Transgenic Sickle Cell Disease Mice Research Articles

Heme-induced loss of renovascular endothelial protein C receptor promotes chronic kidney disease in sickle mice

AbstractChronic kidney disease (CKD) is a major contributor to morbidity and mortality in sickle cell disease (SCD). Anemia, induced by chronic persistent hemolysis, is associated with the progressive deterioration of renal health, resulting in CKD. Moreover, patients with SCD experience acute kidney injury (AKI), a risk factor for CKD, often during vaso-occlusive crisis associated with acute intravascular hemolysis. However, the mechanisms of hemolysis-driven pathogenesis of the AKI-to-CKD transition in SCD remain elusive. Here, we investigated the role of increased renovascular rarefaction and the resulting substantial loss of the vascular endothelial protein C receptor (EPCR) in the progressive deterioration of renal function in transgenic SCD mice. Multiple hemolytic events raised circulating levels of soluble EPCR (sEPCR), indicating loss of EPCR from the cell surface. Using bone marrow transplantation and super-resolution ultrasound imaging, we demonstrated that SCD mice overexpressing EPCR were protective against heme-induced CKD development. In a cohort of patients with SCD, plasma sEPCR was significantly higher in individuals with CKD than in those without CKD. This study concludes that multiple hemolytic events may trigger CKD in SCD through the gradual loss of renovascular EPCR. Thus, the restoration of EPCR may be a therapeutic target, and plasma sEPCR can be developed as a prognostic marker for sickle CKD.

Heme Scavenging by Hemopexin Attenuates Sepsis-Induced Proinflammatory Cytokines and Kidney Insuffciency in a Humanized Mouse Model of Sickle Cell Disease

Sickle cell disease (SCD) is a genetic blood disorder characterized by abnormal hemoglobin production, leading to sickle-shaped red blood cells (RBC). Alteration in RBC shape results in vasoocclusion, chronic hemolysis, and multiple organ damage. One of the severe complications of SCD is an increased susceptibility to infections, including sepsis, which can be life-threatening. Kidney injury, a common and severe complication of sepsis, contributes to the complexity and poor outcomes associated with morbidity and mortality in SCD. However, the mechanisms that underlie septic acute kidney injury (AKI) in SCD are poorly understood. Here, we examined the effects of sepsis in transgenic homozygous SCD (SS; Townes model) mice and their non-sickling (AA) counterparts. We tested the hypothesis that aggravation of inflammation and oxidative stress by free heme released during hemolysis contribute to sepsis-induced kidney insuffciency in SCD. AA and SS mice were subjected to 18 h of sham operation and cecal ligation and puncture (CLP), a pre-clinical model of polymicrobial sepsis, after which kidney function was evaluated by transdermal measurement of glomerular filtration rate (GFR). AKI biomarkers, renal oxidative stress, and cytokines levels were also investigated. We demonstrate that mid- to high-grade CLP (ligation of 60-75% of the cecum and multiple punctures with 21-gauge needles) caused 100% mortality in SS mice. Unlike AA mice (100% survival), SS mice subjected to low-grade CLP (LgCLP; ligation of <45% of the cecum and a single puncture with 21-gauge needles) exhibited 40% survival. LgCLP reduced body weight but did not exacerbate splenomegaly in SS mice. Basal plasma bilirubin, heme, and ferritin were significantly higher in SS than in AA mice. In contrast to AA, LgCLP amplified the increase in plasma bilirubin, heme, and ferritin in SS mice. LgCLP also decreased GFR and increased renal oxidative stress, AKI biomarkers, and kidney-to-body weight ratio in SS mice. Luminex multiplex cytokine analysis indicated that the plasma levels of multiple pro-inflammatory cytokines were elevated in septic SS mice. Heme scavenger hemopexin (HX) diminished LgCLP-induced plasma heme increase. HX also mitigated kidney insuffciency, inflammation, and mortality in the septic SS mice. Our findings suggest that free heme contributes to septic AKI in SCD mice and that HX therapy may attenuate the harmful effect of heme in SCD during sepsis. NHLBI: R01 HL151735. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

20-HETE As an Emerging Target to Improve Renal Health in Sickle Cell Disease

The kidney is one of the most severely affected organs in sickle cell disease (SCD), with glomerular hyperfiltration and microalbuminuria beginning in childhood, and progressing to chronic kidney disease (CKD) in majority of adults. Sickle individuals also suffer from acute kidney injury (AKI) often during characteristic vaso-occlusive crisis. Renal pathology in sickle cell nephropathy includes extensive microvascular congestions and thrombotic microangiopathy. The hypercoagulable and vasoconstrictive environment of SCD, in conjunction with medullary hypoperfusion and cortical hyperperfusion contributes to vaso-occlusive injury and deregulates intrarenal hemostasis leading to CKD development. Ischemic injury increases arachidonic acid hydroxylation producing 20-hydroxyeicosatetraenoic acid (20-HETE), which in turn triggers thrombosis and promotes vasoconstriction in the kidney. However, the role of 20-HETE in regulating renal hemostasis in SCD nephropathy has never been reported. We found that plasma 20-HETE is significantly elevated in SCD patients (n=9-11; p<0.01) compared to normal individuals. Similarly, transgenic SCD mice (SS), homozygous for human hemoglobin S, have increased plasma 20-HETE compared to control mice (AA), homozygous for human hemoglobin A (n=6; p<0.01). We have previously shown that elevated circulating heme mimicking hemolytic crisis triggers clinically relevant acute kidney injury (AKI) in SS mice (Blood (2020) 135(13): 1044-1048). We postulated that hemolytic stress may exacerbate 20-HETE production which in turn aggravates renal microvascular occlusion and vasoconstriction, leading to renal damage in SCD. Immunohistochemistry and biochemical experiments showed that the expression of CYP4A12a, a cytochrome 450-dependent enzyme responsible 20-HETE production in mice, is substantially elevated in the kidneys of SS mice compared to AA mice, associated with increased vascular congestion and renal thrombogenesis. Renal CYP4A12a and plasma 20-HETE were increased substantially following heme-induced AKI in SS mice, associated with a heightened expression of renal angiotensin II receptor 1 (ATIIR1), thus indicating induced vasoconstrictive activity. In vitro, we found that 20-HETE induced ATIIR1 expression in human renal glomerular endothelial cells (hRGEC), which was further exacerbated by low-concentration of heme challenge (5 mM for 24 hour). Nitro-oleic acid (NO 2-OA) is a nitrated fatty acid that binds ATIIR1 and inhibits angiotensin II induced vasoconstriction. Pretreatment with NO 2-OA reduced 20-HETE-dependent ATIIR1 expression in hRGEC, while NO 2-OA prophylaxis during hemin-induced AKI inhibited albuminuria, normalized plasma creatinine levels, and lowered the expression of ATIIR1 in SS mice kidneys. Overall, our data show that elevated 20-HETE levels during hemolytic stress may aggravate renal injury in SCD which can be prevented by NO 2-OA prophylaxis. Ongoing experiments will determine the direct effect of elevated 20-HETE in renal hemostasis and CKD development in SCD.

Enhanced Expression of Heme Oxygenase-1 (HO-1) Among Children with Sickle Cell Disease: Results of the Sickle Cell Disease Genomics of Africa (SickleGenAfrica) Study

Sickle cell disease (SCD) is characterized by intense and chronic intravascular hemolysis that generates multiple erythroid danger associated molecular pattern (eDAMP) molecules in the extracellular space. Extracellular heme is a prototypical eDAMP that causes tissue injury and is associated with multiple complications of SCD including vaso-occlusive crisis, acute kidney injury and acute chest syndrome (ACS). Heme-oxygenase-1 (HO-1) is the rate-limiting enzyme that degrades heme to neutralize its deleterious effects on organ function. Raised extracellular heme is associated with 2.5 higher odds ratio of developing ACS, while HO-1 gene promoter allelic variants that drive robust HO-1 expression are associated with lower ACS-related hospitalization rates among children. Young transgenic homozygous SCD (SS) mice have higher survival rates than adult SS mice from a heme-induced ACS model due to their ability to rapidly degrade excess extracellular heme. While these data suggest that HO-1 may be involved in the well-established higher ACS survival among children, and potentially other ameliorating features of SCD in early development, no study had previously studied HO-1 expression in this patient population. The SickleGenAfrica Network enrolled over 2,000 children and adults with SCD from three sites in Ghana from 2018 to 2021, with consent for use of samples in future studies. The SCD status of participants was confirmed using automated capillary hemoglobin electrophoresis, and serum HO-1 level measured using ELISA. Patients were grouped based on age, using five-year intervals. The median HO-1 level comparisons across groups were performed using the pairwise Wilcoxon test, with Bonferroni correction for multiple comparisons. All analyses were performed using R version 4.3.1. The study included 1,971 patients with SCD, with a median age of 13 years (range 0 - 78), of which 1,067 participants (55%) were female. The majority of patients (1149 ;58%) had HbSS, 589 (30%) had HbSC, 171 (9%) sickle-hereditary persistence of fetal hemoglobin, and 62 (3%) HbS-beta(+). We observed an approximately 3-fold drop-off in median HO-1 levels between the 11-15-year-old (73.9 ng/ml) and 16-20-year-old (25.1 ng/ml) age groups (adj. p<0.001). Furthermore, males had higher average HO-1 levels (85.5 ng/ml) relative to females (63.1 ng/ml) (p<0.001). Our data show for the first time that children with SCD have significantly elevated HO-1 levels relative to adults, with a sharp decline in HO-1 levels during adolescence. HO-1 may serve as a protective SCD factor, contributing to enhanced ACS survival rates in children versus adults. To the best of our knowledge, this is the first study of HO-1 levels in patients with SCD at different ages. Our future work aims to establish whether baseline blood HO-1 levels can predict ACS incidence and severity, in addition to further investigating genetic and epigenetic mechanisms controlling HO-1 expression.

Rhabdomyolysis aggravates renal iron accumulation and acute kidney injury in a humanized mouse model of sickle cell disease

Individuals with sickle cell disease (SCD) are at greater risk of rhabdomyolysis, a potentially life-threatening condition resulting from the breakdown of skeletal muscle fibers. Acute kidney injury (AKI) is one of the most severe complications of rhabdomyolysis. Chronic kidney and cardiovascular disease, which account for SCD mortality, are long-term consequences of AKI. Although SCD elevates the risks of rhabdomyolysis-induced sudden death, the mechanisms that underlie rhabdomyolysis-induced AKI in SCD are unclear. In the present study, we show that, unlike their control non-sickling (AA) counterparts, transgenic homozygous SCD (SS; Townes model) mice exhibited 100% mortality 8–24 h after intramuscular glycerol injection. Five hours after glycerol injection, SS mice showed a more significant increase in myoglobinuria and plasma creatine kinase levels than AA mice. Basal plasma heme and kidney tissue iron levels were significantly higher in SS than in AA mice. In contrast to AA, glycerol-induced rhabdomyolysis aggravated these parameters in SS mice. Rhabdomyolysis also amplified oxidative stress in SS compared to AA mice. Glycerol-treated SS mice exhibited worse renal function, exemplified by a reduction in GFR with a corresponding increase in plasma and urinary biomarkers of early AKI and renal tubular damage. The free radical scavenger and Fenton chemistry inhibitor, TEMPOL, ameliorated rhabdomyolysis-induced AKI in the SS mice. These findings demonstrate that oxidative stress driven by renal iron accumulation amplifies rhabdomyolysis-induced AKI in SCD mice.

Hydroxyurea does not reverse functional alterations of the nitric oxide-cGMP pathway associated with priapism phenotype in corpus cavernosum from sickle cell mouse.

Sickle cell disease (SCD) is a genetic disorder that has been associated with priapism. The role of hydroxyurea, a common SCD therapy, in influencing the nitric oxide (NO)-cGMP pathway and its effect on priapism is unclear. To investigate the effect of hydroxyurea treatment on smooth muscle relaxation of corpus cavernosum induced by stimulation of the NO-cGMP pathway in SCD transgenic mice and endothelial NO synthase gene-deficient (eNOS-/-) mice, which are used as model of priapism associated with the low bioavailability of endothelial NO. Four-month-old wild-type (WT, C57BL/6), SCD transgenic, and eNOS-/- male mice were treated with hydroxyurea (100 mg/Kg/day) or its vehicle (saline) daily for three weeks via intraperitoneal injections. Concentration-response curves for acetylcholine (ACh), sodium nitroprusside (SNP), and electrical field stimulation (EFS) were generated using strips of mice corpus cavernosum. The SCD mice demonstrated an amplified CC relaxation response triggered by ACh, EFS, and SNP. The corpus cavernosum relaxation responses to SNP and EFS were found to be heightened in the eNOS-/- group. However, the hydroxyurea treatment did not alter these escalated relaxation responses to ACh, EFS, and SNP in the corpus cavernosum of the SCD group, nor the relaxation responses to EFS and SNP in the eNOS-/- group. In conclusion, hydroxyurea is not effective in treating priapism associated with SCD. It is likely that excess plasma hemoglobin and reactive oxygen species, which are reported in SCD, are reacting with NO before it binds to GCs in the smooth muscle of the corpus cavernosum, thus preventing the restoration of baseline NO/cGMP levels. Furthermore, the downregulation of eNOS in the penis may impair the pharmacological action of hydroxyurea at the endothelial level in SCD mice. This study emphasize the urgency for exploring alternative therapeutic avenues for priapism in SCD that are not hindered by high plasma hemoglobin and ROS levels.

Reshaping the gut microbiota affects pain and anxiety-like behaviors in a transgenic model of sickle cell disease

Sickle cell disease (SCD) patients experience recurrent episodes of excruciating acute pain and persistent chronic ongoing pain which negatively impacts their quality of life and is the main cause for hospitalizations. Although major progress has been made in understanding the complex SCD pathophysiology, marked deficiencies in the knowledge regarding SCD pain, the factors underlying the transition from acute to chronic pain, pain comorbidities and effective pain management have been realized. Several studies have reported a crucial role of gut microbiota in the pathophysiological features of SCD; however, its role in pain has not been explored. Our study aimed at evaluating the compositional differences in the gut microbiota of SCD transgenic mice and non-sickle control mice and differences in the gut-derived metabolites. We further investigated the role of gut microbiota in SCD pain by utilizing antibiotic-mediated gut microbiota depletion and fecal microbiota transplantation (FMT). We report significant differences in the alpha and beta diversity between the two cohorts. The antibiotic mediated gut microbiota depletion did not affect evoked pain but significantly attenuated ongoing spontaneous pain in SCD mice. FMT from sickle to non-sickle mice resulted in reduced pain threshold for von Frey (0.95±0.17g vs 0.08±0.02g, p<0.001), reduced withdrawal latency for heat hyperalgesia and cold allodynia (15.10±0.79s vs 8.68±1.17s, p<0.01and 2.75±0.26s vs 1.68±0.08s, p<0.01) along with an increase in anxiety-like behaviors tested using elevated-plus maze (EPM) and open field test (OFT). The transfer of gut microbiota from SCD mice to non-sickle control mice was sufficient to evoke anxiety-like behaviors in the recipient mice as evidenced by decreased time spent in the open arm of the EPM and decreased time spent in the central compartment of the open field (76.18±13.18s vs 36.15±7.34s, p<0.05 for EPM and 126.4±35.3s vs 53.98±19.33s for OFT). Additionally, reshaping the gut microbiota of SCD mice with FMT resulted in reduced mechanical allodynia (0.05±0.01g vs 0.25±0.03g, p<0.001) and heat hyperalgesia (5.89±0.67s vs 12.25±0.76s, p<0.001) and reduced anxiety-like behaviors as evidenced by increased amount of time spent in open arms of the elevated plus maze (20.2±6.3s vs 42.1±15.03s) and increased amount of time spent in the central chamber of the open field (44.03±14.49s vs 104.4±[WJ1] 32.79s). These findings provide novel insights into the relationship between gut microbiota and pain in SCD, highlighting the different gut microbial communities which may serve as potential targets for pain management. Funding: This study was supported in part by a grant from the National Heart, Lung, and Blood Institute (NHLBI) R35 HL140021 (Z.W.). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NHLBI or NIH. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neuregulin-1 attenuates hemolysis- and ischemia induced-cerebrovascular inflammation associated with sickle cell disease

ObjectivesIndividuals with sickle cell disease (SCD) are at severely heightened risk for cerebrovascular injury and acute cerebrovascular events, including ischemic and hemorrhagic stroke, potentially leading to impaired development and life-long physical and cognitive disabilities. Cerebrovascular injury specific to SCD includes inflammation caused by underlying conditions of chronic hemolysis and reduced cerebrovascular perfusion. The objectives of this study were to investigate whether expression of neuregulin-1β (NRG-1), an endogenous neuroprotective polypeptide, is increased in SCD or experimental conditions mimicking the hemolysis and ischemic conditions of SCD, and to determine if treatment with exogenous NRG-1 reduces markers of cerebrovascular inflammation. Materials and methodsPlasma and brain-specific NRG-1 levels were measured in transgenic SCD mice. Endogenous NRG-1 levels and response to experimental conditions of excess heme and ischemia were measured in cultured human brain microvascular cells and astrocytes. Pre-treatment with NRG-1 was used to determine NRG-1’s ability to ameliorate resultant cerebrovascular inflammation. ResultsPlasma and brain-specific NRG-1 were elevated in transgenic SCD mice compared to healthy controls. Neuregulin-1 expression was significantly increased in cultured human microvascular cells and astrocytes exposed to excess heme and ischemia. Pre-treatment with NRG-1 reduced inflammatory chemokine (CXCL-1 and CXCL-10) and adhesion molecule (ICAM-1 and VCAM-1) expression and increased pro-angiogenic factors (VEGF-A) in microvascular cells and astrocytes exposed to excess heme and ischemia. ConclusionsElevated NRG-1 in SCD is likely a protective endogenous response to ongoing cerebrovascular insults caused by chronic hemolysis and reduced cerebrovascular perfusion. Administration of NRG-1 to reduce cerebrovascular inflammation may be therapeutically beneficial in SCD and warrants continued investigation.

Endothelial Extracellular Vesicles Regulate Platelet Purinergic Signaling in Sickle Cell Disease

Introduction. Acute chest syndrome (ACS) is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. ACS is often preceded by thrombocytopenia and involves massive thrombosis across pulmonary artery branches. Although, released during hemolysis, adenosine diphosphate (ADP) is known to activate platelets by stimulating their P2Y1 and P2Y12 purinergic receptors, antagonists of P2Y12 have not shown any benefit in ACS therapy, justifying the need for better understanding of purinergic signaling in SCD. CD39 maintains ADP homeostasis by degrading excessive ADP. Though CD39 inhibits ADP-dependent platelet activation, its role in the pathophysiology of SCD is still unidentified. Methods. To evaluate SCD platelet response to ADP in vivo we used a state-of-the-art intravital lung microscopy and a novel in vivo model of ADP-triggered thrombocytopenia in transgenic humanized SCD mice. Additionally, both mouse and human SCD platelet ADP-dependent aggregation was examined using in vitro turbidimetric aggregation assay. Hemin, a major host-derived damage associated molecular pattern associated with SCD, was incubated with Human Lung Micro-Vascular Endothelial Cells (HMVEC-L). Extracellular vesicles (EVs) were obtained from mouse and human plasma samples, and cell culture medium samples using size exclusion chromatography. Obtained EVs were subjected to nanoparticle tracking analysis. EVs CD39 levels and activity were determined using ELISA, western blot and malachite green phosphate assays, respectively. Results. Intravital lung microscopy and in vivo thrombocytopenia studies revealed that intravascular administration of ADP triggered acute pulmonary thrombosis in control but not in SCD mice. In vitro aggregation study confirmed our in vivo findings and demonstrated impaired SCD mouse and human platelet response to ADP, which was, further, significantly augmented by a specific CD39 inhibitor. Hemin triggered shedding of CD39-bearing EVs by HMVEC-L. Indeed, we discovered that isolated from mouse and human plasma endothelial cell-derived SCD EVs expressed higher CD39 levels and activity in comparison to control EVs. Conclusions. Our findings suggest that CD39-bearing EVs prevent ADP-mediated platelet aggregation and pulmonary thrombosis in SCD. Current study explains why P2Y12 blockers are not effective in SCD therapy.

Immunomodulatory actions of a kynurenine-derived endogenous electrophile.

The up-regulation of kynurenine metabolism induces immunomodulatory responses via incompletely understood mechanisms. We report that increases in cellular and systemic kynurenine levels yield the electrophilic derivative kynurenine-carboxyketoalkene (Kyn-CKA), as evidenced by the accumulation of thiol conjugates and saturated metabolites. Kyn-CKA induces NFE2 like bZIP transcription factor 2– and aryl hydrocarbon receptor–regulated genes and inhibits nuclear factor κB– and NLR family pyrin domain containing 3-dependent proinflammatory signaling. Sickle cell disease (SCD) is a hereditary hemolytic condition characterized by basal inflammation and recurrent vaso-occlusive crises. Both transgenic SCD mice and patients with SCD exhibit increased kynurenine and Kyn-CKA metabolite levels. Plasma hemin and kynurenine concentrations are positively correlated, indicating that Kyn-CKA synthesis in SCD is up-regulated during pathogenic vascular stress. Administration of Kyn-CKA abrogated pulmonary microvasculature occlusion in SCD mice, an important factor in lung injury development. These findings demonstrate that the up-regulation of kynurenine synthesis and its metabolism to Kyn-CKA is an adaptive response that attenuates inflammation and protects tissues.

Resveratrol-nitric oxide donor hybrid effect on priapism in sickle cell and nitric oxide-deficient mouse.

BackgroundChildren and adult with sickle cell disease (SCD) display priapism associated with low nitric oxide (NO) bioavailability and oxidative stress in penis.AimThis study aimed to evaluate the effects of hybrid compound RVT-FxMe, derived from resveratrol bearing a NO-donor subunit, on two murine model that display priapism phenotype, SCD transgenic mice and endothelial NO synthase gene-deficient (eNOS-/-) mice.MethodsWild-type, SCD, and eNOS-/- mice were treated with RVT-FxMe (25 mg/kg/d, 2 weeks).OutcomesHematological parameters, concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP), as well as to electrical field stimulation (EFS), were obtained in mice corpus cavernosum strips.ResultsCorpus cavernosum relaxations to SNP and EFS were increased in eNOS-/- group, which were normalized by RVT-FxMe treatment. SCD mice exhibited an excessive CC relaxant response induced by ACh, EFS and SNP RVT-FxMe treatment did not change the increased relaxant responses to ACh, EFS and SNP in corpus cavernosum from SCD group.Clinical translationExcess of plasma hemoglobin in SCD may interfere in pharmacological activity of NO donors compounds.Strength/LimitationsWhile mechanistic data with promising potential is showed, the current study is not without limitations. RVT-FxMe effects in the mid- and long-term warrant complementary studies.ConclusionTreatment with RVT-FxMe reversed the enhanced NO-cGMP-mediated CC relaxations in eNOS-/- mice, but not in SCD mice; it is likely that excess of plasma hemoglobin in SCD mice act to inactivate NO before it reaches soluble guanylyl cyclase, avoiding restoration of NO bioavailability in penis.

Sickle Cell Disease Associated Changes in the Gut Microbiome Contribute to Persistent Pain

Many patients with sickle cell disease (SCD) suffer from chronic pain, the underlying causes of which are unclear. Recent 16s ribosomal RNA sequencing studies revealed differences in the number and types of bacteria in the gastrointestinal tract of patients and mouse models of SCD relative to controls, but it is unclear if or how these changes contribute to symptomology. In these experiments, we used transgenic SCD mice to determine the extent to which disease related gut dysbiosis contributes to persistent pain. Reflexive pain behaviors were first measured in SCD mice following longitudinal probiotic or antibiotic treatment. Vehicle-treated SCD mice displayed significant mechanical allodynia relative to vehicle-treated wildtype mice, and antibiotic treatment further exacerbated mechanical allodynia in both genotypes. In contrast, probiotic treatment completely reversed persistent touch hypersensitivity in SCD mice. Persistent touch pain was also transiently reversed in SCD mice following fecal material transplant from healthy mice. In complementary experiments, wildtype recipient mice developed cold and touch hypersensitivity that persisted for several weeks after fecal material transplant from SCD donors. Using whole-cell patch clamp recordings, we further determined that these behavioral observations were accompanied by altered intrinsic plasticity in a select class of nodose ganglia sensory neurons, the peripheral terminals of which are well positioned to detect sensory information in the gut. Nodose ganglia neurons isolated from animals that received sickle cell fecal material transplants were hyperexcitable relative to those isolated from animals that received control fecal material transplants. These data are the first to suggest that disease-related gut dysbiosis induces pain through changes in vagal nerve activity. Ongoing studies are examining specific bacterial populations and/or metabolites responsible for these functional changes in order to develop novel therapeutics for chronic SCD pain management. Grant support from National Institutes of Health grants K99HL155791 and R01NS070711.

Plasma NTPDase1 Activity Regulates Platelet Purinergic Signaling in Sickle Cell Disease

Acute systemic painful vaso-occlusive episode (VOE) often serves as an antecedent to acute chest syndrome (ACS), which is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. Based on clinical epidemiology, ACS is often preceded by thrombocytopenia and involves massive thrombosis across pulmonary artery branches in 10-20% of ACS patients. Although, released during hemolysis, adenosine diphosphate (ADP) is known to activate platelets by stimulating their P2Y1 and P2Y12 purinergic receptors, antagonists of P2Y12 have not shown any benefit in ACS therapy, justifying the need for better understanding of purinergic signaling in SCD. Ecto-nucleoside-tri-phosphate-diphosphohydrolase-1 (E-NTPDase1; CD39) maintains ADP homeostasis by degrading excessive ADP. Though CD39 inhibits ADP-dependent platelet activation and vascular thrombosis, its role in ASC is still unidentified.Here, we use intravital lung microscopy in transgenic humanized SCD mice to show that intravascular (IV) administration of ADP triggered pulmonary thrombosis in control mice but failed to trigger pulmonary thrombosis in SCD mice. Identical to intravital findings, IV ADP administration also evoked transient thrombocytopenia in control but not SCD mice, while, IV collagen led to comparable drop in platelet count in both SCD and control mice. In vitro turbidimetric aggregation study yet again demonstrated impaired SCD mouse platelet response to ADP, which was significantly augmented by CD39 inhibitor (sodium metatungstate, POM-1). Indeed, we found significantly higher plasma levels and activity of CD39 in SCD mice compared to control mice using ELISA and malachite green assays, respectively. Hemin, a major host-derived damage associated molecular pattern (DAMP) in SCD, was incubated with Human Lung MicroVascular Endothelial Cell (HMVEC-L) to assess CD39 expression using western blotting. Hemin (10 -100 µM) in a dose dependent manner decreased CD39 levels in HMVEC-L.Our current findings suggest that elevated CD39 plasma levels and activity possibly prevents ADP-mediated platelet aggregation and pulmonary thrombosis in SCD. We demonstrated that SCD milieu promotes loss of endothelial CD39, which may be directly associated with increased CD39 plasma levels and activity. Current study explains why P2Y12 blockers are not effective in SCD therapy and warrant the need for further studies to understand the role of purinergic signaling in pathogenesis of ACS. DisclosuresSundd: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring Inc: Research Funding; Bayer: Research Funding.

Oral Administration of the LSD1 Inhibitor OG-S1335 Increases Fetal Hemoglobin in Humanized Transgenic Sickle Cell Disease Mice and in Baboons

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). New and more effective strategies to increase HbF are needed because the current standard of care, hydroxyurea, is not effective in a significant proportion of patients. Although BM transplantation, gene therapy, and gene editing technologies offer the promise of a cure, it is unlikely that these methodologies can be implemented to treat the large numbers of patients projected worldwide, including those in developing nations that could most easily be treated with an effective, orally administered drug therapy that increased HbF. The lysine-specific demethylase 1A (KDM1A, also referred to as LSD1), is a component of co-repressor complexes that repress γ-globin gene expression and a therapeutic target for HbF reactivation (Shi et al, Nat Med 19:291, 2013). Our laboratory has shown that subcutaneous administration of RN-1, a pharmacological inhibitor of LSD1, increased γ-globin expression in SCD transgenic mice (Rivers et al, Exp Hematol 43:546, 2015; Cui et al, Blood 126:386, 2015) and in baboons (Rivers et al, Haematol 101:688, 2016), widely acknowledged as the best animal model to test the activity of HbF-inducing drugs due to conservation of structure and developmental regulation of the β-globin locus among simian primates. The objective of this investigation was to test whether oral administration of a new LSD1 inhibitor, OG-S1335, could increase HbF in SCD transgenic mice and baboons. Oral administration of varying doses (0.25, 0.5, 1.0mg/kg/d; 4d) of OG-S1335 to SCD mice (3 mice per dose) increased γ-globin expression and the percentage of circulating erythrocytes and reticulocytes positive for HbF (F cells and F retics; Table 1). In baboons, dose escalation was initially performed by administering the drug to a single animal in cycles of 3 days, followed by 1-2 weeks of observation to measure effects on blood parameters. Increased levels of F retics were initially observed at a dose of 100μg/kg/d and subsequently 4 additional baboons were treated with OG-S1335 (by oral gavage) at a dose of either 100 or 200μg/kg/d (7 separate experiments in total). The data presented are in the mean+SD format, with “post-dose” indicating the time point when the highest variation from baseline levels was reported. Increased F retics (pre=8.4+8.9%, post=43.0+27.9%; p<0.01), γ-globin chain synthesis (pre=0.05+0.05 γ/γ+β, post=0.28+0.18 γ/γ+β; p<0.01), and γ-globin mRNA (pre=0.02 +0.02 γ/γ+β, post=0.20+0.11 γ/γ+β) were observed with maximal levels achieved 7 days after administration of the first dose (d8). A decrease in the absolute number of reticulocytes (pre=72.8+17.8 X104/μL, post=35.4+17.8 X 104/μL; p<0.05; nadir on d6-8), neutrophils (pre=4634+994/μL, post=2655+1257/μL; p<0.02; nadir on d13-17), and platelets (pre=406+86 X103/μL, post=168+98 X103/μL; p<0.01; nadir on d8) was also reported, together with increased monocytes (pre=200+68/μL, post=541+213/μL; p<0.01; peak on d10-15). A negative correlation between decreased platelet counts and F retics (r= -0.55), γ-globin chain synthesis (r= -0.92), and γ-globin mRNA (r= -0.80) suggests that variability in drug exposure in addition to possible genetic factors regulating γ-globin expression may be responsible for the observed variability between animals. Additional experiments were performed in two phlebotomized anemic baboons to test the effect of the drug in a model with increased relevance to sickle cell disease. OG-S1335 (100μg/kg/d; 3d) induced high levels of F retics (PA8695, pre-dose=24%, post=66.8%; PA8698: pre-dose=13%, post=93.6%), γ-globin chain synthesis (PA8695: pre-dose=0.07 γ/γ+β, post=0.20 γ/γ+β; PA8698: pre-dose=0.02 γ/γ+β, post=0.44 γ/γ+β) and γ-globin mRNA (PA8695: pre-dose=0.06 γ/γ+β, post=0.18 γ/γ+β; PA8698: pre-dose=0.03 γ/γ+β, post=0.33 γ/γ+β) in each animal. We conclude that oral administration of the LSD1 inhibitor OG-S1335 significantly increased F retics, γ-globin chain synthesis, and γ-globin mRNA in SCD mice and in baboons, supporting further efforts toward the development of this drug for SCD therapy. [Display omitted] DisclosuresCiceri:Oryzon Genomics SA: Employment. Cavalcanti:Oryzon Genomics SA: Employment.

Neutrophil Extracellular Traps Promote Lung Arteriole Occlusion in Sickle Cell Disease

Introduction:Sickle cell disease (SCD) is an autosomal-recessive-genetic disorder, which leads to red blood cell sickling and hemolysis. Acute systemic painful vaso-occlusive crisis (VOC) is the predominant pathophysiology requiring emergency medical care by SCD patients. 10-20% of SCD patients hospitalized with VOC tend to develop acute chest syndrome (ACS), a type of lung injury within next few days, suggesting a role for pulmonary vaso-occlusion in ACS. The cellular, molecular and biophysical mechanism of pulmonary vaso-occlusion has just started to unfold and a profound understanding is needed for the development of rescue therapies to halt the progression of VOC to ACS. Our recent findings using quantitative fluorescence intravital lung microscopy (qFILM) revealed that lung vaso-occlusion is enabled by the entrapment of embolic neutrophil-platelet aggregates in the pulmonary arterioles of transgenic humanized SCD mice. Neutrophil extracellular traps (NETs) are web-like structures of decondensed nuclear DNA decorated with citrunilated-histones and neutrophil granule proteins. NETs are released by activated neutrophils under inflammatory conditions and are known to possess pro-inflammatory and pro-thrombotic properties. Recently, hemolysis driven release of cell free hemoglobin or hemin has been shown to promote NETs formation in the pulmonary microcirculation of SCD mice. Nevertheless, how these NETs can serve to facilitate pulmonary vaso-occlusion and promote ACS is still unclear.Materials and Methods:Townes knock-in humanized SS (hα/hα:βS/βS) and AS (hα/hα:βA/βS) mice were used as SCD and control mice, respectively. SS and AS mice were intravascularly (IV) challenged with Oxy-hemoglobin (Oxy-Hb) and hemin to model VOC. Fluorescently-conjugated anti-mouse CD49b and Ly6G mAbs, Sytox Orange, and FITC dextran were administered IV to visualize platelets, neutrophils, extracellular DNA and lung vasculature in the mouse, respectively. Lung microcirculation was visualized in real time in vivo using qFILM and analyzed as described elsewhere. Also, SCD or control human blood with or without treatment with Oxy-Hb or hemin was perfused through in vitro microfluidic microchannels presenting a combination of P-selectin, ICAM-1 and IL-8, and neutrophil-platelet interactions as well as NET formation was visualized using Quantitative Microfluidic Fluorescence Microscopy (qMFM). Fluorescent anti-human mAbs against CD49b, CD16, citrullinated histones (H3-Cit) and neutrophil elastase were added to human blood to visualize platelets, neutrophils, histones, and neutrophil elastase, respectively.Results and Discussion:Our initial findings using qFILMreveal that non-lethal dose of 20 µmole/kg IV hemin or 10 µmole/kg IV Oxy-Hb led to entrapment of neutrophil-platelet embolic aggregates in the lung arteriolar bottlenecks of SCD but not control mice. These aggregates resulted loss of blood perfusion in the lung, followed by shedding of NETs from neutrophils into the blood circulation.Conclusion: Erythroid DAMPs, hemin and Oxy-Hb promote occlusion of lung arterioles by neutrophil-platelet aggregates in SCD mice. These aggregates promote loss of blood flow and release of NETs from neutrophils in the lung arterioles. DisclosuresKato:Bayer: Research Funding; Global Blood Therapeutics: Consultancy; Novartis: Consultancy; MAST Therapeutics: Research Funding.

Enhanced Renal Clearance of Heme Triggers Acute Kidney Injury in Sickle Cell Disease

Circulating heme is scavenged primarily by hemopexin (Hx) for degradation in the liver, while alpha-1-microglobulin (A1M) binds heme for urinary clearance. Individuals with sickle cell disease (SCD) have acquired Hx deficiency. Hitherto, it has not been determined whether the low Hx in SCD results in a disproportionately large amount of heme exposure in the kidneys sufficient to promote acute tissue injury. Acute kidney injury (AKI) is a common downstream complication of acute chest syndrome (ACS) and vaso-occlusive pain crisis (VOC). Emerging evidence indicates acute elevations in circulating heme contributes to both ACS and VOC. Therefore, we tested the hypothesis that acute hemolytic crisis triggers AKI in SCD. We found that steady-state plasma A1M is raised significantly in SCD patients compared to controls (SS: 0.27±0.03, AA: 0.18±0.02 mg/ml p<0.05; n=12), and we determined that the molar A1M/Hx ratio is 14-fold higher in transgenic homozygous SCD (SS) mice compared to control (AA) littermates (n=6; p<0.05). Infusion of a non-lethal dose of purified heme (20 μmoles/kg) raised the concentration of heme in the liver but not in the kidneys of AA mice in 48 hours. On the contrary, the heme infusion over the same duration raised the total heme concentration in the kidneys but not in the liver of the SS mice (p<0.05). The SS mice but not the control AA mice developed severe renal tissue damage, defined by tubular atrophy and cast formation, loss of epithelial brush borders and apoptosis of the proximal tubular epithelium, within 48 hours of the heme infusion. Clinical detection of AKI is typically defined by elevation in serum creatinine and urinary albumin creatinine ratio (uACR). The acute elevation in circulating heme increased the plasma creatinine by 4-fold (p<0.001) with a 2-fold elevation in uACR (p<0.01) exclusively in the SS mice but not in control AA mice. Urinary kidney injury molecule-1 (KIM-1), which is a reliable predictor of AKI increased ~3-fold in the SS mice following the heme infusion, while no alteration of KIM-1 was evident in AA mice treated with the same challenge (n=6). To define the relationship between AKI in SCD and the hepatic and renal mechanisms of heme clearance, we measured plasma A1M and Hx, and urinary KIM-1 in a cohort of Ghanaian SCD patients aged 1-14 years (n=40). The molar ratio of A1M/Hx correlated positively with KIM-1 in this patient population (Pearson r=0.54; p=0.0003). Together, our data supports the conclusion that acute elevation of circulating heme triggers AKI in SCD. Clinically, the relative molar concentrations of plasma A1M and Hx can be developed as a biomarker to assess AKI risk in SCD patients. Finally, our data suggest acute infusions of Hx may protect SCD patients from AKI during episodes of hemolytic crisis. DisclosuresOfori-Acquah:NuvOx Pharma: Patents & Royalties.

Platelet Aggregation Dysfunction in Sickle Cell Disease

Acute systemic painful vaso-occlusive crisis (VOC) is the predominant clinical event requiring sickle cell disease (SCD) patients to seek emergency medical care. VOC often serves as an antecedent to acute chest syndrome (ACS), which is a type of acute lung injury and the leading cause of mortality in SCD. Current treatments for ACS are primarily supportive, and there is a critical need for rescue strategies to prevent the progression of VOC to ACS. ACS involves thrombosis across pulmonary artery branches, but the pathophysiology of thrombosis in SCD is largely unknown. Adenosine diphosphate (ADP) released from lysed erythrocytes can activate platelets by stimulating the purinergic receptors P2Y1 and P2Y12, suggesting that blocking these receptors may prevent VOC. Surprisingly, P2Y12 receptor blockade was unsuccessful in preventing VOC in a recent DOVE trial, justifying the need for better understanding of purinergic signaling in SCD.We used a novel intravital microscopy enabled experimental model of vaso-occlusive pulmonary thrombosis in transgenic SCD mice triggered by an intravenous (IV) administration of ADP. This method allows real time in vivo visualization of platelet aggregation in the lung of live SCD mice using multi-photon-excitation based quantitative fluorescence intravital lung microscopy (qFILM). We discovered that vaso-occlusive thrombosis involves entrapment of large circulating platelet aggregates in the bottle-necks located between the pulmonary arterioles and capillaries. Surprisingly, the dose of ADP that led to the pulmonary vaso-occlusive thrombosis in control mice, failed to induce vaso-occlusive thrombosis in SCD mice. Remarkably, further in vivo and in vitro platelet aggregation studies confirmed that SCD mouse platelets were indeed refractory to ADP but not collagen.Our preliminary findings suggest that therapeutic inhibition of P2Y1 and P2Y12 platelet receptors will not be a successful therapy in SCD. Our findings also warrant the need for further studies to understand the role of purinergic signaling in SCD DisclosuresNo relevant conflicts of interest to declare.

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&amp;lt;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.

Post-translational modification as a response to cellular stress induced by hemoglobin oxidation in sickle cell disease

Intracellular oxidative stress and oxidative modification of sickle hemoglobin (HbS) play a role in sickle cell disease (SCD) pathogenesis. Recently, we reported that Hb-dependent oxidative stress induced post-translational modifications (PTMs) of Hb and red blood cell (RBC) membrane proteins of transgenic SCD mice. To identify the mechanistic basis of these protein modifications, we followed in vitro oxidative changes occurring in intracellular Hb obtained from RBCs and RBC-derived microparticles (MPs) from the blood of 23 SCD patients (HbSS) of which 11 were on, and 12, off hydroxyurea (HU) treatment, and 5 ethnic matched controls. We used mass spectrometry-based proteomics to characterize these oxidative PTMs on a cross-sectional group of these patients (n = 4) and a separate subgroup of patients (n = 2) studied prior to initiation and during HU treatment. Collectively, these data indicated that band-3 and its interaction network involved in MPs formation exhibited more protein phosphorylation and ubiquitination in SCD patients than in controls. HU treatment reversed these oxidative PTMs back to level observed in controls. These PTMs were also confirmed using orthogonal immunoprecipitation experiments. Moreover, we observed specific markers reflective of oxidative stress, including irreversible oxidation of βCys93 and ubiquitination of Hb βLys145 (and βLys96). Overall, these studies strongly suggest that extensive erythrocyte membrane protein phosphorylation and ubiquitination are involved in SCD pathogenesis and provide further insight into the multifaceted effects of HU treatment.

Platelet extracellular vesicles drive inflammasome-IL1β-dependent lung injury in Sickle Cell Disease

Abstract Intraerythrocytic polymerization of hemoglobin S promotes hemolysis and vaso-occlusive events in the microvasculature of sickle cell disease (SCD) patients. Although platelet-neutrophil aggregates-dependent vaso-occlusion is known to occur in the lung and contribute to lung injury, the etiological mechanisms that trigger acute chest syndrome are largely unknown. Here, we use intravital imaging of the lung in transgenic humanized SCD mice and in vitro imaging of SCD patient blood flowing through a microfluidic system to identify the innate-immune mechanism that promotes platelet-neutrophil aggregate-dependent lung vaso-occlusion and injury in SCD. Platelet-inflammasome activation led to generation of IL-1β and caspase-1 carrying platelet extracellular vesicles (EVs) that bind to neutrophils and promote platelet-neutrophil aggregation in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. The inflammasome activation, platelet EV generation and platelet-neutrophil aggregation were enhanced by the presence of lipopolysaccharide at a nanogram dose in SCD but not control human blood. Inhibition of the inflammasome effector caspase-1 or IL-1β pathway attenuated platelet EV generation, prevented platelet-neutrophil aggregation, and restored microvascular blood flow in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. These results are the first to identify that platelet-inflammasome dependent shedding of IL-1β and caspase-1 carrying platelet EVs promote lung vaso-occlusion in SCD. The current findings also highlight the therapeutic potential of targeting the platelet-inflammasome dependent innate immune pathway to prevent acute chest syndrome in SCD.