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. Fe-PyC3A 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.

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.

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.

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.

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.

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 (MR) 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 noninvasively 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, make them strong candidates for clinical translation.