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Cysteine cathepsins are potent proteases implicated in cardiovascular disease for degrading extracellular matrix (ECM) whose structure and integrity determine the mechanical behavior of arteries. Cathepsin knockout mouse models fed atherogenic diets have been used to study their roles in cardiovascular disease, but the impacts of cathepsin knockout on non-atherosclerotic arterial mechanics are scarce. We examine arterial mechanics in several cathepsin knockout mouse lines (CatK

Jasmine Miller-Kleinhenz et al. highlight the risk of science and academia’s general neutrality to discussions around race and social justice. Their collectively-developed course represents a framework to begin these important discussions and improve conversations on race in academia.

Hepatoblastoma (HB) is the most common pediatric liver malignancy. Around 80% of HB demonstrate simultaneous activation of β-catenin and Yes-associated protein 1 (Yap1). The mechanism by which these signaling pathways contribute to HB pathogenesis remain obscure. Recently, mTORC1 activation was reported in human HB cells and in a murine HB model driven by β-catenin and Yap1. Here, we directly investigate the therapeutic impact of mTOR inhibition following HB development in the Yap1-β-catenin model. HB were established by hydrodynamic tail vein injection of Sleeping Beauty transposase and plasmids coding for ΔN90-β-catenin and S127A-Yap1. Five weeks after injection, when HB were evident, mice were randomized into Rapamycin diet-fed or basal-diet-fed groups for 5-weeks. Tumor growth was monitored via ultrasound imaging and mice in both groups were euthanized after 5-weeks for molecular analysis. Transcriptomic analysis showed a strong correlation in gene expression between HB in the Yap1-β-catenin model and HB patient cohorts. Rapamycin treatment decreased HB burden, almost normalizing liver weight to body weight ratio. Ultrasound imaging showed reduction in tumor growth over the duration of Rapamycin treatment as compared to controls. Majority of HB in the controls exhibited crowded fetal or embryonal histology, while remnant tumors in the experimental group showed well-differentiated fetal morphology. Immunohistochemistry confirmed inhibition of mTORC1 in the Rapamycin group. Thus, Rapamycin reduces HB in a clinically relevant model driven by β-catenin and Yap1, supporting use of mTORC1 inhibitors in their therapy. We also show the utility of standard and 3D ultrasound imaging for monitoring liver tumors in mice.

Background aims Mesenchymal stromal cells (MSCs) have shown great promise in the field of regenerative medicine, as many studies have shown that MSCs possess immunomodulatory function. Despite this promise, no MSC therapies have been licensed by the Food and Drug Administration. This lack of successful clinical translation is due in part to MSC heterogeneity and a lack of critical quality attributes. Although MSC indoleamine 2,3-dioxygnease (IDO) activity has been shown to correlate with MSC function, multiple predictive markers may be needed to better predict MSC function. Methods Three MSC lines (two bone marrow-derived, one induced pluripotent stem cell-derived) were expanded to three passages. At the time of harvest for each passage, cell pellets were collected for nuclear magnetic resonance (NMR) and ultra-performance liquid chromatography mass spectrometry (MS), and media were collected for cytokine profiling. Harvested cells were also cryopreserved for assessing function using T-cell proliferation and IDO activity assays. Linear regression was performed on functional data against NMR, MS and cytokines to reduce the number of important features, and partial least squares regression (PLSR) was used to obtain predictive markers of T-cell suppression based on variable importance in projection scores. Results Significant functional heterogeneity (in terms of T-cell suppression and IDO activity) was observed between the three MSC lines, as were donor-dependent differences based on passage. Omics characterization revealed distinct differences between cell lines using principal component analysis. Cell lines separated along principal component one based on tissue source (bone marrow-derived versus induced pluripotent stem cell-derived) for NMR, MS and cytokine profiles. PLSR modeling of important features predicted MSC functional capacity with NMR (R2 = 0.86), MS (R2 = 0.83), cytokines (R2 = 0.70) and a combination of all features (R2 = 0.88). Conclusions The work described here provides a platform for identifying markers for predicting MSC functional capacity using PLSR modeling that could be used as release criteria and guide future manufacturing strategies for MSCs and other cell therapies.

// Adeola O. Adebayo Michael 1, 7, * , Nagib Ahsan 2, 3, * , Valerie Zabala 1 , Heather Francois-Vaughan 1 , Stephanie Post 4 , Kate E. Brilliant 3 , Arthur R. Salomon 3, 5 , Jennifer A. Sanders 1, 6 , Philip A. Gruppuso 1, 5 1 Department of Pediatrics, Rhode Island Hospital and Brown University, Providence, RI, USA 2 Division of Biology and Medicine, Brown University, Providence, RI, USA 3 Center for Cancer Research Development, Proteomics Core Facility, Rhode Island Hospital, Providence, RI, USA 4 Department of Environmental and Evolutionary Biology, Brown University, Providence, RI, USA 5 Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA 6 Department of Pathology and Laboratory Medicine, Brown University, Providence, RI, USA 7 Current address: Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA * These authors contributed equally to this work Correspondence to: Philip A. Gruppuso, email: Philip_Gruppuso@brown.edu Keywords: hepatocellular carcinoma, liver, microdissection, proteomics, pre-neoplasia Received: December 22, 2016 Accepted: January 27, 2017 Published: February 09, 2017 ABSTRACT We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR) complex 1, markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt-Farber) model of hepatocellular carcinoma (HCC) in the rat. In the present study, we characterized the proteome of persistent, pre-neoplastic focal lesions in this model. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and euthanized 4 weeks after the cessation of rapamycin. A second group received placebo pellets. Results were compared to unmanipulated control animals and to animals that underwent an incomplete Solt-Farber protocol to activate hepatic progenitor cells. Regions of formalin-fixed, paraffin-embedded tissue were obtained by laser capture microdissection (LCM). Proteomic analysis yielded 11,070 unique peptides representing 2,227 proteins. Quantitation of the peptides showed increased abundance of known HCC markers (e.g., glutathione S-transferase-P, epoxide hydrolase, 6 others) and potential markers (e.g., aflatoxin aldehyde reductase, glucose 6-phosphate dehydrogenase, 10 others) in foci from placebo-treated and rapamycin-treated rats. Peptides derived from cytochrome P450 enzymes were generally reduced. Comparisons of the rapamycin samples to normal liver and to the progenitor cell model indicated that rapamycin attenuated a loss of differentiation relative to placebo. We conclude that early administration of rapamycin in the Solt-Farber model not only inhibits the growth of pre-neoplastic foci but also attenuates the loss of differentiated function. In addition, we have demonstrated that the combination of LCM and mass spectrometry-based proteomics is an effective approach to characterize focal liver lesions.

BACKGROUND AND AIMS Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.

Hepatoblastoma (HB), the most common pediatric primary liver neoplasm, shows nuclear localization of β-catenin and yes-associated protein 1 (YAP1) in almost 80% of the cases. Co-expression of constitutively active S127A-YAP1 and ΔN90 deletion-mutant β-catenin (YAP1–ΔN90-β-catenin) causes HB in mice. Because heterogeneity in downstream signaling is being identified owing to mutational differences even in the β-catenin gene alone, we investigated if co-expression of point mutants of β-catenin (S33Y or S45Y) with S127A-YAP1 led to similar tumors as YAP1–ΔN90-β-catenin. Co-expression of S33Y/S45Y–β-catenin and S127A-YAP1 led to activation of Yap and Wnt signaling and development of HB, with 100% mortality by 13 to 14 weeks. Co-expression with YAP1–S45Y/S33Y-β-catenin of the dominant-negative T-cell factor 4 or dominant-negative transcriptional enhanced associate domain 2, the respective surrogate transcription factors, prevented HB development. Although histologically similar, HB in YAP1–S45Y/S33Y-β-catenin, unlike YAP1–ΔN90-β-catenin HB, was glutamine synthetase (GS) positive. However, both ΔN90–β-catenin and point-mutant β-catenin comparably induced GS-luciferase reporter in vitro. Finally, using a previously reported 16-gene signature, it was shown that YAP1–ΔN90-β-catenin HB tumors exhibited genetic similarities with more proliferative, less differentiated, GS-negative HB patient tumors, whereas YAP1–S33Y/S45Y-β-catenin HB exhibited heterogeneity and clustered with both well-differentiated GS-positive and proliferative GS-negative patient tumors. Thus, we demonstrate that β-catenin point mutants can also collaborate with YAP1 in HB development, albeit with a distinct molecular profile from the deletion mutant, which may have implications in both biology and therapy.

Simultaneous mutations in CTNNB1 and activation of c-MET occur in 9%‐12.5% of patients with hepatocellular carcinoma (HCC). Coexpression of c-MET-V5 and mutant β-catenin-Myc in mouse liver by sleeping beauty transposon/transposase and hydrodynamic tail vein injection (SB-HTVI) led to the development of HCC with 70% molecular identity to the clinical subset. Using this model, we investigated the effect of EMD1214063, a highly selective c-MET inhibitor. Five weeks after SB-HTVI when tumors were established, EMD1214063 (10 mg/kg) was administered by gastric gavage as a single agent on 5-day-on/3-day-off schedule, compared to vehicle only control. Mice were harvested at 8 or 11 weeks posttreatment. Decreased p-MET, p-AKT, p-STAT3, and p-ERK proved in vivo efficacy of EMD1214063. We observed lower Ki-67, PCNA, V5-tag, and cyclin D1 after EMD1214063 treatment only at 8 weeks. Overall, no significant differences were observed in tumor burden between the groups, although EMD1214063 marginally but significantly improved overall survival by 1.5‐2 weeks. Tumors remained α-fetoprotein+, did not show any differences in inflammation, and lacked fibrosis in either group. In conclusion, c-MET inhibition alone had a minor effect on Met-β-catenin HCC at the early stages of HCC development. Thus, a single therapy with the c-MET inhibitor will be insufficient for sustained response in Met-β-catenin HCC requiring assessment of additional combinations.

Background & Aims Liver fibrosis, hepatocellular necrosis, inflammation, and proliferation of liver progenitor cells are features of chronic liver injury. Mouse models have been used to study the end-stage pathophysiology of chronic liver injury. However, little is known about differences in the mechanisms of liver injury among different mouse models because of our inability to visualize the progression of liver injury in vivo in mice. We developed a method to visualize bile transport and blood-bile barrier (BBlB) integrity in live mice. Methods C57BL/6 mice were fed a choline-deficient, ethionine-supplemented (CDE) diet or a diet containing 0.1% 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) for up to 4 weeks to induce chronic liver injury. We used quantitative liver intravital microscopy (qLIM) for real-time assessment of bile transport and BBlB integrity in the intact livers of the live mice fed the CDE, DDC, or chow (control) diets. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, real-time polymerase chain reaction, and immunoblots. Results Mice with liver injury induced by a CDE or a DDC diet had breaches in the BBlB and impaired bile secretion, observed by qLIM compared with control mice. Impaired bile secretion was associated with reduced expression of several tight-junction proteins (claudins 3, 5, and 7) and bile transporters (NTCP, OATP1, BSEP, ABCG5, and ABCG8). A prolonged (2-week) CDE, but not DDC, diet led to re-expression of tight junction proteins and bile transporters, concomitant with the reestablishment of BBlB integrity and bile secretion. Conclusions We used qLIM to study chronic liver injury, induced by a choline-deficient or DDC diet, in mice. Progression of chronic liver injury was accompanied by loss of bile transporters and tight junction proteins.

[This corrects the article DOI: 10.18632/oncotarget.26668.].

Summary Based on their lobule location, hepatocytes display differential gene expression, including pericentral hepatocytes that surround the central vein, which are marked by Wnt-??-catenin signaling. Activating ??-catenin mutations occur in a variety of liver tumors, including hepatocellular carcinoma (HCC), but no specific therapies are available to treat these tumor subsets. Here, we identify a positive relationship between ??-catenin activation, its transcriptional target glutamine synthetase (GS), and p-mTOR-S2448, an indicator of mTORC1 activation. In normal livers of mice and humans, pericentral hepatocytes were simultaneously GS and p-mTOR-S2448 positive, as were ??-catenin-mutated liver tumors. Genetic disruption of ??-catenin signaling or GS prevented p-mTOR-S2448 expression, while its forced expression in ??-catenin-deficient livers led to ectopic p-mTOR-S2448 expression. Further, we found notable therapeutic benefit of mTORC1 inhibition in mutant-??-catenin-driven HCC through suppression of cell proliferation and survival. Thus, mTORC1 inhibitors could be highly relevant in the treatment of liver tumors that are ??-catenin mutated and GS positive.