Yin-Xiong Li

Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis

In the early stages of nonalcoholic fatty liver disease (NAFLD), triglycerides accumulate in hepatocytes. Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in hepatocyte triglyceride biosynthesis. DGAT2 antisense oligonucleotide (ASO) treatment improved hepatic steatosis dramatically in a previous study of obese mice. According to the 2‐hit hypothesis for progression of NAFLD, hepatic steatosis is a risk factor for nonalcoholic steatohepatitis (NASH) and fibrosis. To evaluate this hypothesis, we inhibited DGAT2 in a mouse model of NASH induced by a diet deficient in methionine and choline (MCD). Six‐week‐old genetically obese and diabetic male db/db mice were fed either the control or the MCD diet for 4 or 8 weeks. The MCD diet group was treated with either 25 mg/kg DGAT2 ASO or saline intraperitoneally twice weekly. Hepatic steatosis, injury, fibrosis, markers of lipid peroxidation/oxidant stress, and systemic insulin sensitivity were evaluated. Hepatic steatosis, necroinflammation, and fibrosis were increased in saline‐treated MCD diet–fed mice compared to controls. Treating MCD diet–fed mice with DGAT2 ASO for 4 and 8 weeks decreased hepatic steatosis, but increased hepatic free fatty acids, cytochrome P4502E1, markers of lipid peroxidation/oxidant stress, lobular necroinflammation, and fibrosis. Progression of liver damage occurred despite reduced hepatic expression of tumor necrosis factor alpha, increased serum adiponectin, and striking improvement in systemic insulin sensitivity. Conclusion: Results from this mouse model would suggest accumulation of triglycerides may be a protective mechanism to prevent progressive liver damage in NAFLD. (HEPATOLOGY 2007.)

Role for Hedgehog signaling in hepatic stellate cell activation and viability

Hepatic stellate cells (HSC) have a complex phenotype that includes both neural and myofibroblastic features. The Hedgehog (Hh) pathway has been shown to direct the fate of neural and myofibroblastic cells during embryogenesis and during tissue remodeling in adults. Therefore, we hypothesized that Hh signaling may regulate the fate of HSC in adults. In this study, we find that freshly isolated stellate cells from adult Patched-lacZ transgenic mice exhibit β-galactosidase activity, indicating Hh pathway activity. Transcripts of Hh ligands, the Hh pathway receptor, and Hh-regulated transcription factors are expressed by stellate cells from mice, rats, and humans. Transfection experiments in a cell line using a Hh-inducible luciferase reporter demonstrate constitutive Hh pathway activity. Moreover, neutralizing antibodies to Hh increase apoptosis, while viability is restored by treatment with Hh ligand. In vitro treatment of primary stellate cells with cyclopamine (Cyc), a pharmacologic inhibitor of the Hh pathway, inhibits activation and slightly decreases cell survival, while a single injection of Cyc into healthy adult mice reduces activation of HSC by more than 50% without producing obvious liver damage. Our findings reveal a novel mechanism, namely the Hh pathway, that regulates the activation and viability of HSC.

Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation

In bile duct-ligated (BDL) rodents, as in humans with chronic cholangiopathies, biliary obstruction triggers proliferation of bile ductular cells that are surrounded by fibrosis produced by adjacent myofibroblastic cells in the hepatic mesenchyme. The proximity of the myofibroblasts and cholangiocytes suggests that mesenchymal–epithelial crosstalk promotes the fibroproliferative response to cholestatic liver injury. Studying BDL mice, we found that bile duct obstruction induces activity of the Hedgehog (Hh) pathway, a system that regulates the viability and differentiation of various progenitors during embryogenesis. After BDL, many bile ductular cells and fibroblastic-appearing cells in the portal stroma express Hh ligands, receptor and/or target genes. Transwell cocultures of an immature cholangiocyte line that expresses the Hh receptor, Patched (Ptc), with liver myofibroblastic cells demonstrated that both cell types produced Hh ligands that enhanced each others viability and proliferation. Further support for the concept that Hh signaling modulates the response to BDL was generated by studying PtcLacZ mice, which have an impaired ability to constrain Hh signaling due to a heterozygous deficiency of Ptc. After BDL, PtcLacZ mice upregulated fibrosis gene expression earlier than wild-type controls and manifested an unusually intense ductular reaction, more expanded fibrotic portal areas, and a greater number of lobular necrotic foci. Our findings reveal that adult livers resurrect developmental signaling systems, such as the Hh pathway, to guide remodeling of the biliary epithelia and stroma after cholestatic injury.

Fetal alcohol exposure impairs hedgehog cholesterol modification and signaling

Consumption of alcohol by pregnant women can cause fetal alcohol spectrum defects (FASD), a congenital disease, which is characterized by an array of developmental defects that include neurological, craniofacial, cardiac, and limb malformations, as well as generalized growth retardation. FASD remains a significant clinical challenge and an important social problem. Although there has been great progress in delineating the mechanisms contributing to alcohol-induced birth defects, gaps in our knowledge still remain; for instance, why does alcohol preferentially induce a spectrum of defects in specific organs and why is the spectrum of defects reproducible and predictable. In this study, we show that exposure of zebrafish embryos to low levels of alcohol during gastrulation blocks covalent modification of Sonic hedgehog by cholesterol. This leads to impaired Hh signal transduction and results in a dose-dependent spectrum of permanent developmental defects that closely resemble FASD. Furthermore, supplementing alcohol-exposed embryos with cholesterol rescues the loss of Shh signal transduction, and prevents embryos from developing FASD-like morphologic defects. Overall, we have shown that a simple post-translational modification defect in a key morphogen may contribute to an environmentally induced complex congenital syndrome. This insight into FASD pathogenesis may suggest novel strategies for preventing these common congenital defects.

Bile ductules and stromal cells express hedgehog ligands and/or hedgehog target genes in primary biliary cirrhosis

Indian Hedgehog (Ihh) regulates tissue morphogenesis. Hedgehog (Hh) activity has been demonstrated in human cholangiocarcinoma and hepatocellular carcinoma lines, and in myofibroblasts and progenitors from adult rodent livers. We evaluated Hh pathway involvement in the response to biliary injury in primary biliary cirrhosis (PBC). Liver sections from 3 PBC patients and 3 controls without liver disease were studied. Immunohistochemistry was used to determine if cells that accumulate in PBC livers express Ihh or Hh‐target genes including the Hh‐receptor, Patched (Ptc), and the Hh‐transcriptional activator glioblastoma (Gli) 2. Positive cells were further identified by costaining for cytokeratin (CK) 19, a biliary marker, or OV6, a hepatic progenitor marker. In all subjects, Gli2 and Ptc expression localized in portal areas. The numbers of Gli2‐ or Ptc‐expressing cells/portal triad were each 10‐fold greater in patients with PBC than in controls (P < 0.05). In PBC livers, some CK19(+) cells coexpressed Gli2 or Ptc. Many stromal fibroblastic cells were also Gli2(+). Strong Ihh expression was detected in most bile ductular cells. Scattered stromal cells also expressed Ihh. The number of Ihh(+) cells/portal triad was 6‐fold greater in PBC livers than controls (P < 0.05). OV6(+) progenitors increased significantly in PBC livers, and some of these cells coexpressed Ihh, Ptc, and/or Gli2. Conclusion: This is the first direct evidence that noncancerous, adult human livers harbor several types of cells that produce and/or respond to Hh ligands. Such Hh‐responsive cells accumulate during the fibroproliferative response to chronic cholestatic liver injury, suggesting a role for Hh signaling in this process. (HEPATOLOGY 2007;45:1091–1096.)

Cardiac neural crest in zebrafish embryos contributes to myocardial cell lineage and early heart function

Myocardial dysfunction is evident within hours after ablation of the cardiac neural crest in chick embryos, suggesting a role for neural crest in myocardial maturation that is separate from its role in outflow septation. This role could be conserved in an animal that does not have a divided systemic and pulmonary circulation, such as zebrafish. To test this hypothesis, we used cell marking to identify the axial level of neural crest that migrates to the heart in zebrafish embryos. Unlike the chick and mouse, the zebrafish cardiac neural crest does not originate from the axial level of the somites. The region of neural crest cranial to somite 1 was found to contribute cells to the heart. Cells from the cardiac neural crest migrated to the myocardial wall of the heart tube, where some of them expressed a myocardial phenotype. Laser ablation of the cardiac premigratory neural crest at the three‐ to four‐somite stage resulted in loss of the neural crest cells migrating to the heart as shown by the absence of AP2‐ and HNK1‐expressing cells and failure of the heart tube to undergo looping. Myocardial function was assessed 24 hr after the cardiac neural crest ablation in a subpopulation of embryos with normal heart rate. Decreased stroke volume, ejection fraction, and cardiac output were observed, indicating a more severe functional deficit in cardiac neural crest‐ablated zebrafish embryos compared with neural crest–ablated chick embryos. These results suggest a new role for cardiac neural crest cells in vertebrate cardiac development and are the first report of a myocardial cell lineage for neural crest derivatives. Developmental Dynamics 226:000–000, 2003.

Diacylglycerol acyltranferase 1 anti‐sense oligonucleotides reduce hepatic fibrosis in mice with nonalcoholic steatohepatitis

Retinyl ester (RE) stores decrease during hepatic stellate cell (HSC) activation and liver fibrosis. Although retinol esterification is mostly catalyzed by lecithin:retinol acyltransferase (LRAT), diacylglycerol acyltransferase (DGAT)1 also does this. In previous reports, LRAT−/− mice had reduced hepatic RE but neither excessive HSC activation nor liver fibrosis, and DGAT1−/− mice had increased liver levels of RE and retinol. We sought to clarify the role of DGAT1 in liver fibrosis. Expression of DGAT1/2 was compared by real time PCR in freshly isolated, primary mouse HSCs and hepatocytes. To induce nonalcoholic steatohepatitis (NASH) and liver fibrosis, adult male db/db mice were fed methionine choline–deficient (MCD) diets. Half were treated with DGAT1 antisense oligonucleotide (ASO); the rest were injected with saline. Results were compared with chow‐fed controls. Inhibition of DGAT1 in liver had no effect on hepatic triglyceride content or liver necroinflammation but reduced HSC activation and liver fibrosis in mice with NASH. To evaluate the role of DGAT1 in HSC activation, HSC were isolated from healthy rats treated with DGAT1 ASO or saline. DGAT1 was expressed at relatively high levels in HSCs. HSC isolated from DGAT1 ASO‐treated rats had reduced DGAT1 expression and increased messenger RNA (mRNA) levels of LRAT and cellular retinol binding protein‐1. During culture, they retained more vitamin A, had repressed collagen a2 (I) transcriptional activity, and expressed less collagen a1 (I) and a2 (I) mRNA. Conclusion: DGAT1 may be a therapeutic target in NASH because inhibiting DGAT1 favorably altered. HSC retinoid homeostasis and inhibited hepatic fibrosis in mice with NASH. (HEPATOLOGY 2007.)

Sustained activation of Rac1 in hepatic stellate cells promotes liver injury and fibrosis in mice

Rac, a small, GTP‐binding protein in the Rho family, regulates several cellular functions, including the activation of NADPH oxidase, a major intracellular producer of reactive oxygen species (ROS). Hepatic stellate cells (HSCs) isolated from mice that are genetically deficient in NADPH oxidase produce less ROS, and their activation during chronic liver injury is abrogated, resulting in decreased liver fibrosis. Therefore, we hypothesized that HSC ROS production and activation would be enhanced, and fibrosis worsened, by increasing Rac expression in HSCs. To achieve this, we used transgenic mice that express constitutively active human Rac1 under the control of the α‐smooth muscle actin (α‐sma) promoter, because α‐sma expression is induced spontaneously during HSC activation. Transgene expression was upregulated progressively during culture of primary Rac‐transgenic HSCs, and this increased HSC ROS production as well as expression of activation markers and collagen. Similarly, Rac mice treated with carbon tetrachloride (CCl4) accumulated greater numbers of activated HSCs and had more liver damage, hepatocyte apoptosis, and liver fibrosis—as well as higher mortality—than CCl4‐treated wild‐type mice. In conclusion, sustained activation of Rac in HSCs perpetuates their activation and exacerbates toxin‐induced liver injury and fibrosis, prompting speculation that Rac may be a therapeutic target in patients with cirrhosis. (HEPATOLOGY 2006;44:1267–1277.)

Hensen’s node gives rise to the ventral midline of the foregut: implications for organizing head and heart development

Patterning of the ventral head has been attributed to various cell populations, including endoderm, mesoderm, and neural crest. Here, we provide evidence that head and heart development may be influenced by a ventral midline endodermal cell population. We show that the ventral midline endoderm of the foregut is generated directly from the extreme rostral portion of Hensens node, the avian equivalent of the Spemann organizer. The endodermal cells extend caudally in the ventral midline from the prechordal plate during development of the foregut pocket. Thus, the prechordal plate appears as a mesendodermal pivot between the notochord and the ventral foregut midline. The elongating ventral midline endoderm delimits the right and left sides of the ventral foregut endoderm. Cells derived from the midline endoderm are incorporated into the endocardium and myocardium during closure of the foregut pocket and fusion of the bilateral heart primordia. Bilateral ablation of the endoderm flanking the midline at the level of the anterior intestinal portal leads to randomization of heart looping, suggesting that this endoderm is partitioned into right and left domains by the midline endoderm, thus performing a function similar to that of the notochord in maintaining left-right asymmetry. Because of its derivation from the dorsal organizer, its extent from the forebrain through the midline of the developing face and pharynx, and its participation in formation of a single midline heart tube, we propose that the ventral midline endoderm is ideally situated to function as a ventral organizer of the head and heart.

A Novel Role for Cardiac Neural Crest in Heart Development

It is well known that cardiac neural crest participates in development of the cardiac outflow septation and patterning of the great arteries. Less well known is that ablation of the cardiac neural crest leads to a primary myocardial dysfunction. Recent data suggests that the myocardial dysfunction occurs because of the absence of an interaction of neural crest and pharyngeal endoderm to alter signaling from the endoderm. Continuation of an FGF-like signal from the endoderm past a precise time in development appears to be detrimental to myocardial maturation.