Enrico M. Novelli

The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe

The intensity of hemolytic anemia has been proposed as an independent risk factor for the development of certain clinical complications of sickle cell disease, such as pulmonary hypertension, hypoxemia and cutaneous leg ulceration. A composite variable derived from several individual markers of hemolysis could facilitate studies of the underlying mechanisms of hemolysis. In this study, we assessed the association of hemolysis with outcomes in sickle cell anemia. A hemolytic component was calculated by principal component analysis from reticulocyte count, serum lactate dehydrogenase, aspartate aminotransferase and total bilirubin concentrations in 415 hemoglobin SS patients. Association of this component with direct markers of hemolysis and clinical outcomes was assessed. As primary validation, both plasma red blood cell microparticles and cell-free hemoglobin concentration were higher in the highest hemolytic component quartile compared to the lowest quartile (P≤0.0001 for both analyses). The hemolytic component was lower with hydroxyurea therapy, higher hemoglobin F, and alpha-thalassemia (P≤0.0005); it was higher with higher systemic pulse pressure, lower oxygen saturation, and greater values for tricuspid regurgitation velocity, left ventricular diastolic dimension and left ventricular mass (all P<0.0001). Two-year follow-up analysis showed that a high hemolytic component was associated with an increased risk of death (hazard ratio, HR 3.44; 95% confidence interval, CI: 1.2–9.5; P=0.02). The hemolytic component reflects direct markers of intravascular hemolysis in patients with sickle cell disease and allows for adjusted analysis of associations between hemolytic severity and clinical outcomes. These results confirm associations between hemolytic rate and pulse pressure, oxygen saturation, increases in Doppler-estimated pulmonary systolic pressures and mortality (Clinicaltrials.gov identifier: NCT00492531).

Echocardiographic Markers of Elevated Pulmonary Pressure and Left Ventricular Diastolic Dysfunction Are Associated With Exercise Intolerance in Adults and Adolescents With Homozygous Sickle Cell Anemia in the United States and United Kingdom

Background— Noninvasively assessed pulmonary pressure elevations and left ventricular (LV) diastolic dysfunction are associated with increased mortality in adults with sickle cell disease, but their relationship to exercise intolerance has not been evaluated prospectively. Methods and Results— Echocardiography, 6-minute walk distance, hemolytic rate, and serum concentrations of ferritin and erythropoietin were evaluated in a cohort of 483 subjects with homozygous hemoglobin S in the US and UK Walk–Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (Walk-PHaSST) study. Tricuspid regurgitation velocity, which reflects systolic pulmonary artery pressure, was 2.7 to <3.0 m/s (mean±SD, 2.8±0.1) in 26% of the subjects and ≥3.0 m/s (mean±SD, 3.4±0.4) in 11%. The LV lateral E/e′ ratio, which has been shown to reflect LV filling pressure in other conditions but has not been studied in sickle cell disease, was significantly higher in the groups with tricuspid regurgitation velocity ≥2.7 m/s. Increased hemolysis (P<0.0001), LV lateral E/e′ ratio (P=0.0001), blood urea nitrogen (P=0.0002), and erythropoietin (P=0.002) were independently associated with an increased tricuspid regurgitation velocity. Furthermore, female sex (P<0.0001), older age (P<0.0001), LV lateral E/e′ ratio (P=0.014), and tricuspid regurgitation velocity (P=0.019) were independent predictors of a shorter 6-minute walk distance. Conclusions— Echocardiography-estimated elevated pulmonary artery systolic pressure and LV lateral E/e′ ratio were independently associated with poor exercise capacity in a large cohort of patients with sickle cell anemia. Controlled trials investigating whether strategies to prevent or delay pulmonary hypertension and/or LV diastolic dysfunction will improve exercise capacity and long-term outcomes in sickle cell anemia should be considered. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00492531.

Gene therapy for lysosomal storage disorders

The lysosomal storage disorders (LSD) are monogenic inborn errors of metabolism with heterogeneous pathophysiology and clinical manifestations. In the last decades, these disorders have been models for the development of molecular and cellular therapies for inherited metabolic diseases. Studies in preclinical in vitro systems and animal models have allowed the successful development of bone marrow transplantation (BMT) and enzyme replacement therapy (ERT) as therapeutic options for several LSDs. However, BMT is limited by poor donor availability and high morbidity and mortality, and ERT is not a life-long cure. Moreover, the neuropathology present in many LSDs responded poorly, if at all, to these treatments. Therefore, gene therapy is an attractive therapeutic alternative. Gene therapy strategies for LSDs have employed ex vivo gene transduction of cellular targets with subsequent transplantation of the enzymatically corrected cells, or direct in vivo delivery of the viral vectors. Oncoretroviral vectors and more recently adeno associated vectors (AAV) and lentiviral vectors have been extensively tested, with some success. This review summarises the main gene therapy strategies which have been employed or are under development for both non-neurological and neuronopathic LSDs. Some of the in vitro and in vivo preclinical studies presented herein have provided the rationale for a gene therapy clinical trial for Gaucher disease Type I.

Pulmonary embolism in sickle cell disease: a case–control study

Introduction: A pulmonary embolism (PE) is a leading cause of mortality in hospitalized patients, yet the prevalence of PE in sickle cell disease (SCD) and its relation to disease severity or intrinsic hypercoagulability are not established.

A double-blind, randomized, multicenter phase 2 study of prasugrel versus placebo in adult patients with sickle cell disease

BackgroundPlatelet activation has been implicated in the pathogenesis of sickle cell disease (SCD) suggesting antiplatelet agents may be therapeutic. To evaluate the safety of prasugrel, a thienopyridine antiplatelet agent, in adult patients with SCD, we conducted a double-blind, randomized, placebo-controlled study.MethodsThe primary endpoint, safety, was measured by hemorrhagic events requiring medical intervention. Patients were randomized to prasugrel 5 mg daily (n = 41) or placebo (n = 21) for 30 days. Platelet function by VerifyNow® P2Y12 and vasodilator-stimulated phosphoprotein assays at days 10 and 30 were significantly inhibited in prasugrel- compared with placebo-treated SCD patients.ResultsThere were no hemorrhagic events requiring medical intervention in either study arm. Mean pain rate (percentage of days with pain) and intensity in the prasugrel arm were decreased compared with placebo. However, these decreases did not reach statistical significance. Platelet surface P-selectin and plasma soluble P-selectin, biomarkers of in vivo platelet activation, were significantly reduced in SCD patients receiving prasugrel compared with placebo. In sum, prasugrel was well tolerated and not associated with serious hemorrhagic events.ConclusionsDespite the small size and short duration of this study, there was a decrease in platelet activation biomarkers and a trend toward decreased pain.

Plasma thrombospondin-1 is increased during acute sickle cell vaso-occlusive events and associated with acute chest syndrome, hydroxyurea therapy, and lower hemolytic rates

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.

Platelet bioenergetic screen in sickle cell patients reveals mitochondrial complex V inhibition, which contributes to platelet activation

Bioenergetic dysfunction, although central to the pathogenesis of numerous diseases, remains uncharacterized in many patient populations because of the invasiveness of obtaining tissue for mitochondrial studies. Although platelets are an accessible source of mitochondria, the role of bioenergetics in regulating platelet function remains unclear. Herein, we validate extracellular flux analysis in human platelets and use this technique to screen for mitochondrial dysfunction in sickle cell disease (SCD) patients, a population with aberrant platelet activation of an unknown mechanism and in which mitochondrial function has never been assessed. We identify a bioenergetic alteration in SCD patients characterized by deficient complex V activity, leading to decreased mitochondrial respiration, membrane hyperpolarization, and augmented oxidant production compared with healthy subjects. This dysfunction correlates with platelet activation and hemolysis in vivo and can be recapitulated in vitro by exposing healthy platelets to hemoglobin or a complex V inhibitor. Further, reproduction of this dysfunction in vitro activates healthy platelets, an effect prevented by attenuation of mitochondrial hyperpolarization or by scavenging mitochondrial oxidants. These data identify bioenergetic dysfunction in SCD patients for the first time and establish mitochondrial hyperpolarization and oxidant generation as potential pathogenic mechanism in SCD as well as a modulator of healthy platelet function.

Risk factors for death in 632 patients with sickle cell disease in the United States and United Kingdom.

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

Thrombospondin-1 Activation of Signal-Regulatory Protein-α Stimulates Reactive Oxygen Species Production and Promotes Renal Ischemia Reperfusion Injury

Ischemia reperfusion injury (IRI) causes tissue and organ injury, in part, through alterations in tissue blood flow and the production of reactive oxygen species. The cell surface receptor signal-regulatory protein-α (SIRP-α) is expressed on inflammatory cells and suppresses phagocytosis, but the function of SIRP-α in IRI has not been determined. We reported previously that the matricellular protein thrombospondin-1 is upregulated in IRI. Here, we report a novel interaction between thrombospondin-1 and SIRP-α on nonphagocytic cells. In cell-free experiments, thrombospondin-1 bound SIRP-α. In vascular smooth muscle cells and renal tubular epithelial cells, treatment with thrombospondin-1 led to phosphorylation of SIRP-α and downstream activation of Src homology domain 2-containing phosphatase-1. Thrombospondin-1 also stimulated phosphorylation of p47(phox) (an organizer subunit for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1/2) and increased production of superoxide, both of which were abrogated by knockdown or antibody blockade of SIRP-α. In rodent aortic rings, treatment with thrombospondin-1 increased the production of superoxide and inhibited nitric oxide-mediated vasodilation in a SIRP-α-dependent manner. Renal IRI upregulated the thrombospondin-1-SIRP-α signaling axis and was associated with increased superoxide production and cell death. A SIRP-α antibody that blocks thrombospondin-1 activation of SIRP-α mitigated the effects of renal IRI, increasing blood flow, suppressing production of reactive oxygen species, and preserving cellular architecture. A role for CD47 in SIRP-α activation in these pathways is also described. Overall, these results suggest that thrombospondin-1 binding to SIRP-α on nonphagocytic cells activates NADPH oxidase, limits vasodilation, and promotes renal IRI.

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. Am. J. Heamtol. 89:1–6, 2014.