Sickle Cell Disease Vasculopathy Research Articles

Membrane bending and sphingomyelinase-associated, sulfatide-dependent hypoxic adhesion of sickle mature erythrocytes.

Abnormal erythrocyte adhesion owing to polymerization of sickle hemoglobin is central to the pathophysiology of sickle cell disease (SCD). Mature erythrocytes constitute >80% of all erythrocytes in SCD; however, the relative contributions of erythrocytes to acute and chronic vasculopathy in SCD are not well understood. Here, we showed that bending stress exerted on the erythrocyte plasma membrane by polymerization of sickle hemoglobin under hypoxia, enhances sulfatide-mediated abnormal mature erythrocyte adhesion. We hypothesized that sphingomyelinase (SMase) activity, which is upregulated by accumulated bending energy, leads to elevated membrane sulfatide availability, and thus, hypoxic mature erythrocyte adhesion. We found that mature erythrocyte adhesion to laminin in controlled microfluidic experiments is significantly greater under hypoxia than under normoxia (1856± 481 vs 78± 23, mean± SEM), whereas sickle reticulocyte (early erythrocyte) adhesion, high to begin with, does not change (1281± 299 vs 1258± 328, mean± SEM). We showed that greater mean accumulated bending energy of adhered mature erythrocytes was associated with higher acid SMase activity and increased mature erythrocyte adhesion (P= .022, for acid SMase activity and P= .002 for the increase in mature erythrocyte adhesion with hypoxia, N= 5). In addition, hypoxia results in sulfatide exposure of the erythrocyte membrane, and an increase in SMase, whereas anti-sulfatide inhibits enhanced adhesion of erythrocytes. These results suggest that the lipid components of the plasma membrane contribute to SCD complications. Therefore, sulfatide and the components of its upregulation pathway, particularly SMase, should be further explored as potential therapeutic targets for inhibiting sickle erythrocyte adhesion.

Near-Infrared Spectroscopy Demonstrates the Benefit of Erythracytapheresis in Sickle Cell Disease Adult Patients with Cerebral Vasculopathy.

Cerebral vasculopathy can induce chronic cerebral hypoperfusion leading to stroke in patients with sickle cell disease (SCD) and is treated by blood exchange transfusion (BET). However, no prospective clinical study has demonstrated the benefit of BET in adults with SCD and cerebral vasculopathy. Near Infrared Spectroscopy (NIRS) is a recent non-invasive method complementary to Magnetic Resonance Imaging (MRI). We evaluated cerebral perfusion using NIRS during erythracytapheresis in patients with SCD with and without steno-occlusive arterial disease. We conducted a monocentric, prospective study in 16 adults with SCD undergoing erythracytapheresis in 2014. Among them, 10 had cerebral steno-occlusive arterial disease. NIRS measured the relative amounts of oxyhemoglobin (OxyHb), deoxyhemoglobin (DeoxyHb) and total hemoglobin (Total Hb) in brain tissue and in muscle. In cerebral hemispheres associated with steno-occlusive arterial disease, we observed a significant increase of OxyHb and Total Hb during BET, without modification of DeoxyHb. Using NIRS during BET showed that BET improves cerebral perfusion in adult patients with SCD with cerebral vasculopathy.

Predictors and clinical complications associated with antiphospholipid antibodies in sickle cell disease.

Although a higher prevalence of antiphospholipid autoantibodies (aPL) has been observed in some cohorts of sickle cell disease (SCD) patients, the clinical risk factors for the development of aPL and its associated complications remain unclear. In a retrospective study of 63 SCD patients, a lower hemoglobin concentration and higher white blood cell count were independently associated with an elevated aPL. SCD patients with elevated aPL had increased pregnancy complications (≥3 miscarriages, preterm delivery, pre-eclampsia) and venous thrombotic events. Our findings suggest that SCD may predispose to the generation of aPL and that aPL itself may contribute to the vasculopathy of SCD. Prospective testing for aPL is warranted in patients with SCD.

Resolvins Modulate Netosis and Autoimmunity in Sickle Cell Disease

Sickle cell disease (SCD) is a genetically determined hematological disease which is characterized by anemia, chronic hemolysis, and multi-organ chronic inflammatory vasculopathy with acute vaso-occlusive painful crises and chronic organ damage. The pathophysiology of SCD is complex and involves several other cell types and metabolic pathways than erythrocytes, including neutrophils and soluble factors such as cytokines or alternative complement pathway which affect the function of vascular endothelium. An inflammatory vasculopathy with chronically activated neutrophils releasing Neutrophil Extracellular Traps (NETs) is a key component of the pathophysiology of sickle cell disease (SCD). Chiang et al. have recently shown that resolvins (Rv) reduce NETS and improve NETs clearance in a model of sepsis by Staphylococus aureus, a known trigger of NETosis (Chiang N et al. Blood 139 (8): 1222, 2022). We previously reported that targeting pro-resolution mechanisms with Rv improves SCD vasculopathy and organ damage in humanized sickle mice. Here, we studied 23 adult patients with SCD, followed by the Comprehensive center for hemoglobinopathies and rare anemias, University of Verona and AOUI Verona from January 2019 to December 2021. For each patient the presence/absence of concomitant rheumatological condition such as rheumatoid arthritis, other inflammatory arthropathies, immune-mediated dermatological conditions, Sicca syndrome and Raynaud's phenomenon, were assessed. Eight age, and ethnicity matched healthy controls were analyzed. Blood was collected for hematological and biochemical assay as part of the standard clinical care for these patients. Additional blood was collected for research studies on whole blood and on plasma cytokines. SCD patients display significant increase in tumor- necrosis-factor (TNF)- α (p = 0.011) and interleukin (IL)-10, suggesting both active inflammatory state and an upregulated counterregulatory mechanism to limit inflammation. We observed that antinuclear antibodies (ANA), but not extractable nuclear antibodies (ENA), were significantly increased in SCD patients (provide specific data here). Previous reports have reported the presence of this type of autoantibodies in the absence of autoimmune clinical manifestations in patients with SCD (Toly-Ndour C et al J Rheumatol 38: 302, 2011). ANA+ SCDsubjects (defined by a cell nuclei homogenous pattern, and positive dilution in the 1:80 to 1: 160 range) had a higher inflammatory and hemolytic status, as demonstrated by increased WBC and neutrophil counts and LDH values compared to ANA- subjects. NETosis plays a key role in modification and externalization of autoantigens involved in production of autoantibodies such as ANA. Here, we found basal NETosis was higher, though not significant, in ANA+ SCD subjects compared to ANA-SCD patients. This was associated with lower expression of receptors for RvD1 and D2, namely ALX, GPR32/DRV1, and GPR18/DRV2 on PMN, monocytes, and lymphocytes from ANA+ SCD patients when compared to ANA- SCD subjects, who were also characterized by relatively low LDH compared to ANA+ SCD patients. Exogenous treatment of SCD neutrophils with RvD1 and D4 significantly dampened NETs release and enhanced NETs clearance by macrophages. RvD4 along with other pro-resolving mediators also stimulated ex vivo clearance of P. aeruginosa and S. aureus by SCD neutrophils. Our findings provide a novel link between autoimmunity and neutrophil-mediated vasculopathy in SCD and highlight the importance of Rv and their immune-pro-resolution effects in limiting disease progression in SCD but also to modulate the host response to infections and sepsis, which is altered in SCD.

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.

Effects of Flavonoid Quercetin on Thrombo-Inflammatory Processes in Patients with Sickle Cell Disease

Introduction:Altered erythrocyte physiology in Sickle Cell Disease (SCD) results in severe vascular complications, including vaso-occlusion (VOC) along with various other manifestations, such as thrombosis and end organ damage. Moreover, platelet activation endothelial dysfunction and fibrin formation, are critical processes implicated in thrombo-inflammatory vasculopathy in SCD. Since flavonoids intake is associated with lowered adverse cardiovascular disease outcomes and cancer associated thrombosis, we are evaluating the effects of the flavonoid Isoquercetin (IQ) on thrombo-inflammatory biomarkers in patients with SCD (NCT:04514510). Here we report the mechanistic effects of Quercetin (Que), an aglycone that differs from IQ by the absence of 3-linked glucoside moiety, on markers of erythrocyte and platelet activation in patients with SCD.Methods:We studied patients with SCD (Genotype SS, mean age: 39 yrs, N=8) and ethnic matched controls (Genotype AA, mean age: 42 yrs, N=8). Erythrocyte reactive oxygen species (ROS) was measured in washed red cells by flow cytometry using the fluorescent probe 2‘, 7‘-dichlorofluorescein diacetate (DCFDA) in the presence or absence of Quercetin (Que) (100 µM). Effects of in vitro erythrocyte ROS induction by hydrogen peroxide (H 2O 2) and ROS inhibition by N-acetyl-L-cysteine (NAC) were evaluated. Baseline and agonist-induced (ADP 20 µM) platelet P-selectin expression in vitro in the presence or absence of Que (100 µM) was assessed by flow cytometry.Results:At baseline, SS erythrocytes demonstrated elevated levels of ROS reflected by greater florescent intensity with DCFDA when compared with erythrocytes from ethnic matched controls (SS mean= 1884, SD: 508.8 vs. AA mean: 1604, SD: 554.5; p value= 0.3798). Stimulation of AA erythrocytes in vitro with H 2O 2 led to accumulation of ROS that approached levels similar to those exhibited by SS erythrocytes at baseline (AA mean: 1890, SD: 748.3). Pre-treatment of SS and AA erythrocytes in vitro with the antioxidant NAC exhibited minimal effects on ROS in SS erythrocytes (SS baseline+NAC mean: 1942, SD: 526.8, p value 0.4609 and SS H 2O 2+NAC mean: 1889, SD: 437.4, p value 0.5469) but lowered ROS levels in both baseline and H 2O 2 treated AA erythrocytes (AA baseline+NAC mean: 1442, SD: 453.9, p value 0.1563 and AA H 2O 2+NAC mean: 1581, SD: 518.7, p value 0.078). Pre-treatment of SS and AA erythrocytes in vitro with Que substantially lowered erythrocyte ROS in patients and controls at the baseline (SS baseline ROS with Que mean: 1582, SD: 542.0, p value 0.0078; AA baseline ROS with Que mean: 1341, SD: 490.9, p value 0.0313; Fig. 1A), and after H 2O 2 stimulation (SS H 2O 2 induced ROS+Que mean: 1677, SD: 572.5, p value 0.0156; AA H 2O 2 induced ROS+Que mean: 1417, SD: 512, p value 0.0078). Pre-treatment of erythrocytes with a combination of Que and NAC did not reveal synergistic anti-oxidant activity (SS NAC+Que mean: 1616, SD: 464.8; AA NAC+Que mean: 1333, SD: 524.1).We simultaneously investigated the effects of Que on platelet P-selectin expression in the same subjects. As expected, baseline platelet surface P-selectin expression was higher in SS patients compared with controls (SS N=5, mean: 1.48%, Max: 4.9%, Min: 0.03%; AA N=4, mean: 0.89%, Max: 2.3%, Min: 0%; p value 0.55). Stimulation of platelet rich plasma (PRP) with ADP increased surface P-selectin expression in comparison with baseline, in both patients and controls (SS N=5, mean: 18.04%, Max: 40.4%, Min: 5.8%, p value 0.0047; AA N=4, mean: 20.60%, Max: 43.8%, Min: 5.4%; p value 0.0140). Pre-treatment of PRP with Que prior to ADP stimulation revealed a trend toward decreased platelet surface P-selectin expression in both patients and controls (SS N=5, mean: 10.49%, Max: 24.8%, Min: 3.1%; AA N=4, mean: 12.09%, Max: 21.9%, Min: 3.4%; Fig 1B) although this did not return to baseline.Conclusion:The flavonoid Quercetin appears to have a beneficial effect in lowering erythrocyte ROS and platelet surface P-selectin and warrant further evaluation as a strategy to ameliorate thrombo-inflammatory pathophysiology in sickle hemoglobinopathies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Vasculopathy in Sickle Cell Disease: From Red Blood Cell Sickling to Vascular Dysfunction.

Sickle cell disease (SCD) is a hereditary disorder that leads to the production of an abnormal hemoglobin, hemoglobin S (HbS). HbS polymerizes in deoxygenated conditions, which can prompt red blood cell (RBC) sickling and leaves the RBCs more rigid, fragile, and prone to hemolysis. SCD patients suffer from a plethora of complications, ranging from acute complications, such as characteristic, frequent, and debilitating vaso-occlusive episodes to chronic organ damage. While RBC sickling is the primary event at the origin of vaso-occlusive processes, other factors that can further increase RBC transit times in the microcirculation may also be required to precipitate vaso-occlusive processes. The adhesion of RBC and leukocytes to activated endothelium and the formation of heterocellular aggregates, as well as increased blood viscosity, are among the mechanisms involved in slowing the progress of RBCs in deoxygenated vascular areas, favoring RBC sickling and promoting vascular occlusion. Chronic inflammatory processes and oxidative stress, which are perpetuated by hemolytic events and ischemia-reperfusion injury, result in this pan cellular activation and some acute events, such as stroke and acute chest syndrome, as well as chronic end-organ damage. Furthermore, impaired vasodilation and vasomotor hyperresponsiveness in SCD also contribute to vaso-occlusive processes. Treating SCD as a vascular disease in addition to its hematological perspective, the present article looks at the interplay between abnormal RBC physiology/integrity, vascular dysfunction and clinical severity in SCD, and discusses existing therapies and novel drugs in development that may ameliorate vascular complications in the disease. © 2021 American Physiological Society. Compr Physiol 11:1785-1803, 2021.

Assessing Dynamic Cerebral Autoregulation in Patients with Sickle Cell Disease Using Near-Infrared Spectroscopy and Paced Breathing

Introduction. Sickle cell disease (SCD) is caused by hemoglobin S polymerization leading to vaso-occlusion, small vessel disease, and reduced tissue oxygen saturation. Noninvasive assessments and screening of small vessel disease and decreased oxygen saturation in select organs, like the brain, are poorly developed. Cerebral autoregulation, a mechanism that ensures cerebral blood flow (CBF) remains constant with changes in mean arterial pressure, is impaired with and is a marker of small vessel disease. Frequency-domain near-infrared spectroscopy (FD-NIRS) can be used as a noninvasive technique to measure cerebral autoregulation by recording cerebral microvascular changes in hemoglobin concentration that are related to cerebral blood flow. Transfer function analysis is a method to noninvasively quantify the relationship between measured physiologic signals, and can be used to estimate cerebral autoregulation. In this study, we hypothesized patients with SCD have impaired cerebral autoregulation and reduced tissue oxygen saturation, which we evaluated via FD-NIRS with transfer function analysis, as compared to healthy controls. Methods. All patients provided informed consent under an IRB protocol approved by the University of Pittsburgh and Carnegie Mellon University. Patients with SCD (n = 11) and healthy, race-matched controls (n =14) were enrolled. All participants sat upright in a chair with probes of a commercial multi-distance FD-NIRS apparatus applied to their forehead in a dark room (Fig. 1A). We used paced breathing to dynamically induce controlled changes in the microvascular tone at 0.1, 0.125, and 0.1667 Hz, spanning normal to more challenging breathing rates, with a visual metronome guiding each paced breathing session (Fig.1.B). To validate each breathing pace, we recorded the participants' respiratory rate via a belt placed around their chest, and their mean arterial pressure through a finger and arm cuff attached to a beat-to-beat finger plethysmographer (Fig.1.A). We used the modified Beer-Lambert law to calculate changes in oxy-hemoglobin (O), deoxy-hemoglobin (D), and total hemoglobin (T) concentrations. Data was bandpass-filtered around the paced breathing frequencies, and the Hilbert transform was applied to calculate the amplitude ratios, |D|/|O|, and phase latency, Arg(D)−Arg(O). The tissue oxygen saturation of hemoglobin, StO2 (mean ±SD), was computed based on an average baseline of two minutes. Results. Arg(D)−Arg(O) and |D|/|O| are shown for controls and patients at all paced breathing frequencies (Fig.1C-D) from the largest source-detector distance. A non-zero phase lag between D and O within both groups indicates influences of cerebral blood volume (CBV) and CBF changes. The phase differences of D and O are therefore related to cerebral autoregulation. We found a greater phase difference (mean range: -320° to -340°) in patients with SCD compared to controls (mean range: -200° to -240°), indicating the presence of vascular obstruction and impaired cerebral autoregulation. Higher amplitude ratios |D|/|O| may indicate slower blood transmit times. We found that patients with SCD had increased amplitude ratios for hemodynamic oscillations at lower paced breathing frequencies compared to healthy controls (Fig. 1D). Finally, we found a significantly reduced cerebral tissue oxygen saturation in patients with SCD (63.1 % ± 7.8 %) in comparison with controls (65.9 % ± 4.9 %, p<0.05, Fig.1E). Discussion. We provide preliminary evidence that FD-NIRS can be used to assess cerebral autoregulation in SCD. Our results confirm and extend those of a prior study by Reinhard et al, 2003, by using the phase latency, Arg(D)−Arg(O), and the amplitude ratios, |D|/|O|, to assess autoregulation. In addition, we detected a noticeable reduction in oxygen saturation in patients with SCD as compared to healthy controls. Further studies may extend our findings to additional frequencies in a larger sample size to more comprehensively assess the impact of mean arterial pressure on autoregulation. Noninvasive measurement of cerebral autoregulation and brain oxygenation in SCD by FD-NIRS may represent a novel biomarker of cerebral vasculopathy in SCD and may help monitor changes in cerebral oxygenation, particularly during treatment with drugs that modulate the hemoglobin oxygen affinity, such as the recently FDA-approved voxelotor. Figure 1 Disclosures No relevant conflicts of interest to declare.

Persistance of Inflammatory Phenotype of Neutrophils in Sickle Cell Disease Children Despite Chronic Exchange Transfusion Program: A Cause of the Progression of Cerebral Vasculopathy?

Sickle cell disease (SCD) is a severe hemoglobinopathy due to the production of abnormal hemoglobin S (HbS). Although red blood cell (RBC) dysfunction is the major contributor to disease, several studies highlighted the important role of polymorphonuclear neutrophils (PMNs), both during acute and chronic complications. One of the most severe complication of SCD is ischemic stroke due to large cerebral artery occlusion. In 1998, the Stroke Prevention (STOP) trial demonstrated that monthly blood transfusions could reduce the risk of stroke by 90% in SCD children with cerebral vasculopathy (CV). However, there is a wide heterogeneity in the course of CV in patients receiving chronic transfusions, since only about half of them improved their CV under transfusion program, while 25% are only stabilized and 29% continue to get worse despite a percentage of HbS permanently below 30%. The aim of our study is to investigate the impact of transfusion programs on neutrophils activation and ageing, in order to identify if inflammation could contribute to the persistence of SCD complications despite red cell transfusion. We performed a prospective study including 58 homozygous SCD children and 10 healthy donors. Of these, 12 had no specific treatment, 11 were on Hydroxyurea (HU) treatment, 21 were on an exchange transfusion program, and 14 were on both an exchange transfusion program and HU treatment for an average of 4.9 years due to persistent CV. Monthly exchange transfusion are carried out either by erythrapheresis or by manual exchanges, consisting of the continuous bleeding of whole blood compensated by simultaneous transfusion of packed red blood cells. Neutrophils were isolated from fresh blood samples before exchange transfusion session and labelled with 8 markers specific of adhesion, activation and ageing. We quantified by flow cytometry the expression of 3 integrins (CD18, CD11a, CD11b), 3 ageing markers (CD182, CD184, CD62L) and 2 adhesion molecules (CD162 and CD66a). We also measured the plasmatic level of elastase, which reflects the NETose activity of PMNs As previously reported, we observed a high leukocytosis and an activated profile of PMNs in the 12 non-transfused SCD patients compared to healthy controls (Figure 1), characterized by an overexpression of the integrin CD18/CD11b (p=0,03) and CD18/CD11a (p=0,02), a higher level of circulating aged PMNs CD184 high/CD62Llow (p=0,04), a higher expression of CD162 (p=0,01) and CD66a (0,01) as well as a higher plasmatic level of elastase (p=0.01). Interestingly, in the PMNs of the 21 patients receiving monthly exchange transfusion, we found an identical expression pattern of integrins, selectins, ageing markers and elastase level compared to those of the PMNs from non-transfused patients. Furthermore, we also observed a persistence of high neutrophilic leukocytosis. This activation pattern was the same for patients on manual exchange or erythrapheresis, even with a tendency towards a more inflammatory profile in patients on erythrapheresis (Figure 1). In the PMNs from the 11 patients receiving HU compared to untreated SCD patients, we found an expected decrease in high leukocytosis and membrane integrin expression CD18/CD11b and CD18/CD11a. The addition of HU therapy in 14 patients in exchange transfusion program allows to alleviate neutrophilic leukocytosis and membrane integrin expression. Our study shows for the first time that replacing sickle RBCs with healthy RBCs is not sufficient to reverse the pathological phenotype of PMNs in SCD. A persistence of the PMNs activation pattern is observed both despite erythrapheresis, where plasma and white blood cells go back to the patient, and in manual exchanges, where the patient is bled from a large volume of whole blood. Given the major role of inflammation in endothelial damage and vasculopathy in SCD, our data could explain the incomplete efficacy of transfusion exchange programs to treat CV. This raises the question to systematically combine anti-inflammatory and anti-white blood cell adhesion treatments such as Hydroxyurea or P-Selectin inhibitors for these patients. Disclosures No relevant conflicts of interest to declare.

Microvasculopathy and biomarkers in sickle cell disease: the promise of non‐invasive real‐time in vivo tools

Microvasculopathy and biomarkers in sickle cell disease: the promise of non‐invasive real‐time <i>in vivo</i> tools

Smooth muscle cytochrome b5 reductase 3 deficiency accelerates pulmonary hypertension development in sickle cell mice

AbstractPulmonary and systemic vasculopathies are significant risk factors for early morbidity and death in patients with sickle cell disease (SCD). An underlying mechanism of SCD vasculopathy is vascular smooth muscle (VSM) nitric oxide (NO) resistance, which is mediated by NO scavenging reactions with plasma hemoglobin (Hb) and reactive oxygen species that can oxidize soluble guanylyl cyclase (sGC), the NO receptor. Prior studies show that cytochrome b5 reductase 3 (CYB5R3), known as methemoglobin reductase in erythrocytes, functions in VSM as an sGC heme iron reductase critical for reducing and sensitizing sGC to NO and generating cyclic guanosine monophosphate for vasodilation. Therefore, we hypothesized that VSM CYB5R3 deficiency accelerates development of pulmonary hypertension (PH) in SCD. Bone marrow transplant was used to create SCD chimeric mice with background smooth muscle cell (SMC)–specific tamoxifen-inducible Cyb5r3 knockout (SMC R3 KO) and wild-type (WT) control. Three weeks after completing tamoxifen treatment, we observed 60% knockdown of pulmonary arterial SMC CYB5R3, 5 to 6 mm Hg elevated right-ventricular (RV) maximum systolic pressure (RVmaxSP) and biventricular hypertrophy in SS chimeras with SMC R3 KO (SS/R3KD) relative to WT (SS/R3WT). RV contractility, heart rate, hematological parameters, and cell-free Hb were similar between groups. When identically generated SS/R3 chimeras were studied 12 weeks after completing tamoxifen treatment, RVmaxSP in SS/R3KD had not increased further, but RV hypertrophy relative to SS/R3WT persisted. These are the first studies to establish involvement of SMC CYB5R3 in SCD-associated development of PH, which can exist in mice by 5 weeks of SMC CYB5R3 protein deficiency.

Thrombomodulin and Endothelial Dysfunction in Sickle Cell Anemia

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

Smooth Muscle Cytochrome b5 Reductase 3 Deficiency Accelerates Pulmonary Hypertension Development in Sickle Cell Mice

Pulmonary and systemic vasculopathy are significant risk factors for early morbidity and death in patients with sickle cell disease (SCD). An underlying mechanism of SCD vasculopathy is vascular smooth muscle (VSM) nitric oxide (NO) resistance, which is mediated by NO scavenging reactions with plasma hemoglobin and reactive oxygen species that can oxidize soluble guanylyl cyclase (sGC), the NO receptor. Prior studies show that cytochrome b5 reductase 3 (CYB5R3), known as methemoglobin reductase in erythrocytes, functions in VSM as an sGC heme iron reductase critical for reducing and sensitizing sGC to NO and generating cGMP for vasodilation. Therefore, we hypothesized that VSM CYB5R3 deficiency accelerates development of pulmonary hypertension (PH) in SCD. Bone marrow transplant was used to create SCD chimeric mice with background smooth muscle cell (SMC) specific tamoxifen-inducible Cyb5r3 knockout (SMC R3 KO) and wild-type (WT) control.Three weeks after completing tamoxifen treatment, we observed 60% knockdown of pulmonary arterial SMC CYB5R3, 5-6 mmHg elevated right ventricular maximum systolic pressure (RVmaxSP) and biventricular hypertrophy in SS chimeras with SMC R3 KO (SS/R3KD) relative to wild-type (SS/R3WT). Right ventricular (RV) contractility, heart rate, hematological parameters and cell-free hemoglobin were similar between groups. When identically generated SS/R3 chimeras were studied 12-weeks after completing tamoxifen treatment, RVmaxSP in SS/R3KD had not increased further, but RV hypertrophy relative to SS/R3WT persisted. These are the first studies to establish involvement of SMC CYB5R3 in SCD-associated development of PH, which can exist in mice by 5 weeks of SMC CYB5R3 protein deficiency. Disclosures Vitturi: Complexa Inc: Consultancy. Ofori-Acquah:Shire Human Genetic Therapies Inc: Other: Financial Relationship. Gladwin:Globin Solutions, Inc: Patents &amp; Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents &amp; Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases . Straub:Bayer Pharmaceuticals: Research Funding.

Stiff Erythrocyte Subpopulations Biomechanically Induce Endothelial Inflammation in Sickle Cell Disease

Background: Originally described as a monogenic hemoglobin disorder resulting in increased red blood cell (RBC) stiffness leading to vaso-occlusion, sickle cell disease (SCD) is now known to be a vasculopathic disease with some semblance to cardiovascular disease in which the endothelium is inflamed. While adhesive RBC-endothelial interactions, inflammatory cytokines, and hemolysis all contribute to SCD vasculopathy, whether the increased stiffness of sickle RBCs directly contributes to endothelial inflammation is unknown. Endothelial cells are now known to mechanotransduce shear forces into biological signals. Pathological alteration of such forces leads to proinflammatory endothelial cell signaling including upregulation of VCAM-1 and E-selectin, which contribute to atherosclerotic plaques leading to myocardial infarction and stroke (Abe, ATVB, 2014). In addition, under normal homeostatic conditions, RBCs do not come into contact with the endothelium due to a cell-free layer created by the Fåhræus-Lindqvist effect. Studies including our own have shown in silico that increasing RBC stiffness diminishes or eliminates the cell-free layer, allowing stiff RBCs to contact the vessel wall (Kumar, Phys Rev E, 2011). This is particularly pertinent in SCD, as all patients have a small population (1-10%) of sickle RBCs that are permanently stiff and misshapen. We therefore hypothesize that purely physical interactions - akin to "scratches" or collisions - between endothelial cells and stiff SCD RBCs breaking through the cell-free layer are sufficient to cause endothelial inflammation in the absence of adhesion or vaso-occlusion (Fig. 1A). Methods: We performed computational direct numerical simulations using the boundary integral method for a binary suspension of flexible biconcave discs and stiff curved prolate spheres modeling healthy RBCs and ISCs, respectively. Experimentally, we leveraged our microfluidic microvasculature models of human umbilical vein endothelial cells cultured throughout each microchannel (Fig. 2). RBCs from SCD patients were "spiked" into normal RBC suspensions to comprise 5 and 10% of the overall population (a representation of ISCs in vivo), suspended in media to 25% hematocrit mimicking conditions seen in SCD patients, and perfused into the microfluidics for 4 hours. Samples of 100% normal RBCs or SCD RBCs were run in parallel. To isolate the stiffness effects of sickle RBCs without confounding hemolytic and adhesive effects, parallel experiments were conducted using nystatin-treated normal RBCs to create artificially stiffened RBC subpopulations, defined by elevated mean corpuscular hemoglobin concentrations (MCHCs), at the same proportion of the overall RBC population (0, 5, 10 and 100%). The endothelialized models were then fixed, permeabilized, and immunostained with antibodies against VCAM-1 and E-selectin. Mean fluorescence intensity was measured to quantify endothelial inflammation. Results: In silico, we observed that ISCs strongly marginate towards the vessel walls due to their stiffness and "pointy" shape, and heterogeneous suspensions with small fractions of stiff, pointy cells (5 and 10%) caused the highest degree of margination (Fig. 1B). Experimentally, endothelium exposed to 5, 10, and 100% SCD RBCs exhibited increased VCAM-1 and E-selectin expression over normal RBCs, and the degree of expression increased with higher percentages of SCD RBCs. While endothelial cells exposed to nystatin-stiffened RBCs also showed increased VCAM-1 and E-selectin expression, those exposed to a lower percentage of stiff cells (5 and 10%) exhibited higher expression than the homogenously stiff (100%) condition (Fig. 3), which is consistent with our computer simulations. Conclusions: Here we demonstrate that purely non-adhesive, physical interactions between endothelial cells and SCD RBCs are sufficient to cause endothelial inflammation. Furthermore, heterogeneous RBC populations, comprised of a small minority of stiff cells, cause more inflammation than uniformly stiff RBCs. Studies elucidating the underlying mechanisms, using different endothelial cell types, and analyzing the effect of vessel curvature are ongoing. Our results introduce a new paradigm for understanding SCD pathophysiology and may help explain how chronic diffuse vasculopathy develops, which could lead to more biophysically-based therapeutic strategies. Disclosures Carden: GBT: Honoraria; NIH: Research Funding. Mannino:Sanguina, LLC: Employment, Equity Ownership. Lam:Sanguina, LLC: Equity Ownership.

Resolution of sickle cell disease–associated inflammation and tissue damage with 17R-resolvin D1

AbstractResolvins (Rvs), endogenous lipid mediators, play a key role in the resolution of inflammation. Sickle cell disease (SCD), a genetic disorder of hemoglobin, is characterized by inflammatory and vaso-occlusive pathologies. We document altered proresolving events following hypoxia/reperfusion in humanized SCD mice. We demonstrate novel protective actions of 17R-resolvin D1 (17R-RvD1; 7S, 8R, 17R-trihydroxy-4Z, 9E, 11E, 13Z, 15E, 19Z-docosahexaenoic acid) in reducing ex vivo human SCD blood leukocyte recruitment by microvascular endothelial cells and in vivo neutrophil adhesion and transmigration. In SCD mice exposed to hypoxia/reoxygenation, oral administration of 17R-RvD1 reduces systemic/local inflammation and vascular dysfunction in lung and kidney. The mechanism of action of 17R-RvD1 involves (1) enhancement of SCD erythrocytes and polymorphonuclear leukocyte efferocytosis, (2) blunting of NF-κB activation, and (3) a reduction in inflammatory cytokines, vascular activation markers, and E-selectin expression. Thus, 17R-RvD1 might represent a new therapeutic strategy for the inflammatory vasculopathy of SCD.

Disrupting the vicious cycle created by NOX activation in sickle erythrocytes exposed to hypoxia/reoxygenation prevents adhesion and vasoocclusion

In sickle cell disease (SCD), recurrent painful vasoocclusive crisis are likely caused by repeated episodes of hypoxia and reoxygenation. The sickle erythrocyte (SSRBC) adhesion plays an active role in vasoocclusion. However, the effect of prolonged reoxygenation after hypoxic stress on the molecular mechanisms in SSRBCs involved in onset of episodic vasoocclusion remain unclear. Exposure of human SSRBCs to hypoxia followed by 2 h reoxygenation, increased reactive oxygen species (ROS) production. Using specific pharmacological inhibitors, we show that excess ROS production in both reticulocytes and mature SSRBCs is regulated by NADPH oxidases (NOXs), the mitogen-activated protein kinase (ERK1/2), and G-protein coupled-receptor kinase 2 (GRK2). Consequently, SSRBC ROS create an intracellular positive feedback loop with ERK1/2 and GRK2 to mediate SSRBC adhesion to endothelium in vitro, and vasoocclusion in a mouse model of vasoocclusion in vivo. Importantly, reducing ROS levels in SSRBCs with redox-active manganese (Mn) porphyrins, commonly known as mimics of superoxide dismutase (SOD), disrupted the cycle created by ROS by affecting NOX and GRK2 activities and ERK1/2 phosphorylation, thus abrogating RBC-endothelial interactions. Inhibition adhesion assays show that LW (ICAM-4, CD242) blood group glycoprotein and CD44 are the RBC adhesion molecules mediating endothelial binding. Conversely, hypoxia/reoxygenation of normal RBCs failed to activate this feedback loop, and adhesion. These findings provide novel insights into the pathophysiological significance of the deleterious cycle created by NOX-dependent ROS, GRK2 and ERK1/2 within SSRBCs activated by hypoxia/reoxygenation, and involved in SSRBC adhesion and vasoocclusion. Thus, this loop in SSRBCs, which can be disrupted by Mn porphyrins, likely drives the profound SCD vasculopathy, and may point to new therapeutic targets to prevent chronic vasoocclusive events.

Hydroxyurea can be used in children with sickle cell disease and cerebral vasculopathy for the prevention of chronic complications? A meta-analysis.

We conducted a systematic review for evaluating the impact of hydroxyurea and chronic blood transfusion in children with sickle cell disease (SCD). A search was done in four databases from inception to 2017. Trials enrolling pediatric patients with SCD and cerebral vasculopathy with or without previous episode of stroke and that reported outcomes of occurrence of stroke and other events were included. Trained reviewers determined eligibility, risk of bias, and abstracted data. Random-effects meta-analysis was conducted. We found that the primary outcome was the occurrence of stroke. We found two trials that recruited 254 patients. No difference was found for confirmed stroke occurrence (risk difference 0.04 [95% CI: -0.03 to 0.03]) and for new-onset neurological deficit (risk difference 0.11 [95% CI: -0.00 to 0.21]). Transfusions provided a significant lower risk of vaso-occlussive crisis (risk difference 0.10 [95% CI: 0.001 to 0.20]). Finally, transfusions provided a lower risk of having high concentrations of abnormal hemoglobin S (mean difference 37.94 [95% CI: 27.55 to 48.32]). As a conclusion, transfusions plus chelation therapy might be used instead of hydroxyurea in children with SCD. There is a lack of high-quality research in the care of children with SCD, and therefore a call for action is needed.

Degree of anemia, indirect markers of hemolysis, and vascular complications of sickle cell disease in Africa

AbstractThe hyperhemolysis paradigm that describes overlapping “hyperhemolytic-endothelial dysfunction” and “high hemoglobin-hyperviscous” subphenotypes of sickle cell disease (SCD) patients is based on North American studies. We performed a transversal study nested in the CADRE cohort to analyze the association between steady-state hemolysis and vascular complications of SCD among sub-Saharan African patients. In Mali, Cameroon, and Ivory Coast, 2407 SCD patients (1751 SS or sickle β-zero-thalassemia [Sβ0], 495 SC, and 161 sickle β+-thalassemia [Sβ+]), aged 3 years old and over, were included at steady state. Relative hemolytic intensity was estimated from a composite index derived from principal component analysis, which included bilirubin levels or clinical icterus, and lactate dehydrogenase levels. We assessed vascular complications (elevated tricuspid regurgitant jet velocity [TRV], microalbuminuria, leg ulcers, priapism, stroke, and osteonecrosis) by clinical examination, laboratory tests, and echocardiography. After adjustment for age, sex, country, and SCD phenotype, a low hemoglobin level was significantly associated with TRV and microalbuminuria in the whole population and with leg ulcers in SS-Sβ0 adults. A high hemolysis index was associated with microalbuminuria in the whole population and with elevated TRV, microalbuminuria, and leg ulcers in SS-Sβ0 adults, but these associations were no longer significant after adjustment for hemoglobin level. In conclusion, severe anemia at steady state in SCD patients living in West and Central Africa is associated with elevated TRV, microalbuminuria, and leg ulcers, but these vascular complications are not independently associated with indirect markers of increased hemolysis. Other mechanisms leading to anemia, including malnutrition and infectious diseases, may also play a role in the development of SCD vasculopathy.

The kidney in sickle hemoglobinopathies .

With improvements in the care of patients with sickle hemoglobinopathies, sickle cell disease (SCD) has evolved from a disease that was fatal in childhood into one in which most survive past their 5th decade and some into old age. As a result, the renal complications of sickle hemoglobinopathies, which are age dependent, have emerged as a common and serious complication of SCD. Approximately 14-18% of mortality in SCD is attributed to chronic kidney disease (CKD), which develops in 1/3 of individuals with SCD and progresses to end-stage renal disease in 4-18% of them. Importantly, the presence of CKD increases the risk of the other systemic complications of SCD, with the median survival of SCD estimated at 51 years, declining to 29 years in those with CKD. The obstructive vasculopathy of SCD affects the glomerulus, tubules, and medulla of the kidney. Albuminuria and inability to concentrate the urine precede the onset of renal failure, and, along with other tubular dysfunctions, are early warning signs of sickle cell nephropathy (SCN). This is a review of the historical background SCN, the pathophysiology of the renal lesions, their varied clinical and pathologic manifestations, and available treatment options. .

Large-Vessel Vasculopathy in Children With Sickle Cell Disease: A Magnetic Resonance Imaging Study of Infarct Topography and Focal Atrophy

BackgroundLarge-vessel vasculopathy (LVV) increases stroke risk in pediatric sickle cell disease beyond the baseline elevated stroke risk in this vulnerable population. The mechanisms underlying this added risk and its unique impact on the developing brain are not established. MethodsWe analyzed magnetic resonance imaging and angiography scans of 66 children with sickle cell disease and infarcts by infarct density heatmaps and Jacobian determinants, a metric utilized to delineate focal volume change, to investigate if infarct location, volume, frequency, and cerebral atrophy differed among hemispheres with and without LVV. ResultsInfarct density heatmaps demonstrated infarct “hot spots” within the deep white matter internal border zone region in both LVV and non–LVV hemispheres, but with greater infarct density and larger infarct volumes in LVV hemispheres (2.2 mL versus 0.25 mL, P < 0.001). Additional scattered cortical infarcts in the internal carotid artery territory occurred in LVV hemispheres, but were rare in non–LVV hemispheres. Jacobian determinants revealed greater atrophy in gray and white matter of the parietal lobes of LVV compared with non–LVV hemispheres. ConclusionLarge-vessel vasculopathy in sickle cell disease appears to increase ischemic vulnerability in the borderzone region, as demonstrated by the increased frequency and extent of infarction within deep white matter, and increased risk of focal atrophy. Scattered infarctions across the LVV–affected hemispheres suggest additional stroke etiologies of vasculopathy (i.e., thromboembolism) in addition to chronic hypoxia-ischemia.