Vaso-occlusion In Sickle Cell Disease Research Articles

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.

Caspase-1 As a Therapeutic Target in Intravascular Hemolysis-Induced Microvascular Inflammation and Occlusive Processes

Hemolytic anemias, such as sickle cell disease (SCD), are a group of acute and acquired conditions characterized by the accelerated destruction of red blood cells (RBC). Intravascular hemolysis (IVH), or RBC lysis within the bloodstream, incurs the release of damage-associated molecular patterns (DAMPs), trigging cell activation and immune responses. Caspase-1 (Casp-1), a protease enzyme, plays a critical role during inflammatory processes by initiating programed cell death and promoting the cleavage of pro-interleukin (IL)-1β into its active form, IL-1β, through inflammasome assembly. Here, we aimed to elucidate the complex mechanisms involved in IVH-induced inflammation, leukocyte-endothelium interactions, and microvascular dynamics. Acute IVH was induced in C57BL/6 mice (HEM) by the injection of sterile water (i.v., 150 µl), and control mice received i.v. saline (CON) (n=3-8 per group). Intravital microscopy was performed at 15 min post IVH induction on the cremaster muscle of mice to quantify venular leukocyte (WBC) rolling and adhesion, and non-invasive laser Doppler flowmetry (LDF) measured blood perfusion in the murine pelvis microvasculature. Blood samples were collected at 1-hour post-IVH for ELISA and flow cytometry analyses. Immortalized human endothelial cells (HUVEC; n=7 per group) were stimulated with heme or S100A8 for 3 hours. HEM mice demonstrated significantly increased plasma heme (P<0.0001) and cell-free hemoglobin (P<0.001), in association with significantly decreased plasma haptoglobin (P<0.05), compared to CON mice, thereby confirming the occurrence of IVH. Furthermore, HEM mice exhibited significantly increased plasma concentrations of the S100A8 alarmin (P<0.001) and IL1-β (P<0.001), as well as augmentations (P<0.001) in plasma TNF-ɑ and IL-6 levels. Flow cytometry was used to measure active Casp-1 in neutrophils and monocytes of mice using the FAM-FLICA probe. FLICA staining was significantly higher in the neutrophils (P<0.05) and monocytes (P<0.05) of HEM mice, compared to CON mice, implying Casp-1 activation in these cell subsets. WBC-endothelium interactions are a hallmark of vaso-occlusion in SCD. HEM mice exhibited elevated venular WBC rolling and adhesion to the endothelium, compared to CON mice (P<0.001), as previously shown (Almeida et al., 2015; PMID: 26019278). In addition, IVH significantly reduced the velocity of microvascular blood flow in mice (P<0.001), and impaired perfusion (P<0.001). To address the role of IVH-induced Casp-1 activation in microvascular dynamics, mice received i.p. administrations of the Casp-1 inhibitor, YVAD, at 1 hour prior to induction of IVH, or not, and were subjected to intravital microscopy or LDF. Interestingly, the inhibition of Casp-1 significantly abrogated IVH-induced WBC rolling (P<0.01) and adhesion to vessel walls (P<0.05) in HEM mice and significantly improved microvascular blood perfusion and flow velocity (P<0.05), suggesting a role for Casp-1 activation in the WBC-endothelium and occlusive interactions observed (See Figure). We then evaluated how heme and S100A8 may induce endothelial cell activation, as these DAMPs were found elevated in the plasma of HEM mice. As expected, compared to unstimulated HUVEC, heme induced endothelial cell activation in vitro, as shown by higher expressions of ICAM-1 (P<0.05), VCAM-1 (P<0.01) and E-selectin (P<0.01), and increased Casp-1 activity (P<0.01). In turn, S100A8 alone significantly elevated ICAM-1 (P<0.05) and E-selectin (P<0.01) expressions on HUVEC, as well as IL-1β release (P<0.001), however no significant increase in Casp-1 activity was observed. HUVEC, when pre-treated with YVAD, displayed a significant reduction in S100A8-induced E-selectin expression (P<0.05), but neither ICAM-1 nor VCAM-1 expression were significantly decreased. In contrast, no changes in the expressions of these adhesion molecules were observed in heme-stimulated cells after YVAD treatment. Taken together, these findings suggest that targeting caspase-1 activation and its associated pathways may represent a potential therapeutic intervention to mitigate the adverse inflammatory and microvascular effects of IVH, a disease mechanism that occurs in severe hemolytic anemias such as SCD.

Evaluation of GBT021601 As a Therapeutic Agent to Restore Bone Marrow Health and Effective Erythropoiesis in a Sickle Mouse Model

Introduction: Hypoxia-mediated damage resulting from vaso-occlusion in sickle cell disease (SCD) leads to abnormal angiogenesis, such as occurs with moyamoya disease and proliferative retinopathy. Abnormal angiogenesis in mouse models of SCD has been observed to impact the bone marrow niche, resulting in a structurally abnormal vascular network. A dysfunctional bone marrow environment results in ineffective erythropoiesis and can damage hematopoietic stem cells (HSCs). Healthy HSCs are essential to the maintenance of hemoglobin (Hb) levels regardless of the rapid turnover of red cells and because of their potential as material for gene therapy. Recently, red cell transfusions were shown to reverse pathogenic angiogenesis in a sickle cell mouse model. However, chronic red cell transfusions are difficult to sustain in individuals with SCD due to complications including iron overload, risk of alloimmunization, and the time and cost involved. GBT021601 is a potent sickle Hb polymerization inhibitor that improves the rheology of red blood cells (RBCs) and has been shown to increase total Hb by as much as 3 g/dL. Here, we explore the effectiveness of GBT021601 for restoring bone marrow health and decreasing proangiogenic factors while maintaining the stemness of marrow cells to preserve erythrocyte maturation. Understanding the impact of GBT021601 on aberrant angiogenesis and the function of HSCs will indicate if it has a role in improving the bone marrow niche and HSC fitness in an era of expanded cell-based therapies for SCD. Methods: Nine-week-old, humanized Townes HbSS SCD mice were split into 3 cohorts (n=8 per group: 4 male, 4 female) and for 12 weeks were fed either standard chow (control) or chow containing 0.1% or 0.2% of GBT021601. After 12 weeks, the mice were phlebotomized and euthanized. Spleens were collected and weighed. Whole bone marrow was collected via centrifugation from both tibias and one femur; the other femur was saved for subsequent 3D confocal microscopy. Bone marrow was analyzed using flow cytometry to measure erythroid cell maturation (Annexin V, TER119, and CD44) and HSC markers (Lin, Sca-1, c-kit). Immunoblot analysis was performed on peripheral plasma to measure hypoxia-induced signaling and markers of angiogenesis (VCAM-1, VEGF-A, ANG-1, and -2). Results: Hb increased by 3 g/dL on average with administration of GBT021601, and spleen weights were significantly reduced ( P=0.0023 and P=0.0015 for control vs 0.1% and 0.2%, respectively). Flow cytometry of the extracted bone marrow revealed a significant increase in mature RBCs and a decrease in erythroid progenitors at all stages ( P<0.001 for RBCs and basophils; P=0.04, P=0.002, and P=0.001 for pro-, poly-, and ortho-chromatic erythroblasts, respectively) (Figure 1). HSCs, defined as Lin-Sca-1 +c-kit +, were not significantly different between groups (control vs 0.1%, control vs 0.2%, and for 0.1% vs 0.2%). Immunoblot analysis revealed a reduction in VCAM-1 and ANG-1 markers with administration of GBT021601 relative to control. However, no difference between ANG-2 and VEGF-A markers was observed between treated and control mice (Figure 2). Conclusions: Treatment with GBT021601 was effective at reducing extramedullary hematopoiesis as determined by the reduction in spleen size compared with control mice. VCAM-1 and ANG-1 are increased in HbSS compared with HbAA mice; treatment with GBT021601 reduced factors that contribute to pathogenic angiogenesis. Treatment with GBT021601 did not change ANG-2 and VEGF-A expression; notably, VEGF-A levels are similar between HbSS and HbAA mice. Our findings support GBT021601 as a sustainable treatment to reduce ineffective erythropoiesis and abnormal angiogenesis-which can otherwise damage the bone marrow niche-thereby suggesting a potential benefit for individuals with SCD participating in future cell-based therapies with curative intent and improved outcomes. More work is planned to examine the ability of GBT021601 to normalize the bone marrow environment and reduce the risk of deleterious effects on allogenic or autologous edited HSCs.

VWF–ADAMTS13 axis dysfunction in children with sickle cell disease treated with hydroxycarbamide vs blood transfusion

AbstractPrevious studies have reported elevated von Willebrand factor (VWF) levels in patients with sickle cell disease (SCD) and demonstrated a key role for the VWF-ADAMTS13 axis in the pathobiology of SCD vaso-occlusion. Although blood transfusion is the gold standard for stroke prevention in SCD, the biological mechanisms underpinning its improved efficacy compared with hydroxycarbamide are not fully understood. We hypothesized that the improved efficacy of blood transfusion might relate to differences in VWF–ADAMTS13 axis dysfunction. In total, 180 children with a confirmed diagnosis of SCD (hemoglobin SS) on hydroxycarbamide (n = 96) or blood transfusion (n = 84) were included. Despite disease-modifying treatment, plasma VWF and VWF propeptide were elevated in a significant proportion of children with SCD (33% and 47%, respectively). Crucially, all VWF parameters were significantly higher in the hydroxycarbamide compared with the blood transfusion cohort (P < .05). Additionally, increased levels of other Weibel-Palade body–stored proteins, including factor VIII (FVIII), angiopoietin-2, and osteoprotegerin were observed, indicated ongoing endothelial cell activation. Children treated with hydroxycarbamide also had higher FVIII activity and enhanced thrombin generation compared with those in the blood transfusion cohort (P < .001). Finally, hemolysis markers strongly correlated with VWF levels (P < .001) and were significantly reduced in the blood transfusion cohort (P < .001). Cumulatively, to our knowledge, our findings demonstrate for the first time that despite treatment, ongoing dysfunction of the VWF–ADAMTS13 axis is present in a significant subgroup of pediatric patients with SCD, especially those treated with hydroxycarbamide.

Crizanlizumab in sickle cell disease.

Vaso-occlusion in sickle cell disease (SCD) leads to a myriad of manifestations driving morbidity and mortality in patients with SCD. Increased leucocyte adhesion and P-selectin expression on platelets and endothelial cells is an inciting eventthat leads to obstruction of microcirculation by adhesion with rigid sickled red blood cells. Crizanlizumab is a first-in-class monoclonal antibody that inhibits P-selectin and has been shown to decrease the frequency of vaso-occlusive pain crises in patients with SCD in clinical trials. The role of crizanlizumab in other manifestations of SCD still needs further investigation.

Oral famotidine reduces the plasma level of soluble P-selectin in children with sickle cell disease.

Plasma histamine levels are increased in patients with sickle cell disease (SCD), potentially promoting endothelial P-selectin expression and vaso-occlusion via histamine type 2 (H2) receptors. We conducted a prospective, non-comparative, single-centre study to determine whether famotidine, a H2 receptor antagonist, reduces P-selectin expression in SCD children. The median plasma P-selectin level was significantly reduced after 29 days of oral famotidine (53.2 ng/mL [IQR: 46.7-63.4] vs. 69.9 ng/mL [IQR: 53.6-84.2], median difference -10.2 ng/mL [IQR: -21.8 to -2.7], p = 0.005) in 28 patients. No effect was observed on other adhesion molecules, inflammation or haemolysis markers, except decreased reticulocyte count. No adverse events deemed related to famotidine were observed. Randomized controlled trials are now needed to assess the efficacy of famotidine in preventing vaso-occlusion in SCD.

Hemolysis dictates monocyte differentiation via two distinct pathways in sickle cell disease vaso-occlusion.

Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by painful vaso-occlusive crises (VOC) and chronic hemolysis. The mononuclear phagocyte system is pivotal to SCD pathophysiology, but the mechanisms governing monocyte/macrophage differentiation remain unknown. This study examined the influence of hemolysis on circulating monocyte trajectories in SCD. We discovered that hemolysis stimulated CSF-1 production, partly by endothelial cells via Nrf2, promoting classical monocyte (CMo) differentiation into blood patrolling monocytes (PMo) in SCD mice. However, hemolysis also upregulated CCL-2 through IFN-I, inducing CMo transmigration and differentiation into tissue monocyte-derived macrophages. Blocking CMo transmigration by anti-P selectin antibody in SCD mice increased circulating PMo, corroborating that CMo-to-tissue macrophage differentiation occurs at the expense of CMo-to-blood PMo differentiation. We observed a positive correlation between plasma CSF-1/CCL-2 ratios and blood PMo levels in patients with SCD, underscoring the clinical significance of these two opposing factors in monocyte differentiation. Combined treatment with CSF-1 and anti-P selectin antibody more effectively increased PMo numbers and reduced stasis compared with single-agent therapies in SCD mice. Altogether, these data indicate that monocyte fates are regulated by the balance between two heme pathways, Nrf2/CSF-1 and IFN-I/CCL-2, and suggest that the CSF-1/CCL-2 ratio may present a diagnostic and therapeutic target in SCD.

Perioperative Complications of Pediatric Orthopaedic Surgery in Sickle Cell Disease.

Vasoocclusion in sickle cell disease can be precipitated by cold temperatures, hypoxia, infection, dehydration, and stress, all of which can occur in the setting of surgery. The purpose of this study was to identify predictors of perioperative complications among pediatric patients with sickle cell disease undergoing orthopaedic surgery. An institutional review board approved single-center retrospective review was conducted of pediatric patients 21 years of age and younger with SCD who underwent any orthopaedic surgery at a single center between 2009 and 2019. Patient data and procedure-specific information were recorded. Preoperative admission for hydration and/or blood transfusion and preoperative laboratory studies were reviewed. The primary study outcome was postoperative complications within 30 days of surgery requiring an ED visit or hospital admission. Ninety-two patients who underwent 118 orthopaedic surgeries were identified. The average age at surgery was 12.0 years (SD 4.8y). Surgical cases were classified as elective (n=82, 70%), infection (n=26, 22%), and trauma (n=9, 8%). The lower extremity was the most frequent surgical site (n=86, 73%). Sixty surgeries (51%) received a preoperative blood transfusion. There were 19 surgeries with postoperative complications (16%) that required an ED visit or hospital readmission within 30 days of surgery. There were significantly more complications following surgery on the hip as compared with other sites (24% vs. 9%, P =0.04). Four or more ED visits in the past year were associated with an OR of 5.7 for a postoperative complication ( P =0.01, 95% CI 1.6-20.5). Patients who had a preoperative blood transfusion had significantly greater rates of complications than those that did not (27% vs. 5%, P <0.01). Children with SCD are at increased risk for complications after orthopaedic surgery, and the current study found an overall postoperative complication rate of 16%. Patients undergoing hip surgery had a disproportionate number of complications, with a 5.8-fold increased risk of a postoperative complication. Patients with 4 or more ED visits in the past year had a 5.7-fold increased risk of a complication. IV Retrospective case series.

5610845 A PHASE 1 STUDY OF CSL888 (HEMOPEXIN) IN ADULT PATIENTS WITH SICKLE CELL DISEASE

Background: Episodes of vaso-occlusive crisis (VOC) are the primary reason for hospitalization among patients with sickle cell disease (SCD). The release of heme from the breakdown of damaged red blood cells is thought to play an important role in contributing to vaso-occlusion in SCD. Free heme promotes activation of endothelium, expression of endothelial cell adhesion molecules and activation of blood cells (neutrophils, monocytes, and platelets) which “stick” to the blood vessel walls. The net result is blockage of blood circulation, resulting in the characteristic pain of VOC. Hemopexin is a 60 kilodalton (kDa) plasma glycoprotein that is mainly expressed in the liver and belongs to the family of acute-phase proteins whose synthesis is induced after an inflammatory event. Hemopexin binds extracellular heme with the highest known affinity (KD < 1pM) of all plasma proteins. In patients with SCD, hemopexin levels are significantly reduced (approximately 85%) compared to healthy controls due to increased consumption. CSL889 is human plasma-derived hemopexin. It is hypothesized that pharmacological administration of CSL889 may decrease heme toxicity, restore normal blood circulation and therefore have beneficial effects in the management of acute VOC in SCD patients. Translation to the clinic is supported by studies performed in vitro demonstrating protection of heme-mediated endothelial cell damage and in vivo using the Townes mouse model of SCD, in which hemopexin was shown ameliorate vaso-occlusion following hemoglobin or hypoxia/reperfusion challenges (Gentinetta et al. 2022). Preclinical repeat dose toxicity studies in animals have evidenced favorable tolerability up to the highest dose level tested. Methods: We describe the design of a Phase 1, first in human, multicenter, open-label study to evaluate the safety, tolerability, and pharmacokinetics (PK) of CSL889 in adult patients with SCD following single intravenous (IV) doses of CSL889 [NCT04285827]. The study will comprise 2 parts. Part A will include patients with SCD in their usual health (not in VOC) who will receive a single dose of CSL889 at one of up to 6 different dose levels. In Part B, patients with SCD who present in VOC requiring intravenous (IV) opioid injections and admission to hospital for further management, will receive a single dose of CSL889 at a level that has been shown to be safe and tolerable in Part A within 36 hours of in-patient admission. All subjects will be followed up for 32 days post-infusion of CSL889. The range of doses to be studied is based on a translational PK model based on data derived from the study of CSL889 in the Townes mouse model of SCD. Results: The primary objective is to determine the safety and tolerability of single doses of CSL889 given to patients with SCD both without (Part A) and with (Part B) active VOC. The secondary objectives are to determine the PK and immunogenicity of CSL889. Exploratory objectives are to determine changes in biomarkers of target engagement (including changes in cell-free heme) and pharmacodynamic biomarkers related to the mechanism of action. Change in pain scores (as measured by Numeric Rating Scale [NRS]) will be additionally evaluated in Part B. Conclusions: Enrollment is in progress. No serious adverse events considered related to the administration of CSL889 have been reported as of 01-Sep-2022. Disclosures: F Wilson, J Jochems, L Lindqvist, K Jung,T Gentinetta, S Costin and GJ Kato are employed by CSL Reference 1. Gentinetta et al, J Clin Med 2022, 11:630

Factor XII contributes to thrombotic complications and vaso-occlusion in sickle cell disease

AbstractA hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMβ2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.

Challenges in the treatment of melanoma with BRAF and MEK inhibitors in patients with sickle cell disease: case report and review of the literature

Patients with sickle cell disease (SCD) suffer from complications due to anemia, inflammation, and vaso-occlusion. Factors that trigger sickling and/or inflammation may initiate such complications, while treatment with hydroxyurea (HU) reduces their emergence and prolongs survival. On the contrary, inhibition of the BRAF-MEK-ERK pathway with BRAF and MEK inhibitors (BRAF/MEKi) has revolutionized treatment of melanoma but their use has been correlated with inflammatory adverse events. Thus, treatment of patients with SCD with BRAF/MEKi may be quite challenging and pyrexia in those patients should be managed as a medical emergency. In this article, intrigued by the case of a 36-year-old female patient with S/β-thal under HU who was treated with dabrafenib and trametinib for melanoma, we analyze the mechanisms underlying inflammation and vaso-occlusion in SCD, the mechanisms of pyrexia and inflammation induced by BRAF/MEKi, their potential interconnections, the shared role of the inflammasome in these two entities, and the protective effect of HU in SCD. Since SCD is the most common inheritable blood disorder, the administration of BRAF/MEKi for melanoma in patients with SCD may be a rather common challenge. Thus, proper treatment with HU may pave the way for an uneventful management of such patients.

Microbial Metabolites Target Histone Deacetylases to Trigger Vascular Inflammation in Sickle Cell Disease

The gut microbiota is a fundamental regulator of host immune responses. Our group previously demonstrated that the depletion of the gut microbiota by broad-spectrum antibiotics (ABX) prevented neutrophil aging (a type of circadian priming) and vaso-occlusion and prolonged survival in a mouse model for sickle cell disease (SCD) (Zhang, D et al. Nature 2015, 570(7570):528-532); the mechanisms involved, however, are as yet unknown. Diverse metabolites are generated by the microbiota from the fermentation of exogenous undigested dietary components and endogenous substrates secreted by the host. Complex carbohydrates are metabolized into short-chain fatty acids (SCFAs), while cholesterol derivatives from the liver are converted into secondary bile acids by intestinal bacteria. The major SCFA producers are Bacteroidetes (gram-negative) and Firmicutes (gram-positive) divisions, and dysbiosis of these bacterial divisions is found in both SCD humans and mice, (Lim, S et al. Am J Hematol 2018, 93(4):E91-E93). Herein, we investigate whether and how microbiota-derived metabolites modulate the dynamics of neutrophils in vaso-occlusive processes in SCD mice. First, to C57BL/6 (wild-type, WT) mice we administered either: 1) one of three main SCFAs, namely sodium butyrate (Na-B), sodium propionate (Na-P), or sodium acetate (Na-A); or 2) a combination of two main secondary bile acids, lithocholic and deoxycholic acids. The number of circulating neutrophils with an aged phenotype (i.e., Ly6Ghi CD11bhi CD62Llo CXCR4pos, Casanova-Acebes, M et al. Cell 2013, 153(5):1025-1035) was assessed by flow cytometry over time. Numbers started to increase at 2h after Na-B administration and we observed a peak of accumulation of total and aged-like neutrophils at 4-5h, with a return to basal after 9h. Consistent with this finding, administration of Na-B to SCD mice led to augmented counts of aged-like neutrophils and impaired survival of mice subjected to inflammation. Interestingly, when SCD mice receiving prolonged ABX treatment (40-45 days) were also given daily Na-B for the last days of ABX treatment, the percentage of aged-like neutrophils in the circulation was higher than that found in the blood of SCD mice under ABX, but without Na-B (55% vs 24%), showing that Na-B by itself is able to reverse the reduction in neutrophil aging promoted by ABX. To address the impact of Na-B in SCD vaso-occlusion, intravital microscopy of the cremaster muscle of SCD mice, after inflammatory stimulus with TNFa, revealed that Na-B abrogated the beneficial effects of ABX and triggered vaso-occlusive processes. We found a 3-fold rise in the adhesion of leukocytes to the endothelium and a 2-fold slower blood flow rate in the microvasculature of mice who received Na-B (see Figure). These results support our hypothesis that the gut microbiota modulates inflammation and vaso-occlusion in SCD through the generation of SCFAs. Because Na-B is known to be a potent inhibitor of histone deacetylase (HDAC) enzymes, we induced HDAC inhibition in WT mice. Vorinostat, a potent synthetic HDAC inhibitor, led to a 3-fold greater expansion of total and aged-like neutrophils in the blood, suggesting a role for HDACs in neutrophil biology. Consistently, in vitro treatment of WT mouse neutrophils with vorinostat or Na-B accelerated the progressive acquisition of an aged-like phenotype, characterized by loss of L-selectin (CD62L). Proteome of neutrophils from WT mice after induction of circadian aging by anti-P and anti-E-selectin antibodies showed that aged neutrophils are similar to neutrophils from vorinostat-treated mice, with down-regulation of genes involved in mRNA processing, protein synthesis, and proteasome complex formation, suggesting that HDAC inhibition is the underlying mechanism driving neutrophil aging. Importantly, the down- and up-regulated pathways found in these neutrophils were the same as those found in SCD mouse neutrophils, and an opposite proteomic profile was observed in neutrophils from ABX-treated SCD mice. Our findings indicate that the intestinal microbiota drives vaso-occlusion and neutrophil aging in SCD mice through the production of metabolites that inhibit HDAC. They also define the microbiota as instigator and innate immunity as effector of vascular disease, such that targeting their crosstalk could be effective at alleviating the devastating symptoms in SCD patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Apohemoglobin-Haptoglobin Complex Prevents Microvascular Dysfunction in Sickle Cell Mice: Potential Therapeutic Approach for Vaso-Occlusive Crisis

Background: Sickle cell disease (SCD) is associated with episodes of vaso-occlusive events (VOE) caused by the effects of hemoglobin S (HbS) polymerization in red blood cells (RBCs) and resultant hemolysis. In the acute setting, treatment for VOE is limited to supportive care such as hydration and aggressive pain management as no specific therapies can alter the course of acute VOE. The apohemoglobin-haptoglobin (apoHb-Hp) complex is a two-protein formulation that is capable of scavenging and neutralizing the toxic effects of cell-free hemoglobin (Hb) and heme released from the hemolysis of polymerized HbS RBCs. The primary objective of this study was to evaluate the microcirculatory effects of apoHb-Hp during hypoxia-induced VOE. Methods: The effects of apoHb-hp was studied in a mouse model of SCD using sickle cell transgenic mice (HbSS-Towne's mice, and compared to mice expressing normal human Hb (HbAA-Towne's mice). Each group consisted of 11 mice, they were initially kept in normal conditions (21% O2) for an hour followed by a hypoxic state (7% O2) for another hour and finally brought back to normal conditions (21% O2) for an hour before being sacrificed. ApoHb-hp (60 mg/mL) and Hp (85 mg/mL) were injected intravenously to yield equimolar total heme and Hb binding capacity between treatments prior to hypoxia exposure. During baseline, hypoxia and normoxia systemic parameters (mean arteriole pressure and heart rate), blood gases (pO2,A, pO2,v, sO2,A, sO2,v), hematological properties (total Hb concentration, hematocrit, and concentration of lactate) and microcirculatory hemodynamics (velocity, diameter, flow and functional capillary density) were measured. After the mice were sacrificed a count of present macrophages, neutrophils and lymphocytes were quantified in the pulmonary tissue. Results: There was no notable changes in blood gas concentrations with any of the treatments. ApoHb-Hp treatment improved microcirculatory hemodynamics of HbSS mice by preventing hypoxia-induced vasoconstriction and reduction of blood flow in arterioles <40µm (P=0.818, P<0.0001), arterioles >40 µm (P>0.9999, P<0.0001) and venules 20<x<60 µm (P=0.150, P=0.330) compared to HbAA mice. Furthermore, the functional capillary density saw favorable recovery to baseline when HbSS mice were treated with apoHb-Hp seeing a 36% drop initially, 50% drop after 24 hours, 35% drop after 48 hours, and 27% drop after 72 hours. For comparison, HbSS mice that didn't receive treatment saw a 50% drop initially, 77% drop after 24 hours, 80% drop after 48 hours, and 72% drop after 72 hours when compared to baseline. Conclusion: These results suggest a role for ApoHb-Hp complex in alleviating microvascular changes occurring during hypoxia mediated acute vaso-occlusion in SCD and merits further study for development as a potential therapeutic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MASP-2 and MASP-3 inhibitors block complement activation, inflammation, and microvascular stasis in a murine model of vaso-occlusion in sickle cell disease

Patients with sickle cell disease (SCD) have ongoing hemolysis that promotes endothelial injury, complement activation, inflammation, vaso-occlusion, ischemia-reperfusion pathophysiology, and pain. Complement activation markers are increased in SCD in steady-state and further increased during vaso-occlusive crisis (VOC). However, the mechanisms driving complement activation in SCD have not been completely elucidated. Ischemia-reperfusion and heme released from hemoglobin during hemolysis, events that characterize SCD pathophysiology, can activate the lectin pathway (LP) and alternative pathway (AP), respectively. Here we evaluated the role of LP and AP in Townes sickle (SS) mice using inhibitory monoclonal antibodies (mAb) to mannose binding lectin (MBL)-associated serine protease (MASP)-2 or MASP-3, respectively. Townes SS mice were pretreated with MASP-2 mAb, MASP-3 mAb, isotype control mAb, or PBS before they were challenged with hypoxia-reoxygenation or hemoglobin. Pretreatment of SS mice with MASP-2 or MASP-3 mAb, markedly reduced Bb fragments, C4d and C5a in plasma and complement deposition in the liver, kidneys, and lungs collected 4 hours after challenge compared to control mAb-treated mice. Consistent with complement inhibition, hepatic inflammation markers NF-ĸB phospho-p65, VCAM-1, ICAM-1, and E-selectin were significantly reduced in SS mice pretreated with MASP-2 or MASP-3 mAb. Importantly, MASP-2 or MASP-3 mAb pretreatment significantly inhibited microvascular stasis (vaso-occlusion) induced by hypoxia-reoxygenation or hemoglobin. These studies suggest that the LP and the AP are both playing a role in promoting inflammation and vaso-occlusion in SCD. Inhibiting complement activation via the LP or the AP might inhibit inflammation and prevent VOC in SCD patients.

Recent advances in "sickle and niche" research - Tribute to Dr. Paul S Frenette.

SummaryIn this retrospective, we review the two research topics that formed the basis of the outstanding career of Dr. Paul S. Frenette. In the first part, we focus on sickle cell disease (SCD). The defining feature of SCD is polymerization of the deoxygenated mutant hemoglobin, which leads to a vicious cycle of hemolysis and vaso-occlusion. We survey important discoveries in SCD pathophysiology that have led to recent advances in treatment of SCD. The second part focuses on the hematopoietic stem cell (HSC) niche, the complex microenvironment within the bone marrow that controls HSC function and homeostasis. We detail the cells that constitute this niche, and the factors that these cells use to exert control over hematopoiesis. Here, we trace the scientific paths of Dr. Frenette, highlight key aspects of his research, and identify his most important scientific contributions in both fields.

Plasma-Derived Hemopexin as a Candidate Therapeutic Agent for Acute Vaso-Occlusion in Sickle Cell Disease: Preclinical Evidence.

People living with sickle cell disease (SCD) face intermittent acute pain episodes due to vaso-occlusion primarily treated palliatively with opioids. Hemolysis of sickle erythrocytes promotes release of heme, which activates inflammatory cell adhesion proteins on endothelial cells and circulating cells, promoting vaso-occlusion. In this study, plasma-derived hemopexin inhibited heme-mediated cellular externalization of P-selectin and von Willebrand factor, and expression of IL-8, VCAM-1, and heme oxygenase-1 in cultured endothelial cells in a dose-responsive manner. In the Townes SCD mouse model, intravenous injection of free hemoglobin induced vascular stasis (vaso-occlusion) in nearly 40% of subcutaneous blood vessels visualized in a dorsal skin-fold chamber. Hemopexin administered intravenously prevented or relieved stasis in a dose-dependent manner. Hemopexin showed parallel activity in relieving vascular stasis induced by hypoxia-reoxygenation. Repeated IV administration of hemopexin was well tolerated in rats and non-human primates with no adverse findings that could be attributed to human hemopexin. Hemopexin had a half-life in wild-type mice, rats, and non-human primates of 80–102 h, whereas a reduced half-life of hemopexin in Townes SCD mice was observed due to ongoing hemolysis. These data have led to a Phase 1 clinical trial of hemopexin in adults with SCD, which is currently ongoing.

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.

Bio-Mechanical and Bio-Rheological Aspects of Sickle Red Cells in Microcirculation: A Mathematical Modelling Approach

Sickle cell disease (SCD) is an inherited monogenic disease characterized by distorted red blood cells that causes vaso-occlusion and vasculopathy. Presently, electrophoresis of haemoglobin and genotyping are used as routine tests for diagnosis of the SCD. These techniques require specialized laboratories and are expensive. The low-cost microfluidics-based diagnostic tool holds a great attention for screening of red blood cell (RBC) deformability. In the present study, lubrication theory has been applied in order to develop a biomechanical model of microcirculation with altered rheological properties of sickle blood in the capillary, which is smaller in size compared to the cell diameter, to explain the multifactorial nature and pathogenesis of vaso-occlusion in SCD. The governing equations have been solved analytically for realistic boundary conditions and simulated using MATLAB. We found that the axial velocity of the cell decreases with a decrease in deformability and compliance. The height of the lubricating film predicts deformation of the cell with respect to local pressure in the microcirculation. Leak back and drag force depend non-linearly on the deformed cell radius with varying viscosity of the plasma and Reynolds number. The modelling predictions of this study is in coherence with experimental results. The analyzed parameters provide unique insights with novel possibilities to design a microfluidics-based effective therapeutic intervention for SCD.

Plasma Concentration of 12-Hydroxyeicosatetraenoic Acid, Single Nucleotide Polymorphisms of 12-Lipooxygenase Gene and Vaso-Occlusion in Sickle Cell Disease.

Background and Novel Aspect of this Work: In the light of previous findings that inflammation predisposes to intercellular adhesion and microvascular occlusion in sickle cell disease (SCD), this study investigated the relationship between the number of vaso-occlusive events in SCD, plasma levels of the pro-inflammatory molecules 12-Hydroxyeicosatetraenoic acid (12-HETE), TNF-α and IL-1β; and single nucleotide polymorphisms (SNPs) in the gene 12-Lipooxygenase (ALOX-12), which encodes the enzyme 12-Lipoxygenase that catalyzes the biosynthesis of 12-HETE. Objective: To evaluate the relationship between vaso-occlusion in SCD and plasma concentrations of 12-HETE, TNF-α, and IL-1β; and single nucleotide polymorphisms (SNPs) in ALOX-12 gene. Participants and Methods: In 50 HbSS patients, the numbers of vaso-occlusive crisis requiring hospital treatment in the previous 1 year and the vaso-occlusive complications of SCD developed to date (e.g stroke) were added to obtain the vaso-occlusive events (VOE) score. In the HbSS patients and 30 healthy sibling control persons, plasma concentrations of 12-HETE, TNF-α and IL-1β were measured by ELISA, the ALOX12 SNPs rs2073438 and rs1126667 detected by DNA sequencing, and the accrued data statistically analyzed. Results: Compared to SCD patients with VOE score 0–1, those with scores ≥3 had higher plasma levels of 12-HETE (p < 0.0001) and TNF-α (p = 0.19), but not IL-1β (p = 0.27). VOE score showed strong direct correlation with plasma level of 12-HETE (r = 0.65, p < 0.0001), but not with TNF-α nor IL-1β. Neither VOE score nor plasma concentration of 12-HETE showed any relationship with the ALOX12 SNPs rs2073438 and rs1126667. Conclusion: The strong direct correlation of VOE score with plasma concentration of 12-HETE suggests that the clinical relevance of this pro-inflammatory molecule in SCD-associated vaso-occlusion needs to be evaluated in further studies.

Heme induces significant neutrophil adhesion in vitro via an NFκB and reactive oxygen species-dependent pathway.

Intravascular hemolysis, a major manifestation of sickle cell disease (SCD) and other diseases, incurs the release of hemoglobin and heme from red blood cells, in turn triggering inflammatory processes. This study investigated the in vitro effects of heme, a major inflammatory DAMP, on the adhesive properties of isolated human neutrophils. Heme (20 and 50µM) significantly increased the adhesion of neutrophils to fibronectin and to recombinant ICAM-1, under static conditions, even more efficiently than the potent pro-inflammatory cytokine, tumor necrosis factor-α (TNF); a microfluidic assay confirmed that heme stimulated neutrophil adhesion under conditions of shear stress. Heme-induced neutrophil adhesion was associated with the increased activities, but not expressions, of the Mac-1 and LFA-1 integrin subunits, CD11b and CD11a, on the cell surface. Notably, heme (50µM) significantly induced NFκB translocation in neutrophils, and inhibition of NFκB activity with the BAY11-7082 molecule abolished heme-induced cell adhesion to fibronectin and significantly decreased CD11a activity. Flow cytometric analysis demonstrated major reactive oxygen species (ROS) generation in neutrophils following heme stimulation that could be inhibited by the antioxidant, α-tocopherol, and by BAY11-7082. Furthermore, co-incubation with α-tocopherol abrogated both heme-stimulated neutrophil adhesion and CD11a/CD11b activation. Thus, our data indicate that heme, at clinically relevant concentrations, is a potent activator of neutrophil adhesion, increasing the ligand affinity of the β2 integrins via a mechanism that may be partially mediated by an NFkB-dependent pathway and the generation of ROS. Given the fundamental role that the adhesion of neutrophils to the vascular wall plays in SCD vaso-occlusion and other vascular inflammatory processes, our findings provide further evidence that cell-free heme is a major therapeutic target in the hemolytic diseases.