Regulation of heme oxygenase 1 expression by miR-27b with stem cell therapy for liver regeneration in rats.

Adipose-derived mesenchymal stem cells (ASCs) have been considered to be attractive and readily available adult mesenchymal stem cells (MSCs) and are becoming increasingly popular for use in regenerating cell therapy. However, recent evidence attributed a fibrotic potential to MSCs which differentiated into myofibroblasts with highly increased α-smooth muscle actin (α-SMA) expression while transplanted into an injured/regenerating liver in mice. In this study, we studied the role of miR-27b in ASCs and their regenerative potential after partial liver resection in rats. ASCs transfected with control siRNA or miR-27b were intravenously injected into autologous rats undergoing 70% partial hepatectomy (PH). Our data showed that the regenerative capacities of ASCs with overexpressed miR-27b were significantly higher compared with control ASCs. However, the enhanced regeneration, hepatic differentiation, and suppressed liver inflammation, as well as fibrotic activity, were significantly reverted by ZnPP coadministration (heme oxygenase-1 [HO-1] inhibitor) indicating an important role of HO-1 in the regenerating and cytoprotective activities of miR-27b-transfected ASCs. We demonstrated that administration of autologous ASCs overexpressed with miR-27b enhances rapid and early liver regeneration and, importantly, preserves function after PH. The ASCs with miR-27b overexpression might offer a viable therapeutic option to facilitate rapid recovery after liver resection.

Significance of portosystemic shunt on graft survival in liver transplantation: a rat model.

AIMS: Distal splenorenal shunt effectively controls bleeding from esophageal and gastric varices but has a different effect on liver transplantation. This study sought to develop an animal model in rats to mimic the recipient with a portosystemic shunt and to investigate its hemodynamic consequences on liver transplantation. METHODS: We prepared 5 groups of allogeneic or syngeneic rat liver transplantation models with versus without portosystemic shunt, to investigate its effects on graft survival and portal flow. To explore the effects of excessive portal flow on graft survival in small-for-size liver transplantation, we transplanted partial liver grafts into syngeneic recipients. RESULTS: In allogeneic combinations, graft survival among the shunt group was shortened compared with their control counterparts. The graft survival of the large shunt group was significantly lower than that of a small shunt or without shunt group in a syngeneic liver transplantation model. Portal blood pressure of the large shunt group was significantly lower than that of the small shunt group. In contrast, excessive portal flow resulted in dysfunction of liver graft in small-for-size liver transplantation. CONCLUSIONS: These results suggested that reduction in portal flow by portosystemic shunt lead to an acceleration of acute rejection and subsequent liver graft dysfunction, but it may be applicable to regulate the excessive portal flow in small-for-size transplantations. This study showed a valuable model mimicking the recipient with a portosystemic shunt.

High-mobility group box 1 protein activates hepatic stellate cells in vitro.

OBJECTIVES: Our previous study noticed remarkably elevated titers of anti-high-mobility group box 1 (HMGB1) antibodies in sera during the tolerance induction phase of a rat tolerogenic orthotopic liver transplantation (OLT) as well as in sera of clinically drug-free patients. We hypothesized that the release of nonhistone nuclear protein HMGB1 during rejection may play a pathogenic role in deteriorating post-OLT graft functions, such as inducing liver fibrosis. This study sought to investigate whether HMGB1 can directly activate hepatic stellate cells (HSCs) and drive them toward fibrogenesis. METHODS: The cultured HSCs were treated with recombinant HMGB1. RT-PCR and Western blotting analysis were used to measure alpha-smooth muscle actin (alpha-SMA) expression. Conditioned media were collected for gelatin zymography to monitor the activities of collagen-degrading matrix metalloproteinases (MMPs). RESULTS: HMGB1 at concentrations > 1 ng/mL significantly stimulated HSC growth as revealed by proliferation and BrdU assays. alpha-SMA gene and protein expression were significantly up-regulated by HMGB1, whereas the MMP-2, but not MMP-9, activity was suppressed by HMGB1 treatment. CONCLUSION: Our data suggested that HMGB1 protein, once released during the rejection phase of OLT, activated HSCs and exhibited profibrogenic effects on liver grafts either by increasing the HSC population and extracellular matrix content in liver grafts, or by transforming HSCs into myofibroblasts. Neutralization with anti-HMGB1 antibody was suggested to be a therapeutic modality applicable to prevent fibrogenesis in post-OLT liver grafts.

Histone H1 vaccine therapy for overcoming acute rejection in experimental organ transplantation.

OBJECTIVE: In a rat tolerogenic orthotopic liver transplantation (OLT) model, the recipient serum (post-OLT serum) shows strong immunosuppressive activity. In our previous reports, we suggested that autoreactive antibody (Ab) against histone H1 is a major immunosuppressive factor in this serum. The present study sought to determine whether up-regulation of anti-histone H1 Ab by histone H1 vaccination led to tolerance. MATERIALS AND METHODS: Using mixed lymphocyte reactions (MLR) and heterotopic heart transplantations (HHT), the alloreactive T-cell responses and allograft survivals of histone H1-immunized rats were compared with those of control rats. Cytokine and cellular profiles were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. RESULTS: The alloreactive T-cell response of histone H1-immunized rats was significantly lower than that of control rats, although there was no difference in nonspecific T-cell activation between the 2 groups. The allograft survival of histone H1-immunized rats was significantly prolonged after HHT. The major histocompatibility complex (MHC) class II and CD25 molecules of histone H1-immunized rats were significantly down-regulated compared with those of control rats. Moreover, the serum cytokine profile was modified by the immunization with histone H1. CONCLUSIONS: These results suggest that histone H1 vaccination of transplant recipients leads to the production of immunosuppressive factors and the modification of cytokine/cellular profiles.

Role of antinuclear antibodies in experimental and clinical liver transplantation.

OBJECTIVE: We recently reported that autoreactive antibodies (Abs) against nuclear histone H1 was transiently induced at an early phase after orthotopic liver transplantation (OLT) in a tolerogenic rat OLT model and possessed immunosuppressive activity. It was also reported that nuclear antigen, high-mobility group box 1 (HMGB1) protein was one of the initiators of the immune reaction. The present study sought to evaluate the role of antinuclear Abs in experimental and clinical liver transplantation. MATERIALS AND METHODS: We prepared 3 animal models: natural tolerance model (DA liver into PVG); acute rejection model (DA liver into LEW); and drug-induced tolerance model (acute rejection model + cyclosporine [CsA]). In addition, we examined clinical samples, including 1 drug-free patient, to measure the antihistone H1/HMGB1 titers at various times after OLT. RESULTS: In a natural tolerance model, antihistone H1 and HMGB1 Ab was induced during the rejection and the tolerance induction phases, respectively. Those Ab responses were also confirmed in a drug-induced tolerance model, whereas no such responses were shown in an acute rejection model. In our clinical drug-free patient, antihistone H1/HMGB1 titer was significantly higher after cessation of CsA than that in healthy volunteers. CONCLUSIONS: Antinuclear Ab is actively expressed in accordance with overcoming rejection episodes with subsequent tolerance induction in both a natural tolerance model and a drug-induced tolerance model. We also observed a similar tendency in our clinical drug-free patient. These results suggested that antinuclear Abs may be useful markers to determine the timing to withdraw immunosuppressants.

Characterization of immunosuppressive factors expressed in serum by rat tolerogenic liver transplantation.

BACKGROUND: In a rat tolerogenic model of orthotopic liver transplantation (OLT), recipient serum after OLT (post-OLT serum) possesses strong immunosuppressive activity. This study aimed to identify immunosuppressive factors present in early post-OLT serum. METHODS: Immunosuppressive activity was evaluated in vitro by inhibition of the mixed-lymphocyte reaction (MLR). Autoantigens recognized by MLR-inhibitory IgG were identified by the internal protein sequencing. RESULTS: Recipient post-OLT serum inhibited MLR, and OLT-inducible IgG was the major immunosuppressive factor. IgG from post-OLT sera (2 to 3 weeks) specifically reacted to 31; 34; and 73-kd autoantigens on spleen cells. The internal sequences of the 31- and 34-kd antigens coincided completely with those of histone H1 molecules. Immunodepletion of anti-histone H1 antibodies (Abs) from early post-OLT serum abolished the MLR-inhibitory activity. Furthermore, rabbit polyclonal Ab-directed histone H1 not only significantly suppressed rat and human MLR but also prolonged survival of heart allografts. Flow-cytometric analysis revealed that some live PVG splenocytes were stained with antihistone H1 Abs, and that these positive cells increased on Con A stimulation. Western blot analysis indicated that several cross-reactive antigens against anti-histone H1 Abs were found in their membrane fraction. CONCLUSIONS: In this study we provide evidence that autoreactive Abs, against histone H1 are a major OLT-induced graft survival factor, and may play at least a part in overcoming the acute rejection phase to establish solid allograft tolerance.