Time-course transcriptome analysis of a double challenge bleomycin-induced lung fibrosis rat model uncovers ECM homoeostasis-related translationally relevant genes.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an irreversible disorder with a poor prognosis. The incomplete understanding of IPF pathogenesis and the lack of accurate animal models is limiting the development of effective treatments. Thus, the selection of clinically relevant animal models endowed with similarities with the human disease in terms of lung anatomy, cell biology, pathways involved and genetics is essential. The bleomycin (BLM) intratracheal murine model is the most commonly used preclinical assay to evaluate new potential therapies for IPF. Here, we present the findings derived from an integrated histomorphometric and transcriptomic analysis to investigate the development of lung fibrosis in a time-course study in a BLM rat model and to evaluate its translational value in relation to IPF. METHODS: Rats were intratracheally injected with a double dose of BLM (days 0-4) and sacrificed at days 7, 14, 21, 28 and 56. Histomorphometric analysis of lung fibrosis was performed on left lung sections. Transcriptome profiling by RNAseq was performed on the right lung lobes and results were compared with nine independent human gene-expression IPF studies. RESULTS: The histomorphometric and transcriptomic analyses provided a detailed overview in terms of temporal gene-expression regulation during the establishment and repair of the fibrotic lesions. Moreover, the transcriptomic analysis identified three clusters of differentially coregulated genes whose expression was modulated in a time-dependent manner in response to BLM. One of these clusters, centred on extracellular matrix (ECM)-related process, was significantly correlated with histological parameters and gene sets derived from human IPF studies. CONCLUSIONS: The model of lung fibrosis presented in this study lends itself as a valuable tool for preclinical efficacy evaluation of new potential drug candidates. The main finding was the identification of a group of persistently dysregulated genes, mostly related to ECM homoeostasis, which are shared with human IPF.

Optimization of M3 Antagonist-PDE4 Inhibitor (MAPI) Dual Pharmacology Molecules for the Treatment of COPD.

Aiming at the inhaled treatment of pulmonary diseases, the optimization process of the previously reported MAPI compound 92a is herein described. The project was focused on overcoming the chemical stability issue and achieving a balanced bronchodilator/anti-inflammatory profile in rats in order to be confident in a clinical effect without having to overdose at one of the biological targets. The chemical strategy was based on fine-tuning of the substitution pattern in the muscarinic and PDE4 structural portions of the dual pharmacology compounds, also making use of the analysis of a proprietary crystal structure in the PDE4 catalytic site. Compound 10f was identified as a chemically stable, potent, and in vivo balanced MAPI lead compound, as assessed in bronchoconstriction and inflammation assays in rats after intratracheal administration. After the in-depth investigation of the pharmacological and solid-state profile, 10f proved to be safe and suitable for development.

SIRT1-dependent restoration of NAD+ homeostasis after increased extracellular NAD+ exposure.

In the last several years, NAD+ supplementation has emerged as an innovative and safe therapeutic strategy for a wide spectrum of disorders, including diabetes and neuropathy. However, critical questions remain as to how NAD+ and its precursors are taken up by cells, as well as the effects of long-lasting intracellular NAD+ (iNAD+) increases. Here, we investigated the kinetics of iNAD+ levels in different cell types challenged with prolonged exposure to extracellular NAD+ (eNAD+). Surprisingly, we found that after the initial increase, iNAD+ contents decreased back to control levels (iNAD+ resetting). Focusing our attention on HeLa cells, we found that oxygen and ATP consumption occurred with similar temporal kinetics after eNAD+ exposure. Using [3H]NAD+ and [14C]NAD+, we determined that NAD+ resetting was not due to increased dinucleotide extrusion but rather due to reduced uptake of cleaved NAD+ products. Indeed, eNAD+ exposure reduced the expression of the ecto-5'-nucleotidase CD73, the nicotinamide adenine mononucleotide transporter solute carrier family 12 member 8, and the nicotinamide riboside kinase. Interestingly, silencing the NAD+-sensor enzyme sirtuin 1 prevented eNAD+-dependent transcriptional repression of ecto-5'-nucleotidase, solute carrier family 12 member 8, and nicotinamide riboside kinase, as well as iNAD+ resetting. Our findings provide the first evidence for a sirtuin 1-mediated homeostatic response aimed at maintaining physiological iNAD+ levels in conditions of excess eNAD+ availability. These data may be of relevance for therapies designed to support the NAD+ metabolome via extracellular supplementation of the dinucleotide or its precursors.

Discovery of M3 Antagonist-PDE4 Inhibitor Dual Pharmacology Molecules for the Treatment of Chronic Obstructive Pulmonary Disease.

In this paper, we report the discovery of dual M3 antagonist-PDE4 inhibitor (MAPI) compounds for the inhaled treatment of pulmonary diseases. The identification of dual compounds was enabled by the intuition that the fusion of a PDE4 scaffold derived from our CHF-6001 series with a muscarinic scaffold through a common linking ring could generate compounds active versus both the transmembrane M3 receptor and the intracellular PDE4 enzyme. Two chemical series characterized by two different muscarinic scaffolds were investigated. SAR optimization was aimed at obtaining M3 nanomolar affinity coupled with nanomolar PDE4 inhibition, which translated into anti-bronchospastic efficacy ex vivo (inhibition of rat trachea contraction) and into anti-inflammatory efficacy in vitro (inhibition of TNFα release). Among the best compounds, compound 92a achieved the goal of demonstrating in vivo efficacy and duration of action in both the bronchoconstriction and inflammation assays in rat after intratracheal administration.

Borderline ovarian tumor in pregnancy: can surgery wait? A case series.

PURPOSE: To study the characteristics of borderline tumors (BOT) diagnosed during pregnancy, as either first diagnosis or relapse, to evaluate safety of expectant management. METHODS: 15 women affected by BOT during pregnancy were included, to evaluate clinical and histo-pathological characteristics. Age of patient, parity, gestational age, follow-up time, size of tumor, surgical approach, type and timing of surgery, FIGO stage, and histologic type were obtained through retrospective review. RESULTS: All patients except one were diagnosed with serous BOT (BOTs). Median follow-up time was 147 ± 57 months. Eight women received first diagnosis of BOT and seven had diagnosis of BOT recurrence during pregnancy, including three with a second relapse and four with a third relapse. BOT were diagnosed at FIGO stage I in most patients (75%) of the first group and in 14.3% of the second group, respectively. Micropapillary pattern was present in 71.4% of patients with first diagnosis of BOT, but only in 14.2% in case of relapse. All relapses were BOTs. No patient with BOT and concomitant pregnancy developed an invasive recurrence later. Overall, 24 relapses occurred in 10 patients (66.7%). Altogether 24 pregnancies occurred during follow-up, with a high livebirth rate (91.6%) and only 2 spontaneous miscarriages. CONCLUSION: According to our experience, an "expectation management" could be a safe option in case of both relapse of BOTs during pregnancy and first suspicion of BOT in pregnant women at advanced gestational age.

Nicotinamide phosphoribosyltransferase (NAMPT) activity is essential for survival of resting lymphocytes.

NAD biosynthesis is emerging as a key regulator of immune cell functions. Accordingly, inhibitors of the NAD-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) have anti-inflammatory effects, counteract hematological malignancies and are being tested in clinical trials. Still, their effect on different cell types still waits to be fully investigated. Here we show that the NAMPT inhibitor FK866 induces NAD depletion in various mouse organs but selectively causes dramatic atrophy of the spleen red pulp. Accordingly, in cultured mouse lymphocytes exposed to FK866, NAD contents drop to 50% of basal values within 2 days, a condition sufficient to prompt complete cell death. Cultures of human lymphocytes are more resistant to FK866 and sustain a 50% NAD reduction for 5 days before dying. Death of both cell types can be prevented by different NAD precursors, indicating critical NAD homeostasis in lymphocytes. Indeed, inhibition of the NAD-consuming enzyme poly(ADP-ribose) polimerase-1 suffices to prevent FK866-induced NAD depletion and death of both lymphocyte types. Poly(ADP-ribose) polymerase-1-null lymphocytes also undergo lower NAD depletion and reduced cell death when exposed to the drug. At variance with other cell types, neither apoptosis nor autophagy are exclusively responsible for lymphocyte death by FK866, consistent with a general impairment of lymphocyte homeostasis following NAD depletion. Data demonstrate a unique sensitivity of resting lymphocytes to NAD-depleting agents, providing new hints of relevance to lymphocyte biology and therapeutic interventions with NAMPT inhibitors.

Neurological basis of AMP-dependent thermoregulation and its relevance to central and peripheral hyperthermia.

Therapeutic hypothermia is of relevance to treatment of increased body temperature and brain injury, but drugs inducing selective, rapid, and safe cooling in humans are not available. Here, we show that injections of adenosine 5'-monophosphate (AMP), an endogenous nucleotide, promptly triggers hypothermia in mice by directly activating adenosine A1 receptors (A1R) within the preoptic area (POA) of the hypothalamus. Inhibition of constitutive degradation of brain extracellular AMP by targeting ecto 5'-nucleotidase, also suffices to prompt hypothermia in rodents. Accordingly, sensitivity of mice and rats to the hypothermic effect of AMP is inversely related to their hypothalamic 5'-nucleotidase activity. Single-cell electrophysiological recording indicates that AMP reduces spontaneous firing activity of temperature-insensitive neurons of the mouse POA, thereby retuning the hypothalamic thermoregulatory set point towards lower temperatures. Adenosine 5'-monophosphate also suppresses prostaglandin E2-induced fever in mice, having no effects on peripheral hyperthermia triggered by dioxymetamphetamine (ecstasy) overdose. Together, data disclose the role of AMP, 5'-nucleotidase, and A1R in hypothalamic thermoregulation, as well and their therapeutic relevance to treatment of febrile illness.

Impact of drug administration route on drug delivery and distribution into the lung: an imaging mass spectrometry approach.

During the last decade, significant technological improvements in mass spectrometry have had a great impact on drug discovery. The development of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has set a new frontier for the study of the distribution of endogenous and exogenous molecules present within a tissue. MALDI-IMS is a surface sampling technique that allows not only the detection of multiple analytes but also gives the spatial distribution of those analytes. Active compounds for pulmonary disease need an optimal and well-studied delivery into the lungs, in order to assure distribution with greater penetration into the peripheral or the alveolar region of the lung to maximize the therapeutic effects. IMS is very useful in the field of drug discovery, showing drug delivery and distribution in the body and organs. In this study, we present a comparison between two different ways of carrying out pulmonary drug administration: inhalation of a nebulized aerosol of aqueous drug solutions and intratracheal administration, which is much simpler, not expensive and commonly used during in vivo screening. Tiotropium bromide is a long-acting anticholinergic medicine used for maintenance treatment of chronic obstructive pulmonary disease. In the present work, tiotropium was administered by nebulization and by intratracheal instillation to guinea pigs at doses able to induce significant anti-bronchoconstrictive activity. Lung samples were dissected, frozen, cryosectioned and coated with matrix (α-hydroxy-cinnamic acid). IMS analyses were performed using a MALDI-LTQ-Orbitrap XL. Using this technique we were able to compare different distributions of the drug depending on the method of administration.

Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis.

During the last several years, evidence that various enzymes hydrolyze NAD into bioactive products prompted scientists to revisit or design strategies able to increase intracellular availability of the dinucleotide. However, plasma membrane permeability to NAD and the mitochondrial origin of the dinucleotide still wait to be clearly defined. Here, we report that intracellular NAD contents increased upon exposure of cell lines or primary cultures to exogenous NAD (eNAD). NAD precursors could not reproduce the effects of eNAD, and they were not found in the incubating medium containing eNAD, thereby suggesting direct cellular eNAD uptake. We found that in mitochondria of cells exposed to eNAD, NAD and NADH as well as oxygen consumption and ATP production were increased. Conversely, DNA repair, a well known NAD-dependent process, was unaltered upon eNAD exposure. We also report that eNAD conferred significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide, or N-methyl-N'-nitro-N-nitrosoguanidine. In particular, eNAD reduced staurosporine-induced loss of mitochondrial membrane potential and ensuing caspase activation. Of importance, pharmacological inhibition or silencing of the NAD-dependent enzyme SIRT1 abrogated the ability of eNAD to provide protection from staurosporine, having no effect on eNAD-dependent protection from C2-ceramide or N-methyl-N'-nitro-N-nitrosoguanidine. Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.

Poly(ADP-ribose) polymerase-1 is a nuclear epigenetic regulator of mitochondrial DNA repair and transcription.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a NAD-consuming enzyme with an emerging key role in epigenetic regulation of gene transcription. Although PARP-1 expression is characteristically restricted to the nucleus, a few studies report the mitochondrial localization of the enzyme and its ability to regulate organelle functioning. Here, we show that, despite exclusive nuclear localization of PARP-1, mitochondrial homeostasis is compromised in cell lines exposed to PARP-1 pharmacological inhibitors or small interfering RNA. PARP-1 suppression reduces integrity of mitochondrial DNA (mtDNA), as well as expression of mitochondria-encoded respiratory complex subunits COX-1, COX-2, and ND-2. Accordingly, PARP-1 localizes at promoters of nuclear genes encoding both the mtDNA repair proteins UNG1, MYH1, and APE1 and the mtDNA transcription factors TFB1M and TFB2M. It is noteworthy that poly(ADP-ribosyl)ation is required for nuclear gene expression of these mitochondrial proteins. Consistent with these findings, PARP-1 suppression impairs mitochondrial ATP production. Our results indicate that PARP-1 plays a central role in mitochondrial homeostasis by epigenetically regulating nuclear genes involved in mtDNA repair and transcription. These data might have important implications in pharmacology of PARP-1 inhibitors as well as clinical oncology and aging.

Inhibition of nicotinamide phosphoribosyltransferase: cellular bioenergetics reveals a mitochondrial insensitive NAD pool.

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly- and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.

Histone deacetylase (HDAC) inhibitors reduce the glial inflammatory response in vitro and in vivo.

Histone deacetylase inhibitors (HDACi) are emerging tools for epigenetic modulation of gene expression and suppress the inflammatory response in models of systemic immune activation. Yet, their effects within the brain are still controversial. Also, whether HDACs are expressed in astrocytes or microglia is unclear. Here, we report the identification of transcripts for HDAC 1-11 in cultured mouse glial cells. Two HDACi such as SAHA and ITF2357 induce dramatic increase of histone acetylation without causing cytotoxicity of cultured cells. Of note, the two compounds inhibit expression of pro-inflammatory mediators by LPS-challenged glial cultures, and potentiate immunosuppression triggered by dexamethasone in vitro. The anti-inflammatory effect is not due to HDACi-induced transcription of immunosuppressant proteins, (including SOCS-1/3) or microRNA-146. Rather, it is accompanied by direct alteration of transcription factor DNA binding and ensuing transcriptional activation. Indeed, both HDACi impair NFkappaB-dependent IkappaBalpha resynthesis in glial cells exposed to LPS, and, among various AP1 subunits and NFkappaB p65, affect the DNA binding activity of c-FOS, c-JUN and FRA2. Importantly, ITF2357 reduces the expression of pro-inflammatory mediators in the striatum of mice iontophoretically injected with LPS. Data demonstrate that mouse glial cells have ongoing HDAC activity, and its inhibition suppresses the neuroinflammatory response because of a direct impairment of the transcriptional machinery.

High expression of indoleamine 2,3-dioxygenase gene in prostate cancer.

Arginase 2, inducible- and endothelial-nitric-oxide synthase (iNOS and eNOS), indoleamine 2,3-dioxygenase (IDO) and TGF-beta, might impair immune functions in prostate cancer (PCA) patients. However, their expression was not comparatively analysed in PCA and benign prostatic hyperplasia (BPH). We evaluated the expression of these genes in PCA and BPH tissues. Seventy-six patients (42 BPH, 34 PCA) were enrolled. Arginase 2, eNOS and iNOS gene expression was similar in BPH and PCA tissues. TGF-beta1 gene expression was higher in BPH than in PCA tissues (p=0.035). IDO gene expression was more frequently detectable (p=0.00007) and quantitatively higher (p=0.00001) in PCA tissues than in BPH. IDO protein, expressed in endothelial cells from both BPH and PCA, was detectable in tumour cells in PCA showing evidence of high specific gene expression. In these patients, IDO gene expression correlated with kynurenine/tryptophan ratio in sera. Thus high expression of IDO gene is specifically detectable in PCA.