Effect of hydroxyurea therapy on intravascular hemolysis and endothelial dysfunction markers in sickle cell anemia patients.

Intravascular hemolysis (IH) contributes to the development of endothelial dysfunction (ED) in sickle cell anemia (SCA), and the effects of hydroxyurea (HU, the only approved drug that decreases the frequency and severity of vaso-oclussive crises) on IH and ED in SCA remain unclear. We evaluated and compared the markers of IH among steady-state adult Brazilians with SCA and HbAA individuals. Overall, this cross-sectional study enrolled 30 SCA patients not receiving HU therapy (HbSS), 25 SCA patients receiving HU therapy (HbSS_HU), and 32 HbAA volunteers (HbAA). The IH markers evaluated were serum Lactate Dehydrogenase (LDH), total heme, plasma hemoglobin (pHb), and soluble CD163 (sCD163). The ED markers analyzed were plasma von Willebrand factor (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo) levels, antigen of VWF-cleaving protease (ADAMTS13:Ag), thrombospondin-1, endothelin-1 levels, and ADAMTS13 Activity (ADAMTS13:Act). The levels of VWF:Ag, VWF:RCo, total heme, thrombospondin-1, and endothelin-1 were significantly higher in SCA patients (HbSS and HbSS_HU) compared to HbAA individuals. Also, pHb, LDH, and thrombospondin-1 levels were significantly higher in the HbSS group than in the HbSS_HU group. Contrarily, the levels of sCD163, ADAMTS13:Ag, and ADAMTS13:Act were significantly lower in both groups of SCA patients than HbAA controls, and ADAMTS13:Act levels were significantly lower in HbSS compared to HbSS_HU patients. The higher ADAMTS13 activity levels in those on HU therapy may be attributed to lower pHb and thrombospondin-1 levels as previously shown by in vitro studies that thrombospondin-1 and pHb are bound to VWF. Thus, VWF is restrained from ADAMTS13 activity and cleavage.

Endothelial Barrier Integrity Is Disrupted In Vitro by Heme and by Serum From Sickle Cell Disease Patients.

Free extracellular heme has been shown to activate several compartments of innate immunity, acting as a danger-associated molecular pattern (DAMP) in hemolytic diseases. Although localized endothelial barrier (EB) disruption is an important part of inflammation that allows circulating leukocytes to reach inflamed tissues, non-localized/deregulated disruption of the EB can lead to widespread microvascular hyperpermeability and secondary tissue damage. In mouse models of sickle cell disease (SCD), EB disruption has been associated with the development of a form of acute lung injury that closely resembles acute chest syndrome (ACS), and that can be elicited by acute heme infusion. Here we explored the effect of heme on EB integrity using human endothelial cell monolayers, in experimental conditions that include elements that more closely resemble in vivo conditions. EB integrity was assessed by electric cell-substrate impedance sensing in the presence of varying concentrations of heme and sera from SCD patients or healthy volunteers. Heme caused a dose-dependent decrease of the electrical resistance of cell monolayers, consistent with EB disruption, which was confirmed by staining of junction protein VE-cadherin. In addition, sera from SCD patients, but not from healthy volunteers, were also capable to induce EB disruption. Interestingly, these effects were not associated with total heme levels in serum. However, when heme was added to sera from SCD patients, but not from healthy volunteers, EB disruption could be elicited, and this effect was associated with hemopexin serum levels. Together our in vitro studies provide additional support to the concept of heme as a DAMP in hemolytic conditions.

Serum folate and cytokines in heterozygous ß-thalassemia.

INTRODUCTION: Folate deficiency is commonly reported in ß-thalassemia. Individuals heterozygous for ß-thalassemia may have higher folate requirements than normal individuals. OBJECTIVES: To document the concentration of serum total folate and its forms in ß-thalassemia heterozygote users (ß-TmU) and nonusers (ß-TmN) of 5 mg folic acid/d; to determine whether folic acid (FA) consumption from fortified foods allows beta-Tm patients, who do not take FA supplements, to meet their dietary folate requirements; and to investigate the association between higher serum unmetabolized folic acid (UMFA) and inflammatory cytokine concentrations. METHODS: Serum total folate and forms were measured in 42 ß-Tm (13 ß-TmU and 29 ß-TmN) and 84 healthy controls. The mononuclear leucocyte mRNA expression of relevant genes and their products and hematological profiles were determined. RESULTS: ß-TmU had higher serum total folate, 5-methyltetrahydrofolate, UMFA, and tetrahydrofolate (THF) compared with ß-TmN. The ß-TmN had lower serum total folate and THF than controls. Plasma total homocysteine (tHcy) was lower in ß-TmU compared with both ß-TmN and controls, while ß-TmN had higher tHcy than controls. ß-TmU had higher IL-8 than their controls while ß-TmN had higher IL-6 and IL-8 than their controls. ß-TmU have higher levels of serum total folate, 5- methyltetrahydrofolate, UMFA, and THF than controls. There was no association between UMFA concentrations and cytokine levels. CONCLUSIONS: Mandatory flour fortification with FA in Brazil may be insufficient for ß-TmN, since they have higher tHcy and lower serum total folate than controls. Furthermore, ß-TmN have higher IL-6 levels than ß-TmU. UMFA was not associated with inflammatory cytokine levels.

Role of innate immunity-triggered pathways in the pathogenesis of Sickle Cell Disease: a meta-analysis of gene expression studies.

Despite the detailed characterization of the inflammatory and endothelial changes observed in Sickle Cell Disease (SCD), the hierarchical relationship between elements involved in the pathogenesis of this complex disease is yet to be described. Meta-analyses of gene expression studies from public repositories represent a novel strategy, capable to identify key mediators in complex diseases. We performed several meta-analyses of gene expression studies involving SCD, including studies with patient samples, as well as in-vitro models of the disease. Meta-analyses were performed with the Inmex bioinformatics tool, based on the RankProd package, using raw gene expression data. Functional gene set analysis was performed using more than 60 gene-set libraries. Our results demonstrate that the well-characterized association between innate immunity, hemostasis, angiogenesis and heme metabolism with SCD is also consistently observed at the transcriptomic level, across independent studies. The enrichment of genes and pathways associated with innate immunity and damage repair-associated pathways supports the model of erythroid danger-associated molecular patterns (DAMPs) as key mediators of the pathogenesis of SCD. Our study also generated a novel database of candidate genes, pathways and transcription factors not previously associated with the pathogenesis of SCD that warrant further investigation in models and patients of SCD.

JAK2 V617F mutation prevalence in myeloproliferative neoplasms in Pernambuco, Brazil.

BACKGROUND: The JAK2 V617F mutation is associated with three myeloproliferative neoplasms (MPNs): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). It generates an unregulated clonal hematopoietic progenitor and leads to abnormal increased proliferation of one or more myeloid lineages. Subjects bearing this mutation may present more frequently with complications such as thrombosis and bleeding, and no specific treatment has yet been developed for BCR-ABL-negative JAK2 V617F-negative MPNs. AIMS: To determine the prevalence of JAK2 V617F in MPNs in Pernambuco, Brazil, and to compare it with previous studies. MATERIAL AND METHODS: 144 blood samples were collected at the Hospital of Hematology of the HEMOPE Foundation and were genotyped by polymerase chain reaction-restriction fragment length polymorphism with BsaXI enzymatic digestion. RESULTS AND DISCUSSION: 88% (46/52) of the patients with PV, 47% (39/81) with ET, and 77% (8/11) with PMF were positive for JAK2 V617F, while more than 35% of the individuals were JAK2 V617F-negative, confirming a high prevalence of this abnormality in MPNs, more frequently with a low mutated allele burden, similar to what has been reported in other Western countries, despite differences among methods used to detect this mutation. Screening for JAK2 V617F may allow specific management of these diseases with JAK2 inhibitors in the future and highlights the need for further studies on the pathogenesis of BCR-ABL-negative JAK2 V617F-negative MPNs.