DGH - Apresentações orais em encontros internacionais
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Browsing DGH - Apresentações orais em encontros internacionais by Subject "Anemia das Células Falciformes"
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- Differential Endothelial VCAM1 Expression and Implications for Sickle Cell Anemia VasculopathyPublication . Silva, Marisa; Coelho, Andreia; Vargas, Sofia; Faustino, PaulaBackground: Vascular disease is systemic in sickle cell anemia (SCA), with profound effects in organs like the brain,where stroke is the most severe end of the cerebral vasculopathy spectrum. Endothelial dysfunction is an important pathobiological mechanism in SCA systemic vasculopathy, with upregulation of adhesion molecules (e.g., VCAM-1), lower nitric oxide bioavailability, and increased oxidative stress. In previous association studies, we found positive associations between the presence of three specific VCAM1 gene promoter haplotypes and i) high blood flow velocities in the median cerebral artery, and ii) a chronic hemolysis biochemical marker. Aims: The aims of our work were: a) to investigate the functional role of those VCAM1 promoter haplotypes in endothelial cell response following endothelial activation through TNF-α stimulation; b) to assess the modulation role of proinflammatory and/or pro-oxidative stimuli on endothelial VCAM1 expression; and, finally, to evaluate how hydroxyurea (HU) treatment would affect that expression. Methods: After molecular cloning of three VCAM1 promoter haplotype constructs, using pGL4 promoterless vectors, haplotype sequence was confirmed, by Sanger sequencing, prior to transfection. Transfection experiments for each construct were performed, with or without TNF-α stimulation, using EAhy926, and HBEC as macrovascular and microvascular endothelial cell models, respectively. Differences in promoter activity were assessed by luciferase reporter assay. RNA was extracted from non-transfected EAhy926 and HBEC cell cultures stimulated or not with TNF-a and/or hemin, and with or without HU treatment. RT-qPCR was performed to analyze VCAM1 expression. HMOX1 and NOS3 were also analyzed for comparison purposes. Results: Our results showed that two VCAM1 promoter haplotypes, previously associated with pediatric cerebral vasculopathy and hemolysis in SCA, increased promoter activity in transfected and TNF-α-stimulated EA.hy926 and HBEC cells, consistent with a higher VCAM1 expression in macro and microvascular settings. In non-transfected cells, we also observed TNF-a-induced VCAM1 overexpression as well as heme-induced overexpression of HMOX1 in both cell models. Heme did not affect VCAM1 nor NOS3 expression and the latter was also not affected by TNF-a stimulus. Hydroxyurea treatment lowered TNF-a-induced VCAM1 and NOS3 expression but did not affect heme-induced HMOX1 expression. Summary/Conclusion: These data further indicate that VCAM1 haplotypes we previously associated with pediatric cerebral vasculopathy and hemolysis in SCA, induce higher VCAM1 expression potentially affecting both cerebral and systemic vasculopathy risk. The differential endothelial expression of VCAM1, NOS3, and HMOX1 after proinflammatory and/or pro-oxidative stimuli also reinforces their genetic modulation role in SCA systemic vasculopathy.
- Endothelial factors and stroke risk in pediatric sickle cell anemia patients: insights from VCAM1 and ITGA4 variantsPublication . Silva, Marisa; Vargas, Sofia; Coelho, Andreia; Mendonça, Joana; Vieira, Luís; Kjollerstrom, Paula; Maia, Raquel; Silva, Rute; Dias, Alexandra; Ferreira, Teresa; Morais, Anabela; Mota Soares, Isabel; Lavinha, João; Faustino, PaulaSickle cell anemia (SCA) arises from homozygosity for the mutation c.20A>T in the HBB gene which originates hemoglobin S (HbS). In hypoxic conditions, HbS polymerizes inside erythrocytes deforming them and ultimately leading to hemolysis and vaso-occlusion. SCA shows a multifactorial-like behaviour with a high heterogeneity of clinical features, with stroke being the most severe of them. This heterogeneity may arise from underlying genetic modifiers, namely those affecting vascular adhesion/endothelial dysfunction. These include genes encoding the VCAM-1 molecule and its ligand VLA-4 (ITGA4 or integrin α4), increasingly studied due to their expression in activated human endothelium and leucocytes/stress reticulocytes, respectively. The aim of this study was to identify putative genetic modulators of stroke risk by analyzing 70 pediatric SCA patients, grouped according to their degree of cerebral vasculopathy. Molecular analysis was performed using Next-Generation Sequencing (NGS) and Sanger Sequencing. R software was used for statistical analyses and association studies. In silico studies were performed using PHASE, TFbind, PROMO and Human Splicing Finder software tools. We identified six different VCAM1 promoter variants and seven haplotypes. The VCAM1 promoter rs1409419_T allele was associated with stroke events (p=0.008; O.R.= 4.33; C.I.95% =1.391-14.257), while one VCAM1 promoter haplotype was found to be protective of stroke (p=0.011; O.R.=0.22; C.I.95% =0.048-0.784). On the ITGA4 gene, forty variants were found, six of them novel. All patients presented with at least one variant in this gene. We observed co-inheritance of specific sets of ITGA4 variants indicating the presence of haplotypes not previously described. Additionally the presence of specific variants seems to result in a predisposition for either high reticulocyte count, elevated lactate dehydrogenase, raised bilirubin levels or increased transcranial Doppler velocity values. Our results reinforce the role of endothelial molecules and blood cell interaction in SCA severity. The association between specific VCAM1, as well as ITGA4, variants with certain cerebral vasculopathy predictors, further enhances their putative modulating effect on pediatric stroke severity and prognosis. These findings provide additional clues on the SCA pathophysiology and uncover features of both genes that may prove to be crucial as potential therapeutic targets.