Fast detection of liver fibrosis with collagen-binding single-nanometer iron oxide nanoparticles via T1-weighted MRI.

SNIO-CBP, a single-nanometer iron oxide (SNIO) nanoparticle functionalized with a type I collagen-binding peptide (CBP), was developed as a T1-weighted MRI contrast agent with only endogenous elements for fast and noninvasive detection of liver fibrosis. SNIO-CBP exhibits 6.7-fold higher relaxivity compared to a molecular gadolinium-based collagen-binding contrast agent CM-101 on a per CBP basis at 4.7 T. Unlike most iron oxide nanoparticles, SNIO-CBP exhibits fast elimination from the bloodstream with a 5.7 min half-life, high renal clearance, and low, transient liver enhancement in healthy mice. We show that a dose of SNIO-CBP that is 2.5-fold lower than that for CM-101 has comparable imaging efficacy in rapid (within 15 min following intravenous injection) detection of hepatotoxin-induced liver fibrosis using T1-weighted MRI in a carbon tetrachloride-induced mouse liver injury model. We further demonstrate the applicability of SNIO-CBP in detecting liver fibrosis in choline-deficient L-amino acid-defined high-fat diet mouse model of nonalcoholic steatohepatitis. These results provide a platform with potential for the development of high relaxivity, gadolinium-free molecular MRI probes for characterizing chronic liver disease.

Increased susceptibility to ischemia causes exacerbated response to microinjuries in the cirrhotic liver.

Fractional laser ablation is a technique developed in dermatology to induce remodeling of skin scars by creating a dense pattern of microinjuries. Despite remarkable clinical results, this technique has yet to be tested for scars in other tissues. As a first step toward determining the suitability of this technique, we aimed to (1) characterize the response to microinjuries in the healthy and cirrhotic liver, and (2) determine the underlying cause for any differences in response. Healthy and cirrhotic rats were treated with a fractional laser then euthanized from 0 h up to 14 days after treatment. Differential expression was assessed using RNAseq with a difference-in-differences model. Spatial maps of tissue oxygenation were acquired with hyperspectral imaging and disruptions in blood supply were assessed with tomato lectin perfusion. Healthy rats showed little damage beyond the initial microinjury and healed completely by 7 days without scarring. In cirrhotic rats, hepatocytes surrounding microinjury sites died 4-6 h after ablation, resulting in enlarged and heterogeneous zones of cell death. Hepatocytes near blood vessels were spared, particularly near the highly vascularized septa. Gene sets related to ischemia and angiogenesis were enriched at 4 h. Laser-treated regions had reduced oxygen saturation and broadly disrupted perfusion of nodule microvasculature, which matched the zones of cell death. Our results demonstrate that the cirrhotic liver has an exacerbated response to microinjuries and increased susceptibility to ischemia from microvascular damage, likely related to the vascular derangements that occur during cirrhosis development. Modifications to the fractional laser tool, such as using a femtosecond laser or reducing the spot size, may be able to prevent large disruptions of perfusion and enable further development of a laser-induced microinjury treatment for cirrhosis.

Imaging kidney inflammation using an oxidatively activated MRI probe.

Imaging tools for kidney inflammation could improve care for patients suffering inflammatory kidney diseases by lessening reliance on percutaneous biopsy or biochemical tests alone. During kidney inflammation, infiltration of myeloid immune cells generates a kidney microenvironment that is oxidizing relative to normal kidney. Here, we evaluated whether magnetic resonance imaging (MRI) using the redox-active iron (Fe) complex Fe-PyC3A as an oxidatively activated probe could serve as a marker of kidney inflammation using mouse models of unilateral ischemia-reperfusion injury (IRI) and lupus nephritis (MRL-lpr mice). We imaged unilateral IRI in gp91phox knockout mice, which are deficient in the nicotinamide oxidase II (NOX2) enzyme required for myeloid oxidative burst, as loss of function control, and imaged MRL/MpJ mice as non-kidney involved lupus control. Gadoterate meglumine was used as a non-oxidatively activated control MRI probe. Fe-PyC3A safety was preliminarily examined following a single acute dose. FePyC3A generated significantly greater MRI signal enhancement in the IRI kidney compared to the contralateral kidney in wild-type mice, but the effect was not observed in the NOX2-deficient control. Fe-PyC3A also generated significantly greater kidney enhancement in MRL-lpr mice compared to MRL/MpJ control. Gadoterate meglumine did not differentially enhance the IRI kidney over the contralateral kidney and did not differentially enhance the kidneys of MRL-lpr over MRL/MpJ mice. Fe-PyC3A was well tolerated at the highest dose evaluated, which was a 40-fold greater than required for imaging. Thus, our data indicate that MRI using Fe-PyC3A is specific to an oxidizing kidney environment shaped by activity of myeloid immune cells and support further evaluation of Fe-PyC3A for imaging kidney inflammation.

A Rationally Designed Complex Replenishes the Transferrin Iron Pool Directly and with High Specificity.

Many forms of anemia are caused or complicated by pathologic restriction of iron (Fe). Chronic inflammation and certain genetic mutations decrease the activity of ferroportin, the only Fe-exporter protein, so that endogenously recycled or nutritionally absorbed Fe cannot be exported to the extracellular Fe carrier protein transferrin for delivery to the bone marrow. Diminished ferroportin activity renders anemia correction challenging as Fe administered intravenously or through nutritional supplementation is trafficked through the ferroportin-transferrin axis. Utilizing judicious application of coordination chemistry principles, we designed an Fe complex (Fe-BBG) with solution thermodynamics and Fe dissociation kinetics optimized to replenish the transferrin-Fe pool rapidly, directly, and with precision. Fe-BBG is unreactive under conditions designed to force redox cycling and production of reactive oxygen species. The BBG ligand has a low affinity for divalent metal ions and does not compete for binding of other endogenously present ions including Cu and Zn. Treatment with Fe-BBG confers anemia correction in a mouse model of iron-refractory iron-deficiency anemia. Repeated exposure to Fe-BBG did not cause adverse clinical chemistry changes or trigger the expression of genes related to oxidative stress or inflammation. Fe-BBG represents the first entry in a promising new class of transferrin-targeted Fe replacement drugs.

Tailored Chemical Reactivity Probes for Systemic Imaging of Aldehydes in Fibroproliferative Diseases.

During fibroproliferation, protein-associated extracellular aldehydes are formed by the oxidation of lysine residues on extracellular matrix proteins to form the aldehyde allysine. Here we report three Mn(II)-based, small-molecule magnetic resonance probes that contain α-effect nucleophiles to target allysine in vivo and report on tissue fibrogenesis. We used a rational design approach to develop turn-on probes with a 4-fold increase in relaxivity upon targeting. The effects of aldehyde condensation rate and hydrolysis kinetics on the performance of the probes to detect tissue fibrogenesis non-invasively in mouse models were evaluated by a systemic aldehyde tracking approach. We showed that, for highly reversible ligations, off-rate was a stronger predictor of in vivo efficiency, enabling histologically validated, three-dimensional characterization of pulmonary fibrogenesis throughout the entire lung. The exclusive renal elimination of these probes allowed for rapid imaging of liver fibrosis. Reducing the hydrolysis rate by forming an oxime bond with allysine enabled delayed phase imaging of kidney fibrogenesis. The imaging efficacy of these probes, coupled with their rapid and complete elimination from the body, makes them strong candidates for clinical translation.

Dual Hydrazine-Equipped Turn-On Manganese-Based Probes for Magnetic Resonance Imaging of Liver Fibrogenesis.

Liver fibrogenesis is accompanied by upregulation of lysyl oxidase enzymes, which catalyze oxidation of lysine ε-amino groups on the extracellular matrix proteins to form the aldehyde containing amino acid allysine (LysAld). Here, we describe the design and synthesis of novel manganese-based MRI probes with high signal amplification for imaging liver fibrogenesis. Rational design of a series of stable hydrazine-equipped manganese MRI probes gives Mn-2CHyd with the highest affinity and turn-on relaxivity (4-fold) upon reaction with LysAld. A dynamic PET-MRI study using [52Mn]Mn-2CHyd showed low liver uptake of the probe in healthy mice. The ability of the probe to detect liver fibrogenesis was then demonstrated in vivo in CCl4-injured mice. This study enables further development and application of manganese-based hydrazine-equipped probes for imaging liver fibrogenesis.

Targeting clinical epigenetic reprogramming for chemoprevention of metabolic and viral hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related mortality with chronic viral hepatitis and non-alcoholic steatohepatitis (NASH) as major aetiologies. Treatment options for HCC are unsatisfactory and chemopreventive approaches are absent. Chronic hepatitis C (CHC) results in epigenetic alterations driving HCC risk and persisting following cure. Here, we aimed to investigate epigenetic modifications as targets for liver cancer chemoprevention. DESIGN: Liver tissues from patients with NASH and CHC were analysed by ChIP-Seq (H3K27ac) and RNA-Seq. The liver disease-specific epigenetic and transcriptional reprogramming in patients was modelled in a liver cell culture system. Perturbation studies combined with a targeted small molecule screen followed by in vivo and ex vivo validation were used to identify chromatin modifiers and readers for HCC chemoprevention. RESULTS: In patients, CHC and NASH share similar epigenetic and transcriptomic modifications driving cancer risk. Using a cell-based system modelling epigenetic modifications in patients, we identified chromatin readers as targets to revert liver gene transcription driving clinical HCC risk. Proof-of-concept studies in a NASH-HCC mouse model showed that the pharmacological inhibition of chromatin reader bromodomain 4 inhibited liver disease progression and hepatocarcinogenesis by restoring transcriptional reprogramming of the genes that were epigenetically altered in patients. CONCLUSION: Our results unravel the functional relevance of metabolic and virus-induced epigenetic alterations for pathogenesis of HCC development and identify chromatin readers as targets for chemoprevention in patients with chronic liver diseases.

Blockade of HIF-1α and STAT3 by hyaluronate-conjugated TAT-chitosan-SPION nanoparticles loaded with siRNA molecules prevents tumor growth.

HIF-1α and STAT3 are two of the critical factors in the growth, proliferation, and metastasis of cancer cells and play a crucial role in inhibiting anti-cancer immune responses. Therefore, we used superparamagnetic iron oxide (SPION) nanoparticles (NPs) coated with thiolated chitosan (ChT) and trimethyl chitosan (TMC) and functionalized with hyaluronate (H) and TAT peptide for delivery of siRNA molecules against STAT3 and HIF-1α to cancer cells both in vivo and in vitro. The results indicated that tumor cell transfection with siRNA-encapsulated NPs robustly inhibited proliferation and migration and induced apoptosis in tumor cells. Furthermore, simultaneous silencing of HIF-1α and STAT3 significantly repressed cancer development in two different tumor types (4T1 breast cancer and CT26 colon cancer) which were associated with upregulation of cytotoxic T lymphocytes and IFN-γ secretion. The findings suggest inhibiting the HIF-1α/STAT3 axis by SPION-TMC-ChT-TAT-H NPs as an effective way to treat cancer.

Serum Angiopoietin-2 Predicts Mortality and Kidney Outcomes in Decompensated Cirrhosis.

Acute kidney injury in decompensated cirrhosis has limited therapeutic options, and novel mechanistic targets are urgently needed. Angiopoietin-2 is a context-specific antagonist of Tie2, a receptor that signals vascular quiescence. Considering the prominence of vascular destabilization in decompensated cirrhosis, we evaluated Angiopoietin-2 to predict clinical outcomes. Serum Angiopoietin-2 was measured serially in a prospective cohort of hospitalized patients with decompensated cirrhosis and acute kidney injury. Clinical characteristics and outcomes were examined over a 90-day period and analyzed according to Angiopoietin-2 levels. Primary outcome was 90-day mortality. Our study included 191 inpatients (median Angiopoietin-2 level 18.2 [interquartile range 11.8, 26.5] ng/mL). Median Model for End-Stage Liver Disease (MELD) score was 23 [17, 30] and 90-day mortality was 41%. Increased Angiopoietin-2 levels were associated with increased mortality (died 21.9 [13.9, 30.3] ng/mL vs. alive 15.2 [9.8, 23.0] ng/mL; P < 0.001), higher Acute Kidney Injury Network stage (stage I 13.4 [9.8, 20.1] ng/mL vs. stage II 20.0 [14.1, 26.2] ng/mL vs. stage III 21.9 [13.0, 29.5] ng/mL; P = 0.002), and need for renal replacement therapy (16.5 [11.3, 23.6] ng/mL vs. 25.1 [13.3, 30.3] ng/mL; P = 0.005). The association between Angiopoietin-2 and mortality was significant in unadjusted and adjusted Cox regression models (P ≤ 0.001 for all models), and improved discrimination for mortality when added to MELD score (integrated discrimination increment 0.067; P = 0.001). Conclusion: Angiopoietin-2 was associated with mortality and other clinically relevant outcomes in a cohort of patients with decompensated cirrhosis with acute kidney injury. Further experimental study of Angiopoietin/Tie2 signaling is warranted to explore its potential mechanistic and therapeutic role in this population.

The farnesoid X receptor agonist EDP-305 reduces interstitial renal fibrosis in a mouse model of unilateral ureteral obstruction.

Farnesoid X receptor (FXR) is a nuclear receptor that has emerged as a key regulator in the maintenance of hepatic steatosis, inflammation, and fibrosis. However, the role of FXR in renal fibrosis remains to be established. Here, we investigate the effects of the FXR agonist EDP-305 in a mouse model of tubulointerstitial fibrosis via unilateral ureteral obstruction (UUO). Male C57Bl/6 mice received a UUO on their left kidney. On postoperative d 4, mice received daily treatment by oral gavage with either vehicle control (0.5% methylcellulose) or 10 or 30 mg/kg EDP-305. All animals were euthanized on postoperative d 12. EDP-305 dose-dependently decreased macrophage infiltration as measured by the F4/80 staining area and proinflammatory cytokine gene expression. EDP-305 also dose-dependently reduced interstitial fibrosis as assessed by morphometric quantification of the collagen proportional area and kidney hydroxyproline levels. Finally, yes-associated protein (YAP) activation, a major driver of fibrosis, increased after UUO injury and was diminished by EDP-305 treatment. Consistently, EDP-305 decreased TGF-ß1-induced YAP nuclear localization in human kidney 2 cells by increasing inhibitory YAP phosphorylation. YAP inhibition may be a novel antifibrotic mechanism of FXR agonism, and EDP-305 could be used to treat renal fibrosis.-Li, S., Ghoshal, S., Sojoodi, M., Arora, G., Masia, R., Erstad, D. J., Ferriera, D. S., Li, Y., Wang, G., Lanuti, M., Caravan, P., Or, Y. S., Jiang, L.-J., Tanabe, K. K., Fuchs, B. C. The farnesoid X receptor agonist EDP-305 reduces interstitial renal fibrosis in a mouse model of unilateral ureteral obstruction.

Codelivery of HIF-1α siRNA and Dinaciclib by Carboxylated Graphene Oxide-Trimethyl Chitosan-Hyaluronate Nanoparticles Significantly Suppresses Cancer Cell Progression.

PURPOSE: Hypoxia-inducible factor (HIF) is one of the critical components of the tumor microenvironment that is involved in tumor development. HIF-1α functionally and physically interacts with CDK1, 2, and 5 and stimulates the cell cycle progression and Cyclin-Dependent Kinase (CDK) expression. Therefore, hypoxic tumor microenvironment and CDK overexpression lead to increased cell cycle progression and tumor expansion. Therefore, we decided to suppress cancer cell expansion by blocking HIF-1α and CDK molecules. METHODS: In the present study, we used the carboxylated graphene oxide (CGO) conjugated with trimethyl chitosan (TMC) and hyaluronate (HA) nanoparticles (NPs) loaded with HIF-1α-siRNA and Dinaciclib, the CDK inhibitor, for silencing HIF-1α and blockade of CDKs in CD44-expressing cancer cells and evaluated the impact of combination therapy on proliferation, metastasis, apoptosis, and tumor growth. RESULTS: The results indicated that the manufactured NPs had conceivable physicochemical properties, high cellular uptake, and low toxicity. Moreover, combination therapy of cancer cells using CGO-TMC-HA NPs loaded with HIF-1α siRNA and Dinaciclib (SCH 727965) significantly suppressed the CDKs/HIF-1α and consequently, decreased the proliferation, migration, angiogenesis, and colony formation in tumor cells. CONCLUSIONS: These results indicate the ability of CGO-TMC-HA NPs for dual drug/gene delivery in cancer treatment. Furthermore, the simultaneous inhibition of CDKs/HIF-1α can be considered as a novel anti-cancer treatment strategy; however, further research is needed to confirm this treatment in vivo. Graphical Abstract The suppression of HIF-1α and CDKs inhibits cancer growth. HIF-1α is overexpressed by the cells present in the tumor microenvironment. The hypoxic environment elevates mitochondrial ROS production and increases p38 MAP kinase, JAK/STAT, ERK, JNK, and Akt/PI3K signaling, resulting in cyclin accumulation and aberrant cell cycle progression. Furthermore, the overexpression of HIF-1α/CDK results in increased expression of genes such as BCL2, Bcl-xl, Ki-67, TGFß, VEGF, FGF, MMP2, MMP9, and, HIF-1α and consequently raise the survival, proliferation, angiogenesis, metastasis, and invasion of tumor cells. In conclusion, HIF-1α-siRNA/Dinaciclib-loaded CGO-TMC-HA NPs can inhibit the tumor expansion by blockage of CDKs and HIF-1α (JAK: Janus kinase, STAT: Signal transducer and activator of transcription, MAPK: mitogen-activated protein kinase, ERK: extracellular signal-regulated kinase, JNK: c-Jun N-terminal kinase, PI3K: phosphatidylinositol 3-kinase).

Molecular Magnetic Resonance Imaging Using a Redox-Active Iron Complex.

We introduce a redox-active iron complex, Fe-PyC3A, as a biochemically responsive MRI contrast agent. Switching between Fe3+-PyC3A and Fe2+-PyC3A yields a full order of magnitude relaxivity change that is field-independent between 1.4 and 11.7 T. The oxidation of Fe2+-PyC3A to Fe3+-PyC3A by hydrogen peroxide is very rapid, and we capitalized on this behavior for the molecular imaging of acute inflammation, which is characterized by elevated levels of reactive oxygen species.  Injection of Fe2+-PyC3A generates strong, selective contrast enhancement of inflamed pancreatic tissue in a mouse model (caerulein/LPS model). No significant signal enhancement is observed in normal pancreatic tissue (saline-treated mice). Importantly, signal enhancement of the inflamed pancreas correlates strongly and significantly with ex vivo quantitation of the pro-inflammatory biomarker myeloperoxidase. This is the first example of using metal ion redox for the MR imaging of pathologic change in vivo. Redox-active Fe3+/2+ complexes represent a new design paradigm for biochemically responsive MRI contrast agents.

Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

The zinc finger transcription factor PW1/PEG3 restrains murine beta cell cycling.

AIMS/HYPOTHESIS: Pw1 or paternally-expressed gene 3 (Peg3) encodes a zinc finger transcription factor that is widely expressed during mouse embryonic development and later restricted to multiple somatic stem cell lineages in the adult. The aim of the present study was to define Pw1 expression in the embryonic and adult pancreas and investigate its role in the beta cell cycle in Pw1 wild-type and mutant mice. METHODS: We analysed PW1 expression by immunohistochemistry in pancreas of nonpregant and pregnant mice and following injury by partial duct ligation. Its role in the beta cell cycle was studied in vivo using a novel conditional knockout mouse and in vitro by lentivirus-mediated gene knockdown. RESULTS: We showed that PW1 is expressed in early pancreatic progenitors at E9.5 but becomes progressively restricted to fully differentiated beta cells as they become established after birth and withdraw from the cell cycle. Notably, PW1 expression declines when beta cells are induced to proliferate and loss of PW1 function activates the beta cell cycle. CONCLUSIONS/INTERPRETATION: These results indicate that PW1 is a co-regulator of the beta cell cycle and can thus be considered a novel therapeutic target in diabetes.

Sources of beta cells inside the pancreas.

The generation of beta(-like) cells to compensate for their absolute or relative shortage in type 1 and type 2 diabetes is an obvious therapeutic strategy. Patients first received grafts of donor islet cells over 25 years ago, but this procedure has not become routine in clinical practice because of a donor cell shortage and (auto)immune problems. Transplantation of differentiated embryonic and induced pluripotent stem cells may overcome some but not all the current limitations. Reprogramming exocrine cells towards functional beta(-like) cells would offer an alternative abundant and autologous source of beta(-like) cells. This review focuses on work by our research group towards achieving such a source of cells. It summarises a presentation given at the 'Can we make a better beta cell?' symposium at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Amin Ardestani and Kathrin Maedler, DOI: 10.1007/s00125-016-3892-9 , and by Heiko Lickert and colleagues, DOI: 10.1007/s00125-016-3949-9 ) and a commentary by the Session Chair, Shanta Persaud (DOI: 10.1007/s00125-016-3870-2 ).

An angiotensin system inhibitor (losartan) potentiates antitumor efficacy of cisplatin in a murine model of non-small cell lung cancer.

Objective: Previous studies have demonstrated synergistic antitumor effects of angiotensin system inhibition (ASI) combined with cisplatin therapy in pancreatic cancer. This study examines whether or not synergistic antitumor effects occur with combination ASI and cisplatin treatment in lung cancer, and whether or not ASI-induced changes in epithelial-mesenchymal transition play a role in the mechanism of this antitumor phenomenon. Methods: A set of lung cancer cell lines representing a spectrum of epithelial to mesenchymal phenotypes were identified and characterized. Response of epithelial-mesenchymal transition markers to losartan was characterized. Cell culture models of lung cancer were next treated with losartan, cisplatin, or combination of both. Markers of epithelial-mesenchymal transition or surrogates of other signaling pathways (AKT, Stat3, and programmed death-ligand), and cell viability were quantified. Findings were confirmed in both allogenic and syngeneic in vivo murine flank tumor models. Results: Losartan treatment significantly increased E-cadherin and reduced vimentin in human lung cancer cell lines. Combination treatment with losartan and cisplatin enhanced epithelial markers, reduced mesenchymal markers, inhibited promesenchymal signaling mediators, and reduced cell viability. Findings were confirmed in vivo in a murine flank tumor model with transition from mesenchymal to epithelial phenotype and reduced tumor size following combination losartan and cisplatin treatment. Conclusions: Combination losartan and cisplatin treatment attenuates the epithelial-mesenchymal transition pathway and enhances the cytotoxic effect of chemotherapy with in vitro and in vivo models of non-small cell lung cancer. This study suggests an important role for ASI therapy in the treatment of lung cancer.

Enhanced maintenance of rat islets of Langerhans on laminin-coated electrospun nanofibrillar matrix in vitro.

Understanding the extracellular matrix (ECM) effect on pancreatic ß cells is critical to optimise the derivation of functional ß cells for transplantation and understand mechanisms that control islet neogenesis and glucose homeostasis. We assessed the effect of natural ECMs [collagen I, collagen IV, laminin and fibronectin (FN)] on rat islets of Langerhans' morphology, adhesion, viability, functionality and islet specific genes expression after 7 days in vitro culture. However, we could not detect a significant difference on the other parameters in these ECMs and islets interaction. To examine islets interactions, we used a synthetic three dimensional surface composed of electrospun polyamide nanofibres. Laminin-coated nanofibrillar surfaces, but not laminin or nanosurface alone, induced comparable expression of the Ins1 and Ins2 genes in adult ß cells. Using a glucose challenge test, a marked response of insulin secretion by islets occurred that were cultured on laminin-coated nanofibrillar surfaces.We contend that the reestablishment of cellular interactions by the combination of nanomaterials and natural ECMs can be useful in maintaining in vitro islet functions.

Precision-Cut Liver Slices as an ex vivo model to evaluate antifibrotic therapies for liver fibrosis and cirrhosis.

Background: Precision-Cut Liver Slices (PCLS) are an ex vivo culture model developed to study hepatic drug metabolism. One of the main benefits of this model is that it retains the structure and cellular composition of the native liver. PCLS also represents a potential model system to study liver fibrosis in a setting that more closely approximates in vivo pathology than in vitro methods. The aim of this study was to assess whether responses to antifibrotic interventions can be detected and quantified with PCLS. Methods: PCLS of 250 µm thickness were prepared from four different murine fibrotic liver models: choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD), thioacetamide (TAA), diethylnitrosamine (DEN), and carbon tetrachloride (CCl4). PCLS were treated with 5 µM Erlotinib for 72 hours. Histology and gene expression were then compared with in vivo murine experiments and TGF-ß1 activated hepatic stellate cells (HSCs). These types of PCLS characterization were also evaluated in PCLS from human cirrhotic liver. Results: PCLS viability in culture was stable for 72 hours. Treatment of erlotinib, an EGFR inhibitor significantly inhibited the expression of profibrogenic genes Il6, Col1a1 and Timp1 in PCLS from CDAHFD-induced cirrhotic mice, and Il6, Col1a1 and Tgfb1 in PCLS from TAA-induced cirrhotic rats. Erlotinib treatment of PCLS from DEN-induced cirrhotic rats inhibited the expression of Col1a1, Timp1, Tgfb1 and Il6, which was consistent with the impact of erlotinib on Col1a1 and Tgfb1 expression in in vivo DEN-induced cirrhosis. Erlotinib treatment of PCLS from CCl4-induced cirrhosis caused reduced expression of Timp1, Col1a1 and Tgfb1, which was consistent with the effect of erlotinib in in vivo CCl4-induced cirrhosis. In addition, in HSCs at PCLS from normal mice, TGF-ß1 treatment upregulated Acta2 (αSMA), while treatment with erlotinib inhibited the expression of Acta2. Similar expression results were observed in TGF-ß1 treated in vitro HSCs. Expression of MMPs and TIMPs, key regulators of fibrosis progression and regression, were also significantly altered under erlotinib treatment in PCLS. Expression changes under erlotinib treatment were also corroborated with PCLS from human cirrhosis samples. Conclusion: The responses to antifibrotic interventions can be detected and quantified with PCLS at the gene expression level. The antifibrotic effects of erlotinib are consistent between PCLS models of murine cirrhosis and those observed in vivo and in vitro. Similar effects were also reproduced in PCLS derived from patients with cirrhosis. PCLS is an excellent model to assess antifibrotic therapies that is aligned with the principles of Replacement, Reduction and Refinement (3Rs).

Molecular magnetic resonance imaging of liver inflammation using an oxidatively activated probe.

Background & Aims: Many liver diseases are driven by inflammation, but imaging to non-invasively diagnose and quantify liver inflammation has been underdeveloped. The inflammatory liver microenvironment is aberrantly oxidising owing in part to reactive oxygen species generated by myeloid leucocytes. We hypothesised that magnetic resonance imaging using the oxidatively activated probe Fe-PyC3A will provide a non-invasive biomarker of liver inflammation. Methods: A mouse model of drug-induced liver injury was generated through intraperitoneal injection of a hepatoxic dose of acetaminophen. A mouse model of steatohepatitis was generated via a choline-deficient, l-amino acid defined high-fat diet (CDAHFD). Images were acquired dynamically before and after intravenous injection of Fe-PyC3A. The contrast agent gadoterate meglumine was used as a non-oxidatively activated negative control probe in mice fed CDAHFD. The (post-pre) Fe-PyC3A injection change in liver vs. muscle contrast-to-noise ratio (ΔCNR) recorded 2 min post-injection was correlated with liver function test values, histologic scoring assigned using the NASH Clinical Research Network criteria, and intrahepatic myeloid leucocyte composition determined by flow cytometry. Results: For mice receiving i.p. injections of acetaminophen, intrahepatic neutrophil composition correlated poorly with liver test values but positively and significantly with ΔCNR (r = 0.64, p <0.0001). For mice fed CDAHFD, ΔCNR generated by Fe-PyC3A in the left lobe was significantly greater in mice meeting histologic criteria strongly associated with a diagnosis NASH compared to mice where histology was consistent with likely non-NASH (p = 0.0001), whereas no differential effect was observed using gadoterate meglumine. In mice fed CDAHFD, ΔCNR did not correlate strongly with fractional composition of any specific myeloid cell subpopulation as determined by flow cytometry. Conclusions: Magnetic resonance imaging using Fe-PyC3A merits further evaluation as a non-invasive biomarker for liver inflammation. Impact and implications: Non-invasive tests to diagnose and measure liver inflammation are underdeveloped. Inflammatory cells such as neutrophils release reactive oxygen species which creates an inflammatory liver microenvironment that can drive chemical oxidation. We recently invented a new class of magnetic resonance imaging probe that is made visible to the scanner only after chemical oxidation. Here, we demonstrate how this imaging technology could be applied as a non-invasive biomarker for liver inflammation.