We therefore harvested primary mouse hepatocytes and exposed confluent and subconfluent cultures to CO or air. CO had no effect on hepatocyte proliferation as assessed by bromodeoxyuridine (BrdU) incorporation (Fig. 5A). We therefore exposed hepatic stellate cells (HSC) to CO or air and measured HGF expression. We observed a significant increase in HGF expression over 24

hours in CO-exposed HSC versus air-treated cells (1,704 ± 540 versus 1,146 ± 293, respectively, P < 0.05). Finally, to assess whether this vectorial HGF was functional, we cocultured HC and HSC ± CO and measured BrdU incorporation. There was a greater PI3K Inhibitor Library high throughput increase in BrdU as well as cell number of HC in CO-treated cocultures versus control (Fig. 5C,D) supporting HSC as a primary target for CO to enhance HGF expression and HC

proliferation (Fig. 5B-F). HGF administered directly to HC (Fig. 5E) in concentrations similar to that generated by CO-exposed HSC increased BrdU incorporation to similar levels as CO. To confirm our hypothesis that CO accelerates liver regeneration after PHTx targeting a Met-HGF axis, we blocked HGF with the HGF receptor antagonist NK4.32 Mice were injected with the NK4 neutralizing antibody prior to PHTx and then divided to receive air or CO. Hepatocyte HTS assay proliferation was assessed at 24 hours by counting Ki-67-positive hepatocytes. As presented in Fig. 1, CO induced proliferation after resection with a greater number of Ki-67-positive hepatocytes versus sham and air controls; however, the CO effect was lost in animals injected with NK4 (Fig. 6A-E). These data strongly support that CO is acting through an HGF-dependent pathway to augment hepatocyte proliferation. Finally, we sought to evaluate the potency of CO to rescue mice in a more severe, massive hepatectomy model. Although in rodent models 70% hepatectomy is well established as a nonlethal model, a massive hepatectomy beyond 70% can result in higher morbidity and mortality.15

上海皓元 Air-treated mice receiving 85% PHTx showed a survival rate of <50%. However, mice exposed to CO for 1 hour prior to hepatectomy demonstrated a 75% survival rate (Fig. 7, P < 0.02). In these studies we describe the effects of CO exposure on liver regeneration after hepatectomy in mice and elucidate a cell and molecular model by which CO enhances early proliferation in the liver. The cytoprotective effects of CO exposure, particularly in the liver, have been demonstrated in several experimental models,16-20, 23, 33 but of particular interest to hepatectomy and transplantation is the ability of CO to prevent IRI,21 where CO exposure has been suggested as a therapy to limit injury.34 Glanemann et al.35 examined the effect of inducing HO-1 on liver regeneration using the 70% hepatectomy model combined with ischemic injury from temporary inflow occlusion (30 minutes).