35. HNF-6 Mediates Growth Hormone Effect on Hepatocyte Function During Cholestasis in Mice

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 191 time from PD to liver-directed therapy of 57 days. The majority (80.3%) of pts underwent a single course of liver-directed therapy, however 20 (30.3%) pts had ⱖ 2 courses. The overall morbidity rate that was attributable to liver-directed therapy was 24%. Post-procedure complications included liver abscess (n⫽12), systemic sepsis (n⫽10), liver insufficiency (n⫽2), bleeding (n⫽2), renal failure (n⫽2), portal vein thrombosis (n⫽1), and radiation hepatitis (n⫽1). Of those pts who developed a liver abscess, 50% (6 out 12) had ⱖ 2 courses of liver-directed therapy. Post-procedure morbidity was also associated with the timing of the liver-directed therapy (simultaneous PD ⫹ liver-directed therapy, 16% vs PD followed by staged liver-directed therapy, 62%; p⫽0.004). One pt died within 30-days of simultaneous PD ⫹ liver resection. The overall median and 5-year survival were 20.5 months and 37.5%, respectively. Pts with primary neuroendocrine tumors tended to have had a better median survival (71.3 months) compared with other tumor histologies (17.1 months) (p⫽0.07). Conclusions: Liver-directed therapy of hepatic metastases plus PD is associated with complications in up to 25% of pts. Furthermore, the incidence of hepatic abscess is significantly increased in pts undergoing PD followed by staged liver-directed therapy. These data may help inform the management of pts with pancreatic head tumors and hepatic metastases. 35. HNF-6 MEDIATES GROWTH HORMONE EFFECT ON HEPATOCYTE FUNCTION DURING CHOLESTASIS IN MICE. Ai-Xuan Holterman, Minhua Wang; U Illinois at Chicago, Chicago, IL Background/Methods: Growth Hormone (GH) pleiotrophic effects on growth and metabolism are classically mediated through peripheral and hepatic IGF-1 production. In the liver, GH also induces the expression of hepatocyte nuclear factor HNF-6, a liver transcription factor which regulates hepatocyte-specific gene expression and hepatocyte proliferation. We test the hypothesis that GH mediates HNF-6 effect on modulating liver regeneration and function during cholestatic liver injury by bile duct ligation (BDL). Four groups of CD1 mice underwent sham surgery (n⫽4) or BDL (n⫽8) along with PBS or 5ug/day GH treatment and were sacrificed after 1 or 7 days. Hepatic functional indices such as serum albumin, cholesterol and liver function enzymes (LFT) were monitored. Hepatocyte proliferation was assessed by BrdU labeling. Whole liver HNF-6 and HNF-6 target gene and protein expression were assessed by real time PCR, western blot and immunostaining. Results: Despite hepatocytic injury (elevated ALT and AST levels), compared to BDL mice, GHtreated BDL mice have less cholestasis and improved liver function with lower serum Alkaline Phosphatase and cholesterol but higher albumin levels at day 7, as well as a higher hepatocyte proliferative response. This is in turn associated with increased HNF-6 expression and its transcriptional activation of albumin, cholesterol 7-a hydroxylase (Cyp7A1), proliferative Cyclin D1. Consistent with Cyp7A1, CyclinD1 and Albumin regulation by HNF-6, liver infected with recombinant adenovirus expressing HNF-6 cDNA showed similar upregulation of these genes. Conclusion: In biliary obstruction, GH treatment diminishes cholestasis, improves liver synthetic function and enhances liver regeneration in mice. GH-mediated HNF-6 upregulation of its proliferative target gene Cyclin D1 and metabolic target genes is one of the mechanisms by which GH treatment enhances hepatocyte proliferation and preserve liver function to attenuate liver injury during the early course of biliary obstruction. 36. SYSTEMIC ADENO ASSOCIATED VIRUS SEROTYPE 8 DELIVERY OF CRE RECOMBINASE MEDIATES CONDITIONAL GENE DELECTION IN MOUSE LIVER. Karen J. Ho1, Caroline Bass2, Alexander Kroemer2, Chunyan Ma2, Nicole M. Nesbitt2, Ernest Terwilliger2, Seth J. Karp2; 1Brigham and Women’s Hospital, Boston, MA; 2Beth Israel Deaconess Medical Center, Boston, MA Background: Viral vectors for hepatocyte-specific gene modulation are promising tools to advance understanding of liver regeneration

GH Mediates HNF6 Effect in the Liver

Serum Alk Phos (U/L) serum Albumin (g/dL) Albumin mRNA* Serum Cholesterol (mg/dL Cyp7A1 mRNA* % hepatocyte proliferation HNF6 mRNA* CyclinD1 mRNA*

PBS/BDL d1

GH/BDL d1

p value

PBS/BDL d7

GH/BDL d7

p value

473⫹/111

462⫹/⫺82

NS

1206⫹/388

550⫹/249

0.001

4.1⫹/⫺0.5

3.8⫹/⫺0.2

NS

2.7⫹/⫺0.4

3.3⫹/⫺0.5

0.05

Not done 247⫹/⫺21

Not done 205⫹/126

Not done NS

0.4⫹/0.17

0.7⫹/0.3

0.01

231⫹/⫺112

113⫹/⫺29

0.005

Not done 0.24⫹/0.17

Not done 1⫹/⫺0.3

Not done 0.001

1.26⫹/1.26

2.3⫹/⫺0.7

0.01

1.9⫹/⫺0.5

3.7⫹/⫺0.5

0.01

0.6⫹/⫺0.1 2.45⫹/⫺1.2

1.5⫹/0.5 4.26⫹/1.25

0.001 0.02

1.6⫹/⫺1.3 3.5⫹/⫺1.7

3.25⫺/1.1 9.93⫹/1.6

0.002 0.005

* mRNA level relative to Sham PBS.

and disease states. These techniques allow in vivo expression of genes and, when combined with transgenic approaches that introduce restriction sites for recombinases, deletion of specific genes. Limitations of previous work in this area include systemic and hepatic toxicity and low levels of gene expression. We hypothesized that adeno-associated virus serotype 8 (AAV8) would be an ideal vector for liver specific gene delivery based on its minimal toxicity and hepatic tropism. Methods: We systemically delivered an AAV8 vector encoding a codon-optimized Cre recombinase (iCre) under the control of the hepatocyte-specfic major urinary protein (MUP) promoter (MUP-icre-AAV8) to ROSA26R reporter mice, which express lacZ after Cre-mediated recombination. Results: We observed diffuse beta-galactosidase expression, restricted to hepatocytes, within 7 days (see Figures 1 and 2). The extent of expression was dose dependent. Cre recombinase protein expression was detectable by 7 days and persisted for at least 31 days. Hepatotoxicity was minimal by histology and liver function tests. The presence of MUP-icreAAV8 did not affect the regenerative potential of hepatocytes after partial hepatectomy as measured by Ki67 staining. Conclusion: MUP-icre-AAV8 may be a useful tool for rapid gene manipulations in vivo by mediating insertion or deletion of specific genes in the liver at specified times, without the need for complex transgenic strategies.

FIGURE 1. 400xLiver-specific recombination in ROSA26R mice. Mice were subject to systemic intravenous injections of vehicle (A) or MUP-icre-AAV8 (B) and whole mount beta-galactosidase staining of organs was assessed after 7days. FIGURE 2. Characterization of MUP-icre-AAV8 recombination in the liver. Analysis of livers 7 days after systemic injection demonstrates diffuse recombination only in hepatocytes. Cells occupying sinusoids (arrowheads) and those comprising the portal mesenchyme are spared. PV, portal vein, HA, hepatic artery, BD, bile duct. Original magnification 400x