Leg ulcers are a debilitating complication of patients with sickle cell disease, and their frequency in North America was reported to be 2.5% by the Cooperative Study of Sickle Cell Disease more than 20 years ago. We sought to determine if the frequency of leg ulcers in sickle cell patients in the United States had declined and to assess which treatments providers use most commonly. We sent an e-mail survey to health professionals belonging to the national Sickle Cell Adult Provider Network. Responses were obtained from 31 of them (26.0%). Most of them (96.0%) reported having some patients with leg ulcers. Providers reported a total of 185 patients with active leg ulcers and 224 in the previous 5 years, for a total of 409 patients. Hb SS (homozygous sickle cell anemia) was the most common genotype of affected individuals, followed by Hb SC (double heterozygote for Hb S [β6(A3)Glu→Val, GAG>GTG; HBB: c.20A>T] and Hb C [β6(A3)Glu→Lys, GAG>AAG; HBB: c.19G>A]). Males showed a 2:1 predominance. Two-thirds of patients were treated with either hydroxyurea (HU) or transfusion therapy and most used compression stockings and topical therapies as directed by wound care services. We conclude that leg ulcers continue to be a debilitating complication of young adults with sickle cell disease, despite improved supportive care and the widespread use of disease modifying agents such HU and transfusion. While some providers offer office-based ulcer care, the majority prefer specialty consultation including podiatry, plastic surgery and dermatology. Despite their frequency, there is no clear consensus among providers as to the best treatment.
Leg Ulcers; Sickle cell disease; Hydroxyurea (HU); Survey
We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate and measure scheme. This is possible by using the fluorescent sterol, dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE non-fluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of non-radiochemical assays to obtain accurate and robust measurement of LCAT esterification activity.
dehydroergosterol; dehydroergosteryl ester; cholesterol oxidase; apolipoprotein A-I mimetic; amphipathic peptide; sickle cell disease
Frequent painful vaso-occlusive crises (VOCs) were associated with mortality in the Cooperative Study of Sickle Cell Disease (CSSCD) over twenty years ago. Modern therapies for sickle cell anemia (SCA) like hydroxyurea are believed to have improved overall patient survival. The current study sought to determine the relevance of the association between more frequent VOCs and death and its relative impact upon overall mortality compared to other known risk factors in a contemporary adult SCA cohort.
Two hundred sixty four SCA adults were assigned into two groups based on patient reported outcomes for emergency department (ED) visits or hospitalizations for painful VOC treatment during the 12 months prior to evaluation.
Higher baseline hematocrit (p = 0.0008), ferritin (p = 0.005), and HDL cholesterol (p = 0.01) were independently associated with 1 or more painful VOCs requiring an ED visit or hospitalization for acute pain. During a median follow-up of 5 years, mortality was higher in the ED visit/hospitalization group (relative risk [RR] 2.68, 95% CI 1.1-6.5, p = 0.03). Higher tricuspid regurgitatant jet velocity (TRV) (RR 2.41, 95% CI 1.5-3.9, p < 0.0001), elevated ferritin (RR 4.00, 95% CI 1.8-9.0, p = 0.001) and lower glomerular filtration rate (RR=2.73, 95% CI 1.6-4.6, p < 0.0001) were also independent risk factors for mortality.
Severe painful VOCs remain a marker for SCA disease severity and premature mortality in a modern cohort along with other known risk factors for death including high TRV, high ferritin and lower renal function. The number of patient reported pain crises requiring healthcare utilization is an easily obtained outcome that could help to identify high risk patients for disease modifying therapies.
ClinicalTrials.gov NCT00011648 http://clinicaltrials.gov/
Vascular dysfunction is an important pathophysiologic manifestation of sickle cell disease (SCD), a condition that increases risk of pulmonary hypertension and stroke. We hypothesized that infrared (IR) imaging would detect changes in cutaneous blood flow reflective of vascular function. We performed IR imaging and conventional strain gauge plethysmography in twenty-five adults with SCD at baseline and during intra-arterial infusions of an endothelium-dependent vasodilator acetylcholine (ACh), an endothelium-independent vasodilator sodium nitroprusside (SNP), and a NOS inhibitor L-NMMA. Skin temperature measured by IR imaging increased in a dose-dependent manner to graded infusions of ACh (+1.1° C, p < 0.0001) and SNP (+0.9° C, p < 0.0001), and correlated with dose-dependent increases in forearm blood flow (ACh: +19.9 mL/min/100mL, p < 0.0001; rs = 0.57, p = 0.003; SNP: +8.6 mL/min/100mL, p < 0.0001; r = 0.70, p = 0.0002). Although IR measurement of skin temperature accurately reflected agonist-induced increases in blood flow, it was less sensitive to decreases in blood flow caused by NOS inhibition. Baseline forearm skin temperature measured by IR imaging correlated significantly with baseline forearm blood flow (31.8±0.2° C, 6.0±0.4 mL/min/100mL; r = 0.58, p = 0.003), and appeared to represent a novel biomarker of vascular function. It predicted a blunted blood flow response to SNP (r = −0.61, p = 0.002), and was independently associated with a marker of pulmonary artery pressure, as well as hemoglobin level, diastolic blood pressure, homocysteine, and cholesterol (R2 = 0.84, p < 0.0001 for the model). IR imaging of agonist-stimulated cutaneous blood flow represents a less cumbersome alternative to plethysmography methodology. Measurement of baseline skin temperature by IR imaging may be a useful new marker of vascular risk in adults with SCD.
Plethysmography; Sickle cell; Temperature; Endothelium; Smooth muscle; Skin; Hemoglobin; Acetylcholine; Nitroprusside; L-NMMA
Rationale: An increased tricuspid regurgitation jet velocity (TRV > 2.5 m/s) and pulmonary hypertension defined by right heart catheterization both independently confer increased mortality in sickle cell disease (SCD).
Objectives: We explored the usefulness of peripheral blood mononuclear cell–derived gene signatures as biomarkers for an elevated TRV in SCD.
Methods: Twenty-seven patients with SCD underwent echocardiography and peripheral blood mononuclear cell isolation for expression profiling and 112 patients with SCD were genotyped for single-nucleotide polymorphisms.
Measurements and Main Results: Genome-wide gene and miRNA expression profiles were correlated against TRV, yielding 631 transcripts and 12 miRNAs. Support vector machine analysis identified a 10-gene signature including GALNT13 (encoding polypeptide N-acetylgalactosaminyltransferase 13) that discriminates patients with and without increased TRV with 100% accuracy. This finding was then validated in a cohort of patients with SCD without (n = 10) and with pulmonary hypertension (n = 10, 90% accuracy). Increased TRV-related miRNAs revealed strong in silico binding predictions of miR-301a to GALNT13 corroborated by microarray analyses demonstrating an inverse correlation between their expression. A genetic association study comparing patients with an elevated (n = 49) versus normal (n = 63) TRV revealed five significant single-nucleotide polymorphisms within GALNT13 (P < 0.005), four trans-acting (P < 2.1 × 10−7) and one cis-acting (P = 0.6 × 10−4) expression quantitative trait locus upstream of the adenosine-A2B receptor gene (ADORA2B).
Conclusions: These studies validate the clinical usefulness of genomic signatures as potential biomarkers and highlight ADORA2B and GALNT13 as potential candidate genes in SCD-associated elevated TRV.
microarray; candidate gene approach; eQTL; pulmonary hypertension
Thermal representations on the surface of a human forearm of underlying perforator vessels have previously been mapped via recovery-enhanced infrared imaging, which is performed as skin blood flow recovers to baseline levels following cooling of the forearm. We noted that the same vessels could also be observed during reactive hyperaemia tests after complete 5-min occlusion of the forearm by an inflatable cuff. However, not all subjects showed vessels with acceptable contrast. Therefore, we applied a thermographic signal reconstruction algorithm to reactive hyperaemia testing, which substantially enhanced signal-to-noise ratios between perforator vessels and their surroundings, thereby enabling their mapping with higher accuracy and a shorter occlusion period.
thermographic signal reconstruction; infrared imaging; reactive hyperemia; perforator vessels; blood flow regulation; skin microcirculation
Priapism is a familiar problem to hematologists, well known for its association with sickle cell disease. It also occurs in a variety of other hematological illnesses, nearly all forms of congenital hemolytic anemia, including other hemoglobinopathies and red blood cell membranopathies and enzymopathies.
Provide urologists with a comprehensive review of priapism in sickle cell disease, with an emphasis on the perspective of a practicing hematologist.
Medline searches through July 2010 were conducted using the terms priapism, erectile dysfunction, and sickle cell.
Main Outcome Measure
Expert opinion was based on review of the medical literature related to this subject matter.
In men with sickle cell disease, large epidemiological studies have linked the risk of priapism to clinical markers of the severity of intravascular hemolysis. Extracellular hemoglobin and arginase released during hemolysis has been implicated in reducing nitric oxide bioavailability, although the relevance of hemolysis to vascular dysfunction has been challenged by some scientists. Consistent with the role of impairment of the nitric oxide axis, mice genetically deficient in nitric oxide production have also been shown to develop priapic activity. Provocative new data indicates that hemolysis-linked dysregulation of adenosine signaling in the penis contributes to priapism in sickle cell mice. Serious questions have arisen regarding the efficacy of mainstays of textbook dogma for treatment of acute severe priapism, including intravenous fluids, alkalinization and exchange transfusion, and there is increasing acceptance for early aspiration and irrigation of the corpus cavernosum.
For sickle cell patients with recurrent priapism, there is very limited evidence for a medical prophylaxis role for hydroxyurea, etilefrine, pseudoephedrine, leuprolide, sildenafil, and other agents. Recent publications have highlighted nitric oxide and adenosine signal transduction pathways as worthy of additional research. Research and clinical management of sickle cell priapism is strengthened by multidisciplinary collaboration between hematologists and urologists.
Priapism; Ischemic Priapism; Stuttering Priapism; Erectile Dysfunction; Sickle cell; Hematology and Priapism
Platelets are activated in sickle cell disease (SCD), and particularly during vaso-occlusive episodes (VOE). Thrombospondin-1 (TSP1), a major secretory product of activated platelets, is increased in the circulation in VOE and binds to sickle red blood cells (RBC) promoting vascular adhesion. Thus, we hypothesized that TSP1 may represent a plasma biomarker of disease severity in SCD. We tested the plasma collected from patients in steady state (n = 27) and VOE (n = 14), as well as healthy controls (n = 17) at the University of Pittsburgh Medical Center (UPMC), and from patients in steady state enrolled in the walk-PHaSST clinical trial (n = 483). We found that TSP1 levels were increased in VOE in the UPMC cohort. Among steady-state patients at UPMC, TSP1 values correlated positively with lifetime history of acute chest syndrome (r = 0.72, P < 0.0001) and hemoglobin concentration (r = 0.49, P = 0.01), and negatively with markers of hemolysis, such as LDH (r = −0.50, P = 0.009). Analysis of the walk-PHaSST cohort also showed a positive association between TSP1 levels and hydroxyurea use (r = 0.14, P = 0.003), and confirmed the negative associations with the severity of hemolysis. Our results suggest that TSP1 levels are associated with more VOE, hydroxyurea use and lower rates of hemolysis. High TSP1 concentrations may indicate higher risk of the viscosity/vaso-occlusion phenotype of SCD.
Pulmonary hypertension (PH) in sickle cell disease (SCD) is an emerging and important clinical problem. In a single-institution adult cohort of 365 patients, we investigated lipid and lipoprotein levels and their relationship to markers of intravascular hemolysis, vascular dysfunction and PH. In agreement with prior studies, we confirm significantly decreased plasma levels of total cholesterol, high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) in SCD vs. ethnically-matched healthy controls. Several cholesterol parameters correlate significantly with markers of anemia, but not endothelial activation or PH. More importantly, serum triglyceride levels are significantly elevated in SCD compared to controls. Elevated triglyceride levels correlate significantly with markers of hemolysis (lactate dehydrogenase and arginase; both p<0.0005), endothelial activation (soluble E-selectin, p<0.0001; soluble P-selectin, p=0.02; soluble vascular cell adhesion molecule-1, p=0.01), inflammation (leukocyte count, p=0.0004; erythrocyte sedimentation rate, p=0.02) and PH (amino-terminal brain natriuretic peptide, p=0.002; prevalence of elevated tricuspid regurgitant velocity (TRV), p<0.001). In a multivariate analysis, triglyceride levels correlate independently with elevated TRV (p=0.002). Finally, forearm blood flow studies in adult patients with SCD demonstrate a significant association between increased triglyceride/HDL-C ratio and endothelial dysfunction (p<0.05). These results characterize elevated plasma triglyceride levels as a potential risk factor for PH in SCD.
The breakdown of senescent or defective red blood cells releases red cell contents, especially hemoglobin, which scavenges nitric oxide (NO) and decomposes to heme and free iron. These are potent oxidants, all of which have promoted the evolution of inducible and vasculoprotective compensatory pathways to rapidly clear and detoxify hemoglobin, heme and iron. Chronic hemolytic red cell disorders as diverse as sickle cell disease, thalassemia, unstable hemoglobinopathy, cytoskeletal defects and enzymopathies have been linked to a clinical constellation of pulmonary hypertension, priapism, leg ulceration and possibly cerebrovascular disease and thrombosis. Besides free hemoglobin, hemolysis has been associated with extracellular arginase that limits substrate availability to NO synthase, endogenous inhibitors of NO synthase activity, and inappropriate activation of hemostatic pathways. This article reviews the hemolytic disorders that have been reported to manifest vascular complications, and explores the speculative possibility that hemolysis mediates some of the vascular complications of inflammation and diabetes.
Sickle cell disease; Thalassemia; Vasculopathy; Hemolysis; Nitric oxide
Diet-induced weight loss in women may be associated with decreases not only in plasma levels of low-density lipoprotein cholesterol (LDL-C), but also in high-density lipoprotein cholesterol (HDL-C). Whether a decrease in HDL-C is associated with altered HDL function is unknown.
One hundred overweight or obese women (age 46 ± 11 years, 60 black; 12 diabetic) were enrolled in the 6 month program of reduced fat and total energy diet and low-intensity exercise. Serum cholesterol efflux capacity was measured in 3H-cholesterol-labeled BHK cells expressing ABCA1, ABCG1 or SR-B1 transporters and incubated with 1% apolipoprotein B-depleted serum. Antioxidant properties of HDL were estimated by paraoxonase-1 (PON1) activity and oxygen radical absorbance capacity (ORAC). Endothelial nitric oxide synthase (eNOS) activation was measured by conversion of L-arginine to L-citrulline in endothelial cells incubated with HDL from 49 subjects. Participants achieved an average weight loss of 2.2 ± 3.9 kg (P<0.001), associated with reductions in both LDL-C (−6 ± 21 mg/dL, P=0.004) and HDL-C (−3 ± 9 mg/dL, P=0.016). Cholesterol efflux capacity by the ABCA1 transporter decreased by 10% (P=0.006); efflux capacities by the ABCG1 and SR-B1 transporters were not significantly altered. ORAC decreased by 15% (P=0.018); neither PON1 activity nor eNOS activation was significantly altered by reduction in HDL-C. Findings were similar for diabetic and non-diabetic subjects.
Diet-induced weight loss in overweight or obese women is associated with a decrease in HDL-C levels, but overall HDL function is relatively spared, suggesting that decrease in HDL-C in this setting is not deleterious to cardiovascular risk.
Subjects at risk for atherosclerosis may have dysfunctional high-density lipoprotein (HDL) despite normal cholesterol content in plasma. We considered whether efflux of excess cellular cholesterol to HDL from obese subjects is associated with impaired arterial endothelial function, a biomarker of cardiovascular risk. Fifty-four overweight (body mass index [BMI] 25 – 29.9 kg/m2) or obese women (BMI ≥ 30 kg/m2), age 46 ± 11 years, were enrolled in a worksite wellness program. HDL cholesterol averaged 57 ± 17 mg/dL and was inversely associated with BMI (r= −0.419, P= 0.002). Endothelial function was assessed by brachial artery flow-mediated dilation (FMD). Cholesterol efflux from 3H-cholesterol-labeled BHK cells transfected with the ATP-binding cassette transporter 1 (ABCA1) showed 8.2 to 22.5% cholesterol efflux over 18 hours when incubated with 1% serum and was positively correlated with FMD (P <0.05), especially in the 34 subjects with BMI ≥ 30 kg/m2 (r= 0.482, P= 0.004). This relation was independent of age, HDL or low-density lipoprotein cholesterol (LDL) concentrations in plasma, blood pressure or insulin resistance by stepwise multiple regression analysis (β= 0.31, R2= 0.21, P= 0.007). Nitration of apoA-I tyrosine residues (by sandwich ELISA) was significantly higher in women with BMI ≥ 30 kg/m2 and the lowest cholesterol efflux than in women with BMI 25 – 29.9 kg/m2 and the highest cholesterol efflux (P= 0.01). We conclude that decreased cholesterol efflux via the ABCA1 transporter is associated with increased nitration of apoA-I in HDL and is an independent predictor of impaired endothelial function in women with BMI ≥ 30 kg/m2. This finding suggests that functional measures of HDL may be better markers for cardiovascular risk than HDL cholesterol levels in this population.
Advances in computer-aided diagnosis (CAD) systems have shown the benefits of using computer-based techniques to obtain quantitative image measurements of the extent of a particular disease. Such measurements provide more accurate information that can be used to better study the associations between anatomical changes and clinical findings. Unfortunately, even with the use of quantitative image features, the correlations between anatomical changes and clinical findings are often not apparent and definite conclusions are difficult to reach. This paper uses nonparametric exploration techniques to demonstrate that even when the associations between two-variables seems weak, advanced properties of the associations can be studied and used to better understand the relationships between individual measurements.
This paper uses quantitative imaging findings and clinical measurements of 85 patients with pulmonary fibrosis to demonstrate the advantages of non-linear dependency analysis. Results show that even when the correlation coefficients between imaging and clinical findings seem small, statistical measurements such as the maximum asymmetry score (MAS) and maximum edge value (MEV) can be used to better understand the hidden associations between the variables.
Erythropoietin is being used more widely in the management of sickle cell disease (SCD, inclusive of homozygous sickle beta, SS, and compound heterozygous sickle beta thalassemia, Sβ0 thal), often in conjunction with hydroxyurea (HU). Herein, we summarize the published experience with erythropoietin use in SCD, including 39 patients (SS, n=30; Sβ0 thal, n=9) who were treated between 1990 and 1996; to which we add 13 patients with Sickle Syndromes (SS, n=12, compound heterozygous SC disease, n=1) who were treated with erythropoietin or darbopoietin (here cumulatively referred to as EPO) at the National Institutes of Health (NIH) since 2002. The dose range of erythropoietin for SCD in the published series, at a median of >200 Units/Kg/dose, is higher than is used in end-stage renal disease. The median duration of erythropoietin therapy in the published series was ≥3 months, with minimal reported side-effects. At the NIH, the median age of Sickle Syndrome patients who received EPO was 51 (24 to 70) years; 12/13 patient had sickle-associated pulmonary hypertension. 11/13 patients were treated with both HU and EPO for > 4 months (median of 11 months on EPO) without complication. 5/13 patients (all HbSS) with pulmonary hypertension were given EPO for reticulocytopenia (<100,000/μL) on HU; 5/13 patients (all HbSS), with pulmonary hypertension, were given EPO and HU concurrently, in light of an estimated glomerular filtration rate of <80 ml/minute. 3/13 patients (2 Hb SS, 1 HbSC) were treated with EPO for miscellaneous reasons. Hematologic responses, detailed herein, were promising. Our experience suggests that EPO may be safe in SCD, when used in conjunction with HU; EPO therapy could allow more aggressive HU dosing in high-risk SCD patients and in the setting of mild renal insufficiency, common to the aging sickle cell population. We outline our current therapeutic strategy for EPO use in SCD.
Sickle Cell Trait (HbAS), the heterozygous state for the sickle hemoglobin beta globin gene is carried by as many as 100 million individuals including up to 25% of the population in some regions of the world (World Health Organization, Provisional agenda item 4.8, EB117/34 (22 December 2005) or World Health Organization, Provisional agenda item 11.4 (24 April 2006)). Persons with HbAS have some resistance to falciparum malaria infection in early childhood (Piel FB, Patil AP, Howes RE, et al., Nat Commun 2010;1104:1–7 and Aidoo M, Terlouw DJ, Kolczak M, et al., Lancet 2002;359:1311–1312) and as a result individuals with HbAS living in malarial endemic regions of Africa have a survival advantage over individuals with HbAA. Reports from the US emphasize possible health risks for individuals with HbAS including increased incidence of renal failure and malignancy, thromboembolic disorders, splenic infarction as a high altitude complication, and exercise-related sudden death. The National Heart, Lung, and Blood Institute, National Institutes of Health convened a workshop in Bethesda, Maryland on June 3–4, 2010, Framing the Research Agenda for Sickle Cell Trait, to review the clinical manifestations of HbAS, discuss the exercise-related sudden death reports in HbAS, and examine the public health, societal, and ethical implications of policies regarding HbAS. The goal of the workshop was to identify potential research questions to address knowledge gaps.
Pulmonary hypertension is a common complication of sickle cell disease (SCD) and a risk factor for early death. Hemolysis may participate in its pathogenesis by limiting nitric oxide (NO) bioavailability and producing vasculopathy. We hypothesized that hemoglobin mutations that diminish hemolysis in SCD would influence pulmonary hypertension susceptibility. Surprisingly, coincident α-thalassemia (OR = 0.95, 95% CI = 0.46 – 1.94, P = NS) was not associated with pulmonary hypertension susceptibility in homozygous SCD. However, pulmonary hypertension cases were less likely to have hemoglobin SC (Odds Ratio [OR] = 0.18, 95% confidence interval [CI] = 0.06 to 0.51, P = 0.0005) or Sβ+ thalassemia (OR = 0.25, 95% CI = 0.06 to 1.16, P = 0.10). These compound heterozygotes may be protected from pulmonary hypertension because of reduced levels of intravascular hemolysis, but develop this complication at a lower rate possibly due to the presence of non-hemolytic risk factors such as renal dysfunction, iron overload and advancing age. Despite this protective association, patients with SC who did develop pulmonary hypertension remained at significant risk for death during 49 months of follow-up (Hazard Ratio=8.20, P=0.0057).
Hemoglobin (Hb) is crucial to the function of the red blood cell. However, when it is released during intravascular hemolysis from the cell into blood plasma, it produces a state of NO depletion, oxidant stress, and vascular dysfunction, including hypertension. In their study reported in this issue of the JCI, Boretti and colleagues used canine and guinea pig models to demonstrate that pharmacological doses of glucocorticoid can increase the plasma levels of haptoglobin (Hp), the principal plasma-binding protein for free Hb (see the related article beginning on page 2271). Hp prevented Hb-induced hypertension and the generation of oxidant damage to the kidney. Neutralization of free Hb appears to be part of the downstream antiinflammatory properties of glucocorticoid.