Simona Inghilleri

The receptor for advanced glycation end products and its ligands: a new inflammatory pathway in lung disease?

The binding of the receptor for advanced glycation end products (RAGE) with its ligands begins a sustained period of cellular activation and inflammatory signal amplification in different tissues and diseases. This binding could represent an as yet uninvestigated pathway of inflammatory reaction in the lung, where the presence of the receptor has been largely documented and advanced glycation end products (AGEs) are produced by nonenzymatic glycation and oxidation of proteins and lipids, driven by smoke and pollutants exposure or inflammatory stress. We immunohistochemically assessed the expression of RAGE and of its major proinflammatory ligands, N-ɛ-carboxy-methyl-lysine, S100B and S-100A12 in normal lung and in non-neoplastic lung disorders including smoke-related airway disease, granulomatous inflammation, postobstructive damage and usual interstitial pneumonia. In normal lung low expression of the receptor was observed in bronchiolar epithelia, type II pneumocytes, macrophages and some endothelia. S100A12 and S100B were expressed, respectively, in granulocytes and in dendritic cells. Carboxy-methyl-lysine was present in bronchiolar epithelia and macrophages. In all pathological conditions associated with inflammation and lung damage overexpression of both the receptor and of AGEs was observed in bronchiolar epithelia, type II alveolar pneumocytes, alveolar macrophages and endothelia. RAGE overexpression was more evident in epithelia associated with inflammatory cell aggregates. Fibroblasts in usual interstitial pneumonia expressed both the receptor and AGEs. The number of S100A12 and S100B immunoreactive inflammatory cells was variable. S100A12 was also expressed in mononuclear inflammatory cells and in activated epithelia. The activation of the inflammatory pathway controlled by the RAGE is not specific of a single lung disease, however, it may be relevant as a nonspecific pathway of sustained inflammation in lung tissue, and on this basis therapeutic approaches based on receptor blockage can be envisaged.

In situ assessment of oxidant and nitrogenic stress in bleomycin pulmonary fibrosis

Reactive oxygen species (ROS) and nitric oxide (NO) have a role in the development of pulmonary fibrosis after bleomycin administration. The ROS production induces an antioxidant response, involving superoxide dismutases (SODs), catalase, and glutathione peroxidases. We compared in situ oxidative burden and antioxidant enzyme activity in bleomycin-injured rat lungs and normal controls. ROS expression and catalase, glucose-6-phosphate-dehydrogenase (G6PHD), and NOS/NADPH-diaphorase activity were investigated by using histochemical reactions. Nitric oxide synthase (e-NOS and i-NOS) and SOD (MnSOD, Cu/ZnSOD, ECSOD) expression was investigated immunohistochemically. After treatment ROS production was enhanced in both phagocytes and in type II alveolar epithelial cells. Mn, Cu/Zn, and ECSOD were overexpressed in parenchymal cells, whereas interstitium expressed ECSOD. Catalase and G6PHD activity was moderately increased in parenchymal and inflammatory cells. NOS/NADPH-d activity and i-NOS expression increased in alveolar and bronchiolar epithelia and in inflammatory cells. It can be suggested that the concomitant activation of antioxidant enzymes is not adequate to scavenge the oxidant burden induced by bleomycin lung damage. Inflammatory cells and also epithelial cells are responsible of ROS and NO production. This oxidative and nitrosative stress may be a substantial trigger in TGF-β1 overexpression by activated type II pneumocytes, leading to fibrotic lesions.

Targeting EGFR in non-small-cell lung cancer: Lessons, experiences, strategies

Cancer is a genetic disease and this concept is now widely exploited by both scientists and clinicians to design new targeted molecules. Indeed many data have already allowed us to ameliorate not only our knowledge about cancer onset, but also about patients treatment. Correlation between mutations in cancer alleles and drug response is a key point to identify drugs that match the genetic profile of each individual tumors. On the other hand, experience derived from inhibition of tyrosine kinase receptors has pointed out that targeted treatment is really successful only in a small subset of tumors. The latter are eventually addicted to those genetic alterations which are responsible for receptors activation and for the continued expression of their signalling. Overall these observations provide a strong rationale for a molecular-based diagnosis and patients selection for targeted therapies. This review analyses the current state of the art of molecularly-tailored pharmacological approach to lung cancer, one of the biggest killers among human solid tumors. Main relevance is addressed to genetic lesions activating the EGFR pathway transducers, focusing on their role as markers of targeted drug response.

Incomplete expression of epithelial–mesenchymal transition markers in idiopathic pulmonary fibrosis

The hypothesis that epithelial-mesenchymal transition (EMT) contributes to the formation of fibroblast foci (FF), which are the histological hallmark and the site of active disease progression of Idiopathic Pulmonary Fibrosis (IPF), has not yet received a conclusive demonstration. Cells undergoing EMT lose epithelial features and acquire mesenchymal markers and morphology. Cadherin expression switch (from E to N) is one of the first events in EMT. We investigated the immunohistochemical expression of E- and N-cadherin, vimentin, fibronectin, laminin-5-γ2, α-smooth muscle actin, and fibroblast-specific protein-1 involved in EMT in 20 IPF lung biopsies, focusing on metaplastic squamous cells of bronchial basal origin, positive for laminin-5-γ2 and ΔNp63/p40, that cover FF. The results were compared with organizing pneumonia, reactive squamous cell metaplasia of bronchiolar epithelia, and squamous cell carcinoma. Bronchiolar basal metaplastic cells in IPF partially lost E-cadherin and expressed vimentin and fibronectin. Hyperplastic pneumocytes in IPF and controls coexpressed E-cadherin and N-cadherin, and were weakly positive for lam5-γ2. Reactive squamous cell metaplasia did not show any mesenchymal markers. Squamous cell carcinoma only expressed lam5-γ2. In IPF lungs, we observed two epithelial cell populations with a different expression profile of markers involved in EMT. Although neither hyperplastic pneumocytes nor bronchial basal cells showed evidence of complete EMT, only the latter seem to be specific for UIP and might have a role in its development.

Thymidylate Synthase and Folyl-Polyglutamate Synthase Are Not Clinically Useful Markers of Response to Pemetrexed in Patients with Malignant Pleural Mesothelioma

Purpose: Thymidylate synthase (TS) is a potential predictor of outcome after pemetrexed (Pem) in patients with malignant pleural mesothelioma (MPM), and assays measuring TS levels are commercially marketed. The goal of this study was to further evaluate the value of TS and to study another potential biomarker of response, the enzyme, folyl-polyglutamate synthase (FPGS), which activates Pem intracellularly. Methods: Levels of TS and FPGS were semi-quantitatively determined immunohistochemically using H-scores on tissue samples from 85 MPM patients receiving Pem as primary therapy. H-score was correlated with radiographic disease control rate (DCR), time to progression (TTP) and overall survival (OS). In addition, expression levels of TS and FPGS in MPM cell lines were determined using immunoblotting and correlated with their sensitivity to Pem-induced cell death. Results: H-scores from patients with disease control versus progressive disease showed extensive overlap. There were no significant correlations of DCR, TTP, or OS to either TS levels (p = 0.73, 0.93, and 0.59, respectively), FPGS levels (p = 0.95, 0.77, and 0.43, respectively) or the ratio of FPGS/TS using the median scores of each test as cutoffs. There was no correlation between TS or FPGS expression and chemosensitivity of mesothelioma cells to Pem in vitro. Conclusions: Although previous retrospective data suggest that TS and FPGS expression might be potential markers of Pem efficacy in MPM, our data indicate these markers lack sufficient predictive value in individual patients and should not be used to guide therapeutic decisions in the absence of prospective studies.

Time course of matrix metalloproteases and tissue inhibitors in bleomycin-induced pulmonary fibrosis

To investigate simultaneously localization and relative activity of MMPs during extracellular matrix (ECM) remodeling in bleomycin-induced pulmonary fibrosis in rat, we analyzed the time course of the expression, activity and/or concentration of gelatinases MMP-2 and MMP-9, collagenase MMP-1, matrylisin MMP-7, TIMP-1 and TIMP-2, both in alveolar space (cellular and extracellular compartments) and in lung tissue. MMP and TIMP expression was detected (immunohistochemistry) in lung tissue. MMP activity (zymography) and TIMP concentration (ELISA) were evaluated in lung tissue homogenate (LTH), BAL supernatant (BALs) and BAL cell pellet (BALp) 3, 7, 14, and 28 days after bleomycin intratracheal instillation. Immunohistochemistry showed an extensive MMP and TIMP expression from day 7 in a wide range of structural and inflammatory cells in treated rats. MMP-2 was present mainly in epithelia, MMP-9 in inflammatory cells. MMP-2 and MMP-9 activity was increased respectively in BAL fluid and BAL cells, with a peak at day 7. TIMP-1 and TIMP-2 concentration (ELISA) enhancement was delayed at day 14. In conclusion gelatinases and their inhibitors are significantly activated during bleomycin-induced pulmonary fibrosis. Marked changes in gelatinases activity are observed early in the alveolar compartment, with a prevailing extracellular activity of MMP-2 and a predominant intracellular distribution of MMP-9, while enzyme activity changes in lung parenchyma were less evident. In the repairing phase the reduction of gelatinases activity is synchronous with a peak of alveolar concentration of their inhibitors.

Antibody-engineered nanoparticles selectively inhibit mesenchymal cells isolated from patients with chronic lung allograft dysfunction.

AIMS Chronic lung allograft dysfunction represents the main cause of death after lung transplantation, and so far there is no effective therapy. Mesenchymal cells (MCs) are primarily responsible for fibrous obliteration of small airways typical of chronic lung allograft dysfunction. Here, we engineered gold nanoparticles containing a drug in the hydrophobic section to inhibit MCs, and exposing on the outer hydrophilic surface a monoclonal antibody targeting a MC-specific marker (half-chain gold nanoparticles with everolimus). MATERIALS & METHODS Half-chain gold nanoparticles with everolimus have been synthesized and incubated with MCs to evaluate the effect on proliferation and apoptosis. RESULTS & DISCUSSION Drug-loaded gold nanoparticles coated with the specific antibody were able to inhibit proliferation and induce apoptosis without stimulating an inflammatory response, as assessed by in vitro experiments. CONCLUSION These findings demonstrate the effectiveness of our nanoparticles in inhibiting MCs and open new perspectives for a local treatment of chronic lung allograft dysfunction.

Factors Influencing Oxidative Imbalance in Pulmonary Fibrosis: An Immunohistochemical Study

Background. Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease of unknown etiology characterized by interstitial fibrosis determining irreversible distortion of pulmonary architecture. Reactive oxygen species (ROS) and markers of oxidative stress play a pivotal role in human IPF pathology, possibly through induction of epithelial-mesenchymal transition (EMT). Methods. We investigated by immunohistochemistry, in UIP and COP tissue samples, the expression of most relevant markers of the molecular interplay involving RAGE, oxidant/antioxidant balance regulation, tissue nitrosylation, and mediators of EMT. Results. In both UIP and COP, the degree of RAGE expression was similarly high, while SODs and i-NOS, diffusely present in COP endoalveolar plugs, were almost absent in UIP fibroblast foci. A lower degree of tissue nitrosilation was observed in UIP than in COP. Conclusions. Fibroblast lesions of UIP and of COP share a similar degree of activation of RAGE, while antioxidant enzyme expression markedly reduced in UIP.

Activation of Oncogenic Pathways in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause. The most recent hypotheses on IPF pathogenesis suggest a central role of epithelial cell damage, followed by a dysregulated molecular cross talk between epithelial cells and fibroblasts. Thus, IPF progression has often been assimilated to that of cancer, and several signaling patterns appear to be disrupted in both diseases. Here, we analyze the expression in an IPF series of a panel of molecules, which are known to play a role in tumorigenic process. Our findings, although preliminary, reveal that IPF landscape is enriched in neoplastic potential expressed in a context of complex genomic polyclonality and cellular heterogeneity. These results provide a rationale for further investigations aimed to exploit—in a similar fashion to cancer—targeted therapies for a “precision medicine” approach to IPF.

The combination of sorafenib and everolimus shows antitumor activity in preclinical models of malignant pleural mesothelioma

BackgroundMalignant Pleural Mesothelioma (MPM) is an aggressive tumor arising from mesothelial cells lining the pleural cavities characterized by resistance to standard therapies. Most of the molecular steps responsible for pleural transformation remain unclear; however, several growth factor signaling cascades are known to be altered during MPM onset and progression. Transducers of these pathways, such as PIK3CA-mTOR-AKT, MAPK, and ezrin/radixin/moesin (ERM) could therefore be exploited as possible targets for pharmacological intervention. This study aimed to identify ‘druggable’ pathways in MPM and to formulate a targeted approach based on the use of commercially available molecules, such as the multikinase inhibitor sorafenib and the mTOR inhibitor everolimus.MethodsWe planned a triple approach based on: i) analysis of immunophenotypes and mutational profiles in a cohort of thoracoscopic MPM samples, ii) in vitro pharmacological assays, ii) in vivo therapeutic approaches on MPM xenografts. No mutations were found in ‘hot spot’ regions of the mTOR upstream genes (e.g. EGFR, KRAS and PIK3CA).ResultsPhosphorylated mTOR and ERM were specifically overexpressed in the analyzed MPM samples. Sorafenib and everolimus combination was effective in mTOR and ERM blockade; exerted synergistic effects on the inhibition of MPM cell proliferation; triggered ROS production and consequent AMPK-p38 mediated-apoptosis. The antitumor activity was displayed when orally administered to MPM-bearing NOD/SCID mice.ConclusionsERM and mTOR pathways are activated in MPM and ‘druggable’ by a combination of sorafenib and everolimus. Combination therapy is a promising therapeutic strategy against MPM.