Genetic Lineage Tracing of Biliary Epithelial Cells.

Lineage tracing of liver cells is a powerful tool to understand liver embryonic development, healthy liver cell homeostasis, tissue repair, and regeneration. Lineage tracing of biliary epithelial cells (BECs) in the adult liver has been used to assess the contribution of the biliary epithelium to liver injury, regeneration, and disease. These studies have shown the contribution of BECs to the expansion of ductular reaction (DR) and liver progenitor cells (LPCs) and eventually the generation of new hepatocytes. Few genetic lineage-tracing mouse models have been proved to trace BECs. This chapter is focused on lineage tracing of BECs in mouse models of liver injury and regeneration. First, we mention different existing approaches to trace the biliary epithelium based on proteins specifically expressed by BECs such as sex-determining region Y-box 9 (SOX9), osteopontin (OPN), and cytokeratin-19 (KRT19). Second, we describe mouse models that can be used to evaluate cell fate during liver injury and regeneration (i.e., partial hepatectomy (PHx), acute liver injury models, and chronic liver damage models such as 3,5-diethoxycarbonyl-1,4-dihydro-collidin (DDC) diet, choline-deficient ethionine-supplemented (CDE) diet, or chronic carbon tetrachloride (CCl4) administration). Third, we suggest possible readouts to assess BECs fate based on immunofluorescence analysis.

Loss of trefoil factor 1 inhibits biliary regeneration but accelerates the hepatic differentiation of progenitor cells in mice.

Although the regeneration of the adult liver depends on hepatic progenitor cells (HPCs), many uncertainties regarding hepatic regeneration in the injured liver remain. Trefoil factor family 1 (TFF1), a secretory protein predominantly expressed in the gastrointestinal tract, is responsible for mucosal restitution. Here, we investigated the role of TFF1 in liver regeneration using a mouse model of hepatic injury (choline-deficient ethionine-supplemented diet and carbon tetrachloride administration) and genetically engineered mice (TFF1 knockout (TFF1-/-)). Immunohistochemistry analysis of human liver samples revealed TFF1 expression in the hepatocytes close to ductular reaction and the regenerating biliary epithelium in injured liver. The number of cytokeratin 19 (CK19)-positive bile ducts was significantly decreased in the TFF1-/- mice after liver injury. Notch pathway in the TFF1-/- mice was also downregulated. HPCs in the control mice differentiated into biliary cells (CK19+/SRY HMG box 9 (SOX9)+) more frequently. In contrast, HPCs in the TFF1-/- mice more frequently differentiated into a hepatic lineage (alpha fetoprotein+/SOX9+) after acute liver damage. Hepatocyte proliferation was upregulated, and the liver weight was increased in TFF1-/- mice in response to chronic liver damage. Thus, TFF1 is responsible for liver regeneration after liver injury by promoting HPC differentiation into a biliary lineage and inhibiting HPC differentiation into a hepatic lineage.

A feasibility study of the toxic responses of human induced pluripotent stem cell-derived hepatocytes to phytochemicals.

Herbal medicines have been increasingly used in the last three decades. Despite their popularity, safety issues with herbal products need to be addressed. We performed a feasibility study of the toxic responses of human induced pluripotent stem cell-derived hepatocytes (iHep cells) to phytochemicals in comparison with hepatoblasoma-derived HepG2 cells and long-term human hepatocytes (LTHHs). The iHep cells expressed typical hepatocyte markers cytochrome P450 3A4 (CYP3A4), hepatocyte nuclear factor 4α, and albumin despite the expression of immature markers α-fetoprotein and cytokeratin 19. We studied the responses of iHep cells to phytochemicals saikosaponin D, triptolide, deoxycalyciphylline B, and monocrotaline with different mode of toxicity employing MTS and lactate dehydrogenase (LDH) assays. Saikosaponin D and triptolide caused dose-dependent cytotoxicity in the iHep cells, which were more sensitive than LTHHs and HepG2 cells. Saikosaponin D-induced cytotoxicity tightly correlated with increased LDH leakage in the iHep cells. Although deoxycalyciphylline B did not exhibit toxic effect on the iHep and HepG2 cells when compared with LTHHs, it decreased CYP3A7 expression in the iHep cells and increased CYP1A2 expression in HepG2 cells. We hereby show the feasibility of using iHep cells to detect toxic effects of phytochemicals.

Baicalein inhibits acinar-to-ductal metaplasia of pancreatic acinal cell AR42J via improving the inflammatory microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers. Recent research has demonstrated that chronic pancreatitis (CP) is associated with an increased risk of PDAC, partly due to acinar-to-ductal metaplasia (ADM). Baicalein has been shown to exert anti-inflammatory and anti-tumor effects for CP or PDAC, respectively. The aim of our study was to investigate the effect of baicalein, and the putative underlying mechanism, on inflammatory cytokines-induced ADM of rat pancreatic acinar cell line AR42J. To investigate ADM and baicalein effects in vitro, AR42J were treated with recombinant rat Tumor Necrosis Factor alpha (rTNFα) with or without baicalein for 5 days. Results showed that rTNFα-induced AR42J cells switched their phenotype from dominantly amylase-positive acinar cells to dominantly cytokeratin 19-positive ductal cells. Moreover, expression of the transcripts for TNFα or Hes-1, a Notch target, was up-regulated in these cells. Interestingly, baicalein reduced the population of ADM as well as cytokines gene expression but not Hes-1. Baicalein inhibited NF-κB activation induced by rTNFα in AR42J, but no effect on Notch 1activation. Moreover, baicalein suppressed the secretion of TNFα and Nitric Oxide (NO) in macrophages stimulated with LPS and further inhibited ADM of conditional medium-treated AR42J cells. Baicalein also suppressed the inflammatory response of LPS-activated macrophages, thereby inhibited ADM of AR42J by altering their microenvironment. Taken together, our study indicates that baicalein reduces rTNFα-induced ADM of AR42J cells by inhibiting NF-κB activation. It also sheds new light on Chinese material medica therapy of pancreatitis and thereby prevention of PDAC.

Gypenosides Ameliorate Carbon Tetrachloride-Induced Liver Fibrosis by Inhibiting the Differentiation of Hepatic Progenitor Cells into Myofibroblasts.

Gypenosides (GPs), the predominant components of Gynostemma pentaphyllum, exert antifibrotic effects; however, the mechanisms underlying their ability to ameliorate liver fibrosis are unclear. Liver fibrosis was induced in C57BL/6 mice via subcutaneous injection of 10% carbon tetrachloride (CCl[Formula: see text] three times a week for two weeks. Then, CCl4 was administered in conjunction with intragastric GPs for another three weeks. For in vitro analyses, WB-F344, hepatatic progenitor cells (HPCs) were treated with transforming growth factor beta 1 (TGF-[Formula: see text]1) with or without GPs for 48[Formula: see text]h. The results showed that alanine aminotransferase (ALT) and aspartate transaminase (AST) activity, deposition of collagen, hydroxyproline content, and expression of alpha-smooth muscle actin ([Formula: see text]-SMA) and collagen type I (Col I) were significantly decreased after treatment with GPs ([Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text]). In the 5M CCl4 group, the expression of HPC markers, Sox9 and cytokeratin 19 (CK19), was significantly increased compared with the normal or GPs-treated group ([Formula: see text], [Formula: see text]). Immunostaining showed that the number of Sox9 and [Formula: see text]-SMA double-positive cells was higher in the 5M CCl4 group than in the normal group, but the addition of GPs caused this cell number to decrease. In WB-F344 cells, the expression of [Formula: see text]-SMA and Col I was significantly increased after treatment with TGF-[Formula: see text], whereas in the GPs treatment group, expression was markedly decreased ([Formula: see text]). The levels of TGF-[Formula: see text] and TGF-[Formula: see text]R1 were markedly reduced after GPs treatment both in vivo and in vitro. In conclusion, GPs ameliorated CCl4-induced liver fibrosis via the inhibition of TGF-[Formula: see text] signaling, consequently inhibiting the differentiation of HPCs into myofibroblasts.

Coexistence of malignant struma ovarii and cervical papillary thyroid carcinoma.

CONTEXT: Struma ovarii is an uncommon monodermal teratoma in which thyroid tissue is the predominant element. Malignant transformation of struma ovarii is an even rarer occurrence. CASE PRESENTATION: We describe a 42-year-old woman who underwent a total abdominal hysterectomy and bilateral salpingo-oophorectomy for a symptomatic left pelvic mass. Histology revealed malignant struma ovarii with classical papillary thyroid carcinoma expression. Ultrasonography of the cervical neck showed thyroid micronodules and a dominant 1-cm nodule in the left thyroid lobe. As the ovarian tumor was large, the patient underwent a total thyroidectomy with the intention of administering ¹³¹I therapy in an adjuvant setting. Histology of the cervical thyroid gland revealed bilateral multifocal papillary thyroid carcinoma with extrathyroidal extension and perithyroidal lymph node metastasis. METHODS: Morphological (microscopy), immunohistochemical (Hector Battifora mesothelial cell 1, cytokeratin-19, galectin-3), and molecular (BRAF V600E, RAS, RET-PTC) characteristics and clonality analysis of the cervical thyroid and ovarian tumors were explored to distinguish them as separate malignancies. RESULTS: The thyroid-type tumors from the cervical gland and ovary were discordant in terms of tissue histology and level of cytokeratin-19 expression. The clinical features and tumor profile results supported the independent existence of these two embryologically related, although topographically distinct, malignancies. CONCLUSION: Our findings provided support for synchronous, albeit distinct, primary tumors in the ovary and cervical thyroid. "Field cancerization" and early genomic instability may explain multifocality in all thyroid-type tissue. In this regard, patients with malignant struma ovarii should undergo imaging of their thyroid gland for coexisting disease and thyroidectomy recommended for suspected malignancy or in preparation for radioiodine therapy.

Alteration in apoptosis and cell cycle by celecoxib and/or fish oil in 7,12-dimethyl benzene (α) anthracene-induced mammary carcinogenesis.

Cyclooxygenase (COX)-2 inhibition by nonsteroidal anti-inflammatory drugs is a useful approach for cancer prevention but has several side effects. A novel approach combining these chemopreventive agents at low doses with dietary elements has been suggested to augment their effects and reduce side effects. Dietary fats, particularly, n-3 polyunsaturated fatty acids (PUFA) also exert cancer chemopreventive effect mediated through COX-2 inhibition. Therefore, the present study was designed to investigate the effect of combined dosage of celecoxib and n-3 PUFA-rich fish oil in experimental mammary carcinogenesis. Female Wistar rats were distributed into control and DMBA-treated groups. The groups were further subdivided based on pretreatment with celecoxib and/or fish oil. The animals were maintained for 90 days before sacrifice. To analyze the role of redox signaling, the two mediators, reactive oxygen species and calcium, and their effects on c-myc expression were evaluated. The chemopreventive effect was assessed by measurement of cell proliferation, apoptosis, and p53 in isolated mammary epithelial cells. Increased redox signaling with enhanced c-myc, p53 expression, and augmented apoptotic and proliferative rate were observed in carcinogen-treated animals. Pretreatment of carcinogen-treated animals with celecoxib and/or fish oil altered redox signaling with reduced c-myc, p53 expression, apoptosis, and proliferation. However, a combination dosage of celecoxib and fish oil had a better chemopreventive effect. The results suggest that a combination of celecoxib and fish oil is more effective in the chemoprevention of experimental mammary carcinogenesis, and this effect can be attributed to the modification of redox signaling.

Expression of specificity protein transcription factors in pancreatic cancer and their association in prognosis and therapy.

Pancreatic cancer is an aggressive malignancy with poor prognosis. Pancreatic adenocarcinoma is one of the leading causes of cancer-related deaths in the United States. Due to the aggressive nature of this malignancy, there is a serious concern for identifying effective targets, and adopting novel strategies for therapy. Members of the Specificity Protein (Sp) family of transcription factors, Sp1, Sp3, and Sp4 regulate the expression of a number of genes associated with cancer cell proliferation, differentiation, and metastasis. Sp1 levels are upregulated in pancreatic cancer cell lines, and surgically resected human pancreatic adenocarcinoma. Sp1 overexpression in tumor tissues is associated with aggressive disease, poor prognosis and inversely correlated with survival. Sp1 is also known to affect angiogenesis by regulating the expression of vascular endothelial growth factor and its receptors. Results from clinical studies suggest Sp1 as new biomarker to identify aggressive pancreatic ductal adenocarcinoma. The pharmacological inhibition of Sp1 using agents such as celecoxib, mithramycin, curcumin, and tolfenamic acid has showed promising results in pre-clinical studies and demonstrated Sp transcription factors as potential targets for pancreatic cancer therapy. This review summarizes studies showing the association of Sp proteins with this malignancy, with a special emphasis on pre-clinical studies that tested strategies to target Sp transcription factors for inhibiting human pancreatic cancer cell proliferation and tumor growth in laboratory animals. The results showed remarkable efficacy and suggest that such approaches have the potential for high success in developing clinically relevant strategies for treating pancreatic cancer.

Atropa belladonna L. water extract: modulator of extracellular matrix formation in vitro and in vivo.

Previously, we found that treatment of cutaneous wounds with Atropa belladonna L. (AB) revealed shortened process of acute inflammation as well as increased tensile strength and collagen deposition in healing skin wounds (Gál et al. 2009). To better understand AB effect on skin wound healing male Sprague-Dawley rats were submitted to one round full thickness skin wound on the back. In two experimental groups two different concentrations of AB extract were daily applied whereas the control group remained untreated. For histological evaluation samples were removed on day 21 after surgery and stained for wide spectrum cytokeratin, collagen III, fibronectin, galectin-1, and vimentin. In addition, in the in vitro study different concentration of AB extract were used to evaluate differences in HaCaT keratinocytes proliferation and differentiation by detection of Ki67 and keratin-19 expressions. Furthermore, to assess ECM formation of human dermal fibroblasts on the in vitro level fibronectin and galectin-1 were visualized. Our study showed that AB induces fibronectin and galectin-1 rich ECM formation in vitro and in vivo. In addition, the proliferation of keratinocytes was also increased. In conclusion, AB is an effective modulator of skin wound healing. Nevertheless, further research is needed to find optimal therapeutic concentration and exact underlying mechanism of action.

Rescue of atypical protein kinase C in epithelia by the cytoskeleton and Hsp70 family chaperones.

Atypical PKC (PKC iota) is a key organizer of cellular asymmetry. Sequential extractions of intestinal cells showed a pool of enzymatically active PKC iota and the chaperone Hsp70.1 attached to the apical cytoskeleton. Pull-down experiments using purified and recombinant proteins showed a complex of Hsp70 and atypical PKC on filamentous keratins. Transgenic animals overexpressing keratin 8 displayed delocalization of Hsp70 and atypical PKC. Two different keratin-null mouse models, as well as keratin-8 knockdown cells in tissue culture, also showed redistribution of Hsp70 and a sharp decrease in the active form of atypical PKC, which was also reduced by Hsp70 knockdown. An in-vitro turn motif rephosphorylation assay indicated that PKC iota is dephosphorylated by prolonged activity. The Triton-soluble fraction could rephosphorylate PKC iota only when supplemented with the cytoskeletal pellet or filamentous highly purified keratins, a function abolished by immunodepletion of Hsp70 but rescued by recombinant Hsp70. We conclude that both filamentous keratins and Hsp70 are required for the rescue rephosphorylation of mature atypical PKC, regulating the subcellular distribution and steady-state levels of active PKC iota.

[Effect of salvianolic acid B on generation and activation of myofibroblast in rat with renal interstitial fibrosis].

OBJECTIVE: To investigate the effect of salvianolic acid B (SA-B) on renal interstitial fibrosis due to unilateral ureteral obstruction. METHOD: Thirty-six SD male rats were randomly divided into 3 groups, 12 in each group, the sham-operated group, the model group and the SA-B treated group. The rat model of renal interstitial fibrosis was successfully established by unilateral ureteral obstruction (UUO). Rats in the SA-B treated group was intragastrically administrated with SA-B (12.5 mg x kg(-1)) daily after modeling. Rats of each group were killed respectively at day 14 and day 21 after UUO. Pathological changes of renal tissue were observed by hematoxylin and eosin (HE) staining. The expression of alpha-smooth muscle actin (alpha-SMA) in kidney was determined with immunohistochemistry. And the expressions of cytokeratinl9 (ck19) mRNA in renal tissue were detected using reverse transcription polymerase chain reaction (RT-PCR). RESULT: Renal interstitial fibrosis was obviously ameliorate in SA-B treated group. The expression of alpha-SMA was significantly decreased in SA-B treated group as compared with that in model group at day 14. And the expression of ck19 was significantly lower than that determined in model group at day 21. CONCLUSION: SA-B could ameliorate renal interstial fibrosis due to UUO, probable by inhibiting epithelial-to-myofibroblast transdifferentiation and the activation of myofibroblast.

Foxl1 is a marker of bipotential hepatic progenitor cells in mice.

UNLABELLED: The liver contains a population of small bipotential facultative progenitor cells that reconstitute liver function when mature hepatocytes or cholangiocytes are unable to proliferate. Mesenchymal markers, including members of the forkhead transcription factor gene family, have been detected in hepatic progenitor cells. The winged helix transcription factor Foxl1 localizes to mesenchymal cells in the intestine; however, its expression in the liver has not been reported. We found that Foxl1 is expressed in rare cells in the normal liver but is dramatically induced in the livers of mice that have undergone bile duct ligation or were fed a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing or choline-deficient, ethionine-supplemented diet. In addition, we employed genetic lineage tracing using a Foxl1-Cre transgenic mouse crossed with the Rosa26R lacZ reporter line to demonstrate that Foxl1-Cre-expressing cells are present within the periportal region shortly after injury. These cells give rise to both hepatocytes [marked by hepatocyte nuclear factor 4 alpha (HNF-4alpha) expression] and cholangiocytes (marked by CK19 expression), indicating that these cells are derived from Foxl1-Cre-expressing cells. Foxl1-Cre-expressing cells are distinct from hepatic stellate cells, portal fibroblasts, and myofibroblasts, although they are located in close proximity to portal fibroblasts. These results demonstrate that the early Foxl1-Cre lineage cell gives rise to both cholangiocytes and hepatocytes after liver injury and suggest the potential for progenitor-portal fibroblast cell interactions. CONCLUSION: We propose that Foxl1 is a bona fide marker of the facultative progenitor cell in the mouse liver.