Manuela Platé

Exploration of a potent PI3 kinase/mTOR inhibitor as a novel anti-fibrotic agent in IPF

Rationale Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal of all fibrotic conditions with no curative therapies. Common pathomechanisms between IPF and cancer are increasingly recognised, including dysfunctional pan-PI3 kinase (PI3K) signalling as a driver of aberrant proliferative responses. GSK2126458 is a novel, potent, PI3K/mammalian target of rapamycin (mTOR) inhibitor which has recently completed phase I trials in the oncology setting. Our aim was to establish a scientific and dosing framework for PI3K inhibition with this agent in IPF at a clinically developable dose. Methods We explored evidence for pathway signalling in IPF lung tissue and examined the potency of GSK2126458 in fibroblast functional assays and precision-cut IPF lung tissue. We further explored the potential of IPF patient-derived bronchoalveolar lavage (BAL) cells to serve as pharmacodynamic biosensors to monitor GSK2126458 target engagement within the lung. Results We provide evidence for PI3K pathway activation in fibrotic foci, the cardinal lesions in IPF. GSK2126458 inhibited PI3K signalling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells with comparable potency. Integration of these data with GSK2126458 pharmacokinetic data from clinical trials in cancer enabled modelling of an optimal dosing regimen for patients with IPF. Conclusions Our data define PI3K as a promising therapeutic target in IPF and provide a scientific and dosing framework for progressing GSK2126458 to clinical testing in this disease setting. A proof-of-mechanism trial of this agent is currently underway. Trial registration number NCT01725139, pre-clinical.

Pseudo-exon activation caused by a deep-intronic mutation in the fibrinogen γ-chain gene as a novel mechanism for congenital afibrinogenaemia

Congenital afibrinogenaemia, characterized by severe fibrinogen deficiency, is caused by mutations within FGA, FGB or FGG. Conventional sequencing of coding regions and splice signals of these three genes did not reveal any mutation in an afibrinogenaemic proband. After confirming disease co‐segregation with the fibrinogen cluster, full intron sequencing was tackled leading to the identification of a novel transvertion within FGG intron 6 (IVS6−320A→T). Its effect on mRNA processing was evaluated in‐vitro: the in‐frame inclusion of a 75‐bp pseudo‐exon carrying a premature stop was found, representing the first report of pseudo‐exon activation as a mechanism leading to afibrinogenaemia.

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

Polyhedral 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 materials 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.

Alterations of mRNA processing and stability as a pathogenic mechanism in von Willebrand factor quantitative deficiencies.

Summary.  Introduction: von Willebrand disease (VWD) is an inherited bleeding disorder due to a deficiency or abnormality of von Willebrand factor (VWF), associated with heterogeneous phenotypes. While VWD mutations acting at the protein level have been deeply investigated, fewer data are available on genetic defects affecting VWF mRNA. Aim: The aim of this study was to characterize the molecular mechanism underlying VWD in three patients. Methods: Mutational screening of the patients (P1–3) was accomplished by DNA sequencing of all VWF exons and splicing junctions. Platelet mRNA was analyzed by reverse‐transcription (RT)‐PCR and real‐time RT‐PCR. Results: P1 is a compound heterozygote for a c.1534‐3C>A transversion in intron 13 and for a nonsense mutation (p.Q77X) in exon 4. P2 is heterozygous for a splicing mutation in intron 9 (c.1109+2T>C). RT‐PCR assays on the patient’s platelet RNA revealed three mRNA populations: (i) wild type; (ii) lacking exon 9; and (iii) lacking exons 8 and 9. P3 showed a novel homozygous splicing mutation in intron 46 (c.7770+1G>T), producing three different mRNA species: (i) retaining the first 25 bp of intron 46; (ii) skipping exon 46; and (iii) skipping exon 46 while retaining 5 bp of intron 45. Whenever possible, the effect of mutations on the levels of VWF transcripts was analyzed, showing that mRNA variants containing a premature termination codon are downregulated, probably by the nonsense‐mediated mRNA decay pathway. Conclusions: The identification of the genetic basis of VWD in three patients confirmed that mutations leading to null alleles in the VWF gene are associated with allele‐specific mRNA degradation.

Clinical and molecular characterisation of 21 patients affected by quantitative fibrinogen deficiency

Fibrinogen is a plasma glycoprotein mainly synthesised by hepatocytes and circulating as a 340-kDa hexamer consisting of two sets of three different polypeptide chains (Aα, Bβ, and γ, encoded by the FGA, FGB, and FGG gene, respectively). Congenital afibrinogenaemia and hypofibrinogenaemia are rare bleeding disorders characterised by abnormally low levels of functional and immunoreactive fibrinogen in plasma, associated with haemorrhagic manifestations of variable severity. While afibrinogenaemia is caused by mutations in the homozygous or compound heterozygous state in one of the three fibrinogen genes, hypofibrinogenaemia is generally due to heterozygous mutations, and is usually characterised by a milder phenotype. The mutational spectrum of these quantitative fibrinogen disorders includes large deletions, point mutations causing premature termination codons, and missense mutations often affecting fibrinogen assembly and/or secretion. Here we report the clinical and molecular characterisation of 13 unrelated afibrinogenaemic and eight hypofibrinogenaemic patients, leading to the identification of 17 different mutations (10 hitherto unknown). All the newly-identified missense and splicing mutations werein vitro expressed to verify their pathogenic role. Our data increase the number of mutations causing quantitative fibrinogen deficiencies by about 7 %. The high number of private mutations identified in the analysed probands indicates that the full mutational screening of the three fibrinogen genes is still required for molecular diagnosis.

Premature termination codon mutations in the Von Willebrand factor gene are associated with allele-specific and position-dependent mRNA decay

Nonsense-mediated mRNA decay (NMD) is an intron-dependent RNA-degradation pathway responsible for depleting transcripts containing premature termination codons (PTCs), presumably to control the synthesis of truncated proteins, potentially deleterious to cells. PTC-bearing (PTC+) mRNAs are unstable only when the PTC is located more than 50–55 nucleotides upstream of the last intron.1 However, not all genes undergo NMD. Among coagulation genes, NMD was demonstrated for factors V, XI, and XIII, whereas it was shown to be inactive for fibrinogen (FGA, FGG) and factor VIII (FVIII) genes (Online Supplementary Table S1). Von Willebrand factor (VWF) is a multimeric glycoprotein, synthesized by endothelial cells and megakaryocytes, promoting both platelet adhesion to the subendothelium at sites of vascular injury, and platelet-platelet interactions in high shear-rate conditions. It also binds and stabilizes FVIII.2 Quantitative VWF deficiency can be classified as partial (VWD1) or complete (VWD3), whereas qualitative defects (VWD2) are subdivided into four main types: VWD2A, VWD2B, VWD2M, VWD2N.2 The aim of this study was to investigate whether PTC-introducing mutations in the VWF gene are associated with NMD. To this purpose, three unrelated Italian probands (P1–P3), heterozygous for at least one truncating mutation, were studied (Figure 1). Their main clinical characteristics are listed in the Online Supplementary Table S2. Figure 1. Functional analysis of the effect of the c.2546+3G>C and c.8155+6T>C splicing mutations on the VWF mRNA. (A, B) Top left: In the box underneath the patient’s ideogram: patient’s VWD type (in bold), VWF:Ag, VWF:RCo, and ... P1 has VWD2N and is compound heterozygous for the previously reported R854Q mutation (c.2561G>A, exon 20)2 and the novel c.2546+3G>C splicing defect (intron 19). P2 has VWD1 caused by compound heterozygosity for two novel mutations: C1927R (c.5779T>C, exon 34) and c.8155+6T>C (intron 50). P3 is heterozygous for the VWD3-causing c.6182delT mutation (exon 36).3 To evaluate the effect of the novel c.2546+3G>C and c.8155+6T>C splicing mutations on VWF pre-mRNA processing, cDNA regions spanning exons 18–21 and 49–52 were amplified by RT-PCR from platelet- and lymphocyte-derived mRNA of each patient. Sequencing of RT-PCR products demonstrated that c.8155+6T>C causes the skipping of exon 50 (Figure 1B), leading to a PTC in exon 51 (for details on methods, see Online Supplementary Appendix). Concerning c.2546+3G>C, a product lacking exon 19 could be amplified and sequenced only after a second semi-nested PCR (Figure 1A); this mutation would lead to the introduction of a PTC in exon 20. A very low amount of the same skipped transcript could be detected also in the control individual, indicating the existence of a “physiological” aberrant splicing event. To investigate whether the two PTC-introducing splicing defects are associated with mRNA degradation, a fragment containing the relevant missense mutation was PCR amplified from genomic DNA and from platelet and lymphocyte cDNAs of P1 and P2, and sequenced. Concerning P1, the product obtained from genomic DNA resulted heterozygous for R854Q, whereas platelet- and lymphocyte-derived RT-PCR products were homozygous for this missense substitution (Figure 2A), confirming that the PTC+ allele was degraded. As for patient P2, the C1927R mutation was detected in the heterozygous state both on genomic DNA and on cDNA (Figure 2A), suggesting that the PTC+ allele is not subjected to NMD, as expected for a PTC located 23 bp upstream of the last exon-exon junction. Figure 2. Missense mutations and 1-bp deletion analyses. The central panel shows a schematic representation of part of the VWF gene (introns 18–21 and introns 33–37; exons are represented by boxes, introns by lines, and are not drawn to scale). ... Patient P3 was similar to P1 in that the genomic sequence was heterozygous for the T deletion, whereas the cDNA sequences appeared wild type (Figure 2B), suggesting a selective degradation of the mutant transcript. To calculate the extent of the PTC+ mRNA degradation, the fragment containing the T deletion was also PCR amplified using a (6-Fam)-labeled primer. PCR reactions were separated on an ABI-3130XL sequencer and the peak areas measured by the GeneMapper v4.0 software. On genomic DNA, the wild-type/mutant ratio was equal to ~1, as expected. Conversely, a degradation of 91.2% and 86.1% of the PTC+ mRNA was calculated in platelets and lymphocytes (Figure 2B). To summarize, our data consistently demonstrate that truncated VWF proteins are unlikely to be produced as a result of mRNA degradation, a topic on which conflicting data were reported in the literature.4–6 Moreover, we confirm that NMD susceptibility of VWF transcripts is PTC-position dependent. Last but not least, we were able to measure the extent of degradation of the c.6182delT transcript (~90%). Given the effects of VWF inhibitors, i.e. ineffectiveness of replacement therapy and anaphylactic reactions to treatment,2 it would be important to establish if NMD might be a modulator of inhibitor development. Considering that some PTC-introducing mutations escape degradation even in genes known to be targets of NMD7,8 it would be interesting to analyze a larger group of VWD3 patients with truncating mutations, to verify if mRNA degradation represents a general rule for truncating mutations in the VWF gene.

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.

Congenital hypofibrinogenemia : characterization of two missense mutations affecting fibrinogen assembly and secretion

Congenital hypofibrinogenemia is a rare bleeding disorder characterized by abnormally low levels of fibrinogen in plasma, generally due to heterozygous mutations in one of the three fibrinogen genes (FGA, FGB, and FGG, coding for Aalpha, Bbeta, and gamma chain, respectively). Hypofibrinogenemic patients are usually asymptomatic, whereas individuals bearing similar mutations in the homozygous or compound heterozygous state develop a severe bleeding disorder: afibrinogenemia. The mutational spectrum of these quantitative fibrinogen disorders includes large deletions, point mutations causing premature termination codons, and missense mutations affecting fibrinogen assembly or secretion, distributed throughout the 50-kb fibrinogen gene cluster. In this study, we report the mutational screening of two unrelated hypofibrinogenemic patients leading to the identification of two missense mutations, one hitherto unknown (alphaCys45Phe), and one previously described (gammaAsn345Ser). The involvement of alphaCys45Phe and gammaAsn345Ser in the pathogenesis of hypofibrinogenemia was investigated by in-vitro expression experiments. Both mutations were demonstrated to cause a severe impairment of intracellular fibrinogen processing, either by affecting half-molecule dimerization (alphaCys45Phe) or by hampering hexamer secretion (gammaAsn345Ser).

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.

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>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>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>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>A polymorphism was found to increase F2R expression by 2.6-fold (P < 0.001). The rs2227744G>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>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.