Hepatic stellate cells promote hepatocyte engraftment in rat liver after prostaglandin-endoperoxide synthase inhibition

Abstract

Background & aims: Hepatic inflammation occurs immediately after cells are transplanted to the liver, but the mechanisms that underlie this process are not fully defined. We examined cyclooxygenase pathways that mediate hepatic inflammation through synthesis of prostaglandins, prostacyclins, thromboxanes, and other prostanoids following transplantation of hepatocytes.

Methods: We transplanted F344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient F344 rats. Changes in cyclooxygenase pathways were analyzed, and specific pathways were blocked pharmacologically; the effects on cell engraftment and native liver cells were determined.

Results: Transplantation of hepatocytes induced hepatic expression of prostaglandin-endoperoxide synthases 1 and 2, which catalyze production of prostaglandin H2, as well as the downstream factor thromboxane synthase, which produces thromboxane A2 (a regulator of vascular and platelet responses in inflammation). Transplanted hepatocytes were in proximity with liver cells that expressed prostaglandin-endoperoxide synthases. The number of engrafted hepatocytes increased in rats given naproxen or celecoxib before transplantation but not in rats given furegrelate (an inhibitor of thromboxane synthase) or clopodigrel (an antiplatelet drug). Naproxen and celecoxib did not prevent hepatic ischemia or activation of neutrophils, Kupffer cells, or inflammatory cytokines, but they did induce hepatic stellate cells to express cytoprotective genes, vascular endothelial growth factor and hepatocyte growth factor, and matrix-type metalloproteinases and tissue inhibitor of metalloproteinase-1, which regulate hepatic remodeling.

Conclusions: Activation of cyclooxygenase pathways interferes with engraftment of transplanted hepatocytes in the liver. Pharmacologic blockade of prostaglandin-endoperoxide synthases stimulated hepatic stellate cells and improved cell engraftment.

Figure 1. Cell transplantation and perturbations in cyclooxygenase pathways

(A-D) Histochemical staining for DPPIV showing transplanted cells (arrows, red color) in the liver 6 h, 1 d, 3 d and 7 d after transplantation. Note transplanted cells in sinusoids and portal radicles after 6 h 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 d and 7 d (arrows, C, D), indicating transplanted cells had integrated in liver parenchyma. Orig. mag. x400; 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.

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,E). Orig. mag. x400; toluidine blue counterstain.

Figure 3. Changes in cell engraftment after treatment with PTS1 and PTS2 blockers

(A, 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 (G) and multiple doses of these drugs (H). Asterisks = p<0.05 versus controls, ANOVA with Tukey test.

Figure 4. Changes in cyclooxygenase genes after drug treatments

Shown are western blots with decreased expression of PTGS1, PTGS2 and TXAS proteins 1 d and 3 d after cell transplantation. Lanes 1, 2, transplantation of hepatocytes alone; lanes 3, 4, 6 mg/kg naproxen once before cell transplantation; lanes 5, 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.

Figure 5. Cumulative morphometric analysis of myeloperoxidase-positive neutrophils and Kupffer cells in liver tissue 6 h 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. Asterisks = p<0.05 versus untreated controls.

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 d after cell transplantation with interspersed HSC in periportal area (arrows). (C and D) Show rats 3 d after cell transplantation with 6 mg/kg naproxen or 40 mg/kg celecoxib with extensive increases in desmin-stained HSC. Orig. mag. x400; hematoxylin counterstain. (E) RT-PCR showing expression of HGF and VEGF mRNAs 3 d after cell transplantation with and without 6 mg/kg naproxen. Lane 1, untreated control liver; lane 2, cell transplantation alone; lane 3, cell transplantation after naproxen. (F) RT-PCR showing expression of MMP-3, -9 and -13, and TIMP-1 mRNAs. Lanes 1, 2, untreated controls; lanes 3, 4, cell transplantation alone; lanes 5, 6, cell transplantation after naproxen.

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 μM, 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 μM naproxen under corresponding conditions, p<0.05, ANOVA.

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.