These findings established that cell transplantation activated hepatic cyclooxygenase pathways during early clearance of transplanted cells from the liver. We found that mechanisms in cyclooxygenase-dependent cell clearance were complex, involving activation of PTGS1 and PTGS2 in native cells, whereas TXA2 or platelets were not involved. Sham-treatment with intrasplenic injection of vehicle alone also induced expression of cyclooxygenase genes, although it should be noteworthy that these components of intravascular perturbations cannot be completely separated from the procedure of cell transplantation. Blockade of PTGS1 and PTGS2 improved cell engraftment in the liver. This effect was mediated through HSC without reversal of ischemia-reperfusion events or phagocyte and macrophage responses after cell transplantation, which was unexpected, and opens new windows in the cellular roles of cyclooxygenase pathways.
After transplantation, engraftment of hepatocytes faces multiple hurdles, including microcirculatory alterations,6
with hepatic parenchymal disruption, loss of gap junctions in ischemic areas, and GGT expression as a reflection of oxidative stress and glutathione depletion.16
Since cyclooxygenase mechanisms may regulate hepatic vasculature through local production of PGI2
, or impart cytoprotection against ischemia-induced changes, we examined alterations in these mechanisms. Naproxen or celecoxib did not prevent ischemic injury and GGT expression, indicating that hepatic changes induced by cell transplantation differed from other types of hepatic ischemia, where naproxen or celecoxib showed therapeutic efficacy. 20
The absence of an effect of furegrelate and clopidogrel in doses capable of inhibiting TXAS activity and platelet function, respectively, 21,22
on cell engraftment indicate the insignificance of TXA2 pathways in transplanted cell clearances.
We expected that naproxen and celecoxib will exert their beneficial effects, at least in part, by suppressing hepatic inflammation, especially since these drugs controlled inflammatory liver injury. 23,24
Activation of neutrophils and Kupffer cells, and expression of multiple cytokines-chemokines following cell transplantation was in agreement with hepatic inflammation. As PTGS1 and PTGS2 were often expressed in cells adjacent to transplanted hepatocytes, including HSC, this suggested opportunities for cell-cell interactions, along with potential for amplification of inflammatory perturbations. The underlying mechanisms regulating interactions between transplanted and native cells have not been established, although these would likely extend from changes in the hepatic environment to release of soluble signals from cells themselves. For instance, hypoxia after vascular occlusion, decreased ATP levels, release of mediators, such as endothelin-1, cytokines, MMP-9, or other molecules, from inflammatory cells, LSEC, parenchymal cells, and transplanted hepatocytes themselves, could have stimulated HSC. 25-28
It should be noteworthy that transplanted hepatocytes were previously demonstrated to express VEGF, 3
which plays major roles in activating HSC. 29
Inflammatory mediators, e.g., inducers of TNF-α or other signaling, 25,26,28
constitute plausible candidates for activating HSC. After cell transplantation in the liver, TNF-α expression was significantly upregulated, as also shown here; however, desmin expression in HSC was not prevented in cell transplantation studies with etanercept to block TNF-α. 8
Therefore, delineation of critical mechanisms in activation of HSC after cell transplantation requires more work.
Stimulation of HSC should offer an appropriate explanation for the efficacy of naproxen and celecoxib in improving hepatocyte engraftment in the liver since HSC were identified as regulators of cell engraftment. 8
Naproxen and celecoxib increased desmin expression in HSC after cell transplantation. This desmin-positive phenotype of HSC was similar to previous cell transplantation studies, where desmin was expressed without α-smooth muscle actin, presumably because this stimulus was transient and nonfibrogenic. Our studies with CFSC-8B cells verified that naproxen stimulated VEGF and HGF expression. In previous studies, activation of HSC produced expression of multiple cytokines, growth factors and MMPs associated with HSC, e.g., bFGF, HGF, TGF-β1, TNF-α, and VEGF, as well as MMP-3, -9 and -13, in particular, and TIMP-1, as well. 8
Cytoprotective molecules, e.g., HGF and VEGF should be of particular relevance in improving survival of transplanted hepatocytes, as shown by our findings of hepatocyte protection from cytokine toxicity by VEGF-containing CFSC-8B conditioned medium. Similarly, release of VEGF and MMPs would promote disruption of LSEC required for the entry of transplanted hepatocytes into the space of Disse and subsequently into the liver parenchyma followed by the involvement of TIMP-1 and other molecules in coordinating tissue remodeling during cell engraftment ().
Working model of cyclooxygenase pathways and hepatocyte engraftment in the liver
These findings of hepatic HGF, VEGF and MMP expression were different from other systems, where naproxen or celecoxib were found to inhibit these genes. For instance, celecoxib impaired HGF signaling in esophagus to delay ulcer healing,30
suppressed VEGF expression to impair growth of pancreatic adenocarcinoma,31
and of retinal pigment epithelial cells. 32
Similarly, celecoxib or naproxen decreased expression of MMPs-1, -2, -3, and -9 in chondrocytes, articular cartilage, meniscus and synovial cells.33,34
Therefore, cyclooxygenase pathways may subserve variable biological effects under tissue- and cell type-specific contexts.
This improvement in engraftment of transplanted hepatocytes by widely used PTGS1 and PTGS2 inhibitors, naproxen and celecoxib, will advance clinical strategies for cell therapy. Use of naproxen in a single dose before cell transplantation should be convenient. Relatively small drug doses were effective in our studies and no further advantage was found of administering drugs repeatedly over a longer period, suggesting that early and initial decrease in expression of PTGS1 and PTGS2 was sufficient. The lesser efficacy of celecoxib indicates that blocking PTGS2 alone will not be as effective for cell therapy.