Risk Of Stroke In Sickle Cell Disease Research Articles

Abstract TMP7: Disruption of Blood Brain Barrier is Associated With Silent Cerebral Infarcts in Patients With Sickle Cell Disease

Background: Microvascular thromboinflammation is a major pathomechanism underlying vaso-occlusive crisis in patients with sickle cell disease (SCD), yet its role underlying the elevated stroke risk in SCD is unclear. Tissue-based blood brain barrier (BBB) imaging, as a metric of cerebral thromboinflammation, has not been well studied in SCD. We hypothesized that BBB permeability would be increased in patients with SCD and associated with ischemic burden. Methods: Ten young adults with SCD and 17 controls underwent dynamic contrast-enhanced and multimodal MRI to quantify BBB gadolinium leakage rates (Ktrans), white matter microstructural disruption (increased mean diffusivity, MD), and volume of silent cerebral infarcts (SCI). Infarct voxels were removed to avoid the confounding effects of measuring Ktrans in previously infarcted tissue. Whole brain mean Ktrans measures were compared between SCD vs. controls. Bivariate Spearman’s rank correlations measured relationships between Ktrans and SCI volume / MD. Results: BBB permeability, measured by whole brain Ktrans, was elevated in SCD vs. controls (Fig A). Compared to controls, Ktrans was elevated in SCD with infarcts (B), while SCD without infarcts did not differ from controls. In participants with infarcts, Ktrans, measured within non-infarcted tissue, positively correlated with SCI volume (Rho=0.6, p =0.015, C). Furthermore, whole brain Ktrans, positively correlated with MD in normal appearing white matter (Rho=0.5, p =0.01). Conclusion: Our preliminary data suggest BBB permeability is elevated and associated with stroke and microstructural injury in young adults with SCD. We are enrolling more participants. Children with SCD may also have BBB injury but will require a novel non-contrasted MR approach to assess. BBB permeability may provide new insight into the increased stroke risk in SCD. Targeting thromboinflammation and preserving BBB integrity may lead to novel neuroprotective pathways.

A Genetic Variant in GOLGB1 Affects Golgi Function and Stroke Risk in Patients with Sickle Cell Disease

Background: Five to ten percent of patients with sickle cell disease (SCD) have a high risk of suffering an ischemic stroke before they are 10 years of age. The clinical consequences of stroke are severe yet our current mechanistic understanding of the causes of stroke in SCD is limited. There is strong evidence that there is genomic contribution to the risk of stroke, other than just the SCD gene mutation. By whole exome sequencing, we have shown that a specific coding variant in the GOLGB1 gene, Y1212C, is significantly associated with decreased risk of stroke. GOLGB1 encodes a conserved Golgi membrane protein that is essential for maintaining Golgi structure and associated protein transport. To elucidate how the GOLGB1 Y1212C variant is protective against stroke risk in SCD, we performed morphological analysis of Golgi in cultured primary macrophages and whole proteome analyses of peripheral immune cells isolated from SCD patients.Methods: For primary macrophage preparations, we collected whole blood from non-SCD controls (n=11) and pediatric SCD patients (n=12). Monocytic CD14 positive cells were isolated and cultured for nine days with granulocyte monocyte stimulating colony factor, fixed and sequentially stained with antibodies against GOLGB1, TGN-46 and GM130. DAPI was used as a nuclear stain. High resolution images were acquired on Zeiss LSM 780 laser scanning confocal microscope with a 63X oil objective. Golgi morphological analysis was performed using a MATLAB script for cell segmentation, estimation of individual cell area, quantification of Golgi fragments per cell, and Golgi volume and organelle spread index. For the proteomic analysis, we isolated peripheral blood mononuclear cells from 7 patients with SCD. These cells were immediately flash-frozen and stored at -80oC until proteomic processing. We used a label-free protocol using dual-pH reverse phase fractionation and peptide sequencing of digested samples on a Thermo Scientific Orbitrap mass spectrometer. Protein abundances in each sample were estimated by using the intensity-based absolute quantification algorithm and total protein normalization was done with a fraction of total score (iFOT). Data were evaluated using GraphPad Prism and statistical analyses for differences between SCD and non-SCD samples or between samples with and without the GOLGB1 variant were performed with unpaired Student's t-test or Kruskal-Wallis test; analyses for multiple group comparisons were performed with the one-way ANOVA method. Statistical significance was set at P < 0.05.Results: In cultured primary macrophages, SCD individuals with the GOLGB1 Y1212C variant had altered Golgi morphologies compared to individuals with wild-type GOLGB1. Overall, macrophages derived from patients with SCD (n=12) were significantly larger (average cell size 11,728 vs. 7,598; p<0.001) yet had smaller Golgi (average Golgi volume 69.6µm vs. 75.4µm, p<0.01) and with more detected Golgi fragments (12.3 vs. 10.2 per cell, p<0.01) compared to non-SCD controls (n=11). For SCD patients with the GOLGB1 variant (n=5 heterozygote and 1 homozygote), their Golgi apparatus were more compact (average spread index 0.087 vs. 0.079; p<0.05) and had less Golgi fragments per cell (average 10.9 vs. 13.9; p<0.05) compared to SCD patients wild-type for GOLGB1 (n=6). From the proteomics analysis of peripheral blood immune cells, we identified 73 proteins that were differentially expressed (p<0.05) between SCD patients with (n=3) and without (n=4) the GOLGB1 variant. Of these proteins, 64 (90%) had lower levels in patients with the GOLGB1 Y1212C variant. Pathway analysis of the identified proteins showed that there strong clustering of proteins involved in platelet activation, regulation of actin cytoskeleton, and COPI-independent Golgi-to-ER retrograde trafficking (Table 1).Conclusions: Our study has shown that a coding variant in GOLGB1, identified as protective against risk of stroke in patients with SCD, has significant effects on Golgi function in SCD samples. We observed that having the GOLGB1 Y1212C variant resulted in more compact and less fragmented Golgi apparatus. Proteomic analysis showed that SCD patients with the GOLGB1 variant also had significantly lower levels of proteins involved in platelet activation and Golgi trafficking. Our findings suggest a novel role for the Golgi apparatus in controlling protein flux that modulates risk of stroke in SCD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mast Cells Contribute to Brain Microvascular Permeability in Sickle Cell Disease

Mast cells are in close proximity to the vasculature and cause endothelial activation, plasma extravasation, and vascular dysfunction (Gupta & Harvima, Immunol Rev 2018). Vascular dysfunction in sickle cell disease (SCD) is accompanied by increased expression of P-selectin. Treatment with Crizanlizumab, an antibody against P-selectin, led to significantly less sickle cell-related pain crises (Ataga et al., NEJM 2017) highlighting the role of endothelial P-selectin in vasoocclusive crises (VOC). Earlier studies demonstrated that mast cell activation with morphine or ischemia/reperfusion stimulates endothelial E- and/or P-selectin expression. However, it is unknown how mast cells stimulate endothelial selectin expression in SCD. Endothelial dysfunction contributes significantly to the pathobiology of SCD including VOC and may play a critical role in increased blood-brain barrier (BBB) permeability, which may contribute to stroke, another major comorbidity of SCD. One of the known triggers of endothelial dysfunction, inflammation and oxidative stress is endoplasmic reticulum (ER) stress. We hypothesize that in a sickle microenvironment, mediators derived from activated mast cells stimulate endothelial P-selectin expression via ER stress leading to increased BBB permeability. We examined the ability of mast cells to stimulate P-selectin expression and BBB permeability via ER stress in a sickle microenvironment.We isolated MCs from HbAA-BERK and HbSS-BERK, control and sickle mice, respectively; incubated them in vitro and collected mast cell conditioned media (MCCM) from HbAA MCs and HbSS MCs. Normal mouse brain microvascular endothelial cells (mBMECs) were treated with unconditioned MCCM, HbAA MCCM, or HbSS MCCM to examine the effect of mast cell activation on endothelium. We observed increased mast cell activity in HbSS mice evinced by significantlyhigher plasma and skin histamine levels, compared to HbAA mice (p< 0.02 for both). Mast cells from HbSS mouse skin showed significantly increased expression of histamine compared to HbAA skin mast cells (p< 0.04). mBMECs incubated with HbAA and HBSS MCCM exhibited about 3- and 6-fold fold increases in P-selectin expression, compared to unconditioned culture medium, respectively (p< 0.0001 for both). Therefore, mast cells in culture release substances that stimulate P-selectin expression which is further increased by mast cells from sickle (HbSS) microenvironment. Preincubation of mBMEC with 5 microM salubrinal, an inhibitor of dephosphorylation of elongation initiation factor-a, which reduces ER stress, significantly inhibited HbSS MCCM-induced P-selectin expression on mBMEC (p< 0.0001) to the level induced by HbAA-MCCM. In contrast, salubrinal did not inhibit HbAA-MCCM-induced P-selectin expression on mBMEC, suggesting that in a sickle microenvironment mast cells contribute to P-selectin expression via ER stress.We next examined mast cell activity on endothelial permeability in vitro on mBMEC monolayers and in vivo in the brain of HbSS mice. mBMECs incubated with HbSS MCCM showed a significant increase in Evans blue leakage compared to unconditioned or HbAA MCCM (p<0.0001 for both), which was inhibited by preincubation of mBMEC with 5 microM salubrinal prior to incubation with HbSS MCCM (p< 0.0001). In vivo female HbSS mice showed a significantly increased leakage in the brain of FITC-dextran injected through tail vein compared to HbAA mice (p< 0.01). HbSS mice treated with 1 mg/kg salubrinal demonstrated inhibition of FITC-dextran leakage in the brain compared to vehicle (p< 0.05). Thus, ER stress contributes to increased BBB permeability in HbSS mice. We observed activated degranulating mast cells in the brain parenchyma of HbSS mice. In HbAA mice, quiescent mast cells were confined to the meninges of the brain but not seen in the parenchyma. Together, these data suggest that mast cell activation contributes to BBB permeability in a sickle microenvironment via ER stress-mediated P-selectin expression. In turn, this mast cell-initiated activity in the brain may underlie the pathobiology of stroke in SCD. Inhibitors of mast cells and P-selectin have been tested clinically leading to reduced VOC in SCD without known adverse events. Therefore, mast cell activation-induced P-selectin via ER stress may serve as a treatable target for reducing the risk of stroke in SCD. DisclosuresGupta:Novartis: Honoraria; Tau tona: Consultancy.

Abstract TMP57: Cerebral Oxygen Metabolism as a Biomarker to Stratify Stroke Risk in Young Adults with Sickle Cell Disease

Background: While imaging biomarkers guide stroke prevention strategies in children with sickle cell (SC) disease, none have been adequately studied in adults. High oxygen extraction (OEF) predicts stroke in non-SC adults with carotid occlusion, while low oxygen metabolism (CMRO 2 ) predicts tissue at imminent risk in acute ischemic stroke. We hypothesized that metrics of cerebral metabolism: (1) differ between SC adults with and without stroke and (2) correlate with infarct burden. Methods: A prospective MRI study enrolled 37 adults (28 ± 8 yr) from SC clinic into 4 groups: (1) 9 age/race matched healthy controls, (2) 6 SC adults without infarcts, (3) 15 SC adults with infarcts (infarct volume 7.4 ± 17.5 ml), and (4) 7 SC adults on chronic transfusions (Tx) (infarct volume 3.6 ± 6.6 ml). Arterial spin labelling and asymmetric spin echo measured voxel-wise cerebral blood flow (CBF) and OEF. CMRO 2 = CBF x OEF x blood oxygen content. Infarcts were delineated on FLAIR. OEF, CBF, and CMRO 2 (excluding infarcted tissue) were compared: between groups 1-3 (Kruskal-Wallis) and in group 4 between pre- and post-tx scans (Signed Rank). An ROI defined by high OEF within the deep white matter (a region at high stroke risk in SC) was applied to group 3. OEF, CBF, and CMRO 2 within the ROI were correlated with hemispheric infarct volume (IV) (Spearman’s ρ ). Results: Whole brain OEF showed a stepwise increase from controls, to SC adults without stroke, to SC adults with stroke (P&lt;.001). SC adults on chronic Tx had intermediate OEF, with lowering of OEF post-Tx (Fig A). CBF and CMRO 2 were similar for SC adults with and without stroke (Fig B, C). High OEF and low CBF/CMRO 2 in the ROI correlated with hemispheric infarct burden: IV vs. OEF ( ρ =.40, P=.043); IV vs. CBF ( ρ =-.61, P=.002); and IV vs. CMRO 2 ( ρ =-.50, P=.016). Conclusion: Global OEF holds promise to stratify stroke risk in SC disease. Regional metrics of cerebral oxygen metabolism may indicate tissue-specific metabolic stress at imminent risk of infarction.

First Report on Elevated Transcranial Doppler Ultrasound Velocities As First Stroke Risk and Their Association with Hemoglobin F Expression in Saudi Arabian Pediatric Sickle Cell Patients

Introduction: Sickle cell disease (SCD) is one of the most common inherited hemoglobinopathies in Middle East and specifically Saudi Arabia which leads to vaso-occlusive, hematological and infectious crises, including stroke as one of the common complications (1,2). Primary prevention of the first stroke is carried out by risk assessment using transcranial Doppler (TCD) ultrasound and providing blood transfusions to SCD patients with higher than 199 cm/sec TCD velocities (3). Furthermore, althoughhigher HbF expression is also associated with less severity of the SCD, no studies are available about association of HbF with TCD flow velocities and hence risk of stroke in SCD (4). This study has been carried out to identify SCD patients at high risk of stroke based on TCD results, and to determine the association between Hb-F levels and blood velocity using TCD results.Patients and Methods: Cross-sectional study included 56 pediatric SCD patients. TCD ultrasounds were performed at diagnosis for assessment of first stroke risk and then after every 6 months for follow-up studies. Patients with elevated TCD velocities given monthly packed RBCs transfusions to have HbS level around 40% (5,6). Data analysis was performed by "Statistical Package for the Social Sciences (SPSS, Version 17.0)".Results:In our patient population, male to female ratio was 1.33:1 (32 males, 24 females) and mean age was 7.5 ± 3.42 years (range: 2-14 years). Seven (12.5%) patients had TCD velocities higher than or equal to 200 cm/second, with mean age of 4.7±1.4 years and mean HbF 7.5±3.8, which were significantly different from patient group with normal flow velocities (mean age of 7.8±3.4 years; mean HbF 20±15.9). Patients with higher TCD velocities receiving blood transfusion got velocities in normal range within 6-12 months. Most importantly, normal TCD flow velocities were significantly associated with higher HbF expression and vice versa. As protective role of HbF in sickle cell patients is well established (11;13) and inducing the HbF expression in such patients using medications like hydroxyurea (20) or by genetically modifying the silencing of fetal γ-globin gene have been long standing goal in treatment of sickle cell disease (21), our findings about correlation of higher TCD flow velocities with lower HbF expression (and vice versa) and hence higher risk of first stroke in sickle cell patients provides a new direction for assessing the risk for vasculopathy in SCD patients.Discussion/Conclusions:Low prevalence of SCD patients with higher TCD velocities in our study population is in accordance with previous studies (5). Risk of first stroke was clinically manageable by 6-12 months of regular PRBCs transfusions, per established clinical practice (6). Moreover, normal TCD flow velocities and hence lower risk of stroke was significantly associated with the higher hemoglobin F expression and vice versa. As protective role of HbF in sickle cell patients is well established (4) and inducing the HbF expression in such patients have been long standing goal in treatment of sickle cell disease (7), our findings about correlation of higher TCD flow velocities with lower HbF expression (and vice versa) and hence higher risk of first stroke in sickle cell patients provides a new direction for assessing the risk for vasculopathy in SCD patients.

Suppression of the Hemodynamic Response Function Demonstrates Altered Cerebral Vasoreactivity in Sickle Cell Disease

Introduction: Children with sickle cell disease (SCD) face progressive cognitive decline compared to their unaffected siblings, even prior to the occurrence of stroke, suggesting damage to the underlying functional brain organization. Hence, functional MRI (fMRI) studies are needed to help characterize the pathophysiological basis of cognitive decline in SCD.Functional MRI techniques measure the blood oxygenation level dependent (BOLD) signal, which is sensitive to changes in the ratio of oxy- to deoxy-hemoglobin driven by local changes in neural activity. Regional brain activity raises local oxygen consumption that is overly compensated for by increases in local blood flow, bringing an excess of oxygenated blood and elevating the ratio of oxy- to deoxy-hemoglobin in the active brain regions. This stereotyped neurovascular coupling sequence unfolds over 20 seconds and gives rise to the canonical hemodynamic response function (HRF). Given the severe chronic anemia with attendant hypoxia, alterations in oxygen binding, increased cerebral blood flow (CBF), and prevalence of vasculopathy associated with SCD, careful comparison of the HRF between children with SCD and controls must occur prior to pursuing fMRI analyses.Children with SCD have increased CBF and oxygen extraction fraction at baseline when compared to controls, suggesting that they require these compensatory mechanisms to meet the brain's metabolic demands at rest, which could affect their ability to further increase blood flow in response to local neural activity. Thus, we hypothesized that children with SCD would have a blunted HRF with lower peak amplitudes when compared to sibling controls. To test our hypothesis, we conducted the gold-standard visual task-driven fMRI paradigm for measuring HRF in children with SCD and their siblings.Methods: Children with HbSS or HbS beta thalassemia null disease and healthy, sibling controls between the ages of 5 and 21 years were recruited from St. Louis Children's Hospital. Participants were excluded for history of overt stroke, vasculopathy, stem cell transplant or chronic exchange transfusion therapy. Participants completed a fMRI task that required them to press a button with their right index fingers at the onset of a 30 Hz counterphase-flickering checkerboard and to press a button with their left index finger at its offset. Nine time points (TR = 2.2 s) were included in the general linear model used to describe the HRF in response to each stimulus. No assumptions were made regarding the shape of the HRF. A peak-finding algorithm extracted regions of interest (ROI) from the group fMRI activation map. Activations were averaged across all voxels within each ROI to generate region-wise time courses for each participant. Results are reported as median [interquartile range], and comparisons are made using a Mann Whitney U test or Pearson chi-square test.Results: Fourteen children with SCD and 13 healthy sibling controls were included in the analysis. There was not a statistically significant difference in age (10.0 [9.8-15.5] years vs. 13.0 years [11.0-14.5]; p = 0.66) or gender (p = 0.31) between the SCD and control cohorts. Eighty-six percent of SCD participants were treated with hydroxyurea at a dose of 30.3 [28.6-33.1] mg/kg/day. The baseline hemoglobin of the SCD cohort was 8.8 [8.3-10.1] g/dL. Two children with SCD had a history of silent cerebral infarcts.The peak amplitude of the HRF in the primary visual cortex (V1) was significantly reduced in children with SCD (right: 0.29 [0.23-0.50]% and left: 0.29 [0.20-0.53]%) when compared to sibling controls (right: 0.56 [0.53-0.95]%, p < 0.001 and left: 0.52 [0.38-0.90]%, p = 0.01). While the HRF amplitude was reduced by almost 50%, there was no difference in the timing of the BOLD response (i.e. lag) between groups, both demonstrating a time-to-peak of 8.8 seconds (Figure).Conclusion: The striking decrease in HRF amplitude suggests that patients with SCD have altered cerebral vasoreactivity to neural stimuli. Further investigation into the pathophysiology behind and clinical implications of the blunted hemodynamic response in SCD is warranted. We are currently investigating the relationship between HRF suppression, cognitive outcomes and stroke risk in SCD, and the effects of HRF suppression on functional connectivity networks using resting-state functional connectivity MRI collected simultaneously in the same cohorts. [Display omitted] DisclosuresFields:NeuroPhage Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. King:NIH - BMTCTN: Research Funding; NIH - NHLBI: Research Funding; HRSA: Research Funding. Dosenbach:NeuroPhage Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Abstract WMP97: Regional Cerebral Oxygen Extraction is Elevated in Tissue at High Risk of Stroke in Pediatric Sickle Cell Disease

Introduction: Children with sickle cell disease (SCD) are at high risk of stroke. Hemispheric oxygen extraction fraction (OEF) is a predictor of stroke in adults with carotid occlusion, but OEF has not been evaluated as a predictor of stroke in children with SCD. Hypothesis: OEF is elevated in SCD children compared to controls within a region at high risk of stroke as defined by an infarct heat-map created from a separate retrospective SCD cohort. Methods: A prospective MRI study enrolled 37 children aged 5-21: 17 with SCD and no stroke, 12 with SCD and silent infarcts (median infarct volume=0.3ml), and 8 sibling controls. None were on transfusions or had overt stroke history. Voxel-wise OEF was measured using an asymmetric spin echo sequence. In a separate retrospective cohort of 67 SCD children with overt and silent stroke, infarct regions on FLAIR were manually outlined and coregistered to an average T1 map to create an infarct heat-map (Fig A) which was used to define a “high risk” ROI (defined by &gt;3% infarct density). This ROI was aligned to individual OEF maps from the prospective cohort (Fig B, average OEF map). OEF within the “high risk” ROI was compared between SCD children and controls; and between SCD children with and without infarction using Mann Whitney U tests. Results: The infarct heat-map from the retrospective cohort (Fig A) and the average OEF map from the prospective SCD cohort (Fig B) demonstrate striking co-localization of infarct density and elevated OEF. Within the “high risk” ROI, OEF was higher in SCD children compared to controls (39% [36, 46] vs. 23% [22, 27], p&lt;0.0001 (Fig C). OEF within this “high risk” ROI did not differ between SCD children with and without infarcts (40% [38, 47] vs. 38% [35, 46], p=0.6). Conclusion: OEF in SCD children is elevated in the internal borderzone, a region with high stroke risk in SCD. Regional OEF may be a marker of cerebral metabolic stress that could be exploited to stratify stroke risk in this vulnerable population.

Adenylyl Cyclase 9 Polymorphisms Reveal Potential Link to HDL Function and Cardiovascular Events in Multiple Pathologies: Potential Implications in Sickle Cell Disease.

Adenylyl cyclase 9 (ADCY9) mediates β2-adrenoceptor (β2-AR) signalling. Both proteins are associated with caveolae, specialized cholesterol-rich membrane substructures. Apolipoprotein A1 (ApoA1), the major protein component of high-density lipoprotein (HDL), removes cholesterol from cell membrane and caveolae and may thereby influence β2-AR signalling, shown in vitro to be modulated by cholesterol. Patients with Sickle Cell Disease (SCD) typically have low HDL and ApoA1 levels. In patients, mainly of African origin, with SCD, β2-AR activation may trigger adhesion of red blood cells to endothelial cells, leading to vascular occlusive events. Moreover, ADCY9 polymorphism is associated with risk of stroke in SCD. In recent clinical trials, ADCY9 polymorphism was found to be a discriminant factor associated with the risk of cardiovascular (CV) events in Caucasian patients treated with the HDL-raising compound dalcetrapib. We hypothesize that these seemingly disparate observations share a common mechanism related to interaction of HDL/ApoA1 and ADCY9 on β2-AR signalling. This review also raises the importance of characterizing polymorphisms that determine the response to HDL-raising and -mimicking agents in the non-Caucasian population at high risk of CV diseases and suffering from SCD. This may facilitate personalized CV treatments.