Genetic Abolishment of Hepatocyte Proliferation Activates Hepatic Stem Cells

Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer

Pharmacological evaluation of [I-123]-CLINDE: A radioiodinated imidazopyridine-3-acetamide for the study of peripheral benzodiazepine binding sites (PBBS)

Purpose The study aims to evaluate the iodinated imidazopyridine, N′,N′-diethyl-6-Chloro-(4′-[123I]iodophenyl)imidazo [1,2-a]pyridine-3-acetamide ([123I]-CLINDE) as a tracer for the study of peripheral benzodiazepine binding sites (PBBS). Materials and methods In vitro studies were performed using membrane homogenates and sections from kidney, adrenals, and brain cortex of Sprague–Dawley (SD) rats and incubated with [123I]-CLINDE. For in vivo studies, the rats were injected with [123I]-CLINDE. In competition studies, PBBS-specific drugs PK11195 and Ro 5-4864 and the CBR specific drug Flumazenil were injected before the radiotracer. Results In vitro binding studies in adrenal, kidney, and cortex mitochondrial membranes indicated that [123I]-CLINDE binds with high affinity to PBBS, Kd = 12.6, 0.20, and 3.84 nM, respectively. The density of binding sites was 163, 5.3, and 0.34 pmol/mg protein, respectively. In vivo biodistribution indicated high uptake in adrenals (5.4), heart (1.5), lungs (1.5), kidney (1.5) %ID/g at 6 h p.i. In the central nervous system (CNS), the olfactory bulbs displayed the highest uptake; up to six times the activity in blood. Preadministration of unlabeled CLINDE, PK11195 and Ro 5–4864 (1 mg/kg) reduced the uptake of [123I]-CLINDE by 70–55% in olfactory bulbs. In the kidney and heart, a reduction of 60–80% ID/g was observed, while an increase was observed in the adrenals requiring 10 mg/kg for significant displacement. Flumazenil had no effect on uptake in peripheral organs and brain. Metabolite analysis indicated >90% of the radioactivity in the above tissues was intact [123I]-CLINDE. Conclusion [123I]-CLINDE displays high and selective uptake for the PBBS and warrants further development as a probe for imaging PBBS using single photon emission computed tomography (SPECT)