RNA interference of cholangiocyte expression of the alpha2beta1 integrin reduces susceptibility to rhesus rotavirus infection

S50

Pediatric Surgery

J Am Coll Surg

CONCLUSIONS: ACE-I/PEG reduced the severity of DSSinduced colitis; most likely by a decline in EC apoptosis and reduced TNF-alpha expression. The higher TNF-alpha expression at higher ACE-I/PEG doses may be due to more preserved mucosa (the source of TNF-alpha). The optimal effect was between 14.5micrograms/day to 145micrograms/day. ACE-I/PEG may be a potential new option for treating inflammatory bowel disease.

Factor-VII transcription correlates with hepatocyte proliferation and hepatocyte growth factor expression in a rodent extra-hepatic portal vein obstruction model Bill Chiu MD, Hector Melin-Aldana MD, Srikumar Pillai MD, Fei Chu MD, PhD, Riccardo Superina MD Children’s Memorial Hospital, Northwestern University, Chicago, IL INTRODUCTION: Extrahepatic portal vein obstruction (EHPVO) is clinically associated with decreased liver-dependant coagulation factors and small liver volumes. We developed a rodent EHPVO model to define the mechanisms for coagulation factor regulation and liver homeostasis. METHODS: Fifteen rats underwent controlled narrowing of portal vein (PV) at the hilum, 15 underwent sham operations. Three animals from each group were sacrificed at post-operative-days(POD) 1,2,4,7,21. PV sections were taken to measure vein diameters. Livers were studied for apoptosis, proliferation, and hepatocyte-growth-factor (HGF) expression using TUNEL assays, Ki-67, and HGF antibody. Liver total-RNA was harvested for RT-PCR, using primers for factor-V, VII, VIII, X, protein-C, and anti-thrombin. Student’s t-test was used for analysis. RESULTS: Experimental group had 59⫾17% decrease in distal PV diameter. Proportional body and liver weight was equivalent in both groups, but spleen weight was higher in the experimental group at POD4,7,21(p⬍0.05). Percentage of apoptotic cells in experimental group was increased from that of control at POD4(p⬍0.05). Percentage of proliferating cells, HGF expression, and factor-VII transcription in experimental group were lower than those of control at POD2(p⬍0.05) but higher than those of control at POD7(p⬍0.05). Ki-67/TUNEL double-staining showed no grouping of apoptotic and proliferating cells. In contrast, factor-V transcription in the experimental group was increased at POD2 compared to controls(p⬍0.05). Transcription of factor-VIII, X, protein-C, and anti-thrombin was unchanged. Experiment

Control

P Value

Proportional Spleen Weight POD4

0.42⫾0.008%

0.36⫾0.03%

P⬍0.05

Proportional Spleen Weight POD7 Proportional Spleen Weight POD21 Apoptosis POD4 Proliferation POD2 Proliferation POD7 HGF Expression POD2 HGF Expression POD7 Factor-VII Transcription POD2 Factor-VII Transcription POD7 Factor-V Transcription POD2

0.54⫾0.09% 0.4⫾0.08% 0.6⫾0.25% 0.96⫾0.92% 2.69⫾1.12% 40⫾5% 97⫾3% 0.85⫾0.27 1.25⫾0.22 1.62⫾0.2

0.42⫾0.13% 0.36⫾0.05% 0.27⫾0.13% 2.99⫾0.75% 0.58⫾0.55% 87⫾13% 80⫾9% 1 1 1

P⬍0.05 P⬍0.05 P⬍0.05 P⬍0.05 P⬍0.05 P⬍0.05 P⬍0.05 P⬍0.05 P⬍0.05 P⬍0.05

CONCLUSIONS: Our EHPVO rodent model demonstrated an early increase in hepatocyte apoptosis, impairment of factor-VII transcription, and decrease in proliferative indices. These data support the hypothesis that EHPVO leads to widespread dysregulation of hepatic homeostasis.

RNA interference of cholangiocyte expression of the alpha2beta1 integrin reduces susceptibility to rhesus rotavirus infection Mubeen A Jafri MD, Bryan Donnelly BS, Steve Allen MD, Greg Tiao MD Cincinnati Children’s Hospital Medical Center, Cincinnati, OH INTRODUCTION: Biliary atresia is a disease of newborns which results in obliteration of the biliary tree. A model has been established in which infection with rhesus rotavirus(RRV) leads to a cholangiopathy mirroring human disease in newborn mice, and a cytopathic effect in a cholangiocyte cell line. We have previously demonstrated that viral attachment was significantly greater in cholangiocytes than hepatocytes and this relation correlated with expression of the integrin alpha2beta1. We sought to further investigate this by evaluating in vivo expression of alpha2beta1 and blocking of integrin expression in cell culture. METHODS: Cholangiocyte and hepatocyte cell lines along with liver and biliary samples were evaluated for alpha2beta1 expression using western blotting and flow cytometry. Immunohistochemistry was used to evaluate localization of the integrin. Alpha2beta1 was down-regulated using short interfering RNA(siRNA). Rotavirus binding and replication assays were conducted in the setting of integrin downregulation. RESULTS: Alpha2beta1 expression could be demonstrated in dissected samples with western blotting. Localization to biliary epithelium was demonstrated with immunohistochemistry. siRNA decreased alpha2beta1 expression by 50% along with viral binding and replication by 49% and 47% respectively(p⬍0.001) in cell culture. CONCLUSIONS: RRV attached with greater affinity and replicated more efficiently in cholangiocytes than hepatocytes. Bile duct epithelium as well as cholangiocyte cell lines expressed alpha2beta1 and downregulation using RNA interference significantly reduced attachment and resulting viral yields. Alpha2beta1 expression may be an important mechanism confering cholangiocyte susceptibility to RRV infection and a target for therapeutic interventions in biliary atresia.

AML-1/Angiopoietin signaling contributes to hepatoblastoma recurrence during VEGF blockade Joey C Papa MD, Angela Kadenhe-Chiweshe MD, Kimberly McCrudden MD, Jianzhong Huang MD, Darrell Yamashiro MD, PhD, Jessica Kandel MD Columbia University, New York, NY INTRODUCTION: Blockade of Vascular Endothelial Growth Factor (VEGF), a key mediator of angiogenesis, has been validated as a