PI3K p110γ overexpression in idiopathic pulmonary fibrosis lung tissue and fibroblast cells: in vitro effects of its inhibition.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease whose molecular pathogenesis remains unclear. In a recent paper, we demonstrated a key role for the PI3K pathway in both proliferation and differentiation into myofibroblasts of normal human lung fibroblasts treated with TGF-ß. In this research, we assessed the expression of class I PI3K p110 isoforms in IPF lung tissue as well as in tissue-derived fibroblast cell lines. Moreover, we investigated the in vitro effects of the selective inhibition of p110 isoforms on IPF fibroblast proliferation and fibrogenic activity. IHC was performed on normal and IPF lung tissue. Expression levels of PI3K p110 isoforms were evaluated by western blot and flow cytometry analysis. Fibroblast cell lines were established from both normal and IPF tissue and the effects of selective pharmacological inhibition as well as specific gene silencing by small interfering RNAs were studied in vitro. No significant differences between normal and IPF tissue/tissue-derived fibroblasts were observed for the expression of PI3K p110 α, ß and δ isoforms whereas p110γ was more greatly expressed in both IPF lung homogenates and ex vivo fibroblast cell lines. Myofibroblasts and bronchiolar basal cells in IPF lungs exhibited strong immunoreactivity for p110γ. Positive staining for the markers of proliferation proliferating cell nuclear antigen and cyclin D1 was also shown in cells of fibrolastic foci. Furthermore, both p110γ pharmacological inhibition and gene silencing were able to significantly inhibit proliferation rate as well as α-SMA expression in IPF fibroblasts. Our data suggest that PI3K p110γ isoform may have an important role in the etio-pathology of IPF and can be a specific pharmacological target.

Thymosin ß4 protects C57BL/6 mice from bleomycin-induced damage in the lung.

BACKGROUND: Thymosin ß4 (Tß4) was recently found at high concentration in the bronchoalveolar lavage fluid (BALF) of scleroderma patients with lung involvement. It has been hypothesized that Tß4 may exert a cyto-protective effect during lung injury because lower Tß4 levels were associated with interstitial lung disease progression. Moreover, Tß4 treatment prevented profibrotic gene expression in cardiac cells in vitro and in vivo. MATERIALS AND METHODS: In this study, we explored a putative Tß4 protective role in lung damage by utilizing a well-known in vivo model of lung fibrosis. C57BL/6 mice were treated with bleomycin (BLEO, 1 mg/kg) in the absence or presence of Tß4 (6 mg/kg delivered intraperitoneally on the day of BLEO treatment and for two additional doses). After sacrifice 1 week later, measurement of fluid and collagen content in the lung, BALF analysis, myeloperoxidase (MPO) activity assay, lung histology and IHC were performed. RESULTS: Compared with BLEO-treated mice, BLEO-treated mice who received Tß4 did not lose as much weight and had a higher survival rate. Moreover, BLEO-induced inflammation and lung damage were substantially reduced by Tß4 treatment, as demonstrated by the significant reduction in oedema, total collagen content, lung infiltration by leucocytes, MPO activity in lung homogenates, and histological evidence of the ongoing lung fibrosis. Results of IHC show a strong reactivity for Tß4 in the lung tissue of Tß4-treated mice. CONCLUSIONS: This is the first report that shows a Tß4 protective role in lung toxicity associated with BLEO in a mouse model. Future studies are needed to assess its putative antifibrotic properties.

Mediterranean Diet and Particulate Matter Exposure Are Associated With LINE-1 Methylation: Results From a Cross-Sectional Study in Women.

Emerging evidence suggests that air pollution increases the risk of cardiovascular disease (CVD) and metabolic disorders, adding to the global burden of disease attributable to lifestyle and behavioral factors. Although long interspersed nucleotide elements 1 (LINE-1) methylation has been associated with these disorders, no studies have simultaneously examined the effects of diet and air pollution exposure on DNA methylation. Herein, we evaluated the association of particulate matter (PM with aerodynamic diameters of less than 10 mm) exposure and adherence to Mediterranean Diet (MD) with LINE-1 methylation. Healthy women (n = 299), aged 15 to 80 years, were enrolled in a cross-sectional study. Dietary data and adherence to MD were assessed by a Food Frequency Questionnaire (FFQ) and Mediterranean Diet Score (MDS). PM10 levels during 1-month before recruitment were recorded by monitoring stations and assigned to each woman based on their residential address and day of recruitment. LINE-1 methylation in blood samples was assessed by pyrosequencing and reported as percentage of 5-methylcytosine (5mC). The Mann-Whitney U test, Spearman's rank correlation test and linear regression models were applied. Our results demonstrated, for the first time, an inverse association between adherence to MD and exposure to PM10 with LINE-1 methylation: while higher monthly PM10 exposure decreases LINE-1 methylation level (ß = -0.121; p = 0.037), the adherence to MD increases it (ß = 0.691; p < 0.001). MDS seemed to interact with PM10 levels (p = 0.002) on LINE-1 methylation, as such we confirmed that the effect of MD decreased with increasing PM10 levels (ß = 0.657; p < 0.001 in the first tertile; ß = 0.573; p < 0.001 in the second tertile; ß = 0.551; p < 0.001 in the third tertile). Thus, we suggest that LINE-1 methylation is a possible mechanism underpinning environment-related health effects, and encourage further research to evaluate whether the adherence to the MD could counteract the negative effect of PM10 exposure.

Integrated approach of nutritional and molecular epidemiology, mineralogical and chemical pollutant characterisation: the protocol of a cross-sectional study in women.

INTRODUCTION: Environmentally-related health and disease are the result of the exposome, the totality of a person's environmental exposures, from all sources and routes, across their lifespan. Epigenetic phenomena, including DNA methylation, can be potentially modified by environmental and lifestyle factors, and result in environmental reprogramming of the genome for exposed individuals and for future generations of offspring. OBJECTIVE: The objective of the project is to evaluate the risk of DNA hypomethylation due to air pollution, Mediterranean diet adherence, folate intake, and demographic and socioeconomic factors, in healthy women living in the metropolitan area of Catania, Italy. METHODS AND ANALYSIS: Non-pregnant healthy women will be enrolled in a cross-sectional study. Sociodemographic, lifestyle and dietary intake information will be collected. LINE-1 methylation will be measured by pyrosequencing. The participants' home addresses will be geocoded and each woman will be assigned to the closest monitoring station for particulate matter (PM) exposure assessment. Mineralogical-chemical characterisation of PM and cellular model assays will be performed. An integrated approach will be designed to estimate the combined possible effect of air pollution, Mediterranean diet adherence, folate intake and other lifestyle characteristics on LINE-1 methylation levels. ETHICS AND DISSEMINATION: The project has been approved by the ethics committees of the involved institution and funded by the University of Catania (Finanziamento della Ricerca, FIR 2014). All participants will be fully informed of the purpose and procedures of the study, and signed written consents will be obtained. All the data collected will be treated confidentially and analysed in an aggregate and anonymous way. The results will be published in peer-reviewed journals and communicated to local public health agencies, in order to provide essential information for timely and effective public health action.

Preventive and therapeutic effects of thymosin ß4 N-terminal fragment Ac-SDKP in the bleomycin model of pulmonary fibrosis.

In this study, the bleomycin model of pulmonary fibrosis was utilized to investigate putative anti-fibrotic activity of Ac-SDKP in vivo. Male CD-1 mice received intra-tracheal bleomycin (BLEO, 1 mg/kg) instillation in the absence or presence of Ac-SDKP (a dose of 0.6 mg/kg delivered intra-peritoneally on the day of BLEO treatment, d0, followed by bi-weekly additional doses). To evaluate therapeutic effects in a subset of mice, Ac-SDKP was administered one week after BLEO instillation (d7). Animals were sacrificed at one, two, or three weeks later. Measurement of fluid and collagen content in the lung, Broncho Alveolar Lavage Fluid (BALF) analysis, lung histology, immunohistochemistry (IHC), and molecular analysis were performed. Compared to BLEO-treated mice, animals that received also Ac-SDKP (at both d0 and d7) had significantly decreased mortality, weight loss, inflammation (edema, and leukocyte lung infiltration), lung damage (histological evidence of lung injury), and fibrosis (collagen histological staining and soluble collagen content in the lung) at up to 21 days. Moreover, IHC and quantitative RT-PCR results demonstrated a significant decrease in BLEO-induced IL-17 and TGF-ß expression in lung tissue. Importantly, α-SMA expression, the hallmark of myofibroblast differentiation, was also decreased. This is the first report showing not only a preventive protective role of Ac-SDKP but also its significant therapeutic effects in the bleomycin model of pulmonary fibrosis, thus supporting further preclinical and clinical studies.

Anti-inflammatory and antifibrotic effects of resveratrol in the lung.

Resveratrol, a natural polyphenolic molecule with several biological activities, is a well recognized anti-oxidant, anti-aging and cancer chemopreventive agent. Moreover, resveratrol anti-inflammatory and antifibrotic properties have been demonstrated both in vitro and in different animal models of inflammatory pathologies, including bowel and liver diseases. We review the evidence of resveratrol protective role in respiratory diseases such as acute lung injury, asthma, chronic obstructive pulmonary disease and lung fibrosis. We conclude that resveratrol and its derivatives may act as a therapeutic agents in respiratory diseases and pertinent clinical trials should be performed.

Effects of thymosin ß4 and its N-terminal fragment Ac-SDKP on TGF-ß-treated human lung fibroblasts and in the mouse model of bleomycin-induced lung fibrosis.

UNLABELLED: Thymosin ß4 (Tß4) and its amino-terminal fragment comprising N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) have been reported to act as anti-inflammatory and anti-fibrotic agents in vitro and in vivo. In recent papers, we have shown that Tß4 exerts a widely protective role in mice treated with bleomycin, and in particular, we have demonstrated its inhibitory effects on both inflammation and early fibrosis. OBJECTIVES: In this study, the putative anti-proliferative and anti-fibrogenic effects of Tß4 and Ac-SDKP were evaluated in vitro. In addition, the effects of Tß4 up to 21 days were evaluated in the bleomycin mouse model of lung fibrosis. METHODS: We utilized both control and TGF-ß-stimulated primary human lung fibroblasts isolated from both idiopathic pulmonary fibrosis (IPF) and control tissues. The in vivo effects of Tß4 were assessed in CD1 mice treated with bleomycin. RESULTS: In the in vitro experiments, we observed significant anti-proliferative effects of Ac-SDKP in IPF fibroblasts. In those cells, Ac-SDKP significantly inhibited TGF-ß-induced α-SMA and collagen expression, hallmarks of fibroblast differentiation into myofibroblasts triggered by TGF-ß. In vivo, despite its previously described protective role in mice treated with bleomycin at 7 days, Tß4 failed to prevent fibrosis induced by the drug at 14 and 21 days. CONCLUSION: We conclude that, compared to Tß4, Ac-SDKP may have greater potential as an anti-fibrotic agent in the lung. Further in vivo experiments are warranted.

Effect of pirfenidone on proliferation, TGF-ß-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts.

Pirfenidone is an orally active small molecule that has been shown to inhibit the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Although pirfenidone exhibits well documented antifibrotic and antiinflammatory activities, in vitro and in vivo, its molecular targets and mechanisms of action have not been elucidated. In this study, we investigated the effects of pirfenidone on proliferation, TGF-ß-induced differentiation and fibrogenic activity of primary human lung fibroblasts (HLFs). Pirfenidone reduced fibroblast proliferation and attenuated TGF-ß-induced α-smooth muscle actin (SMA) and pro-collagen (Col)-I mRNA and protein levels. Importantly, pirfenidone inhibited TGF-ß-induced phosphorylation of Smad3, p38, and Akt, key factors in the TGF-ß pathway. Together, these results demonstrate that pirfenidone modulates HLF proliferation and TGF-ß-mediated differentiation into myofibroblasts by attenuating key TGF-ß-induced signaling pathways.

Thymosin ß4 reduces IL-17-producing cells and IL-17 expression, and protects lungs from damage in bleomycin-treated mice.

Thymosin ß4 (Tß4) is a highly conserved peptide with immunomodulatory properties. In this research we investigated the effects of Tß4 on the bleomycin-induced lung damage in CD-1 mice and the changes in the number of IL-17-producing cells as well as the IL-17 expression in the lung. Male CD-1 mice were treated with bleomycin (1mg/kg) in the absence or the presence of Tß4 (6mg/kg delivered intra-peritoneally on the day of bleomycin treatment and for 2 additional doses). After sacrifice one week later, lung histology, measurement of collagen content of the lung, Broncho Alveolar Lavage Fluid (BALF) analysis, evaluation of IL17-producing cells in the blood as well as RT-PCR and IHC in the lung tissue were performed. As expected, bleomycin-induced inflammation and lung damage were substantially reduced by Tß4 treatment in CD-1 mice, as shown by the significant reduction of (i) leukocytes in BALF, (ii) histological evidence of the lung damage, and (iii) total collagen content in the lung. Importantly, the bleomycin-induced increase in the number of IL17-producing cells in the blood was significantly blocked by Tß4. Accordingly, IHC and RT-PCR results demonstrated that Tß4 substantially inhibited bleomycin-induced IL-17 over-expression in the lung tissue. This is the first report showing that a decreased amount of IL17-producing cells and inhibited IL-17 expression in the lung with Tß4 treatment correlate with its anti-inflammatory and anti-fibrotic effects.

Protective effects of thymosin ß4 in a mouse model of lung fibrosis.

Thymosin ß4 (Tß4) has been found to have several biological activities related to antiscarring and reduced fibrosis. For example, the anti-inflammatory properties of Tß4 and its splice variant have been shown in the eye and skin. Moreover, Tß4 treatment prevents profibrotic gene expression in cardiac and in hepatic cells in vitro and in vivo. In a recent study on scleroderma patients it was hypothesized that Tß4 may exert a protective effect during human lung injury. In an ongoing study, we have explored the putative Tß4 protective role in the lung context by utilizing a well-known in vivo model. We have observed significant protective effects of Tß4 on bleomycin-induced lung damage, the main outcomes being the halting of the inflammatory process and a substantial reduction of histological evidence of lung injury.

Inhibition of PI3K prevents the proliferation and differentiation of human lung fibroblasts into myofibroblasts: the role of class I P110 isoforms.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease characterized by an accumulation of fibroblasts and myofibroblasts in the alveolar wall. Even though the pathogenesis of this fatal disorder remains unclear, transforming growth factor-ß (TGF-ß)-induced differentiation and proliferation of myofibroblasts is recognized as a primary event. The molecular pathways involved in TGF-ß signalling are generally Smad-dependent yet Smad-independent pathways, including phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), have been recently proposed. In this research we established ex-vivo cultures of human lung fibroblasts and we investigated the role of the PI3K/Akt pathway in two critical stages of the fibrotic process induced by TGF-ß: fibroblast proliferation and differentiation into myofibroblasts. Here we show that the pan-inhibitor of PI3Ks LY294002 is able to abrogate the TGF-ß-induced increase in cell proliferation, in α- smooth muscle actin expression and in collagen production besides inhibiting Akt phosphorylation, thus demonstrating the centrality of the PI3K/Akt pathway in lung fibroblast proliferation and differentiation. Moreover, for the first time we show that PI3K p110δ and p110γ are functionally expressed in human lung fibroblasts, in addition to the ubiquitously expressed p110α and ß. Finally, results obtained with both selective inhibitors and gene knocking-down experiments demonstrate a major role of p110γ and p110α in both TGF-ß-induced fibroblast proliferation and differentiation. This finding suggests that specific PI3K isoforms can be pharmacological targets in IPF.