Injury In Sickle Cell Disease Research Articles

Mildronate ameliorates kidney injury and reduces inflammation in Sickle Cell Disease mice

INTRODUCTION: Sickle cell disease (SCD) is a hereditary hemoglobin disorder characterized by hemolytic anemia, vaso-occlusive crisis and chronic inflammation that affects multiple organs including kidney, liver, brain, spleen, and heart. Renal disease, the most common complication in SCD, is a progressive disease that begins in childhood as glomerular hyperfiltration, then advances to albuminuria, following by a fast decline in glomerular filtration. It may further progress to a renal failure in adults. More than 50% of SCD patients develop chronic kidney disease (CKD), and 14-18% progress to the end-stage kidney disease. Renal injury in SCD results from a cascade of events starting with chronic red blood cells sickling and hemolysis leading to increased blood viscosity, microvascular obstruction and dilation, hypoxia in the medulla, oxidative stress, and inflammation. Mildronate (meldonium) is an anti-ischemic and anti-inflammatory medication approved in Eastern Europe. HYPOTHESIS: We hypothesized that mildronate administration reduces renal medullary hypoxia and decreases oxidative stress and inflammation ameliorating renal injury in SCD mice. METHODS: The study was approved by the Howard University IACUC. Townes SCD mice, expressing human HbS, and control mice, expressing human HbA, were injected with mildronate (400 mg/kg in saline) intraperitoneally or saline as vehicle control over the course of ten days. Blood was collected and used for hematological parameters analysis. Cytokines were measured by Bio-Plex suspension array in plasma. Hypoxyprobe Biotin kit (Hypoxyprobe Inc) was used for visualization of organ hypoxia. The effect of Mildronate on systemic and renal oxidative stress was measured by malondialdehyde (MDA) using ELISA (Abcam). Urinary KIM-1 was measured by TIM-1/KIM-1 ELISA kit (R&D Systems). RESULTS: Mildornate injections did not affect hematological parameters of SCD mice including hematocrit, MCV, MCH, CMCH, erythrocytes and hemoglobin. Mildronate administration resulted in a significant reduction of the circulating pro-inflammatory cytokines IFN-γ, IL-1β and TNF-α and a trend toward reduction of IL-6, and IL-17 in SCD mice. We found higher level of hypoxia in the medulla of SCD mice compared to control mice. Mildronate injections did not reduced hypoxia in renal medulla of SCD mice. Also, mildronate injections did not change MDA levels in plasma or kidney of SCD mice. We found a significant reduction of proteinuria in SCD mice injected with mildronate compared to SCD mice injected with vehicle. Analysis of H&E-stained kidneys showed significant reduction of glomerular size and medullar congestion areas in the mildronate injected SCD mice. Urinary KIM-1 levels were reduced by mildronate administration. CONCLUSIONS: Our results demonstrate that mildronate ameliorates glomerular and tubular injury in SCD mice and reduces capillary congestion. Mildronate improves renal function, in part by reducing inflammation but not through the reduction of hypoxia or oxidative stress. In future, we plan to assess the mildronate’s impact on kidney function and vessel inflammation. This work was supported by NIH grants R01HL125005, U54MD007597, and SC1HL150685. We thank Xiaomei Niu for helping with the Bio-Plex assay. 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.

Mechanisms of Iron-Mediated Renal Injury in a Mouse Model of Sickle Cell Disease

Background: Sickle cell disease (SCD) is a hematologic disorder characterized by polymerization of the mutated hemoglobin S and sickling of red blood cells, leading to hemolysis, release of free heme/iron, and subsequent tissue iron accumulation. The kidney is one of the most frequently affected organs by iron overload, and chronic hemolysis is associated with chronic kidney disease severity in SCD. The potential role of iron overload in causing the observed renal injury in SCD has never been established. The goal of our study is to identify the impact of iron accumulation on the renal phenotype in SCD. We hypothesized that renal iron accumulation causes injury by oxidative stress dependent ferroptosis and/or mitochondrial dysfunction and subsequent kidney damage, that is worse in males than in females. Methods: We used 20-week-old male and female humanized SCD mice (HbSS) and age-matched genetic controls (HbAA). Mice were treated with either vehicle or the iron chelator Deferiprone (DFP, 100 mg/kg/day) for 8 weeks. Urinary excretion of protein and albumin as markers of renal injury were measured. Glomerular filtration rate (GFR) was measured transdermally after injection of FITC-sinistrin. Thiobarbituric acid reactive substance (TBARS) assay was performed for assessment of lipid peroxidation. Mitochondrial bioenergetics was assessed by Oroboros O2k Fluorometer of permeabilized renal cortices. Results: We previously demonstrated comparable iron accumulation in the kidney of male and female mice. However, HbSS males developed a more severe renal injury than females at 20 weeks of age, evidenced by proteinuria (2.52 ± 0.16 vs. 0.55 ± 0.16 mg/24hrs, p=0.0008) and albuminuria (76.44 ± 6.85 vs. 33.9 ± 8.81 μg/24hrs, p=0.0005). There was higher lipid peroxidation, a hallmark of ferroptosis, in the renal cortex of female HbSS than HbAA mice (1.31 ± 0.07 vs. 1.07 ± μM/mg, p=0.04). Bioenergetics studies revealed significantly lower state 3 oxygen consumption rate (108.2 ± 18.1 vs 164.8 ± 9.3 pmol/(s*mg), p=0.05), signifying lower oxidative phosphorylation capacity in HbSS males. In addition, there was significant reduction in state 4◦ respiration (36.4 ± 3.4 vs. 52.8 ± 3.4 pmol/(s*mg), p=0.03) in male HbSS mice. I also detected a decrease in maximum complex IV activity in male HbSS mice (325.5 ± 63.8 vs. 639.5 ± 51.3 pmol/(s*mg), p=0.02). Treatment with DFP improved proteinuria (2.66 ± 0.09 vs. 1.3 ± 0.14 mg/24hrs, p=0.0011) and GFR (165.3 ± 9.4 vs. 236.6 ± 12.6 μl/min, p=0.0058) in male HbSS mice. Conclusion: Iron accumulation causes renal injury which may be attributed to ferroptosis and mitochondrial dysfunction in SCD mice. Funding: NHLBI—R00HL144817 and ASN Norman Siegel Research Grant to MK. 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.

Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis-induced hepatobiliary injury in sickle cell disease.

Sickle cell disease (SCD)-associated chronic hemolysis promotes oxidative stress, inflammation, and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell-free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD-related hepatobiliary injury have not been fully elucidated yet. Here we show that chronic liver injury in SCD patients is associated with elevated levels of hepatic membrane-bound CD163. Hemolysis and increase in hepatic heme, hemoglobin, and iron levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that heme oxygenase-1 (HO-1) positively regulates membrane-bound CD163 expression independent of nuclear factor erythroid 2-related factor 2 (NRF2) signaling in SCD liver. We further demonstrate that the interaction between CD163 and HO-1 is not dependent on CD163-hemoglobin binding. These findings indicate that CD163 is a potential biomarker of SCD-associated hepatobiliary injury. Understanding the role of HO-1 in membrane-bound CD163 regulation may help identify novel therapeutic targets for hemolysis-induced chronic liver injury.

Impaired Hemoglobin Clearance By Liver Sinusoidal Endothelium Promotes Liver Damage in Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic disorder that affects 100,000 African Americans and millions of people worldwide. Intra-erythrocytic polymerization of sickle hemoglobin (HbS) promotes erythrocyte sickling, impaired rheology, ischemia and hemolysis, leading to the development of progressive liver injury in SCD. Liver resident macrophages and monocytes are known to enable the clearance of HbS, however, the role of liver sinusoidal endothelial cells (LSECs) in HbS clearance and liver injury in SCD remains unknown. Using real-time intravital ( in vivo) imaging of the liver as well as primary culture of human and mice LSECs, we show for the first time that liver injury is associated with accumulation of HbS and iron in the LSECs, leading to the development of senescence in LSECs. Hepatic monocytes were observed to attenuate LSEC-senescence by accelerating HbS clearance in the liver of SS mice, however, this protection was impaired in P-selectin-deficient SCD mice secondary to reduced monocyte recruitment in the liver. These findings are the first to suggest that LSECs contribute to HbS clearance and LSEC-senescence promotes progressive liver injury in SS mice. Our results reveal a novel insight into the pathogenesis of hemolysis induced chronic liver injury in SCD caused by LSEC senescence. Identifying the regulators of LSEC mediated HbS clearance may lead to new therapies to prevent the progression of liver and other organ injuries in SCD.

Hemoglobin Receptor CD163 As a Potential Biomarker of Hemolysis Induced Hepatobiliary Injury in Sickle Cell Disease

Sickle cell disease (SCD) associated chronic hemolysis promotes oxidative stress, inflammation and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD related hepatobiliary injury has not been fully elucidated yet. Here, we show that chronic liver injury in SCD mice and patients is associated with elevated levels of hepatic CD163. Hemolysis and increase in hepatic heme, hemoglobin, iron and haptoglobin levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that HO-1 positively regulates CD163 expression and that HO-1 and NRF2 form a complex with CD163 in the SCD liver. Finally, we demonstrate that blocking CD163 activity promotes hemolysis induced liver injury in SCD. These findings indicate that CD163 is a mediator and a potential biomarker of SCD associated hepatobiliary injury. Understanding the role of HO-1 in CD163 regulation may help identify novel therapeutic targets for hemolysis induced chronic liver injury.

Circulating NETs Promote Platelet-TLR9 Dependent Acute Chest Syndrome in Sickle Cell Disease

Acute chest syndrome (ACS), a type of acute lung injury is one of the primary reasons for mortality among Sickle Cell Disease (SCD) patients. Although new evidence suggests that circulating neutrophil extracellular traps (cNETs) promote lung injury in SCD by triggering occlusion of pulmonary arterioles by neutrophil-platelet-erythrocyte thrombo-inflammatory aggregates, the molecular mechanism underlying cNETs-dependent lung vaso-occlusion remains unknown. Surprisingly, platelets are one of the few cell types that express Toll-like receptor 9 (TLR9), a receptor for double-stranded DNA on the surface (rather than in endosomes), however, the role of platelet-TLR9 in promoting pulmonary thrombo-inflammation in SCD has never been studied. Using intravital (in vivo) lung microscopy in live SCD mice and imaging flow cytometry (in vitro) of SCD mice plasma, we show for the first time that lung vaso-occlusion and elevated levels of cNETs in knock-in humanized SCD mice challenged with 10 μmol/kg oxy-hemoglobin (oxy-Hb) is associated with significant upregulation of TLR9 surface expression in platelets. Importantly, TLR9 inhibition using a function blocking Ab led to significant reduction by ~6 and ~4 fold in the frequency and size of lung vaso-occlusions in SCD mice challenged with IV oxy-Hb. Identical to SCD mice, TLR9 surface expression was also significantly higher in SCD than control human platelets, and the expression was further upregulated following treatment of SCD patient platelet-rich-plasma (PRP) with oxy-Hb. Remarkably, elevated TLR9 expression in SCD patient platelets was concomitant to increased phosphorylation of both downstream TLR9 pathway effector tank-binding-kinase-1 (TBK-1) and the TBK-1 substrate interferon regulatory factor-3 (IRF3), which is the major transcription factor driving IFN-1 response. Taken together, our current findings suggest for the first time that activation of nucleic acid receptor TLR9 on the surface of platelets by cNETs contributes to lung vaso-occlusion and acute chest syndrome in SCD.

Hemolysis Induced Induction of Matrix Metalloproteinases in Sickle Cell Disease

Sickle Cell Disease (SCD) is a monogenic disorder that impacts around 100,000 people in the United States and 20 million people worldwide. SCD is caused by a point mutation in the β-globin gene. SCD can lead to complications, such as hemolysis, vaso-occlusion, oxidative stress, organ injury, and erythrocyte sickling. Organ injury, specifically liver injury, is one of the main causes of mortality in SCD patients. Hemolysis induced accumulation of hemoglobin, heme and iron acts as potent damage associated molecular pattern (DAMP) and can cause chronic liver injury and inflammation. Previous studies in the lab have demonstrated exacerbated injury and inflammation in SCD liver. Chronic inflammation is known to activate matrix metalloproteinases (MMPs) which can then induce the expression of cytokines and chemokines. In this study, we examined the role of hemolysis-induced induction of MMPs using blood and liver samples from humanized townes SCD mouse model. Proteomic analysis using SCD mouse blood sample revealed increased expression of 8 MMPs in SCD mice following oxyhemoglobin treatment compared to their baseline expression. We next examined the mRNA expression of these MMPs in the liver by doing qRT-PCR. Interestingly, two MMPs- Steap3 and Timp3, showed significant upregulation in SCD mice liver compared to the littermate control, which positively correlated with hemolysis and the age of the mouse. Studies are currently underway to understand the role of Steap3 and Timp3 in hemolysis-induced chronic liver injury and their potential usage as novel biomarkers of hemolysis-induced chronic organ injury in SCD

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.

Impaired hemoglobin clearance by sinusoidal endothelium promotes vaso-occlusion and liver injury in sickle cell disease.

Sickle cell disease (SCD) is a monogenic disorder that affects 100,000 African-Americans and millions of people worldwide. Intra-erythrocytic polymerization of sickle hemoglobin (HbS) promotes erythrocyte sickling, impaired rheology, ischemia and hemolysis, leading to the development of progressive liver injury in SCD. Liver-resident macrophages and monocytes are known to enable the clearance of HbS; however, the role of liver sinusoidal endothelial cells (LSEC) in HbS clearance and liver injury in SCD remains unknown. Using real-time intravital (in vivo) imaging in mice liver as well as flow cytometric analysis and confocal imaging of primary human LSEC, we show for the first time that liver injury in SCD is associated with accumulation of HbS and iron in the LSEC, leading to senescence of these cells. Hemoglobin uptake by LSEC was mediated by micropinocytosis. Hepatic monocytes were observed to attenuate LSEC senescence by accelerating HbS clearance in the liver of SCD mice; however, this protection was impaired in P-selectin-deficient SCD mice secondary to reduced monocyte recruitment in the liver. These findings are the first to suggest that LSEC contribute to HbS clearance and HbS-induced LSEC senescence promotes progressive liver injury in SCD mice. Our results provide a novel insight into the pathogenesis of hemolysis-induced chronic liver injury in SCD caused by LSEC senescence. Identifying the regulators of LSEC-mediated HbS clearance may lead to new therapies to prevent the progression of liver injury in SCD.

White-matter abnormalities and cognitive dysfunction are linked to astrocyte activation in sickle mice.

White-matter injury in sickle-cell disease (SCD) includes silent cerebral infarction diagnosed by diffusion tensor imaging (DTI), a complication associated with cognitive dysfunction in children with SCD. The link between white-matter injury and cognitive dysfunction has not been fully elucidated. The goal of this study was to define whether cerebrovascular lesions and cognitive function in SCD are linked to neuroaxonal damage and astrocyte activation in humanized Townes' SCD mice homozygous for human sickle hemoglobin S (SS) and control mice homozygous for human normal hemoglobin A (AA). Mice underwent MRI with DTI and cognitive testing, and histology sections from their brains were stained to assess microstructural tissue damage, neuroaxonal damage, and astrocyte activation. Fractional anisotropy, showing microstructural cerebrovascular abnormalities identified by DTI in the white matter, was significantly associated with neuronal demyelination in the SS mouse brain. SS mice had reduced learning and memory function with a significantly lower discrimination index compared with AA control mice in the novel object recognition tests. Neuroaxonal damage in the SS mice was synchronously correlated with impaired neurocognitive function and activation of astrocytes. The interplay between astrocyte function and neurons may modulate cognitive performance in SCD.

Silent infarction in sickle cell disease is associated with brain volume loss in excess of infarct volume.

Sickle cell disease (SCD) increases cerebral infarct risk, but reported effects on brain volume have varied. More detailed information using larger cohorts and contemporary methods could motivate the use of longitudinal brain volume assessment in SCD as an automated marker of disease stability or future progression. The purpose of this study was to rigorously evaluate whether children and young adults with SCD have reduced gray matter volume (GMV) and white matter volume (WMV) compared to healthy controls using high-resolution MRI. We tested the hypotheses that (i) elevated CBF, a marker of cerebral hemodynamic compensation in SCD, is associated with global and regional brain atrophy, and (ii) silent cerebral infarct burden is associated with brain atrophy in excess of infarct volume. Healthy controls (n = 49) and SCD participants without overt stroke (n = 88) aged 7-32 years completed 3 T brain MRI; pseudocontinuous arterial spin labeling measured CBF. Multivariable linear regressions assessed associations of independent variables with GMV, WMV, and volumes of cortical/subcortical regions. Reduced hemoglobin was associated with reductions in both GMV (p = 0.032) and WMV (p = 0.005); reduced arterial oxygen content (CaO2) was also associated with reductions in GMV (p = 0.035) and WMV (p = 0.006). Elevated gray matter CBF was associated with reduced WMV (p = 0.018). Infarct burden was associated with reductions in WMV 30-fold greater than the infarct volume itself (p = 0.005). Increased GM CBF correlated with volumetric reductions of the insula and left and right caudate nuclei (p = 0.017, 0.017, 0.036, respectively). Infarct burden was associated with reduced left and right nucleus accumbens, right thalamus, and anterior corpus callosum volumes (p = 0.002, 0.002, 0.009, 0.002, respectively). We demonstrate that anemia and decreased CaO2 are associated with reductions in GMV and WMV in SCD. Increased CBF and infarct burden were also associated with reduced volume in subcortical structures. Global WMV deficits associated with infarct burden far exceed infarct volume itself. Hemodynamic compensation via increased cerebral blood flow in SCD seems inadequate to prevent brain volume loss. Our work highlights that silent cerebral infarcts are just a portion of the brain injury that occurs in SCD; brain volume is another potential biomarker of brain injury in SCD.

Insulin-like Growth Factor-1 Prevents Hypoxia/Reoxygenation-Induced White Matter Injury in Sickle Cell Mice

Occlusion of cerebral blood vessels causes acute cerebral hypoxia-an important trigger of ischemic white matter injury and stroke in sickle cell disease (SCD). While chronic hypoxia triggers compensatory neuroprotection via insulin-like growth factor-1 (IGF-1) and hypoxia inducible factor-1α (HIF-1α), severe bouts of acute hypoxia and subsequent restoration of blood flow (hypoxia/reoxygenation, H/R) overwhelm compensatory mechanisms and cause neuroaxonal damage-identified as white matter lesions-in the brain. The neuroprotective role of IGF-1 in the pathogenesis of white matter injury in SCD has not been investigated; however, it is known that systemic IGF-1 is reduced in individuals with SCD. We hypothesized that IGF-1 supplementation may prevent H/R-induced white matter injury in SCD. Transgenic sickle mice homozygous for human hemoglobin S and exposed to H/R developed white matter injury identified by elevated expression of non-phosphorylated neurofilament H (SMI32) with a concomitant decrease in myelin basic protein (MBP) resulting in an increased SMI32/MBP ratio. H/R-challenge also lowered plasma and brain IGF-1 expression. Human recombinant IGF-1 prophylaxis significantly induced HIF-1α and averted H/R-induced white matter injury in the sickle mice compared to vehicle-treated mice. The expression of the IGF-1 binding proteins IGFBP-1 and IGFBP-3 was elevated in the IGF-1-treated brain tissue indicating their potential role in mediating neuroprotective HIF-1α signaling. This study provides proof-of-concept for IGF-1-mediated neuroprotection in SCD.

Platelet TLR9 Promotes Pulmonary Thrombo-Inflammation in Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic disorder, which affects millions of people worldwide. Erythrocyte sickling, chronic hemolysis and ischemia-reperfusion injury promote sterile inflammation in SCD.Recent evidence suggests that activation of innate immune pathways in platelets contribute to development of acute chest syndrome (ACS), a type of acute lung injury in SCD by promoting occlusion of lung arterioles by neutrophil-platelet-erythrocyte aggregates (pulmonary thrombo-inflammation). Although, TLR9 is functionally expressed on the surface of activated platelets and may bind to circulating double stranded DNA, the role of platelet TLR-9 in promoting pulmonary thrombo-inflammation in SCD remains elusive. Our initial findings using intravenous lipopolysaccharide (0.1 μg/kg LPS) or oxy-hemoglobin (10 μmole/kg oxy-Hb) triggered model of vaso-occlusive crisis in transgenic-humanized SCD mice reveal that pulmonary vaso-occlusion is associated with elevated surface-expression of platelet TLR-9 in SCD mice. Unlike SCD mice, surface-expression of TLR9 was undetectable in the platelets of control mice challenged with either IV LPS or oxy-Hb. These findings suggest a potential role for platelet TLR9 in promoting lung vaso-occlusion and injury in SCD. Currently, studies are in progress to identify the innate immune signaling downstream to platelet TLR9, contributing to pulmonary thrombo-inflammation in SCD.

Complement Drives Acute Chest Syndrome in a Model of Sickle Cell Disease

Background: Despite being the first genetic disease described, sickle cell disease (SCD) continues to afflict millions of individuals worldwide. Patients with SCD can experience severe complications, including vasoocclusive crisis (VOC), which can progress to acute chest syndrome (ACS), a form of acute lung injury. While VOC can cause significant morbidity, ACS is the leading cause of mortality in patients with SCD. Despite the devastating consequences of ACS, no interventions exist that directly treat this complication. As a result, when patients present with ACS, options often remain limited to supportive care. The lack of optimal treatment options for patients with SCD experiencing ACS directly contributes to their morbidity and mortality. Using a combined approach of prospective observational studies in patients with SCD that experienced ACS and a novel preclinical model specifically designed to define the role of complement in SCD pathophysiology, we tested the hypothesis that complement plays a central role in the pathophysiology of ACS. Methods: Samples from patients with SCD experiencing ACS were compared to baseline measurements for the same patient obtained at outpatient follow up 4-6 weeks later. ELISA based assays were used to measure Ba, Bb, C3a, C5a, C5b-9 (membrane attack complex (MAC)) in each sample. SCD mice (HbSS) and control mice (HbAA) were injected with 7.5 units of cobra venom factor (CVF), a potent complement activator, followed by evaluation of hematocrit (Hct) measured by a Sysmex veterinary hematology analyzer, free heme by calorimetric assay, and complement deposition on red blood cells (RBCs) by flow cytometry or pulmonary endothelial cells by confocal analysis. Respiratory rate and O2 saturation were measured using a MouseOx pulse oximeter. HbSS or HbAA mice were crossed with complement component 3 (C3) knock out (KO) mice to generate HbSS x C3 KO and HbAA x C3 KO mice. Hemin (70 to 210 mmol/kg concentration) or CVF (7.5 units/mouse) were injected into HbSS, HbAA, HbSS x C3 KO or HbAA x C3 KO followed by evaluation of hemolysis, C3 deposition and pulmonary function. A one-way ANOVA with a Tukey's post hoc with a p value <0.05 was considered significant. Results: During ACS in patients, significant elevations in Ba and Bb, complement activation products unique to the alternative pathway, and downstream activation of complement, including C3a, C5a and MAC, were observed (p<0.0001). Changes in complement activation were accompanied by a drop in hemoglobin during ACS when compared to baseline (p<0.001), suggesting that complement activation results in hemolysis. To formally test this, the impact of complement activation in a preclinical model of SCD was defined. Injection of CVF or hemin resulted in rapid C3 deposition on RBCs and pulmonary endothelium in HbSS, but not HbAA, recipients. In addition, CVF or hemin injection was accompanied by increased hemolysis and ACS manifested by increased respiratory rate, drop in oxygen saturation and death within 2 hours in the majority of recipients (p<0.001). In contrast, control Hb AA mice were unaffected by CVF or hemin injection. Given the role of free heme in the development of ACS, we next defined the role of complement in hemin-induced lung injury. To accomplish this, SCD mice were crossed with C3 KO mice to generate HbSS x C3 KO. HbSS x C3 KO mice displayed a mild increase in Hb values at baseline (p<0.01), suggesting a role of C3 even in steady-state SCD-related hemolysis. Equally important, injection of HbSS x C3 KO mice with CVF or hemin failed to result in the same levels of C3 deposition, hemolysis, compromised pulmonary function, or mortality (p<0.001) as HbSS recipients. Discussion: These results demonstrate a critical role for C3 in the development of acute lung injury in SCD. The sensitivity of HbSS RBCs to complement-induced hemolysis and C3 deposition observed following hemin injection in SCD recipients suggests that C3 plays a key role in decreased hematocrits and pulmonary injury that accompanies ACS. As treatment of ACS largely relies on supportive care, these results suggest that approaches aimed at targeting complement activation may represent a useful prophylactic or treatment strategy for ACS. In doing so, these results hold promise in providing a more direct approach at reducing one of the most severe complications of patients with SCD.

Neutrophils as drivers of vascular injury in sickle cell disease.

While neutrophils are the main effectors of protective innate immune responses, they are also key players in inflammatory pathologies. Sickle cell disease (SCD) is a genetic blood disorder in which red blood cells (RBCs) are constantly destroyed in the circulation which generates a highly inflammatory environment that culminates in vascular occlusions. Vaso-occlusion is the hallmark of SCD and a predictor of disease severity. Neutrophils initiate and propagate SCD-related vaso-occlusion through adhesive interactions with the activated and dysfunctional endothelium, sickle RBCs, and platelets, leading to acute and chronic complications that progress to irreversible organ damage and ultimately death. The use of SCD humanized mouse models, in combination with in vivo imaging techniques, has emerged as a fundamental tool to understand the dynamics of neutrophils under complex inflammatory contexts and their contribution to vascular injury in SCD. In this review, we discuss the various mechanisms by which circulating neutrophils sense and respond to the wide range of stimuli present in the blood of SCD patients and mice. We argue that the central role of neutrophils in SCD can be rationalized to develop targets for the management of clinical complications in SCD patients.

Molecular mechanisms of hepatic dysfunction in sickle cell disease: lessons from Townes mouse model.

Sickle cell disease (SCD) is an autosomal recessive genetic disorder that affects ∼100,000 Americans and millions of people worldwide. Erythrocyte sickling, vaso-occlusion, sterile inflammation, and hemolysis are the major pathophysiological pathways leading to liver injury in SCD. Although hepatic dysfunction affects up to 10%-40% of patients with SCD, therapeutic approaches to prevent liver injury in SCD are not known, and the molecular mechanisms promoting progressive liver injury in SCD remain poorly understood. Animal models have been beneficial in bridging the gap between preclinical and translational research in SCD. Recent advances in methodology have allowed the development of several humanized animal models to address various aspects of SCD-related liver diseases. This review provides an overview of current knowledge of the molecular mechanisms and potential therapeutic options of SCD-associated liver dysfunction using the Townes mouse model.

Liver-to-lung microembolic NETs promote gasdermin D–dependent inflammatory lung injury in sickle cell disease

AbstractAcute lung injury, referred to as the acute chest syndrome, is a major cause of morbidity and mortality in patients with sickle cell disease (SCD), which often occurs in the setting of a vaso-occlusive painful crisis. P-selectin antibody therapy reduces hospitalization of patients with SCD by ∼50%, suggesting that an unknown P-selectin–independent mechanism promotes remaining vaso-occlusive events. In patients with SCD, intraerythrocytic polymerization of mutant hemoglobin promotes ischemia-reperfusion injury and hemolysis, which leads to the development of sterile inflammation. Using intravital microscopy in transgenic, humanized mice with SCD and in vitro studies with blood from patients with SCD, we reveal for the first time that the sterile inflammatory milieu in SCD promotes caspase-4/11–dependent activation of neutrophil–gasdermin D (GSDMD), which triggers P-selectin–independent shedding of neutrophil extracellular traps (NETs) in the liver. Remarkably, these NETs travel intravascularly from liver to lung, where they promote neutrophil-platelet aggregation and the development of acute lung injury. This study introduces a novel paradigm that liver-to-lung embolic translocation of NETs promotes pulmonary vascular vaso-occlusion and identifies a new GSDMD-mediated, P-selectin–independent mechanism of lung injury in SCD.

Cortactin loss protects against hemin-induced acute lung injury in sickle cell disease.

In patients with sickle cell disease (SCD), acute chest syndrome (ACS) is a common form of acute lung injury and a major cause of morbidity and mortality. The pathophysiology of ACS is complex, and hemin, the prosthetic moiety of hemoglobin, has been implicated in endothelial cell (EC) activation and subsequent acute lung injury (ALI) and ACS in vitro and in animal studies. Here, we examined the role of cortactin (CTTN), a cytoskeletal protein that regulates EC function, in response to hemin-induced ALI and ACS. Cortactin heterozygous (Cttn+/-) mice (n = 8) and their wild-type siblings (n = 8) were irradiated and subsequently received bone marrow cells (BMCs) extruded from the femurs of SCD mice (SS) to generate SS Cttn+/- and SS CttnWT chimeras. Following hemoglobin electrophoretic proof of BMC transplantation, the mice received 35 µmol/kg of hemin. Within 24 h, surviving mice were euthanized, and bronchoalveolar fluid (BAL) and lung samples were analyzed. For in vitro studies, human lung microvascular endothelial cells (HLMVECs) were used to determine hemin-induced changes in gene expression and reactive oxygen species (ROS) generation in cortactin deficiency and control conditions. When compared with wild-type littermates, the mortality for SS Cttn+/- mice trended to be lower after hemin infusion and these mice exhibited less severe lung injury and less necroptotic cell death. In vitro studies confirmed that cortactin deficiency is protective against hemin-induced injury in HMLVECs, by decreasing protein expression of p38/HSP27, improving cell barrier function, and decreasing the production of ROS. Further studies examining the role of CTTN in ACS are warranted and may open a new avenue of potential treatment for this devastating disease.

Abstract WP260: The Response Of Annexin A1 In Astrocyte Activation And White Matter Injury- A New Target For Stroke In Sickle Cell Disease

Introduction: Ischemic stroke is common in sickle cell disease (SCD). White matter injuries, incorporated into silent cerebral infarcts, are major risk factors of stroke in SCD. The pathogenesis of cerebral infarction in SCD is not known. Induced thrombogenesis may cause impaired astrocyte that stimulates demyelination leading to white matter injury. Annexin A1 (Anxa1) can regulate astrocyte function by inhibiting intracellular phospholipase A2 (PLA2). We hypothesized that astrocyte activation during thrombogenic events promotes white matter injury in SCD. We tested astrocyte dysfunction and white matter injury in a clinically relevant mouse model of SCD. Methods: Cerebral Infarctions induced by de novo thrombogenesis were identified by TTC staining in transgenic sickle (SS) and control (AA) mice with human sickle and normal hemoglobin respectively. Cerebral microvessels and astrocytes were isolated from SS and AA mice. Elisa assays were used to measure thrombin, PLA2 and Anxa1. Immunofluorescence staining was performed to determine astrocyte activation and demyelination. Results: Thrombogenesis within cerebral microvessels was elevated by ~2-fold (n=5, p&lt;0.05), and cerebral infarction became prevalent in SS mice compared to AA mice (n=7, p&lt;0.001). The infarcted brain tissue from SS mice showed neuronal cell death, whereas the brain tissue from the AA mice appeared normal. The SS mice had increased glial fibrillary acid protein positive (GFAP + ) activated astrocytes with significant increase in PLA2 accumulation. Relative abundance of non-phosphorylated neurofilament H (SMI32) and myelin basic protein (MBP) (SMI32/MBP), indicating white matter injury, was substantially induced in the SS compared to the AA. Expression of Anxa1 reduced significantly in plasma as well as within astrocytes’ end feet attached to the isolated cerebral microvessels of the SS mice. Recombinant Anxa1 prophylaxis to SS mice reduced the GFAP + astrocytes, PLA2 accumulation, demyelination and infarct score (n=6, p&lt;0.01) compared to SS mice treated with vehicle. Conclusion: White matter injury in SS mice is associated with astrocyte dysfunction and lack of Anxa1, which may be developed as potential therapeutic targets to prevent and/or treat stroke in SCD.

P-Selectin Deficiency in Sickle Cell Disease Mice Leads to Senescence Induced Organ Injury

Sickle cell disease (SCD) is caused by a homozygous mutation in the β-globin gene, which leads to erythrocyte sickling, vaso-occlusion, and premature hemolysis . Vaso-occlusion and hemolysis are the two predominant pathophysiologic events in SCD that contribute to chronic organ damage and acute systemic painful vaso-occlusive episode (VOE). Previous studies both in vivo and in vitro have confirmed the role of endothelial and platelet adhesion marker P-selectin in sickle cell vasoocclusive crisis. Moreover, a phase 2 study showed a significant reduction in painful vaso-occlusive episodes among SCD patients receiving the P-selectin-blocking antibody crizanlizumab . However, the long-term effect of crizanlizumab in SCD and associated organ injuries is not known. To understand the roles and regulation of P-selectin dependent vasoocclusion in SCD associated organ injuries, we have introduced the first SCD mice genetically lacking P-selectin in hematopoietic and nonhematopoietic compartments. Using this model (SS-Selp −/−) we have shown that P-selectin deficiency protects SCD mice from lung vaso-occlusion. Here we have used this model to study the liver injury in SCD. Using quantitative liver intravital imaging (qLIM) technique, we show that P-selectin deficiency protects SCD mice from liver vaso-occlusion. However, we found persistent hepatobiliary injury in SS-Selp −/− mice. Mechanistically, we show that blocking P-selectin causes significant enrichment of circulating inflammatory cells however the organ specific recruitment of inflammatory cells was drastically impaired. Remarkably, impairment of organ specific recruitment of inflammatory cells exacerbated cellular senescence and injury in sickle mouse. We found significant enrichment of senescent markers including P21, P16 and phosphor P53 using both western blot and immunohistochemistry. Colocalization analysis confirmed that hepatocytes, cholangiocytes and macrophages were susceptible to senescence. Moreover, we also found impaired iron trafficking in in SS-Selp −/− mice. Work is currently underway to understand how p-selectin loss promotes liver senescence and impaired iron trafficking in the liver. In summary, our results reveal a significant defect in iron homeostasis and exacerbated senescence in the liver of SS-Selp −/− mice suggesting that increased liver senescence might impair iron homeostasis which then contribute to hepatobiliary injury in SCD. Thus, the efficacy of P-selectin inhibition in preventing SCD warrants further studies to determine whether long term inhibition of P-selectin would lead to end stage complications. DisclosuresSundd: Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring Inc: Research Funding.