Masaaki Kataoka

Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats

BACKGROUNDnThe aim of this study was to investigate regulatory mechanisms of sinusoidal endothelial cell (SEC) proliferation after hepatectomy in rats.nnnMETHODSnWe investigated expressions of vascular endothelial cell growth factor (VEGF) and its receptors, flt-1 and KDR/flk-1, in regenerating liver after 70% hepatectomy. Proliferation of both hepatocytes and SECs was also monitored by evaluating the proliferating cell nuclear antigen (PCNA) labeling index. Furthermore, VEGF production by cultured hepatocytes isolated at different times after hepatectomy was measured in vitro.nnnRESULTSnThe expression of VEGF mRNA was increased markedly between 48 and 72 h after hepatectomy, and thereafter decreasing gradually. The immunohistochemical staining revealed that expression of VEGF started to increase 24 h after hepatectomy, with a peak at 72 h, and the majority of the VEGF-positive cells were hepatocytes located in periportal areas. Meanwhile, expression of flt-1 and KDR/flk-1 was observed along the sinusoids even before hepatectomy, but was increased between 72 and 120 h. Furthermore, VEGF production by cultured hepatocytes isolated 72 h after hepatectomy was significantly increased. The PCNA labeling index of the SECs exhibited a delayed and slower regenerative response in comparison to the hepatocytes, reaching a peak at 72 h.nnnCONCLUSIONSnThese data strongly suggest that VEGF secreted by proliferating hepatocytes may represent an important stimulator of SEC proliferation.

Effect of cold-ischemia time on C-X-C chemokine expression and neutrophil accumulation in the graft liver after orthotopic liver transplantation in rats.

Background. The precise mechanisms leading to polymorphonuclear neutrophil (PMN) recruitment and activation in the extended cold-preserved liver after transplantation are not yet fully understood. Methods. We histologically evaluated the number of accumulated PMNs in graft livers, with varying time periods of cold ischemia (1, 6, and 24 hr in University of Wisconsin solution at 4°C), after liver transplantation in rats. Intragraft expression of macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) mRNA, as well as immunohistochemical expression of MIP-2 and CINC in the graft liver, were investigated after reperfusion. The levels of MIP-2 and CINC in the hepatic vein, and tumor necrosis factor (TNF)-&agr;, which stimulates these chemokine production, were also monitored. Results. The number of accumulated PMNs in sinusoids significantly increased in the 24-hr cold-ischemia group within 3 hr after reperfusion, compared with the 1-hr and 6-hr groups. Serum MIP-2 levels in the 24-hr group significantly increased at 3, 6, and 12 hr after reperfusion, compared with the other groups. Intragraft MIP-2 mRNA was also up-regulated to a greater extent in the 24-hr group. Similarly, serum CINC levels in the 24-hr group significantly increased at 3 hr, compared with the 1-hr group. CINC mRNA also increased as cold-ischemia time was prolonged. Immunohistochemical staining revealed that hepatocytes were the main source of both MIP-2 and CINC protein. In addition, TNF-&agr; in the hepatic vein was detected only in the 24-hr group after reperfusion. Conclusion. Extended cold preservation of the graft liver might up-regulate MIP-2 and CINC expression of hepatocytes, most probably through elevated TNF-&agr;, and might contribute to PMN recruitment and activation after reperfusion.

Donor-specific antigen transfusion-mediated skin-graft tolerance results from the peripheral deletion of donor-reactive CD8+ T cells

Background. The mechanism of donor-specific transfusion (DST)-induced long-term skin-graft survival is examined in 2CF1 (2C×dm2) transgenic and B6F1 (C57BL/6×dm2) nontransgenic mice in which CB6F1 (Balb/c×B6) DST and donor skin grafts differ from 2CF1 or B6F1 recipients only at major histocompatibility complex class I Ld. Methods. Saline (control) or allogeneic CB6F1 spleen cells were injected intravenously into 2CF1 and B6F1 mice. One week later, CB6F1 tail skin was transplanted onto the dorsum of these mice. Fluorescence-activated cell sorter analysis (flow cytometric analysis) of peripheral blood was performed 2 days before DST, 5 days after DST, and 7, 14, 21, 28, and 75 days after skin grafting. Splenocyte responsiveness was measured by in vitro mixed lymphocyte culture and cytotoxic T lymphocyte. Cytokine protein production (interleukin [IL]-2 and interferon-&ggr;) was measured by enzyme-linked immunosorbent assay. Results. Whereas all CB6F1 skin grafts in control saline-treated 2CF1 and B6F1 mice were rejected, 100% of 2CF1 and B6F1 pretreated with CB6F1 DST accepted the class I Ld disparate donor skin indefinitely. DST followed by a CB6F1 skin graft led to a significant deletion of donor-reactive CD8+ T cells by fluorescence-activated cell sorter analysis and decreased production of the inflammatory cytokines IL-2 and interferon-&ggr;. The hyporesponsiveness of residual CD8+ T cells in mixed lymphocyte culture and cytotoxic T lymphocyte to Ld after DST was restored to normal by IL-2. Conclusion. These findings demonstrate that administration of DST uniformly results in long-term Ld+ skin-allograft acceptance. This tolerance induction is related to both a significant decrease in donor-reactive CD8+ transgenic T cells and anergy of the residual CD8+ T cells.