Clare Selden

Human hepatocyte cell lines proliferating as cohesive spheroid colonies in alginate markedly upregulate both synthetic and detoxificatory liver function

BACKGROUND/AIMS Bio-artificial liver support systems for treatment of hepatic failure require maintained expression of hepatocyte function in vitro. We studied cultures of human hepatocyte cell-lines proliferating within alginate beads, investigating the hypothesis that 3-dimensional cohesive colonies of hepatocyte cell-lines would achieve polarity and cell-to-cell contact resulting in upregulation of function. METHODS HepG2 and HHY41 human cell lines in alginate beads were cultured for >20 days. RESULTS Proliferation was maintained for 20 days. Production of albumin, prothrombin, fibrinogen, alpha-1-acid glycoprotein and alpha-1-antitrypsin was maintained throughout, maximal at days 8-10, when upregulation was 300-1100% compared with monolayer cultures at similar cell number per unit volume. Detoxificatory functions: ethoxyresorufin deethylase activity, androstenedione metabolism, and urea synthesis from arginine was also increased several-fold. Function returned to pre-freezing levels within 18 h of thawing after cryopreservation of cells in alginate. Electron microscopy revealed spherical colonies of cells of cuboidal shape, with cell-to-cell contact via desmosomes and junctional complexes, abundant microvilli, and cytoplasmic appearances suggesting transcriptionally active hepatocytes. CONCLUSION Hepatocyte cell-lines, proliferating in alginate express a range of liver-specific functions at levels approaching those found in vivo, relevant to their use in liver support systems.

The role of non-parenchymal cells in liver growth

The main non-parenchymal cells of the liver, Kupffer cells, sinusoidal endothelial cells and stellate cells, participate in liver growth with respect to both their own proliferation, and effects on hepatocyte proliferation. In the well-characterised paradigm of 70% partial hepatectomy, they undergo DNA synthesis and cell division 20-24h later than the hepatocyte population. They exert both positive and negative influences on hepatocyte proliferation, including provision of an extracellular matrix-bound reservoir of hepatocyte growth factor that is activated after damage; priming of hepatocytes for DNA synthesis through rapid generation of TNF-alpha and IL-6; and generation of factors at later time points that curb hepatocyte DNA synthesis (IL-1, TGF-beta) and initiate reconstruction and reformation of matrix proteins.

Increased Sensitivity of Histidinemic Mice to UVB Radiation Suggests a Crucial Role of Endogenous Urocanic Acid in Photoprotection

Urocanic acid (UCA) is produced by the enzyme histidase and accumulates in the stratum corneum of the epidermis. In this study, we investigated the photoprotective role of endogenous UCA in the murine skin using histidinemic mice, in which the gene encoding histidase is mutated. Histidase was detected by immunohistochemistry in the stratum granulosum and stratum corneum of the normal murine skin but not in the histidinemic skin. The UCA content of the stratum corneum and the UVB absorption capacity of aqueous extracts from the stratum corneum were significantly reduced in histidinemic mice as compared with wild-type mice. When the shaved back skin of adult mice was irradiated with 250 mJ cm(-2) UVB, histidinemic mice accumulated significantly more DNA damage in the form of cyclobutane pyrimidine dimers than did wild-type mice. Furthermore, UVB irradiation induced significantly higher levels of markers of apoptosis in the epidermis of histidinemic mice. Topical application of UCA reversed the UVB-photosensitive phenotype of histidinemic mice and increased UVB photoprotection of wild-type mice. Taken together, these results provide strong evidence for an important contribution of endogenous UCA to the protection of the epidermis against the damaging effects of UVB radiation.

Intrahepatic cholestasis of pregnancy levels of sulfated progesterone metabolites inhibit farnesoid X receptor resulting in a cholestatic phenotype

Intrahepatic cholestasis of pregnancy (ICP) is the most prevalent pregnancy‐specific liver disease and is associated with an increased risk of adverse fetal outcomes, including preterm labor and intrauterine death. The endocrine signals that cause cholestasis are not known but 3α‐sulfated progesterone metabolites have been shown to be elevated in ICP, leading us to study the impact of sulfated progesterone metabolites on farnesoid X receptor (FXR)‐mediated bile acid homeostasis pathways. Here we report that the 3β‐sulfated progesterone metabolite epiallopregnanolone sulfate is supraphysiologically raised in the serum of ICP patients. Mice challenged with cholic acid developed hypercholanemia and a hepatic gene expression profile indicative of FXR activation. However, coadministration of epiallopregnanolone sulfate with cholic acid exacerbated the hypercholanemia and resulted in aberrant gene expression profiles for hepatic bile acid‐responsive genes consistent with cholestasis. We demonstrate that levels of epiallopregnanolone sulfate found in ICP can function as a partial agonist for FXR, resulting in the aberrant expression of bile acid homeostasis genes in hepatoma cell lines and primary human hepatocytes. Furthermore, epiallopregnanolone sulfate inhibition of FXR results in reduced FXR‐mediated bile acid efflux and secreted FGF19. Using cofactor recruitment assays, we show that epiallopregnanolone sulfate competitively inhibits bile acid‐mediated recruitment of cofactor motifs to the FXR‐ligand binding domain. Conclusion: Our results reveal a novel molecular interaction between ICP‐associated levels of the 3β‐sulfated progesterone metabolite epiallopregnanolone sulfate and FXR that couples the endocrine component of pregnancy in ICP to abnormal bile acid homeostasis. (HEPATOLOGY 2013;)

Hepatotropin mRNA expression in human foetal liver development and in liver regeneration.

A 569 bp probe against the β‐chain of hepatotropin was used to examine expression of RNA for this growth factor in human adult and foetal liver, foetal kidney and pancreas, and rat liver after partial hepatectomy. Low level expression of a 6kb RNA occurred in human adult and normal rat liver. 70% hepatectomy increased expression, peaking at 10 h and returning to near normal levels 24 h after resection. The 6 kb band was strongly expressed in human foetal liver, as compared with adult, but not in foetal kidney or pancreas, suggesting a major role for hepatotropin in both foetal development and regeneration of the liver.

Alginate-encapsulated HepG2 Cells in a Fluidized Bed Bioreactor Maintain Function in Human Liver Failure Plasma

Alginate-encapsulated HepG2 cells cultured in microgravity have the potential to serve as the cellular component of a bioartificial liver. This study investigates their performance in normal and liver failure (LF) human plasma over 6-8 h in a fluidized bed bioreactor. After 8 days of microgravity culture, beads containing 1.5 x 10(9) cells were perfused for up to 8 h at 48 mL/min with 300 mL of plasma. After exposure to 90% LF plasma, vital dye staining showed maintained cell viability, while a 7% increase in lactate dehydrogenase activity indicated minimal cell damage. Glucose consumption, lactate production, and a 4.3-fold linear increase in alpha-fetoprotein levels were observed. Detoxificatory function was demonstrated by quantification of bilirubin conjugation, urea synthesis, and Cyp450 1A activity. These data show that in LF plasma, alginate-encapsulated HepG2 cells can maintain viability, and metabolic, synthetic, and detoxificatory activities, indicating that the system can be scaled-up to form the biological component of a bioartificial liver.

Is the Filaggrin–Histidine–Urocanic Acid Pathway Essential for Stratum Corneum Acidification?

TO THE EDITOR Acidification of the surface of the stratum corneum (SC), the acid mantle, was initially thought to be important in the defense against infection. The growth of pathogenic microorganisms, such as Staphylococcus aureus and Streptococcus pyogenes, is inhibited by an acidic skin pH whereas the growth of resident (normal) skin flora is stimulated (Puhvel et al., 1975; Korting et al., 1990, 1992). However, recent studies have shown that acidification of the SC has additional functions, including regulating several key SC functions. A major function of the skin is to form a permeability barrier between the dry external environment and the moist interior of the body (Elias, 2007). This permeability barrier resides in the extracellular lipid membranes of the SC, and studies have shown that an acidified SC is required for the formation of a functionally competent permeability barrier (Mauro et al., 1998; Fluhr et al., 2001; Hachem et al., 2003). Specifically, in the SC b-glucocerebrosidase and acid sphingomyelinase metabolize glucosylceramides and sphingomyelin, respectively, to ceramides, which is the major family of lipids in the extracellular membranes that mediate permeability barrier function (Feingold, 2007). Both the enzymes require an acidic milieu for optimal enzymatic activity; hence, when the pH of the SC increases, the metabolism of glucosylceramides and sphingomyelin to ceramides is impaired, resulting in abnormal permeability barrier homeostasis (Holleran et al., 1992, 1993; Feingold, 2007). In addition, an acidic SC pH inhibits the activity of serine proteases thereby maintaining the cohesiveness and integrity of the SC (Hachem et al., 2005). With an increase in SC pH, the activities of these serine proteases are stimulated resulting in the degradation of corneodesmosomes and a decrease in SC integrity and cohesion (Fluhr et al., 2004b; Hachem et al., 2005). Thus, an acidic SC is important in regulating the metabolism and function of the SC, and alterations in SC pH could have numerous adverse effects. A variety of different pathways are postulated to contribute to the acid mantle Abbreviation: SC, stratum corneum

Inhibition of Na+-Taurocholate Co-transporting Polypeptide-mediated Bile Acid Transport by Cholestatic Sulfated Progesterone Metabolites

Sulfated progesterone metabolite (P4-S) levels are raised in normal pregnancy and elevated further in intrahepatic cholestasis of pregnancy (ICP), a bile acid-liver disorder of pregnancy. ICP can be complicated by preterm labor and intrauterine death. The impact of P4-S on bile acid uptake was studied using two experimental models of hepatic uptake of bile acids, namely cultured primary human hepatocytes (PHH) and Na+-taurocholate co-transporting polypeptide (NTCP)-expressing Xenopus laevis oocytes. Two P4-S compounds, allopregnanolone-sulfate (PM4-S) and epiallopregnanolone-sulfate (PM5-S), reduced [3H]taurocholate (TC) uptake in a dose-dependent manner in PHH, with both Na+-dependent and -independent bile acid uptake systems significantly inhibited. PM5-S-mediated inhibition of TC uptake could be reversed by increasing the TC concentration against a fixed PM5-S dose indicating competitive inhibition. Experiments using NTCP-expressing Xenopus oocytes confirmed that PM4-S/PM5-S are capable of competitively inhibiting NTCP-mediated uptake of [3H]TC. Total serum PM4-S + PM5-S levels were measured in non-pregnant and third trimester pregnant women using liquid chromatography-electrospray tandem mass spectrometry and were increased in pregnant women, at levels capable of inhibiting TC uptake. In conclusion, pregnancy levels of P4-S can inhibit Na+-dependent and -independent influx of taurocholate in PHH and cause competitive inhibition of NTCP-mediated uptake of taurocholate in Xenopus oocytes.

Augmentation of the early phase of liver regeneration after 70% partial hepatectomy in rats following selective Kupffer cell depletion

BACKGROUND/AIMS Kupffer cells are located in the liver sinusoids adjacent to hepatocytes and elaborate a range of growth regulatory molecules involved in regulating hepatocyte proliferation. In vitro observations imply the potential for Kupffer cells to exert both stimulatory and inhibitory influences on hepatocyte DNA synthesis. We aimed to determine the overall effect of Kupffer cell activity during the early regenerative processes after partial hepatectomy. METHODS We investigated hepatocyte DNA synthesis, induced by partial hepatectomy in rats, following selective elimination of Kupffer cells by liposome encapsulated dichlormethylene bisphosphonate (Cl2MBP). RESULTS We demonstrate that the early phase of liver regeneration was enhanced following Kupffer depletion, as indicated by a greater proportion of hepatocytes undergoing DNA synthesis, and a higher mitotic index. This was associated with an alteration in the balance of growth factors in the liver; HGF and TGFbeta mRNA were reduced in Kupffer cell-depleted animals, and IL-1beta mRNA was absent. In addition, in the absence of partial hepatectomy, the selective depletion of Kupffer cells leads to an increase in the proliferation of hepatocytes in resting liver undergoing DNA synthesis. CONCLUSION The overall effect of depleting the liver of Kupffer cells is to enhance the proliferation rate of hepatocytes, both after partial hepatectomy and in the resting state.

Hepatocyte progenitors in man and in rodents - multiple pathways, multiple candidates

In severe injury, liver‐cell progenitors may play a role in recovery, proliferating, and subsequently differentiating into mature liver cells. Identifying these progenitors has major therapeutic potential for ex vivo pharmaceutical testing, bioartificial liver support, tissue engineering and gene therapy protocols. Potential liver‐cell progenitors have been identified from bone marrow, peripheral blood, cord blood, foetal liver, adult liver and embryonic stem cells. Differences and similarities are found among cells isolated from rodents and humans. This review will discuss identifying markers and differentiation potential in in vitro and in vivo models of these putative progenitors in both humans and rodents.