Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS). We examined the oxidative effects of sickle cell disease on hematopoietic stem cell function in a sickle mouse model. In vitro colony-forming assays showed a significant decrease in progenitor colony formation derived from sickle compared to control bone marrow (BM). Sickle BM possessed a significant decrease in the KSL (c-kit+, Sca-1+, Lineage−) progenitor population, and cell cycle analysis showed that there were fewer KSL cells in the G0 phase of the cell cycle compared to controls. We found a significant increase in both lipid peroxidation and ROS in sickle-derived KSL cells. In vivo analysis demonstrated that normal bone marrow cells engraft with increased frequency into sickle mice compared to control mice. Hematopoietic progenitor cells derived from sickle mice, however, demonstrated significant impairment in engraftment potential. We observed partial restoration of engraftment by n-acetyl cysteine (NAC) treatment of KSL cells prior to transplantation. Increased intracellular ROS and lipid peroxidation combined with improvement in engraftment following NAC treatment suggests that an altered redox environment in sickle mice affects hematopoietic progenitor and stem cell function.

Cerebrovascular abnormalities are a common feature of sickle cell disease that may be associated with risk of vaso-occlusive pain crises, microinfarcts, and cognitive impairment. An activated endothelium and adhesion factors, VCAM-1 and P-selectin, are implicated in sickle cell vasculopathy, including abnormal hemodynamics and leukocyte adherence. This study examined the association between cerebral expression of these adhesion factors and cortical microvascular blood flow dynamics by using in-vivo two-photon microscopy. We also examined the impact of blood transfusion treatment on these markers of vasculopathy. Results showed that sickle cell mice had significantly higher maximum red blood cell (RBC) velocity (6.80 ± 0.25 mm/sec, p ≤ 0.01 vs. 5.35 ± 0.35 mm/sec) and more frequent blood flow reversals (18.04 ± 0.95 /min, p ≤ 0.01 vs. 13.59 ± 1.40 /min) in the cortical microvasculature compared to controls. In addition, sickle cell mice had a 2.6-fold (RFU/mm2) increase in expression of VCAM-1 and 17-fold (RFU/mm2) increase in expression of P-selectin compared to controls. This was accompanied by an increased frequency in leukocyte adherence (4.83 ± 0.57 /100 μm/min vs. 2.26 ± 0.37 /100 μm/min, p ≤ 0.001). We also found that microinfarcts identified in sickle cell mice were 50% larger than in controls. After blood transfusion, many of these parameters improved, as results demonstrated that sickle cell mice had a lower post-transfusion maximum RBC velocity (8.30 ± 0.98 mm/sec vs. 11.29 ± 0.95 mm/sec), lower frequency of blood flow reversals (12.80 ± 2.76 /min vs. 27.75 ± 2.09 /min), and fewer instances of leukocyte adherence compared to their pre-transfusion imaging time point (1.35 ± 0.32 /100 μm/min vs. 3.46 ± 0.58 /100 μm/min). Additionally, we found that blood transfusion was associated with lower expression of adhesion factors. Our results suggest that blood transfusion and adhesion factors, VCAM-1 and P-selectin, are potential therapeutic targets for addressing cerebrovascular pathology, such as vaso-occlusion, in sickle cell disease.

Sickle cell disease (SCD) is associated with repeated bouts of vascular insufficiency leading to organ dysfunction. Deficits in revascularization following vascular injury are evident in SCD patients and animal models. We aimed to elucidate whether enhancing nitric oxide bioavailability in SCD mice improves outcomes in a model of vascular insufficiency. Townes AA (wild type) and SS (sickle cell) mice were treated with either L-Arginine (5% in drinking water), L-NAME (N(ω)-nitro-L-arginine methyl ester; 1 g/L in drinking water) or NO-generating hydrogel (PA-YK-NO), then subjected to hindlimb ischemia via femoral artery ligation and excision. Perfusion recovery was monitored over 28 days via LASER Doppler perfusion imaging. Consistent with previous findings, perfusion was impaired in SS mice (63 ± 4% of non-ischemic limb perfusion in AA vs 33 ± 3% in SS; day 28; P < 0.001; n = 5–7) and associated with increased necrosis. L-Arginine treatment had no significant effect on perfusion recovery or necrosis (n = 5–7). PA-YK-NO treatment led to worsened perfusion recovery (19 ± 3 vs. 32 ± 3 in vehicle-treated mice; day 7; P < 0.05; n = 4–5), increased necrosis score (P < 0.05, n = 4–5) and a 46% increase in hindlimb peroxynitrite (P = 0.055, n = 4–5). Interestingly, L-NAME worsened outcomes in SS mice with decreased in vivo lectin staining following ischemia (7 ± 2% area in untreated vs 4 ± 2% in treated mice, P < 0.05, n = 5). Our findings demonstrate that L-arginine and direct NO delivery both fail to improve postischemic neovascularization in SCD. Addition of NO to the inflammatory, oxidative environment in SCD may result in further oxidative stress and limit recovery.

Background: The pathogenesis of albuminuria in SCD remains incompletely understood. We evaluated the association of albuminuria with measures of endothelial function, and explored associations of both albuminuria and measures of endothelial function with selected biological variables (vascular endothelial growth factor [VEGF], endothelin-1 [ET-1], soluble fms-like tyrosine kinase-1 [sFLT-1], soluble vascular cell adhesion molecule-1 [soluble VCAM-1] and plasma hemoglobin). Methods: Spot urine measurements for albumin-creatinine ratio (UACR) and 24-hour urine protein were obtained. Endothelial function was assessed using brachial artery ultrasound with measurements of flow-mediated dilation (FMD), nitroglycerin-mediated dilation (NTMD) and hyperemic velocity. Results: Twenty three subjects with varying degrees of albuminuria were evaluated. UACR was significantly correlated with FMD (ρ = -0.45, p = 0.031). In univariate analysis, UACR was correlated with VEGF (ρ = -0.49; 95% CI: -0.75 –-0.1, p = 0.015), plasma hemoglobin (ρ = 0.50; 95% CI: 0.11–0.75, p = 0.013) and ET-1 (ρ = 0.40; 95% CI: -0.03–0.69, p = 0.06). Multivariable analysis showed significant associations of ET-1 (estimate: 455.1 [SE: 198.3], p = 0.02), VEGF (estimate: -1.1 [SE: 0.53], p = 0.04) and sFLT-1 (estimate: -1.14 [SE: 0.49], p = 0.02) with UACR. Only ET-1 (estimate: -8.03 [SE: 3.87], p = 0.04) was significantly associated with FMD in multivariable analyses. Finally, UACR was correlated with both 24-hour urine protein (ρ = 0.90, p < 0.0001) and urine aliquots for albumin-creatinine ratio obtained from the 24-hour urine collection (ρ = 0.97, p < 0.0001). Conclusion: This study provides more definitive evidence for the association of albuminuria with endothelial dysfunction in SCD. Elevated circulating levels of ET-1 may contribute to SCD-related glomerulopathy by mediating endothelial dysfunction.

The intestinal microbiome has emerged as a potential contributor to the severity of sickle cell disease (SCD). We sought to determine whether SCD mice exhibit intestinal barrier dysfunction, inflammation, and dysbiosis. Using the Townes humanized sickle cell mouse model, we found a 3-fold increase in intestinal permeability as assessed via FITC-dextran (4 kDa) assay in SS (SCD) mice compared to AA (wild type) mice (n = 4, p < 0.05). This was associated with 25 to 50% decreases in claudin-1, 3, and 15 and zonula occludens-1 gene expression (n = 8–10, p < 0.05) in the small intestine. Increased Ly6G staining demonstrated more neutrophils in the SS small intestine (3-fold, n = 5, p < 0.05) associated with increased expression of TNFα, IL-17A, CXCL1, and CD68 (2.5 to 5-fold, n = 7–10, p < 0.05). In addition, we observed 30 to 55% decreases in superoxide dismutase-1, glutathione peroxidase-1, and catalase antioxidant enzyme expression (n = 7–8, p < 0.05) concomitant to an increase in superoxide (2-fold, n = 4, p < 0.05). Importantly, all significant observations of a leaky gut phenotype and inflammation were limited to the small intestine and not observed in the colon. Finally, characterization of the composition of the microbiome within the small intestine revealed dysbiosis in SS mice compared to their AA littermates with 47 phyla to species-level significant alterations in amplicon sequence variants. We conclude that the intestinal barrier is compromised in SCD, associated with decreased gene expression of tight junction proteins, enhanced inflammation, oxidative stress, and gut microbiome dysbiosis, all specific to the small intestine.

Background: There are sparse data on the long-term and late effects of hematopoietic cell transplantation (HCT) for sickle cell disease (SCD). Objective: This study aims to establish an international registry of long-term outcomes post-HCT for SCD and demonstrate the feasibility of recruitment at a single site in the United States. Methods: The Sickle Cell Transplantation Evaluation of Long-Term and Late Effects Registry (STELLAR) was designed to enroll patients with SCD ≥1 year post-HCT, their siblings without SCD, and nontransplanted controls with SCD to collect web-based participant self-reports of health status and practices by using the Bone Marrow Transplant Survivor Study (BMTSS) surveys, health-related quality of life (HRQOL) using the Patient-Reported Outcomes Measurement Information System (PROMIS) Pediatric Profile-25 or Pediatric Profile-29 survey, chronic graft-versus-host disease (cGVHD) using the symptom scale survey, daily pain using an electronic pain diary, the economic impact of HCT using the financial hardship survey, sexual function using the PROMIS Sexual Function SexFSv2.0 survey, and economic productivity using the American Time Use Survey (ATUS). We also piloted retrieval of clinical data previously submitted to the Center for International Blood and Marrow Transplant Research (CIBMTR); recorded demographics, height, weight, blood pressure, waist and hip circumferences, timed up and go (TUG) test, and handgrip test; and obtained blood for metabolic screening, gonadal function, fertility potential, and biorepository of plasma, serum, RNA, and DNA. Results: Of 100 eligible post-HCT patients, we enrolled 72 (72%) participants aged 9-38 (median 17) years. We also enrolled 19 siblings aged 5-32 (median 10) years and 28 nontransplanted controls with SCD aged 4-46 (median 22) years. Of the total 119 participants, 73 (61%) completed 85 sets of surveys and 41 (35%) contributed samples to the biorepository. We completed ATUS interviews of 28 (24%) participants. We successfully piloted retrieval of data submitted to the CIBMTR and expanded recruitment to multiple sites in the United States, Canada, the United Kingdom, and Nigeria. Conclusions: It is feasible to recruit subjects and conduct study procedures for STELLAR in order to determine the long-term and late effects of HCT for SCD.

Background and Purpose-The effects of lytic stroke therapy in patients with sickle cell anemia are unknown, although a recent study suggested that coexistent sickle cell anemia does not increase the risk of cerebral hemorrhage. This finding calls for systemic analysis of the effects of thrombolytic stroke therapy, first in humanized sickle mice, and then in patients. There is also a need for additional predictive markers of sickle cell anemia-associated vasculopathy. Methods-We used Doppler ultrasound to examine the carotid artery of Townes sickle mice tested their responses to repetitive mild hypoxia-ischemia- and transient hypoxia-ischemia-induced stroke at 3 or 6 months of age, respectively. We also examined the effects of tPA (tissue-type plasminogen activator) treatment in transient hypoxia-ischemia-injured sickle mice. Results-Three-month-old sickle cell (SS) mice showed elevated resistive index in the carotid artery and higher sensitivity to repetitive mild hypoxia-ischemia-induced cerebral infarct. Six-month-old SS mice showed greater resistive index and increased flow velocity without obstructive vasculopathy in the carotid artery. Instead, the cerebral vascular wall in SS mice showed ectopic expression of PAI-1 (plasminogen activator inhibitor-1) and P-selectin, suggesting a proadhesive and prothrombotic propensity. Indeed, SS mice showed enhanced leukocyte and platelet adherence to the cerebral vascular wall, broader fibrin deposition, and higher mortality after transient hypoxia-ischemia. Yet, post-transient hypoxia- ischemia treatment with tPA reduced thrombosis and mortality in SS mice. Conclusions-Sickle mice are sensitive to hypoxia/ischemia-induced cerebral infarct but benefit from thrombolytic treatment. An increased resistive index in carotid arteries may be an early marker of sickle cell vasculopathy. Visual Overview-An online visual overview is available for this article.

Sickle cell disease (SCD)-associated pulmonary hypertension (PH) causes significant morbidity and mortality. Here, we defined the role of endothelial specific peroxisome proliferator-activated receptor g (PPARg) function and novel PPARg/HUWE1/miR-98 signaling pathways in the pathogenesis of SCD-PH. PH and right ventricular hypertrophy (RVH) were increased in chimeric Townes humanized sickle cell (SS) mice with endothelialtargeted PPARg knockout (SSePPARgKO) compared with chimeric littermate control (SSLitCon). Lung levels of PPARg, HUWE1, and miR-98 were reduced in SSePPARgKO mice compared with SSLitCon mice, whereas SSePPARgKO lungs were characterized by increased levels of p65, ET-1, and VCAM1. Collectively, these findings indicate that loss of endothelial PPARg is sufficient to increase ET-1 and VCAM1 that contribute to endothelial dysfunction and SCD-PH pathogenesis. Levels of HUWE1 and miR-98 were decreased, and p65 levels were increased in the lungs of SS mice in vivo and in hemin-treated human pulmonary artery endothelial cells (HPAECs) in vitro. Although silencing of p65 does not regulate HUWE1 levels, the loss of HUWE1 increased p65 levels in HPAECs. Overexpression of PPARg attenuated hemininduced reductions of HUWE1 and miR-98 and increases in p65 and endothelial dysfunction. Similarly, PPARg activation attenuated baseline PH and RVH and increased HUWE1 and miR-98 in SS lungs. In vitro, hemin treatment reduced PPARg, HUWE1, and miR-98 levels and increased p65 expression, HPAEC monocyte adhesion, and proliferation. These derangements were attenuated by pharmacological PPARg activation. Targeting these signaling pathways can favorably modulate a spectrum of pathobiological responses in SCD-PH pathogenesis, highlighting novel therapeutic targets in SCD pulmonary vascular dysfunction and PH.

Platelets express a variety of membrane and secreted glycoproteins, but the importance of glycosylation to platelet functions is poorly understood. To explore the importance of O-glycosylation, we generated mice with a targeted deletion of Cosmc in murine endothelial/hematopoietic cells (EHC) (EHC Cosmc−/y). X-linked Cosmc encodes an essential chaperone that regulates protein O-glycosylation. This targeted mutation resulted in lethal perinatal hemorrhage in the majority of mice, and the surviving mice displayed severely prolonged tail-bleeding times and macrothrombocytopenia. EHC Cosmc−/y platelets exhibited a marked decrease in GPIb-IX-V function and agonist-mediated integrin αIIbβ3 activation, associated with loss of interactions with von Willebrand factor and fibrinogen, respectively. Significantly, three O-glycosylated glycoproteins, GPIbα, αIIb, and GPVI normally on platelet surfaces that play essential roles in platelet functions, were partially proteolyzed in EHC Cosmc−/y platelets. These results demonstrate that extended O-glycans are required for normal biogenesis of the platelets as well as the expression and functions of their essential glycoproteins, and that variations in O-glycosylation may contribute to altered hemostasis.

Increased frequency and risk of infection is one of the well described complications of sickle cell anemia (SCA). Dietary supplementation in children with SCA and growth retardation improved growth and decreased incidence of infection. We investigated the impact of a high protein diet on weight gain, hematological profile, and immune cytokine levels in the Berkeley model of SCA, 16 of which were randomized to either regular mouse diet with 20% of calories from protein (n = 8) or a test feed with 35% of calories from protein (n = 8). Control mice (C57BL/6, n = 16) were correspondingly randomized, and were all feed ad libitum for three months with actual intake estimated by subtracting the weight of gnaw waste from that of the feed given. Blood was collected at sacrifice by cardiac puncture and plasma levels of T helper cell 1 (TH1) and TH2 associated cytokines were measured using a multiplex antibody immobilized bead assay. SCA mice receiving the 35% protein diet had modest improvements in weight, red blood cell count, and hemoglobin level, with a slight decrease in reticulocyte count compared with SCA mice on the regular mouse diet. Furthermore, they also had significantly higher plasma levels of cytokines tumor necrosis factor (TNF)-α (P = 0.02), interferon (IFN)-γ (P = 0.01), interleukin 10 (IL-10; P = 0.02), and IL-4 (P = 0.02) compared with those that received the 20% protein diet. We conclude that providing additional protein calories to transgenic SCA mice increased the plasma levels of acute inflammatory cytokines associated with immune response to infection, which might partly explain decreased episodes of infection observed among supplemented children with SCA.