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HbSC disease, a less severe form of sickle cell disease, affects the retina more frequently and patients have higher rates of proliferative retinopathy that can progress to vision loss. This study aimed to identify differences in the expression of endothelial cell-derived molecules associated with the pathophysiology of proliferative sickle cell retinopathy (PSCR). RNAseq was used to compare the gene expression profile of circulating endothelial colony-forming cells from patients with SC hemoglobinopathy and proliferative retinopathy (n = 5), versus SC patients without retinopathy (n = 3). Real-time polymerase chain reaction (qRT-PCR) was used to validate the RNAseq results. A total of 134 differentially expressed genes (DEGs) were found. DEGs were mainly associated with vasodilatation, type I interferon signaling, innate immunity and angiogenesis. Among the DEGs identified, we highlight the most up-regulated genes ROBO1 (log2FoldChange = 4.32, FDR = 1.35E-11) and SLC38A5 (log2FoldChange = 3.36 FDR = 1.59E-07). ROBO1, an axon-guided receptor, promotes endothelial cell migration and contributes to the development of retinal angiogenesis and pathological ocular neovascularization. Endothelial SLC38A5, an amino acid (AA) transporter, regulates developmental and pathological retinal angiogenesis by controlling the uptake of AA nutrient, which may serve as metabolic fuel for the proliferation of endothelial cells (ECs) and consequent promotion of angiogenesis. Our data provide an important step towards elucidating the molecular pathophysiology of PSCR that may explain the differences in ocular manifestations between individuals with hemoglobinopathies and afford insights for new alternative strategies to inhibit pathological angiogenesis.
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Proteínas do Tecido Nervoso , Receptores Imunológicos , Neovascularização Retiniana , Proteínas Roundabout , Adulto , Feminino , Humanos , Masculino , Angiogênese , Células Endoteliais/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologiaRESUMO
BACKGROUND: Iron overload (IO) is a complex condition in which clinical, behavioral and genetic factors contribute to the phenotype. In multiethnic and non-Caucasian populations, mutations in HFE gene alone cannot explain IO in most of the cases, and additional genetic and environmental factors must be investigated. Bone Morphogenetic Proteins (BMPs) play a central role in iron homeostasis by modulating HAMP transcription through the signaling pathway that includes SMAD and HJV. In this study, we aimed to explore the clinical relevance of BMP6 mutations in a cohort of Brazilian patients with IO. METHODS: 41 patients with IO were evaluated. Blood samples were collected to analyze BMP6 mutations through New Sequence Generations (NGS). Frequency of variants and mutations were analyzed and correlated with clinical and environmental characteristics. RESULTS: We identified BMP6 mutations in three patients with IO. The p.Arg257His mutation was identified in two patients and the p.Leu71Val mutation was identified in one patient. Two of these patients had additional risk factors for IO (HFE mutations and diabetes mellitus). CONCLUSION: BMP6 mutations, when combined to other genetic and clinical risk factors, may contribute to IO. Functional studies and THE evaluation of large cohorts are necessary to fully address BMP6 role in IO.
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Ischemic stroke (IS) is one of the most impairing complications of sickle cell anemia (SCA), responsible for 20% of mortality in patients. Rheological alterations, adhesive properties of sickle reticulocytes, leukocyte adhesion, inflammation and endothelial dysfunction are related to the vasculopathy observed prior to ischemic events. The role of the vascular endothelium in this complex cascade of mechanisms is emphasized, as well as in the process of ischemia-induced repair and neovascularization. The aim of the present study was to perform a comparative transcriptomic analysis of endothelial colony-forming cells (ECFCs) from SCA patients with and without IS. Next, to gain further insights of the biological relevance of differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction network (PPI) construction and in silico prediction of regulatory factors were performed. Among the 2469 DEGs, genes related to cell proliferation (AKT1, E2F1, CDCA5, EGFL7), migration (AKT1, HRAS), angiogenesis (AKT1, EGFL7) and defense response pathways (HRAS, IRF3, TGFB1), important endothelial cell molecular mechanisms in post ischemia repair were identified. Despite the severity of IS in SCA, widely accepted molecular targets are still lacking, especially related to stroke outcome. The comparative analysis of the gene expression profile of ECFCs from IS patients versus controls seems to indicate that there is a persistent angiogenic process even after a long time this complication has occurred. Thus, this is an original study which may lead to new insights into the molecular basis of SCA stroke and contribute to a better understanding of the role of endothelial cells in stroke recovery.
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Anemia Falciforme , Acidente Vascular Cerebral , Humanos , Células Endoteliais/metabolismo , Transcriptoma , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/complicações , Anemia Falciforme/complicações , Isquemia , Perfilação da Expressão Gênica , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Família de Proteínas EGF/genética , Família de Proteínas EGF/metabolismoRESUMO
Among sickle cell anemia (SCA) complications, proliferative sickle cell retinopathy (PSCR) is one of the most important, being responsible for visual impairment in 10-20% of affected eyes. The aim of this study was to identify differentially expressed genes (DEGs) present in pathways that may be implicated in the pathophysiology of PSCR from the transcriptome profile analysis of endothelial progenitor cells. RNA-Seq was used to compare gene expression profile of circulating endothelial colony-forming cells (ECFCs) from HbSS patients with and without PSCR. Furthermore, functional enrichment analysis and protein-protein interaction (PPI) networks were performed to gain further insights into biological functions. The differential expression analysis identified 501 DEGs, when comparing the groups with and without PSCR. Furthermore, functional enrichment analysis showed associations of the DEGs in 200 biological processes. Among these, regulation of mitogen-activated protein (MAP) kinase activity, positive regulation of phosphatidylinositol 3-kinase (PI3K), and positive regulation of Signal Transducer and Activator of Transcription (STAT) receptor signaling pathway were observed. These pathways are associated with angiogenesis, cell migration, adhesion, differentiation, and proliferation, important processes involved in PSCR pathophysiology. Moreover, our results showed an over-expression of VEGFC (vascular endothelial growth factor-C) and FLT1 (Fms-Related Receptor Tyrosine Kinase 1) genes, when comparing HbSS patients with and without PSCR. These results may indicate a possible association between VEGFC and FLT1 receptor, which may activate signaling pathways such as PI3K/AKT and MAPK/ERK and contribute to the mechanisms implicated in neovascularization. Thus, our findings contain preliminary results that may guide future studies in the field, since the molecular mechanisms of PSCR are still poorly understood.
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Células Progenitoras Endoteliais , Doenças Retinianas , Humanos , Células Progenitoras Endoteliais/metabolismo , Transcriptoma/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Perfilação da Expressão GênicaRESUMO
STAT3 is a cytokine-signaling transcription factor critical for gene regulation. Gain-of-function (GOF) mutations in STAT3 are associated with lymphoproliferation, autoimmune cytopenias, increased susceptibility to infection, early-onset solid-organ autoimmunity, short stature, and eczema. We studied the JAK/STAT signaling pathway gene expression and the cytokine profile in two families carrying STAT3-GOF variants to shed light on the STAT3-GOF-associated variable expressivity, including the identification of disease markers. Considering 92 target genes, KIT and IL2RA were downregulated only in patients with high clinical penetrance, while CXCL8 was markedly downregulated for all of them. Unlike previous studies, SOCS3-a STAT3 inhibitor-was not upregulated in patients. In addition, low levels of IL-2 and a reduced numbers of Tregs cells were strikingly prevalent in patients. This study shows a disruptive role of STAT3-GOF variants in the regulatory axis activities CXCL8/STAT3, KIT/STAT3, IL2/CD25/Treg, which, by slightly different mechanisms, underlie the broad clinical spectrum seen in the studied patients. In addition, we suggest the investigation of CXCL8 as a biomarker for identifying STAT3-GOF mutation.
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Mutação com Ganho de Função , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-2/metabolismo , Interleucina-8/metabolismo , Fator de Transcrição STAT3/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Adulto , Pré-Escolar , Hibridização Genômica Comparativa , Citocinas/sangue , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Mutação em Linhagem Germinativa , Heterozigoto , Humanos , Imunofenotipagem , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
ABSTRACT Introduction Mutations affecting genes involved in oxidative and signaling pathways may be associated with kidney disease in sickle cell anemia. We determined the allele and genotype frequencies of some polymorphisms in the promoter regions of the Heme Oxygenase-1 (HMOX1) [rs2071746 (A > T) and (GT)n repeats, short (S) and long (L) alleles] and Bone Morphogenetic Protein Receptor type-1B (BMPR1B) [rs17022863 (A > G), rs4331783 (A > G) and rs1470409 (A > G)] genes in 75 adult patients with sickle cell anemia and 160 healthy controls and investigated whether these polymorphisms may influence the estimated glomerular filtration rate for the patients. Methods The single nucleotide polymorphisms were genotyped using the TaqMan assays, the HMOX1(GT)n repeats were determined by polymerase chain reaction fragment size analysis and the estimated glomerular filtration rate was calculated by the Modification of Diet in Renal Disease formula. Results Regarding the HMOX1rs2071746, the estimated glomerular filtration rate median was significantly higher in TT patients (p = 0.019), including when TT was compared with AT + AA (p = 0.009); for the (GT)n repeats, the estimated glomerular filtration rate medians of SS, SL and LL significantly differed (p = 0.009), being the LL estimated glomerular filtration rate median significantly higher, when compared with the LS + SS (p = 0.005). These results suggest that both the homozygotes, TT for rs2071746 and LL for (GT)n repeats, lead to a higher risk of developing renal complications. Concerning the BMPR1B, the frequencies of GG for rs17022863 and AA for rs4331783 were significantly higher in patients than in controls (p = 0.002 and p = 0.008, respectively), however no association with estimated glomerular filtration rate was found. Conclusion These results contribute to a better understanding of the genetic factors related to the development of nephropathy in sickle cell anemia patients.
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Humanos , Masculino , Feminino , Polimorfismo Genético , Estresse Oxidativo , Heme Oxigenase-1 , Taxa de Filtração Glomerular , Anemia FalciformeRESUMO
INTRODUCTION: Mutations affecting genes involved in oxidative and signaling pathways may be associated with kidney disease in sickle cell anemia. We determined the allele and genotype frequencies of some polymorphisms in the promoter regions of the Heme Oxygenase-1 (HMOX1) [rs2071746 (A>T) and (GT)n repeats, short (S) and long (L) alleles] and Bone Morphogenetic Protein Receptor type-1B (BMPR1B) [rs17022863 (A>G), rs4331783 (A>G) and rs1470409 (A>G)] genes in 75 adult patients with sickle cell anemia and 160 healthy controls and investigated whether these polymorphisms may influence the estimated glomerular filtration rate for the patients. METHODS: The single nucleotide polymorphisms were genotyped using the TaqMan assays, the HMOX1(GT)n repeats were determined by polymerase chain reaction fragment size analysis and the estimated glomerular filtration rate was calculated by the Modification of Diet in Renal Disease formula. RESULTS: Regarding the HMOX1rs2071746, the estimated glomerular filtration rate median was significantly higher in TT patients (p=0.019), including when TT was compared with AT+AA (p=0.009); for the (GT)n repeats, the estimated glomerular filtration rate medians of SS, SL and LL significantly differed (p=0.009), being the LL estimated glomerular filtration rate median significantly higher, when compared with the LS+SS (p=0.005). These results suggest that both the homozygotes, TT for rs2071746 and LL for (GT)n repeats, lead to a higher risk of developing renal complications. Concerning the BMPR1B, the frequencies of GG for rs17022863 and AA for rs4331783 were significantly higher in patients than in controls (p=0.002 and p=0.008, respectively), however no association with estimated glomerular filtration rate was found. CONCLUSION: These results contribute to a better understanding of the genetic factors related to the development of nephropathy in sickle cell anemia patients.
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PURPOSE: To determine the various haptoglobin genotypes and their influence on the clinico-laboratory manifestations among young Nigerian sickle cell anemia (SCA) patients. PATIENTS AND METHODS: A total of 101 SCA patients and 64 controls were studied. SCA was diagnosed by polymerase chain reaction (PCR). Haptoglobin genotype was determined by PCR followed by agarose gel electrophoresis. The patients' laboratory and clinical parameters were differentiated by haptoglobin genotypes. RESULTS: The Hp1 and Hp2 alleles frequencies were 0.62 and 0.38 in the patients and 0.73 and 0.27 in the controls, respectively, and these did not differ significantly (p>0.05). The haptoglobin genotype distribution among the patients and controls were Hp1-1, 43 (42.6%); Hp2-1, 40 (39.6%); Hp2-2, 18 (17.8%) and Hp1-1, 35 (54.7%); Hp2-1, 24 (37.5%); Hp2-2, 5 (7.8%), respectively, with no difference between the two groups (P>0.05). No significant difference was found in the clinical events and laboratory parameters of the patients when partitioned according to the various haptoglobin genotypes (P> 0.05). CONCLUSION: This study found that haptoglobin gene polymorphism does not have a significant influence on the clinico-laboratory manifestations among SCA patients.
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Sickle cell disease (SCD), one of the most common hemoglobinopathies worldwide, is characterized by a chronic inflammatory component, with systemic release of inflammatory cytokines, due to hemolysis and vaso-occlusive processes. Patients with SCD demonstrate dysfunctional T and B lymphocyte responses, and they are more susceptible to infection. Although dendritic cells (DCs) are the main component responsible for activating and polarizing lymphocytic function, and are able to produce pro-inflammatory cytokines found in the serum of patients with SCD, minimal studies have thus far been devoted to these cells. In the present study, we identified the subpopulations of circulating DCs in patients with SCD, and found that the bloodstream of the patients showed higher numbers and percentages of DCs than that of healthy individuals. Among all the main DCs subsets, inflammatory DCs (CD14+ DCs) were responsible for this rise and correlated with higher reticulocyte count. The patients had more activated monocyte-derived DCs (mo-DCs), which produced MCP-1, IL-6, and IL-8 in culture. We found that a CD14+ mo-DC subset present in culture from some of the patients was the more activated subset and was mainly responsible for cytokine production, and this subset was also responsible for IL-17 production in co-culture with T lymphocytes. Finally, we suggest an involvement of heme oxygenase in the upregulation of CD14 in mo-DCs from the patients, indicating a potential mechanism for inducing inflammatory DC differentiation from circulating monocytes in the patients, which correlated with inflammatory cytokine production, T lymphocyte response skewing, and reticulocyte count.