unique disease dominated by hemolytic anemia and vaso-occlusive events. The latter trigger a version of ischemia/reperfusion (I/R) pathobiology that is singular in its origin, cyclicity, complexity, instability, perpetuity, and breadth of clinical consequences. Specific clinical features are probably attributable to local I/R injury (e.g., stroke syndromes) or remote organ injury (e.g., acute chest syndrome) or the systematization of inflammation (e.g., multifocal arteriopathy). Indeed, by fashioning an underlying template of endothelial dysfunction and vulnerability, the robust inflammatory systematization no doubt contributes to all sickle pathology. In this Review, we highlight I/R-targeting therapeutics shown to improve microvascular blood flow in sickle transgenic mice undergoing I/R, and we suggest how such insights might be translated into human therapeutic strategies.

Antithrombotic effects of heme-degrading and heme-binding proteins.

In the murine venous thrombosis model induced by ligation of the inferior vena cava (IVCL), genetic deficiency of heme oxygenase-1 (HO-1) increases clot size. This study examined whether induction of HO-1 or administration of its products reduces thrombosis. Venous HO-1 upregulation by gene delivery reduced clot size, as did products of HO activity, biliverdin, and carbon monoxide. Induction of HO-1 by hemin reduced clot formation, clot size, and upregulation of plasminogen activator inhibitor-1 (PAI-1) that occurs in the IVCL model, while leaving urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) expression unaltered. The reductive effect of hemin on clot size required HO activity. The IVCL model exhibited relatively high concentrations of heme that peaked just before maximum clot size, then declined as clot size decreased. Administration of hemin decreased heme concentration in the IVCL model. HO-2 mRNA was induced twofold in the IVCL model (vs. 40-fold HO-1 induction), but clot size was not increased in HO-2-/- mice compared with HO-2+/+ mice. Hemopexin, the major heme-binding protein, was induced in the IVCL model, and clot size was increased in hemopexin-/- mice compared with hemopexin+/+ mice. We conclude that in the IVCL model, the heme-degrading protein HO-1 and HO products inhibit thrombus formation, as does the heme-binding protein, hemopexin. The reductive effects of hemin administration require HO activity and are mediated, in part, by reducing PAI-1 upregulation in the IVCL model. We speculate that HO-1, HO, and hemopexin reduce clot size by restraining the increase in clot concentration of heme (now recognized as a procoagulant) that otherwise occurs.NEW & NOTEWORTHY This study provides conclusive evidence that two proteins, one heme-degrading and the other heme-binding, inhibit clot formation. This may serve as a new therapeutic strategy in preventing and treating venous thromboembolic disease.

Decreased erythrocyte binding of Siglec-9 increases neutrophil activation in sickle cell disease.

Oxidative stress and inflammation promote vaso-occlusion in sickle cell disease (SCD). CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids inhibit innate immune cell functions. We have shown that Siglec-9 on human neutrophils interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactive oxygen species (ROS). We hypothesized that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and thus a decreased neutrophil oxidative burst. SS erythrocytes express significantly more sialic acid than AA erythrocytes (p = 0.02). SS erythrocytes displayed significantly less Siglec-9-Fc binding 39% ± 11 (mean ± SEM) compared to AA erythrocytes 78% ± 5 (p = 0.009). Treatment of AA erythrocytes with sialidase to remove sialic acid decreased binding to 3% ± 7.9 (p ≤ 0.001). When freshly isolated neutrophils were incubated with AA erythrocytes, neutrophils achieved 16% ± 6 of the oxidative burst exhibited by a stimulated neutrophil without erythrocytes. In contrast, neutrophils incubated with SS erythrocytes achieved 47% ± 6 of the oxidative burst (AA versus SS, p = 0.03). Stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation while with SS erythrocytes NETs increased. SS erythrocytes are deficient in binding to neutrophil Siglec-9 which may contribute to the increased oxidative stress in SCD.

Thrombin activation of PAR-1 contributes to microvascular stasis in mouse models of sickle cell disease.

Vaso-occlusive crisis (VOC) is the primary cause of morbidity and hospitalization in sickle cell disease (SCD); however, only 4 therapies (hydroxyurea, l-glutamine, crizanlizumab, and voxeletor) are currently approved in SCD. These agents limit the duration, severity, and frequency of crises. Activation of coagulation is a hallmark of SCD. Studies in animal models of SCD have shown that coagulation contributes to the chronic inflammation and end-organ damage associated with the disease; however, it is unknown whether coagulation directly contributes to the microvascular stasis that causes VOC. Herein, we demonstrate that inhibition of tissue factor (TF) and the downstream coagulation proteases factor Xa and thrombin significantly attenuates heme-induced microvascular stasis in mouse models of VOC. Pharmacologic inhibition of the principal thrombin receptor, protease activated receptor-1 (PAR-1), as well as deficiency of PAR-1 in all nonhematopoietic cells, also reduces stasis in sickle mice. PAR-1 deficiency was associated with reduced endothelial von Willebrand factor expression, which has been shown to mediate microvascular stasis. In addition, TF inhibition reduces lung vaso-occlusion in sickle mice mediated by arteriolar neutrophil-platelet microemboli. In sum, these results suggest that prophylactic anticoagulation might attenuate the incidence of VOC.

Endothelial TLR4 Expression Mediates Vaso-Occlusive Crisis in Sickle Cell Disease.

Heme, released from red blood cells in sickle cell disease (SCD), interacts with toll-like receptor 4 (TLR4) to activate NF-κB leading to the production of cytokines and adhesion molecules which promote inflammation, pain, and vaso-occlusion. In SCD, TLR4 inhibition has been shown to modulate heme-induced microvascular stasis and lung injury. We sought to delineate the role of endothelial verses hematopoietic TLR4 in SCD by developing a TLR4 null transgenic sickle mouse. We bred a global Tlr4-/- deficiency state into Townes-AA mice expressing normal human adult hemoglobin A and Townes-SS mice expressing sickle hemoglobin S. SS-Tlr4-/- had similar complete blood counts and serum chemistries as SS-Tlr4+/+ mice. However, SS-Tlr4-/- mice developed significantly less microvascular stasis in dorsal skin fold chambers than SS-Tlr4+/+ mice in response to challenges with heme, lipopolysaccharide (LPS), and hypoxia/reoxygenation (H/R). To define a potential mechanism for decreased microvascular stasis in SS-Tlr4-/- mice, we measured pro-inflammatory NF-κB and adhesion molecules in livers post-heme challenge. Compared to heme-challenged SS-Tlr4+/+ livers, SS-Tlr4-/- livers had lower adhesion molecule and cytokine mRNAs, NF-κB phospho-p65, and adhesion molecule protein expression. Furthermore, lung P-selectin and von Willebrand factor immunostaining was reduced. Next, to establish if endothelial or hematopoietic cell TLR4 signaling is critical to vaso-occlusive physiology, we created chimeric mice by transplanting SS-Tlr4-/- or SS-Tlr4+/+ bone marrow into AA-Tlr4-/- or AA-Tlr4+/+ recipients. Hemin-stimulated microvascular stasis was significantly decreased when the recipient was AA-Tlr4-/- . These data demonstrate that endothelial, but not hematopoietic, TLR4 expression is necessary to initiate vaso-occlusive physiology in SS mice.

A novel, highly potent and selective phosphodiesterase-9 inhibitor for the treatment of sickle cell disease.

The most common treatment for patients with sickle cell disease (SCD) is the chemotherapeutic hydroxyurea, a therapy with pleiotropic effects, including increasing fetal hemoglobin (HbF) in red blood cells and reducing adhesion of white blood cells to the vascular endothelium. Hydroxyurea has been proposed to mediate these effects through a mechanism of increasing cellular cGMP levels. An alternative path to increasing cGMP levels in these cells is through the use of phosphodiesterase-9 inhibitors that selectively inhibit cGMP hydrolysis and increase cellular cGMP levels. We have developed a novel, potent and selective phosphodiesterase-9 inhibitor (IMR-687) specifically for the treatment of SCD. IMR-687 increased cGMP and HbF in erythroid K562 and UT-7 cells and increased the percentage of HbF positive erythroid cells generated in vitro using a two-phase liquid culture of CD34+ progenitors from sickle cell blood or bone marrow. Oral daily dosing of IMR-687 in the Townes transgenic mouse SCD model, increased HbF and reduced red blood cell sickling, immune cell activation and microvascular stasis. The IMR-687 reduction in red blood cell sickling and immune cell activation was greater than that seen with physiological doses of hydroxyurea. In contrast to other described phosphodiesterase-9 inhibitors, IMR-687 did not accumulate in the central nervous system, where it would inhibit phosphodiesterase-9 in neurons, or alter rodent behavior. IMR-687 was not genotoxic or myelotoxic and did not impact fertility or fetal development in rodents. These data suggest that IMR-687 may offer a safe and effective oral alternative for hydroxyurea in the treatment of SCD.

Heme oxygenase-2 protects against ischemic acute kidney injury: influence of age and sex.

Heme oxygenase (HO) activity is exhibited by inducible (HO-1) and constitutive (HO-2) proteins. HO-1 protects against ischemic and nephrotoxic acute kidney injury (AKI). We have previously demonstrated that HO-2 protects against heme protein-induced AKI. The present study examined whether HO-2 is protective in ischemic AKI. Renal ischemia was imposed on young and aged HO-2+/+ and HO-2-/- mice. On days 1 and 2 after renal ischemia, there were no significant differences in renal function between young male HO-2+/+ and HO-2-/- mice, between young female HO-2+/+ and HO-2-/- mice, or between aged female HO-2+/+ and HO-2-/- mice. However, in aged male mice, HO-2 deficiency worsened renal function on days 1 and 2 after ischemic AKI, and, on day 2 after ischemia, such deficiency augmented upregulation of injury-related genes and worsened histological injury. Renal HO activity was markedly decreased in unstressed aged male HO-2-/- mice and remained so after ischemia, despite exaggerated HO-1 induction in HO-2-/- mice after ischemia. Such exacerbation of deficiency of HO-2 protein and HO activity may reflect phosphorylated STAT3, as activation of this proinflammatory transcription factor was accentuated early after ischemia in aged male HO-2-/- mice. This exacerbation may not reflect impaired induction of nephroprotectant genes, since the induction of HO-1, sirtuin 1, and ß-catenin was accentuated in aged male HO-2-/- mice after ischemia. We conclude that aged male mice are hypersensitive to ischemic AKI and that HO-2 mitigates such sensitivity. We speculate that this protective effect of HO-2 may be mediated, at least in part, by suppression of phosphorylated STAT3-dependent signaling.

Critical role of C5a in sickle cell disease.

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD.

Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles.

The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes-sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent ß-globin posttranslational modifications, including the irreversible oxidation of ßCys93 and the ubiquitination of ßLys96 and ßLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, ßCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients.