PI3K inhibition reduces murine and human liver fibrogenesis in precisioncut liver slices

Background: Liver fibrosis results from continuous inflammation and injury. Despite its high prevalence worldwide, no approved antifibrotic therapies exist. Omipalisib is a selective inhibitor of the PI3K/mTOR pathway that controls nutrient metabolism, growth and proliferation. It has shown antifibrotic properties in vitro. While clinical trials for idiopathic pulmonary fibrosis have been initiated, an in-depth preclinical evaluation is lacking. We evaluated omipalisib's effects on fibrogenesis using the ex vivo model of murine and human precision-cut tissue slices (PCTS). Methods: Murine and human liver and jejunum PCTS were incubated with omipalisib up to 10 mu M for 48 h. PI3K pathway activation was assessed through protein kinase B (Akt) phosphorylation and antifibrotic efficacy was determined via a spectrum of fibrosis markers at the transcriptional and translational level. Results: During incubation of PCTS the PI3K pathway was activated and incubation with omipalisib prevented Akt phosphorylation (IC50 = 20 and 1.5 nM for mouse and human, respectively). Viability of mouse and human liver PCTS was compromised only at the high concentration of 10 and 1-5 mu M, respectively. However, viability of jejunum PCTS decreased with 0.1 (mouse) and 0.01 mu M (human). Spontaneously increased fibrosis related genes and proteins were significantly and similarly suppressed in mouse and in human liver PCTS. Conclusions: Omipalisib has antifibrotic properties in ex vivo mouse and human liver PCTS, but higher concentrations showed toxicity in jejunum PCTS. While the PI3K/mTOR pathway appears to be a promising target for the treatment of liver fibrosis, PCTS revealed likely side effects in the intestine at higher doses

Transcriptomic characterization of culture-associated changes in murine and human precision-cut tissue slices

Our knowledge of complex pathological mechanisms underlying organ fibrosis is predominantly derived from animal studies. However, relevance of animal models for human disease is limited; therefore, an ex vivo model of human precision-cut tissue slices (PCTS) might become an indispensable tool in fibrosis research and drug development by bridging the animal-human translational gap. This study, presented as two parts, provides comprehensive characterization of the dynamic transcriptional changes in PCTS during culture by RNA sequencing. Part I investigates the differences in culture-induced responses in murine and human PCTS derived from healthy liver, kidney and gut. Part II delineates the molecular processes in cultured human PCTS generated from diseased liver, kidney and ileum. We demonstrated that culture was associated with extensive transcriptional changes and impacted PCTS in a universal way across the organs and two species by triggering an inflammatory response and fibrosis-related extracellular matrix (ECM) remodelling. All PCTS shared mRNA upregulation of IL-11 and ECM-degrading enzymes MMP3 and MMP10. Slice preparation and culturing activated numerous pathways across all PCTS, especially those involved in inflammation (IL-6, IL-8 and HMGB1 signalling) and tissue remodelling (osteoarthritis pathway and integrin signalling). Despite the converging effects of culture, PCTS display species-, organ- and pathology-specific differences in the regulation of genes and canonical pathways. The underlying pathology in human diseased PCTS endures and influences biological processes like cytokine release. Our study reinforces the use of PCTS as an ex vivo fibrosis model and supports future studies towards its validation as a preclinical tool for drug development

RNAi screen for NRF2 inducers identifies targets that rescue primary lung epithelial cells from cigarette smoke induced radical stress

Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the 'druggable' genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism

Modulation of vascular contraction via soluble guanylate cyclase signaling in a novel ex vivo method using rat precision‐cut liver slices

Abstract Fibrotic processes in the liver of non‐alcoholic steatohepatitis (NASH) patients cause microcirculatory dysfunction in the organ which increases blood vessel resistance and causes portal hypertension. Assessing blood vessel function in the liver is challenging, necessitating the development of novel methods in normal and fibrotic tissue that allow for drug screening and translation toward pre‐clinical settings. Cultures of precision cut liver slices (PCLS) from normal and fibrotic rat livers were used for blood vessel function analysis. Live recording of vessel diameter was used to assess the response to endothelin‐1, serotonin and soluble guanylate cyclase (sGC) activation. A cascade of contraction and relaxation events in response to serotonin, endothelin‐1, Ketanserin and sGC activity could be established using vessel diameter analysis of rat PCLS. Both the sGC activator BI 703704 and the sGC stimulator Riociguat prevented serotonin‐induced contraction in PCLS from naive rats. By contrast, PCLS cultures from the rat CCl4 NASH model were only responsive to the sGC activator, thus establishing that the sGC enzyme is rendered non‐responsive to nitric oxide under oxidative stress found in fibrotic livers. The role of the sGC pathway for vessel relaxation of fibrotic liver tissue was identified in our model. The obtained data shows that the inhibitory capacities on vessel contraction of sGC compounds can be translated to published preclinical data. Altogether, this novel ex vivo PCLS method allows for the differentiation of drug candidates and the translation of therapeutic approaches towards the clinical use

Induction of CSE stress tolerance in HBEpC cell cultures by KEAP1 knock down.

<p>Cell cultures of primary human bronchial epithelial cells were transfected for 48h with positive and negative control siRNAs prior to stimulation with 3% CSE-conditioned medium. After stimulation for 24h cells were analyzed either by Zeiss Axioplan (20× magnification) microscopy (A) or for their vitality by the Cell Titer Glo assay (B). Downregulation of KEAP1 greatly improves the survival of cells after CSE exposure. An ATP standard was used for the calculation of ATP concentration per well. Data are represented as means of 3 independent biological replicates +SD. Significant differences of siKEAP1 compared to siControl treated samples are highlighted (* p≤0.05, ** p≤0.01, students T-test).</p

RNAi screen to detect activators of NRF-2.

<p>(A) HEK-hNQ cells were transfected with siRNA pools (3 siRNAs per gene) in 384-well format. 48 hours post transfection, cells on three replicate plates were lysed and luminescence was measured. Cells on identical triplicate plates were stained with Hoechst dye and images were acquired using an automated microscope. (B) Performance of control siRNA pools. Average and standard deviations of normalized values for control siRNA pools on all plates. (C) Frequency distribution of coefficients of variance between three replicates indicates high reproducibility of screen results. Solid line, normalized luminescence measurements, dashed line, normalized number of nuclei. (D) Performance of control siRNA pools. Average and standard deviations of normalized values for control siRNA pools on all plates.</p

Activation of the NRF2 transcription factor reporter system by different agents of radical stress or KEAP1 modifier.

<p>(A) Cell cultures of HEK293-hNQ cells were treated for 24 h with tBHQ (3, 6, 12, 18, 25, 37, 50, 75, 100 [μM]), CSE (0.04, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5, 5, 10 [% (v/v)]) or Paraquat (25, 50, 75, 100, 125, 150, 200, 250, 300 [μM]). Luciferase activity was expressed as % of untreated samples (Control). (B) Cultures of HEK293-hNQ and HEK293-hNQ-del cells were transfected with positive and negative control siRNAs and lysed 48 h later for the determination of luciferase activity. Treatment of these cells with the NRF2 activator tBHQ at 50μM was performed for 24h. All results were normalized to untreated HEK293-hNQ samples. Values are represented as means of independent biological experiments ± SD (n = 6).</p

Activation of the NRF2 transcription factor reporter system correlates with the induction of the NRF2 responsive gene NQO1.

<p>siRNAs of hit candidates of the primary RNAi screen were transfected into HEK-hNQ cells and luciferase activity or hNQO1 mRNA levels were determined 48 h post transfection. The log2 ratios of specific luciferase activity were plotted versus log2 ratios of normalized expression levels of human NQO1 of identical siRNA treated samples. Only results of significant differences (p≤0.05) in both readouts are shown. SiKEAP1 treated samples are highlighted in black.</p