Roberto Loi

Progenitor-derived hepatocellular carcinoma model in the rat†

Human hepatocellular carcinoma (HCC) is a heterogeneous disease of distinct clinical subgroups. A principal source of tumor heterogeneity may be cell type of origin, which in liver includes hepatocyte or adult stem/progenitor cells. To address this issue, we investigated the molecular mechanisms underlying the fate of the enzyme‐altered preneoplastic lesions in the resistant hepatocyte (RH) model. Sixty samples classified as focal lesions, adenoma, and early and advanced HCCs were microdissected after morphological and immunohistochemical evaluation and subjected to global gene expression profiling. The analysis of progression of the persistent glutathione S‐transferase (GSTP)+ focal lesions to fully developed HCC showed that approximately 50% of persistent nodules and all HCCs expressed cytokeratin 19 (CK19), whereas 14% of remodeling nodules were CK19+. Unsupervised hierarchical clustering of the expression profiles also grouped the samples according to CK19 expression. Furthermore, supervised analysis using the differentially expressed genes in each cluster combined with gene connectivity tools identified 1308 unique genes and a predominance of the AP‐1/JUN network in the CK19+ lesions. In contrast, the CK19‐negative cluster exhibited only limited molecular changes (156 differentially expressed genes versus normal liver) consistent with remodeling toward differentiated phenotype. Finally, comparative functional genomics showed a stringent clustering of CK19+ early lesions and advanced HCCs with human HCCs characterized by poor prognosis. Furthermore, the CK19‐associated gene expression signature accurately predicted patient survival (P < 0.009) and tumor recurrence (P < 0.006). Conclusion: Our data establish CK19 as a prognostic marker of early neoplastic lesions and strongly suggest the progenitor derivation of HCC in the rat RH model. The capacity of CK19‐associated gene signatures to stratify HCC patients according to clinical prognosis indicates the usefulness of the RH model for studies of stem/progenitor‐derived HCC. (HEPATOLOGY 2010.)

Comparative Decellularization and Recellularization of Normal versus Emphysematous Human Lungs

Acellular whole human lung scaffolds represent a unique opportunity for ex vivo tissue engineering. However, it remains unclear whether lungs from individuals with chronic lung diseases such as chronic obstructive pulmonary disease (COPD) can be appropriately decellularized and recellularized. To assess this, cadaveric human lungs from normal (non-smoking) patients and from patients with COPD (smoking history) were decellularized and found by histochemical and immunohistochemical staining, electron microscopy, and mass spectrometry to retain characteristic histological architecture and extracellular matrix components (ECM) reflecting either normal or COPD, particularly emphysematous, origin. Inoculation of human bronchial epithelial cells, endothelial progenitor cells, bone marrow-derived mesenchymal stem cells, and lung fibroblasts via airway or vascular routes into small, excised segments of the decellularized lungs demonstrated that normal lung scaffolds robustly supported initial engraftment and growth of each cell type for up to one month. In contrast, despite initial binding, all cell types inoculated into decellularized emphysematous lungs did not survive beyond one week. However, cell attachment and proliferation on solubilized ECM homogenates of decellularized normal and emphysematous lungs coated onto tissue culture plates was comparable and not impaired, suggesting that the 3-dimensional decellularized emphysematous scaffolds may lack the necessary ECM architecture to support sustained cell growth.

Early Increase in Cyclin-D1 Expression and Accelerated Entry of Mouse Hepatocytes into S Phase after Administration of the Mitogen 1,4-Bis(2- (3,5-Dichloropyridyloxy)) Benzene

We have previously demonstrated that hepatocyte proliferation induced by the mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) is independent of changes in cytokines, immediate early genes, and transcription factors that are considered to be necessary for regeneration of the liver after partial hepatectomy (PH) or necrosis. To further investigate the differences between mitogen-induced mouse hepatocyte proliferation and liver regeneration after PH, we have measured the expression of cyclin D1, cyclin D3, cyclin E, and cyclin A and of the cyclin-dependent kinases CDK2, CDK4, and CDK6. The involvement of the cyclin-dependent kinase inhibitors p21 and p27 and of the oncosuppressor gene p53 was also examined at different times after stimulation of hepatocyte proliferation. Results showed that a single administration of TCPOBOP caused a very rapid increase in the levels of cyclin D1, a G1 protein, when compared with two thirds PH (8 hours versus 30 hours). The early increase in cyclin D1 protein levels was associated with a faster onset of increased expression of S-phase-associated cyclin A (24 hours versus 36 hours with PH mice). Accordingly, measurement of bromodeoxyuridine (BrdU) incorporation revealed that, although approximately 8% of hepatocytes were BrdU-positive as early as 24 hours after TCPOBOP, no significant changes in BrdU incorporation were observed at the same time point after two thirds PH. The expression of other proteins involved in cell cycle control, such as cyclin-dependent kinases (CDK4, CDK2, CDK6), was also analyzed. Results showed that expression of CDK2 was induced much more rapidly in TCPOBOP-treated mice (2 hours) than in mice subjected to PH (36 hours). A different pattern of expression in the two models of hepatocyte proliferation, although less dramatic, was also observed for CDK4 and CDK6. Expression of the CDK inhibitors p21 and p27 and the oncosuppressor gene p53 variably increased after two thirds PH, whereas basically no change in protein levels was found in TCPOBOP-treated mice. The results demonstrate that profound differences in many cell cycle-regulatory proteins exist between direct hyperplasia and compensatory regeneration. Cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by the primary mitogen TCPOBOP and suggests that a direct effect of the mitogen on this cyclin may be responsible for the rapid onset of DNA synthesis observed in TCPOBOP-induced hyperplasia.

The effect of age and emphysematous and fibrotic injury on the re-cellularization of de-cellularized lungs.

Use of de-cellularized cadaveric lungs as 3-dimensional scaffolds for ex vivo lung tissue generation offers a new potential therapeutic approach for clinical lung transplantation. However, it is likely that some of the available cadaveric human lungs may be from older donors or from donors with previously existing structural lung diseases such as emphysema or pulmonary fibrosis. It is not known whether these lungs will be suitable for either de-cellularization or re-cellularization. To investigate this, we assessed the effects of advanced age, representative emphysematous and fibrotic injuries, and the combination of advanced age and emphysematous injury and found significant differences both in histologic appearance and in the retention of extracellular matrix (ECM) and other proteins, as assessed by immunohistochemistry and mass spectrometry, between the different conditions. However, despite these differences, binding, retention and growth of bone marrow-derived mesenchymal stromal cells (MSCs) over a 1-month period following intratracheal inoculation were similar between the different experimental conditions. In contrast, significant differences occurred in the growth of C10 mouse lung epithelial cells between the different conditions. Therefore, age, lung injury, and the cell type used for re-cellularization may significantly impact the usefulness of de-cellularized whole lungs for ex vivo lung tissue regeneration.

The effects of storage and sterilization on de-cellularized and re-cellularized whole lung.

Despite growing interest on the potential use of de-cellularized whole lungs as 3-dimensional scaffolds for ex vivo lung tissue generation, optimal processing including sterilization and storage conditions, are not well defined. Further, it is unclear whether lungs need to be obtained immediately or may be usable even if harvested several days post-mortem, a situation mimicking potential procurement of human lungs from autopsy. We therefore assessed effects of delayed necropsy, prolonged storage (3 and 6 months), and of two commonly utilized sterilization approaches: irradiation or final rinse with peracetic acid, on architecture and extracellular matrix (ECM) protein characteristics of de-cellularized mouse lungs. These different approaches resulted in significant differences in both histologic appearance and in retention of ECM and intracellular proteins as assessed by immunohistochemistry and mass spectrometry. Despite these differences, binding and proliferation of bone marrow-derived mesenchymal stromal cells (MSCs) over a one month period following intratracheal inoculation was similar between experimental conditions. In contrast, significant differences occurred with C10 mouse lung epithelial cells between the different conditions. Therefore, delayed necropsy, duration of scaffold storage, sterilization approach, and cell type used for re-cellularization may significantly impact the usefulness of this biological scaffold-based model of ex vivo lung tissue regeneration.

Mesenchymal Stromal Cells Mediate Aspergillus Hyphal Extract-Induced Allergic Airway Inflammation by Inhibition of the Th17 Signaling Pathway

Systemic administration of mesenchymal stromal cells (MSCs) suppresses airway inflammation and methacholine‐induced airway hyper‐responsiveness (AHR) in mouse models of T helper cell (Th) type 2‐mediated eosinophilic allergic airway inflammation (AAI); however, the efficacy of MSCs in mouse models of severe Th17‐mediated neutrophilic AAI has not yet been demonstrated. We assessed MSC effects in a mouse model of mixed Th2/Th17 AAI produced by mucosal exposure to Aspergillus fumigatus hyphal extract (AHE). Following sensitization produced by oropharyngeal AHE administration, systemic (tail vein) administration of syngeneic MSCs on the first day of challenge significantly reduced acute AHR predominantly through reduction of Th17‐mediated airway inflammation. In parallel experiments, MSCs also mitigated AHR when administered during recurrent challenge 10 weeks after initial sensitization and challenge through reduction in systemic Th17‐mediated inflammation. Investigation into potential mechanistic actions of MSCs in this model demonstrated that although T regulatory cells were increased in all AHE‐treated mice, MSC administration did not alter T regulatory cell numbers in either the acute or recurrent model. Differential induction of interleukin‐17a secretion was observed in ex vivo restimulation of mediastinal lymph node mixed‐cell cytokine analyses. Although the mechanisms by which MSCs act to decrease inflammation and AHR in this model are not yet fully elucidated, decrease in Th17‐mediated airway inflammation appears to play a significant role. These results provide a basis for further investigations of MSC administration as a potential therapeutic approach for severe refractory neutrophilic asthma.

Local hypothyroidism favors the progression of preneoplastic lesions to hepatocellular carcinoma in rats.

Thyroid hormone receptors (TRs) are ligand‐dependent transcription factors that mediate most of the effects elicited by the thyroid hormone, 3,5,3′‐L‐triiodothyronine (T3). TRs have been implicated in tumorigenesis, although it is unclear whether they act as oncogenes or tumor suppressors, and at which stage of tumorigenesis their dysregulation occurs. Using the resistant‐hepatocyte rat model (R‐H model), we found down‐regulation of TRβ1 and TRα1 and their target genes in early preneoplastic lesions and hepatocellular carcinoma (HCCs), suggesting that a hypothyroid status favors the onset and progression of preneoplastic lesions to HCC. Notably, TRβ1 and, to a lesser extent, TRα1 down‐regulation was observed only in preneoplastic lesions positive for the progenitor cell marker, cytokeratin‐19 (Krt‐19) and characterized by a higher proliferative activity, compared to the Krt‐19 negative ones. TRβ1 down‐regulation was observed also in the vast majority of the analyzed human HCCs, compared to the matched peritumorous liver or to normal liver. Hyperthyroidism induced by T3 treatment caused up‐regulation of TRβ1 and of its target genes in Krt‐19+ preneoplastic rat lesions and was associated with nodule regression. In HCC, TRβ1 down‐regulation was not the result of hypermethylation of its promoter, but was associated with an increased expression of TRβ1‐targeting microRNAs ([miR]‐27a, ‐181a, and ‐204). An inverse correlation between TRβ1 and miR‐181a was also found in human cirrhotic peritumoral tissue, compared to normal liver. Conclusion: Down‐regulation of TRs, especially TRβ1, is an early and relevant event in liver cancer development and is species and etiology independent. The results also suggest that a hypothyroid status of preneoplastic lesions may contribute to their progression to HCC and that the reversion of this condition may represent a possible therapeutic goal to interfere with the development of this tumor. (Hepatology 2015;61:249–259)

Endogenous Distal Airway Progenitor Cells, Lung Mechanics, and Disproportionate Lobar Growth Following Long‐Term Postpneumonectomy in Mice

Using a model of postpneumonectomy (PNY) compensatory lung growth in mice, we previously observed an increase in numbers of a putative endogenous distal airway progenitor cell population (CCSPpos/pro‐SPCpos cells located at bronchoalveolar duct junctions [BADJs]), at 3, 7, and 14 days after pneumonectomy, returning to baseline at 28 days post‐PNY. As the origin of these cells is poorly understood, we evaluated whether bone marrow cells contributed to the pool of these or other cells during prolonged post‐PNY lung regrowth. Naïve and sex‐mismatched chimeric mice underwent left PNY and were evaluated at 1, 2, and 3 months for numbers of BADJ CCSPpos/pro‐SPCpos cells and presence of donor‐derived marrow cells engrafted as airway or alveolar epithelium. Nonchimeric mice were also examined at 12 months after PNY for numbers of BADJ CCSPpos/pro‐SPCpos cells. Notably, the right accessory lobe (RAL) continued to grow disproportionately over 12 months, a novel finding not previously described. Assessment of lung mechanics demonstrated an increase in lung stiffness following PNY, which significantly diminished over 1 year, but remained elevated relative to 1‐year‐old naïve controls. However, the number of CCSPpos/pro‐SPCpos BADJ cells ≥1‐month following PNY was equivalent to that found in naïve controls even after 12 months of continued RAL growth. Notably, no donor bone marrow‐derived cells engrafted as airway or alveolar epithelial cells, including those at the BADJ, up to 3 months after PNY. These studies suggest that lung epithelial cells, including CCSPpos/pro‐SPCpos cells, are not replenished from marrow‐derived cells during post‐PNY lung growth in mice. STEM Cells2013;31:1330–1339

Chronic treatment with SCH 23390 increases the production rate of dopamine D1 receptors in the nigro-striatal system of the rat

The effects of chronic treatment with the selective dopamine D1 receptor antagonist, SCH 23390, on the steady-state densities and turnover rates of these receptors were investigated in the striatum and substantia nigra of the rat. To this aim, we assessed the repopulation kinetics of [3H]SCH 23390 binding sites after irreversible inactivation of dopamine D1 receptors induced by a single dose of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 10 mg/kg s.c.) in rats chronically treated with SCH 23390. The receptor repopulation was analyzed using a theoretical model that assumes a constant rate of receptor production and a first-order receptor degradation rate. The repeated administration of SCH 23390 (0.05 mg/kg s.c., thrice daily for 21 days) enhanced the steady-state density of dopamine D1 receptors in the striatum (+30%) and substantia nigra (+24%). This treatment also increased the production rates of dopamine D1 receptors in the striatum (+44%) and substantia nigra (+54%). By contrast, the rate constants of dopamine D1 receptor degradation were unchanged in both brain areas. These results suggest that the up-regulation of dopamine D1 receptors induced by chronic treatment with SCH 23390 is determined by modifications in the processes that control the rate of receptor production but not of receptor degradation.

Derangement of intestinal epithelial cell monolayer by dietary cholesterol oxidation products

Abstract The emerging role of the diet in the incidence of intestinal inflammatory diseases has stimulated research on the influence of eating habits with pro‐inflammatory properties in inducing epithelial barrier disturbance. Cholesterol oxidation products, namely oxysterols, have been shown to promote and sustain oxidative/inflammatory reactions in human digestive tract. This work investigated in an in vitro model the potential ability of a combination of dietary oxysterols representative of a hyper‐cholesterol diet to induce the loss of intestinal epithelial layer integrity. The components of the experimental mixture were the main oxysterols stemming from heat‐induced cholesterol auto‐oxidation, namely 7‐ketocholesterol, 5&agr;,6&agr;‐and 5&bgr;,6&bgr;‐epoxycholesterol, 7&agr;‐ and 7&bgr;‐hydroxycholesterol. These compounds added to monolayers of differentiated CaCo‐2 cells in combination or singularly, caused a time‐dependent induction of matrix metalloproteinases (MMP)‐2 and ‐9, also known as gelatinases. The hyperactivation of MMP‐2 and ‐9 was found to be associated with decreased levels of the tight junctions zonula occludens‐1 (ZO‐1), occludin and Junction Adhesion Molecule‐A (JAM‐A). Together with such a protein loss, particularly evident for ZO‐1, a net perturbation of spatial localization of the three tight junctions was observed. Cell monolayer pre‐treatment with the selective inhibitor of MMPs ARP100 or polyphenol (‐)‐epicathechin, previously shown to inhibit NADPH oxidase in the same model system, demonstrated that the decrease of the three tight junction proteins was mainly a consequence of MMPs induction, which was in turn dependent on the pro‐oxidant property of the oxysterols investigated. Although further investigation on oxysterols intestinal layer damage mechanism is to be carried on, the consequent ‐ but incomplete ‐ prevention of oxysterols‐dependent TJs alteration due to MMPs inhibition, avoided the loss of scaffold protein ZO‐1, with possible significant recovery of intestinal monolayer integrity. Graphical abstract No caption available. HighlightsOxysterols produced in cholesterol‐rich foods alter intestinal epithelial layer.Matrix metalloproteinases (MMPs)‐2 and ‐9 are activated by dietary oxysterols.Oxysterols affect MMP‐2 and ‐9 when used in combination as they are in food.Oxy‐mix disrupts intestinal tight junctions mainly through MMP‐2 and‐9 induction.These effects depend on the pro‐oxidant/inflammatory properties of dietary oxysterols.