Berkeley Mice Research Articles

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34+ hematopoietic stem and progenitor cells (HSPC). This LVV contains locus control region cores expressing an anti-sickling βAS3-globin gene and two microRNA adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbAAS3 + HbF), while concurrently reducing sickle hemoglobin (HbS). The reduction in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34+ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo. With high titer and enhanced transduction of HSPC at low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to reduce costs and help improve accessibility to gene therapy for SCD.

Red Blood Cell Pyruvate Kinase Properties in Sickle Cell Disease - of Mice and Men

Background: Townes and Berkeley mouse models of sickle cell disease (SCD) are commonly used to study SCD pathophysiology and to develop novel therapies. Although the pathophysiology in these models is similar to the human disease, there are distinct differences. For example, Townes SS mice have higher ATP and lower 2,3-diphosphyglycerate (2,3-DPG) levels compared to Townes AA mice. This contrasts with SCD patients, who show lower ATP and higher 2,3-DPG levels, suggesting potential differences between human and murine red blood cell (RBC) metabolism. Pyruvate kinase (PK), a key ATP-generating enzyme in glycolysis, is a target for novel SCD therapies. While PK activators have been shown to increase hemoglobin in clinical trials, such treatments do not consistently lead to the same hematological change in mouse models, further indicating that PK properties may differ between SCD mouse models and human patients. A detailed comparative characterization of PK and glycolytic metabolites in murine SCD models and SCD patients is therefore important. Aim: To study PK properties in murine and human SCD RBCs. Methods: Wild-type (C57BL/6J), Townes (AA and SS) and Berkeley mice (non-sickling and sickling) were used. Human blood was obtained from healthy controls (HbAA) and untreated SCD patients (HbSS). Hematological parameters were measured using the Sysmex hematology analyzer. PK (Vmax, 5mM substrate) and hexokinase (HK) activity, and PK thermostability (53°C) were measured in purified RBCs. Levels of ATP, 2,3-DPG, as well as reduced glutathione (GSH) were measured by targeted LC-MS/MS and spectrophotometry, respectively. P50 (oxygen tension at which 50% of hemoglobin is saturated with oxygen) was measured with the Hemox analyzer (TCS). Statistical analysis was performed in Graphpad Prism 9.4.1. by Welch's t-test. Results: Samples from 17 wild-type mice, 21 Townes mice (10 AA, 11 SS), 15 Berkeley mice (7 non-sickling, 8 sickling) and 12 human subjects (6 HbAA, 6 HbSS) were included. Both Townes and Berkeley SCD mice showed increased PK activity (Table 1; p<0.0001). This may be attributed to their high reticulocyte counts (Townes SS vs. AA: 43% vs. 4%; Berkeley sickling vs. non-sickling: 30% vs. 6%), because when normalizing PK activity to that of HK, another RBC age-dependent enzyme, no genotype differences were found in these mouse models. This lack of PK/HK difference was in sharp contrast with our finding in human controls vs. SCD patients (Table 1). Notably, both Berkeley sickling and non-sickling mice had lower PK/HK ratio than their Townes counterparts (p<0.0001). Mouse PK thermostability was markedly reduced compared to that of human, and PK from the two sickling mice was less stable than their non-sickling controls (Fig. 1). Moreover, both sickling mice showed higher ATP/2,3-DPG ratio (Table 1; p<0.01). This was again in direct contrast with our observation using human blood, where SCD patients had lower ATP/2,3-DPG ratio than healthy controls (p<0.05). Even though there was no difference in ATP/2,3-DPG ratio between the two SCD models, Townes SS mice had significantly lower 2,3-DPG and higher ATP levels than Townes AA mice, whereas Berkeley sickling mice only had significantly higher ATP level compared to their controls. Furthermore, sickling mice had lower P50 value compared to non-sickling mice, reflecting a higher oxygen affinity. This also contrasted with human SCD patients, who had higher P50 value than healthy controls (Table 1). GSH levels were increased in both SCD patients and sickling mice, although the elevation was more pronounced in sickling mice (Table 1). Conclusions: This study reveals important distinctions between mouse models and humans: no differences in PK/HK ratio between sickling and non-sickling mice, and an overall lower PK thermostability in mice. Furthermore, this study shows significant changes in 2,3-DPG content (important for oxygen affinity), energy level (ATP), oxygen affinity (P50) and antioxidant level (GSH) when comparing sickling to non-sickling mice. Sickling mice also have higher ATP/2,3-DPG ratio, which contrasts with SCD patients. These differences should be considered when studying PK activators in SCD mouse models and extrapolating results from SCD mouse models to humans.

Ellagic Acid Exerts Dual Action to Curb the Pathophysiological Manifestations of Sickle Cell Disease and Attenuate the Hydroxyurea-Induced Myelosuppression in Berkeley Mice.

The use of adjuvant therapy is an attractive approach to manage sickle cell disease (SCD) symptomatically. The present study aimed to investigate the potential of ellagic acid as an adjuvant therapy with hydroxyurea (HU), a key drug for SCD with myelosuppressive toxic effects. A panel of experiments was performed using SCD patient's blood (ex vivo) and transgenic mice model of SCD (in vivo). Ellagic acid exhibited the following beneficial pharmacological actions: (a) potent anti-sickling, polymerization inhibitory, and inherent non-hemolytic activity; (b) pronounced action to abrogate HU-induced neutropenia and to improve key hematological parameters during SCD (RBC, Hb, platelet levels); (c) considerable action to foster vascular tone (L-proline); (d) marked attenuating effect against oxidative stress (nitrotyrosine, hypoxanthine, MDA, GSH); (e) substantial inhibitory role against inflammation (analgesic activity and regulation of hemin, TNF-α, IL-1β, NF-κB/IκBα); (f) remarkable outcome of declining vaso-occlusive crisis (P-selectin, ERK1/2); (g) notable shielding deed against elevated biochemical marker for organ toxicity (creatinine); (h) noticeably prevented histopathological alterations of the spleen. Additionally, the pharmacokinetic study results of HU in the presence and absence of ellagic acid using a mouse model demonstrate that ellagic acid could be safely co-administered with HU. Overall findings suggest that ellagic acid is a promising candidate for adjuvant therapy in SCD based on its own significant ability against SCD and potentiating capability of HU action via targeting improvement at the various stages of pathophysiological complications during SCD and minimizing HU-induced toxicological manifestations.

Flurbiprofen inhibits heme induced NLRP3 inflammasome in Berkeley sickle cell disease mice.

Sickle cell disease (SCD) is accompanied by several complications, which emanate from the sickling of erythrocytes due to a point mutation in the β-globin chain of hemoglobin. Sickled erythrocytes are unable to move smoothly through small blood capillaries and therefore, cause vaso occlusion and severe pain. Apart from pain, continuous lysis of fragile sickled erythrocytes leads to the release of heme, which is a strong activator of the NLRP3 inflammasome, thus producing chronic inflammation in sickle cell disease. In this study, we identified flurbiprofen among other COX-2 inhibitors to be a potent inhibitor of heme-induced NLRP3 inflammasome. We found that apart from being a nociceptive agent, flurbiprofen exerts a strong anti-inflammatory effect by suppressing NF-κB signaling, which was evidenced by reduced levels of TNF-α and IL-6 in wild-type and sickle cell disease Berkeley mice models. Our data further demonstrated the protective effect of flurbiprofen on liver, lungs, and spleen in Berkeley mice. The current sickle cell disease pain management regime relies mainly on opiate drugs, which is accompanied by several side effects without modifying the sickle cell disease-related pathology. Considering the potent role of flurbiprofen in inhibiting NLRP3 inflammasome and other inflammatory cytokines in sickle cell disease, our data suggests that it can be explored further for better sickle cell disease pain management along with the possibility of disease modification.

Metabolic correlates to critical speed in murine models of sickle cell disease.

Introduction: Exercise intolerance is a common clinical manifestation in patients with sickle cell disease (SCD), though the mechanisms are incompletely understood. Methods: Here we leverage a murine mouse model of sickle cell disease, the Berkeley mouse, to characterize response to exercise via determination of critical speed (CS), a functional measurement of mouse running speed upon exerting to exhaustion. Results: Upon observing a wide distribution in critical speed phenotypes, we systematically determined metabolic aberrations in plasma and organs-including heart, kidney, liver, lung, and spleen-from mice ranked based on critical speed performances (top vs. bottom 25%). Results indicated clear signatures of systemic and organ-specific alterations in carboxylic acids, sphingosine 1-phosphate and acylcarnitine metabolism. Metabolites in these pathways showed significant correlations with critical speed across all matrices. Findings from murine models were thus further validated in 433 sickle cell disease patients (SS genotype). Metabolomics analyses of plasma from 281 subjects in this cohort (with HbA < 10% to decrease confounding effects of recent transfusion events) were used to identify metabolic correlates to sub-maximal exercise test performances, as measure by 6min walking test in this clinical cohort. Results confirmed strong correlation between test performances and dysregulated levels of circulating carboxylic acids (especially succinate) and sphingosine 1-phosphate. Discussion: We identified novel circulating metabolic markers of exercise intolerance in mouse models of sickle cell disease and sickle cell patients.

Blockade of endothelin-1 receptor B regulates molecules of the major histocompatibility complex in sickle cell disease.

Major Histocompatibility Complex (MHC) molecules have been proposed to play a role in Sickle Cell Disease (SCD) pathophysiology. Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion, and excess levels of endothelin-1 (ET-1) contributing to vaso-occlusive crises. ET-1 activates endothelial cells, induces oxidative stress and inflammation, and alters erythrocyte volume homeostasis. However, the role of ET-1 on MHC regulation in SCD is unclear. We first studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βS-Antilles and Berkeley (BERK) mice. We observed significant increases in H2-Aa mRNA levels in spleens, lungs, and kidneys from transgenic sickle mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). Mice treated for 14 days with ET-1 receptor antagonists significantly reduced H2-Aa mRNA levels. We characterized the effect of ET-1 on MHC class II expression in the human endothelial cell line EA.hy926. We observed dose-dependent increases in the expression of MHC class II (HLA-DRA) and MHC transcription factor (CIITA) that were significantly blocked by treatment with BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the HLA-DRA promoter, an event blocked by BQ788 treatment. These results implicate ET-1 as a novel regulator of MHC class II molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD.

Oxycodone Produces Antinociception, but Worsens Functional Behaviors, in Humanized Sickle Cell Disease Mice

Introduction: Sickle Cell Disease (SCD) is a major cause of morbidity and mortality worldwide, and affects more than 100,000 people in the US. Acute, vaso-occlusive pain crises are the hallmark of the disease and the primary cause of hospitalization. Recurrent, acute pain episodes compound and result in chronic pain for many patients, though the mechanisms of this transition are poorly understood. Opioids remain the standard of care for chronic SCD pain, despite their adverse side effects (e.g., constipation, respiratory depression, abuse liability, dependence) and the fact that prolonged opioid treatment results in tolerance. Thus, a pressing need exists to identify effective non-opioid analgesics to reduce SCD chronic pain. Humanized mouse models of SCD, such as the Berkeley (BERK) model, provide a useful tool to investigate disease pathophysiology and evaluate novel therapeutic strategies. Though oxycodone is one of the most commonly prescribed analgesics for SCD chronic pain, it has yet to be evaluated in this SCD mouse model. Therefore, we assess whether oxycodone would ameliorate phenotypic hypernociception and behavioral deficits in the BERK mouse model of chronic pain. Methods: Male and female HbSS-BERK (sickle) and HbAA-BERK (control) mice were used as subjects. Stimulus-evoked nociceptive behaviors were assessed using the Von Frey and Hot Plate; and functional non-evoked behaviors were assessed using Grip Strength, Inverted Screen, and Nesting tests. Oxycodone (0, 1.25, 2.5, 5, and 10 mg/kg, s.c.) was administered one hour prior to testing. Data were analyzed as Student's t-test, one- and two-way ANOVAs followed by Tukey or Sidak post-hoc analysis when appropriate (p < 0.05 considered significant). Results: Oxycodone (2.5, 5, and 10 mg/kg) fully attenuated mechanical allodynia in HbSS-BERK mice; and produced significant antinociception in HbAA-BERK mice at the highest dose (10 mg/kg). Oxycodone (1.25 and 2.5 mg/kg) partially and fully attenuated thermal hyperalgesia in HbSS-BERK mice, respectively. 5 and 10 mg/kg of oxycodone in HbSS-BERK and 2.5, 5, and 10 mg/kg in HbAA-BERK mice produced significant antinociception. In the grip strength assay, oxycodone had no significant effect in HbSS-BERK mice, but worsened performance in HbAA-BERK mice. Conversely, oxycodone worsened inverted screen deficits in HbSS-BERK mice, with no significant effect in HbAA-BERK mice. Finally, oxycodone (2.5 and 5 mg/kg) worsened nest-building behavior regardless of genotype. Conclusion: Oxycodone, a gold standard analgesic for pain management, acutely reverses the hyperalgesic phenotype in BERK mice. However, oxycodone not only produces complete antinociception in those same assays, but also worsens functional deficits in the HbSS-BERK mice along with their controls. These findings demonstrate that oxycodone is suboptimal for restoring normal pain sensation as well as daily functioning.

O-13: INHIBITION OF THE ENDOCANNABINOID REGULATING ENZYME MONOACYLGLYCEROL LIPASE AMELIORATES THE HYPER-NOCICEPTIVE PHENOTYPE IN THE BERKELEY MOUSE MODEL OF SICKLE CELL DISEASE

Purpose: Sickle Cell Disease (SCD) is a major cause of morbidity and mortality worldwide, and affects more than 100,000 people in the US. Recurrent episodes of acute, vaso-occlusive pain crisis, a hallmark of the disease, lead to chronic pain for many patients, though the mechanisms of this transition are poorly understood. Although opioids remain the standard of care to treat SCD chronic pain, their myriad adverse side effects (e.g., constipation, respiratory depression, abuse liability, dependence) as well as the fact that SCD chronic pain requires prolonged opioid treatment that results in tolerance, severely limit their therapeutic utility. Thus, a pressing need exists to identify effective non-opioid analgesic strategies to reduce SCD chronic pain. Humanized mouse models of SCD, such as the Berkeley (BERK) model, provide a useful tool to investigate disease pathophysiology and evaluate novel therapeutic strategies. Dorsal Root Ganglion (DRG) neurons are peripheral sensory neurons essential for the transmission of nociceptive afferents to the CNS. In rodent models of neuropathic pain, harvested DRG neurons show hyperexcitability. Inhibitors of the major degradative enzyme of 2-arachidonoylglycerol, monoacylglycerol lipase (MAGL), reduce nociceptive behavior in neuropathic and inflammatory preclinical models of pain through cannabinoid receptor-dependent and -independent mechanisms. As MAGL inhibitors have yet to be tested in BERK mice, here we test whether this approach will ameliorate the hyper-nociceptive phenotype of HbSS-BERK mice. Materials and methods: Male and female HbSS-BERK (sickle) and HbAA-BERK (control) mice were used as subjects for these experiments. Nociceptive behaviors were assessed using the von Frey and Hot Plate tests. Motor-functional behavior was assessed using the Grip Strength and Nesting assays. Neuronal hyperexcitability was assessed using whole cell patch clamp electrophysiology of L4-S1 DRG neurons. Data were analyzed as two- and three-way ANOVAs followed by Tukey post-hoc analysis when appropriate (p < 0.05 considered significant). Results: HbSS-BERK mice displayed profound mechanical, thermal and deep tissue/musculoskeletal hypersensitivity. HbSS-BERK mice also exhibited deficits in nest-building behavior. Patch clamp studies revealed that DRG neurons harvested from HbSS-BERK mice displayed extremely hyperexcitability. MJN-110 dose-dependently reduced mechanical allodynia and thermal hyperalgesia, and ameliorated deficits in grip strength in HbSS-BERK mice. Importantly, seven days of daily injections of MJN-110 (5 mg/kg) continued to ameliorate the hyper-nociceptive phenotype of HbSS-BERK mice, which did not undergo tolerance. Moreover, DRG harvested from the MJN-treated HbSS-BERK mice showed similar DRG neuronal activity as seen in control mice. Conclusion: These findings validate that HbSS-BERK mice show hypersensitive responses to mechanical and heat stimuli, and exhibit motor-functional deficits in grip strength and nest building behavior. Additionally, this work demonstrates hyperexcitability of sensory neurons from the lower lumbar region of the spine of HbSS-BERK mice. Finally, the observations that MJN110 reduces these hyper-nociceptive behaviors and ameliorates neuronal hyperexcitability in HbSS-BERK mice suggest that MAGL inhibition represents a viable strategy to reduce chronic pain related to sickle cell disease. The authors do not declare any conflict of interest

Limitations of mouse models for sickle cell disease conferred by their human globin transgene configurations.

ABSTRACTWe characterized the human β-like globin transgenes in two mouse models of sickle cell disease (SCD) and tested a genome-editing strategy to induce red blood cell fetal hemoglobin (HbF; α2γ2). Berkeley SCD mice contain four to 22 randomly arranged, fragmented copies of three human transgenes (HBA1, HBG2-HBG1-HBD-HBBS and a mini-locus control region) integrated into a single site of mouse chromosome 1. Cas9 disruption of the BCL11A repressor binding motif in the γ-globin gene (HBG1 and HBG2; HBG) promoters of Berkeley mouse hematopoietic stem cells (HSCs) caused extensive death from multiple double-strand DNA breaks. Long-range sequencing of Townes SCD mice verified that the endogenous Hbb genes were replaced by single-copy segments of human HBG1 and HBBS including proximal but not some distal gene-regulatory elements. Townes mouse HSCs were viable after Cas9 disruption of the HBG1 BCL11A binding motif but failed to induce HbF to therapeutic levels, contrasting with human HSCs. Our findings provide practical information on the genomic structures of two common mouse SCD models, illustrate their limitations for analyzing therapies to induce HbF and confirm the importance of distal DNA elements in human globin regulation. This article has an associated First Person interview with the first author of the paper.

Monoacylglycerol Lipase Inhibition: A Strategy to Treat Chronic Pain in a Humanized Sickle Cell Mouse Model

Introduction: Sickle Cell Disease (SCD) is a major cause of morbidity and mortality worldwide, and affects more than 100,000 people in the US. Acute, vaso-occlusive pain crises are the hallmark of the disease and the primary cause of hospitalization. Recurrent, acute pain episodes compound and result in chronic pain for many patients, though the mechanisms of this transition are poorly understood. Although opioids remain the standard of care to treat SCD chronic pain, their myriad adverse side effects (e.g., constipation, respiratory depression, abuse liability, dependence) as well as the fact that SCD chronic pain requires prolonged opioid treatment that results in tolerance, severely limit their therapeutic utility. Thus, a pressing need exists to identify effective non-opioid analgesic strategies to reduce SCD chronic pain. Humanized mouse models of SCD, such as the Berkeley (BERK) model, provide a useful tool to investigate disease pathophysiology and evaluate novel therapeutic strategies. Dorsal Root Ganglion neurons are peripheral sensory neurons essential for pain preclinical investigations. DRG neurons from rodents in neuropathic pain models show hyperexcitability. Inhibitors of the major degradative enzyme of 2-arachidonoylglycerol, monoacylglycerol lipase (MAGL), reduce nociceptive behavior in neuropathic and inflammatory preclinical models of pain through cannabinoid receptor-dependent and -independent mechanisms. MAGL inhibitors have yet to be tested in BERK mice and we hypothesize that they will ameliorate pain-like behavior and neuronal hyperexcitability. Here we evaluate the effects of MJN110 in a series of pain-related behaviors in BERK mice.Methods: Male and female HbSS-BERK (sickle) and HbAA-BERK (control) mice were used as subjects for these experiments. Pain-like behaviors were assessed using the Von Frey, Hot Plate, and Grip Strength tests. Neuronal hyperexcitability was assessed using whole cell patch clamp electrophysiology of L4-S1 dorsal root ganglia (DRG) neurons. For the dose-response experiments, MJN-110 (1.25, 2.5, 5, and 10 mg/kg, i.p.) was administered one hour prior to testing. Data were analyzed as Student's t-test, one- and two-way ANOVAs followed by Tukey or Sidak post-hoc analysis when appropriate (p < 0.05 considered significant).Results: HbSS-BERK mice displayed profound mechanical allodynia, as well as thermal and deep tissues/musculoskeletal hyperalgesia. HbSS-BERK mice possess extremely hyperexcitable DRG neurons. MJN-110 dose-dependently reduced mechanical allodynia and thermal hyperalgesia in HbSS-BERK mice. Importantly, BERK mice given seven days of daily injections of MJN-110 (5 mg/kg) displayed sustained antinociception that did not undergo tolerance. Ongoing studies are aimed to determine whether MJN110 ameliorates the hyperexcitability of DRG neurons.Conclusion: These initial findings, not only validate that BERK mice show hypersensitive responses to mechanical and heat stimuli, but also demonstrate that neurons innervating the distal parts of BERK mice are hyperexcitable. Additionally, MJN110 reduces these hyper-nociceptive behaviors suggesting that MAGL inhibition represents a viable strategy to reduce chronic pain behaviors in the BERK mouse model. DisclosuresGupta: Tautona Group: Consultancy, Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; 1910 Genetics: Other: Grantee; Grifols: Other: Grantee; SCDAA: Membership on an entity's Board of Directors or advisory committees; CSL Behring LLC: Honoraria; NIH: Other: Grantee; University of Minnestoa Foundation: Other: Philanthropic Funding; Southern California Institute for Research and Education Foundation: Other: Philanthropic Funding; Cyclerion: Research Funding; UCI Foundation: Other: Philanthropic Funding. Smith: Emmaus Pharmaceuticals: Consultancy; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis Pharmaceuticsl: Consultancy, Honoraria; Novo-Nordisk: Other: DSMB; Pfizer: Consultancy, Research Funding; Imara: Research Funding; Forma Therapeutics: Consultancy, Research Funding; Agios: Research Funding.

Neutrophil DREAM Promotes Neutrophil Recruitment in Vascular Inflammation Via Nuclear Factor Kappa B-Dependent and Independent Mechanisms

The interaction between neutrophils and endothelial cells (ECs) is critical for the pathogenesis of vascular inflammation. Neutrophil recruitment to inflamed tissues is initiated by rolling on activated ECs through the interactions between P-/E-selectins and their ligands. Subsequently, activated integrins (mainly αLβ2 and αMβ2) and chemokine receptors bind to their ligands on ECs and mediate slow-rolling, adhesion, crawling, and transmigration of neutrophils. Although many neutrophil adhesion receptors have been identified, the regulation of their ligand-binding function remains not fully understood. Using real-time intravital microscopy with mice lacking downstream regulatory element antagonist modulator (DREAM) and their bone marrow chimeric mice, we demonstrated that hematopoietic cell DREAM contributes to neutrophil recruitment to sites of vascular inflammation induced by TNF-α- but not a G protein-coupled receptor ligand, MIP-2 or fMLP. Our studies using adoptive neutrophil transfers and flow chamber assays revealed that neutrophil DREAM positively regulates the neutrophil recruitment processes under TNF-α-induced inflammatory conditions. Using RNA-seq and biochemical and cell biological studies, we found that neutrophil DREAM upregulates numerous pro-inflammatory molecules and down-regulates anti-inflammatory molecules after TNF-α treatment. In particular, neutrophil DREAM repressed expression of A20, a negative regulator of NF-κB signaling, and enhanced phosphorylation of IκB kinase (IKK) in response to TNF-α, suggesting the role of neutrophil DREAM in NF-κB activity. Furthermore, we observed that DREAM deletion and IKK inhibition significantly diminishes the ligand-binding activity of β2 integrins in neutrophils after short-term treatment with TNF-α and that deletion of neutrophil DREAM does not affect the expression of other neutrophil adhesion receptors, such as PSGL-1, L-selectin, CD44, CXCR2, and CXCR4. As assessed by flow cytometry using conformation-specific reporter antibodies, knockdown of DREAM in neutrophil-like HL-60 cells decreased TNF-α-induced activation of β2 integrins. Neutrophil DREAM promoted degranulation through IKK-mediated SNAP-23 phosphorylation after short-term treatment with TNF-α, implying the role of neutrophil DREAM-IKK signaling in NF-κB-independent signaling. Using intravital microscopy with Berkeley mice (a mouse model of sickle cell disease) deficient in hematopoietic or nonhematopoietic DREAM, we demonstrated that hematopoietic cell DREAM is crucial for inducing intravascular cell-cell aggregation and vaso-occlusive events in microvessels following the TNF-α challenge. Furthermore, infusion of DREAM KO neutrophils, compared with WT neutrophils, significantly reduced neutrophil recruitment and vaso-occlusive events in TNF-α-challenged SCD mice. These results demonstrate that neutrophil DREAM positively regulates β2 integrin function and promotes neutrophil recruitment during sterile inflammation via NF-κB-dependent and independent mechanisms. Our study provides evidence that targeting DREAM might be a novel therapeutic strategy to reduce excessive neutrophil recruitment in inflammatory diseases. DisclosuresNo relevant conflicts of interest to declare.

Olinciguat, a stimulator of soluble guanylyl cyclase, attenuates inflammation, vaso-occlusion and nephropathy in mouse models of sickle cell disease.

Background and PurposeReduced bioavailability of NO, a hallmark of sickle cell disease (SCD), contributes to intravascular inflammation, vasoconstriction, vaso‐occlusion and organ damage observed in SCD patients. Soluble guanylyl cyclase (sGC) catalyses synthesis of cGMP in response to NO. cGMP‐amplifying agents, including NO donors and phosphodiesterase 9 inhibitors, alleviate TNFα‐induced inflammation in wild‐type C57BL/6 mice and in ‘humanised’ mouse models of SCD.Experimental ApproachEffects of the sGC stimulator olinciguat on intravascular inflammation and renal injury were studied in acute (C57BL6 and Berkeley mice) and chronic (Townes mice) mouse models of TNFα‐induced and systemic inflammation associated with SCD.Key ResultsAcute treatment with olinciguat attenuated increases in plasma biomarkers of endothelial cell activation and leukocyte‐endothelial cell interactions in TNFα‐challenged mice. Co‐treatment with hydroxyurea, an FDA‐approved SCD therapeutic agent, further augmented the anti‐inflammatory effect of olinciguat. In the Berkeley mouse model of TNFα‐induced vaso‐occlusive crisis, a single dose of olinciguat attenuated leukocyte‐endothelial cell interactions, improved blood flow and prolonged survival time compared to vehicle‐treated mice. In Townes SCD mice, plasma biomarkers of inflammation and endothelial cell activation were lower in olinciguat‐ than in vehicle‐treated mice. In addition, kidney mass, water consumption, 24‐h urine excretion, plasma levels of cystatin C and urinary excretion of N‐acetyl‐β‐d‐glucosaminidase and neutrophil gelatinase‐associated lipocalin were lower in Townes mice treated with olinciguat than in vehicle‐treated mice.Conclusion and ImplicationsOur results suggest that the sGC stimulator olinciguat attenuates inflammation, vaso‐occlusion and kidney injury in mouse models of SCD and systemic inflammation.

Increased Endoplasmic Reticulum Amino Peptidase‐1 in Sickle Cell Disease: Role of Hypoxia

Sickle cell disease (SCD) is a genetic disorder caused by a single amino acid substitution in the β‐globin gene that results in hemoglobin polymerization, deformed erythrocytes and endothelial cell activation leading to chronic inflammation, anemia, hypoxemia and vaso‐occlusive crisis (VOC). These events contribute to the development of acute and often severe and debilitating, painful episodes in patients with SCD through unclear mechanisms. In SCD, chronic hypoxia and subacute inflammation contribute to endothelial cell activation that is associated with increased inflammatory cytokine markers such as endothelin‐1 (ET‐1), tumor necrosis factor‐α (TNFα), and TNFα receptor‐1 (TNFR1). Endoplasmic Reticulum Aminopeptidase‐1 (ERAP1) is a ubiquitously expressed zinc‐metallopeptidase involved in regulating immune function and inflammation that cleaves cytokine cell surface receptors such as TNFR1 and plays an important role in the pathophysiology of ankylosing spondylitis. However, the relationship between ERAP1 and SCD is, to the best of our knowledge, unexplored. We first studied the effects of deoxygenation (5% O2) on ERAP1 mRNA expression levels by qRT‐PCR in the human endothelial cell line, EA.hy926. We observed a 3‐fold increase in the expression of ERAP1 after 4 hrs of hypoxia (p&lt;0.05, n=4) and 4‐fold increase after 8 hrs of hypoxia (p&lt;0.05, n=4); events that were associated with a 3.5‐fold increase in the hypoxia‐inducible factor 1‐α mRNA levels at these time points. Consistent with these results, activation of EA.hy926 cells with agonist such as angiotensin II was likewise associated with increased ERAP1 mRNA (p&lt;0.05, n=4). We then studied two sickle transgenic knockout mouse models expressing human sickle hemoglobin, the βSAntilles and Berkeley (BERK) mice. We observed significant increases in ERAP1 mRNA levels (n=7 each; p&lt;0.01) in kidneys from sickle transgenic mice when compared to control transgenic knockout mice expressing human hemoglobin A (HbA). In addition, and as previously documented in other sickle mouse models, our sickle mice had increased circulating TNFα by ELISA as compared to HbA mice (p&lt;0.05, n=6–10 of each mouse type). In conclusion, increases in ERAP1 levels represent a novel target in SCD that is likely secondary to hypoxia and activated endothelial cells.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of Mature Erythrocyte Proteasomal Machinery Under Hypoxia

Proteasome machinery is a conserved cellular component to maintain normal protein homeostasis. Hypoxia is well known to control hypoxia inducible factor levels by proteasomal machinery in nucleated cells. However, the specific targets and regulation of proteasomal machinery in non-nucleated mature erythrocytes under hypoxia remain poorly understood. To determine if hypoxia regulates erythrocyte proteasomal machinery, we conducted Western blot to detect total ubiquitinated and K48 specific ubiquitinated proteins on the erythrocyte membrane in both human and mice with or without sickle cell disease (SCD), a hemolytic genetic disease with a high mortality, morbidity and frequently facing hypoxia. We found that ubiquitinated, especially K48 specific ubiquitinated proteins were significantly accumulated in both SCD Berkeley mice and humans compared to WT mice and normal controls, indicating that proteasomal machinery is impaired in SCD. Next, to determine specific ubiquitinated proteins accumulated on the membrane of human sickle erythrocytes (sRBC), we conducted immunoprecipitation of sRBC membrane proteins with total ubiquitin antibody followed by an robust and nonbiased proteomic profiling. We found significant accumulation of several categories of ubiquitinated proteins on human mature sRBC membrane, including cytoskeleton proteins (Spectrin, Actin, Ankryin), glycolytic enzymes (GAPDH, 2,3-BPG mutase, Pyruvate Kinase, G6PD), transporters (Band3, large neutral AA transporter, calcium transporter, ENT1), reactive oxygen species (ROS) related enzyme (catalase), components of proteasome machinery [E2, E3 ligases, and valosin-containing protein (p97)]. Mechanistically, we revealed that the impaired proteasomal machinery found in mature sRBC was due to the blockage of trafficking of p97 bound ubiquitinated-proteins from membrane to cytosolic proteasome. As such, inhibition of p97 by CB-5083 or proteasome by MG132 led to further induction of hypoxia-induced ubiquitinated membrane proteins and sickling in cultured human sRBC. Given the fact that sphingosine-1-phosphate (S1P) contributes to sickling by binding with deoxygenated sickle Hb (deoxy-HbS), triggering deoxy-HbS anchoring membrane and releasing glycolytic enzymes, we immediately hypothesized that S1P may be involved in proteasomal machinery by regulating trafficking of p97-bound ubiquitinated proteins from membrane to cytosol in sRBC. To test this intriguing possibility, we generated SCD/Sphk1-/- mouse. Intriguingly, we found that the genetic deletion of SphK1 attenuated impaired proteasomal machinery in sRBC with less accumulation of p97 and ubiquitinated proteins on sRBC membrane, indicating that elevated S1P is detrimental in sRBC by inducing accumulation of p97 and ubiquitinated proteins on the membrane. Moreover, to determine if S1P-regulated p97 trafficking from membrane to the cytosol is unique to sRBC, we exposed wild type and SphK1-/- mice to 8% hypoxia up to 72 hours. In contrast to sRBC, we found that genetic deletion of SphK1 abolished p97 trafficking from membrane to cytosol in normal erythrocytes under hypoxia. Finally, we conducted in vitro proof-of-principle genetic studies to determine if S1P directly involves in translocation of membrane anchored p97 to cytosol using inverted ghost membrane (IGM). We demonstrated that S1P treatment induces deoxy-HbA translocation from cytosol to membrane and in turn restoring p97 release from membrane to the cytosol in IGM isolated from SphK1-/- mice only under hypoxia but not normoxia. Thus, we have provided both human and mouse genetic evidence supporting a working model: in normal individuals under hypoxia, S1P is a key factor regulating the efficient proteasomal machinery by binding deoxy-HbA, promoting deoxy-HbA anchoring membrane and in turn triggering release of p97-ubiqutinated proteins to cytosol for its degradation. With mutation in HbS, S1P promotes deoxy-HbS anchoring the membrane and forms polymers, thus blocks membrane bound p97 release and impairs proteasomal machinery in sRBC. Overall, our findings identified that S1P is a missing key component of proteasome machinery by its differential mechanism regulating p97 trafficking from membrane to cytosol in normal erythrocyte physiology under hypoxia and the pathophysiology of SCD that open up new and differential therapies for the SCD and normal individuals facing hypoxia. DisclosuresNo relevant conflicts of interest to declare.

PD49-08 INVESTIGATING CORPORAL GENE EXPRESSION PATTERNS IN ISCHEMIC PRIAPISM USING A BERK MOUSE MODEL

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology II1 Apr 2018PD49-08 INVESTIGATING CORPORAL GENE EXPRESSION PATTERNS IN ISCHEMIC PRIAPISM USING A BERK MOUSE MODEL Amarnath Mukherjee, Augene Park, Moses Tar, Henny Billet, and Kelvin Davies Amarnath MukherjeeAmarnath Mukherjee More articles by this author , Augene ParkAugene Park More articles by this author , Moses TarMoses Tar More articles by this author , Henny BilletHenny Billet More articles by this author , and Kelvin DaviesKelvin Davies More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2324AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Ischemic priapism is a sustained erection lasting more than 4 hours, and can lead to permanent penile scarring. It is estimated that between 44-90% of priapic episodes lasting more than 24 hours lead to erectile dysfunction (ED). Individuals with hematological disorders, especially those with sickle-cell anemia, are at higher risk of developing priapism. Using sickle transgenic-knockout Berkeley (BERK) mice as our model for sickle-cell anemia, we compared gene expression patterns in the corpora cavernosa between sickle-cell mice and control mice in order to identify genes of interest for future research. METHODS BERK mice and control C57 mice from two different age groups (5-weeks and 12-weeks of age) were sacrificed and their corporal RNA was isolated using Trizol. The 5-week group was considered to be at a “pre-priapic” stage for the BERK mice, and the 12-week group was considered to be at a “priapic” stage for the BERK mice. The RNA was quantified and evaluated for purity. The RNA labeling, microarray hybridization, and quality control were performed by using Affymetrix Clariom S mouse microarray probe sets. The analysis of the genome array data was performed using Affymetrix TAC and EC software following manufacturer's protocol. Changes in gene expression between the BERK and control mice of corresponding age groups were determined and statistically significant results from each age group were then compared to each other to find any genes that were common to both data sets. RESULTS Out of a total of 424 genes identified in the 5-week data set and 437 genes identified in the 12-week data set, 84 genes were found in both sets. Among those common to both sets were genes such as Adh6b (alcohol dehydrogenase 6b), Hspb6 (heat shock protein beta-6), Rxfp1 (relaxin/insulin-like family peptide receptor 1), and Snca (synuclein-alpha). Some genes that appeared in both data sets showed different trends in expression between the two age groups. For example, while Adh6b was upregulated by 3.94-fold in 5-week old BERK mice, it was downregulated by 3.14-fold in 12-week old BERK mice. CONCLUSIONS We have conducted a global screen of progressive changes in gene expression which occur in the sickle cell mice. This screen will identify novel markers of priapism that may be useful targets for preventing or treating priapism in sickle cell patients. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e968 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Amarnath Mukherjee More articles by this author Augene Park More articles by this author Moses Tar More articles by this author Henny Billet More articles by this author Kelvin Davies More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Patent highlights December 2016-January 2017.

Patent highlights December 2016-January 2017.

Specific Inhibition of AKT with ARQ 092, an Orally-Available Selective AKT Inhibitor, Attenuates Acute Vaso-Occlusive Events in Sickle Cell Disease

▪Previous studies have identified the Ser/Thr protein kinase, AKT, as a therapeutic target for thromboinflammatory diseases. Recently, we have shown that the basal levels of AKT phosphorylation were significantly increased in neutrophils and platelets isolated from sickle cell disease (SCD) patients compared with those cells from healthy donors and that specific inhibition of AKT2 impaired neutrophil-endothelial cell and neutrophil-platelet interactions in venules of Berkeley (SCD) mice (Li et al. J Clin Invest 2014; Barazia et al. Blood 2015). Although our results suggest that AKT2 inhibition may be a novel therapy to treat a vaso-occlusive crisis in SCD patients, no AKT2 specific inhibitor is available in clinic. Using ARQ 092, an orally-available, allosteric AKT inhibitor that is currently in Phase I clinical trials in oncology indications and AKT-driven overgrowth disease, Proteus syndrome, here we report that specific inhibition of AKT attenuates the adhesive function of neutrophils and platelets from sickle cell disease (SCD) patients in vitro and heterotypic cell-cell interactions in the Berkeley mouse model of SCD in vivo. Studies using neutrophils and platelets isolated from SCD patients revealed that treatment with 50-500 nM ARQ 092 significantly blocks αMβ2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Consistently, treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Using in vivo real-time fluorescence intravital microscopy, we demonstrated that short-term oral administration of ARQ 092 (100 mg/kg body weight) or hydroxyurea (HU, 250 mg/kg body weight), a main therapy for the treatment of SCD, diminishes neutrophil-endothelial cell and neutrophil-platelet interactions in venules of Berkeley mice challenged with TNF-α to induce a vaso-occlusive crisis. Co-administration of HU and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) in the cremaster vessel wall, and improved survival in these mice. Ex vivo studies in Berkeley mice suggested that co-administration of HU and ARQ 092 efficiently blocks neutrophil and platelet activation and reactive oxygen species generation. The synergistic effects of both inhibitors resulted from increased NO production by HU and specific inhibition of AKT by ARQ 092. Our results provide important evidence that ARQ 092 has potential as a novel drug to be developed to prevent or treat the acute vaso-occlusive complications in SCD patients. DisclosuresAbbadessa:ArQule, Inc: Employment. Yu:ArQule, Inc: Employment. Schwartz:ArQule, Inc: Employment.

Endothelin-1 Regulates Molecules of the Major Histocompatibility Complex: Role in Sickle Cell Disease

Molecules of the Major Histocompatibility Complex (MHC), and in particular specific human leukocyte antigen (HLA) alleles, have been proposed in the pathophysiology of immune and vascular alterations leading to vasoocclusive crises (VOC) and stroke in Sickle Cell Disease (SCD). Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion and excess levels of the potent mitogen, endothelin-1 (ET-1). ET-1 activates endothelial cells, induces oxidative stress and inflammation in the vascular wall and regulates erythrocyte homeostasis. However, the role of ET-1 on MHC regulation in SCD is not clear. We first characterized the effect of ET-1 on MHC expression in the human endothelial cell line, EA.hy926. We observed dose-dependent increases in the expression of MHC class I (HLA-A2 4.8 ± 2.1 folds p<0.01 n=4), MHC class II (HLA-DR 4.4 ± 1.7 folds p<0.01 n=4) and MHC transcription factor (CIITA 3.5 ± 1.8 folds p<0.05 n=4) in EA.hy926 cells. ET-1-stimulated expression of HLA-A2, HLA-DR and CIITA were significantly blocked by pre-incubation of cells with 10 µM BQ788, a selective blocker of ET-1 type B receptors (p<0.01, n=4). Chromatin immunoprecipitation (ChIP) studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the promoter region within MHC molecules (HLA-A2 62% ± 5%, HLA-DRB 33% ± 18%, p<0.01, n=4); an event that was likewise blocked by BQ788. We then studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βSAntilles and Berkeley (BERK) mice, respectively. We observed significant increases in MHC molecule, H2-Aa mRNA levels (n=7; p<0.01) in spleens from sickle transgenic mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). We then treated BERK, βSAntilles and HbA mice for 14 days with ET-1 receptor antagonists and observed significant reductions in H2-Aa mRNA levels in spleen tissue from sickle transgenic mice but not in HbA mice (n=7; p<0.05). These results implicate ET-1 as a novel regulator of MHC molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD. DisclosuresNo relevant conflicts of interest to declare.

A Novel, Highly Potent and Selective PDE9 Inhibitor for the Treatment of Sickle Cell Disease

▪BackgroundHydroxyurea (HU), a chemotherapeutic agent and an approved therapy for sickle cell disease (SCD), increases cGMP levels and thereby increases fetal hemoglobin (HbF) levels in RBC, which generally correlates with decreased hemolysis and reduced disease severity in subjects with SCD. In addition, increased cGMP levels are anti-inflammatory and reduce the adhesion of WBC to the vascular endothelium. However, potential adverse effects of HU such as infertility, susceptibility to infections, or teratogenic effect have been the subject of concerns. Phosphodiesterase-9 inhibitors (PDE9i) may provide an alternative to HU because of their ability to inhibit the degradation of cGMP and increase cellular cGMP levels. Previously described PDE9i were developed to inhibit neuronal PDE9 for neurologic diseases, like Alzheimer's. A PDE9i with low brain penetrance may be preferable for the chronic treatment of SCD adults and children. IMR-687 is a safe, potent, PDE9i being developed specifically for SCD without these liabilities of HU or the high brain penetrance of other PDE9is.HypothesisWe hypothesized that PDE9i's will increase cellular cGMP levels in hematopoietic cells, increase HbF in red blood cells (RBC), inhibit RBC sickling, decrease inflammation (leukocytosis), and inhibit vaso-occlusion in murine models of SCD.Results & MethodsIMR-687, is a potent inhibitor of PDE9A (IC50 <10 nM) and demonstrates >100-fold higher IC50 with other phosphodiesterases. To determine the ability of IMR-687 to induce cGMP, K562 erythroid cells were exposed to increasing concentrations of IMR-687 or HU and cGMP assessed at 6 hours. IMR-687, at much lower drug concentrations, induced higher levels of cGMP than HU at 6 hours (Fig. Left Panel).We next tested IMR-687 in the Berkeley (Berk) mouse model of SCD. Groups of 7-8 Berk SCD mice were dosed by gavage with either vehicle, 30 mg/kg/day of IMR-687, or 100 mg/kg/day of HU. After 30 days of treatment, both IMR-687 and HU resulted in statistically significant decreases in the percentage of sickled RBCs (Fig. Center Left Panel) and increases in the percentage of HbF positive RBCs relative to controls. In addition, both compounds led to statistically significant decreases in total bilirubin, spleen weight and total leucocyte counts (Fig. Center Right Panel).The ability of IMR-687 to reduce microvascular stasis (% non-flowing venules) was assessed in HbSS-Townes transgenic sickle mice after transient hypoxia and re-oxygenation. Groups of 3 mice/group were dosed orally via drinking water with IMR-687 (30 mg/kg/day), HU (100 mg/kg/day) or vehicle for 10 days. After 10 days of treatment, the mice were exposed to transient hypoxia. Compared with controls, 30 mg/kg/day IMR-687 and 100 mg/kg HU produced statistically significant reductions in microvascular stasis at both 1-hour (Fig. Center Right Panel) and 4-hour time points post-hypoxia. In addition, IMR-687 significantly reduced the proportion of sickled RBCs, increased the number of HbF positive red cells, and reduced the white count.Lastly, low brain penetration of IMR-687 was confirmed after IV dosing in the rat, with brain concentrations >20 times lower than those in the plasma at all time points assessed. Consistent with this, treatment of C57Bl/6J mice with 10 mg/kg/day IMR-687 for 5 days displayed no effect on locomotor activity or classical fear conditioning (an animal model of learning and memory). In contrast, treatment with a brain penetrant PDE9i, developed for the treatment of neurologic diseases, significantly increased conditioned fear responses in these mice.ConclusionsIMR-687 increases HbF levels, and reduces red cell sickling, leukocytosis and microvascular stasis, without the observed toxicities of HU. IMR-687 may offer a once a day, oral, safe replacement for HU in the treatment of SCD. [Display omitted] DisclosuresMcArthur:Imara: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: Patent; Cydan Development: Employment, Equity Ownership. Maciel:Imara: Research Funding. Chen:Imara: Research Funding. Fricot:Imara: Research Funding. Kobayashi:Imara: Equity Ownership; Cydan Development: Employment. Nguyen:Imara: Research Funding. Parachova:Imara: Patents & Royalties. Abdulla:Imara: Research Funding. Vercellotti:Imara: Research Funding. Svenstrup:Imara: Consultancy, Patents & Royalties. Belcher:Imara: Research Funding; CSL-Behring: Research Funding.

Co-Administration of Hydroxyurea and a Specific AKT2 Inhibitor Has Beneficial Effects on Acute Vaso-Occlusive Events and Survival in Sickle Cell Disease Mice

We recently demonstrated that the basal levels of AKT phosphorylation were significantly increased in neutrophils and platelets isolated from sickle cell disease (SCD) patients compared with those cells from healthy donors and that specific inhibition of AKT2 impaired neutrophil-endothelial cell and neutrophil-platelet interactions in venules of Berkeley (SCD) mice (Li et al. J Clin Invest 2014). Although hydroxyurea (HU), an inducer of fetal hemoglobin, is the main therapy for treatment of SCD, it is unclear whether the short-term treatment with HU has immediate benefits on acute vaso-occlusive events in SCD. Using real-time fluorescence intravital microscopy, we demonstrated that co-administration of HU (100 microg/g body weight, iv injection) and Akti XII (3 microg/g body weight, iv injection), an AKT2 inhibitor, efficiently reduced neutrophil adhesion and platelet-neutrophil aggregation in cremaster muscle venules of TNF-α-challenged Berkeley mice. Importantly, compared with HU or Akti XII treatment alone, treatment with both agents significantly improved blood flow rates and survival in the mice. Further, similar results were obtained in Berkeley mice challenged with hypoxia (8% oxygen for 3 hours) and subsequent reoxygenation (room air for 3 hours). As determined by histochemistry of cremaster muscle sections following intravital microscopy of Berkeley mice, the expression of endothelial E-selectin and intercellular adhesion molecule 1 (ICAM-1) was significantly decreased by treatment with either HU or Akti XII. Leukocyte transmigration in the lung section of Berkeley mice was also inhibited by either agent, and the inhibitory effect was potentiated by co-administration of HU and Akti XII. Using the plasma and isolated cells of Berkeley mice, we found that the level of plasma nitric oxide species (NOx) was significantly elevated by treatment with HU but not Akti XII and that AKT2 phosphorylation levels in activated neutrophils and platelets were reduced by treatment with Akti XII but not HU. Taken together, these results suggest that short-term treatment of Berkeley mice with either HU or Akti XII inhibits inflammatory conditions: treatment with HU significantly increases plasma NOx levels, treatment with Akti XII decreases AKT2 phosphorylation in neutrophils and platelets without affecting plasma NOx levels, and administration of either agent reduces the surface expression of ICAM-1 and E-selectin on activated endothelial cells. Thus, our results provide evidence that co-administration of HU and a specific AKT2 inhibitor has immediate benefits on acute vaso-occlusive events and survival in SCD mice exceeding those seen for single therapy. DisclosuresNo relevant conflicts of interest to declare.