Volume 136, Issue 7, Pages 2356-2364 (June 2009) Hepatic Stellate Cells Promote Hepatocyte Engraftment in Rat Liver After Prostaglandin- Endoperoxide Synthase Inhibition  Yuta Enami, Sriram Bandi, Sorabh Kapoor, Natan Krohn, Brigid Joseph, Sanjeev Gupta  Gastroenterology  Volume 136, Issue 7, Pages 2356-2364 (June 2009) DOI: 10.1053/j.gastro.2009.03.003 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Cell transplantation and perturbations in cyclooxygenase pathways. (A–D) Histochemical staining for DPPIV showing transplanted cells (arrows, red) in the liver 6 hours, 1 day, 3 days, and 7 days after transplantation. Note that transplanted cells in sinusoids and portal radicles after 6 hours show diffuse membranous staining of DPPIV (A). By contrast, fewer transplanted cells were present at later times (B–D), and DPPIV staining was linear after 3 days and 7 days (arrows, C and D), indicating transplanted cells had integrated in liver parenchyma. Original magnification, 400×; methylgreen counterstain; Pa, portal area. (E) Real-time quantitative RT-PCR data showing changes in expression of PTGS1, PTGS2, and TXAS genes compared with untreated control animals and sham-treated animals as indicated. Expression of PTGS2 mRNA increased most. (F) Western blots showing protein analysis. Lane 1, unmanipulated normal liver; lane 2, sham-operated liver; lanes 3–6, livers after hepatocyte transplantation. Gastroenterology 2009 136, 2356-2364DOI: (10.1053/j.gastro.2009.03.003) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Immunohistological studies of PTGS1 expression. PTGS1 in normal rat liver (A) was expressed occasionally and was more frequently expressed after cell transplantation (arrows, B–F), including HSC (inset, B). Combined staining for DPPIV and PTGS1 showed that transplanted cells (thick arrows) and cells expressing PTGS1 (thin arrows) were often in proximity to one another (C and E). Original magnification, 400×; toluidine blue counterstain. Gastroenterology 2009 136, 2356-2364DOI: (10.1053/j.gastro.2009.03.003) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Changes in cell engraftment after treatment with PTS1 and PTS2 blockers. (A and B) Charts show morphometric quantitation of cell engraftment after single doses of 2, 4, and 6 mg/kg naproxen or 20, 30, and 40 mg/kg celecoxib (A) and multiple doses of these drugs (B). *P < .05 vs controls, ANOVA with Tukey test. Gastroenterology 2009 136, 2356-2364DOI: (10.1053/j.gastro.2009.03.003) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Changes in cyclooxygenase genes after drug treatments. Shown are Western blots with decreased expression of PTGS1, PTGS2, and TXAS proteins 1 day and 3 days after cell transplantation. Lanes 1 and 2, transplantation of hepatocytes alone; lanes 3 and 4, 6 mg/kg naproxen once before cell transplantation; lanes 5 and 6, 40 mg/kg celecoxib once before cell transplantation. Note that PTGS1 expression declined slightly after drug treatments, whereas PTGS2 and TXAS expression decreased much more. Gastroenterology 2009 136, 2356-2364DOI: (10.1053/j.gastro.2009.03.003) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Cumulative morphometric analysis of myeloperoxidase-positive neutrophils and Kupffer cells in liver tissue 6 hours after cell transplantation. (A) Shows changes in the number of myeloperoxidase-positive neutrophils. (B) Shows numbers of carbon-containing Kupffer cells with grading as described in the text. *P < .05 vs untreated controls. Gastroenterology 2009 136, 2356-2364DOI: (10.1053/j.gastro.2009.03.003) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Desmin staining to identify HSC. (A) Shows normal untreated rat without cell transplantation with desmin staining in major vessels in portal area as well as occasional desmin-positive HSC (arrow). (B) Shows rat 3 days after cell transplantation with interspersed HSC in periportal area (arrows). (C and D) Show rats 3 days after cell transplantation with 6 mg/kg naproxen or 40 mg/kg celecoxib with extensive increases in desmin-stained HSC (arrows). Original magnification, 400×; hematoxylin counterstain. (E) RT-PCR showing expression of HGF and VEGF mRNAs 3 days after cell transplantation with and without 6 mg/kg naproxen. Expression of HGF and VEGF mRNA increased after naproxen. Lane 1, untreated control liver; lane 2, sham treatment; lane 3, cell transplantation alone; lane 4, cell transplantation after naproxen. (F) RT-PCR showing expression of MMP-3, -9, and -13 and TIMP-1 mRNAs. Lane 1, untreated control lane 2, sham-treated; lane 3, cell transplantation alone; lane 4, cell transplantation after naproxen. Gastroenterology 2009 136, 2356-2364DOI: (10.1053/j.gastro.2009.03.003) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Effects of naproxen on cytoprotective gene expression in CFSC-8B rat stellate cells cultured under normoxia and hypoxia conditions. (A) Shows RT-PCR for cellular mRNAs indicating increases in expression of HGF and VEGF mRNAs with naproxen, up to 1 μmol/L, especially under hypoxia conditions. (B) Shows VEGF protein levels in culture medium, which increased from 409 ± 10 pg/mL and 424 ± 5 pg/mL under basal normoxia and hypoxia conditions, respectively, to 648 ± 26 pg/mL (1.8-fold) and 1133 ± 42 pg/mL (2.8-fold) after culture with 1 μmol/L naproxen under corresponding conditions, P < .05, ANOVA. Gastroenterology 2009 136, 2356-2364DOI: (10.1053/j.gastro.2009.03.003) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 Working model of cyclooxygenase pathways and hepatocyte engraftment in the liver. Cell transplantation induced hepatic expression of PTGS1, PTGS2, and other genes, although prostanoid limbs represented by TXA2 and PGE2 did not regulate cell engraftment. The studies showed that naproxen and celecoxib affected native liver cells, including HSC. Hepatic expression of pleiotropic factors, such as HGF and VEGF, which are both capable of cytoprotection, as well as of genes regulating extracellular matrix (ECM) remodeling provide relevant mechanisms to improve survival and engraftment of transplanted cells. Gastroenterology 2009 136, 2356-2364DOI: (10.1053/j.gastro.2009.03.003) Copyright © 2009 AGA Institute Terms and Conditions