Damage In Sickle Cell Disease Research Articles

Time-restricted feeding does not improve nephropathy in humanized sickle cell disease mice

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by a point mutation in the hemoglobin gene. This substitution results in a “sickling” of red blood cells and subsequent vascular and organ damage. Current treatments, while increasing the lifespan of sickle cell patients, has resulted in a higher prevalence of chronic complications like SCD nephropathy. Our group has previously shown that time-restricted feeding (TRF) in chronic high fat fed mice reduces kidney damage. Given the increased kidney damage in SCD, we hypothesize that TRF in humanized SCD mice will attenuate sickle cell nephropathy. To test this hypothesis, we studied 18-22-week-old male SCD mice. Mice underwent 3 weeks of TRF, only having access to food during the active, dark period (Zeitgeber Time (ZT) 12 to 24). The control group had access to food ad libitum (ad-lib). At the end of the 3 weeks, at 21-25 week old ages, 12 hour urine was collected in metabolic cages as well as kidneys and plasma collection at ZT3 (inactive period). Excretion of urinary kidney damage markers (albumin and NGAL-1) showed no difference between TRF and ad-lib in both the active and inactive collection periods. Urinary excretion of endothelin-1 is known to be upregulated in SCD patients; however, TRF did not change this marker in SCD mice. TRF did not change plasma levels of cytokines or soluble adhesion molecules. Histological analysis of kidneys was conducted as an additional marker of kidney damage. We analyzed iron levels in the kidney with Prussian blue staining as well as fibrosis with Masson’s trichrome. TRF did not change kidney iron accumulation or kidney fibrosis. Immunohistochemical staining for CD3+ T cells did not show a difference in glomerular T cell infiltration in the TRF group compared to the ad-lib group. In conclusion, TRF does not appear to have an effect on renal inflammation or kidney function in SCD mice, but future studies need to be completed. R01 DK134562. 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.

Role of hydroxyurea therapy in the prevention of organ damage in sickle cell disease: a systematic review and meta-analysis

Background Hydroxyurea is an affordable drug that reduces vaso-occlusive crises and transfusion requirements in sickle cell disease. However, its effectiveness in preventing chronic organ damage is still unclear. This systematic review and meta-analysis aimed to evaluate the role of hydroxyurea in preventing organ morbidity.Method We included original articles published in English from 1st January 1990 to 31st January 2023, reporting hydroxyurea therapy and organ damage from PubMed, Google Scholar, Scopus, and CrossRef databases. A total of 45 studies with 4681 sickle cell disease patients were evaluated for organ damage.ResultsOur analysis showed that hydroxyurea intervention significantly lowered transcranial Doppler and tricuspid regurgitant velocity, with a standardized mean difference of − 1.03 (− 1.49; − 0.58); I2 = 96% and − 1.37 (CI − 2.31, − 0.42); I2 = 94%, respectively. Moreover, the pooled estimate for albuminuria showed a beneficial effect post-hydroxyurea therapy by reducing the risk of albuminuria by 58% (risk ratio of 0.42 (0.28; 0.63); I2 = 28%).ConclusionOur study found that a hydroxyurea dose above 20 mg/kg/day with a mean rise in HbF by 18.46% post-hydroxyurea therapy had a beneficial role in reducing transcranial doppler velocity, tricuspid regurgitant velocity, albuminuria, and splenic abnormality.Systematic review registrationPROSPERO CRD42023401187.

Hemoglobin Genotype, Hemolytic Activity, Fetal Hemoglobin and Retinal Changes on Optic Coherence Tomography in Children with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) affects multiple organs, including the eye. Optical coherence tomography (OCT) is a quick, noncontact test that provides high-resolution images of distinctive retinal layers. OCT has revolutionized ophthalmology examination and offers valuable new information on retinal damage in SCD. Our earlier studies show that OCT has a higher detection rate for retinal changes in SCD, offering earlier diagnosis than fundoscopy alone. Additionally, abnormal retinal OCT correlates with cerebrovascular disease (CVD) strongly suggesting that retinal OCT may aid in detecting and monitoring SCD related brain injury. One of the characteristics of SCD is the marked variability in patients' clinical manifestations. SCD genotype impacts the presentation of sickle cell retinopathy (SCR). Though OCT has revealed a higher frequency of SCR and more extensive retinal changes associated with the more severe SCD genotypes SS and Sβ0 as compared to SC, significant heterogeneity remains even among patients with the same genotype. Early identification of factors predictive of organ damage, such as SCR and CVD, will allow initiation of individualized disease-modifying treatment before organ systems suffer repeated hypoxic-ischemic events, thus improving long-term outcomes. Degree of hemolysis and fetal hemoglobin (FHb) are among the most important factors affecting SCD severity. There have been limited studies on their effect on ocular health in children with SCD. Methods: The Delaware Comprehensive Sickle Cell Research Center performed a prospective longitudinal cohort study in which all participants underwent complete ophthalmologic examination and OCT imaging. Posterior pole volume scans centered at approximately 3 mm temporal to the foveal were obtained. Areas of retinal thinning were identified using the thickness map. Medical history, brain imaging and laboratory data were collected. All available FHb values were recorded to examine the changing trend with age. Retinal findings were correlated with brain imaging and clinical data. Analysis was performed using IBM SPSS Statistics 27 (IBM, Armonk, NY). The Chi-square test was used for categorical data, and the nonparametric test for continuous variable analysis. The relationship between FHb, age at FHb testing and OCT was examined using multiple regression analysis. Results: From July 2015 to April 2023, 181 (88 males) patients aged 5.35 - 20.39 (12.72 ± 4.29) years with SCD (108 SS, 56 SC, 10 Sβ+ thalassemia, 5 Sβ0 thalassemia and 2 other) enrolled in the study. Visual acuity ranged 20/20 - 20/40. On funduscopic exams, 23 of 181 (12.71%) showed signs of SCR, whereas 107 (59.12%) had retinal injuries on OCT. Seventy-six patients with SS/Sβ0 genotype had brain MRI, and 28 had abnormal findings (Table). Twenty-four of the 28 patients (85.71%) also had abnormal OCT (p = 0.021). Children with SC and SS/Sβ0 disease with normal retinal OCT had FHb values significantly higher than those with abnormal OCT (Z =-2.61 and -4.16, p = 0.009 and 0.000). Children with SS/Sβ0 disease, abnormal OCT had more elevated reticulocyte counts than those with normal OCT (Z = -2.848, p = 0.004). SS genotype were evaluated for FHb values. As expected, FHb levels decline with age, and the rate of descent diminishes (i.e., more rapid fall at an early age). Interestingly, the decrease in FHb is significantly more profound before age 15 for patients with abnormal OCT than those with normal OCT (Figure). The effect of hydroxyurea (HU) in preventing SCR is inconclusive. Children with normal OCT had similar FHb levels without and with HU. However, HU raises FHb levels among those with abnormal OCT. Conclusions: 1) Retinal and brain injury correlates in SS/Sβ0, and OCT may help to select patients for MRI. 2) Abnormal OCT is associated with increased reticulocyte count, indicating heightened hemolysis. While AST and indirect bilirubin levels are not significantly different between normal and abnormal OCT groups in the current dataset, we will repeat the analysis of these hemolytic markers with a larger sample. 3) Early serial measurement of FHb levels throughout childhood can identify patients at high risk for subsequent severe complications such as SCR and CVD who might benefit from early disease modifying treatments. These important findings and the impact of hydroxyurea require further study in a larger patient population undergoing serial exams.

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.

Optimizing Albuminuria Assessment in Patients with Sickle Cell Disease: Insights from the Sikamic Study

Background Patients with sickle cell disease (SCD) are at increased risk of developing renal complications, making early detection and management of these issues critical. Albuminuria has been identified as a relevant biomarker associated with chronic kidney disease (CKD) progression in SCD, as demonstrated in several studies (Gonzalez et al., 2017; Guasch et al., 2018). Quantification of albuminuria is typically performed using the urine albumin-to-creatinine ratio (ACR). However, this measurement is not without its challenges, as variability in results can occur due to several factors, including analytical method, varying sampling conditions, and biological and medical factors such as physical activity, diurnal changes, diet, and hydration, which require the collection of multiple consecutive first morning samples. Studies have shown that the use of ACR from first morning urine samples provides results comparable to the gold standard 24-hour urine collection, with improved precision and less intra-individual variability than random urine samples typically collected during clinic visits (Witte 2009, Jermendy 2001, Lambers 2008). Collecting more urine samples generally improves precision and statistical power, but Kröpelin et al (2025) showed that collecting more than three consecutive urine samples does not significantly improve precision. Material and method The ongoing SIKAMIC study is a phase IIb, international (France and Africa), double-blind, randomized, placebo-controlled trial. The study aims to determine the effect of hydroxycarbamide on albuminuria level after 6 months of treatment in SCD adult patients. Here, we analyzed the variability of ACR in all patients pre-selected for the study. Microalbuminuria is commonly considered the first clinical indicator of glomerular damage in SCD. As per the KDIGO (Kidney Disease: Improving Global Outcomes), microalbuminuria is characterized by an ACR ranging from 3 to 30 mg/mmol, while macroalbuminuria refers to an ACR greater than 30 mg/mmol. For SIKAMIC study, patients with an ACR above 3 mg/mmol and below 100 mg/mmol were included. ACR was measured in first morning urine samples over three consecutive days. Patients were instructed to collect these samples at home, which were either collected by a hospital courier or brought to the hospital/laboratory by the patient for analysis. Estimated glomerular filtration rate (eGFR) was calculated using the CKD EPI formula without ethnic criteria. Means and standard deviations of the three measurements were calculated per patient, and a reproducibility test was performed using Mixed Effect Model and Bland Altman analyses (Graphpad Prism 9). Results Out of the initial 179 patients pre-selected for the study (59 from France and 120 from Africa) 99 patients (34 in France and 65 in Africa) met the inclusion criteria and were enrolled in the SIKAMIC study. Pre-selected patients from Africa were younger than patients from France with a mean age of 28.3 years and 33.8 years respectively (p<0.001). However, the mean ACR did not differ between groups (11.8 vs 12.7 mg/mmol; p=0.8) as observed for the mean eGFR value (123 vs 120.9; p=0.75). Additionally, the sex ratio was similar between groups (0.62 vs 0.67). For all patients, the mean ACR was 14.6, 17.3 and 15.6 mg/mmol on days 1, 2 and 3, respectively, with no significant difference (p=0.43; Figure 1 : A). The mean standard deviation of ACR between day 1, 2 and 3 was 6.8 mg/mmol, suggesting good consistency and reproducibility of the ACR measurements over the three days. Bland-Altman plots are shown in Figure 1 for the total population (n=161; 1B), microalbuminuric (1C; n=86) and macroalbuminuric (1D; n=11) patients, illustrating the agreement of ACR measurements within these subgroups. Conclusion : The SIKAMIC study contributes valuable insights into the evaluation of albuminuria in SCD patients. The reproducibility of the ACR appears to be sufficient to allow satisfactory monitoring on a single morning measurement in SCD routine care, both in Africa and Europe population of SCD patients.

Plasma Proteome Profiling of Sickle Cell Patients Reveals Activation of Inflammatory Pathways during Steady State and Vaso-Occlusive Crisis

Background The pathophysiology of Sickle Cell Disease (SCD) is characterized by chronic intravascular hemolysis and ischemia-reperfusion, resulting in oxidative stress and chronic inflammation. Although inflammation seems to play an important role in the development of tissue damage in SCD, little is known about the various cellular and cytokine pathways involved in the pathophysiology of SCD. This study aims to further elucidate the inflammatory pathways in SCD using targeted plasma protein profiling both in steady state and during vaso-occlusive crisis (VOC). Methods In this prospective study, SCD patients with the genotypes HbSS and HbSβ 0-thalassemia and healthy ethnicity matched controls were included. Plasma levels of 92 proteins involved in various inflammatory pathways were measured in EDTA plasma using Olink Proximity Extension Assay (PEA) technology. In brief, pairs of oligonucleotide-labeled antibody probes were added to 20 μL of plasma, resulting in the binding of these antibody probes to their targeted protein. Subsequently DNA polymerase was added, leading to a proximity-dependent DNA polymerization event, generating a unique PCR target sequence. The final assay read-out is presented in Normalized Protein eXpression (NPX) values, which is an arbitrary unit on a log2-scale with an NPX difference (estimate) of 1 meaning a doubling of protein concentration. RStudio version 4.2.1 was used for data analysis and visualization. Differences between subgroups were tested using an unpaired or paired t-test as appropriate. Results were corrected for multiple testing using the Benjamini-Hochberg method. Results with p < 0.05 were considered significant. Furthermore Over-representation Analysis (ORA) was performed to investigate the association of the differentially expressed proteins with specific biological processes. Results Plasma samples were collected from 43 SCD patients in steady state (median age: 25 years (IQR: 20 - 32)) and 13 healthy ethnicity matched controls (median age: 29 (IQR: 28-39)). From 15 SCD patients plasma samples were collected during both steady state and VOC (median age: 27 (IQR: 19 - 34)). Comparison of protein levels between healthy controls and SCD patients in steady state identified 38 proteins (IL-18, ANGPT2, Gal-9, CD4, HGF, HO-1, CCL4, ARG1, ADA, GZMA, PD-L1, CCL23, LAG3, TNFRSF4, CCL3, CXCL11, CRTAM, IL-10, GZMB, CD27, FGF2, IL-8, IL-6, TNF, PGF, NCR1, TNFSF14, CASP-8, IL-7, FASLG, TNFRSF9, LAP TGF-beta-1, PD-L2, CCL19, CD83, GZMH, MCP-4, CD244, TIE2) that were significantly higher in SCD patients as shown in Figure 1. Furthermore soluble CD70 was significantly lower in SCD patients in steady state compared to healthy controls (estimate: -0.34, p < 0.05). Over-representation Analysis revealed a significant upregulation of proteins involved in the regulation of hematopoiesis, the IL-18 signaling pathway, T-cell activation, leukocyte differentiation and leukocyte adhesion (Figure 2). Pairwise comparison of protein levels between steady state and VOC in SCD patients revealed 6 proteins (TWEAK, VEGFR-2, CD244, PDCD1, GZMA, TNFRSF4) which were significantly lower and 12 proteins (IL-6, TNFSF14, ADA, IL-15, HGF, IL-10, IL-8, Gal-9, CSF-1, CCL20, MMP7, VEGFA) which were significantly higher during VOC as compared to steady state. During VOC, pathways involved in the regulation of cell death (IL-6, TNFSF14, ADA, HGF, IL-10, PDCD1, VEGFA) and tissue development (IL-6, ADA, HGF, IL-10, VEGFA) were significantly enriched. Summary/conclusion The results of these proteomic analyses provide new insights into the pathways that regulate inflammation in SCD, both during steady state and VOC. These findings might contribute to a better understanding of SCD pathophysiology and identifying potential new targets for therapeutic interventions in SCD patients. Our results warrant further in-depth investigation of the different activated pathways.

Association of haemolysis markers, blood viscosity and microcirculation function with organ damage in sickle cell disease in sub-Saharan Africa (the BIOCADRE study).

Sickle cell anaemia (SCA) is a monogenic disease with a highly variable clinical course. We aimed to investigate associations between microvascular function, haemolysis markers, blood viscosity and various types of SCA-related organ damage in a multicentric sub-Saharan African cohort of patients with SCA. In a cross-sectional study, we selected seven groups of adult patients with SS phenotype in Dakar and Bamako based on the following complications: leg ulcer, priapism, osteonecrosis, retinopathy, high tricuspid regurgitant jet velocity (TRV), macro-albuminuria or none. Clinical assessment, echocardiography, peripheral arterial tonometry, laboratory tests and blood viscosity measurement were performed. We explored statistical associations between the biological parameters and the six studied complications. Among 235 patients, 58 had high TRV, 46 osteonecrosis, 43 priapism, 33 leg ulcers, 31 retinopathy and 22 macroalbuminuria, whereas 36 had none of these complications. Multiple correspondence analysis revealed no cluster of complications. Lactate dehydrogenase levels were associated with high TRV, and blood viscosity was associated with retinopathy and the absence of macroalbuminuria. Despite extensive phenotyping of patients, no specific pattern of SCA-related complications was identified. New biomarkers are needed to predict SCA clinical expression to adapt patient management, especially in Africa, where healthcare resources are scarce.

Retinal Damage in Sickle Cell Disease in Abidjan (Ivory Coast)

Retinal Damage in Sickle Cell Disease in Abidjan (Ivory Coast)

5613401 SICKLE CELL DISEASE GENOMICS OF AFRICA (SICKLEGENAFRICA) NETWORK: QUALITATIVE INTERVIEW FINDINGS ON REASONS FOR REFUSAL OF BROAD CONSENT

Background: Sickle Cell Disease Genomics of Africa (SickleGenAfrica) Network is a collaborative project to examine the overarching premise that genetic variation affects the body’s defence against intravascular haemolysis and the development of organ damage in sickle cell disease. In addition, SickleGenAfrica employs community engagement to address fundamental ethical issues relating to genomics research, and this study focussed on broad consent incorporated in the process of informed consent. Objectives: To identify the reasons why some SickleGenAfrica participants refused to provide broad consent for future use of their biological samples and information for other genomics research. Methods: Qualitative research using one-on-one telephone interviews Setting: SickleGenAfrica study sites in Nigeria and Tanzania Participants: SickleGenAfrica site investigators based in Abuja, Kano, Lagos and Dar es Salaam study sites Analysis: Inductive thematic analysis Results: Overall, findings from the SickleGenAfrica project revealed that broad consent for future genomics research was obtained from all participants at two study sites in Accra and Kumasi (Ghana). Nonetheless, a small number of participants at four study sites in Nigeria and Tanzania refused broad consent stating reasons including concerns about confidentiality, unavailable updated information for future research, and lack of immediate benefits from genomic research. In addition, psychosocial factors such as gender differences, family dynamics with autonomous decision-making, and beliefs about possible spiritual uses of blood samples. Furthermore, insufficient information and limited understanding about genomics research emerged as reasons for refusal of broad consent. Conclusions: Continuous education to address ethical issues in genomics research, culturally sensitive psychosocial interventions for participants, and recommendations from beneficiaries of genomics research are required to help obtain broad consent in Africa.

Macrophage metabolic rewiring improves heme-suppressed efferocytosis and tissue damage in sickle cell disease

AbstractSickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory condition, which is contributed by heme-activated proinflammatory macrophages. Although previous studies addressed heme ability to stimulate macrophage inflammatory skewing through Toll-like receptor4 (TLR4)/reactive oxygen species signaling, how heme alters cell functional properties remains unexplored. Macrophage-mediated immune cell recruitment and apoptotic cell (AC) clearance are relevant in the context of SCD, in which tissue damage, cell apoptosis, and inflammation occur owing to vaso-occlusive episodes, hypoxia, and ischemic injury. Here we show that heme strongly alters macrophage functional response to AC damage by exacerbating immune cell recruitment and impairing cell efferocytic capacity. In SCD, heme-driven excessive leukocyte influx and defective efferocytosis contribute to exacerbated tissue damage and sustained inflammation. Mechanistically, these events depend on heme-mediated activation of TLR4 signaling and suppression of the transcription factor proliferator-activated receptor γ (PPARγ) and its coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α). These changes reduce efferocytic receptor expression and promote mitochondrial remodeling, resulting in a coordinated functional and metabolic reprogramming of macrophages. Overall, this results in limited AC engulfment, impaired metabolic shift to mitochondrial fatty acid β-oxidation, and, ultimately, reduced secretion of the antiinflammatory cytokines interleukin-4 (IL-4) and IL-10, with consequent inhibition of continual efferocytosis, resolution of inflammation, and tissue repair. We further demonstrate that impaired phagocytic capacity is recapitulated by macrophage exposure to plasma of patients with SCD and improved by hemopexin-mediated heme scavenging, PPARγ agonists, or IL-4 exposure through functional and metabolic macrophage rewiring. Our data indicate that therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PGC1α/PPARγ modulation significantly ameliorates tissue damage associated with SCD pathophysiology.

Development and Validation of the Patient/Caregiver Reported Hydroxyurea Evaluation of Adherence for Life (HEAL) Scale.

Hydroxyurea reduces the incidence of vaso-occlusive episodes, stroke, and respiratory, cardiac, and renal damage in sickle cell disease by increasing fetal hemoglobin. However, because suboptimal adherence to hydroxyurea limits its effectiveness, understanding patient-specific barriers to hydroxyurea adherence could help improve adherence and health outcomes in patients with sickle cell disease. The aim of this single-site, prospective, IRB-approved study was to validate a 24-item patient- and caregiver-reported hydroxyurea treatment adherence questionnaire, the Hydroxyurea Evaluation of Adherence for Life (HEAL) scale. A sample of 24 adults with sickle cell disease and 16 caregivers of children with sickle cell disease completed the HEAL scale, and a subset of the original sample provided a second HEAL scale for test-retest reliability. HEAL scale results were validated against global adherence ratings from participants and health-care providers, records of access to pill bottles, and laboratory values for fetal hemoglobin and absolute neutrophil count. Results demonstrated excellent internal consistency for the HEAL Total score and eight (3-item) subscale scores (Dose, Remember, Plan, Cost, Understand, Effectiveness, Laboratory, and Pharmacy), as well as strong test-retest reliability for all HEAL scores except the Cost subscale. HEAL Total scores correlated significantly with validity measures, including global adherence ratings and lab values. The HEAL scale offers significant clinical potential for understanding adherence in individual sickle cell disease patients and significant research potential for characterizing adherence in persons with sickle cell disease who are treated with hydroxyurea.

Heme and Iron Aggravate Tissue Damage in Sickle Cell Disease By Driving Macrophage Functional Reprogramming through the Coordinated Suppression of Efferocytosis and Mitochondrial Metabolism

Introduction: Sickle cell disease (SCD) is hallmarked by an underlying chronic inflammatory status, which is in part driven by pro-inflammatory macrophages. Macrophages are highly plastic immune cells which integrate and respond to a variety of signals they are exposed to in the surrounding microenvironment. One of these stimuli is represented by heme released upon hemolysis, which acts as damage-associated molecular pattern to stimulate macrophage inflammatory phenotypic switching through TLR4 signaling and ROS production (Vinchi et al., Blood 2016). Heme-induced pro-inflammatory activation program in macrophages promotes sterile inflammation and aggravates hepatic fibrosis in SCD, contributing to the inflammatory landscape and organ damage typically associated with this disease. While previous studies addressed heme ability to induce inflammatory cytokine production in macrophages, how heme alters cell functional properties remains unexplored. Macrophage functions such as immune cell recruitment ability and apoptotic cell clearance are relevant in the context of SCD, where tissue damage and cell apoptosis occur frequently due to vaso-occlusive episodes, hypoxia and ischemic injury. Methods: Here we analyzed macrophage response to apoptotic stimuli in vivo, in mouse models of heme overload and SCD, as well as in vitro, in bone-marrow-derived macrophages (BMDMs), to unveil the impact of hemolysis on macrophage functional properties. Results: Our results demonstrate that, in addition to inflammatory activation, heme severely alters macrophage functional response to apoptotic cell damage by exacerbating their immune cell recruitment ability and impairing their efferocytic capacity. We show that exposure to heme excess drives defective efferocytosis of apoptotic neutrophils in BMDMs and mouse model of heme overload. This is fully recapitulated in SCD mice, where limited efferocytosis contributes to impaired apoptotic cell clearance and exacerbated tissue damage. Mechanistically, altered efferocytosis depends on heme-driven activation of TLR4 signaling pathway and the suppression of the nuclear transcription factor PPARγ and its coactivator PGC1α, which lead to reduced expression of efferocytic receptors and impaired mitochondrial dynamics and functions. This results in limited recognition/engulfment of apoptotic cells and decreased shift to aerobic mitochondrial fatty acid β-oxidation and anti-inflammatory IL-4 and IL-10 release, with consequent inhibition of continual efferocytosis, inflammation resolution and tissue repair. Heme-driven changes are associated with cell metabolic skewing towards glycolysis and pentose phosphate pathway and reduced reliance on mitochondrial fatty acid oxidation and TCA cycle. Macrophages pre-loaded with fatty acids showed decreased oxygen consumption rate and ATP production after heme exposure, indicating that heme-altered mitochondrial dynamics impair lipid catabolism via limited mitochondrial oxidative phosphorylation. Overall, heme induces macrophage phenotypic and functional reprogramming through the coordinated suppression of efferocytosis, via efferocytosis receptor downregulation, and fatty acid β-oxidation, via altered mitochondrial dynamics. We further demonstrate that heme-impaired efferocytosis and tissue damage promote autoimmunity through anti-nuclear antibody production against 'self' secondary necrotic cells, potentially explaining the increasing evidence of autoimmune diseases in SCD patients. Finally, hemopexin-mediated heme scavenging and anti-inflammatory IL-4 treatment improve tissue damage in hemolytic mice through macrophage rewiring. Defective efferocytosis is reproduced in vitro by macrophage exposure to SCD patients' plasma and rescued by heme scavenging via haptoglobin/hemopexin and PPARγ/PGC1α modulation via PPARα agonist or IL-4. Conclusion: Our data indicate that the therapeutic improvement of heme-altered macrophage functional properties via heme scavenging or PPARγ/PGC1α modulation significantly promotes the resolution of inflammation and ameliorates tissue damage associated with SCD pathophysiology. Thus, macrophage functional rewiring offers potentially valuable therapeutic strategies for SCD patients to improve tissue damage resolution upon vaso-occlusive crisis and ischemic events and prevent autoimmune conditions.

What does it mean to be affiliated with care?: Delphi consensus on the definition of "unaffiliation" and "specialist" in sickle cell disease.

Accruing evidence reveals best practices for how to help individuals living with Sickle Cell Disease (SCD); yet, the implementation of these evidence-based practices in healthcare settings is lacking. The Sickle Cell Disease Implementation Consortium (SCDIC) is a national consortium that uses implementation science to identify and address barriers to care in SCD. The SCDIC seeks to understand how and why patients become unaffiliated from care and determine strategies to identify and connect patients to care. A challenge, however, is the lack of agreed-upon definition for what it means to be unaffiliated and what it means to be a "SCD expert provider". In this study, we conducted a Delphi process to obtain expert consensus on what it means to be an "unaffiliated patient" with SCD and to define an "SCD specialist," as no standard definition is available. Twenty-eight SCD experts participated in three rounds of questions. Consensus was defined as 80% or more of respondents agreeing. Experts reached consensus that an individual with SCD who is unaffiliated from care is "someone who has not been seen by a sickle cell specialist in at least a year." A sickle cell specialist was defined as someone with knowledge and experience in SCD. Having "knowledge" means: being knowledgeable of the 2014 NIH Guidelines, "Evidence-Based Management of SCD", trained in hydroxyurea management and transfusions, trained on screening for organ damage in SCD, trained in pain management and on SCD emergencies, and is aware of psychosocial and cognitive issues in SCD. Experiences that are expected of a SCD specialist include experience working with SCD patients, mentored by a SCD specialist, regular attendance at SCD conferences, and obtains continuing medical education on SCD every 2 years." The results have strong implications for future research, practice, and policy related to SCD by helping to lay a foundation for an new area of research (e.g., to identify subpopulations of unaffiliation and targeted interventions) and policies that support reaffiliation and increase accessibility to quality care.

Intravascular Hemolysis Leads to Increased Esophagus Smooth Muscle Contractions in Mice: Implication for Dysphagia and Odynophagia

Introduction:The nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling pathway is well established as a regulatory pathway of smooth muscle relaxation and contraction. Intravascular hemolysis is a hallmark of some hemolytic anemia, such as paroxysmal nocturnal hemoglobinuria (PNH). NO scavenging by excess plasma hemoglobin (Hb) has been associated with clinical manifestation of intravascular hemolysis. PNH patients display odynophagia and dysphagia, particularly during hemolytic exacerbations, and appear to be due to esophageal spasm. In this study, since plasma Hb excess leads to lower NO bioavailability, we hypothesized that exaggerated intravascular hemolysis may impair the esophageal function. The aim of this study was to evaluate the esophageal function in phenylhydrazine (PHZ)-treated mice, a murine model of intravascular hemolysis. The magnitude of intravascular hemolysis in sickle cell disease (SCD) is a controversial issue. For comparative purpose, SCD mice were also studied. We have focused on the role of the NO signaling pathway and contractile mechanisms. Moreover, the effects of iron chelator deferoxamine treatment on esophagus contractile mechanisms in PHZ mice were also evaluated.Methods: C57BL/6 mice (control) and Berkeley SCD transgenic male mice (3-4 mo old) were used. Intravascular hemolysis was induced in C57BL/6 mice by intraperitoneal injection of 2 doses of PHZ at 50 mg/Kg 8h apart. Mice were euthanized in isoflurane chamber 4 days after the second PHZ injection. The esophagus smooth muscle rings were mounted in isolated organ baths. Concentration-response curves to muscarinic agonist carbachol (CCh; 10 nM -100 μM) and potassium chloride (KCl) were obtained in esophagus from control, PHZ and SCD mice. Control and PHZ mice were treated with deferoxamine (150 mg/kg/day) or vehicle (saline) from the second to the 4th day via i.p. injection.Results: Hematological parameters (RBC [x 10-6/μL], Hb [g/dL], HCT [%]) show that PHZ (2.8±0.2, 6±0.4 and 17±1) and SCD mice (5.6±0.5, 6.1±0.3 and 28±3) have severe anemia, compared to control mice (8.8±0.1, 12.4±0.1 and 45±1). Plasma Hb was higher in SCD and PHZ compared to control mice (2.1- and 3.3 fold, respectively, P<0.05). Moreover, plasma Hb was 57% higher (P<0.05) in PHZ compared to SCD mice. CCh induced concentration-dependent esophagus contractions in control, PHZ and SCD mice. The maximal responses (Emax) were higher (P<0.05) in the esophagus of PHZ (0.75±0.3 mN/mg) compared to control mice (0.50±0.03 mN/mg).In order to evaluate the esophagus contractions independently of receptor stimulation, a concentration-response curve to KCl was constructed. Again, Emax induced by KCl were greater (P<0.05) in PHZ esophagus (0.45±0.01 mN/mg) compared to control mice (0.28±0.02 mN/mg). In SCD mice esophagus, contractile responses induced by CCh and KCl remained unchanged. In vitro preincubation of esophagus with the NO-independent heme-dependent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 (1 μM) fully reversed the higher Emax of CCh and KCl in PHZ mice to control values, without changing the responses in control mice. No changes in the contractile responses for CCh and KCl were observed with the preincubation with NO donor sodium nitroprusside (SNP; 300 μM) in the PHZ and control group. Treatment with deferoxamine did not prevent the elevation of contractile responses in PHZ mice esophagus.Conclusion: Our data show that exaggerated intravascular hemolysis can lead to enhanced esophagus contractions in PHZ mice. BAY 41-2272 is likely to counteract the elevation of contractile responses by stimulating sGC, elevating cGMP levels by stimulating the sGC mostly via NO-independent mechanisms. NO donor failed to modify the increased esophagus contractions in PHZ mice. It is likely that Hb or increased O2- production in esophagus of PHZ mice act to inactivate NO before it reaches sGC. Deferoxamine treatment failed to avoid the esophageal dysfunction, suggesting that excess iron may not be associated with this disorder. SCD mice do not display esophageal disorder, indicating that excess of plasma Hb is not abundant enough to cause esophagus damage in SCD. To the best of our knowledge this is the first study in an experimental model that supports the hypothesis that esophageal clinical symptoms in PNH are secondary to impairment of NO-cGMP pathway caused by intravascular hemolysis.Financial Support: FAPESP. DisclosuresFertrin:Alexion Pharmaceuticals: Consultancy.

Role of heme oxygenase 1 in cardiac ferroptosis in sickle cell disease

Decreased red blood cell (RBC) half-life in sickle cell disease (SCD) results in increased hemolysis and is associated with stroke and other cardiovascular complications although the exact mechanisms remain unexplored. We hypothesized that increased circulating heme in SCD promotes cardiotoxicity through ferroptosis by upregulating heme oxygenase 1 (Hmox1). Our data demonstrated that sickling Townes mice had higher serum heme, lower serum levels of the heme scavenger protein hemopexin and increased cardiotoxicity and that supplementation with hemopexin decreased cardiotoxicity in sickling mice. We also demonstrated that cardiac expression of Hmox1 was increased in SCD mice; inhibition or induction of Hmox1 decreased or worsened cardiac damage, respectively. We also demonstrated that labile redox-active iron, a product of heme degradation by Hmox1, was increased in SCD. Subsequently, we evaluated if ferroptosis, an iron-dependent non-apoptotic cell death mechanism, resulted from increased Hmox1 and drove cardiotoxicity in SCD. We found an increase in lipid peroxidation and ferroptotic markers in the cardiac tissue of SCD mice, which were corrected by hemopexin and Hmox1 inhibition whereas Hmox1 induction exacerbated lipid peroxidation and cardiac ferroptosis. Finally, inhibitors or inducers of ferroptosis decreased or exacerbated SCD cardiotoxicity respectively. Together, our results identify a novel mechanism for heme-associated cardiac damage in SCD.

Sickle Cell Disease: Role of Oxidative Stress and Antioxidant Therapy.

Sickle cell disease (SCD) is the most common hereditary disorder of hemoglobin (Hb), which affects approximately a million people worldwide. It is characterized by a single nucleotide substitution in the β-globin gene, leading to the production of abnormal sickle hemoglobin (HbS) with multi-system consequences. HbS polymerization is the primary event in SCD. Repeated polymerization and depolymerization of Hb causes oxidative stress that plays a key role in the pathophysiology of hemolysis, vessel occlusion and the following organ damage in sickle cell patients. For this reason, reactive oxidizing species and the (end)-products of their oxidative reactions have been proposed as markers of both tissue pro-oxidant status and disease severity. Although more studies are needed to clarify their role, antioxidant agents have been shown to be effective in reducing pathological consequences of the disease by preventing oxidative damage in SCD, i.e., by decreasing the oxidant formation or repairing the induced damage. An improved understanding of oxidative stress will lead to targeted antioxidant therapies that should prevent or delay the development of organ complications in this patient population.

Abnormal Ambulatory Blood Pressure and Early Renal Dysfunction in Sickle Cell Disease.

There is sparsity of studies evaluating blood pressure in children with sickle cell disease (SCD), which have shown inconsistent results. Few of the studies have documented lower office blood pressure (BP) in SCD patients, whereas, others have shown presence of masked hypertension and abnormal ambulatory blood BP monitoring (ABPM). Thus, the present study was conducted to examine 24h ABPM parameters and renal dysfunction in children with SCD and compare them with healthy controls. A cross-sectional study was conducted on 56 children (30 children having SCD and 26 controls). ABPM and evaluation of renal functions including serum creatinine, serum urea, urinary creatinine, urinary protein and specific gravity was performed. Spot urinary protein to creatinine ratio was found to be higher in patients with SCD (63.3%) as compared to controls (p< 0.001). Proteinuria was observed in 1/4th of the SCD patients less than ten years of age. Masked hypertension was present in 2 (6.6%) patients, ambulatory hypertension in 4 (13.3%), ambulatory pre-hypertension in 1 (3.3%) and abnormal dipping in 60%. A statistically significant correlation of BMI for age Z-score and standard deviation score (SDS/Z) of 24h systolic BP (r = 0.56, p= 0.002); estimated glomerular filtration rate (eGFR) with 24h diastolic BP SDS (r = -0.52; p= 0.038) and age with e GFR (r = 0.54; p= 0.025) was found in the present study. The present study corroborates that ABPM abnormalities (ambulatory hypertension, non-dipping pattern, ambulatory prehypertension) and early onset proteinuria are significant findings in patients with SCD. This underscores the importance of regular screening for proteinuria and ABPM in routine care, for early detection and prevention of progressive renal damage in SCD.

Circulating Extracellular Vesicles and Endothelial Damage in Sickle Cell Disease.

Endothelial damage is central to the pathogenesis of many of the complications of sickle cell disease. Circulating extracellular vesicles (EVs) have been implicated in modulating endothelial behavior in a variety of different, diseases with vascular pathologies. As seen in other hemolytic diseases, the plasma of sickle cell patients contains EVs of different sizes and cellular sources. The medium-sized vesicles (microparticles) primarily derive from mature red blood cells and platelets; some of these EVs have procoagulant properties, while others stimulate inflammation or endothelial adhesiveness. Most of the small EVs (including exosomes) derive from erythrocytes and erythrocyte precursors, but some also originate from platelets, white blood cells, and endothelial cells. These small EVs may alter the behavior of target cells by delivering cargo including proteins and nucleic acids. Studies in model systems implicate small EVs in promoting vaso-occlusion and disruption of endothelial integrity. Thus, both medium and small EVs may contribute to the increased endothelial damage in sickle cell disease. Development of a detailed understanding of the composition and roles of circulating EVs represents a promising approach toward novel predictive diagnostics and therapeutic approaches in sickle cell disease.

Erythrocyte adaptive metabolic reprogramming under physiological and pathological hypoxia.

The erythrocyte is the most abundant cell type in our body, acting as both a carrier/deliverer and sensor of oxygen (O2). Erythrocyte O2 delivery capacity is finely regulated by sophisticated metabolic control. In recent years, unbiased and robust human metabolomics screening and mouse genetic studies have advanced erythroid research revealing the differential role of erythrocyte hypoxic metabolic reprogramming in normal individuals at high altitudes and patients facing hypoxia, such as sickle cell disease (SCD) and chronic kidney disease (CKD). Here we summarize recent progress and highlight potential therapeutic possibilities. Initial studies showed that elevated soluble CD73 (sCD73, converts AMP to adenosine) results in increased circulating adenosine that activates the A2B adenosine receptor (ADORA2B). Signaling through this axis is co-operatively strengthened by erythrocyte-specific synthesis of sphingosine-1-phosphate (S1P). Ultimately, these mechanisms promote the generation of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific allosteric modulator that decreases haemoglobin--O2-binding affinity, and thus, induces deoxygenated sickle Hb (deoxyHbS), deoxyHbS polymerization, sickling, chronic inflammation and tissue damage in SCD. Similar to SCD, plasma adenosine and erythrocyte S1P are elevated in humans ascending to high altitude. At high altitude, these two metabolites are beneficial to induce erythrocyte metabolic reprogramming and the synthesis of 2,3-BPG, and thus, increase O2 delivery to counteract hypoxic tissue damage. Follow-up studies showed that erythrocyte equilibrative nucleoside transporter 1 (eENT1) is a key purinergic cellular component controlling plasma adenosine in humans at high altitude and mice under hypoxia and underlies the quicker and higher elevation of plasma adenosine upon re-ascent because of prior hypoxia-induced degradation of eENT1. More recent studies demonstrated the beneficial role of erythrocyte ADORA2B-mediated 2,3-BPG production in CKD. Taken together, these findings revealed the differential role of erythrocyte hypoxic metabolic reprogramming in normal humans at high altitude and patients with CKD vs. SCD patients and immediately suggest differential and precision therapies to counteract hypoxia among these groups.

Calpain-1 Contributes to Pain and Organ Damage in Sickle Cell Disease

Sickle cell disease (SCD) is complex with disabling and life-threatening consequences including pain and organ damage. We hypothesized that calpain-1, a calcium-dependent protease may contribute to both organ damage and pain in SCD because deletion of calpain-1 in HbSS Townes mice ameliorated hyperalgesia (Nwankwo et al., Haematologica 2016), and BDA-410, a selective calpain-1 inhibitor, significantly improved age-associated organ damage in a mouse model of aging (Nabeshima et al., Sci Reports 2014). We performed a randomized double-blind placebo-controlled trial in homozygous female HbSS BERK (sickle) mice to examine the effect of BDA-410 on pain and organ pathology. Mice were treated daily intraperitoneally with vehicle (50 μl DMSO in 950 μl sesame oil) and BDA-410 at 30 mg/kg or 100 mg/kg for 2 weeks. Mechanical-, thermal- (heat and cold), and deep hyperalgesia were assessed at baseline (before treatment), during treatment (1-hour post-injection, as well as on days 4, 8, 12), and after discontinuation of treatment (days 16, 20, 24, and 28). Using 2-way repeated measures ANOVA with Tukey's correction, we observed a significant decrease in mechanical, heat, and cold hyperalgesia in sickle mice treated with BDA-410. Compared to baseline, we observed a significant decrease in, [a] mechanical hyperalgesia, on day 8 with 30 mg/kg (p=0.0312), and days 8, 12, 16, 20, and 24 with 100 mg/kg dose (p=0.0012; p&amp;lt;0.0001; p&amp;lt;0.001; p=0.0006 and p=0.0007, respectively); [b] heat hyperalgesia, on days 8, 12, 16, and 20 with 30 mg/kg (p=0.044, 0.0027, 0.0172 and 0.0326, respectively), and on day 12 with 100 mg/kg (p=0.0138); and [c] cold hyperalgesia on days 8, 12, 16, 20, 24, and 28 with both 30 mg/kg (p= 0.0254, 0.0155, 0.001, 0.003, 0.035 and 0.008, respectively) and 100 mg/kg (p= 0.0054, 0.0035, 0.005, 0.009, 0.011 and 0.003, respectively). No significant difference in deep hyperalgesia was observed with either treatment. No significant effect of vehicle was found on any measures. Although BDA-410 treatment was discontinued after 2 weeks, analgesic effect was maintained for a significantly longer time, suggesting amelioration of the pathobiology of pain and/or inhibition of neural transmission of pain mechanisms. Therefore, BDA-410 is effective in reducing chronic pain in sickle mice without causing tolerance. In addition to pain, extensive Purkinje cell damage in the cerebellum and liver infarcts have been demonstrated in BERK sickle mice (Manci et al., Blood 2006). We observed reduced liver infarction in number and area as well as a significant decrease in Purkinje cell damage in sickle mice treated with 30 mg/kg and 100 mg/kg BDA-410 vs. vehicle (p= 0.0007 &amp; 0.0233, respectively). Thus, BDA-410 is effective at significantly reducing Purkinje cell damage in the brain, which is associated with motor dysfunction as well as pain. We therefore examined gait parameters using Mouse Walker equipment which captures natural walking gait based on optical total internal reflection (TIR). Foot contacts disrupt the effect causing frustrated total internal reflection (fTIR) resulting in illuminated points of contact detected by a high-speed camera (Lumenera Lt425C) and a high-performance recording software (StreamPix 7, Norpix). Gait-related parameters were extracted using MouseWalker software. Sickle mice showed significantly lower walking speed (p=0.0062), increased stance duration and stance instability (p= 0.001 and 0.030, respectively) compared to control mice. Gait parameters correlated positively for mechanical hyperalgesia with stance duration (r=0.72, p=0.004) and negatively for grip force with stance instability and stance duration (r = -0.70, p=0.0075 and r = -0.84, p=0.0002). Since decreased grip force demonstrates increased hyperalgesia, deep hyperalgesia also correlates positively with stance instability and stance duration. Thus, Purkinje cell damage is associated with alterations in gait and pain in sickle mice. Since BDA-410 inhibited Purkinje cell damage and reduced pain, it demonstrates the potential of targeting calpain-1 as a treatable target to develop novel therapeutics for treating pain and organ damage in SCD. These data also suggest that gait measures may serve as biomarkers for Purkinje cell damage and pain. We speculate that gait measures may have utility as a diagnostic and prognostic tool in the progression of SCD. BDA-410 and funding in part were provided by 1910 Genetics. Disclosures Pagare: 1910 Genetics: Employment. Nwankwo:1910 Genetics: Equity Ownership. Gupta:1910 Genetics: Research Funding.