Telomerase and Telomere Length in Pulmonary Fibrosis

In addition to its expression in stem cells and many cancers,telomerase activity is transiently induced inmurine bleomycin (BLM)induced pulmonary fibrosis with increased levels of telomerase transcriptase (TERT) expression, which is essential for fibrosis. To extend these observations to human chronic fibrotic lung disease,we investigated the expression of telomerase activity in lung fibroblasts from patients with interstitial lung diseases (ILDs), including idiopathic pulmonaryfibrosis (IPF).The resultsshowedthat telomerase activity was induced in more than 66% of IPF lung fibroblast samples, in comparison with less than 29% from control samples,some of which were obtained from lung cancer resections. Less than 4%of the humanIPF lung fibroblast samples exhibited shortened telomeres,whereas less than 6% of peripheral blood leukocyte samples from patients with IPF or hypersensitivity pneumonitis demonstrated shortened telomeres. Moreover, shortened telomeres in lategeneration telomerase RNA component knockout mice did not exert a significant effect on BLM-induced pulmonary fibrosis. In contrast, TERT knockout mice exhibited deficient fibrosis that was independent of telomere length. Finally, TERT expression was up-regulated by a histone deacetylase inhibitor, while the induction of TERT in lung fibroblastswasassociatedwiththebindingofacetylatedhistoneH3K9to the TERT promoter region. These findings indicate that significant telomerase inductionwas evident in fibroblasts from fibroticmurine lungs and a majority of IPF lung samples, whereas telomere shortening was not a common finding in the human blood and lung fibroblast samples. Notably, the animal studies indicated that the pathogenesis of pulmonary fibrosis was independent of telomere length

Calmodulin-binding transcription activator 1 (CAMTA1) alleles predispose human episodic memory performance

Little is known about the genes and proteins involved in the process of human memory. To identify genetic factors related to human episodic memory performance, we conducted an ultra-high-density genome-wide screen at > 500000 single nucleotide polymorphisms (SNPs) in a sample of normal young adults stratified for performance on an episodic recall memory test. Analysis of this data identified SNPs within the calmodulin-binding transcription activator 1 (CAMTA1) gene that were significantly associated with memory performance. A follow up study, focused on the CAMTA1 locus in an independent cohort consisting of cognitively normal young adults, singled out SNP rs4908449 with a P-value of 0.0002 as the most significant associated SNP in the region. These validated genetic findings were further supported by the identification of CAMTA1 transcript enrichment in memory-related human brain regions and through a functional magnetic resonance imaging experiment on individuals matched for memory performance that identified CAMTA1 allele-specific upregulation of medial temporal lobe brain activity in those individuals harboring the ‘at-risk' allele for poorer memory performance. The CAMTA1 locus encodes a purported transcription factor that interfaces with the calcium-calmodulin system of the cell to alter gene expression patterns. Our validated genomic and functional biological findings described herein suggest a role for CAMTA1 in human episodic memor

Identification of a Cell-of-Origin for Fibroblasts Comprising the Fibrotic Reticulum in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of the middle aged and elderly with a prevalence of one million persons worldwide. The fibrosis spreads from affected alveoli into contiguous alveoli, creating a reticular network that leads to death by asphyxiation. Lung fibroblasts from patients with IPF have phenotypic hallmarks, distinguishing them from their normal counterparts: pathologically activated Akt signaling axis, increased collagen and α-smooth muscle actin expression, distinct gene expression profile, and ability to form fibrotic lesions in model organisms. Despite the centrality of these fibroblasts in disease pathogenesis, their origin remains uncertain. Here, we report the identification of cells in the lungs of patients with IPF with the properties of mesenchymal progenitors. In contrast to progenitors isolated from nonfibrotic lungs, IPF mesenchymal progenitor cells produce daughter cells manifesting the full spectrum of IPF hallmarks, including the ability to form fibrotic lesions in zebrafish embryos and mouse lungs, and a transcriptional profile reflecting these properties. Morphological analysis of IPF lung tissue revealed that mesenchymal progenitor cells and cells with the characteristics of their progeny comprised the fibrotic reticulum. These data establish that the lungs of patients with IPF contain pathological mesenchymal progenitor cells that are cells of origin for fibrosis-mediating fibroblasts. These fibrogenic mesenchymal progenitors and their progeny represent an unexplored target for novel therapies to interdict fibrosis

Melting and differentiation of early-formed asteroids: The perspective from high precision oxygen isotope studies

A number of distinct methodologies are available for determining the oxygen isotope composition of minerals and rocks, these include laser-assisted fluorination, secondary ion mass spectrometry (SIMS)and UV laser ablation. In this review we focus on laser-assisted fluorination, which currently achieves the highest levels of precision available for oxygen isotope analysis. In particular, we examine how results using this method have furthered our understanding of early-formed differentiated meteorites. Due to its rapid reaction times and low blank levels, laser-assisted fluorination has now largely superseded the conventional externally-heated Ni “bomb” technique for bulk analysis. Unlike UV laser ablation and SIMS analysis, laser-assisted fluorination is not capable of focused spot analysis. While laser fluorination is now a mature technology, further analytical improvements are possible via refinements to the construction of sample chambers, clean-up lines and the use of ultra-high resolution mass spectrometers. High-precision oxygen isotope analysis has proved to be a particularly powerful technique for investigating the formation and evolution of early-formed differentiated asteroids and has provided unique insights into the interrelationships between various groups of achondrites. A clear example of this is seenin samples that lie close to the terrestrial fractionation line (TFL). Based on the data from conventional oxygen isotope analysis, it was suggested that the main-group pallasites, the howardite eucrite diogenite suite (HEDs) and mesosiderites could all be derived from a single common parent body. However,high precision analysis demonstrates that main-group pallasites have a Δ17O composition that is fully resolvable from that of the HEDs and mesosiderites, indicating the involvement of at least two parent bodies. The range of Δ17O values exhibited by an achondrite group provides a useful means of assessing the extent to which their parent body underwent melting and isotopic homogenization. Oxygen isotope analysis can also highlight relationships between ungrouped achondrites and the more well-populated groups. A clear example of this is the proposed link between the evolved GRA 06128/9 meteorites and the brachinites. The evidence from oxygen isotopes, in conjunction with that from other techniques, indicates that we have samples from approximately 110 asteroidal parent bodies (∼60 irons, ∼35 achondrites and stony-iron, and ∼15 chondrites) in our global meteorite collection. However, compared to the likely size of the original protoplanetary asteroid population, this is an extremely low value. In addition, almost all of the differentiated samples (achondrites, stony-iron and irons) are derived from parent bodies that were highly disrupted early in their evolution. High-precision oxygen isotope analysis of achondrites provides some important insights into the origin of mass-independent variation in the early Solar System. In particular, the evidence from various primitive achondrite groups indicates that both the slope 1 (Y&R) and CCAM lines are of primordial significance. Δ17O differences between water ice and silicate-rich solids were probably the initial source of the slope 1 anomaly. These phases most likely acquired their isotopic composition as a result of UV photo-dissociation of CO that took place either in the early solar nebula or precursor giant molecular cloud. Such small-scale isotopic heterogeneities were propagated into larger-sized bodies, such as asteroids and planets, as a result of early Solar System processes, including dehydration, aqueous alteration,melting and collisional interactions

Fibrotic Myofibroblasts Manifest Genome-Wide Derangements of Translational Control

Background: As a group, fibroproliferative disorders of the lung, liver, kidney, heart, vasculature and integument are common, progressive and refractory to therapy. They can emerge following toxic insults, but are frequently idiopathic. Their enigmatic propensity to resist therapy and progress to organ failure has focused attention on the myofibroblast–the primary effector of the fibroproliferative response. We have recently shown that aberrant beta 1 integrin signaling in fibrotic fibroblasts results in defective PTEN function, unrestrained Akt signaling and subsequent activation of the translation initiation machinery. How this pathological integrin signaling alters the gene expression pathway has not been elucidated. Results: Using a systems approach to study this question in a prototype fibrotic disease, Idiopathic Pulmonary Fibrosis (IPF); here we show organized changes in the gene expression pathway of primary lung myofibroblasts that persist for up to 9 sub-cultivations in vitro. When comparing IPF and control myofibroblasts in a 3-dimensional type I collagen matrix, more genes differed at the level of ribosome recruitment than at the level of transcript abundance, indicating pathological translational control as a major characteristic of IPF myofibroblasts. To determine the effect of matrix state on translational control, myofibroblasts were permitted to contract the matrix. Ribosome recruitment in control myofibroblasts was relatively stable. In contrast, IPF cells manifested large alterations in the ribosome recruitment pattern. Pathological studies suggest an epithelial origin for IPF myofibroblasts through the epithelial to mesenchymal transition (EMT). In accord wit

FoxO3a (Forkhead Box O3a) deficiency protects Idiopathic Pulmonary Fibrosis (IPF) fibroblasts from type I polymerized collagen matrix-induced apoptosis via caveolin-1 (cav-1) and Fas.

Idiopathic Pulmonary Fibrosis is a lethal fibrotic disease characterized by the unrelenting proliferation and persistence of fibroblasts in a type I collagen-rich matrix that result in an expanding reticular network of fibrotic tissue. However, the underlying mechanism responsible for the persistence of myofibroblasts in IPF remains unclear. During normal tissue repair, unwanted fibroblasts are eliminated during collagen-matrix contraction by a mechanism whereby high PTEN activity suppresses Akt. We have previously found that FoxO3a, a transcriptional activator of apoptosis-inducing proteins, is inactivated in IPF fibroblasts resulting from aberrantly high PI3K/Akt activity due to inappropriately low PTEN activity. Here we demonstrate that this low FoxO3a activity confers IPF fibroblasts with resistance to collagen-mediated apoptosis. We show that the mechanism by which low FoxO3a activity confers IPF fibroblasts with an apoptotic resistant phenotype involves suppression of Fas expression as a result of down regulation of cav-1 expression via a PTEN/Akt-dependent pathway. We demonstrate that PTEN over-expression or Akt inhibition increases FoxO3a expression in IPF fibroblasts, resulting in up-regulation of caveolin-1. We show that FoxO3a binds to the cav-1 promoter region and ectopic expression of FoxO3a transcriptionally increases cav-1 mRNA and protein expression. In turn, we show that overexpression of caveolin-1 increases Fas levels and caspase-3/7 activity and promotes IPF fibroblast apoptosis on polymerized type I collagen. We have found that the expression of caveolin-1, Fas and cleaved caspase-3 proteins in fibroblasts within the fibroblastic foci of IPF patient specimens is low. Our data indicate that the pathologically altered PTEN/Akt axis inactivates FoxO3a down-regulating cav-1 and Fas expression. This confers IPF fibroblasts with an apoptosis-resistant phenotype and may be responsible for IPF progression

Forced expression of FoxO3a increases Fas expression via cav-1 in IPF fibroblasts and promotes apoptosis on polymerized collagen matrix.

<p>A. IPF fibroblasts infected with adenovirus over-expressing FoxO3a (F3), cav-1 (C-1) or empty vector GFP were cultured on polymerized collagen for 48 h in serum free medium. Left panel, Fas, FoxO3a, cav-1 and GAPDH expression were measured by Western blot analysis. Three independent assays are represented. Right panel. Fas/GAPDH protein expression ratio was quantified. *<i>p</i> = 0.02 versus GFP. **<i>p</i> = 0.05 versus GFP. B. IPF fibroblasts infected with adenovirus over-expressing FoxO3a (F3), cav-1 (C-1) or empty vector GFP were cultured on polymerized collagen for 48 h and caspase-3/7 activity was measured. Results were obtained from triplicates. C. IPF fibroblasts infected with adenovirus over-expressing FoxO3a (F3), cav-1 (C1), FoxO3a in the presence of 10 nM of Cav-1 siRNA (F3+C-1S), or empty vector GFP with 10 nM control siRNA (GFP/Con) were cultured on polymerized collagen for 48 hrs. Fas, FoxO3a, cav-1 and GAPDH expression were examined by Western blot analysis. Blot represents 3 independent assays. D. IPF fibroblasts infected with adenovirus expressing FoxO3a alone (F3) or IPF fibroblasts over-expressing FoxO3a in the presence of 10 nM of Cav-1 siRNA (F3+C-1S) or GFP/Control siRNA (GFP/Con) were cultured on polymerized collagen for 48 hrs. Left panel, Fas expression was quantified. *<i>p</i> = 0.03 versus GFP/Con. Results were obtained from 3 independent assays. Middle panel, Caspase-3/7 activity was measured. *<i>p</i> = 0.01 versus GFP/Con. Results were obtained from triplicates. Right panel, viable cells were quantified. *<i>p</i> = 0.01, **<i>p</i> = 0.05 versus GFP/Con, respectively. Results were obtained from quadruplicates. E–F. 2×10⁵ IPF fibroblasts infected with adenovirus over-expressing FoxO3a (F3), cav-1 (C1) or empty vector GFP were cultured on polymerized collagen in the presence of 10 nM Fas or control siRNA for 48 h in serum free medium. Fas, FoxO3a, cav-1 and GAPDH expression were measured by Western blot analysis (E). Cell viability (F, upper panel) and caspase-3/7 activity (F, lower panel) were measured as described in Materials and Methods. Results were obtained from triplicates.</p

Aberrant function of the PTEN/Akt axis inactivates FoxO3a and suppresses cav-1 expression.

<p><b>A</b>. Left panel. IPF and control fibroblasts infected with adenovirus expressing hyperactive Akt (HA), dominant negative Akt (DA) or empty vector GFP were cultured on polymerized collagen and cav-1 protein expression was examined by Western blot analysis. GAPDH is shown as a loading control. Right panel, cav-1/GAPDH expression ratio was quantified by densitometry. *<i>p</i> = 0.017, **<i>p</i> = 0.008 versus GFP control. <b>B</b>. Control fibroblasts infected with adenovirus co-expressing either wild type FoxO3a & hyperactive Akt or empty GFP & hyperactive Akt were cultured on polymerized collagen in serum free medium for 24 h, and Western analysis was carried out to measure cav-1, p-Akt, FoxO3a and GAPDH levels. Note that cav-1 level is increased when FoxO3a and hyperactive Akt were co-expressed. <b>C</b>. Left, IPF fibroblasts infected with adenovirus expressing wild type PTEN (WP) or empty vector GFP were cultured on polymerized collagen for 24 h and cav-1 protein expression was examined by Western blot analysis. GAPDH is shown as loading control. Right, cav-1/GAPDH levels were quantified by densitometry. <b>D</b>. Left, control fibroblasts infected with adenovirus expressing mutant PTEN (MP) or empty vector GFP were cultured on polymerized collagen for 24 h and cav-1 protein levels were examined by Western blot analysis. GAPDH is shown as loading control. Right, cav-1/GAPDH levels were quantified by densitometry. All blots represent at least 3 independent assays.</p