Proteasome inhibition induces both antioxidant and hb f responses in sickle cell disease via the nrf2 pathway.

Oxidant stress is implicated in the manifestations of sickle cell disease including hemolysis and vascular occlusion. Strategies to induce antioxidant response as well as Hb F (α2γ2) have the potential to ameliorate the severity of sickle cell disease. Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2) is a transcription factor that regulates antioxidant enzymes as well as γ-globin transcription. The Nrf2 in the cytoplasm is bound to its adapter protein Keap-1 that targets Nrf2 for proteasomal degradation, thereby preventing its nuclear translocation. We examined whether inhibiting the 26S proteasome using the clinically applicable proteasome inhibitors bortezomib and MLN 9708 would promote nuclear translocation of Nrf2, and thereby induce an antioxidant response and as well as Hb F in sickle cell disease. Proteasome inhibitors induced reactive oxygen species (ROS) and thereby increased Nrf2-dependent antioxidant enzyme transcripts, elevated cellular glutathione (GSH) levels and γ-globin transcripts as well as Hb F levels in the K562 cell line and also in erythroid burst forming units (BFU-E) generated from peripheral blood mononuclear cells of sickle cell disease patients. These responses were abolished by siRNA-mediated knockdown of Nrf2. Proteasome inhibitors, especially newer oral agents such as MLN9708 have the potential to be readily translated to clinical trials in sickle cell disease with the dual end points of antioxidant response and Hb F induction.

Definitions of the phenotypic manifestations of sickle cell disease.

Sickle cell disease (SCD) is a pleiotropic genetic disorder of hemoglobin that has profound multiorgan effects. The low prevalence of SCD ( approximately 100,000/US) has limited progress in clinical, basic, and translational research. Lack of a large, readily accessible population for clinical studies has contributed to the absence of standard definitions and diagnostic criteria for the numerous complications of SCD and inadequate understanding of SCD pathophysiology. In 2005, the Comprehensive Sickle Cell Centers initiated a project to establish consensus definitions of the most frequently occurring complications. A group of clinicians and scientists with extensive expertise in research and treatment of SCD gathered to identify and categorize the most common complications. From this group, a formal writing team was formed that further reviewed the literature, sought specialist input, and produced definitions in a standard format. This article provides an overview of the process and describes 12 body system categories and the most prevalent or severe complications within these categories. A detailed Appendix provides standardized definitions for all complications identified within each system. This report proposes use of these definitions for studies of SCD complications, so future studies can be comparably robust and treatment efficacy measured. Use of these definitions will support greater accuracy in genotype-phenotype studies, thereby achieving a better understanding of SCD pathophysiology. This should nevertheless be viewed as a dynamic rather than final document; phenotype descriptions should be reevaluated and revised periodically to provide the most current standard definitions as etiologic factors are better understood, and new diagnostic options are developed.

Neuropsychological dysfunction and neuroimaging abnormalities in neurologically intact adults with sickle cell anemia.

CONTEXT: Sickle cell anemia (SCA) is a chronic illness causing progressive deterioration in quality of life. Brain dysfunction may be the most important and least studied problem affecting individuals with this disease. OBJECTIVE: To measure neurocognitive dysfunction in neurologically asymptomatic adults with SCA vs healthy control individuals. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study comparing neuropsychological function and neuroimaging findings in neurologically asymptomatic adults with SCA and controls from 12 SCA centers, conducted between December 2004 and May 2008. Participants were patients with SCA (hemoglobin [Hb] SS and hemoglobin level < or = 10 mg/dL) aged 19 to 55 years and of African descent (n = 149) or community controls (Hb AA and normal hemoglobin level) (n = 47). Participants were stratified on age, sex, and education. MAIN OUTCOME MEASURES: The primary outcome measure was nonverbal function assessed by the Wechsler Adult Intelligence Scale, third edition (WAIS-III) Performance IQ Index. Secondary exploratory outcomes included performance on neurocognitive tests of executive function, memory, attention, and language and magnetic resonance imaging measurement of total intracranial and hippocampal volume, cortical gray and white matter, and lacunae. RESULTS: The mean WAIS-III Performance IQ score of patients with SCA was significantly lower than that of controls (adjusted mean, 86.69 for patients with SCA vs 95.19 for controls [mean difference, -5.50; 95% confidence interval {CI}, -9.55 to -1.44]; P = .008), with 33% performing more than 1 SD (<85) below the population mean. Among secondary measures, differences were observed in adjusted mean values for global cognitive function (full-scale IQ) (90.47 for patients with SCA vs 95.66 for controls [mean difference, -5.19; 95% CI, -9.24 to -1.13]; P = .01), working memory (90.75 vs 95.25 [mean difference, -4.50; 95% CI, -8.55 to -0.45]; P = .03), processing speed (86.50 vs 97.95 [mean difference, -11.46; 95% CI, -15.51 to -7.40]; P < .001), and measures of executive function. Anemia was associated with poorer neurocognitive function in older patients. No differences in total gray matter or hippocampal volume were observed. Lacunae were more frequent in patients with SCA but not independently related to neurocognitive function. CONCLUSION: Compared with healthy controls, adults with SCA had poorer cognitive performance, which was associated with anemia and age.

A novel high-throughput screening assay for sickle cell disease drug discovery.

Although the pathophysiology and molecular basis of sickle cell disease (SCD) were described more than half a century ago, an effective and safe therapy is not yet available. This may be explained by the lack of a suitable high-throughput technique that allows rapid screening of thousands of compounds for their antisickling effect. The authors have thus developed a novel high-throughput screening (HTS) assay based on detecting the ability of red blood cells (RBC) to traverse a column of tightly packed Sephacryl chromatography beads. When deoxygenated, sickle RBC are rigid and remain on the top of the column. However, when deoxygenated and treated with an effective antisickling agent, erythrocytes move through the Sephacryl media and produce a red dot on the bottom of the assay tubes. This approach has been adapted to wells in a 384-well microplate. Results can be obtained by optical scanning: The size of the red dot is proportional to the antisickling effect of the test molecule. The new assay is simple, inexpensive, reproducible, requires no special reagents, and should be readily adaptable to robotic HTS systems. It has the potential to identify novel drug candidates, allowing the development of new therapeutic options for individuals affected with SCD.

Sickle cell disease summit: from clinical and research disparity to action.

The American Society of Pediatric Hematology/Oncology Sickle Cell Summit brought together a broad range of constituencies to identify a unified approach to healthcare and research disparities for sickle cell disease. Recommendations included the following: (1) speak with a unified voice representing all constituencies; (2) optimize access to care from knowledgeable health care providers and create a medical home for all individuals with the disease; (3) utilize population-based surveillance to measure outcomes; (4) develop overall approaches to basic, translational, clinical, and health services research; (5) enhance the community role in advocacy, education, service, and fundraising. Taskforces were identified to effect implementation.

Development and validation of SIMS: an instrument for measuring quality of life of adults with sickle cell disease.

In recent years, there has been a growing interest in the assessment of quality of life (QOL) issues, particularly in chronic debilitating conditions. Several instruments have been developed, tested, and validated in the general population and in other chronic diseases; however, few studies have examined QOL issues in adults with sickle cell disease (SCD). We developed Sickle Cell Impact Measurement Scale (SIMS), an instrument for measuring the QOL of adults with SCD. The 142-item multi-dimensional SIMS questionnaire was developed using 4 validated instruments and additional questions based upon recommendations of patient focus groups. The SIMS was self-administered to 106 SCD and 45 rheumatoid arthritis (RA) patients over 4 sites. SIMS was evaluated on measures of both internal consistency and construct validity. Item reduction was performed based on results of factor analysis. The SIMS achieved good internal consistency, with a Cronbach's alpha coefficient reported of 0.86, and distinguished between patients with SCD and RA. Overall, QOL did not differ significantly among SCD and RA patients. However, SCD patients scored higher in both physical and social domains, which was expected and reflected the differences in the pathophysiology of each disease. The SIMS is a reliable, valid, and responsive questionnaire, which functions well as a discriminative instrument for the measure of health-related QOL (HRQOL) of adults with SCD. The SIMS is currently being administered to adults with SCD across several centers for further validation to become a disease-specific, global QOL instrument.

Outcome of sickle cell anemia: a 4-decade observational study of 1056 patients.

Based on a prospective cohort study of 1056 patients with sickle cell anemia (Hb SS) initiated in 1959, we investigated the influence of calendar era, age, sex, and prior medical conditions on the subsequent development of irreversible organ damage and survival using the Cox regression model with time-dependent covariates adjusting for all prior occurrences. We studied 30 acute clinical events, and focused on 8 prototypic forms of irreversible organ damage. Childhood survival to age 20 years has improved from 79% for those born before 1975 to 89% for children born in or after 1975. Bone infarction was a significant risk factor for avascular necrosis (p = 0.01), and infantile dactylitis was a significant risk factor for stroke (p = 0.01). Prior hospitalized vaso-occlusive sickle crisis in adults was significantly associated with the increased rate of avascular necrosis (p < 0.001), leg ulcers (p < 0.001), sickle chronic lung disease (p < 0.001), renal failure (p < 0.005), and early death (p < 0.001). The diagnosis of clearly evident clinical conditions such as leg ulcer, osteonecrosis, and retinopathy predicted an increased likelihood of developing a more lethal form of organ damage and earlier death: 77% of patients with chronic lung disease, 75% of those with renal insufficiency, and 51% of those with stroke had a prior chronic condition. Of the 232 patients who died, 73% had 1 or more clinically recognized forms of irreversible organ damage. By the fifth decade, nearly one-half of the surviving patients (48%) had documented irreversible organ damage. End-stage renal disease (glomerulosclerosis), chronic pulmonary disease with pulmonary hypertension, retinopathy, and cerebral microinfarctions are manifestations of arterial and capillary microcirculation obstructive vasculopathy. The current study underscores the need for preventive therapy to ameliorate the progression of the sickle vasculopathy.

L-glutamine therapy reduces endothelial adhesion of sickle red blood cells to human umbilical vein endothelial cells.

BACKGROUND: We have previously demonstrated that therapy with orally administered L-glutamine improves nicotinamide adenosine dinucleotide (NAD) redox potential of sickle red blood cells (RBC). On further analysis of L-glutamine therapy for sickle cell anemia patients, the effect of L-glutamine on adhesion of sickle RBC to human umbilical vein endothelial cells (HUVEC) was examined. METHODS: The first part of the experiment was conducted with the blood samples of the 5 adult sickle cell anemia patients who had been on L-glutamine therapy for at least 4 weeks on a dosage of 30 grams per day compared to those of patient control group. In the second part of the experiment 6 patients with sickle cell anemia were studied longitudinally. Five of these patients were treated with oral L-glutamine 30 grams daily and one was observed without treatment as the control. t-test and paired t-test were used for determination of statistical significance in cross-sectional and longitudinal studies respectively. RESULTS: In the first study, the mean adhesion to endothelial cells with the autologous plasma incubated cells were 0.97 +/- 0.45 for the treated group and 1.91 +/- 0.53 for the nontreated group (p < 0.02). Similarly with lipopolysaccharide (LPS) incubated cells the mean adhesion to endothelial cells were 1.39 +/- 0.33 for the treated group and 2.80 +/- 0.47 for the untreated group (p < 0.001). With the longitudinal experiment, mean decrease in the adhesion to endothelial cells was 1.13 +/- 0.21 (p < 0.001) for the 5 treated patients whereas the control patient had slight increase in the adhesion to endothelial cells. CONCLUSION: In these studies, oral L-glutamine administration consistently resulted in improvement of sickle RBC adhesion to HUVEC. These data suggest positive physiological effects of L-glutamine in sickle cell disease.

Arterial blood pressure and hyperviscosity in sickle cell disease.

Abnormal rheologic behavior of sickle cells is the result of increased viscosity of the blood caused by the polymerization of hemoglobin S and the resultant production of dense, dehydrated sickle erythrocytes. As the viscosity of sickle cells increases, there is a negative impact on blood flow, which contributes to the vascular occlusion process, the hallmark of the sickling disorders. Blood flow is directly proportional to the blood pressure and inversely proportional to the blood viscosity. Blood flow has important implications for the diagnosis and management of hypertension in sickle cell patients and for transfusion therapy for the acute and chronic complications of this disease.

The acute chest syndrome.

Recent large clinical studies of the acute chest syndrome (ACS) have improved our understanding of its pathophysiology and epidemiology. However, there is still a need for better methods of distinguishing vaso-occlusion from fibrin or fat embolism, for rapid diagnostic tests to make positive identifications of microbial infection, for adjunctive therapies that would affect prognosis, and for identification of factors that influence prognosis. The difference in clinical course and severity between children and adults supports the results of current studies indicating multiple causes for ACS. The mainstay of successful treatment remains high-quality supportive care. The judicious use of transfusion therapy has a major role in preventing mortality in the absence of a specific therapy that consistently improves the clinical course.

A novel role of hypoxia-inducible factor in cobalt chloride- and hypoxia-mediated expression of IL-8 chemokine in human endothelial cells.

Tissue hypoxemia is common in several pathological diseases, including vaso-occlusion in sickle cell disease and myocardial infarction. One finds increased presence of leukocytes during lung injury and at sites of inflammation in vascular endothelium. In this study, we used human pulmonary microvascular endothelial cells and human dermal microvascular endothelial immortalized cell line to delineate the cellular signaling mechanism of hypoxia- and CoCl2 (a mimetic of hypoxia)-induced IL-8 expression, and the latter's role in chemotaxis of polmorphonuclear neutrophils. We show that hypoxia- and CoCl2-induced IL-8 mRNA and protein expression involved activation of PI3K/Akt and p38 MAPK, but not MEK kinase. Analysis of some transcription factors associated with IL-8 promoter revealed that hypoxia and CoCl2 increased DNA-binding activity of hypoxia-inducible factor-1alpha (HIF-1alpha), NF-kappaB, and AP-1. In addition, we show that hypoxia- and CoCl2-induced IL-8 expression requires activation of HIF as demonstrated by the following: 1) EMSA; 2) transfection studies with IL-8 promoter reporter constructs with mutation in HIF-1alpha binding site; 3) attenuation of IL-8 expression by both HIF-1alpha small interfering RNA and R59949; 4) augmentation of IL-8 expression by either transfection with HIF-prolyl hydroxylase-2 small interfering RNA or overexpression of HIF-1alpha; and 5) chromatin immunoprecipitation analysis. Moreover, conditioned medium from hypoxia-treated endothelial cells augmented chemotaxis of neutrophils, due to release of IL-8. These data indicate that hypoxia-induced signaling in vascular endothelium for transcriptional activation of IL-8 involves PI3K/Akt, p38 MAPK, and HIF-1alpha. Pharmacological agents, which inhibit HIF-1alpha, may possibly ameliorate inflammation associated with hypoxia in pathological diseases.

Rheologic behavior of sickle and normal red blood cell mixtures in sickle plasma: implications for transfusion therapy.

BACKGROUND: Guidelines for transfusion in sickle cell disease usually define an upper hematocrit (Hct) limit of 0.30 to 0.35 to avoid blood hyperviscosity. In vitro viscosity studies of normal (AA) and sickle (SS) red blood cell (RBC) mixtures in buffer appear to confirm that this Hct limit is optimal for oxygen delivery to vascular beds as judged by the ratio of Hct to viscosity, with this ratio often termed "oxygen or RBC transport effectiveness." In the absence of plasma, however, effects due to RBC-RBC interactions mediated by plasma proteins cannot be assessed. STUDY DESIGNS AND METHODS: To investigate the optimal Hct-to-viscosity ratio of RBCs in plasma, the rheologic effects of Hct (0.20-0.40), the proportion of SS RBCs (0-100%), and shear rate (1-1000/sec) for mixtures of oxygenated and deoxygenated SS and AA RBCs were evaluated in sickle plasma at 37 degrees C. RESULTS: RBC suspension viscosity was shear-dependent (i.e., viscosity decreased with increasing shear rate) and increased with Hct and proportion of SS RBCs. An "optimal" Hct level (defined as a maximal of the Hct-to-viscosity ratio) was seen only at shear rates above 50/sec. At lower shear rates (e.g., 5/sec), where plasma-mediated RBC-RBC interactions predominate, any increment in Hct was offset by a proportionally greater increase in viscosity, thus leading to a lower Hct-to-viscosity ratio. CONCLUSION: These results indicate the importance of plasma-mediated RBC interactions and suggest that the benefits of transfusion may vary depending on local flow rates (i.e., shear rates) and organ-specific hemodynamics.

Bone marrow embolism in sickle cell disease: a review.

The fat embolism syndrome is an important complication of patients with sickle cell hemoglobinopathies because of severe morbidity and mortality. Our recent experience with three cases that survived with intensive supportive care and prompt use of transfusion stimulates this review. A high index of suspicion, prompt use of diagnostic tools, and aggressive clinical management are the keys to a successful outcome.

Myocardial injury or infarction associated with fat embolism in sickle cell disease: a report of three cases with survival.

Fat embolism represents a dread complication of sickle cell hemoglobinopathies. We present the details of three cases that, in addition to an acute chest syndrome, had serological and clinical parameters consistent with myocardial damage. A favorable outcome was obtained with judicious use of blood transfusion.

Outcome in hemoglobin SC disease: a four-decade observational study of clinical, hematologic, and genetic factors.

Over the past 40 years, we observed 284 subjects with hemoglobin SC disease (Hb SC) for 2,837 person-years. We examined the association of the course of clinical events with hematologic and genetic factors. The mean entry age was 21 years, although 15% entered before one year of age. The mean Hb concentration was 11.3 g/dL, the mean fetal hemoglobin was 2.5%, and the mean MCV was 84.4 fL. Twenty-five subjects died at a median age of 37 years. Chronic organ-specific complications occurred in 112 subjects (39.4%), with advanced retinopathy in 65 subjects (22.9%) and osteonecrosis (avascular necrosis) in 42 subjects (14.8%). We identified the beta-globin haplotypes in 82 subjects and the alpha-gene status in 79. Twenty-nine percent had alpha-thalassemia-2. The beta(CI) haplotype was present in 85.4%. We found a decreased incidence of retinopathy in the beta(CI) subjects compared to the non-beta(CI) subjects (33% vs. 67%; P = 0.049) with a later mean onset age (29 years vs. 21 years; log-rank test, P= 0.026). We also found a consistent pattern of decreased morbidity in subjects who had alpha-thalassemia-2 in comparison to those who did not. We found a reduced risk of chronic organ-specific complications (log-rank test, P= 0.003), lower incidence of sickle crisis (48% vs. 80%, P= 0.001), later onset of gallbladder disease (age of onset: 55 years vs. 34 years; P= 0.055), and lower risk of osteonecrosis (log-rank test, P= 0.024). Our findings suggest that Hb SC subjects who have not inherited alpha-thalassemia-2 might benefit from erythrocyte rehydration therapy.

Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor.

Monocytes from patients with sickle cell disease (SCD) are in an activated state. However, the mechanism of activation of monocytes in SCD is not known. Our studies showed that placenta growth factor (PlGF) activated monocytes and increased mRNA levels of cytokines (tumor necrosis factor-alpha [TNF-alpha] and interleukin-1beta [IL-1beta]) and chemokines (monocyte chemotactic protein-1 [MCP-1], IL-8, and macrophage inflammatory protein-1beta [MIP-1beta]) in both normal monocytes and in the THP-1 monocytic cell line. This increase in mRNA expression of cytochemokines was also reflected in monocytes derived from subjects with SCD. We studied the PlGF-mediated downstream cellular signaling events that caused increased transcription of inflammatory cytochemokines and chemotaxis of THP-1 monocytes. PlGF-mediated cytochemokine mRNA and protein expression was inhibited by PD98059 and wortmannin, inhibitors of mitogen-activated protein kinase kinase (MAPK/MEK) kinase and phosphatidylinositol-3 (PI3) kinase, respectively, but not by SB203580, a p38 kinase inhibitor. PlGF caused a time-dependent transient increase in phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2), which was completely inhibited by wortmannin, indicating that activation of PI3 kinase preceded MEK activation. PlGF also induced transient phosphorylation of AKT. MEK and PI3 kinase inhibitors and antibody to Flt-1 abrogated PlGF-induced chemotaxis of THP-1 monocytes. Overexpression of a dominant-negative AKT or a dominant-negative PI3 kinase p85 subunit in THP-1 monocytes attenuated the PlGF-mediated phosphorylation of ERK-1/2, cytochemokine secretion, and chemotaxis. Taken together, these data show that activation of monocytes by PlGF occurs via activation of Flt-1, which results in activation of PI3 kinase/AKT and ERK-1/2 pathways. Therefore, we propose that increased levels of PlGF in circulation play an important role in the inflammation observed in SCD via its effects on monocytes.