Tohru Adachi

NAD(P)H oxidase plays a crucial role in PDGF-induced proliferation of hepatic stellate cells.

The proliferation of hepatic stellate cells (HSCs) is a critical step in hepatic fibrogenesis. Platelet‐derived growth factor (PDGF) is the most potent mitogen for HSCs. We investigated the role of nonphagocytic NAD(P)H oxidase–derived reactive oxygen species (ROS) in PDGF‐induced HSC proliferation. The human HSC line, LI‐90 cells, murine primary‐cultured HSCs, and PDGF‐BB were used in this study. We examined the mechanism of PDGF‐BB‐induced HSC proliferation in relation to the role of a ROS scavenger and diphenylene iodonium, an inhibitor of NAD(P)H oxidase. We also measured ROS production with the aid of chemiluminescence. We showed that PDGF‐BB induced proliferation of HSCs through the intracellular production of ROS. We also demonstrated that HSCs expressed key components of nonphagocytic NAD(P)H oxidase (p22phox, gp91phox, p47phox, and p67phox) at both the messenger RNA and protein levels. Diphenylene iodonium suppressed PDGF‐BB–induced ROS production and HSC proliferation. Coincubation of H2O2 and PDGF‐BB restored the proliferation of HSCs that was inhibited by diphenylene iodonium pretreatment. Phosphorylation of the mitogen‐activated protein kinase (MAPK) family constitutes a signal transduction pathway of cell proliferation. Our data demonstrate that NAD(P)H oxidase–derived ROS induce HSC proliferation mainly through the phosphorylation of p38 MAPK. Moreover, an in vivo hepatic fibrosis model also supported the critical role of NAD(P)H oxidase in the activation and proliferation of HSCs. In conclusion, NAD(P)H oxidase is expressed in HSCs and produces ROS via activation of NAD(P)H oxidase in response to PDGF‐BB. ROS further induce HSC proliferation through the phosphorylation of p38 MAPK. (HEPATOLOGY 2005;41:1272–1281.)

Differentiation of bone marrow cells into cells that express liver-specific genes in vitro: implication of the Notch signals in differentiation

Bone marrow (BM) stem cells have been shown to differentiate into liver cells. It remains difficult to sort and culture BM stem cells, and the gene expression of liver-specific proteins in these cells has not been fully investigated. We used a negative selective magnetic cell separation system to obtain stem cell-enriched BM cells. The cells obtained were cultured with hepatocytes or with hepatocyte growth factor (HGF), and the differentiation of BM cells into cells expressing liver-specific genes, hepatocyte nuclear factor (HNF) 1alpha, cytokeratin (CK) 8, alpha-fetoprotein (AFP), and albumin was investigated by the reverse transcription-polymerase chain reaction. We also investigated the gene expressions of Notch receptor-1 (Notch-1) and its ligand Jagged-1 in BM cell differentiation. Sorted BM cells showed positive for Sca-1 (Ataxin-1) by immunofluorescence staining. Fluorescence activated cell sorter analysis showed that 32.6% of sorted BM cells had a high level of expression of the hematopoietic stem cell marker CD90 (Thy-1). When cultured with hepatocytes, these cells expressed the liver-specific genes HNF1alpha and CK8 on culture day 3, AFP and albumin on culture day 7. When cultured with HGF (20ng/ml), the cells expressed HNF1alpha on day 3 and CK8 on day 7. Gene expressions of Notch-1 and Jagged-1 were detected in cultured BM cells on day 3. These results suggest that the negative selective magnetic cell separation system is useful for the rapid preparation of stem cell-enriched BM cells, and that the Notch signaling pathway plays a role in BM cell differentiation into a hepatocyte lineage in vitro.

Mucosal sulfhydryl compounds evaluation by in vivo electron spin resonance spectroscopy in mice with experimental colitis.

Background: Sulfhydryl (SH) compounds are essential in maintaining mucosal integrity in the gastrointestinal tract. A decrease in colonic mucosal SH compounds affects the redox status of the mucosa, resulting in vulnerability to further attacks. Therefore, there is a strong need for in vivo evaluation of SH compounds in the colonic mucosa. Aims: The aim of the current study was to establish a method of evaluating levels of SH compounds in the colonic mucosa of live animals before and after induction of colitis. Methods: Murine experimental colitis was induced by instillation of trinitrobenzene sulphonic acid (TNBS) dissolved in 50% ethanol into the colon via the anus. For evaluation of mucosal SH compounds in the colon, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL), a stable nitroxide radical, was instilled into the colonic lumen of live mice and the spin clearance rate was measured by L-band electron spin resonance (ESR) spectroscopy. Results: Morphological study showed that mucosal damage was severe one or two days after TNBS instillation. The colonic mucosa started to regenerate at four days, and looked normal at seven days, after induction of colitis. The spin clearance rate of carbamoyl-PROXYL decreased significantly at 0.5, 1, 2, and 4 days after induction of colitis compared with mice before TNBS instillation. Surprisingly, although the colonic mucosa looked normal seven days after TNBS administration, the spin clearance rate still remained significantly slow. The spin clearance rate returned to normal 14 days after induction of colitis. The change in in vivo spin clearance rate was consistent with the time dependent change in mucosal reduced glutathione, a major component of SH compounds. Conclusion: The spin clearance rate obtained by L-band ESR spectroscopy in combination with carbamoyl-PROXYL can give an estimate of the level of colonic mucosal SH compounds in live animals and is useful for evaluating the mucosal defence system against oxidative stress.