Darlene Allen

Lipid levels in sickle-cell disease associated with haemolytic severity, vascular dysfunction and pulmonary hypertension

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 haemolysis, 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 versus ethnically‐matched healthy controls. Several cholesterol parameters correlated significantly with markers of anaemia, but not endothelial activation or PH. More importantly, serum triglyceride levels were significantly elevated in SCD compared to controls. Elevated triglyceride levels correlated significantly with markers of haemolysis (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 (leucocyte 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 correlated independently with elevated TRV (P = 0·002). Finally, forearm blood flow studies in adult patients with SCD demonstrated 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.

Severe painful vaso-occlusive crises and mortality in a contemporary adult sickle cell anemia cohort study.

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/

Iron, inflammation, and early death in adults with sickle cell disease

Rationale: Patients with sickle cell disease (SCD) have markers of chronic inflammation, but the mechanism of inflammation and its relevance to patient survival are unknown. Objective: To assess the relationship between iron, inflammation, and early death in SCD. Methods and Results: Using peripheral blood mononuclear cell transcriptome profile hierarchical clustering, we classified 24 patients and 10 controls in clusters with significantly different expression of genes known to be regulated by iron. Subsequent gene set enrichment analysis showed that many genes associated with the high iron cluster were involved in the toll-like receptor system (TLR4, TLR7, and TLR8) and inflammasome complex pathway (NLRP3, NLRC4, and CASP1). Quantitative PCR confirmed this classification and showed that ferritin light chain, TLR4, and interleukin-6 expression were >100-fold higher in patients than in controls (P<0.001). Further linking intracellular iron and inflammation, 14 SCD patients with a ferroportin Q248H variant that causes intracellular iron accumulation had significantly higher levels of interleukin-6 and C-reactive protein compared with 14 matched SCD patients with the wild-type allele (P<0.05). Finally, in a cohort of 412 patients followed for a median period of 47 months (interquartile range, 24–82), C-reactive protein was strongly and independently associated with early death (hazard ratio, 3.0; 95% confidence interval, 1.7–5.2; P<0.001). Conclusions: Gene expression markers of high intracellular iron in patients with SCD are associated with markers of inflammation and mortality. The results support a model in which intracellular iron promotes inflammatory pathways, such as the TLR system and the inflammasome, identifying important new pathways for additional investigation.

A GCH1 haplotype confers sex-specific susceptibility to pain crises and altered endothelial function in adults with sickle cell anemia.

GTP cyclohydrolase (GCH1) is rate limiting for tetrahydrobiopterin (BH4) synthesis, where BH4 is a cofactor for nitric oxide (NO) synthases and aromatic hydroxylases. GCH1 polymorphisms are implicated in the pathophysiology of pain, but have not been investigated in African populations. We examined GCH1 and pain in sickle cell anemia where GCH1 rs8007267 was a risk factor for pain crises in discovery (n = 228; odds ratio [OR] 2.26; P = 0.009) and replication (n = 513; OR 2.23; P = 0.004) cohorts. In vitro, cells from sickle cell anemia subjects homozygous for the risk allele produced higher BH4. In vivo physiological studies of traits likely to be modulated by GCH1 showed rs8007267 is associated with altered endothelial dependent blood flow in females with SCA (8.42% of variation; P = 0.002). The GCH1 pain association is attributable to an African haplotype with where its sickle cell anemia pain association is limited to females (OR 2.69; 95% CI 1.21–5.94; P = 0.01) and has the opposite directional association described in Europeans independent of global admixture. The presence of a GCH1 haplotype with high BH4 in populations of African ancestry could explain the association of rs8007267 with sickle cell anemia pain crises. The vascular effects of GCH1 and BH4 may also have broader implications for cardiovascular disease in populations of African ancestry. Am. J. Hematol. 89:187–193, 2014.

Hydroxyurea-Increased Fetal Hemoglobin Is Associated with Less Organ Damage and Longer Survival in Adults with Sickle Cell Anemia

Background Adults with sickle cell anemia (HbSS) are inconsistently treated with hydroxyurea. Objectives We retrospectively evaluated the effects of elevating fetal hemoglobin with hydroxyurea on organ damage and survival in patients enrolled in our screening study between 2001 and 2010. Methods An electronic medical record facilitated development of a database for comparison of study parameters based on hydroxyurea exposure and dose. This study is registered with ClinicalTrials.gov, number NCT00011648. Results Three hundred eighty-three adults with homozygous sickle cell disease were analyzed with 59 deaths during study follow-up. Cox regression analysis revealed deceased subjects had more hepatic dysfunction (elevated alkaline phosphatase, Hazard Ratio = 1.005, 95% CI 1.003–1.006, p<0.0.0001), kidney dysfunction (elevated creatinine, Hazard Ratio = 1.13, 95% CI 1.00–1.27, p = 0.043), and cardiopulmonary dysfunction (elevated tricuspid jet velocity on echocardiogram, Hazard Ratio = 2.22, 1.23–4.02, p = 0.0082). Sixty-six percent of subjects were treated with hydroxyurea, although only 66% of those received a dose within the recommended therapeutic range. Hydroxyurea use was associated with improved survival (Hazard Ratio = 0.58, 95% CI 0.34–0.97, p = 0.040). This effect was most pronounced in those taking the recommended dose of 15–35 mg/kg/day (Hazard Ratio 0.36, 95% CI 0.17–0.73, p = 0.0050). Hydroxyurea use was not associated with changes in organ function over time. Further, subjects with higher fetal hemoglobin responses to hydroxyurea were more likely to survive (p = 0.0004). While alkaline phosphatase was lowest in patients with the best fetal hemoglobin response (95.4 versus 123.6, p = 0.0065 and 96.1 versus 113.6U/L, p = 0.041 at first and last visits, respectively), other markers of organ damage were not consistently improved over time in patients with the highest fetal hemoglobin levels. Conclusions Our data suggest that adults should be treated with the maximum tolerated hydroxyurea dose, ideally before organ damage occurs. Prospective studies are indicated to validate these findings.

Elevated Transpulmonary Gradient and Cardiac Magnetic Resonance-Derived Right Ventricular Remodeling Predict Poor Outcomes in Sickle Cell Disease

Adults with sickle cell disease have a high prevalence of pulmonary hypertension. This picture is often complicated by concurrent left ventricular diastolic dysfunction and anemia-related changes in hemodynamics. The change in pressure across the pulmonary circulation reflected by the transpulmonary gradient is less clouded by anemia-related adaptations. We characterized the association of elevated transpulmonary gradient with right ventricular structure and function, exercise capacity and mortality. Data from 84 patients (age 41+/-13 years, 55% female [n=46], 82% hemoglobin SS [n=69]) with right heart catheterization from the Bethesda Sickle Cell Cohort were analyzed. Of the 84 patients, forty-one underwent cardiac magnetic resonance imaging within two days of right heart catheterization. Patients with a catheterization-derived transpulmonary gradient ≥12mmHg had more severe symptoms (p=0.013), shorter 6-minute walk distance (p=0.006), lower cardiac index (p<0.001), reduced right ventricular ejection fraction (p=0.002) and cardiac magnetic resonance imaging markers of adverse morphologic adaptation. An RVEF <32% derived from cardiac magnetic resonance predicted decreased survival (HR 3.70, 95% CI 1.04-13.12, p=0.030) and higher New York Heart Association classification (OR 9.29, p=0.018). In a multivariate model controlling for age and phenotype, transpulmonary gradient ≥12mmHg and right ventricular ejection fraction <32% were independently predictive of increased mortality (HR 5.47, 95% CI 1.13-26.42, p=0.035 and HR 5.11, 95% CI 1.13-23.13, p=0.034). Patients with sickle cell disease and elevated transpulmonary gradient have findings of maladaptive RV remodeling on cardiac magnetic resonance imaging. Elevated transpulmonary gradient and cardiac magnetic resonance-derived RV dysfunction independently predict higher mortality in sickle cell disease. Cardiac magnetic resonance may have a useful role in the clinical evaluation and non-invasive prognostication of adults with sickle cell disease and suspected pulmonary hypertension. This clinical trial was registered at clinicaltrials.gov identifier: [NCT00011648][1] NCT00081523, [NCT00023296][2], and [NCT00352430][3]. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00011648&atom=%2Fhaematol%2Fearly%2F2015%2F11%2F13%2Fhaematol.2015.125229.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00023296&atom=%2Fhaematol%2Fearly%2F2015%2F11%2F13%2Fhaematol.2015.125229.atom [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00352430&atom=%2Fhaematol%2Fearly%2F2015%2F11%2F13%2Fhaematol.2015.125229.atom

Abnormal Ventilation–Perfusion Scan is Associated with Pulmonary Hypertension in Sickle Cell Adults

Pulmonary hypertension (PH) in adults with sickle cell disease (SCD) is associated with early mortality. Chronic thromboembolic PH (CTEPH) is an important complication and contributor to PH in SCD but is likely underappreciated. Guidelines recommend ventilation–perfusion (V/Q) scintigraphy as the imaging modality of choice to exclude CTEPH. Data on V/Q scanning are limited in SCD. Our objective was to compare the performance of V/Q scanning with that of CT pulmonary angiography (CTPA) and to report clinical outcomes associated with abnormal V/Q findings. Methods: Laboratory data, echocardiography, 6-min-walk testing, V/Q scanning, CTPA, and right heart catheterization (RHC) were prospectively obtained. High-probability and intermediate-probability V/Q findings were considered to be abnormal. Included for analysis were 142 SCD adults (aged 40.1 ± 13.7 y, 83 women, 87% hemoglobin SS) in a stable state enrolled consecutively between March 13, 2002, and June 8, 2017. Results: V/Q results were abnormal in 65 of 142 patients (45.8%). CTPA was positive for pulmonary embolism in 16 of 60 (26.7%). RHC confirmed PH (mean pulmonary artery pressure ≥ 25 mmHg) in 46 of 64 (71.9%), of whom 34 (73.9%) had abnormal V/Q findings. Among those without PH by RHC (n = 18), 2 of 18 patients had abnormal V/Q findings. Thirty-three patients had a complete dataset (V/Q scanning, CTPA, and RHC); 29 of 33 had abnormal RHC findings, of whom 26 had abnormal V/Q findings, compared with 11 who had abnormal CTPA findings. There was greater concordance between V/Q findings and RHC (κ-value = 0.53; P < 0.001) than between CTPA and RHC (κ-value = 0.13; P = 0.065). The sensitivity and specificity for V/Q scanning was 89.7% and 75.0%, respectively, whereas CTPA had sensitivity of 37.3% and specificity of 100%. Abnormal V/Q finding swere associated with hemodynamic severity (mean pulmonary artery pressure, 35.2 ± 9.6 vs. 26.9 ± 10.5 mm Hg, P = 0.002; transpulmonary gradient, 21.5 ± 9.7 vs. 12.16 ± 11 mmHg, P = 0.005; and pulmonary vascular resistance, 226.5 ± 135 vs. 140.7 ± 123.7 dynes⋅s⋅cm−5, P = 0.013) and exercise capacity (6-min-walk distance, 382.8 ± 122.3 vs. 442.3 ± 110.6 m, P < 0.010). Thirty-four deaths were observed over 15 y. All-cause mortality was higher in the abnormal-V/Q group (21 [61.8%]) than in the normal-V/Q group (13 [38.2%]) (log-rank test, P = 0.006; hazard ratio, 2.54). Conclusion: V/Q scanning is superior to CTPA in detecting thrombotic events in SCD. Abnormal V/Q findings are associated with PH, worse hemodynamics, lower functional capacity, and higher mortality. Despite high sensitivity in detecting CTEPH, V/Q scanning is underutilized. We recommend the use of V/Q scanning in the evaluation of dyspnea in adult SCD patients given the important implications toward management.

GlycA is not a useful biomarker of inflammation in sickle cell disease

Sickle cell disease (SCD) is a multisystemic disorder, the pathology being driven by recurrent inflammation particularly during a vaso‐occlusive crisis. GlycA, a composite measure of protein glycation, is a sensitive biomarker for disorders associated with vascular inflammation. We determined the utility of GlycA as a biomarker of inflammation in SCD.

The platelet NLRP3 inflammasome is upregulated in sickle cell disease via HMGB1/TLR4 and Bruton tyrosine kinase

A key inflammatory mechanism recently identified in platelets involves the Nod-like receptor nucleotide-binding domain leucine-rich repeat containing protein 3 (NLRP3) and Bruton tyrosine kinase (BTK), which control activation of caspase-1 within inflammasome complexes. We investigated platelet caspase-1 activity in the context of sickle cell disease (SCD) directly in platelets isolated from SCD patients (n = 24) and indirectly by incubating platelets from healthy subjects with plasma obtained from SCD patients (n = 20), both in steady state and during an acute pain crisis (paired samples). The platelet NLRP3 inflammasome was upregulated in SCD patients under steady state conditions compared with healthy controls, and it was further upregulated when patients experienced an acute pain crisis. The results were consistent with indirect platelet assays, in which SCD plasma increased caspase-1 activity of platelets from healthy subjects in an NLRP3-dependent fashion. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) was elevated in plasma of SCD subjects compared with healthy controls and correlated with caspase-1 activity in platelets. Pharmacological or antibody-mediated inhibition of HMGB1, Toll-like receptor 4, and BTK interfered with sickle plasma-induced platelet caspase-1 activation. In Townes SCD mice, caspase-1 activity and aggregation of circulating platelets were elevated, which was suppressed by IV injection of an NLRP3 inhibitor and the BTK inhibitor ibrutinib. Activation of the platelet NLRP3 inflammasome in SCD may have diagnostic and therapeutic implications.