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Fig. A1. From the top to the bottom: staining for AV, for CD61 and for AV & CD61 (PMP). Gating area using the methodology by Shet et al. (16) (red, right) and this by Biró et al. (23) (black, left). No clear demarcation is seen between MP and noise.
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Introduction
Circulating microparticles (MP) have been described in sickle cell anaemia (SCA); however, their interaction with endothelial markers remains unclear. We investigated the relationship between MP, protein C (PC), free protein S (PS), nitric oxide (NO), endothelin-1 (ET-1) and adrenomedullin (ADM) in a large cohort of paediatric patients...
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Background:
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Citations
... [9][10][11][12][13] During episodes of acute chest syndrome, HbSC disease is associated with higher levels of nitric oxide compared with patients with HbSS. 14 Nitric oxide reduces cell adhesion and inflammation in the endothelium which ameliorates nitric oxide pathology in sickle cell mice models. 15 These findings have resulted in this subtype of SCD often being labelled as 'less severe'. ...
Sickle cell disease (SCD) includes a group of heterogenous disorders that result in significant morbidities. HbSS is the most common type of SCD and HbSC is the second most common type of SCD. The prevalence of HbSC disease in the United States and United Kingdom is ~1 in 7174 births and 1 in 6174 births respectively. Despite its frequency, however, HbSC disease has been insufficiently studied and was historically categorized as a more ‘mild’ form of SCD. We conducted this study of HbSC disease as part of the NHLBI funded Sickle Cell Disease Implementation Consortium (SCDIC). The SCDIC registry included 2282 individuals with SCD, ages 15–45 years of whom 502 (22%) had HbSC disease. Compared with people with sickle cell anaemia (SCA), the study found that people with HbSC disease had a higher frequency of splenomegaly (n (%) = 169 (33.7) vs. 392 (22.1)) and retinopathy (n (%) = 116 (23.1) vs. 189 (10.6)). A Many people with HbSC also had avascular necrosis (n (%) = 112 (22.3)), pulmonary embolism (n (%) = 43 (8.6)) and acute chest syndrome (n (%) = 228 (45.4)) demonstrating significant disease severity. HbSC disease is more clinically severe than was previously recognized and deserves additional evaluation and targeted treatments.
... Hemoglobin S polymerizes in hypoxic conditions, which are also associated with the upregulation of Hypoxia-Inducible-Factor-α genes [8], such as Endothelin-1 [9]. As well as affecting their oxygen-carrying capacity and elasticity, recurrent sickling of RBCs due to polymerized hemoglobin may damage them, leading to hemolysis [10] and the release of free hemoglobin, heme, and extracellular vesicles (microparticles). ...
Executive function and processing speed difficulties are observed in children living with sickle cell anemia (SCA). The influence of processing speed on executive function is not well understood. We recruited 59 children living with SCA and 24 matched controls aged 8–18 years between 2010 and 2016 from clinics in the UK. Children completed tests in processing speed and cognitive flexibility, subdomains of executive function. MRI scans were conducted within one year of testing; oxygen saturation was obtained on the day of testing. Hemoglobin levels were obtained from medical records. Caregivers completed the executive function questionnaire. Hierarchical linear regressions found that hemoglobin, oxygen saturation, age, infarct status, and processing speed were not independent predictors for any model. However, for all cognitive flexibility tests, there was a significant interaction between infarct status and processing speed; children without silent cerebral infarction (SCI) with faster processing speed had better cognitive flexibility. Our findings indicate that, when interpreting executive function difficulties, it is important to account for the relationship between SCI status and processing speed. More research is needed to elucidate the mechanisms, but clinically, including executive function testing as part of clinic visits by embedding psychologists within the healthcare team would appear to be a critical step.
... 10,11 Ischemia-reperfusion injury and hemolysis contribute to an inflammatory milieu 12,13 and endothelial activation. 14 Hypercoagulability results from complex interactions between the coagulation system and the endothelium, including von Willebrand factor release, nitric oxide depletion, upregulation of vascular adhesion molecules like P-selectin, 15 interactions with microparticles, and alterations in the activity of intrinsic anticoagulants like protein C and protein S. [16][17][18][19] Chronic vasculitis and hypercoagulability in the carotid, vertebral, and cerebral arteries lead to acute infarctive and ischemic stroke, as well as chronic complications like carotid stenosis and moyamoya syndrome. 20 Stroke and other complications of cerebrovascular disease represent significant sources of morbidity and mortality for people with SCD. ...
The importance of protecting brain function for people with sickle cell disease (SCD) cannot be overstated. SCD is associated with multiple cerebrovascular complications that threaten neurocognitive function and life. Without screening and preventive management, 11% of children at 24% of adults with SCD have ischemic or hemorrhagic strokes. Stroke screening in children with SCD is well-established using transcranial Doppler ultrasound (TCD). TCD velocities above 200 cm/s significantly increase the risk of stroke, which can be prevented using chronic red blood cell (RBC) transfusion. RBC transfusion is also the cornerstone of acute stroke management and secondary stroke prevention. Chronic transfusion requires long-term management of complications like iron overload. Hydroxyurea can replace chronic transfusions for primary stroke prevention in a select group of patients or in populations where chronic transfusions are not feasible. Silent cerebral infarction (SCI) is even more common than stroke, affecting 39% of children and more than 50% of adults with SCD; management of SCI is individualized and includes careful neurocognitive evaluation. Hematopoietic stem cell transplant prevents cerebrovascular complications, despite the short- and long-term risks. Newer disease-modifying agents like voxelotor and crizanlizumab, as well as gene therapy, may treat cerebrovascular complications, but these approaches are investigational.
... The hallmark complication associated with SCD is the vaso-occlusive painful crisis, leading to chronic hemolytic anemia [18]. However, the pathophysiology of vasoocclusive complications in SCD is only partially understood [19]. Several studies reported the activation of coagulation as a prominent feature of SCD [20] and proposed the involvement of circulating cell-derived MVs in pathophysiological processes of SCD including coagulation, inflammation, and abnormal cellular adherence [15]. ...
... Our findings are in consistency with a recent study by Kasar et al. [18] and Tantawy et al. [24] who reported a higher number of MVs in crisis versus steady state and control. However, our results were incongruent with findings in two other studies [19,25], with a relevant discrepancy on absolute MVs numbers in SCD crises and a significant increase in crises in our study. Also, in another study, in contrast to our finding, Nouboussie et al. [25] recently depicted higher number of MVs in steady state. ...
Introduction
Thrombotic complication is one of the features of sickle cell disease (SCD), characterized by appearance of phosphatidylserine on the outer membrane of sickle-shaped red blood cells and most abundantly on membrane protrusions called microvesicles (MVs). However, the exact mechanism by which MVs may enhance coagulant activity in SCD patients has not been fully addressed. The aim of this study was to further investigate the procoagulant activity of circulating MVs in sickle cell crises.
Materials and Methods
Subjects included in this cross-sectional study were 47 patients with SCD and 25 normal subjects with written informed consent obtained from all the participants. MV analysis was conducted by using CD61, CD235α, and Annexin-V monoclonal antibodies. The coagulant activity of MVs was determined by an ELISA-based procoagulant activity assay.
Results
The majority of MVs were originated from platelets (CD61+) and erythrocytes (CD235+). These MVs demonstrated significantly enhanced levels during the painful crisis when compared with the steady-state period (p < 0.001) and controls (p < 0.001). Also, the procoagulant activity of MVs was significantly higher in crisis compared to those of steady state (p < 0.001) and positively correlated with the number of Annexin-V+ MVs (p < 0.001). Significant correlations were found between erythrocyte-derived MVs with hemolysis marker (r = 0.51, p < 0.001) and the hemoglobin level (r = −0.63, p < 0.001).
Conclusion
The numbers of platelet- and erythrocyte-derived MVs are related to painful crisis, and their quantification in SCD may be helpful for identifying cases at increased risk of thrombotic complications.
... In response, an inflammatory reaction is triggered, which may lead to the narrowing of vessels, including intracranial and extracranial arteries ( Figure 2). This increases the risk of vascular nitric oxide release, endothelial activation, and adherence of red and white cells, platelets and microparticles, especially as contemporaneous reduction of protein S and C shifts the blood towards a more prothrombotic condition [15][16][17], increasing the risk of arterial, as well as venous, thrombosis [18]. ...
Sickle cell disease (SCD) is the most common inherited single-gene disease. Complications include chronic anaemia, reduced oxygen-carrying capability, and cerebral vasculopathy, resulting in silent cerebral infarction, stroke, and cognitive dysfunction with impairments in measures of executive function, attention, reasoning, language, memory, and IQ. This systematic review aims to investigate the association between neuroimaging findings and cognition in children with SCD. Searches of PubMed and Embase were conducted in March 2022. Studies were included if participants were <18 years, if original data were published in English between 1960 and 2022, if any genotype of SCD was included, and if the relationship between cognition and neuroimaging was examined. Exclusion criteria included case studies, editorials, and reviews. Quality was assessed using the Critical Appraisal Skills Programme Case Control Checklist. A total of 303 articles were retrieved; 33 met the eligibility criteria. The presence of overt or silent strokes, elevated blood flow velocities, abnormal functional connectivity, and decreased fMRI activation were associated with neuropsychological deficits in children with SCD when compared to controls. There is a critical need to address the disease manifestations of SCD early, as damage appears to begin at a young age. Most studies were cross-sectional, restricting the interpretation of the directionality of relationships. Future research employing longitudinal neuroimaging and neuropsychological assessments could improve our understanding of the cumulative consequences of SCD on the developing brain.
... This finding validates and confirms previous studies that have already shown that RBCEV (also called microparticles) levels are higher in SCD than in healthy controls (HbAA) and that RBCEV levels change during crises. 2,3 Moreover, the presence of other extracellular vesicles from endothelial origin further confirms that endothelial activation is present in SCD. The presence of sickle cell RBCEVs have also been reported to cause direct micro-occlusion in the kidney in a mouse model. ...
... This is also in keeping with similar findings of reduced naturally occurring anticoagulant (Protein C and Protein S) and vaso-occlusion in SCD. 2,10 In our opinion, this study has a strong scientific value, since it has been conducted in both in vitro and in vivo studies, in a mouse model with SCD. It will now be important to validate these findings in human trials, possibly in a large cohort of patients with SCD. ...
... Sickle RBCs (SS RBCs) are prone to extra-and intravascular haemolysis. 1 During haemolysis, SS RBCs release damage-associated molecular patterns (DAMPs) including haemoglobin/haeme and extracellular vesicles (EVs). [2][3][4][5][6] The DAMPs cumulatively promote a pro-inflammatory milieu with decreased levels of natural occurring anticoagulant proteins (i.e., protein C and protein S), nitric oxide (NO) bioavailability, and ADAMTS13, and with increased levels of von Willebrand factor (VWF). 7 The decreased of levels of natural anticoagulant proteins may promote endothelial damage, 8,9 the decreased NO bioavailability decreases its effect in impairing bloodendothelial cell (EC) adhesion, 10 and the increased levels of VWF favours RBC adhesion on endothelium. 11 Consequently, these DAMP-associated endothelial damages intermittent vasoocclusion of the microvasculature in SCD. ...
... To demonstrate that the observed RBC adhesion was induced by REV-mediated HPMEC activation, we investigated the impact of the haeme-binding protein haemopexin on REV-mediated RBC adhesion. Pre-incubating REVs with haemopexin reduced the HPMEC activation indicated by reduced number of adherent SS RBCs ( Figure 2G, mean [SD] 157 [42] vs. 14 [8], p < 0.05). ...
... Insets are closer view of RBCs adhered to HPMECs. (G) Interaction between SS RBC and SS REV activated HPMECs is significantly stronger than the other test groups, and this interaction is reduced by haemopexin and ADAMTS13 (mean [SD]: SS REV-SS RBC = 157 [42], AA REV-SS RBC = 16 [12], AA REV-SS RBC = 19 [12], AA REV-AA RBC 5 [4], SS REV-SS RBC with haemopexin = 14 [8], SS REV-SS RBC with ADAMTS13 = 19 [10], and haeme-SS RBC = 140 [49], p < 0.05 for all groups except for haeme-SS RBC, n = five in each group, two-group t-test). HPMECs, human pulmonary microvascular endothelial cells; RBC, red blood cell; REVs, RBC-derived extracellular vesicles; VWF, von Willebrand factor. ...
Endothelial activation and sickle red blood cell (RBC) adhesion are central to the pathogenesis of sickle cell disease (SCD). Quantitatively, RBC‐derived extracellular vesicles (REVs) are more abundant from SS RBCs compared with healthy RBCs (AA RBCs). Sickle RBC‐derived REVs (SS REVs) are known to promote endothelial cell (EC) activation through cell signalling and transcriptional regulation at longer terms. However, the SS REV‐mediated short‐term non‐transcriptional response of EC is unclear. Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 h. Compared with AA REVs, SS REVs promoted human pulmonary microvascular ECs (HPMEC) activation indicated by increased von Willebrand factor (VWF) expression. Under microfluidic conditions, we found abnormal SS RBC adhesion to HPMECs exposed to SS REVs. This enhanced SS RBC adhesion was reduced by haeme binding protein haemopexin or VWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs. Consistent with these observations, haemin‐ or SS REV‐induced microvascular stasis in SS mice with implanted dorsal skin‐fold chambers that was inhibited by ADAMTS13. The adhesion induced by SS REVs was variable and was higher with SS RBCs from patients with increased markers of haemolysis (lactate dehydrogenase and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Our results emphasise the critical contribution made by REVs to the pathophysiology of SCD by triggering acute microvascular EC activation and abnormal RBC adhesion. These findings may help to better understand acute pathophysiological mechanism of SCD and thereby the development of new treatment strategies using VWF as a potential target.
... 3 Free hemoglobin, released during hemolysis, scavenges nitric oxide (NO) which leads to vaso-constriction, exacerbating vaso-occlusion. 4,5 Universal newborn screening (NBS) and preventative treatments have effectively eliminated early childhood mortality in the US, although people with SCD continue to experience increased morbidity and early mortality. 6,7 Prior to 2017 there were limited treatment options to prevent the complications of SCD; hydroxyurea (HU) was the only FDA-approved treatment. ...
... [13][14][15][16] HU may act as a NO donor which improves blood flow. 4,5 Recent results suggest improved cognition after 1 year of HU use in a pediatric cohort 17 and a neuroprotective effect in persons with SCD 5 years and older. 18 Despite well-documented benefits, HU is underutilized in the United States. ...
Introduction
Hydroxyurea reduces the incidence of vaso-occlusive episodes, stroke, and respiratory, cardiac, and renal damage in sickle cell disease by increasing fetal hemoglobin. However, because suboptimal adherence to hydroxyurea limits its effectiveness, understanding patient-specific barriers to hydroxyurea adherence could help improve adherence and health outcomes in patients with sickle cell disease. The aim of this single-site, prospective, IRB-approved study was to validate a 24-item patient- and caregiver-reported hydroxyurea treatment adherence questionnaire, the Hydroxyurea Evaluation of Adherence for Life (HEAL) scale.
Methods
A sample of 24 adults with sickle cell disease and 16 caregivers of children with sickle cell disease completed the HEAL scale, and a subset of the original sample provided a second HEAL scale for test-retest reliability. HEAL scale results were validated against global adherence ratings from participants and health-care providers, records of access to pill bottles, and laboratory values for fetal hemoglobin and absolute neutrophil count.
Results and Discussion
Results demonstrated excellent internal consistency for the HEAL Total score and eight (3-item) subscale scores (Dose, Remember, Plan, Cost, Understand, Effectiveness, Laboratory, and Pharmacy), as well as strong test-retest reliability for all HEAL scores except the Cost subscale. HEAL Total scores correlated significantly with validity measures, including global adherence ratings and lab values. The HEAL scale offers significant clinical potential for understanding adherence in individual sickle cell disease patients and significant research potential for characterizing adherence in persons with sickle cell disease who are treated with hydroxyurea.
... In SCD, the concentration of the two most commonly identified MPs subtypes, RBC-, and platelet-MPs is increased, compared to healthy controls [128,129] (Table 2). HU treatment impact on MPs concentration is controversial, since several reports showed decreases [128][129][130], unchanged [14,131], or increased [132,133] levels. These conflicting results could be accounted for by the large interindividual variation in MPs concentration in SCD. ...
... A positive history of osteonecrosis of the femoral head [135], leg ulcers [136], acute chest syndrome, and pulmonary hypertension [129] has been associated with elevated concentration of MPs from various cell types. During VOC, the concentration of PLT-and RBC-MPs was also reported to be increased in crosssectional settings [132,137,138], and in longitudinal studies including 17 SCD patients [139] or 32 SCA patients [140]. Our group reported that SCA patients with frequent VOCs had increased levels of PLT-MPs, compared to SCA patients with rare crises [141]. ...
Sickle cell disease (SCD) is the commonest hemoglobinopathy worldwide. It is characterized by an impairment of shear stress-mediated vasodilation, a pro-coagulant and a pro-adhesive state orchestrated among others by the depletion of the vasodilator nitric oxide, by the increased phosphatidylserine exposure and tissue factor expression and by the increased interactions of erythrocytes with endothelial cells that mediate the overexpression of adhesion molecules such as VCAM-1, respectively. Extracellular vesicles (EVs) have been shown to be novel actors involved in SCD pathophysiological processes. Medium-sized EVs, also called microparticles, which exhibit increased plasma levels in this pathology, were shown to induce the activation of endothelial cells, thereby increasing neutrophil adhesion, a key process potentially leading to the main complication associated with SCD, vaso-occlusive crises (VOCs). Small-sized EVs, also named exosomes, which have also been reported to be overrepresented in SCD, were shown to potentiate interactions between erythrocytes and platelets, and to trigger endothelial monolayer disruption, two processes also known to favor the occurrence of VOCs. In this review we provide an overview of the current knowledge about EVs concentration and role in SCD.
... Solovey et al. [19] indicated that sickle cell anemia involves an abnormal and enhanced antiapoptotic tendency for endothelial cells and VEGF was responsible for this behavior. Bottomley et al. [20] reported that overexpression of VEGF prompts high expression levels of intercellular adhesion molecule-1 (ICAM-1) and high expression levels of VEGF and that was measured in the plasma of SCD patients. Several studies have reported that an abnormal adherent of RBCs to endothelial cells and their adhesiveness correlates with clinical severity in SCD patients [21]. ...
Recent studies have indicated that microRNA and VEGF are considered to be genetic modifiers and are associated with elevated levels of fetal haemoglobin HbF, and thus they reduce the clinical impact of sickle haemoglobin (HbS) patients. This cross-sectional study was performed on clinical confirmed subjects of SCD cases. miR-423-rs6505162 C>T and VEGF-2578 C>A genotyping was conducted by ARMS-PCR in SCD and healthy controls. A strong clinical significance was reported while comparing the association of miR-423 C>T genotypes between SCD patients and controls (p = 0.031). The microRNA-423 AA genotype was associated with an increased severity of SCD in codominant model with odd ratio (OR = 2.36, 95% CI, (1.15–4.84), p = 0.018) and similarly a significant association was observed in recessive inheritance model for microRNA-423 AA vs (CC+CA) genotypes (OR = 2.19, 95% CI, (1.32–3.62), p < 0.002). The A allele was associated with SCD severity (OR = 1.57, 95% CI, (1.13–2.19), p < 0.007). The distribution of VEGF-2578 C>A genotypes between SCD patients and healthy controls was significant (p < 0.013). Our results indicated that in the codominant model, the VEGF-2578-CA genotype was strongly associated with increased SCD severity with OR = 2.56, 95% CI, (1.36–4.82), p < 0.003. The higher expression of HbA1 (65.9%), HbA2 (4.40%), was reported in SCD patients carrying miR-423-AA genotype than miR-423 CA genotype in SCD patients carrying miR-423 CA genotype HbA1 (59.98%), HbA2 (3.74%) whereas SCD patients carrying miR-423 CA genotype has higher expression of HbF (0.98%) and HbS (38.1%) than in the patients carrying AA genotype HbF (0.60%), HbS (36.1%). ARMS-PCR has been proven to be rapid, inexpensive and is highly applicable to gene mutation screening in laboratories and clinical practices. This research highlights the significance of elucidating genetic determinants that play roles in the amelioration of the HbF levels that is used as an indicator of severity of clinical complications of the monogenic disease. Further well-designed studies with larger sample sizes are necessary to confirm our findings.
