Little is known about the relationship between quality of life (QOL) and food insecurity (FI) among patients with sickle cell disease (SCD). We hypothesized FI is associated with lower QOL in ...children and young adults with SCD. Overall (N = 99), 22% screened positive for FI. Supplemental Nutrition Assistance Program (SNAP) enrollment was 50 and 71% among people from food secure and FI households, respectively. A higher FI score was correlated with lower overall QOL (r = −0.22, p = .03), specifically lower QOL in worry and communication domains. Interventions for FI beyond SNAP may be important for QOL among people living with SCD.
Summary
Sickle cell anaemia (SCA) is the most frequent genetic haemoglobinopathy, which exhibits a highly variable clinical course characterized by hyper‐coagulable and pro‐inflammatory states, as ...well as endothelial dysfunction. Extracellular microvesicles are released into biological fluids and play a role in modifying the functional phenotype of target cells. We hypothesized that potential differences in plasma‐derived extracellular microvesicles (EV) function and cargo from SCA patients may underlie divergent clinical trajectories. Plasma EV from SCA patients with mild, intermediate and severe clinical disease course were isolated, and primary endothelial cell cultures were exposed. Endothelial cell activation, monocyte adhesion, barrier disruption and exosome cargo (microRNA microarrays) were assessed. EV disrupted the endothelial barrier and induced expression of adhesion molecules and monocyte adhesion in a SCA severity‐dependent manner compared to healthy children. Microarray approaches identified a restricted signature of exosomal microRNAs that readily distinguished severe from mild SCA, as well as from healthy children. The microRNA candidates were further validated using quantitative real time polymerase chain reaction assays, and revealed putative gene targets. Circulating exosomal microRNAs may play important roles in predicting the clinical course of SCA, and in delineation of individually tailored, mechanistically‐based clinical treatment approaches of SCA patients in the near future.
Background
Acute Chest Syndrome (ACS) is one of the leading causes of death among children with Sickle Cell Disease (SCD). Disruption of microvascular integrity is critical to the pathophysiology of ...ACS, but the factors governing its phenotypic variability are incompletely understood. Because circulating exosomes have been implicated in vascular dysfunction in various diseases, we hypothesized that exosomes induce endothelial dysfunction in patients who experience ACS.
Procedure
Cross‐sectional cohort study including 33 outpatients with SCD (without new health‐related complaints or recent transfusions) and a cohort of control patients. Exosomes were isolated from platelet‐free plasma.
Results
ImageStream showed that exosome counts were greatly increased in patients with SCD compared with controls, but there were few differences in the concentrations of exosomes between patients who had experienced ACS (ACS(+)) and those who had not (ACS(−)). Exosomes were added to human microvascular endothelial cells, and the exosomal effects on monolayer integrity was determined using Electric Cell‐substrate Impedance Sensing (ECIS). Exosomes from SCD patients without ACS differed minimally from control patients; however, exosomes from ACS(+) decreased endothelial cell resistance compared to ACS(−), (Relative resistance: ACS(+): 0.981 ± 0.055 vs ACS(−): 1.124 ± 0.042; P = 0.006). Treatment of endothelial cultures with exosomes from ACS(−) patients increased endothelial Nitric Oxide Synthase (eNOS) mRNA expression, while ACS(+)‐derived exosomes were not able to increase eNOS expression above that of controls.
Conclusions
These findings demonstrate that patients with SCD have circulating exosomes that produce differential effects that may contribute to the pathophysiology of ACS and may serve as risk‐related biomarkers.
We previously found that the plasma of patients with sickle cell disease (SCD) contains large numbers of small extracellular vesicles (EVs) and that the EVs disrupt the integrity of endothelial cell ...monolayers (especially if obtained during episodes of acute chest syndrome, ACS). The present study was designed to test the generality of this finding to other complications of SCD, specifically to evaluate the possibility that circulating EVs isolated during a vaso-occlusive crises (VOC) also cause damage to the intercellular connections between endothelial cells. Plasma was obtained from nine pediatric subjects at baseline and during VOC episodes. EVs isolated from these samples were added to cultures of microvascular endothelial cells. Immunofluorescence microscopy was employed to assess monolayer integrity and to localize two intercellular junction proteins (VE-cadherin and connexin43). The EVs isolated during VOC caused significantly greater monolayer disruption than those isolated at baseline. The extent of disruption varied between different episodes of VOC or ACS in the same patient. The VOC EVs disrupted the integrity of both junction proteins at appositional membranes. These results suggest that circulating EVs may be involved in modulating endothelial integrity contributing to the pathogenesis of different complications of SCD.
Intercellular junctions maintain the integrity of the endothelium. We previously found that the adherens and tight junctions between endothelial cells are disrupted by plasma extracellular vesicles ...from patients with sickle cell disease (especially those with Acute Chest Syndrome). In the current study, we evaluated the effects of these vesicles on endothelial gap junctions. The vesicles from sickle cell patients (isolated during episodes of Acute Chest Syndrome) disrupted gap junction structures earlier and more severely than the other classes of intercellular junctions (as detected by immunofluorescence). These vesicles were much more potent than those isolated at baseline from the same subject. The treatment of endothelial cells with these vesicles led to reduced levels of connexin43 mRNA and protein. These vesicles severely reduced intercellular communication (transfer of microinjected Neurobiotin). Our data suggest a hierarchy of progressive disruption of different intercellular connections between endothelial cells by circulating extracellular vesicles that may contribute to the pathophysiology of the endothelial disturbances in sickle cell disease.
Introduction: Acute chest syndrome (ACS) is one of the leading causes of mortality in patients with sickle cell disease (SCD). Despite this, there are no biomarkers that can distinguish patients who ...present with a painful vaso-occlusive episode (VOE) and may rapidly develop ACS. Endothelial damage is a critical component of the pathophysiology of SCD. Small extracellular vesicles (EVs) are endocytic-derived vesicles that package protein and nucleic acid cargo for communication between cells. We and others have demonstrated that EVs have unique properties in patients with SCD. We have shown that EVs from SCD patients differentially affect endothelial integrity in vitro. We have previously performed miRNA sequencing and identified 15 miRNA that were differentially expressed based on a history of ACS. In this study we sought to closely assess how three of these miRNA, miR-let7c-5p, miR-122-5p, and miR-369-5p, differ across the timeline of patients with sickle cell, ie. at baseline, during ACS and during VOE. Methods: The SCD biobank at the University of Chicago Comer Children's Hospital and LaRabida Children's Hospital was queried for patients who had multiple blood samples over many years that included at least one each of a baseline (> 4 weeks from an acute event), a hospitalization for ACS and a hospitalization for a VOE. All subjects had been prospectively enrolled, with informed consent provided by parents or patients aged >18 years and assent obtained from subjects aged 9-18 years. Control samples (n=5) were obtained from patients without SCD who received bloodwork for routine check-ups or for follow-up of iron-deficiency anemia. Equal volumes of control samples were pooled for consistency across experiments. EVs were isolated via Total Exosome Isolation Kit (Thermo Fisher Scientific) per the manufacturer's guidelines. RNA was purified from samples using the miRNeasy Micro Kit (Qiagen). For quantitative PCR, complementary DNA was generated with the TaqMan Advanced miRNA cDNA Synthesis kit (Thermo Fisher Scientific). Specified miRNAs (hsa-miR-122-5p, hsa-miR-99a-5p, hsa-miR-369-5p, hsa-miR-let-7c-5p) were analyzed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and TaqMan Advanced miRNA Assay primers (Thermo Fisher Scientific). Samples were analyzed in triplicate on a 7500 Fast Real-Time PCR machine (Applied Biosystems) and threshold cycle values were normalized to cel-miR-39 controls. Relative changes in expression for ACS and VOE samples were calculated using the respective baseline samples or non-SCD controls as reference values and theΔΔ quantification cycle method. Results: To determine whether exosomal miRNA levels correlated with the disease status of patients, normalized samples from all patients were aggregated and then stratified based on sample type (baseline, VOE, ACS, or ACS post transfusion). While there were no statistically significant differences in exosomal expression of miR-let-7c-5p and miR-122-5p between sample types, there was a promising trend for miR-122-5p. There was a 2.6-fold increase in miR-122-5plevels, relative to baseline, when patients experienced a vaso-occlusive episode. In contrast, miR-122-5p levels decreased 2.3-fold, relative to baseline, when patients were hospitalized with acute chest syndrome. Extravesicular miR-369-5p levels were differentially regulated and correlated with the disease status of patients. When patients were hospitalized with a VOE or ACS, there was a 2.9-fold and 10-fold increase in miR-369-5p levels, respectively, compared to their baseline. In addition, there was a statistically significant difference between VOE and ACS samples, which suggests that miR-369-5p levels can distinguish between VOE and ACS events (Fig. 3C). Interestingly, when ACS samples were obtained post-transfusion (ACS ptx), miR-369-5p levels decreased significantly to values near that of VOE samples. Conclusions: In this study, we report that the miRNA-369-5p levels in small EVs are differentially up-regulated in SCD patients during an ACS event. Furthermore, we show that these levels can distinguish patients presenting with a VOE from those who eventually develop ACS. Our findings demonstrate that it is possible to utilize extracellular vesicles and their contents to predict ACS. Further studies should be done so there is a tool to predict ACS and improve the clinical outcomes for patients with SCD.
OBJECTIVES/GOALS: We have shown that small extracellular vesicles (exosomes) isolated from patients with a history of ACS disrupt the endothelium in vitro. Sequencing of miRNA contents of these ...vesicles suggested that miR let-7c was differentially expressed. The current study was designed to determine the relationship between miR let-7c levels and ACS. METHODS/STUDY POPULATION: We identified 16 subjects from the SCD Lungomics biobank at the University of Chicago Comer and La Rabida Childrens Hospitals who had samples obtained at baseline. Among them, 9 had a history of ACS (ACS(+)) and 7 did not (ACS(-)). For all subjects, we reviewed clinical data relevant to their SCD and laboratory data (including hemoglobin, absolute reticulocyte count, white blood cell count) obtained at the same time as the baseline samples. RNA was isolated from the plasma and miR let7c was quantified using quantitative RT-PCR. RESULTS/ANTICIPATED RESULTS: Subjects were similar clinically, except that those with a history of ACS were more likely to be on hydroxyurea (p<0.05) and to have obstructive sleep apnea (p<0.05). Hematologic laboratory values were similar irrespective of ACS history. The mean miR let-7c level was 2-fold less in subjects with a history of ACS than in those who had never had ACS (p<0.05). A plasma miR let-7c level < 1 (normalized to the control subjects) had a positive predictive value of 0.78 for history of ACS and a sensitivity of 78% and specificity of 71%. Among subjects with a history of ACS, the let7c levels did not correlate with time since the last ACS event. However, among subjects who developed ACS following the baseline samples, higher miR let-7c levels correlated with increased length of time to next ACS event (R=0.8). DISCUSSION/SIGNIFICANCE: Our results in a group of subjects with SCD show that plasma miR let-7c levels are decreased in subjects with a history of ACS. They suggest that miR let-7c may be protective against development of ACS and that measurement of its levels could be a useful biomarker to assess or predict risk for this complication of SCD.
Introduction: Sickle-cell disease (SCD) is an inherited hemoglobinopathy frequently complicated by vaso-occlusive episodes (VOE) and acute chest syndrome (ACS). Likely due to chronic hemolysis and ...inflammation, roughly 10% of patients with SCD develop pulmonary hypertension (PH) (Gordeuk et al., 2016). Early signs of PH in patients with SCD often present as elevated pulmonary pressures and right ventricular (RV) dysfunction, which could be exacerbated by VOE or ACS (Dessap et al., 2007). Multiple studies have shown that ACS and PH are individually established causes of increased mortality in patients with SCD, but less is known about the relationship between concurrent ACS and PH and the effects on clinical outcomes. The diagnostic criteria for PH were recently updated, and the impact of those changes on outcomes in SCD remain unknown. We sought to evaluate clinical outcomes in patients with SCD who met PH diagnostic criteria when hospitalized for ACS. Methods: We retrospectively collected clinical information from the electronic medical record (EMR) for all ACS admissions from 2017-2021 at the University of Chicago. Patients were assessed if they met PH diagnostic criteria based on available right heart catheterization (RHC) and transthoracic echocardiography (TTE) reports prior to the ACS admission date. PH was defined as having a mean pulmonary artery pressure >20 mmHg on RHC, tricuspid regurgitation velocity (TRV) >3.4 m/s, or TRV 2.9-3.4 m/s with two additional concerning TTE findings. ACS was defined as a new pulmonary opacity on imaging and at least one diagnostic symptom such as fever, chest pain, or hypoxemia. Depending on frequencies, chi-square or Fisher's exact tests were used to assess associations between PH and clinical outcomes: intensive care unit (ICU) transfer within 8 days of ACS diagnosis, 28-day readmission, and 28-day mortality. Two-sample t-test was used to assess the association between length of stay (LOS) and PH. Results: There were 231 ACS admissions from 2017-21, but 1 admission was excluded from analysis due to lack of past medical history within the EMR. In total, 10 out of 230 ACS admissions (4%) met PH diagnostic criteria prior to admission, comprised of 8 out of 127 patients (6%). There were no significant associations between PH and clinical outcomes except for LOS (14.5 days for PH vs 9.1 days for no PH, p=0.04). Conclusions: If a patient with SCD and PH was admitted for ACS, their LOS was 5 days longer than a patient without PH. An increased LOS may be due to a higher degree of complications such as elevated pain scores, prolonged oxygen requirements, or greater hemodynamic compromise. Future directions of the study will assess for causes of an increased LOS in the setting of PH. Only 6% of this cohort of patients with ACS admissions had PH, and this was likely under identified due to lack of adequate work-up (RHC or TTE). Diagnosis of PH prior to ACS admission may help with future prognostication and clinical decision-making regarding early intervention. The study highlights the increased healthcare requirements accompanying PH and a continued need to study PH, specifically in patients with SCD who may be at high risk for adverse clinical outcomes during an ACS episode.
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