Transgenic Sickle Mice Research Articles

Dimethyl Fumarate Induces Cytoprotection and Inhibits Inflammation and Vaso-Occlusion in Transgenic Sickle Mice

Patients with sickle cell disease have unrelenting hemolysis leading to the release of hemoglobin and heme into the vasculature that promote oxidative stress, inflammation, vaso-occlusive pain crises, ischemia-reperfusion injury and organ damage. Induction of the cytoprotective heme metabolizing enzyme heme oxygenase-1 (HO-1), the iron-binding protein ferritin heavy chain or administration of the HO-1 metabolite CO induces cytoprotective responses that inhibit oxidative stress, inflammation, vaso-occlusion and organ damage in transgenic sickle mice expressing human βS globins. The master regulator of these anti-oxidative and cytoprotective responses is nuclear factor erythroid 2-related factor (Nrf2). Nrf2 activity is controlled, in part, by the cytosolic protein, kelch-like ECH-associated protein 1 (Keap1). Nrf2 is anchored in the cytoplasm through binding with Keap1 which results in its ubiquitination and subsequent proteosomal degradation. Upon exposure to stress stimuli, such as reactive oxygen species and electrophiles, Nrf2 is stabilized and able to translocate to the nucleus where it trans-activates target genes that possess an antioxidant responsive element (ARE) in their promoter regions. Recently the FDA and their European and Canadian counterparts approved dimethyl fumarate (Tecfidera) for the treatment of relapsing multiple sclerosis. Dimethyl fumarate (DMF) and its metabolite monomethyl fumarate alkylate a critical reactive thiol Cys-151 on Keap1 causing release of Nrf2, nuclear localization and activation of cellular anti-oxidant and cytoprotective responses. Based on our previous results showing cytoprotection through HO-1 and it products, we evaluated DMF responses in NY1DD transgenic sickle mice. DMF (15mg/kg) or vehicle (0.08% methyl cellulose) was administered by oral gavage BID X 3 days to NY1DD mice. On the first day of treatment, mice were implanted with dorsal skin-fold chambers. One hour after the last treatment, 20-30 flowing subcutaneous venules were selected and mapped in the dorsal skin-fold chamber window followed by infusion of heme (3.2 µmols/kg) into the tail vein. All of the selected venules were re-examined at 1 and 4 hours post-infusion and the number of static (no flow) venules were counted and expressed as percent stasis. After the 4 hour stasis measurement, blood was collected and organs were removed and flash frozen. In sickle mice treated with vehicle, microvascular stasis was 29% and 24% at 1 and 4 hours, respectively (Fig. 1). In contrast, in sickle mice treated with DMF, stasis was 6% and 4% at 1 and 4 hours (p<0.001). There also was a marked increase in nuclear Nrf2 in liver nuclear extracts of DMF-treated sickle mice on Western blots. Analysis of liver mRNA by qRT-PCR revealed that there was a 2.4 to 4.4-fold enrichment of mRNA coding for putative Nrf2-responsive antioxidant proteins in DMF-treated sickle mice compared to vehicle treated mice (Table 1). This was accompanied by a 3.3-fold increase in HO-1 activity (p<0.05), a distinct increase in cytoplasmic ferritin heavy chain on Western blot and a marked decrease in nuclear NF-κB phospho-p65 activation on Western blot of liver nuclear extracts of DMF treated sickle mice versus vehicle controls. Red blood cells and white blood cells isolated from DMF-treated sickle mice had significantly decreased adhesion to resting and heme- and TNF-activated HUVEC compared to blood cells from mice treated with vehicle. We conclude that DMF may be an effective agent to prevent sickle crises through induction of HO-1, ferritin and other antioxidant proteins, and inhibition of vaso-occlusion. These data combined with prior evidence that monomethyl fumarate can induce hemoglobin F make it an idea candidate for clinical trials in SCD patients.Table 1Enrichment of mRNA for Antioxidant Proteins in DMF-treated LiversmRNAFold enrichmentP valueHO-12.40.08Hemopexin2.50.023Ferritin Heavy Chain2.6<0.001Haptoglobin2.70.046Nrf22.70.004Ferroportin3.1<0.001Glutamate#Cysteine Ligase Catalytic Subunit3.3<0.001Glutathione S-Transferase A23.40.032NAD(P)H Dehydrogenase [Quinone] 13.80.008Glutathione S-Transferase Mu 13.8<0.001Multidrug Resistance-associated Protein 24.4<0.001 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MEK Inhibitors, Novel Anti-Adhesive Molecules, Reverse Sickle Red Blood Cell Adhesion and Vaso-Occlusion in Vivo

In sickle cell disease (SCD), painful vaso-occlusive crises and end-organ damage are caused by obstruction of the vessels due in large part to sickle erythrocyte (SSRBC) adherence to both the endothelium and leukocytes. Despite the contribution of SSRBC adhesiveness to vaso-occlusion, we lack treatments that specifically target intracellular signaling mechanisms critical for SSRBC cell-cell interactions. We have previously shown that the mitogen-activated protein (MAP) kinase ERK1/2 is aberrantly activated in SSRBCs. ERK1/2 signaling activation is critical for SSRBC interactions with the vascular endothelium and leukocytes. We have now evaluated the anti-adhesive properties of RDEA119, an inhibitor of MEK1/2, the kinase that activates the MAP kinase ERK1/2, in in vitro adhesion assays and in vivo in both nude mouse and humanized models of vaso-occlusion analyzed by intravital microscopy. We used RDEA119 because this dryg has previously been developed for human use. We found that RDEA119 caused a dose-dependent inhibition of human SSRBC adhesion to cultured TNFα-activated human micro-vascular endothelial cells (HMVECs) in vitro with a half-maximum inhibitory concentration (IC50) &lt; 2 nmol/L. This suggests that RDEA119 would be effective at very low doses in reducing SSRBC adhesion and vaso-occlusion in vivo. To determine if RDEA119 prevents vaso-occlusion in nude mice, animals were injected with TNFα followed by infusion of RDEA119 (or placebo control), and then human SSRBCs. Intravital microscopy observations of enflamed vessels visible through dorsal skin-fold window chamber implants immediately after human SSRBC infusion showed persistent human SSRBC adhesion to both small and large vessels, with mean diameter of 29±3.5 μm, and vaso-occlusion in placebo-treated nude mice for at least 2 h, promoting blood stasis. However, treatment of nude mice with either 0.2 mg/kg or 2 mg/kg RDEA119 markedly reduced human SSRBC adhesion and mitigated vaso-occlusion. While human SSRBC adhered to 68.5±4.6% of total vessels recorded in placebo-treated animals, SSRBCs occupied only 33 ± 6.6% (p&lt;0.0001) and 25 ± 3.7% (p&lt;0.0001) of vessels in 0.2 and 2 mg/kg RDEA119-treated nude mice, respectively. As a result, normal blood flow was restored in 95±3.5% and 76±6.2% of vessels in 0.2 and 2 mg/kg RDEA119-treated animals, respectively, compared to 43±5.8% of vessels with normal blood flow in placebo-treated mice (p&lt;0.001). Nude mice treated with 0.2 mg/kg RDEA119 also exhibited reduced human SSRBC trapping in the lungs, liver and spleen (p&lt;0.0001), and an increased number of human SSRBCs remaining in circulation. RDEA119 was also administered to nude mice after onset of vaso-occlusion for a more clinically relevant outcome. Infusion of a single dose of 1 mg/kg RDEA119 to nude mice reduced human SSRBC adhesion and vaso-occlusion within 5 min of RDEA119 administration, and adhesion progressively decreased thereafter compared to SSRBC adhesion and vaso-occlusion in these same vessel segments prior to drug administration (p&lt;0.0001). RDEA119 led to a 72% reduction in the percentage of vessels occupied by adherent human SSRBCs (p&lt;0.001) and improvement in blood flow in 89±2.4% of vessels (p&lt;0.001). Similar therapeutic benefits were obtained when RDEA119 was administered after the inflammatory trigger of TNFα to induce vaso-occlusion in transgenic sickle mice. RDEA119 resulted in a significant decrease in adherent sickle cells in enflamed vessels of sickle mice treated with only one dose of RDEA119 at 0.1, 0.05 or 0.025 mg/kg (p&lt;0.001). RDEA119 at 0.1, 0.05 or 0.025 mg/kg also significantly reduced adhesion of leukocytes to the vascular endothelium compared with placebo-treated sickle mice (p&lt;0.001). The effect of RDEA119 on both sickle RBCs and leukocyte adhesion was rapid, because a reduction in adhesion of the cells was already detectable within the first 15 minutes after drug infusion, and adhesion further decreased thereafter. In contrast, adherence of leukocytes increased progressively over time in placebo-treated sickle mice. Treatment of sickle mice with RDEA119 led to improved microcirculatory blood flow in enflamed vessels visible through the dorsal skin-fold window chamber and improved survival. These results suggest that RDEA119 may represent a valuable novel therapeutic intervention for acute pain crises that should be further evaluated in humans with SCD. Disclosures No relevant conflicts of interest to declare.

Therapeutic Amelioration of Vaso-Occlusion and Blood Flow Impairment in Transgenic Sickle Mice By the Antioxidant Flavonoid Quercetin

Sickle cell disease (SCD) is characterized by painful vaso-occlusive crises, which are, at least in part, due to an interaction of the sickle RBC (sRBC) with the vascular endothelium. Abnormal red blood cells (RBCs) impair blood flow and contribute to microcirculatory complications. Oxidative stress and/or oxidants generated via hemoglobin S (HbS) auto-oxidation play a vital role in the vaso-occlusive event in SCD. Antioxidant therapy mediated free radical scavenging and attenuation of oxidative stress may reduce red cell sickling and be beneficial for SCD. Several studies have described an antioxidant effect of flavonoids on the attenuation of free radical mediated biological membrane damage and the consumption of flavonoids reduces the prevalence of vascular diseases. Among flavonoids, quercetin (QUE) pentahydroxy flavone is the major representative. In vitro, QUE is a strong antioxidant with alkoxyl and peroxy radical scavenging ability. Due to the high susceptibility of sickle RBC to oxidation, QUE could be a useful therapy for SCD. Based on this concept, we examined the potential effect of QUE to improve microvascular function in a murine model of SCD.Methods: To confirm the protective effect of quercetin in vivo, we used Berkeley (Berk) sickle transgenic mice which express exclusively human α- and βS-globins with low levels of γ-globin (∼ 3-5%) generated by Paszty et al 1997. C57BL /6J were used as control wild type. We injected a single dose of QUE at different concentrations (50, 100, 200mg/kg body weight) intraperitoneally under normoxic conditions. Three hours after QUE administration, in vivo intra-vital microscopic observation of post-capillary venules in cremaster muscle was performed. The luminal diameters of the venules (∼ 20-40 µm diameter), centerline red blood cell velocity (Vrbc), adherent, emigrated and rolling leukocytes were measured by the technique described by Kaul et al 2004. Wall shear rate was calculated by Lipowsky et al, 1980.Results: QUE treatment restored blood flow, as evidenced by complete disappearance of vaso-occlusion in the postcapillary venules of Berk mice (Figure 1). However, no significant differences in venular diameter were noted with QUE treatment at any of the dose levels tested (50, 100, 200mg/kg) when compared to untreated Berk and wild type mice. But, when compared to untreated Berk mice, a significant increase in the RBC velocity was demonstrated in a dose dependent fashion (treated: 1.74 ±1.3 mm/sec, 3.02± 1.2 mm/sec, 3.4±0.90 mm/sec for 50, 100, 200 mg/kg dosing respectively vs. untreated 1.01± 1.05mm/sec, p<0.05). A dose of 200 mg level completely neutralized the vaso-occlusion. Increases in wall shear rate (650.01± 252.05 s-1 vs. 180.12± 165.02 s-1, p<6.03x10-6) was also observed in QUE treated vs. untreated Berk. This improvement of blood flow in the postcapillary venules correlated well with observed decreases in leukocyte adhesion (Figure 2A) and leukocyte emigration (Figure 2B) in QUE treated Berk mice (for doses 50, 100, and 200mg/kg) when compared to untreated Berk mice. Leukocyte rolling was also decreased for doses 100 and 200mg/kg (p<0.007, p<0.0002 respectively) after treatment with QUE when compared to untreated Berk and wild type. [Display omitted] [Display omitted] [Display omitted] Conclusion: We observed an improvement in RBC velocity and wall shear rate, as well as a complete attenuation of leukocyte adhesion, rolling and emigration at the highest dose of QUE treated transgenic sickle Berk mice. We suggest that these effects may be due to a decreased sickle RBC interaction with the vascular bed. Our present data provide a strong basis for the therapeutic application of flavonoids in SCD. Further studies are needed to better understand the mechanism of action in vivo for therapeutic effect in SCD. DisclosuresThangaswamy:AMI Life Sciences Private Ltd: Drug supplied Other. Billett:Selexys Pharmaceuticals: Research Funding.

Targeted Disruption of Nrf2 in Non-Hematopoietic Tissues Exacerbates Hemolysis and Accelerates Chronic Lung Disease in Transgenic Sickle Cell Anemia Mice

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates the cellular defense mechanism by mediating a coordinate induction of cytoprotective antioxidant responsive element-driven genes. Nrf2 agonists augment fetal hemoglobin expression in hematopoietic progenitors and have emerged as a new class of drugs for therapeutic induction of fetal hemoglobin in sickle cell anemia (SCA). However, the cytoprotective effect of Nrf2 on the pathobiology of SCA has not been previously defined. To investigate the role and mechanism of Nrf2 in SCA independent of globin gene modulation, we generated chimeric mice with disruption of Nrf2 in non-hematopoietic tissues. A total of twenty-six Nrf2-/- mice were transplanted with bone marrow harvested from the Berkeley sickle and hemizygous (Hemi) mice and the Townes sickle (SS) and control (AA) mice. All sickle/Nrf2-null chimeras (SSNHNrf2-/-; n=13) developed classical hematological and intravascular hemolysis features of SCA after eight weeks of transplantation. Despite the presence of erythrocyte Nrf2, SSNHNrf2-/- chimeras developed more severe intravascular hemolysis than SS wild-type (SSWT) donor-litter-mates. The concentration of plasma cell-free Hb (p<0.01), plasma heme (p<0.05) and lactate dehydrogenase (p<0.05) were 1.5 to 2.0-fold higher in the chimeras than in the SSWT mice. The chimeras had markedly higher concentrations of soluble vascular cell adhesion molecule-1 (p<0.01) and soluble P-selectin in their plasma (p<0.05), and more oxidizing extracellular redox status (p<0.05). The latter finding was corroborated by markedly lower reduced-glutathione in the SSNHNrf2-/- mouse lungs (19.07±0.32 vs 26.5 ±1.5 nmol/mg protein, p<0.01). The worsening of hemolysis, vascular inflammation and oxidative stress in the SSNHNrf2-/- chimeras was unrelated to the transplantation procedure since none of these effects were seen in the AANHNrf2-/- and HemiNHNrf2-/- controls. Next, we found that the SSNHNrf2-/- chimeras had hypoxemia at baseline (oxygen saturation, 95.8±1.7%) while age-matched SSWT mice had normal oxygen saturation (99.2±0.2%, p<0.05). Consistent with these disparate pulmonary phenotypes, the SSNHNrf2-/- but not the SSWT mice developed pulmonary edema at a relatively young age, which was confirmed by histological evidence of alveolar edema. Remarkably, the severely damaged SSNHNrf2-/- mouse lungs contained a similar quantity of heme oxygenase-1 (HO-1) mRNA and protein as the relatively less inflamed SSWT lungs. In addition, the amount of HO-1 in the plasma was also similar in these animals. These results indicate that other transcription factors modulate HO-1 induction, and that HO-1 may play a lesser role in mitigating the chronic effects of SCA. To test the latter idea, we performed a longitudinal study in a cohort of thirty-two SS and AA mice of both sexes. At 8 weeks of age, plasma HO-1 was 3-fold higher in both male and female SS mice than in the AA mice (p<0.001). The HO-1 values did not change at 12, 24 and 40 weeks in any group, however intravascular hemolysis, vascular inflammation and endothelial barrier integrity deteriorated progressively only in the SSWT mice. Overall, our results demonstrate that the elevated HO-1 expression typical of SCA is insufficient to blunt many of the chronic effects of the disease in mice. However, other Nrf2 target genes in non-hematopoietic tissues can slow down this process. Such genes offer an attractive prophylactic therapeutic strategy to preserve organ function in adults with SCA. DisclosuresArcher:Global Blood Therapeutics: Consultancy.

Regulation of Na+/Mg2+ Exchange in Sickle Erythrocytes By Endothelin-1

Excess levels of endothelin-1 (ET-1), erythrocyte sickling and chronic inflammation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have shown that ET-1 receptor antagonists improve hematological parameters by reducing Gardos channel activity in two transgenic mouse models of SCD while reducing oxidant stress by decreasing circulating levels of protein disulfide isomerase. Magnesium (Mg2+) deficiency, mediated in part via increased erythrocyte Na+/Mg2+ exchanger activity, has been demonstrated to contribute to erythrocyte dehydration, K+ loss and sickling in SCD. However, the relationship between ET-1 and the Na+/Mg2+ exchanger in SCD remains unclear. We measured Na+/Mg2+ exchange activity in ex vivo red cells and observed increased activity following in vitro incubation of human (2.2 ± 0.2 to 3.2 ± 0.1 mmol/1013 cell x h, P<0.03, n=5) and mouse red blood cells with ET-1 (P<0.001, n=5); events that were significantly blocked by pre-incubation of cells with 1 μM BQ788, a selective inhibitor of ET-1 type B receptors. In addition, in vitro deoxygenation of sickle red cells led to increased exchanger activity that was inhibited by impramine, a Na+/Mg2+ exchange inhibitor, and associated with reduced deoxygenation-stimulated sickle cell dehydration. These results suggest an important role for ET-1 and cellular magnesium homeostasis in sickle cell disease. To this end, we studied Na+/Mg2+ exchange activity in ex vivo erythrocytes from three transgenic sickle mouse models and observed increased activity in these cells when compared to red cells from either Hb A transgenic or C57BL/J6 wild-type mice (P<0.03, n=4). We then tested the in vivo effects of ET-1 receptor antagonists on erythrocyte Na+/Mg2+ exchange activity in the BERK mouse, a transgenic model of SCD. We blocked ET-1 receptors type A and B by in vivo treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed lower erythrocyte exchanger activity when compared to cells from vehicle treated BERK mice (P<0.02, n=6). Thus our results suggest that ET-1 receptor blockade represents an important therapeutic approach to control erythrocyte volume and magnesium homeostasis that may lead to improved inflammatory and vascular complications observed in SCD. Supported by NIH R01HL090632 to AR. DisclosuresNo relevant conflicts of interest to declare.

H-ferritin ferroxidase induces cytoprotective pathways and inhibits microvascular stasis in transgenic sickle mice

Hemolysis, oxidative stress, inflammation, vaso-occlusion, and organ infarction are hallmarks of sickle cell disease (SCD). We have previously shown that increases in heme oxygenase-1 (HO-1) activity detoxify heme and inhibit vaso-occlusion in transgenic mouse models of SCD. HO-1 releases Fe2+ from heme, and the ferritin heavy chain (FHC) ferroxidase oxidizes Fe2+ to catalytically inactive Fe3+ inside ferritin. FHC overexpression has been shown to be cytoprotective. In this study, we hypothesized that overexpression of FHC and its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic SCD mice in response to plasma hemoglobin. We utilized a Sleeping Beauty (SB) transposase plasmid to deliver a human wild-type-ferritin heavy chain (wt-hFHC) transposable element by hydrodynamic tail vein injections into NY1DD SCD mice. Control SCD mice were infused with the same volume of lactated Ringer’s solution (LRS) or a human triple missense FHC (ms-hFHC) plasmid with no ferroxidase activity. 8 weeks later, LRS-injected mice had ~40% microvascular stasis (% non-flowing venules) 1 h after infusion of stroma-free hemoglobin, while mice overexpressing wt-hFHC had only 5% stasis (p < 0.05), and ms-hFHC mice had 33% stasis suggesting vascular protection by ferroxidase active wt-hFHC. The wt-hFHC SCD mice had marked increases in splenic hFHC mRNA and hepatic hFHC protein, ferritin light chain (FLC), 5-aminolevulinic acid synthase (ALAS), heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), and HO-1 activity and protein. There was also a decrease in hepatic activated nuclear factor-kappa B (NF-κB) phospho-p65 and vascular cell adhesion molecule-1 (VCAM-1). Inhibition of HO-1 activity with tin protoporphyrin demonstrated HO-1 was not essential for the protection by wt-hFHC. We conclude that wt-hFHC ferroxidase activity enhances cytoprotective Nrf2-regulated proteins including HO-1, thereby resulting in decreased NF-κB-activation, adhesion molecules, and microvascular stasis in transgenic SCD mice.

MicroRNA‐301b reduces PPARγ expression in transgenic sickle mice and in hemin‐treated human pulmonary artery endothelial cells (1089.22)

Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis resulting in the release of hemin into the blood. Our preliminary microarray data indicate that PPARγ expression is significantly reduced in hemin‐treated human pulmonary artery endothelial cells (HPAECs). To examine post‐transcriptional mechanisms of PPARγ regulation in pulmonary hypertension in SCD (SCD‐PH), Townes knockin SCD mice aged 12‐weeks were gavaged daily with vehicle or rosiglitazone (RSG, 10 mg/kg/day) for 10 d. PPARγ was decreased and ET‐1 expression was increased in sickle mice (SS) compared to littermate control mice (AA). Using an in vitro model that mimics acute extracellular hemin release, HPAECs were treated with DMSO or hemin (5 μM) for 72 h ± RSG (10 μM) for the final 24 h. Similarly, hemin reduced PPARγ and increased ET‐1 expression in HPAECs. MicroRNA (miR‐301b), which negatively regulates PPARγ, was increased in SCD mice and in hemin‐treated HPAECs. In contrast, PPARγ activation attenuates increases in miR‐301b levels in SS mice and in hemin‐induced HPAECs. These findings suggest that hemolysis in SCD increases miR‐301b to reduce PPARγ and increase ET‐1 levels. These results suggest novel pathways for SCD‐PH pathogenesis and therapy.Grant Funding Source: Supported by AHA‐SDG 13SDG14150004,CEB F16788‐00, Atlanta VA 1I01BX001910, NIH HL074518, DK102167

Sickle cell transgenic (HbSS) mice lungs produce greater inflammatory mediators with downregulation of dilator proteins (1175.10)

Sickle cell chronic lung disease is one of the notable causes of death in patients with sickle cell disease (SCD). Prevention and treatment of acute chest syndrome (ACS) constitutes a major concern in clinical management of SCD. Some lung complications in SCD result from direct or indirect effect of pulmonary microvascular occlusion by sickle erythrocytes and their adhesion to the vascular endothelium. Vasoactive eicosanoids are released resulting in inflammatory response. Studies with HbSS mice have enhanced knowledge of SCD pathology, but the biochemical mechanisms of initiation and progression of ACS is not well understood. We hypothesized that lungs of SCD patients are under greater inflammatory stress due to decreased expression of anti‐inflammatory proteins and metabolites, but greater inflammatory proteins and metabolites. We studied baseline expression of 5‐Lox, cysLT1 as well as PKA, COX1 and COX2 and CREB and measured Leukotrienes and PGI2 release by lungs of HbSS mice and transgenic HbAA mice. HbSS mice lungs expressed 40‐60% more 5‐Lox and CysLT1, but 40‐70% less COX1/COX2 and CREB proteins; produced 68% greater LTs, but PGI2 was not detected. Expression of PKA was not different. The data suggest that SCD lungs are under greater inflammatory stress which may trigger ACS and sustain sickle cell chronic lung disease in SCD patients.Grant Funding Source: LA Biomed Seed grant 520522

Sildenafil promotes eNOS activation and inhibits NADPH oxidase in the transgenic sickle cell mouse penis.

Sickle cell disease (SCD)-associated vasculopathy in the penis is characterized by aberrant nitric oxide and phosphodiesterase (PDE) 5 signaling, and by increased oxidative stress. Preliminary clinical trials show that continuous treatment with PDE5 inhibitor sildenafil unassociated with sexual activity decreases priapic activity in patients with SCD. However, the mechanism of its vasculoprotective effect in the penis remains unclear. We evaluated whether continuous administration of PDE5 inhibitor sildenafil promotes eNOS function at posttranslational levels and decreases superoxide-producing enzyme NADPH oxidase activity in the sickle cell mouse penis. SCD transgenic mice were used as an animal model of SCD. WT mice served as controls. Mice received treatment with the PDE5 inhibitor sildenafil (100 mg/kg/day) or vehicle for 3 weeks. eNOS phosphorylation on Ser-1177 (positive regulatory site), eNOS interactions with heat-shock protein 90 (HSP90) (positive regulator), phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177), an NADPH oxidase catalytic subunit gp91(phox), and a marker of oxidative stress (4-hydroxy-2-nonenal [HNE]) were measured by Western blot. Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse. Continuous treatment with sildenafil reversed (P < 0.05) the abnormalities in protein expressions of P-eNOS (Ser-1177), eNOS/HSP90 interaction, P-AKT, protein expression of gp91(phox), and 4-HNE, in the sickle cell mouse penis. Sildenafil treatment of WT mice did not affect any of these parameters. Our findings that sildenafil enhances eNOS activation and inhibits NADPH oxidase function in the sickle cell mouse penis offers a vasculoprotective molecular basis for the therapeutic effect of sildenafil in the penis in association with SCD.

Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease

AbstractTreatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4β1, or αVβ3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4−/− mice transplanted with TLR4+/+ sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4−/− ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.

Morphine stimulates platelet-derived growth factor receptor-β signalling in mesangial cells in vitro and transgenic sickle mouse kidney in vivo

Pain and renal dysfunction occur in sickle cell disease. Morphine used to treat pain also co-activates platelet-derived growth factor receptor-β (PDGFR-β), which can adversely affect renal disease. We examined the influence of morphine in mesangial cells in vitro and in mouse kidneys in vivo. > Mouse mesangial cells treated with 1 μM morphine in vitro or kidneys of transgenic homozygous or hemizygous sickle or control mice (n=3 for each), treated with morphine (0.75, 1.4, 2.14, 2.8, 3.6, and 4.3 mg kg(-1) day(-1) in two divided doses during the first, second, third, fourth, fifth, and sixth weeks, respectively), were used. Western blotting, bromylated deoxy uridine incorporation-based cell proliferation assay, reverse transcriptase-polymerase chain reaction, immunofluorescent microscopy, and blood/urine chemistry were used to analyse signalling, cell proliferation, opioid receptor (OP) expression, and renal function. Morphine stimulated phosphorylation of PDGFR-β and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) to the same extent as induced by platelet-derived growth factor-BB (PDGF-BB) and promoted a two-fold increase in mesangial cell proliferation. The PDGFR-β inhibitor, AG1296, OP antagonists, and silencing of μ- and κ-OP abrogated morphine-induced MAPK/ERK phosphorylation and proliferation by ~100%. Morphine treatment of transgenic mice resulted in phosphorylation of PDGFR-β, MAPK/ERK, and signal transducer and activator of transcription 3 (Stat3) in the kidneys. Morphine inhibited micturition and blood urea nitrogen (BUN) clearance and increased BUN and urinary protein in sickle mice. Morphine stimulates mitogenic signalling leading to mesangial cell proliferation and promotes renal dysfunction in sickle mice.

Targeted Treatment Of Acute Chest Syndrome In Transgenic Sickle Mice

Acute chest syndrome (ACS) is a major cause of morbidity and mortality in sickle cell disease (SCD). The diagnosis, prevention and treatment of ACS pose major clinical concerns in SCD partly because the mechanism underlying the pathogenesis of this syndrome remains elusive. Our group first reported that excess intravascular hemin causes a lethal acute lung injury (ALI) in transgenic SCD mice reminiscent of ACS (Ghosh and Ofori-Acquah, Blood 116 Suppl 1:944, 2010). Subsequently, large-scale genomics studies by Bean et al., (Blood 120:3822-8, 2012) and Galarneau et al., (Blood, 122:434-42, 2013) have implicated hemin catabolism and inflammation in the pathogenesis of ACS. In addition, we have reported recently that raised plasma free hemin increases the odds of ACS in children with SCD (Adisa et al., Br J Haematol. 2013). Collectively, these studies support a new theme of ACS pathogenesis involving extracellular hemin. In the current study, we validated the respiratory dysfunction of this ACS model, tested the hypothesis that toll-like receptor 4 (TLR4) mediates the associated lung injury, and examined the efficacy of two strategies to treat the condition in mice. Arterial blood gas analysis of SS mice with the ACS-like disease confirmed severe hypoxemia (PaO2; 40.23 ±3.85 mmHg, SO2; 58.72±6.6%, p<0.001), and revealed worsening hypercapnia (PaCO2; 56.08±3.64 mmHg) and acidosis (mean pH; 7.21), which are all typical of severe ALI. Blood gas parameters remained normal in control AA mice exposed to the same concentration of extracellular hemin. Next, we generated two types of sickle bone marrow chimeric mice that lack expression of TLR4 in non-hematopoietic cells (SSNHTLR4-/-) or express TLR4 on all cell types (SSTLR4+/+). Induction of extracellular hemin crisis with 70 micromoles/kg of hemin resulted in rapid oxygen desaturation, reduced breath rate, pulmonary infiltration, high lung wet/dry weight ratio and sudden death in the SSTLR4+/+ chimeras (n=6). Although all the SSNHTLR4-/- mice acquired a similar phenotype of hemin crisis as the SSTLR4+/+ mice, defined by acute intravascular hemolysis (∼1g/dl drop in Hb, ∼1g/dl increase in cell-free Hb), exhaustion of plasma hemopexin, ∼3-fold increase in plasma free hemin, they did not develop any respiratory symptoms (n=7). Next, we tested two potential therapies, recombinant human hemopexin and TAK-242 a small molecule inhibitor of TLR4. Both agents protected SS mice from developing ACS when they were administered immediately after the induction of hemin crisis. When SS mice were treated after developing respiratory distress evident by significant reductions in oxygen saturation and breath rate, only the recombinant human hemopexin was effective in preventing respiratory failure. In conclusion, we show that TLR4 expressed by the vessel wall, most likely the endothelium, mediates the inflammatory response to excess free hemin associated with severe ACS in mice. Pharmacological inhibition of TLR4 signaling and hemin sequestration may be effective in preventing the development of ACS. In addition, hemin sequestration offers a targeted approach that may halt the progression of ACS in those with more advanced disease. Disclosures:No relevant conflicts of interest to declare.

Fetal Hemoglobin In Sickle Cell Anemia: A Glass Half Full?

HbF modulates the phenotype of sickle cell anemia by inhibiting deoxyHbS polymerization. HbF is confined to erythrocytes called F-cells that can be detected by FACS when these cells contain sufficient HbF. Measuring the amount of HbF/F-cell is difficult and not clinically available. African-Americans with sickle cell anemia have 2-80% F-cells with an average HbF/F-cell of 6.4±1.6 pg. The distribution of HbF/F-cell is highly individual regardless of HbF level. People with HbS-gene deletion hereditary persistence of HbF (HPFH) have a mean HbF of 30%, and HbF is evenly distributed among their erythrocytes. Polymer is not present in these cells either experimentally or after calculating the HbS polymer fraction at 70% O2 saturation. Therefore, each cell contains about 10 pg. of HbF. DeoxyHbS polymerization is prevented at physiologic venous and capillary O2 saturations of 40-70% when HbF/F-cell is 9-12 pgs. We call this the “protective” level of HbF. F-cells need not contain “protective” levels of HbF. Some β-globin gene cluster haplotypes are associated with high HbF. Carriers of these haplotypes can have milder disease. Nevertheless, even patients with high HbF can have frequent painful episodes, acute chest syndrome and osteonecrosis. Patients with HbS-δβ thalassemia have 15 to 25% HbF but are anemic and have vasoocclusive complications, albeit less often than in sickle cell anemia. Hydroxyurea reduces the morbidity and mortality of sickle cell anemia, an effect likely to be mediated by its induction of HbF. Patients treated with hydroxyurea are better and probably live longer, but adults are anemic and rarely asymptomatic. In all these patient groups, HbF is unevenly distributed among erythrocytes. In contrast, people with HbS-HPFH are nearly asymptomatic and not anemic. The failure of HbF to modulate uniformly all complications of sickle cell disease might be related to the heterogeneous concentration of HbF in sickle erythrocytes. HbF is associated with protection from the development of certain disease subphenotypes but has limited prognostic value in individuals. In many cross-sectional studies, high HbF was associated with a reduced rate of acute painful episodes, fewer leg ulcers, less osteonecrosis, less frequent acute chest syndromes and reduced disease severity. HbF had a weak or no clear association with priapism, urine albumin excretion, stroke and silent cerebral infarction, systemic blood pressure and tricuspid regurgitant velocity. Perhaps this is because intravascular hemolysis of cells with little or no HbF causes nitric oxide scavenging, or because these complications are less dependent on HbS polymerization. No study provides information on the concentration of HbF/F-cell other than providing the relatively meaningless calculated mean value. Rather than the total number of F-cells or the concentration of HbF in the hemolysate, HbF/F-cell and the proportion of F-cells that have “protective” HbF is the most critical predictor of the likelihood of some disease subphenotypes. Hypothetical distributions of HbF-cells with different levels of HbF/F-cell can be plotted for different concentrations of HbF. With mean HbF levels of 5%, 10% and 20%, and HbF content per cell of 1.5, 3 and 6 pg., assuming a fixed mean, the variance was changed to show how the distribution of HbF per cell can greatly vary, even if the mean is constant. For example, with 20% HbF, as few as 1% and as many as 24% of cells have “protective” HbF. When HbF is lower, few or no “protected” cells can be present. Due to the heterogeneous concentrations of HbF, HbS can polymerize in some F-cells that have sub-polymer inhibiting concentrations of HbF. Inducing high levels of HbF is one approach to treating sickle cell disease. Inactivating BCL11A, a repressor of γ-globin gene expression, abrogates sickle cell disease in transgenic sickle mice. Their HbF was distributed homogeneously, and their phenotype mimicked HbS-HPFH. If it becomes possible in humans to target BCL11A or its pathway with agents that affect gene transcription, will it result in pancellular HbF? Broadening the distribution of HbF amongst sickle erythrocytes with drugs like hydroxyurea that effect the kinetics of erythropoiesis, coupled with an agent whose primary mechanism of action is to increase the transcription of the γ-globin genes, might be the most fruitful approach to HbF induction therapy and more efficacious than single agent treatment. Disclosures: No relevant conflicts of interest to declare.

Fucosylation Inhibitor, 2-Fluorofucose, Inhibits NF-κB Activation and Vaso-Occlusion In Transgenic Sickle Mice

Polymorphonuclear cells (PMNs) roll along vessel walls by binding to E- and P-selectins on the endothelial surface. These vascular adhesion molecules are involved in the initial tethering of PMNs on endothelium rather than their firm adhesion. Endothelial cell–expressed P- and E-selectins interact with leukocyte counterreceptors P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1). These glycoprotein counterreceptors are active only when modified posttranslationally by fucosylated oligosaccharides represented by the sialyl Lewis x tetrasaccharide (sLex) or its structural variants. Because of the important role that fucose plays in protein glycosylation and cellular functions, specific inhibitors of its incorporation could have significant applications in basic research and therapy. One such inhibitor, 2-fluorofucose peracetate, was recently reported to block fucosylation of cellular proteins and to inhibit binding of PMNs to P- and E-selectin. One potential application is the inhibition of leukocyte recruitment in pro-inflammatory disease states such as sickle cell disease (SCD). P-selectin expression on endothelium has been demonstrated to play an important role in vaso-occlusion, a hallmark of SCD. Inhibition of P-selectin binding inhibits vaso-occlusion in transgenic sickle mice. Therefore, we hypothesized that 2-fluorofucose would inhibit tissue inflammation and vaso-occlusion in SCD mice. MethodsNY1DD sickle mice were given plain water or 20 or 100 mM 2-fluorofucose in their drinking water ad libitum. The first cohort of mice (n=8/group) was treated for 7 days. On day 7 the mice were sacrificed with CO2, an EDTA blood sample was collected from the heart and the liver was removed and frozen in liquid nitrogen. Total white blood cell counts and differentials were performed by manual counting using a hemocytometer and Wright-stained blood smears, respectively. Nuclear extracts were prepared from nuclei isolated from liver homogenates. Western blots of the nuclear extracts were immunostained with antibodies to NF-ĸB phospho-p65 a marker of NF-ĸB activation. The second cohort of mice (n=4/group) was also treated for 7 days. On day 4, dorsal skin fold chambers were implanted onto the mice. On day 7, flowing venules in the dorsal skin-fold chamber window were selected and mapped using intravital microscopy. Thereafter, the mice were infused via the tail vein with human stroma-free hemoglobin (0.32 µmols heme/kg), a known inducer of vascular stasis in SCD mice. At 1 and 4 hours after infusion the same venules were re-examined and the percentage of vessels that had become static (no flow) was recorded. A positive control group of NY1DD mice (n=3) with implanted dorsal skin-fold chambers was given water to drink and pretreated with hemin (40 µmols/kg X 3 days, i.p.), a known inhibitor of vascular stasis. ResultsWhite blood cells counts were 16.3±3.2 (K/µL, mean±SD) in SCD mice treated with water. The white counts increased to 22.1±5.2 in SCD mice treated with 20 mM 2-fluorofucose and 34.2±7.2 in SCD mice treated with 100 mM 2-fluorofucose, respectively (p<0.05 for all pairwise comparisons). NF-ĸB in liver and other organs is activated in SCD mice compared to normal C57BL/6 mice. Nuclear NF-ĸB phospho-p65 was partially diminished in mice treated with 20 mM 2-fluorofucose and markedly decreased in mice treated with 100 mM 2-fluorofucose or pretreated with heme (Fig. 1).Infusion of hemoglobin induced ∼30% microvascular stasis at 1 and 4 hours in SCD mice treated with water (Fig. 2). Treatment with 20 mM 2-fluorofucose partially inhibited stasis at 1 and 4 hours (p<0.05 compared to water). Treatment with 100 mM 2-fluorofucose or heme inhibited stasis to 6.7% or less at 1 and 4 hours (p<0.025 compared to water). [Display omitted] [Display omitted] ConclusionsOral administration of 2-fluorofucose has significant anti-inflammatory and anti-vaso-occlusive properties in SCD mice. Disclosures:Belcher:Seattle Genetics, Inc.: Research Funding. Chen:Seattle Genetics, Inc.: Research Funding. Nguyen:Seattle Genetics, Inc.: Research Funding. Okeley:Seattle Genetics, Inc.: Employment. Senter:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc.: Employment. Vercellotti:Seattle Genetics, Inc.: Research Funding.

Determinants Of Heme-Oxygenase-1 Upregulation In Patients With Sickle Cell Disease

Inflammation is a cardinal component of the pathogenesis of sickle cell disease (SCD). Increased plasma concentration of the inflammatory agonist hemin increases the odds of acute chest syndrome (ACS) in children with SCD (Adisa et al., Br. J Haematol, 2013). In addition, free hemin promotes the development of a lethal ACS-like disease in transgenic sickle mice (Ghosh et al., J Clin Invest, 2013). Hemin degradation is controlled by the rate-limiting enzyme heme oxygenase-1 (HO-1). Polymorphism of a (GT)n dinucleotide repeat in the HO-1 promoter, which enhances expression of the gene, is associated with lower rates of hospitalization for ACS in children. Over-expression of HO-1 reduces stasis in a mouse model of SCD vaso-occlusion. However, the role of plasma HO-1 in SCD patients is entirely unknown. In this study, we measured steady-state plasma HO-1 in two cohorts of patients. Cohort 1 in Atlanta (n=98) consisted of children with a mean age of 10.07±0.42 years (range 2-19 years) and cohort 2 from Accra (n=80) consisted of older patients (mean age 25.30±1.0 years, range 13-58 years). The mean plasma HO-1 of both cohorts was significantly higher compared to the mean value of age- and ethnic-matched individuals with normal adult Hb; Atlanta: 10.19±5.80 vs. 2.08± 1.16, p<0.0001 and Accra: 13.7±8.14 vs. 2.57± 0.82, p<0.0001. Plasma HO-1 varied by 25-fold in both cohorts and it correlated with the white blood cell count (Atlanta: r=0.3361, p<0.0001, Accra: r=0.25, p=0.02). Fifty-four percent (n=53) of subjects in the Atlanta cohort were on hydroxyurea. The mean plasma HO-1 of this subgroup was lower (8.1 ± 4.5) compared to the hydroxyurea naïve Accra cohort (p=<0.0001). Further studies of the Accra cohort revealed significant correlations between HO-1 and multiple markers of vascular inflammation; sICAM-1(r=0.2794, p=0.03, n=60), sE-selectin (r= 0.4209, p=0.0017, n=58) and sP-selectin (r=0.3855, p=0.0028, n=58). The number of the (GT)n dinucleotide in the HO-1 promoter ranged 17 to 45; the distribution was trimodal with peaks at 23, 30 and 41 repeats. The overwhelming majority of patients had medium and large size alleles that are generally hypo-response to induction. Plasma HO-1 level correlated with the length of the (GT)n dinucleotide repeat (p=0.003, n=80). In a multivariable regression model, WBC, sICAM-1, sE-selectin and sP-selectin accounted for 13.4% of the total variance of plasma HO-1 level, and the (GT)n polymorphism accounted for 9.8%. In conclusion, the concentration of plasma HO-1 is generally raised among SCD patients at steady-state. However, a large proportion of patients have a relatively modest level that is probably inadequate to counter the severity of inflammation typical of SCD, due in part to a hypo-responsive HO-1 promoter. Therapeutic strategies that complement induction of the endogenous HO-1 gene may be critical to ameliorate inflammation in SCD. Disclosures:No relevant conflicts of interest to declare.

Interference With TNFα Using Long-Term Etanercept In S+SAntilles Sickle Transgenic Mice Ameliorates Abnormal Endothelial Activation, Vasoocclusion, and Pulmonary Hypertension Including Its Pulmonary Arterial Wall Remodeling

A systemic inflammatory state is a prominent feature of sickle cell anemia. Using sickle transgenic mice, we previously identified a blood monocyte-TNF-endothelium activation axis using multiple complementary approaches. For example, endothelial activation (identified by abnormal expression of VCAM-1 and tissue factor (TF) by pulmonary vessel endothelium) was evident in unstressed BERK and S+SAntilles sickle mice. In NY1DD sickle mice, this was triggered by transient enhanced sickling and vasoocclusion resulting from transient exposure to hypoxia/reoxygenation (H/R). Endothelial activation depended upon Egr-1 and NFkB(p50) in peripheral blood mononuclear cells, and transfusion experiments clarified that the peripheral blood monocytes (PBM) were the responsible effecter cells. Finally, interference with TNFa using etanercept (Etn) prevented this PBM-induced endothelial activation. Based on these background data, we have now tested the “clinical” efficacy of long-term, anti-inflammatory TNFa interference via treatment of S+SAntilles sickle mice with Etn (started at weaning, 3 mg/kg sc, once/wk x 6 wk). In parallel, littermate controls received equimolar scrambled peptide version of Etn's TNF-receptor moiety. In response we observed significant improvement in multiple parameters relevant to inflammation-induced vessel wall disease in this model, as follows (data shown in inset). Etn decreased blood markers of inflammation: plasma levels of sVCAM-1 and SAP (by ELISA), and MCP-1 and TNFa (by FACS cytobead assay). Etn also decreased the % of PBM positive for TNFa (by FACS). Etn diminished the abnormal pulmonary vessel endothelial expression of VCAM-1 and TF (by histochemical and immunofluorescent staining). Etn eliminated the enhanced vasoocclusion seen as dermal microvascular stasis in the live mouse (using the dorsal skin fold chamber) measured at 1 and 4 hours post-H/R (7%O2x1hr, followed by 21%O2x 1-4hr). Instructively, Etn did not significantly improve the similar stasis induced by heme infusion, a TNF-independent direct endothelial stressor. Finally, Etn diminished evidence for pulmonary hypertension, assessed via three of its hallmark features: elevated right ventricular mean systolic pressure (by RV catheterization), abnormal peri-vascular inflammatory cell aggregates (by immuno-histochemical staining for CD11b), and abnormal pulmonary arterial wall muscularization (by immuno-histochemical staining for SM actin). Remarkably, Etn actually normalized RVSP, and Etn's partial improvement in pulmonary arterial muscularization resulted not only from prevention of its progression over duration of this study but also from some reversal of the abnormal remodeling that had already developed by the time Etn was started. In parallel, Etn reduced the number of abnormal peri-vascular aggregates of CDllb+ cells in the pulmonary arterial system.These results enable several conclusions. In the S+SAntilles sickle model, endothelial activation and aspects of vascular wall pathobiology derive from TNFa produced by activated PBM, a monocyte-TNF-endothelial activation axis. Interference with TNFa using etanercept exerts multiple salubrious vascular effects that predict possible clinical benefit. And results suggest that inflammation involving TNFa plays a proximate, generative role in development of pulmonary hypertension in this model. Assuming these benefits are independently corroborated, a controlled test of etanercept's efficacy for aspects of human sickle vascular pathobiology perhaps should be considered. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Pharmacokinetic Evaluation Of Oral Administered Carbon Monoxide Instilled In a Liquid Formulation (CO-LF) To Rats To Determine Carbon Monoxide Hemoglobin Levels With Potential Efficacy In Patients With Sickle Cell Disease (SCD)

There is a large published literature presenting information regarding the toxicology of CO in humans. There is information showing that CO-Hb levels below 15% are not associated with clinical adverse signs or symptoms in healthy individuals and smokers have CO-Hb levels around 6%, but may be above 10% on occasion.It has also been found that CO at low levels may have positive effects in SCD. It has been reported that non-toxic levels of CO extend the red cell life span in SCD patients (Beutler E, 1975.). Further, CO might play a role in preventing sickle cell formation by preventing the polymerization of hemoglobin (Sirs JA, 1963), and it has been reported that CO might have a preventative effect on the occurrence of clinical symptoms of SCD (Yallop D et al, 2007). Inhaled CO administered in low concentrations to transgenic sickle mice for one hour per day, 3 days per week for 10 weeks, was non-toxic and substantially decreased white cell counts and cellular inflammatory signals (Beckman J et al, 2009).This research program hypothesizes that CO, when administered orally at non-toxic levels, results in CO-Hb levels that will result in benefit to patients with SCD through two mechanisms: reducing the sickling phenomenon of red cells and by reducing the inflammatory response in these patients. It also hypothesizes that the safest and most efficient mode of administration of CO is by the oral route via a solution with a known dose of CO. MethodsThe carbon monoxide (CO) is obtained from a commercial source. The liquid formulation (LF) is composed of a mixture of the following components: water, proteins, lipids and carbohydrates ( PLC Mixture). The CO is instilled into this LF and tested for CO concentration. The principle of the method used for CO measurement is to allow the CO and added Hb to interact and to measure the CO-Hb formed by using the dithionite reduction method employing a spectrophotometer. (A.H. Chalmers 1991).The container is weighed before and after CO instillation to determine the occurrence of CO gas leakage.The container for the CO-LF is of low gas permeability. Once CO instillation in the LF is complete, the containers of CO-LF are sealed and labeled. The prepared containers are stored at 2-8°C for no longer than 72 hours.The CO content in blood is expressed as ‘%' of total hemoglobin (CO-Hb) and mg/L in the CO-LF.CO-LF was administered via esophageal-placed gavage using Sprague-Dawley rats with jugular venous catheters. ResultsIn the first study, there were 5 groups of 4 non-anemic rats studied, with each group comprising of a different LF made with the following percent of PLC Mixture combined with water: 50% PLC Mixture, 20% PLC Mixture, 10% PLC Mixture, 5% PLC Mixture, and 0% PLC Mixture (water only).Two 3 mL doses of CO-LF were administered to each rat one hour apart. Data generated showed levels of CO-Hb at baseline varying between 0 and 1.1 %. After the administration of the test article, the highest CO-Hb levels were 6.1% in one animal at 1 hour after the first dose, and 9.6% one hour after the second dose in one animal.The levels of CO-Hb fell at different rates depending upon the dose and formulation. The PK data for the first study is shown in Table 1. Differences among the test articles were not statistically different.Table 1PK Data for the CO-LF Non-Anemic Rat Studies.ExperimentTerminal half-life (hours)AUC 0-inf (h-%)0% (water only)0.510.0855% PLC Mixture0.650.07610% PLC Mixture0.810.0720% PLC Mixture0.50.0950% PLC Mixture0.640.11The PK parameters evaluated over 24 hours were not impacted by the administered PLC Mixture, but animals receiving CO-50% PLC Mixture showed an initial fall to baseline levels, then a second rise in CO-Hb levels peaking in one animal at 3.1% at 48 hours. Such late increments were not seen with other CO-LF's.The second PK study was comprised of 2 groups of 4 anemic rates, and employed CO-0% and CO-10% PLC Mixture. This study was carried out on rats made anemic by blood draw to an estimated 50% blood volume. The PK characteristics show data similar to that seen in non-anemic rats. ConclusionThis research has documented the feasibility in a proof-of-concept rat model that orally administered CO via a CO liquid formulation can produce CO-Hb levels that would be expected to impact the pathophysiology of the Sickle Cell Disease-associated micro-vasculature. The terminal half-life of orally administered CO in the rat is similar to that reported via inhaled CO. Disclosures:Gomperts:Hillhurst Biopharmaceuticals Inc: Equity Ownership, Patents & Royalties. Forman:Hillhurst Biopharmaceuticals Inc: Equity Ownership, Patents & Royalties.

Aldosterone Stimulates Neutrophils Leading To Increased β-Glucuronidase, Protein Disulfide Isomerase and Myeloperoxidase Secretion

Aldosterone (ALDO) has been shown to play an important role in inflammatory responses in addition to its well described effects on sodium homeostasis via activation of the mineralocorticoid receptor (MR). However, its effects on polymorphonuclear leukocytes (PMNC) are not well described. We isolated untouched circulating human PMNC by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% of PMNC were positive for the myeloid-neutrophil markers, CD45, CD16 and CD66b. We show that PMNC express MR by western blot and RT-PCR analyses. We incubated PMNC with ALDO (10–9–10–7M) for 30 min and observed a dose-dependent rise in β–glucuronidase release with an EC50 of 6.11 nM (P<0.001, n=3), an event that was blocked by pre-incubation of cells with 1μM canrenoic acid (CA), an MR antagonist (P<0.04, n=3). In addition, our results show that incubation of human PMNC with 10-8M ALDO likewise led to increases in myeloperoxidase ([MPO], P<0.05, n=3) and protein disulfide isomerase ([PDI], P<0.01, n=4), a multifunctional enzyme of the thioredoxin superfamily that mediates redox modifications, regulates KCNN4 channel and erythrocyte volume and is up-regulated under hypoxic conditions (Prado, 2013 FASEB J). We then studied the effects of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 5 days with 1.3% DMSO. Our results likewise show an increase in MPO responses upon 10–8M ALDO stimulation as compared to vehicle (AUC: 1090±147 to 505±48, P<0.02, n=3). We have recently reported that aldosterone stimulates increases of striatin, a scaffolding protein that interacts with caveolin-1, and co-precipitates with striatin and as such may facilitate cross talk of signaling complexes. As there are no pharmacological inhibitors of striatin we used a molecular approach to reduce striatin levels. In differentiated HL-60 cells, siRNA against striatin led to reduced MPO responses (AUC: 590±14 to 528±13, P<0.05, n=3) that were associated with significantly reduced striatin mRNA levels but not when cells were transfected with scrambled siRNA as determined by quantitative RT-PCR with ABI TaqMan detection probes and β-microglobulin used as an endogenous control (P<0.01, n=3). These results suggest that striatin plays an important role in ALDO-stimulated degranulation responses. Of importance we also observed that incubation with ALDO (10–9–10–7M) in differentiated HL60 cells led to increases in the oxidative-respiratory burst [superoxide production] in a dose- and time-dependent manner (P<0.01, n=4). Consistent with these results, we observed that ALDO likewise led to significant increases in the oxidative-respiratory burst in human PMNC (P<0.01, n=3). As there is evidence that activated neutrophils, MPO and PDI are elevated in Sickle Cell Disease, we studied the in vivo effects of MR blockade in BERK sickle transgenic mice, a model of increased oxidative stress. Sickle mice were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR receptor antagonist, and tap water ad libitum for 14 days at which time the mice were sacrificed and blood collected. We observed that mice on eplerenone had significantly lower plasma PDI activity than mice on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units [RFU]; P<0.005, n=6 and 9) and lower MPO levels (AUC: 214±11 to 73±20, P<0.03, n=3); events that were associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). Thus, our results suggest that MR activation by ALDO is a novel mechanism for neutrophil stimulation and as such represents a novel therapeutic target aimed at ameliorating the vascular complications of Sickle Cell Disease. Supported by NIH R01HL090632 (AR) and R01HL096518 (JRR). Disclosures:No relevant conflicts of interest to declare.

Neuropathy, neuropathic pain, and sickle cell disease

Neuropathy, neuropathic pain, and sickle cell disease

MP4CO, a pegylated hemoglobin saturated with carbon monoxide, is a modulator of HO-1, inflammation, and vaso-occlusion in transgenic sickle mice

AbstractTransgenic sickle mice expressing βS hemoglobin have activated vascular endothelium in multiple organs that exhibits enhanced expression of NF-ĸB and adhesion molecules and promotes microvascular stasis in sickle, but not normal, mice in response to hypoxia/reoxygenation (H/R), or heme. Induction of heme oxygenase-1 (HO-1) or administration of its products, carbon monoxide (CO) or biliverdin, inhibits microvascular stasis in sickle mice. Infusion of human hemoglobin conjugated with polyethylene glycol and saturated with CO (MP4CO) markedly induced hepatic HO-1 activity and inhibited NF-ĸB activation and H/R-induced microvascular stasis in sickle mice. These effects were mediated by CO; saline or MP4 saturated with O2 (MP4OX) had little to no effect on H/R-induced stasis, though unmodified oxyhemoglobin exacerbated stasis. The HO-1 inhibitor, tin protoporphyrin, blocked MP4CO protection, consistent with HO-1 involvement in the protection afforded by MP4CO. MP4CO also induced nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), an important transcriptional regulator of HO-1 and other antioxidant genes. In a heterozygous (hemoglobin-AS) sickle mouse model, intravenous hemin induced cardiovascular collapse and mortality within 120 minutes, which was significantly reduced by MP4CO, but not MP4OX. These data demonstrate that MP4CO induces cytoprotective Nrf2 and HO-1 and decreases NF-ĸB activation, microvascular stasis, and mortality in transgenic sickle mouse models.