Membrane particles from mesenchymal stromal cells reduce the expression of fibrotic markers on pulmonary cells

Background: Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with limited treatment options in which the telomere shortening is a strong predictive factor of poor prognosis. Mesenchymal stromal cells (MSC) administration is probed in several experimental induced lung pathologies; however, MSC might stimulate fibrotic processes. A therapy that avoids MSC side effects of transformation would be an alternative to the use of living cells. Membranes particles (MP) are nanovesicles artificially generated from the membranes of MSC containing active enzymes involved in ECM regeneration. We aimed to investigate the anti-fibrotic role of MP derived from MSC in an in vitro model of pulmonary fibrosis. Methods: Epithelial cells (A549) and lung fibroblasts, from IPF patients with different telomere length, were co-cultured with MP and TGF-β for 48h and gene expression of major pro-fibrotic markers were analyzed. Results: About 90% of both types of cells effectively took up MP without cytotoxic effects. MP decreased the expression of profibrotic proteins such as Col1A1, Fibronectin and PAI-1, in A549 cells. In fibroblasts culture, there was a different response in the inhibitory effect of MP on some pro-fibrotic markers when comparing fibroblast from normal telomere length patients (FN) versus short telomere length (FS), but both types showed an inhibition of Col1A1, Tenascin-c, PAI-1 and MMP-1 gene expression after MP treatment

Role of transcription factor Sp1 and CpG methylation on the regulation of the human podocalyxin gene promoter

BACKGROUND: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene. RESULTS: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first ~600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines. CONCLUSION: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl

Lung Transplant Improves Survival and Quality of Life Regardless of Telomere Dysfunction

Trasplante de pulmón; Fibrosis pulmonar; Trastornos de los telómerosTrasplantament pulmonar; Fibrosi pulmonar; Trastorns dels telòmersLung transplantation; Pulmonary fibrosis; Telomere disordersIntroduction: Fibrotic interstitial lung diseases (ILDs) are the first indication for lung transplantation (LT). Telomere dysfunction has been associated with poor post-transplant outcomes. The aim of the study was to evaluate the morbi-mortality and quality of life in fibrotic ILDs after lung transplant depending on telomere biology. Methods: Fibrotic ILD patients that underwent lung transplant were allocated to two arms; with or without telomere dysfunction at diagnosis based on the telomere length and telomerase related gene mutations revealed by whole-exome sequencing. Post-transplant evaluation included: (1) short and long-term mortality and complications and (2) quality of life. Results: Fifty-five percent of patients that underwent LT carried rare coding mutations in telomerase-related genes. Patients with telomere shortening more frequently needed extracorporeal circulation and presented a higher rate of early post-transplant hematological complications, longer stay in the intensive care unit (ICU), and a higher number of long-term hospital admissions. However, post-transplant 1-year survival was higher than 80% regardless of telomere dysfunction, with improvement in the quality of life and oxygen therapy withdrawal. Conclusions: Post-transplant morbidity is higher in patients with telomere dysfunction and differs according to elapsed time from transplantation. However, lung transplant improves survival and quality of life and the associated complications are manageable.This study was funded by Instituto de Salud Carlos III through project PI18/00367 (Co-funded by European Regional Development Fund, ERDF, a way to build Europe), Spanish Society of Respiratory (SEPAR), Barcelona Respiratory Network (BRN), and Fundació Ramón Pla Armengol. RP laboratory was funded by grants PI20-00335 (Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain supported by FEDER funds). MM-M was funded by grants PI18/00367 (Fondo de Investigaciones Sanitarias, ISCIII, Spain, supported by FEDER funds), AC19/00006 (Projects of International Programs, ISCIII, Spain, supported by FEDER funds), Cohorte FPI CIBERES-ISCIII, Barcelona Respiratory Network-Fundation Ramon Pla Armengol, Spanish Society of Respiratory (SEPAR), and Catalan Society of Respiratory (SOCAP-FUCAP). CF was funded by Ministerio de Ciencia e Innovación (grant RTC-2017-6471-1; AEI/FEDER, UE), and by Cabildo Insular de Tenerife (CGIEU0000219140)

PTP-1B is an essential positive regulator of platelet integrin signaling

Outside-in integrin αIIbβ3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein–tyrosine phosphatase (PTP)–1B in this process. In resting platelets, c-Src forms a complex with αIIbβ3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to αIIbβ3 triggers PTP-1B recruitment to the αIIbβ3–c-Src–Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B–deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from αIIbβ3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B–deficient platelets are defective in outside-in αIIbβ3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in αIIbβ3 signaling in platelets

Genetic analyses of aplastic anemia and idiopathic pulmonary fibrosis patients with short telomeres, possible implication of DNA-repair genes

Aplastic anemia; DNA repair; Pulmonary fibrosisAnèmia aplàstica; Reparació d'ADN; Fibrosi pulmonarAnemia aplástica; Reparación de ADN; Fibrosis pulmonarBACKGROUND: Telomeres are nucleoprotein structures present at the terminal region of the chromosomes. Mutations in genes coding for proteins involved in telomere maintenance are causative of a number of disorders known as telomeropathies. The genetic origin of these diseases is heterogeneous and has not been determined for a significant proportion of patients. METHODS: This article describes the genetic characterization of a cohort of patients. Telomere length was determined by Southern blot and quantitative PCR. Nucleotide variants were analyzed either by high-resolution melting analysis and Sanger sequencing of selected exons or by massive sequencing of a panel of genes. RESULTS: Forty-seven patients with telomere length below the 10% of normal population, affected with three telomeropathies: dyskeratosis congenita (4), aplastic anemia (22) or pulmonary fibrosis (21) were analyzed. Eighteen of these patients presented known pathogenic or novel possibly pathogenic variants in the telomere-related genes TERT, TERC, RTEL1, CTC1 and ACD. In addition, the analyses of a panel of 188 genes related to haematological disorders indicated that a relevant proportion of the patients (up to 35%) presented rare variants in genes related to DNA repair or in genes coding for proteins involved in the resolution of complex DNA structures, that participate in telomere replication. Mutations in some of these genes are causative of several syndromes previously associated to telomere shortening. CONCLUSION: Novel variants in telomere, DNA repair and replication genes are described that might indicate the contribution of variants in these genes to the development of telomeropathies. Patients carrying variants in telomere-related genes presented worse evolution after diagnosis than the rest of patients analyzed.Funded by grants PI14–01495 and PI17–01401 (Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain supported by FEDER funds) and by one ACCI project from CIBERER and one grant to the FPI cohort from CIBERES

The importance of platelet glycoside residues in the haemostasis of patients with immune thrombocytopaenia

Loss of sialic acid from the carbohydrate side chains of platelet glycoproteins can affect platelet clearance, a proposed mechanism involved in the etiopathogenesis of immune thrombocy-topaenia (ITP). We aimed to assess whether changes in platelet glycosylation in patients with ITP affected platelet counts, function, and apoptosis. This observational, prospective, and transversal study included 82 patients with chronic primary ITP and 115 healthy controls. We measured platelet activation markers and assayed platelet glycosylation and caspase activity, analysing samples using flow cytometry. Platelets from patients with ITP with a platelet count <30 × 103/µL presented less sialic acid. Levels of α1,6-fucose (a glycan residue that can directly regulate antibody-dependent cellular cytotoxicity) and α-mannose (which can be recognised by mannose-binding-lectin and acti-vate the complement pathway) were increased in the platelets from these patients. Platelet surface exposure of other glycoside residues due to sialic acid loss inversely correlated with platelet count and the ability to be activated. Moreover, loss of sialic acid induced the ingestion of platelets by human hepatome HepG2 cells. Changes in glycoside composition of glycoproteins on the platelets’ surface impaired their functional capacity and increased their apoptosis. These changes in platelet glycoside residues appeared to be related to ITP severity.This research was funded by FIS-Fondos FEDER PI19/00631, FIS-Fondos FEDER PI19/00772 and Platelet Disorder Support Associatio

Evidence of telomere attrition and a potential role for DNA damage in systemic sclerosis

[Background]: To investigate the role of cell senescence in systemic sclerosis (SSc), we analyzed telomere shortening (TS) in SSc patients and the effect of targeting DNA damage in the bleomycin model of skin fibrosis. [Results]: Telomere length (TL) in blood leukocytes of 174 SSc patients and 68 healthy controls was measured by Southern blot, and we found shorter age-standardized TL in SSc patients compared to healthy controls. TL was shorter in SSc patients with ILD compared to those without ILD and in anti-topoisomerase I positive compared to anti-centromere positive patients. To analyze the potential role of DNA damage in skin fibrosis, we evaluated the effects of the DNA protective GSE4 peptide in the bleomycin mouse model of scleroderma and the fibrotic response of cultured human dermal fibroblasts. Administration of GSE4-nanoparticles attenuated bleomycin-induced skin fibrosis as measured by Masson’s staining of collagen and reduced Acta2 and Ctgf mRNA expression, whereas transduction of dermal fibroblasts with a lentiviral GSE4 expression vector reduced COL1A1, ACTA2 and CTGF gene expression after stimulation with bleomycin or TGF-β, in parallel to a reduction of the phospho-histone H2A.X marker of DNA damage. [Conclusions]: SSc is associated with TS, particularly in patients with lung disease or anti-topoisomerase I antibodies. Administration of GSE4 peptide attenuated experimental skin fibrosis and reduced fibroblast expression of profibrotic factors, supporting a role for oxidative DNA damage in scleroderma.The authors received financial support from Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (PI19/01129, PI20/00335, and RIER network RD16/0012 RETICS program), co-financed by the European Regional Development Fund (FEDER)

Lung Transplant Improves Survival and Quality of Life Regardless of Telomere Dysfunction

Introduction: Fibrotic interstitial lung diseases (ILDs) are the first indication for lung transplantation (LT). Telomere dysfunction has been associated with poor post-transplant outcomes. The aim of the study was to evaluate the morbi-mortality and quality of life in fibrotic ILDs after lung transplant depending on telomere biology. Methods: Fibrotic ILD patients that underwent lung transplant were allocated to two arms; with or without telomere dysfunction at diagnosis based on the telomere length and telomerase related gene mutations revealed by whole-exome sequencing. Post-transplant evaluation included: (1) short and long-term mortality and complications and (2) quality of life. Results: Fifty-five percent of patients that underwent LT carried rare coding mutations in telomerase-related genes. Patients with telomere shortening more frequently needed extracorporeal circulation and presented a higher rate of early post-transplant hematological complications, longer stay in the intensive care unit (ICU), and a higher number of long-term hospital admissions. However, post-transplant 1-year survival was higher than 80% regardless of telomere dysfunction, with improvement in the quality of life and oxygen therapy withdrawal. Conclusions: Post-transplant morbidity is higher in patients with telomere dysfunction and differs according to elapsed time from transplantation. However, lung transplant improves survival and quality of life and the associated complications are manageable

Next-generation sequencing in bone marrow failure syndromes and isolated cytopenias: experience of the spanish network on bone marrow failure sundromes

© 2021 the Author(s).Inherited bone marrow failure syndromes (IBMFSs) are a group of congenital rare diseases characterized by bone marrow failure, congenital anomalies, high genetic heterogeneity, and predisposition to cancer. Appropriate treatment and cancer surveillance ideally depend on the identification of the mutated gene. A next-generation sequencing (NGS) panel of genes could be 1 initial genetic screening test to be carried out in a comprehensive study of IBMFSs, allowing molecular detection in affected patients. We designed 2 NGS panels of IBMFS genes: version 1 included 129 genes and version 2 involved 145 genes. The cohort included a total of 204 patients with suspected IBMFSs without molecular diagnosis. Capture-based targeted sequencing covered > 99% of the target regions of 145 genes, with more than 20 independent reads. No differences were seen between the 2 versions of the panel. The NGS tool allowed a total of 91 patients to be diagnosed, with an overall molecular diagnostic rate of 44%. Among the 167 patients with classified IBMFSs, 81 patients (48%) were diagnosed. Unclassified IBMFSs involved a total of 37 patients, of whom 9 patients (24%) were diagnosed. The preexisting diagnosis of 6 clinically classified patients (6%) was amended, implying a change of therapy for some of them. Our NGS IBMFS gene panel assay is a useful tool in the molecular diagnosis of IBMFSs and a reasonable option as the first tier genetic test in these disorders

Next-generation Sequencing in Bone Marrow Failure Syndromes and Isolated Cytopenias : Experience of the Spanish Network on Bone Marrow Failure Syndromes

Inherited bone marrow failure syndromes (IBMFSs) are a group of congenital rare diseases characterized by bone marrow failure, congenital anomalies, high genetic heterogeneity, and predisposition to cancer. Appropriate treatment and cancer surveillance ideally depend on the identification of the mutated gene. A next-generation sequencing (NGS) panel of genes could be 1 initial genetic screening test to be carried out in a comprehensive study of IBMFSs, allowing molecular detection in affected patients. We designed 2 NGS panels of IBMFS genes: version 1 included 129 genes and version 2 involved 145 genes. The cohort included a total of 204 patients with suspected IBMFSs without molecular diagnosis. Capture-based targeted sequencing covered > 99% of the target regions of 145 genes, with more than 20 independent reads. No differences were seen between the 2 versions of the panel. The NGS tool allowed a total of 91 patients to be diagnosed, with an overall molecular diagnostic rate of 44%. Among the 167 patients with classified IBMFSs, 81 patients (48%) were diagnosed. Unclassified IBMFSs involved a total of 37 patients, of whom 9 patients (24%) were diagnosed. The preexisting diagnosis of 6 clinically classified patients (6%) was amended, implying a change of therapy for some of them. Our NGS IBMFS gene panel assay is a useful tool in the molecular diagnosis of IBMFSs and a reasonable option as the first tier genetic test in these disorders