Transgenic Sickle Mice Research Articles

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

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

Targeting AnxA1/Formyl Peptide Receptor 2 Pathway Affords Protection against Pathological Thrombo-Inflammation.

Stroke is a leading cause of death and disability globally and is associated with a number of co-morbidities including sepsis and sickle cell disease (SCD). Despite thrombo-inflammation underlying these co-morbidities, its pathogenesis remains complicated and drug discovery programs aimed at reducing and resolving the detrimental effects remain a major therapeutic challenge. The objective of this study was to assess whether the anti-inflammatory pro-resolving protein Annexin A1 (AnxA1) was able to reduce inflammation-induced thrombosis and suppress platelet activation and thrombus formation in the cerebral microvasculature. Using two distinct models of pathological thrombo-inflammation (lipopolysaccharide (LPS) and sickle transgenic mice (STM)), thrombosis was induced in the murine brain using photoactivation (light/dye) coupled with intravital microscopy. The heightened inflammation-induced microvascular thrombosis present in these two distinct thrombo-inflammatory models was inhibited significantly by the administration of AnxA1 mimetic peptide AnxA1Ac2-26 (an effect more pronounced in the SCD model vs. the endotoxin model) and mediated by the key resolution receptor, Fpr2/ALX. Furthermore, AnxA1Ac2-26 treatment was able to hamper platelet aggregation by reducing platelet stimulation and aggregation (by moderating αIIbβ3 and P-selectin). These findings suggest that targeting the AnxA1/Fpr2/ALX pathway represents an attractive novel treatment strategy for resolving thrombo-inflammation, counteracting e.g., stroke in high-risk patient cohorts.

Nonhematopoietic Nrf2 Deficiency Worsens Chronic Intravascular Hemolysis but Not Cardiomyopathy in Transgenic Sickle Cell Mice

Background: Cardiopulmonary complications are the leading causes of death in sickle cell disease (SCD). Cardiac complications in SCD include left ventricular (LV) hypertrophy and LV systolic or diastolic dysfunction. In SCD, intravascular hemolysis (IVH) contributes to multiorgan damage, but its role in cardiac injury is unknown. We previously reported that nonhematopoietic deficiency of nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor that controls antioxidative and anti-inflammatory genes, worsens the severity of IVH in SCD mice. Here, we first sought to determine if Townes transgenic SCD mice develop an abnormal cardiac phenotype, utilizing a cross-sectional study of varying-aged mice to test the hypothesis that these mice develop an age-related cardiomyopathy. Second, we determined whether IVH associated with absence of Nrf2 in the nonhematopoietic system worsens SCD cardiomyopathy. Methods: To identify a SCD cardiac phenotype, we performed echocardiography in a cross sectional cohort of Townes transgenic sickle cell mice (SS) and matched AA controls of varying ages (1-10 months). LV echocardiography dimensions were normalized to body weight. To determine the role of IVH and Nrf2 on the SCD cardiac phenotype, we transplanted whole bone marrow cells from SS mice to irradiated Nrf2-/- mice or C57BL/6 (Nrf2+/+) mice, to generate bone marrow chimeric sickle mice with Nrf2 deficiency in nonhematopoietic tissues (SSNHNrf2-/-) or chimeric sickle controls (SSNrf2+/+), respectively. Serial echocardiography was performed for up to 10 months, and hearts were harvested post-mortem for histology and molecular studies. Results: Cross-sectional study: Echocardiography showed cardiac abnormalities, most pronounced by 10 months, in the SS (n=8-9) compared to AA mice (n=9-13), respectively: LV hypertrophy was evidenced by a larger LV internal diameter (0.16 ± 0.01 vs. 0.13 ± 0.10 mm/g, p=0.005), higher LV mass index (5.2 ± 0.5 vs. 4.0 ±0.2 mg/g, p=0.02) and higher post-mortem heart weight (7.8 ± 0.3 vs. 5.2 ± 0.2 mg/g, p<0.0001) in SS mice. Compared to AA mice, SS mice had a lower ejection fraction (52 ± 4% vs 69 ± 2%, p=0.006) and fractional shortening (27 ± 2% vs. 39 ± 2%, p=0.001), consistent with impaired systolic function in SCD. Trichome-stained histological heart sections revealed myocardial fibrosis in SS but not in AA mice. Longitudinal study: Ten months after bone marrow transplant engraftment, SSNHNrf2-/- mice (n=6) had a significantly higher total plasma heme (p=0.008), lactate dehydrogenase (p=0.038), and plasma hemoglobin (p=0.008), confirming an exacerbation of IVH compared to SSNrf2+/+ controls (n=3). Surprisingly, compared to SSNrf2+/+ mice, SSNHNrf2-/- mice had a higher hematocrit (27.0 ± 1.0 vs. 23.1 ± 0.2%, p=0.035), lower reticulocyte count (57.5 ± 2.2% vs. 68.6 ± 4.8%, p=0.04), and a trend towards higher RBC count (p=0.05). Serial echo revealed that SSNHNrf2-/- mice, compared to SSNrf2+/+ controls, had a lower LV internal diameter (0.17 ± 0.01 vs 0.21 ± 0.01 mm/g, p=0.001) and LV mass index (4.7 ± 0.2 vs. 6.8 ± 0.2 mg/g, p=0.0004), consistent with less LV hypertrophy in the SSNHNrf2-/- mice. These findings were confirmed postmortem, where SSNHNrf2-/- mice had lower heart weight (p=0.0002) and cardiac mRNA expression of BNP (p=0.045) compared to SSNrf2+/+ controls. There were no significant differences in systolic or diastolic function parameters between SSNrf2+/+ controls and SSNHNrf2-/- mice. There was, however, a trend towards less myocardial fibrosis in SSNHNrf2-/- mice based on cardiac mRNA expression of Collagens 1 and 3. Conclusions: Townes transgenic sickle mice exhibit an age-dependent cardiomyopathy, with LV eccentric hypertrophy, systolic dysfunction, and myocardial fibrosis by 10 months of age. We reaffirm that Nrf2 deficiency in non-hematopoietic tissue worsens markers of IVH in SCD mice and unveil an unexpected decoupling phenomenon whereby despite increasing IVH in SSNHNrf2-/- mice, anemia improves. The amelioration of the SCD cardiac phenotype in SSNHNrf2-/- mice suggests that chronic IVH conferred by Nrf2 deficiency does not principally drive SCD-associated cardiomyopathy. While the improvement in anemia that we observed with Nrf2 deficiency may have played a role in amelioration of cardiomyopathy, further study is warranted to explore a possible cardiac protective effect of Nrf2 deficiency in SCD mice. Disclosures No relevant conflicts of interest to declare.

Spatiotemporal Alterations in Gait in Humanized Transgenic Sickle Mice.

Sickle cell disease (SCD) is a hemoglobinopathy affecting multiple organs and featuring acute and chronic pain. Purkinje cell damage and hyperalgesia have been demonstrated in transgenic sickle mice. Purkinje cells are associated with movement and neural function which may influence pain. We hypothesized that Purkinje cell damage and/or chronic pain burden provoke compensatory gait changes in sickle mice. We found that Purkinje cells undergoe increased apoptosis as shown by caspase-3 activation. Using an automated gait measurement system, MouseWalker, we characterized spatiotemporal gait characteristics of humanized transgenic BERK sickle mice in comparison to control mice. Sickle mice showed alteration in stance instability and dynamic gait parameters (walking speed, stance duration, swing duration and specific swing indices). Differences in stance instability may reflect motor dysfunction due to damaged Purkinje cells. Alterations in diagonal and all stance indices indicative of hesitation during walking may originate from motor dysfunction and/or arise from fear and/or anticipation of movement-evoked pain. We also demonstrate that stance duration, diagonal swing indices and all stance indices correlate with both mechanical and deep tissue hyperalgesia, while stance instability correlates with only deep tissue hyperalgesia. Therefore, objective analysis of gait in SCD may provide insights into neurological impairment and pain states.

Integrin VLA-4 as a PET imaging biomarker of hyper-adhesion in transgenic sickle mice

AbstractIn sickle cell disease (SCD), very late antigen-4 (VLA-4 or integrin α4β1) mediates the adhesion of reticulocytes to inflamed, proinflammatory endothelium, a key process in promoting vaso-occlusive episodes (VOEs). We hypothesized that a radionuclide tracer targeting VLA-4 could be harnessed as a positron emission tomography (PET) imaging biomarker of VOEs. We tested the VLA-4 peptidomimetic PET tracer 64Cu-CB-TE1A1P-PEG4-LLP2A (64Cu-LLP2A) for imaging hyper-adhesion–associated VOEs in the SCD Townes mouse model. With lipopolysaccharide (LPS)-induced VOEs, 64Cu-LLP2A uptake was increased in the bone marrow of the humeri and femurs, common sites of VOEs in SCD mice compared with non-SCD mice. Treatment with a proven inhibitor of VOEs (the anti-mouse anti-P-selectin monoclonal antibody [mAb] RB40.34) during LPS stimulation led to a reduction in the uptake of 64Cu-LLP2A in the humeri and femurs to baseline levels, implying blockade of VOE hyper-adhesion. Flow cytometry with Cy3-LLP2A demonstrated an increased percentage of VLA-4–positive reticulocytes in SCD vs non-SCD mice in the bone and peripheral blood after treatment with LPS, which was abrogated by anti-P-selectin mAb treatment. These data, for the first time, show in vivo imaging of VLA-4–mediated hyper-adhesion, primarily of SCD reticulocytes, during VOEs. PET imaging with 64Cu-LLP2A may serve as a valuable, noninvasive method for identifying sites of vaso-occlusion and may provide an objective biomarker of disease severity and anti-P-selectin treatment efficacy in patients with SCD.

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

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

Significant Quantitative Differences in Orexin Neuronal Activation After Pain Assessments in an Animal Model of Sickle Cell Disease.

Sickle cell disease is a hemoglobinopathy that causes sickling of red blood cells, resulting in vessel blockage, stroke, anemia, inflammation, and extreme pain. The development and treatment of pain, in particular, neuropathic pain in sickle cell disease patients is poorly understood and impedes our progress toward the development of novel therapies to treat pain associated with sickle cell disease. The orexin/hypocretin system offers a novel approach to treat chronic pain and hyperalgesia. These neuropeptides are synthesized in three regions: perifornical area (PFA), lateral hypothalamus (LH), and dorsomedial hypothalamus (DMH). Data suggest that orexin–A neuropeptide has an analgesic effect on inflammatory pain and may affect mechanisms underlying the maintenance of neuropathic pain. The purpose of this study was to determine whether there are neuronal activation differences in the orexin system as a result of neuropathic pain testing in a mouse model of sickle cell disease. Female transgenic sickle mice that express exclusively (99%) human sickle hemoglobin (HbSS-BERK) and age-/gender-matched controls (HbAA-BERK mice; n = 10/group, 20–30 g) expressing normal human hemoglobin A were habituated to each test protocol and environment before collecting baseline measurements and testing. Four measures were used to assess pain-related behaviors: thermal/heat hyperalgesia, cold hyperalgesia, mechanical hyperalgesia, and deep-tissue hyperalgesia. Hypothalamic brain sections from HbAA-BERK and HbSS-BERK mice were processed to visualize orexin and c-Fos immunoreactivity and quantified. The percentage of double labeled neurons in the PFA was significantly higher than the percentage of double labeled neurons in the LH orexin field of HbAA-BERK mice (*p < 0.05). The percentages of double labeled neurons in PFA and DMH orexin fields are significantly higher than those neurons in the LH of HbSS-BERK mice (*p < 0.05). These data suggest that DMH orexin neurons were preferentially recruited during neuropathic pain testing and a more diverse distribution of orexin neurons may be required to produce analgesia in response to pain in the HbSS-BERK mice. Identifying specific orexin neuronal populations that are integral in neuropathic pain processing will allow us to elucidate mechanisms that provide a more selective, targeted approach in treating of neuropathic pain in sickle cell disease.

Effects of hypoxia-reoxygenation stimuli on renal redox status and nuclear factor erythroid 2-related factor 2 pathway in sickle cell SAD mice.

What is the central question of this study? What are the effects of repeated subclinical vaso-occlusions on nuclear factor erythroid 2 related factor 2 (Nrf2) and oxidative stress balance regulation in the kidney of transgenic SAD mice? What is the main finding and its importance? In response to hypoxia-reoxygenation, nuclear Nrf2 protein expression decreased in the kidney of SAD mice while haem oxygenase transcripts were increased. This suggest that in SAD mice, other transcription factors than Nrf2 could be involved in renal antioxidant gene regulation in response to hypoxia-reoxygenation. Hypoxia-reoxygenation (H/R) stress is known to increase oxidative stress in transgenic sickle mice and can cause organ failure. Here we described the effects of H/R on nuclear factor erythroid 2-related factor 2 (Nrf2) as a putative regulator of redox status in the kidneys of SAD mice investigating Nrf2-regulated antioxidant enzymes. Transgenic SAD mice and healthy C57Bl/6J mice were exposed to 4h of hypoxia followed by various times of reoxygenation at ambient air (2 or 6h). Regardless of the conditions (i.e. normoxia or H/R), SAD mice expressed higher renal oxidative stress levels. Nuclear Nrf2 protein expression decreased after 2h post-hypoxia only in the medulla region of the kidney and only in SAD mice. Simultaneously, haem oxygenase transcripts were affected by H/R stimulus with a significant enhancement after 2h post-hypoxia. Similarly, hypoxia inducible factor-1α staining increased after 2h post-hypoxia in SAD mice in both cortex and medulla areas. Our data confirm that the kidneys are organs that are particularly sensitive to H/R stimuli in sickle cell SAD mice. Also, these results suggest an effect of the duration of recovery period (short vs. long) and specific responses according to kidney areas, medulla vs. cortex, on Nrf2 expression in response to H/R stimuli in SAD mice.

Thrombomodulin and Endothelial Dysfunction in Sickle Cell Anemia

Thrombomodulin (THBD) is a transmembrane protein that regulates endothelial function by 1) binding thrombin and inhibiting its interaction with fibrinogen, 2) augmenting protein C activation, and 3) down-regulating complement activation by inactivating C3a and C5a (PMID 29866818). Reduced THBD function has been implicated in several vasculopathies (PMID 29866818). Oxidative damage increases cleaved non-functional THBD in circulation and increased circulating non-functional THBD predicts the severity of organ damage in thrombotic microangiopathies (PMID 11190905). Vascular endothelial dysfunction is a hallmark feature of sickle cell disease (SCD) that leads to acute and chronic organ damage and may, in part, be mediated by hemolysis (PMID 28248201). Hemolysis leads to release of cell-free hemoglobin which may lead to vasculopathy through consumption of NO, activation of TLR4 pathways, and direct oxidative damage. We investigated whether decreased THBD activity may be implicated in the pathophysiology of SCD vasculopathy in endothelial cells, transgenic sickle mice, and in a prospective cohort of patients with sickle cell anemia. In vitro: We exposed endothelial cells (EA.hy926, ATTC® CRL-2922TM; Manassas, VA) to incremental doses of cell-free hemoglobin. With higher doses of cell-free hemoglobin we observed reduced surface endothelial cell THBD activity at 6 hours of incubation, assessed by cleavage of chromogenic substrate for activated protein C in the presence of thrombin (Figure 1A). In conjunction with the reduced endothelial THBD activity, there were increased concentrations of cleaved THBD in the supernatant by ELISA (R&amp;D Systems, Minneapolis, MN) (Figure 1B). Transgenic sickle mice: At 6 months of age, transgenic sickle mice (Townes model, Jackson Laboratory; Bar Harbor, Maine) had higher plasma concentrations of cleaved non-functional THBD versus hemoglobin AA mice (Figure 2A). Furthermore, staining of the glomerular microvasculature demonstrated decreased endothelial-bound THBD (Figure 2B). Patients with sickle cell anemia: We evaluated whether plasma concentrations of cleaved non-functional THBD are predictive of acute multiorgan failure syndrome (PMID 8109600) in a cohort of 103 SCD patients recruited into a longitudinal kidney cohort study. Clinical and laboratory variables and plasma samples were obtained at the time of enrolment and the patients were monitored prospectively for acute multiorgan failure syndrome. Hemoglobinuria was defined by urine dipstick positive for blood with &amp;lt; 2 red blood cells/high power field. The median age of this cohort was 35 years (interquartile range, 28 - 44 years), 46% were female, 87% had hemoglobin SS genotype, and 47% were on hydroxyurea at the time of enrolment. In cross-sectional analysis, plasma THBD concentrations were greater in patients with hemoglobinuria, a marker of intravascular hemolysis-derived cell-free hemoglobin in circulation exceeding scavenging capability and filtering through the glomerulus, versus without hemoglobinuria (6.1 ± 0.6 µg/mL vs. 3.6 ± 0.4 µg/mL, P = 0.004) (Figure 3A). With a median follow up of 5.5 years (interquartile range, 1.4 - 5.9 years), 18 (17%) SCD patients had a multiorgan failure event. SCD patients with a multiorgan failure event were older (43 vs. 34 years, P = 0.01) but without significant differences in sex, SCD genotype, or hydroxyurea therapy. After adjusting for age, baseline THBD concentrations predicted a greater risk for multi-organ failure syndrome (log-transformed OR 4.0, 95% CI: 1.2 - 13.3; P = 0.01) (Figure 3B). In conclusion, circulating non-functional THBD, a protein that normally functions to reduce vasculopathy when bound to the endothelium, is increased after endothelial cell exposure to incremental doses of hemoglobin in vitro, in hemoglobin SS vs. AA mice, and in SCD patients with versus without hemoglobinuria. Furthermore, circulating THBD is a biomarker that predicted the risk for multi-organ failure syndrome on longitudinal follow up in SCD patients. Future studies investigating the role of THBD in SCD vasculopathy may help improve our understanding for the catastrophic multiorgan failure syndrome and therapies to augment endothelial cell THBD function may guide future intervention practices. Figure 1 Disclosures Gordeuk: Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Modus Therapeutics: Consultancy, Honoraria; Pfizer: Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding. Saraf:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding.

Targeted Therapy of Acute Chest Syndrome with Recombinant Heme Oxygenase-1 Revealed By the Resistance of Young Transgenic Sickle Cell Mice to Heme-Induced Acute Lung Injury

Acute chest syndrome (ACS) is a major pulmonary complication of sickle cell disease (SCD) with currently no mechanistic-based therapies. Although ACS is more frequent in childhood, survival among children 1-9 years is ~10-fold higher compared to adults 20 years and older. Hitherto, mechanisms responsible for the high ACS survival in children have not been defined. We previously reported that acute elevation of circulating heme causes a TLR4-dependant lethal acute lung injury (ALI) in transgenic mice with homozygous SCD (SS) reminiscent of severe ACS (Ghosh et al., J Clin Invest, 2013). In the current study we discovered that young (4-6 weeks) and adult (12 weeks) SS mice suffer comparable severity of hemolytic crises when challenged with intravenous hemin (35 μmoles/kg bw). However, virtually none of the young mice develop ALI while nearly all the adults succumb with respiratory failure. We discovered that young SS mice rapidly clear excess heme from the circulation suggesting this was the reason for their resistance to intravenous hemin. Interestingly, young SS mice have significantly lower plasma hemopexin levels than adult SS mice, which excludes the classical heme scavenging pathway as the reason for their heme resistance. Previous studies have linked genetically high heme oxygenase-1 (HO-1) expression with low-risk of ACS, and raised steady-state levels of plasma heme with high-risk ACS in children. To explore these associations further, we studied a large cohort of patients and discovered for the first time that the concentration of plasma HO-1 in SCD children 1-9 yrs is 2-fold higher than adults 20 yrs and older (23.6±1.1, n=191 versus mean 10.7±0.6, n=67); these findings were phenocopied in transgenic SS mice. Plasma fractionization experiments revealed that HO-1 co-localizes with the enzymatic and co-factor machinery required for heme degradation indicating that heme can be degraded in the blood circulation. To test this idea ~3 weeks old SS mice were treated with the HO-1 inhibitor tin protoporphyrin (SnPP) or vehicle, and challenged with intravenous hemin five days later to induce ALI/ACS. Plasma HO activity declined by ~76% in SnPP-treated mice and was unchanged in the vehicle treated animals (n=12-14, p&amp;lt;0.001). A significant majority (10/12; 83%) of the vehicle-treated mice survived, while a majority of the SnPP-treated mice (10/14; 71%) developed lethal ALI/ACS. In another series of experiments young SS mice were treated for three months with Nrf2 activator to block age-dependent HO-1 decline, and then treated with vehicle or SnPP prior to ALI/ACS induction. The SnPP-treated group had 58% reduction in plasma HO-1 (p&amp;lt;0.05), failed to clear heme and developed lethal ALI/ACS, while a majority (63%) of the vehicle treated SS mice with persistently raised HO-1 activity in adulthood survived. Finally, adult SS mice were treated acutely with novel truncated human HO-1 recombinant proteins or a carrier vehicle immediately prior to induction of ALI/ACS. Recombinant variant 4 (V4) possessing an Fc component that enhances protein half-life provided 100% protection while 70% of vehicle treated mice succumbed with ALI/ACS (Mantel-Cox log-rank p&amp;lt;0.001). While the arterial oxygen saturation level (% SpO2) in vehicle treated SS mice declined sharply reflecting severe respiratory distress, V4-treated mice maintained normal SpO2 (p&amp;lt;0.001). In conclusion, we have determined that HO-1 activity in blood plasma declines with aging in SCD patients and identify this phenomenon as the basis of poor survival among adult SS mice with induced ALI/ACS. Furthermore, we provide proof-of-principle in mice that a novel human recombinant HO-1 protein can attenuate lung injury and improve survival from ALI/ACS in SCD. Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship.

Pharmacological Increase of Hb-O2 Affinity with a Voxelotor Analog Does Not Decrease Brain Tissue pO2 or Limit O2 Extraction in Brain Tissues of Sickle Cell Mice

Sickle Cell Disease (SCD) is characterized by hemolytic anemia, vaso-occlusion, and progressive end-organ damage. The underlying mechanism of SCD is the polymerization of sickle hemoglobin (HbS) that occurs when sickle erythrocytes (SS RBCs) are partially deoxygenated in microcirculation, leading to SCD pathophysiologic features. One of the most devastating complications of SCD occurs in the central nervous system (CNS), where overt stroke or repeated silent cerebral infarcts lead to significant physical and neurocognitive consequences. In SCD, the brain's response to insufficient oxygen (O2) delivery is balanced by increased blood flow to preserve O2 supply. However, the systemic endothelial dysfunction in SCD limits the capacity for vascular regulatory and compensatory changes to preserve appropriate tissue oxygenation, especially in tissues with high O2 demand like the brain. Low hemoglobin (Hb) levels and increased cerebral blood flow (CBF) are associated with increased stroke risk, suggesting that anemia-induced tissue hypoxia is an important factor contributing to subsequent morbidity in SCD patients. Voxelotor (GBT440) is a small molecule, HbS polymerization inhibitor being developed by Global Blood Therapeutics (GBT) for the treatment of SCD. By addressing the underlying mechanism of SCD, voxelotor has the potential to be disease-modifying and alleviate the clinical manifestations of SCD. Mechanistically, voxelotor increases Hb-O2 affinity and delays the transition from oxyHb to deoxyHb under hypoxic conditions. This study assessed the impact of a pharmacologically mediated increase in Hb-O2 affinity on brain tissue oxygenation under both normoxic and hypoxic conditions in Townes transgenic sickle mice (SCD mice). Two compounds that increase the Hb-O2 affinity with similar potency, voxelotor and an analog to voxelotor, GBT1118, were considered for the study. The target for Hb occupancy with test compounds was ≥30% based on the therapeutic target occupancies observed with voxelotor in clinical studies. The effects of increased Hb-O2 affinity on brain tissue oxygenation were assessed directly with O2-specific microelectrodes in a cranial window and indirectly with hypoxyprobe staining (pimonidazole) of brain tissue. Unique to this SCD model, the targeted Hb occupancy (≥30%) could not be consistently achieved by voxelotor. In contrast, repeat oral dosing of GBT1118 at 200 mg/kg/day for 2 weeks in SCD mice achieved steady state concentrations of 802 ± 81 µM (mean ± SD; n=5), corresponding to a Hb occupancy of 44 ± 5%. Consequently, GBT1118 decreased the p50 (partial pressure of O2 at which Hb is 50% saturated) values of SCD mouse blood from 39 ± 0.8 mmHg (Vehicle-dosed) to 21 ± 1.6 mmHg (GBT1118-dosed). While we could not achieve the desired Hb occupancy (≥30%) with voxelotor in this model, the Hb occupancy and change in p50 achieved with GBT1118 afforded us the opportunity to ask whether significantly increasing Hb-O2 affinity affects brain O2 tension. Measurements of cortical O2 tension (pO2) showed no difference in pO2 values under normoxia (21% O2) (Figure A), and slightly higher pO2 values under hypoxia (10% O2) (Figure B) for GBT1118-dosed SCD mice compared with vehicle-dosed SCD mice. Collectively across all brain tissues, the GBT1118-induced increase in Hb-O2 affinity reduced tissue hypoxia in SCD mice under hypoxia as measured by pimonidazole staining (Figure C). Together, these results indicate that a pharmacological increase of Hb-O2 affinity does not decrease cortical tissue pO2 in SCD mice and may reduce brain hypoxia under hypoxic conditions. Figure Disclosures Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Lucas:Global Blood Therapeutics: Research Funding. Muller:Global Blood Therapeutics: Research Funding. Williams:Global Blood Therapeutics: Research Funding. Zhang:Global Blood Therapeutics: Employment, Equity Ownership. Rademacher:Global Blood Therapeutics: Employment, Equity Ownership. Alt:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Cabrales:Global Blood Therapeutics: Research Funding.

Effects of Individualized Treadmill Endurance Training on Oxidative Stress in Skeletal Muscles of Transgenic Sickle Mice.

Oxidative stress is a key feature in the pathophysiology of sickle cell disease. Endurance training has been shown to reduce oxidative stress in the heart and the liver of sickle mice. However, the effects of endurance training on skeletal muscles, which are major producers of reactive oxygen species during exercise, are currently unknown. The aim of this study was to evaluate the effect of sickle genotype on prooxidant/antioxidant response to individualized endurance training in skeletal muscles of sickle mice. Healthy and homozygous Townes sickle mice were divided into trained or sedentary groups. Maximal aerobic speed and V̇O2 peak were determined using an incremental test on a treadmill. Trained mice ran at 40% to 60% of maximal aerobic speed, 1 h/day, 5 days/week for 8 weeks. Oxidative stress markers, prooxidant/antioxidant response, and citrate synthase enzyme activities were assessed in the gastrocnemius, in the plantaris, and in the soleus muscles. Maximal aerobic speed and V̇O2 peak were significantly reduced in sickle compared to healthy mice (-57% and -17%; p < 0.001). NADPH oxidase, superoxide dismutase, and catalase activities significantly increased after training in the gastrocnemius of sickle mice only. A similar trend was observed for citrate synthase activity in sickle mice (p = 0.06). In this study, we showed an adaptive response to individualized endurance training on the prooxidant/antioxidant balance in the gastrocnemius, but neither in the plantaris nor in the soleus of trained sickle mice, suggesting an effect of sickle genotype on skeletal muscle response to endurance treadmill training.

Morphine promotes neovascularizing retinopathy in sickle transgeneic mice

AbstractNeovascularizing retinopathy is a significant complication of sickle cell disease (SCD), occurring more frequently in HbSC than HbSS disease. This risk difference is concordant with a divergence of angiogenesis risk, as identified by levels of pro- vs anti-angiogenic factors in the sickle patient's blood. Because our prior studies documented that morphine promotes angiogenesis in both malignancy and wound healing, we tested whether chronic opioid treatment would promote retinopathy in NY1DD sickle transgenic mice. After 10 to 15 months of treatment, sickle mice treated with morphine developed neovascularizing retinopathy to a far greater extent than either of the controls (sickle mice treated with saline and wild-type mice treated identically with morphine). Our dissection of the mechanistic linkage between morphine and retinopathy revealed a complex interplay among morphine engagement with its μ opioid receptor (MOR) on retinal endothelial cells (RECs); morphine-induced production of tumor necrosis factor α and interleukin-6 (IL-6), causing increased expression of both MOR and vascular endothelial growth factor receptor 2 (VEGFR2) on RECs; morphine/MOR engagement transactivating VEGFR2; and convergence of MOR, VEGFR2, and IL-6 activation on JAK/STAT3-dependent REC proliferation and angiogenesis. In the NY1DD mice, the result was increased angiogenesis, seen as neovascularizing retinopathy, similar to the retinal pathology occurring in humans with SCD. Therefore, we conclude that chronic opioid exposure, superimposed on the already angiogenic sickle milieu, might enhance risk for retinopathy. These results provide an additional reason for development and application of opioid alternatives for pain control in SCD.

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.

Iron Deficiency Induces Faster Sickling Kinetics in Transgenic Sickle Mice

Sickle cell disease (SCD) is caused by polymerization of deoxygenated hemoglobin‐S (HbS) within the red blood cell (RBC). The kinetics of hemoglobin polymerization are dependent on the intra‐erythrocytic concentration of HbS; specifically, the time from deoxygenation to polymerization is inversely related to HbS concentration30. We hypothesized that iron‐deficient erythropoiesis would produce RBCs with decreased intracellular Hb concentration and prolonged sickling times which would reduce the severity of SCD.To interrogate this hypothesis, we studied Townes mice that express humanized alpha globin, gamma globin, and either beta‐S (Townes‐SCD) or normal beta globin (Townes‐Control). RBCs from these Townes‐SCD mice have similar sickling kinetics to RBCs from human volunteers with SCD. To induce iron deficiency, we fed mice either an iron‐restricted diet (3 ppm iron) or iron‐normal diet (48 ppm iron) introduced at the time of weaning. After two months on the experimental diets, we measured RBC parameters using an Advia 2120 hematology analyzer and measured in vitro RBC sickling kinetics at 3% oxygen. Analysis of RBC parameters revealed that SCD mice on the iron‐restricted diet had lower hematocrit (32% vs 42.2%, P &lt;0.0001), and there were trends toward lower hemoglobin, mean corpuscular volume, mean cell hemoglobin, and mean corpuscular hemoglobin concentration. Surprisingly, the in vitro sickling kinetic assay showed a significantly shortened time to 50% sickled in the Iron Restricted group (t50: 96.6 vs 136.5 min., p&lt;0.0001).At this two‐month time point, this study provides insights into the hematologic changes that occur during the initial onset of iron deficiency anemia in Townes‐SCD mice. We were surprised to find that the rate of sickling increased in the iron‐restricted mice; however, at two months of iron deficient diet, we have yet to see a substantial fall in the MCHC, a major determinant of the sickling delay time. Furthermore, these mice may have experienced compensatory changes in the red cell that adversely affected sickling such as a decrease in hemoglobin F levels, or a shift in the Hb‐ O2 dissociation curve that favors releasing oxygen at lower partial pressures. To address these questions, we will investigate the hemoglobin composition and the oxygen dissociation curve at the remaining four‐ and six‐month timepoints. We aim to determine the intermediate and long‐term potential of iron deficiency to delay sickling and reduce the severity of organ damage in the context of SCD. This may inform a strategy of optimizing iron intake as a potential treatment for SCD.Support or Funding InformationSupport from NIAID Intramural funding and NIH Division of Veterinary ResourcesScholarship Award from the Saudi Arabian Cultural Mission (SACM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mn Porphyrin-Based Redox Active Drugs As Novel Anti-Adhesive and Anti-Inflammatory Agents Targeting Sickle Red Blood Cell Oxidative Damage In Vivo

In sickle cell disease (SCD), painful vaso-occlusive crises and end-organ damage are caused by occlusion of the vessels due largely to sickle red blood cell (RBC) adhesion to both the endothelium and adherent leukocytes. RBC oxidative damage caused by continuous endogenous and exogenous oxidative stress may participate in the occurrence of vaso-occlusive crises.We have evaluated the effects of scavenging reactive oxygen species (ROS) in sickle RBCs on cell adhesion and vaso-occlusion in a humanized mouse model of vaso-occlusion in vivo analyzed by intravital microscopy. To scavenge RBC ROS, we used our manganese porphyrin-based superoxide dismutase (SOD) mimics MnTnBuOE-2-PyP5+ (MnBuOE) and MnTE-2-PyP5+ (MnE), powerful catalysts of superoxide dismutation, and reductants of peroxynitrite, peroxide and hypochlorite. Intravital microscopy observations of enflamed vessels visible through dorsal skin-fold window chamber implants was performed after the inflammatory trigger of tumor necrosis factor alpha (TNFα) to induce vaso-occlusion in transgenic sickle mice followed by subcutaneous injection of MnBuOE at 0.1, 0.2 or 2 mg/kg, or MnE at 0.5 or 2 mg/kg.Treatment of sickle mice with only one dose of 0.1, 0.2 and 2 mg/kg MnBuOE decreased dose-dependently adhesion of both sickle cells and leukocytes in enflamed vessels by 68±4% (p<0.01), 85±2.3% (p<0.01) and 89±4.3% (p<0.01), respectively, compared with vehicle-treated sickle mice. MnBuOE at 0.1, 0.2 and 2 mg/kg also caused significant and dose-dependent reduction in leukocyte rolling flux (p<0.05). Similar inhibitory benefits were obtained when MnE was administered to TNFa-treated sickle mice. MnE at 0.5 and 2 mg/kg significantly decreased the number of adherent sickle cells and leukocytes by 76±8.6% (p<0.01) and 92±2.5% (p<0.01), respectively, and leukocyte rolling flux (p<0.01) compared to vehicle-treated animals. The effect of these two SOD mimics on sickle RBCs and leukocyte adhesion, and leukocyte rolling flux was rapid, because a decline in cell adhesion and leukocyte rolling flux were already detectable within the first 15 minutes after injection of the compounds. In contrast, cell adhesion and leukocyte rolling flux were already pronounced 15 minutes following vehicle injection. Reduced cell adhesion to the endothelium by the SOD mimics resulted in improved microcirculatory blood flow in sickle mice. These favorable effects on cell adhesion and vaso-occlusion following SOD mimic treatment were indeed due at least to the significant decrease in sickle RBC ROS levels compared to vehicle-treated mice (p<0.001).The long-term anti-adhesive and anti-inflammatory effects of MnBuOE and MnE in sickle mice were next examined. Subcutaneous administration for 28 days of MnBuOE at 0.1 and 0.5 mg/kg inhibited significantly adhesion of RBCs and leukocytes in enflamed venules by 34±13% (p<0.05) and 69±3.5% (p<0.001), respectively, and leukocyte rolling flux (p<0.001) compared to vehicle-treated sickle mice. Subcutaneous injection of MnE at 0.5 and 1 mg/kg for 28 days also had significant effect on sickle cell and leukocyte adhesion (p<0.01), and leukocyte rolling flux (p<0.01). In addition, venous blood gases were significantly improved by the SOD mimics. The levels of partial pressure of Carbon dioxide (pCO2), partial pressure of oxygen (pO2), base excess of the extracellular fluid (BEecf), bicarbonate (HCO3-) concentration, total CO2 (TCO2) concentration, and the indicators of hypoxia, hemoglobin saturation of oxygen (sO2) and lactate, became close to or within the normal ranges (p<0.05) in sickle mice treated with 1 mg/kg MnE. MnBuOE at 0.1 mg/kg showed only a trend toward an increase in venous blood gases, with a significant decrease in lactate (p<0.05). Leukocytosis in sickle mice treated with the SOD mimics was also alleviated. A significant drop in leukocyte (p<0.05), neutrophil (p<0.01), lymphocyte (p<0.05) and monocyte (p<0.05) counts was detected in sickle mice treated with either 0.1 mg/kg MnBuOE or 1 mg/kg MnE. These beneficial therapeutic outcomes induced by the SOD mimics were due at least in part to a decline in RBC ROS levels (p<0.001) and RBC phosphatidylserine surface exposure (p<0.05), an eryptosis marker.These results suggest that our SOD mimics may represent a valuable novel therapeutic intervention for not only vaso-occlusive crises, but inflammation as well, that should be further evaluated in patients with SCD. DisclosuresNo relevant conflicts of interest to declare.

Effect of Chronic Opioid Therapy on Pain and Survival in a Mouse Model of Sickle Cell Disease

Pain is one of the major comorbidities of sickle cell disease (SCD) requiring chronic opioid therapy (COT). However, COT has been associated with reduced survival and opioid-induced hyperalgesia (OIH), which can exacerbate the chronic pain and increased mortality already inherent to SCD. It is challenging to examine pain, opioid use and survival in populations with SCD due to significant heterogeneity in multiple clinicopathologic factors. To determine the effect of COT we used transgenic HbSS BERK sickle mice, which show pain characteristics similar to those observed in patients with SCD, including sensitivity to thermal and mechanical stimuli, increased opioid requirement, and higher levels of circulating substance P and tryptase (Tran et al., Blood 2017). Additionally, similar to female patients with SCD, female BERK sickle mice show more pain than males. Therefore, we performed a randomized double-blind placebo-controlled trial to examine the effect of COT on pain and survival in female homozygous HbSS BERK (sickle) and HbAA BERK (control) mice, expressing >99% human sickle hemoglobin and normal human hemoglobin A, respectively. Mice were injected subcutaneously each day with either morphine sulfate at a starting dose of 20 mg/kg, which was increased to 25 mg/kg, 30 mg/kg, 35 mg/kg, and 40 mg/kg after weeks 12, 18, 28, 30, and 38 respectively or placebo (equal volume of saline) until the end of survival. Dose of morphine was selected based on the effective analgesic dose for these mice (Kohli et al., Blood 2010). Mechanical-, thermal- (heat and cold), and musculoskeletal/deep hyperalgesia were analyzed in all mice biweekly before and after drug treatments. We used Kaplan-Meier and log-rank test with Sidak correction for survival; 2-way repeated measures analysis of variance with Bonferroni's correction to compare hyperalgesia between time points among control and sickle mice and Cox proportional-hazards regression analysis for the association of hyperalgesia with survival.We observed significantly decreased survival of saline-treated sickle mice compared to saline-treated control mice (P = 0.0009). Compared to saline, morphine treatment led to a significant decrease in survival in control mice (P = 0.035) but not in sickle mice (P > 0.05). Furthermore, we did not observe an association between hyperalgesia and survival in either control or sickle mice. However, we discerned a significant increase in mechanical, cold, and heat hyperalgesia in control mice after 4 weeks of morphine treatment (P < 0.02, compared to day '0') which continued to increase up to 12 weeks (P <0.05 compared to week 4). Similarly, in sickle mice we observed an increase in mechanical hyperalgesia after 4 weeks (P <0.02, compared to day '0') of morphine treatment which continued to increase up to 12 weeks (P < 0.0001). These data suggest COT leads to OIH in both control and sickle mice. It is noteworthy that morphine treatment continued to show an analgesic effect over the course of 12 weeks in spite of an increase in hyperalgesia in both groups of mice. Thus, mice did not develop tolerance to morphine analgesia. Since mice were treated under uniform conditions, the effect of chronic morphine treatment could be observed without any confounding factors. Our findings in control mice recapitulate the clinical observations that COT is associated with reduced lifespan in non-sickle patients. COT lead to OIH in sickle mice but provided analgesia without causing tolerance or reducing survival. OIH may contribute may exacerbate a vicious cycle of chronic pain and opioid use in SCD. Differences exist between humans and mice including morphine metabolism between the two but due to several similarities of HbSS BERK sickle mice with clinical pain characteristics and biology, these observations have a significant translational potential following clinical trials for optimizing the treatment of pain in SCD. These data highlight the critical need to develop analgesic strategies devoid of OIH. DisclosuresGupta:Tau tona: Consultancy; Novartis: Honoraria.

Progressive glomerular and tubular damage in sickle cell trait and sickle cell anemia mouse models

Homozygosity for the hemoglobin (Hb) S mutation (HbSS, sickle cell anemia) results in hemoglobin polymerization under hypoxic conditions leading to vaso-occlusion and hemolysis. Sickle cell anemia affects 1:500 African Americans and is a strong risk factor for kidney disease, although the mechanisms are not well understood. Heterozygous inheritance (HbAS; sickle cell trait) affects 1:10 African Americans and is associated with an increased risk for kidney disease in some reports. Using transgenic sickle mice, we investigated the histopathologic, ultrastructural, and gene expression differences with the HbS mutation. Consistent with progressive glomerular damage, we observed progressively greater urine protein concentrations (P = 0.03), glomerular hypertrophy (P = 0.002), and glomerular cellularity (P = 0.01) in HbAA, HbAS, and HbSS mice, respectively. Ultrastructural studies demonstrated progressive podocyte foot process effacement, glomerular basement membrane thickening with reduplication, and tubular villous atrophy with the HbS mutation. Gene expression studies highlighted the differential expression of several genes involved in prostaglandin metabolism (AKR1C18), heme and iron metabolism (HbA-A2, HMOX1, SCL25A37), electrolyte balance (SLC4A1, AQP6), immunity (RSAD2, C3, UBE2O), fatty acid metabolism (FASN), hypoxia hall-mark genes (GCK, SDC3, VEGFA, ETS1, CP, BCL2), as well as genes implicated in other forms of kidney disease (PODXL, ELMO1, FRMD3, MYH9, APOA1). Pathway analysis highlighted increased gene enrichment in focal adhesion, extracellular matrix-receptor interaction, and axon guidance pathways. In summary, using transgenic sickle mice, we observed that inheritance of the HbS mutation is associated with glomerular and tubular damage and identified several candidate genes and pathways for future investigation in sickle cell trait and sickle cell anemia-related kidney disease.

A monocyte-TNF-endothelial activation axis in sickle transgenic mice: Therapeutic benefit from TNF blockade.

Elaboration of tumor necrosis factor (TNF) is a very early event in development of ischemia/reperfusion injury pathophysiology. Therefore, TNF may be a prominent mediator of endothelial cell and vascular wall dysfunction in sickle cell anemia, a hypothesis we addressed using NY1DD, S+SAntilles, and SS‐BERK sickle transgenic mice. Transfusion experiments revealed participation of abnormally activated blood monocytes exerting an endothelial activating effect, dependent upon Egr‐1 in both vessel wall and blood cells, and upon NFκB(p50) in a blood cell only. Involvement of TNF was identified by beneficial impact from TNF blockers, etanercept and infliximab, with less benefit from an IL‐1 blocker, anakinra. In therapeutic studies, etanercept ameliorated multiple disturbances of the murine sickle condition: monocyte activation, blood biomarkers of inflammation, low platelet count and Hb, vascular stasis triggered by hypoxia/reoxygenation (but not if triggered by hemin infusion), tissue production of neuro‐inflammatory mediators, endothelial activation (monitored by tissue factor and VCAM‐1 expression), histopathologic liver injury, and three surrogate markers of pulmonary hypertension (perivascular inflammatory aggregates, arteriolar muscularization, and right ventricular mean systolic pressure). In aggregate, these studies identify a prominent—and possibly dominant—role for an abnormal monocyte‐TNF‐endothelial activation axis in the sickle context. Its presence, plus the many benefits of etanercept observed here, argue that pilot testing of TNF blockade should be considered for human sickle cell anemia, a challenging but achievable translational research goal.

Transcriptomic analysis of gene signatures associated with sickle pain

Pain is a hallmark feature of sickle cell disease (SCD). Recurrent and unpredictable acute pain due to vaso-oclussive crises (VOC) is unique to SCD; and can be superimposed on chronic pain. To examine the mechanisms underlying pain in SCD, we performed RNA sequencing of dorsal root ganglion (DRG) of transgenic sickle mice and their age-matched control mice expressing normal human hemoglobin A, at 2 and 5 months of age. Sickle and control mice of both ages were equally divided into hypoxia/reoxygenation (to simulate VOC) and normoxia treatment, resulting in eight groups of mice. Each group had at least six mice. RNA isolated from the DRG was sequenced and paired-end 50 bp sequencing data were generated using Illumina’s HiSeq 2000. This large dataset can serve as a resource for examining transcriptional changes in the DRG that are associated with age and hypoxia/reoxygenation associated signatures of nociceptive mechanisms underlying chronic and acute pain, respectively.