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2.  Risk Factors for Death in 632 Patients with Sickle Cell Disease in the United States and United Kingdom 
PLoS ONE  2014;9(7):e99489.
Background
The role of pulmonary hypertension as a cause of mortality in sickle cell disease (SCD) is controversial.
Methods and Results
We evaluated the relationship between an elevated estimated pulmonary artery systolic pressure and mortality in patients with SCD. We followed patients from the walk-PHaSST screening cohort for a median of 29 months. A tricuspid regurgitation velocity (TRV)≥3.0 m/s cuttof, which has a 67–75% positive predictive value for mean pulmonary artery pressure ≥25 mm Hg was used. Among 572 subjects, 11.2% had TRV≥3.0 m/sec. Among 582 with a measured NT-proBNP, 24.1% had values ≥160 pg/mL. Of 22 deaths during follow-up, 50% had a TRV≥3.0 m/sec. At 24 months the cumulative survival was 83% with TRV≥3.0 m/sec and 98% with TRV<3.0 m/sec (p<0.0001). The hazard ratios for death were 11.1 (95% CI 4.1–30.1; p<0.0001) for TRV≥3.0 m/sec, 4.6 (1.8–11.3; p = 0.001) for NT-proBNP≥160 pg/mL, and 14.9 (5.5–39.9; p<0.0001) for both TRV≥3.0 m/sec and NT-proBNP≥160 pg/mL. Age >47 years, male gender, chronic transfusions, WHO class III–IV, increased hemolytic markers, ferritin and creatinine were also associated with increased risk of death.
Conclusions
A TRV≥3.0 m/sec occurs in approximately 10% of individuals and has the highest risk for death of any measured variable.
The study is registered in ClinicalTrials.gov with identifier
NCT00492531
doi:10.1371/journal.pone.0099489
PMCID: PMC4079316  PMID: 24988120
3.  Clinical correlates of acute pulmonary events in children and adolescents with sickle cell disease* 
Objectives
We aimed to identify risk factors for acute pulmonary events in children and adolescents in the Pulmonary Hypertension and the Hypoxic Response in SCD (PUSH) study.
Methods
Patients with hemoglobin SS (n=376) and other sickle cell genotypes (n=127) aged 3-20 years were studied at four centers in a cross-sectional manner. A sub-group (n=293) was followed for a median of 21 months (range 9-35).
Results
A patient-reported history of one or more acute pulmonary events, either acute chest syndrome (ACS) or pneumonia, was obtained in 195 hemoglobin SS patients (52%) and 51 patients with other genotypes (40%). By logistic regression, history of acute pulmonary events was independently associated with patient-reported history of asthma (p<0.0001), older age (p=0.001), >3 severe pain episodes in the preceding 12 months (p=0.002), higher tricuspid regurgitation velocity (TRV) (p=0.028), and higher white blood cell (WBC) count (p=0.043) among hemoglobin SS patients. History of acute pulmonary events was associated with >3 severe pain episodes (p=0.009) among patients with other genotypes. During follow-up, 43 patients (15%) had at least one new ACS episode including 11 without a baseline history of acute pulmonary events. History of acute pulmonary events (odds ratio 5.4; p<0.0001) and younger age (odds ratio 0.9; p=0.010) were independently associated with developing a new episode during follow-up.
Conclusions
Asthma history, frequent pain and higher values for TRV and WBC count were independently associated with history of acute pulmonary events in hemoglobin SS patients and frequent pain was associated in those with other genotypes. Measures to reduce pain episodes and control asthma may help to decrease the incidence of acute pulmonary events in SCD.
doi:10.1111/ejh.12118
PMCID: PMC3689858  PMID: 23560516
sickle cell disease; acute chest syndrome; vaso-occlusive crisis; asthma; pain
4.  Computed tomography correlates with cardiopulmonary hemodynamics in pulmonary hypertension in adults with sickle cell disease 
Pulmonary Circulation  2014;4(2):319-329.
Our objective was to determine whether computed tomography angiography (CTA) measurements of pulmonary artery size can noninvasively assess hemodynamics and diagnose pulmonary hypertension (PH) secondary to sickle cell disease (SCD). Twenty SCD patients with confirmed PH were compared with 20 matched controls. Diameters of the pulmonary artery trunk and branches were measured with CTA and a semiautomatic segmentation algorithm. Measurements were normalized by body size and correlated (Spearman rank) with hemodynamic markers from right-heart catheterization. Receiver operating characteristic (ROC) curves were used to investigate the role of pulmonary artery sizes in diagnosing PH. Analysis of pulmonary artery sizes adjusted for body surface area (BSA) resulted in the most significant discrimination between subjects with PH secondary to SCD and controls (P < 0.001); PH was diagnosed accurately with an area under the ROC curve of 0.99. There was significant correlation between pulmonary artery sizes and body mass index (BMI) and BSA only in controls (r = 0.46–0.68, P < 0.04 for all). The most significant correlations with hemodynamic markers were found between BMI-adjusted pulmonary artery sizes and high systolic pulmonary arterial pressure, high pulmonary vascular resistance, high systemic vascular resistance, and low cardiac output (r = 0.47, 0.62, 0.61, and 0.66, respectively; P < 0.04 for all). BMI-adjusted CTA measures of the pulmonary artery relate to high pulmonary vascular resistance and reduced cardiac output in patients with SCD and PH. CTA with quantitative image analysis is a powerful noninvasive diagnostic tool for PH in SCD and shows promise as estimator of hemodynamic markers.
doi:10.1086/675997
PMCID: PMC4070777  PMID: 25006451
CT angiography; cardiopulmonary hemodynamics; pulmonary hypertension; sickle cell disease; arterial size; quantitative imaging
5.  Elevated sphingosine-1-phosphate promotes sickling and sickle cell disease progression 
The Journal of Clinical Investigation  2014;124(6):2750-2761.
Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S1P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S1P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S1P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte sphingosine kinase 1 (SPHK1) underlies sickling and disease progression by increasing S1P levels in the blood. Additionally, we observed elevated SPHK1 activity in erythrocytes and increased S1P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK1-mediated production of S1P on sickling that was independent of S1P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK1-mediated elevation of S1P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD.
doi:10.1172/JCI74604
PMCID: PMC4089467  PMID: 24837436
6.  Understanding the Erythrocyte Storage Lesion 
Anesthesiology  2012;117(6):1159-1161.
doi:10.1097/ALN.0b013e318272d8ac
PMCID: PMC3504349  PMID: 23168421
7.  Anemia, Age, Desaturation and Impaired Neurocognition in Sickle Cell Anemia 
Pediatric blood & cancer  2012;59(5):773-774.
doi:10.1002/pbc.24276
PMCID: PMC3442134  PMID: 22949104
8.  Vasculopathy, inflammation, and blood flow in leg ulcers of patients with sickle cell anemia 
Chronic leg ulcers are frequent and debilitating complications of sickle cell anemia. Inadequate blood supply has been postulated to be an important factor in their occurrence and delayed healing. Little is known about their microcirculatory and histopathological changes. We evaluated the microcirculation of lower extremity ulcers with laser speckle contrast imaging and infrared thermography and obtained clinical and laboratory characteristics in 18 adults with sickle cell anemia and chronic leg ulcers. Skin biopsies were obtained in four subjects. Subjects had markers of severe disease, anemia, high degree of hemolysis, inflammation, and thrombophilia. The highest blood flow was present in the ulcer bed, progressively less in the immediate periwound area, and an unaffected control skin area in the same extremity. Microscopic examination showed evidence of venostasis, inflammation, and vasculopathy. Blood vessels were increased in number, had activated endothelium and evidence of thrombosis/recanalization. High blood flow may be due to chronic inflammation, cutaneous vasodilatation, venostasis, and in situ thrombosis. These changes in skin microcirculation are similar to chronic venous ulcers in the non-sickle cell disease (SCD) population, thus suggesting that leg ulcers may be another end-organ complication with endothelial dysfunction that appears in patients with SCD at a younger age and with higher frequency than in the general population.
doi:10.1002/ajh.23571
PMCID: PMC3946883  PMID: 23963836
9.  LEG ULCERS IN SICKLE CELL DISEASE: CURRENT PATTERNS AND PRACTICES 
Hemoglobin  2013;37(4):10.3109/03630269.2013.789968.
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.
doi:10.3109/03630269.2013.789968
PMCID: PMC3864012  PMID: 23600469
Leg Ulcers; Sickle cell disease; Hydroxyurea (HU); Survey
10.  TRV: A Physiological Biomarker in Sickle Cell Disease 
Pediatric blood & cancer  2011;58(6):831-832.
doi:10.1002/pbc.23399
PMCID: PMC3511045  PMID: 22180092
11.  A Fluorescence Method to Detect and Quantitate Sterol Esterification by Lecithin: Cholesterol Acyltransferase 
Analytical biochemistry  2013;441(1):10.1016/j.ab.2013.06.018.
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.
doi:10.1016/j.ab.2013.06.018
PMCID: PMC3822574  PMID: 23851343
dehydroergosterol; dehydroergosteryl ester; cholesterol oxidase; apolipoprotein A-I mimetic; amphipathic peptide; sickle cell disease
12.  Severe Painful Vaso-Occlusive Crises and Mortality in a Contemporary Adult Sickle Cell Anemia Cohort Study 
PLoS ONE  2013;8(11):e79923.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
Trial Registration
ClinicalTrials.gov NCT00011648 http://clinicaltrials.gov/
doi:10.1371/journal.pone.0079923
PMCID: PMC3818240  PMID: 24224021
13.  Infrared Imaging of Nitric Oxide-Mediated Blood Flow in Human Sickle Cell Disease 
Microvascular research  2012;84(3):262-269.
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.
doi:10.1016/j.mvr.2012.06.011
PMCID: PMC3483464  PMID: 22784510
Plethysmography; Sickle cell; Temperature; Endothelium; Smooth muscle; Skin; Hemoglobin; Acetylcholine; Nitroprusside; L-NMMA
14.  A Novel Molecular Signature for Elevated Tricuspid Regurgitation Velocity in Sickle Cell Disease 
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.
doi:10.1164/rccm.201201-0057OC
PMCID: PMC3443809  PMID: 22679008
microarray; candidate gene approach; eQTL; pulmonary hypertension
15.  Priapism in Sickle Cell Disease: A Hematologist’s Perspective 
Introduction
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.
Aim
Provide urologists with a comprehensive review of priapism in sickle cell disease, with an emphasis on the perspective of a practicing hematologist.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1111/j.1743-6109.2011.02287.x
PMCID: PMC3253142  PMID: 21554552
Priapism; Ischemic Priapism; Stuttering Priapism; Erectile Dysfunction; Sickle cell; Hematology and Priapism
16.  Endothelin receptor antagonists for pulmonary hypertension in adult patients with sickle cell disease 
British journal of haematology  2009;147(5):737-743.
Pulmonary Hypertension is a serious complication of sickle cell disease (SCD), with a high risk of morbidity and mortality. Endothelin-1, a potent vasoconstrictor peptide known to be elevated in SCD, acts through two receptors: ETR-A and ETR-B. Bosentan and ambrisentan are ETR blockers approved for use in primary pulmonary hypertension. We report on the use of ETR blocking agents in a cohort of 14 high-risk adult patients with SCD and pulmonary hypertension. Patients underwent right heart catheterization, 6-min walk test, echocardiogram, physical examination and laboratory studies prior to starting ETR blocking agents. Six patients received bosentan and two ambrisentan as initial therapy. Six additional patients were already on sildenafil when ETR blocking agents were added. Over at least six months of therapy, sequential measurements of 6-min walk distance increased significantly (mean ± standard error baseline 357 ± 22 m, 1–2 months 367 ± 17 m, 3–4 months 387 ± 16 m, 5–6 months 398 ± 18 m, n=12, p<0.05, ANOVA with repeated measures). Nonsignificant downward trends were also observed for amino-terminal brain natriuretic peptide and tricuspid regurgitant velocity. Pulmonary artery mean pressures decreased in the three patients that had a repeat right heart catheterization from a mean of 44 to 38 mmHg, although this was not statistically significant. Adverse events while on ETR blocking therapy were similar to the ones reported on patients with primary PH: increase in serum alanine aminotransferase (2), increased peripheral edema (3), rash (4), and headache. Therapy was stopped in two patients, who were switched to the other ETR blocking agent, with resolution of symptoms. These data suggest preliminary evidence for benefit from the use of bosentan and ambrisentan and supports their use in pulmonary hypertension in SCD.
doi:10.1111/j.1365-2141.2009.07906.x
PMCID: PMC3225273  PMID: 19775299
17.  LIPID LEVELS IN SICKLE-CELL DISEASE ASSOCIATED WITH HEMOLYTIC SEVERITY, VASCULAR DYSFUNCTION AND PULMONARY HYPERTENSION 
British journal of haematology  2010;149(3):436-445.
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.
doi:10.1111/j.1365-2141.2010.08109.x
PMCID: PMC3212812  PMID: 20230401
18.  Pleiotropic Effects of Intravascular Hemolysis on Vascular Homeostasis 
British journal of haematology  2009;148(5):690-701.
Summary
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.
doi:10.1111/j.1365-2141.2009.08004.x
PMCID: PMC3210728  PMID: 19958359
Sickle cell disease; Thalassemia; Vasculopathy; Hemolysis; Nitric oxide
19.  Reconstruction of Thermographic Signals to Map Perforator Vessels in Humans 
Quantitative infrared thermography journal  2012;9(2):10.1080/17686733.2012.737157.
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.
doi:10.1080/17686733.2012.737157
PMCID: PMC3650860  PMID: 23667389
thermographic signal reconstruction; infrared imaging; reactive hyperemia; perforator vessels; blood flow regulation; skin microcirculation
20.  Expression of Regulatory Platelet MicroRNAs in Patients with Sickle Cell Disease 
PLoS ONE  2013;8(4):e60932.
Background
Increased platelet activation in sickle cell disease (SCD) contributes to a state of hypercoagulability and confers a risk of thromboembolic complications. The role for post-transcriptional regulation of the platelet transcriptome by microRNAs (miRNAs) in SCD has not been previously explored. This is the first study to determine whether platelets from SCD exhibit an altered miRNA expression profile.
Methods and Findings
We analyzed the expression of miRNAs isolated from platelets from a primary cohort (SCD = 19, controls = 10) and a validation cohort (SCD = 7, controls = 7) by hybridizing to the Agilent miRNA microarrays. A dramatic difference in miRNA expression profiles between patients and controls was noted in both cohorts separately. A total of 40 differentially expressed platelet miRNAs were identified as common in both cohorts (p-value 0.05, fold change>2) with 24 miRNAs downregulated. Interestingly, 14 of the 24 downregulated miRNAs were members of three families - miR-329, miR-376 and miR-154 - which localized to the epigenetically regulated, maternally imprinted chromosome 14q32 region. We validated the downregulated miRNAs, miR-376a and miR-409-3p, and an upregulated miR-1225-3p using qRT-PCR. Over-expression of the miR-1225-3p in the Meg01 cells was followed by mRNA expression profiling to identify mRNA targets. This resulted in significant transcriptional repression of 1605 transcripts. A combinatorial approach using Meg01 mRNA expression profiles following miR-1225-3p overexpression, a computational prediction analysis of miRNA target sequences and a previously published set of differentially expressed platelet transcripts from SCD patients, identified three novel platelet mRNA targets: PBXIP1, PLAGL2 and PHF20L1.
Conclusions
We have identified significant differences in functionally active platelet miRNAs in patients with SCD as compared to controls. These data provide an important inventory of differentially expressed miRNAs in SCD patients and an experimental framework for future studies of miRNAs as regulators of biological pathways in platelets.
doi:10.1371/journal.pone.0060932
PMCID: PMC3625199  PMID: 23593351
21.  Plasma thrombospondin-1 is increased during acute sickle cell vaso-occlusive events and associated with acute chest syndrome, hydroxyurea therapy, and lower hemolytic rates 
American journal of hematology  2012;87(3):326-330.
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.
doi:10.1002/ajh.22274
PMCID: PMC3619659  PMID: 22318901
22.  Diet-induced Weight Loss in Overweight or Obese Women and Changes in High-density Lipoprotein Levels and Function 
Obesity (Silver Spring, Md.)  2012;20(10):2057-2062.
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.
doi:10.1038/oby.2012.56
PMCID: PMC3374067  PMID: 22402736
24.  Endogenous Nitric Oxide Synthase Inhibitors in Sickle Cell Disease: Abnormal Levels and Correlations with Pulmonary Hypertension, Desaturation, Hemolysis, Organ Dysfunction and Death 
British journal of haematology  2008;145(4):506-513.
Pulmonary hypertension (PH) in patients with sickle cell disease (SCD) is linked to intravascular hemolysis, impaired nitric oxide bioavailability, renal dysfunction, and early mortality. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthases (NOS), is associated with vascular disease in other populations. We determined the plasma concentrations for several key arginine metabolites and their relationships to clinical variables in 177 patients with SCD and 29 control subjects: ADMA, symmetric dimethylarginine (SDMA), NG-monomethyl L-arginine (L-NMMA), N-omega-hydroxy-L-arginine (NOHA), arginine and citrulline. The median ADMA was significantly higher in SCD than controls (0.94 vs. 0.31 μmol/L, p<0.001). Patients with homozygous SCD had a remarkably lower ratio of arginine to ADMA (50 vs. 237, p<0.001). ADMA correlated with markers of hemolysis, low oxygen saturation and soluble adhesion molecules. PH was associated with high levels of ADMA and related metabolites. Higher ADMA level was associated with early mortality, remaining significant in a multivariate analysis. Subjects with homozygous SCD have high systemic levels of ADMA, associated with PH and early death, implicating ADMA as a functional NOS inhibitor in these patients. These defects and others converge on the nitric oxide pathway in homozygous SCD with vasculopathy.
doi:10.1111/j.1365-2141.2009.07658.x
PMCID: PMC2935697  PMID: 19344390
Sickle Cell Disease; ADMA; SDMA; NOHA; Arginine; Pulmonary Hypertension
25.  High-Density Lipoprotein Cholesterol Efflux, Nitration of Apolipoprotein A-I and Endothelial Function in Obese Women 
The American Journal of Cardiology  2011;109(4):527-532.
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.
doi:10.1016/j.amjcard.2011.10.008
PMCID: PMC3270119  PMID: 22105786

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