Global gene expression reveals an increase of HMGB1 and APEX1 proteins and their involvement in oxidative stress, apoptosis and inflammation pathways among beta-thalassaemia intermedia and major phenotypes.

Beta-thalassaemia (BT) is classified according to blood transfusion requirement as minor (BTMi), intermedia (BTI) and major (BTM). BTM is the most severe form, requiring regular transfusions while transfusion need is only occasional in BTI. Differential gene expression between patients has not been assessed so far. Here, we evaluated the global gene expression profiles during differentiation of human erythroid cells of two patients carrying the same mutation [CD39, (C → T)], though displaying different phenotypes (BTI and BTM). Considering the role of reactive oxygen species (ROS) in the pathophysiology of thalassaemia, we focused on differentially expressed genes involved in metabolic pathways triggered by ROS, such as inflammation and apoptosis, and, from these, we selected the Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) and High Mobility Group Box1 (HMGB1) genes, whose role in BT is not well established. An in-depth expression analysis of transcriptional and protein levels in patients carrying a range of mutations associated with BT phenotypes indicated that APEX1 was increased in both BTI and BTM. Furthermore, higher amounts of HMGB1 was found in the plasma of BTI patients. Our findings suggest that these proteins have important roles in BT and could represent new targets for further studies aiming to improve the management of the disease.

Gene expression analysis of the Brazilian type of hereditary persistence of fetal hemoglobin: identification of genes that could be related to γ-globin activation.

Increased γ-globin production and consequent fetal hemoglobin (Hb F, α2γ2) formation is an important modulator of the clinical and hematological features of hemolytic anemias, such as sickle cell disease and ß-thalassemia (ß-thal). Hb F genes are genetically regulated, but despite numerous studies, the molecular basis of hemoglobin (Hb) switching is not completely understood. Hereditary persistence of fetal Hb (HPFH) is a consequence of impaired switching in adult life, which results in the continued expression of the γ-globin gene. This study was undertaken to identify genes that could be involved in Hb switching and/or maintenance of elevated Hb F levels. Two libraries were constructed using reticulocytes from normal donors and from Brazilian HPFH subjects. Results suggest that the maintenance of Hb F levels could be associated with some gene/protein expression modifications, such as low expression of KLF1, a transcription factor known to contribute to the regulation and modulation of Hb switching, decreased expression of MIER1, known for the recruitment of chromatin remodeling enzymes, and decreased expression of HOOK3. These data suggest new genes that may play a role in globin gene regulation, γ-globin gene expression and augmentation of Hb F levels, and may represent newly-defined cellular pathways for the control of Hb switching in erythroid cells.

Endothelial activation by platelets from sickle cell anemia patients.

Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1ß and a tendency towards elevated expressions of P-selectin and activated αIIbß3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association with other therapies for SCD.

Identification of target genes using gene expression profile of granulocytes from patients with chronic myeloid leukemia treated with tyrosine kinase inhibitors.

Differential gene expression analysis by suppression subtractive hybridization with correlation to the metabolic pathways involved in chronic myeloid leukemia (CML) may provide a new insight into the pathogenesis of CML. Among the overexpressed genes found in CML at diagnosis are SEPT5, RUNX1, MIER1, KPNA6 and FLT3, while PAN3, TOB1 and ITCH were decreased when compared to healthy volunteers. Some genes were identified and involved in CML for the first time, including TOB1, which showed a low expression in patients with CML during tyrosine kinase inhibitor treatment with no complete cytogenetic response. In agreement, reduced expression of TOB1 was also observed in resistant patients with CML compared to responsive patients. This might be related to the deregulation of apoptosis and the signaling pathway leading to resistance. Most of the identified genes were related to the regulation of nuclear factor κB (NF-κB), AKT, interferon and interleukin-4 (IL-4) in healthy cells. The results of this study combined with literature data show specific gene pathways that might be explored as markers to assess the evolution and prognosis of CML as well as identify new therapeutic targets.