Molecular characterization of the first missense mutation in the fibrinogen Aalpha-chain gene identified in a compound heterozygous afibrinogenemic patient

AbstractCongenital afibrinogenemia is a rare coagulopathy characterized by extremely low levels of functional and immunoreactive fibrinogen in plasma, associated with a hemorrhagic phenotype of variable severity. It is transmitted as an autosomal recessive trait and is invariantly associated with mutations affecting 1 of the 3 fibrinogen genes (FGA, FGB, and FGG, coding for Aα, Bβ, and γ chain, respectively). Most genetic defects causing afibrinogenemia are truncating mutations, whereas only few missense mutations (6) have been identified so far, all located in FGB.In this study, the mutational screening of an afibrinogenemic Italian male identified the first missense mutation (Met51Arg) in FGA leading to afibrinogenemia. The patient was a compound heterozygote for a previously described frameshift mutation (1215delT) in the same gene. Met51Arg involves a residue located at the very beginning of the coiled-coil domain, in a region demonstrated to play a pivotal role in hexamer formation. In-vitro expression experiments showed that Met51Arg strongly reduces secretion of hexameric fibrinogen, whereas traces of not completely assembled trimeric intermediate were found in conditioned media. Western blot analysis on the proband's plasma confirmed the presence in vivo of the trimeric fibrinogen, supporting the hypothesis that Met51Arg prevents the final step of fibrinogen assembly

TGFβ upregulates PAR-1 expression and signalling responses in A549 lung adenocarcinoma cells

The major high-affinity thrombin receptor, proteinase activated receptor-1 (PAR-1) is expressed at low levels by the normal epithelium but is upregulated in many types of cancer, including lung cancer. The thrombin-PAR-1 signalling axis contributes to the activation of latent TGFβ in response to tissue injury via an avβ6 integrin-mediated mechanism. TGFβ is a pleiotropic cytokine that acts as a tumour suppressor in normal and dysplastic cells but switches into a tumour promoter in advanced tumours. In this study we demonstrate that TGFβ is a positive regulator of PAR-1 expression in A549 lung adenocarcinoma cells, which in turn increases the sensitivity of these cells to thrombin signalling. We further demonstrate that this effect is Smad3-, ERK1/2-and Sp1-dependent. We also show that TGFβ-mediated PAR-1 upregulation is accompanied by increased expression of integrin av and β6 subunits. Finally, TGFβ pre-stimulation promotes increased migratory potential of A549 to thrombin. These data have important implications for our understanding of the interplay between coagulation and TGFβ signalling responses in lung cancer

Impact of a functional polymorphism in the PAR-1 gene promoter in COPD and COPD exacerbations

Proteinase-activated receptor-1 (PAR-1) plays a key role in mediating the interplay between coagulation and inflammation in response to injury. The aim of this study was to investigate the role of the promoter single-nucleotide polymorphism (SNP) rs2227744G&gt;A in modulating PAR-1/ F2R gene expression in the context of chronic obstructive pulmonary disease (COPD) and COPD exacerbations. The function of the rs2227744G&gt;A SNP was investigated by using reporter gene assays. The frequency of the polymorphism in the UK population was assessed by genotyping 8,579 healthy individuals from the Whitehall II and English Longitudinal Study of Ageing cohorts. The rs2227744G&gt;A SNP was genotyped in a carefully phenotyped cohort of 203 COPD cases and matched controls. The results were further replicated in two different COPD cohorts. The minor allele of the rs2227744G&gt;A polymorphism was found to increase F2R expression by 2.6-fold ( P &lt; 0.001). The rs2227744G&gt;A SNP was not significantly associated with COPD, or with lung function, in all cohorts. The minor allele of the SNP was found to be associated with protection from frequent exacerbations ( P = 0.04) in the cohort of COPD patients for which exacerbation frequency was available. Considering exacerbations as a continuous variable, the presence of the minor allele was associated with a significantly lower COPD exacerbation rate (3.03 vs. 1.98 exacerbations/year, Mann-Whitney U-test P = 0.04). Taken together, these data do not support a role for the rs2227744G&gt;A F2R polymorphism in the development of COPD but suggest a protective role for this polymorphism from frequent exacerbations. Studies in separate cohorts to replicate these findings are warranted. </jats:p

Surface modification of a POSS-nanocomposite material to enhance cellular integration of a synthetic bioscaffold

AbstractPolyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) is a versatile nanocomposite biomaterial with growing applications as a bioscaffold for tissue engineering. Integration of synthetic implants with host tissue can be problematic but could be improved by topographical modifications. We describe optimization of POSS-PCU by dispersion of porogens (sodium bicarbonate (NaHCO3), sodium chloride (NaCl) and sucrose) onto the material surface, with the principle aim of increasing surface porosity, thus providing additional opportunities for improved cellular and vascular ingrowth. We assess the effect of the porogens on the material's mechanical strength, surface chemistry, wettability and cytocompatibilty. Surface porosity was characterized by scanning electron microscopy (SEM). There was no alteration in surface chemistry and wettability and only modest changes in mechanical properties were detected. The size of porogens correlated well with the porosity of the construct produced and larger porogens improved interconnectivity of spaces within constructs. Using primary human bronchial epithelial cells (HBECs) we demonstrate moderate in vitro cytocompatibility for all surface modifications; however, larger pores resulted in cellular aggregation. These cells were able to differentiate on POSS-PCU scaffolds. Implantation of the scaffold in vivo demonstrated that larger pore sizes favor cellular integration and vascular ingrowth. These experiments demonstrate that surface modification with large porogens can improve POSS-PCU nanocomposite scaffold integration and suggest the need to strike a balance between the non-porous surfaces required for epithelial coverage and the porous structure required for integration and vascularization of synthetic scaffolds in future construct design

Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds

Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3–8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs

Understanding the burden of interstitial lung disease post-COVID-19: the UK Interstitial Lung Disease-Long COVID Study (UKILD-Long COVID)

Introduction The COVID-19 pandemic has led to over 100 million cases worldwide. The UK has had over 4 million cases, 400 000 hospital admissions and 100 000 deaths. Many patients with COVID-19 suffer long-term symptoms, predominantly breathlessness and fatigue whether hospitalised or not. Early data suggest potentially severe long-term consequence of COVID-19 is development of long COVID-19-related interstitial lung disease (LC-ILD). Methods and analysis The UK Interstitial Lung Disease Consortium (UKILD) will undertake longitudinal observational studies of patients with suspected ILD following COVID-19. The primary objective is to determine ILD prevalence at 12 months following infection and whether clinically severe infection correlates with severity of ILD. Secondary objectives will determine the clinical, genetic, epigenetic and biochemical factors that determine the trajectory of recovery or progression of ILD. Data will be obtained through linkage to the Post-Hospitalisation COVID platform study and community studies. Additional substudies will conduct deep phenotyping. The Xenon MRI investigation of Alveolar dysfunction Substudy will conduct longitudinal xenon alveolar gas transfer and proton perfusion MRI. The POST COVID-19 interstitial lung DiseasE substudy will conduct clinically indicated bronchoalveolar lavage with matched whole blood sampling. Assessments include exploratory single cell RNA and lung microbiomics analysis, gene expression and epigenetic assessment. Ethics and dissemination All contributing studies have been granted appropriate ethical approvals. Results from this study will be disseminated through peer-reviewed journals. Conclusion This study will ensure the extent and consequences of LC-ILD are established and enable strategies to mitigate progression of LC-ILD

Molecular characterization of 7 patients affected by dys- or hypo-dysfibrinogenemia: Identification of a novel mutation in the fibrinogen Bbeta chain causing a gain of glycosylation

Fibrinogen is a hexameric glycoprotein consisting of two sets of three polypeptides (the Aα, Bβ, and γ chains, encoded by the three genes FGA, FGB, and FGG). It is involved in the final phase of the coagulation process, being the precursor of the fibrin monomers necessary for the formation of the hemostatic plug. Rare inherited fibrinogen disorders can manifest as quantitative deficiencies, qualitative defects, or both. In particular, dysfibrinogenemia and hypo-dysfibrinogenemia are characterized by reduced functional activity associated with normal or reduced antigen levels, and are usually determined by heterozygous mutations affecting any of the three fibrinogen genes. In this study, we investigated the genetic basis of dys- and hypo-dysfibrinogenemia in seven unrelated patients. Mutational screening disclosed six different variants, two of which novel (FGB-p.Asp185Asn and FGG-p.Asn230Lys). The molecular characterization of the FGG-p.Asn230Lys mutation, performed by transient expression experiments of the recombinant mutant protein, demonstrated that it induces an almost complete impairment in fibrinogen secretion, according to a molecular mechanism often associated with quantitative fibrinogen disorders. Conversely, the FGB-p.Asp185Asn variant was demonstrated to be a gain-of-glycosylation mutation leading to a hyperglycosylation of the Bβ chain, not affecting fibrinogen assembly and secretion. To our knowledge, this is the second gain-of-glycosylation mutation involving the FGB gene

Genome-wide posttranscriptional dysregulation by microRNAs in human asthma as revealed by Frac-seq.

MicroRNAs are small noncoding RNAs that inhibit gene expression posttranscriptionally, implicated in virtually all biological processes. Although the effect of individual microRNAs is generally studied, the genome-wide role of multiple microRNAs is less investigated. We assessed paired genome-wide expression of microRNAs with total (cytoplasmic) and translational (polyribosome-bound) mRNA levels employing subcellular fractionation and RNA sequencing (Frac-seq) in human primary bronchoepithelium from healthy controls and severe asthmatics. Severe asthma is a chronic inflammatory disease of the airways characterized by poor response to therapy. We found genes (i.e., isoforms of a gene) and mRNA isoforms differentially expressed in asthma, with novel inflammatory and structural pathophysiological mechanisms related to bronchoepithelium disclosed solely by polyribosome-bound mRNAs (e.g., IL1A and LTB genes or ITGA6 and ITGA2 alternatively spliced isoforms). Gene expression (i.e., isoforms of a gene) and mRNA expression analysis revealed different molecular candidates and biological pathways, with differentially expressed polyribosome-bound and total mRNAs also showing little overlap. We reveal a hub of six dysregulated microRNAs accounting for ∼90% of all microRNA targeting, displaying preference for polyribosome-bound mRNAs. Transfection of this hub in bronchial epithelial cells from healthy donors mimicked asthma characteristics. Our work demonstrates extensive posttranscriptional gene dysregulation in human asthma, in which microRNAs play a central role, illustrating the feasibility and importance of assessing posttranscriptional gene expression when investigating human disease