Suberanilohydroxamic acid prevents TGF-β1-induced COX-2 repression in human lung fibroblasts post-transcriptionally by TIA-1 downregulation

Cyclooxygenase-2 (COX-2), with its main antifibrotic metabolite PGE, is regarded as an antifibrotic gene. Repressed COX-2 expression and deficient PGE have been shown to contribute to the activation of lung fibroblasts and excessive deposition of collagen in pulmonary fibrosis. We have previously demonstrated that COX-2 expression in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) is epigenetically silenced and can be restored by epigenetic inhibitors. This study aimed to investigate whether COX-2 downregulation induced by the profibrotic cytokine transforming growth factor-β1 (TGF-β1) in normal lung fibroblasts could be prevented by epigenetic inhibitors. We found that COX-2 protein expression and PGE production were markedly reduced by TGF-β1 and this was prevented by the pan-histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) and to a lesser extent by the DNA demethylating agent Decitabine (DAC), but not by the G9a histone methyltransferase (HMT) inhibitor BIX01294 or the EZH2 HMT inhibitor 3-deazaneplanocin A (DZNep). However, chromatin immunoprecipitation assay revealed that the effect of SAHA was unlikely mediated by histone modifications. Instead 3'-untranslated region (3'-UTR) luciferase reporter assay indicated the involvement of post-transcriptional mechanisms. This was supported by the downregulation by SAHA of the 3'-UTR mRNA binding protein TIA-1 (T-cell intracellular antigen-1), a negative regulator of COX-2 translation. Furthermore, TIA-1 knockdown by siRNA mimicked the effect of SAHA on COX-2 expression. These findings suggest SAHA can prevent TGF-β1-induced COX-2 repression in lung fibroblasts post-transcriptionally through a novel TIA-1-dependent mechanism and provide new insights into the mechanisms underlying its potential antifibrotic activity

Effect of epigenetic inhibitors on lung fibroblast phenotype change in idiopathic pulmonary fibrosis

Introduction and objectives: Idiopathic Pulmonary Fibrosis (IFP) is a fatal interstitial lung disease with unknown aetiology. Lung myofibroblasts (activated fibrobalsts) are the major effector cells in the pathogenesis of IPF. Transforming growth factor-β (TGF-β1) is a potent activator of fibroblasts. Lack of effective treatment options necessitates novel therapeutic approaches. Epigenetic drugs, by inhibiting chromatin modifying enzymes involved in gene expression control, represent promising agents capable of modulating the cellular phenotype. We previously demonstrated that the cyclooxygenase-2 (COX-2) gene is epigenetically silenced in lung fibroblasts from IPF patients (F-IPF)[1] and epigenetic inhibitors and restore COX-2 expression. However, whether epigenetic inhibitors can alter fibroblast phenotype remains unknown. This study aimed to investigate the effect of four different epigenetic enzyme inhibitors on fibroblast phenotype change in IPF. Methods: F-IPF and fibroblasts from non-fibrotic lung (F-NL) treated with TGF-β1 were cultured to test the effects of the epigenetic inhibitors BIX01294 (BIX, G9a histone methyltransferase inhibitor), 3- deazaneplanocin A (DZNep, EZH2 histone methyltransferase inhibitor), SAHA (histone deacetylases inhibitor) and Decitabine (DAC, DNA demethylating agent), in comparison with the COX-2 products prostaglandin E2 (PGE2). The expression of COX-2 and myofibroblast markers collagen 1 (COL1) and α- smooth muscle actin (α-SMA) was assessed. The COX-2 DNA promoter methylation level was analysed by bisulfite sequencing. Results: TGF-β1 induced a myofibroblast phenotype in F-NL characterised by COL1 and α-SMA upregulation and COX-2 downregulation, similar to F-IPF. PGE2 and SAHA were able to maintain/restore COX-2 expression in TGF-β1-induced myofibroblasts and F-IPF. DAC demonstrated similar effect in TGF-β1 treated F-NL only. SAHA also reduced COL1 and α-SMA expression. But DZNep and BIX showed no effect. No differences in the COX-2 promoter methylation was detected between F-NL and F-IPF. Conclusions: Among the epigenetic inhibitors tested, SAHA shows a promising antifibrotic effect by inhibiting fibroblast activation and the underlying molecular mechanisms are currently under investigation

Specific Biomarkers Are Associated with Docetaxeland Gemcitabine-Resistant NSCLC Cell Lines

AbstractFive-year survival rate for lung cancer is limited to 10% to 15%. Therefore, the identification of novel therapeutic prognostic factors is an urgent requirement. The aim of this study is thus to highlight specific biomarkers in chemoresistant non-small cell lung cancer cell lines. Therefore, we checked—in the control condition as well as after short-term pharmacological treatment with either docetaxel or gemcitabine—the expression of genes such as tumor suppressor genes (CDKN2A, DAPK, FHIT, GSTP1, MGMT, RARβ2, RASSF1A, and TIMP3), genes associated with drug resistance (BRCA1, COX2, ERCC1, IGFBP3, RRM1, and TUBB3), and stemness-related genes (CD133, OCT4, and SLUG) in two cellular models of squamous carcinoma (CAEP) and adenocarcinoma (RAL) of the lung originally established. Their promoter methylation profile was also evaluated. Drug-related genes were upregulated. Cisplatin resistance matched with high levels of BRCA1 and ERCC1 in both cell lines; docetaxel sensitivity of CAEP cells was associated to levels of TUBB3 lower than RAL cells. Although CAEP cells were more sensitive to gemcitabine, both cell lines showed high levels of RRM1. Stemness-related genes were downregulated in the control condition but became upregulated in docetaxel-resistant cells, indicating the selection of a population with stemness features. We did not find an unequivocal correspondence between gene expression and respective DNA promoter methylation status, suggesting the involvement of additional mechanisms of gene expression regulation. These results highlight specific biomarkers consistent with the different responses of the two cell lines to standard pharmacological treatments and indicate specific molecular traits for their chemoresistance

Priming adult stem cells by hypoxic pretreatments for applications in regenerative medicine

The efficiency of regenerative medicine can be ameliorated by improving the biological performances of stem cells before their transplantation. Several ex-vivo protocols of non-damaging cell hypoxia have been demonstrated to significantly increase survival, proliferation and post-engraftment differentiation potential of stem cells. The best results for priming cultured stem cells against a following, otherwise lethal, ischemic stress have been obtained with brief intermittent episodes of hypoxia, or anoxia, and reoxygenation in accordance with the extraordinary protection afforded by the conventional maneuver of ischemic preconditioning in severely ischemic organs. These protocols of hypoxic preconditioning can be rather easily reproduced in a laboratory; however, more suitable pharmacological interventions inducing stem cell responses similar to those activated in hypoxia are considered among the most promising solutions for future applications in cell therapy. Here we want to offer an up-to-date review of the molecular mechanisms translating hypoxia into beneficial events for regenerative medicine. To this aim the involvement of epigenetic modifications, microRNAs, and oxidative stress, mainly activated by hypoxia inducible factors, will be discussed. Stem cell adaptation to their natural hypoxic microenvironments (niche) in healthy and neoplastic tissues will be also considered

S52 Suberanilohydroxamic acid (SAHA) inhibits collagen deposition in a transforming growth factor β1-driven precision cut lung slice (PCLS) model of pulmonary fibrosis

Introduction and objectives Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive interstitial lung disease that is refractory to current treatment options. Transforming growth factor (TGF)-β1 is a key pro-fibrotic cytokine that plays a crucial role in IPF pathogenesis. Our group previously demonstrated distinct epigenetic modifications involved in repression of the antifibrotic gene cyclooxygenase-2 (COX-2) in fibroblasts from IPF (F-IPF) lungs compared with fibroblasts from non-fibrotic lungs (F-NL). Epigenetic drugs capable of inhibiting DNA and histone modifications may, therefore, represent a putative novel therapy. The aim of this study was to investigate the ability of 4 epigenetic inhibitors to regulate TGF-β-driven fibrosis in ex vivo mouse lung. Methods A precision-cut lung slice (PCLS) model of fibrosis was established using the previously described1 CC10-tTS-rtTA-TGFβ1 transgenic (tgTGF-β1) mouse. The model was first assessed by investigating PCLS overexpression of TGF-β1 in response to stimulation of the transgene by doxycycline treatment. Gene expression of COX-2 and fibrotic markers including collagen were assessed after 4 days of treatment. The anti-fibrotic potential of 4 epigenetic inhibitors; BIX01294 (BIX, inhibitor of G9a histone methyltransferase), 3-deazaneplanocin A (DZNep, inhibitor of EZH2 histone methyltransferase), SAHA (inhibitor of histone deacetylases, HDACs) and Decitabine (DAC, DNA demethylating agent) was investigated. Viability of PCLS was assessed by MTT and Prestoblue® assay. Results Treatment of PCLS from tgTGF-β1 mice with doxycycline induced a concentration-dependent increase in global TGF-β1, pro-fibrotic markers including collagen and pro-inflammatory COX-2, which was comparable to recombinant TGF-β1 treatment. Treatment with three of the epigenetic inhibitors BIX01294, DZNep and DAC did not reduce the pro-fibrotic response following doxycycline treatment. However SAHA demonstrated a significant suppressive effect on COX-2 and collagen expression, while not directly affecting TGF-β1 transgene expression. Conclusions The data suggests that SAHA has the potential to reduce fibrosis in a TGF-β1 driven model of pulmonary fibrosis. Further work is currently underway to assess the anti-fibrotic potential of this drug in tgTGF-β1 animals

Suberanilohydroxamic acid (SAHA) inhibits collagen deposition in a transforming growth factor β1-driven precision cut lung slice (PCLS) model of pulmonary fibrosis

Introduction and Objectives: Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive interstitial lung disease that is refractory to current treatment options. Transforming growth factor (TGF)-β1 is a key pro-fibrotic cytokine that plays a crucial role in IPF pathogenesis. Our group previously demonstrated distinct epigenetic modifications involved in repression of the antifibrotic gene cyclooxygenase-2 (COX-2) in fibroblasts from IPF (F-IPF) lungs compared with fibroblasts from non-fibrotic lungs (F-NL). Epigenetic drugs capable of inhibiting DNA and histone modifications may, therefore, represent a putative novel therapy. The aim of this study was to investigate the ability of 4 epigenetic inhibitors to regulate TGF-β-driven fibrosis in ex vivo mouse lung. Methods: A precision-cut lung slice (PCLS) model of fibrosis was established using the previously described [1] CC10-tTS-rtTA-TGFβ1 transgenic (tgTGF-β1) mouse. The model was first assessed by investigating PCLS overexpression of TGF-β1 in response to stimulation of the transgene by doxycycline treatment. Gene expression of COX-2 and fibrotic markers including collagen were assessed after 4 days of treatment. The anti-fibrotic potential of 4 epigenetic inhibitors; BIX01294 (BIX, inhibitor of G9a histone methyltransferase), 3-deazaneplanocin A (DZNep, inhibitor of EZH2 histone methyltransferase), SAHA (inhibitor of histone deacetylases, HDACs) and Decitabine (DAC, DNA demethylating agent) was investigated. Viability of PCLS was assessed by MTT and Prestoblue® viability assay. Results: Treatment of PCLS from tgTGF-β1 mice with doxycycline induced a concentration-dependent increase in global TGF-β1, pro-fibrotic markers including collagen and pro-inflammatory COX-2, which was comparable to recombinant TGF-β1 treatment. Treatment with three of the epigenetic inhibitors BIX01294, DZNep and DAC did not reduce the pro-fibrotic response following doxycycline treatment. However SAHA demonstrated a significant suppressive effect on COX-2 and collagen expression, while not directly affecting TGF-β1 transgene expression. Conclusions: The data suggests that SAHA has the potential to reduce fibrosis in a TGF-β1 driven model of pulmonary fibrosis. Further work is currently underway to assess the anti-fibrotic potential of this drug in tgTGF-β1 animals

Quotients of incidence geometries

We develop a theory for quotients of geometries and obtain sufficient conditions for the quotient of a geometry to be a geometry. These conditions are compared with earlier work on quotients, in particular by Pasini and Tits. We also explore geometric properties such as connectivity, firmness and transitivity conditions to determine when they are preserved under the quotienting operation. We show that the class of coset pregeometries, which contains all flag-transitive geometries, is closed under an appropriate quotienting operation.Comment: 26 pages, 5 figure

Imbalanced Prostanoid Release Mediates Cigarette Smoke-Induced Human Pulmonary Artery Cell Proliferation

BackgroundPulmonary hypertension is a common and serious complication of chronic obstructive pulmonary disease (COPD). Studies suggest that cigarette smoke can initiate pulmonary vascular remodelling by stimulating cell proliferation; however, the underlying cause, particularly the role of vasoactive prostanoids, is unclear. We hypothesize that cigarette smoke extract (CSE) can induce imbalanced vasoactive prostanoid release by differentially modulating the expression of respective synthase genes in human pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs), thereby contributing to cell proliferation.MethodsAqueous CSE was prepared from 3R4F research-grade cigarettes. Human PASMCs and PAECs were treated with or without CSE. Quantitative real-time RT-PCR and Western blotting were used to analyse the mRNA and protein expression of vasoactive prostanoid syhthases. Prostanoid concentration in the medium was measured using ELISA kits. Cell proliferation was assessed using the cell proliferation reagent WST-1.ResultsWe demonstrated that CSE induced the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostanoid synthesis, in both cell types. In PASMCs, CSE reduced the downstream prostaglandin (PG) I synthase (PGIS) mRNA and protein expression and PGI2 production, whereas in PAECs, CSE downregulated PGIS mRNA expression, but PGIS protein was undetectable and CSE had no effect on PGI2 production. CSE increased thromboxane (TX) A synthase (TXAS) mRNA expression and TXA2 production, despite undetectable TXAS protein in both cell types. CSE also reduced microsomal PGE synthase-1 (mPGES-1) protein expression and PGE2 production in PASMCs, but increased PGE2 production despite unchanged mPGES-1 protein expression in PAECs. Furthermore, CSE stimulated proliferation of both cell types, which was significantly inhibited by the selective COX-2 inhibitor celecoxib, the PGI2 analogue beraprost and the TXA2 receptor antagonist daltroban.ConclusionsThese findings provide the first evidence that cigarette smoke can induce imbalanced prostanoid mediator release characterized by the reduced PGI2/TXA2 ratio and contribute to pulmonary vascular remodelling and suggest that TXA2 may represent a novel therapeutic target for pulmonary hypertension in COPD

Red Blood Cell Morphologic Abnormalities in Patients Hospitalized for COVID-19

Peripheral blood smear is a simple laboratory tool, which remains of invaluable help for diagnosing primary and secondary abnormalities of blood cells despite advances in automated and molecular techniques. Red blood cells (RBCs) abnormalities are known to occur in many viral infections, typically in the form of mild normo-microcytic anemia. While several hematological alterations at automated complete blood count (including neutrophilia, lymphopenia, and increased red cell distribution width—RDW) have been consistently associated with severity of COVID-19, there is scarce information on RBCs morphological abnormalities, mainly as case-reports or small series of patients, which are hardly comparable due to heterogeneity in sampling times and definition of illness severity. We report here a systematic evaluation of RBCs morphology at peripheral blood smear in COVID-19 patients within the first 72 h from hospital admission. One hundred and fifteen patients were included, with detailed collection of other clinical variables and follow-up. A certain degree of abnormalities in RBCs morphology was observed in 75 (65%) patients. Heterogenous alterations were noted, with spiculated cells being the more frequent morphology. The group with >10% RBCs abnormalities had more consistent lymphopenia and thrombocytopenia compared to those without abnormalities or <10% RBCs abnormalities (p < 0.018, and p < 0.021, respectively), thus underpinning a possible association with an overall more sustained immune-inflammatory “stress” hematopoiesis. Follow-up analysis showed a different mortality rate across groups, with the highest rate in those with more frequent RBCs morphological alterations compared to those with <10% or no abnormalities (41.9%, vs. 20.5%, vs. 12.5%, respectively, p = 0.012). Despite the inherent limitations of such simple association, our results point out towards further studies on erythropoiesis alterations in the pathophysiology of COVID-19

Interplay between EZH2 and G9a regulates CXCL10 gene repression in idiopathic pulmonary fibrosis

Selective repression of the antifibrotic gene CXCL10 contributes to tissue remodelling in idiopathic pulmonary fibrosis (IPF). We have previously reported that histone deacetylation and histone H3 lysine 9 (H3K9) methylation are involved in CXCL10 repression. This study explored the role of H3K27 methylation and the interplay between the two histone lysine methyltransferases, Enhancer of Zest Homolog 2 (EZH2) and G9a, in CXCL10 repression in IPF. By applying chromatin immunoprecipitation (ChIP), Re-ChIP and proximity ligation assays, we demonstrated that, like G9a-mediated H3K9 methylation, EZH2-mediated H3K27me3 was significantly enriched at the CXCL10 promoter in fibroblasts from IPF lungs (F-IPF) compared with fibroblasts from non-fibrotic lungs (F-NL) and that EZH2 and G9a physically interacted with each other. EZH2 knockdown reduced not only EZH2 and H3K27me3 but also G9a and H3K9me3 and G9a knockdown reduced not only G9 and H3K9me3 but also EZH2 and H3K27me3. Depletion and inhibition of EZH2 and G9a also reversed histone deacetylation and restored CXCL10 expression in F-IPF. Furthermore, treatment of F-NL with the profibrotic cytokine TGF-β1 increased EZH2, G9a, H3K27me3, H3K9me3 and histone deacetylation at the CXCL10 promoter, similar to that observed in F-IPF, which was correlated with CXCL10 repression and was prevented by EZH2 and G9a knockdown. These findings suggest that a novel and functionally interdependent interplay between EZH2 and G9a regulates histone methylation-mediated epigenetic repression of the antifibrotic CXCL10 gene in IPF. This interdependent interplay may prove to be a target for epigenetic intervention to restore the expression of CXCL10 and other anti-fibrotic genes in IPF