Recurrence-free survival more than 10 years after liver resection for hepatocellular carcinoma.

BACKGROUND: High recurrence rates after liver resection with curative intent for hepatocellular carcinoma (HCC) remain a problem. The characterization of long-term survivors without recurrence after liver resection may help improve the therapeutic strategy for HCC. METHODS: A nationwide Japanese database was used to analyse 20 811 patients with HCC who underwent liver resection with curative intent. RESULTS: The 10-year recurrence-free survival rate after liver resection for HCC with curative intent was 22.4 per cent. Some 281 patients were recurrence-free after more than 10 years. The HCCs measured less than 5 cm in 83.2 per cent, a single lesion was present in 91.7 per cent, and a simple nodular macroscopic appearance was found in 73.3 per cent of these patients; histologically, most HCCs showed no vascular invasion or intrahepatic metastases. Multivariable analysis revealed tumour differentiation as the strongest predictor of death from recurrent HCC within 5 years. CONCLUSION: Long-term recurrence-free survival is possible after liver resection for HCC, particularly in patients with a single lesion measuring less than 5 cm with a simple nodular appearance and low tumour marker levels.

Reduced stability of retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat protein overexpressed in hepatomas.

Hepatocellular carcinoma (HCC) is one of the most common cancers in Asia and Africa, where hepatitis virus infection and exposure to specific liver carcinogens are prevalent. Although inactivation of some tumor suppressor genes such as p53 and p16INK4Ahas been identified, no known oncogene is commonly activated in hepatocellular carcinomas. Here we have isolated genes overexpressed in hepatocellular carcinomas by cDNA subtractive hybridization, and identified an oncoprotein consisting of six ankyrin repeats (gankyrin). The expression of gankyrin was increased in all 34 hepatocellular carcinomas studied. Gankyrin induced anchorage-independent growth and tumorigenicity in NIH/3T3 cells. Gankyrin bound to the product of the retinoblastoma gene (RB1), increasing its phosphorylation and releasing the activity of the transcription factor E2F-1. Gankyrin accelerated the degradation of RB1 in vitro and in vivo, and was identical to or interacted with a subunit of the 26S proteasome. These results demonstrate the importance of ubiquitin-proteasome pathway in the regulation of cell growth and oncogenic transformation, and indicate that gankyrin overexpression contributes to hepatocarcinogenesis by destabilizing RB1.

Strategies to induce marked prolongation of secondary skin allograft survival in alloantigen-primed mice.

Alloreactive memory T cells mediate accelerated rejection. We investigated the effect of polyclonal anti-T-cell antibody (ALS) and rapamycin (RAPA) on skin allograft survival in naïve or alloantigen-primed mice. ALS prolonged graft survival in both naïve and alloantigen-primed mice. T-cell depletion by ALS was associated with increased CD4(+)CD44(hi)OX40(+) and CD8(+)CD44(hi)CD122(+) memory T cells. Addition of RAPA to ALS extended graft survival in naïve mice, but had no effect on secondary allograft survival in alloantigen-primed mice. In adoptive transfer experiments, RAPA inhibited alloantigen-stimulated proliferation and allograft rejection by naïve T cells. In contrast, alloantigen-primed memory T cells, particularly CD4(+)CD44(hi)OX40(+) and CD8(+)CD44(hi)CD122(+) T cells, were resistant to RAPA in response to alloantigen and mediated accelerated rejection in the presence of RAPA. Resistance to RAPA by alloantigen-primed mice was overcome by the use of high-dose ALS, which achieved marked prolongation of secondary skin allograft survival (>100 days). Inhibition of CD122(+) T cells and/or OX40/OX40L costimulation blockade, combined with low-dose ALS and RAPA, was also effective. These results demonstrate that tolerance may be achieved in allosensitized individuals by T-cell depletion- and RAPA-based strategies employing high-dose ALS or targeting CD122(+)CD8(+) T cells and/or the OX40/OX40L costimulatory pathway.

Aurora kinase B is a predictive factor for the aggressive recurrence of hepatocellular carcinoma after curative hepatectomy.

BACKGROUND: Patterns of cancer recurrence hold the key to prognosis after curative resection. This retrospective study aimed to identify a predictor and therapeutic candidate for aggressive recurrence of hepatocellular carcinoma (HCC). METHODS: Primary HCC tissues from 107 patients who had curative resection were analysed. Genome-wide gene expression profiles were investigated using a microarray technique, and clustering analysis was carried out based on the first diagnosis of recurrence according to the Milan criteria. Immunohistochemical expression and array-based comparative genomic hybridization (array-CGH) were also assessed. RESULTS: Microarray analysis revealed overexpression of Aurora kinase B, a chromosome passenger protein kinase, as the most significant predictor of the aggressive recurrence of HCC. Aurora kinase B protein expression was significantly associated with aggressive recurrence (P < 0.001) and prognosis (P < 0.001). Multivariable analysis identified Aurora kinase B as the only independent predictor of aggressive recurrence of HCC (P = 0.031). Array-CGH analysis showed that genomic instability was closely related to Aurora kinase B expression (P = 0.011). CONCLUSION: Aurora kinase B is an effective predictor of aggressive HCC recurrence, in relation to the genomic instability. It might be worth considering as a molecular target for the adjuvant therapy of HCC.

Differentiation-dependent expression of I and sialyl I antigens in the developing lung of human embryos and in lung cancers.

The localization of two carbohydrate antigens, I and sialyl I antigens, in the lungs of developing human embryos was investigated using specific monoclonal antibodies and compared with the distribution patterns of the known embryonic antigen, stage-specific embryonic antigen-1 (Lex hapten). When the future bronchi were actively developing from the bronchial buds in the lungs of 50- to 53-day-old embryos, the immature bronchial bud cells were I-, Lex+, while the fully differentiated epithelial cells of the larger bronchus were I+, Lex-. When the bronchiolar bud cells matured into bronchiolar epithelial cells in the lung of a 12-week-old embryo, the immature bronchiolar bud cells were I-,Lex+, while the fully differentiated epithelial cells of the bronchioles were I+,Lex-. Sialylated forms of the antigens finally appeared in the lungs of 18-week-old embryos, when the terminal bud cells actively proliferated and underwent the differentiation process into epithelial cells of alveoli and alveolar ducts. The immature terminal bud cells at this stage were I-, sialyl I-, Lex+, sialyl Lex-i+, while the fully differentiated alveolar epithelial cells were I+, sialyl I+, Lex-, sialyl Lex-i-. After 8 months, the flattened mature alveolar epithelial cells were strongly positive for both I and sialyl I antigens, the strong expression of which continued after birth and even into the adult stage. These distribution patterns indicate that the I and sialyl I antigens are specific markers for the differentiated type cells in each stage of development, while Lex and related embryonic antigens were specific to the immature bud cells in every stage. The above-described differentiation-dependent expression patterns of these antigens seem to be reflected in the distribution of these antigens in human lung cancers, i.e., I and sialyl I antigens were expressed in lung cancer cells more weakly than in normal lung cells, while the Lex and sialyl Lex-i were expressed in cancer cells much more strongly than in normal lung cells. This was further reflected in the serum levels of these antigens in the patients with respiratory disorders. The distribution pattern of the serum levels of these antigens in patients with lung cancers showed sialyl Lex-i greater than sialyl I, indicating that these serum antigens originated from the lung cancer lesion where sialyl Lex-i is much more dominant than sialyl I antigen.(ABSTRACT TRUNCATED AT 400 WORDS)

sFlt-1 gene-transfected fibroblasts: a wound-specific gene therapy inhibits local cancer recurrence.

Local recurrence occurs frequently at the site of injury after surgical resection. On the other hand, fibroblasts have been shown to accumulate in the injured area to heal and remodel the damaged tissues. Therefore, fibroblasts are likely to be useful as wound-specific vectors for delivery of genes to sites of surgically injury. The present study was performed to investigate wound-specific migration of exogenously administered fibroblasts and efficacy of gene therapy using genetically engineered fibroblasts in an i.p. wound recurrence model in rats. We demonstrated that fibroblasts transfected with the GFP gene accumulated specifically around the site of injury immediately after i.p. injection. Then, fibroblasts transfected with an adenovirus designated as AdFex that encoded the soluble form of Flt-1 (sFlt-1), a vascular endothelial growth factor receptor, were administered i.p. to the rats to examine inhibition of tumor growth. At day 16 after implantation, a significantly smaller tumor volume and less microvessel density in wound sites were observed in the AdFex/fibroblast-treated rats than in controls. Furthermore, this treatment also resulted in an improved survival rate. In conclusion, autologous fibroblasts show promise as a wound-specific vector for gene therapy, and administration of sFlt-1 gene-engineered fibroblasts contributed to local control of the tumor around the injured tissue.

Enhanced expression of multiple protein tyrosine phosphatases in the regenerating mouse liver: isolation of PTP-RL10, a novel cytoplasmic-type phosphatase with sequence homology to cytoskeletal protein 4.1.

To elucidate the role that protein tyrosine phosphatase (PTPs) may play in liver regeneration, PTPs expressed in the mouse liver after partial hepatectomy (PH) were investigated by a PCR-based cloning method. Sequencing of 115 cDNA clones identified 10 different sequences including MPTP (T cell PTP), PTP-1B, PTP-P19, mR-PTP mu, R-PTP alpha, PTP NE-3 (PTP-P1), R-PTP-kappa and the murine homologue of human LAR. The remaining two sequences, PTP-RL9 and PTP-RL10, encoded novel PTPs. PTP-RL10 cDNA contained an open reading frame of 1176 amino acids with no apparent membrane-spanning region. The amino-terminal region had sequence homology to those of human erythrocyte protein 4.1 and ezrin, cytoskeletal proteins. In the regenerating liver, the levels of five PTP gene mRNAs (MPTP, PTP-P19, R-PTP alpha, LAR homologue, and PTP-RL9) increased within 6 h, decreased to the normal level by 24 h, and increased again at 48 to 72 h after PH. The levels of PTP-1B and R-PTP-kappa mRNAs peaked within 6 h, decreased gradually, and returned to the normal level by 168 h after PH. In contrast, the levels of two PTP mRNAs (mR-PTP mu and PTP-RL10) peaked at 48 to 72 h, and returned to the normal level by 168 h after PH. No expression of PTP NE-3 was detected in the liver by Northern blotting. The differential expression of multiple PTPs during the pre-replicative and post-replicative stages of liver regeneration suggests that PTPs are involved in the regulation of growth and differentiation of liver cells.

Enhancement of angiogenesis, tumor growth, and metastasis by transfection of vascular endothelial growth factor into LoVo human colon cancer cell line.

The expression of vascular endothelial growth factor (VEGF), a highly potent angiogenic molecule, is thought to be correlated with the development of colon cancer; however, direct evidence for a role of VEGF in metastasis is lacking. This study was designed to more directly establish the role of VEGF in the growth and metastasis of human colon cancer using a genetically engineered cancer cell line. A stable VEGF gene-transfected human colon cancer cell line, LoVo, was made by genetic manipulation using eukaryotic expression constructs designed to express the complete VEGF121 cDNA in the sense orientation. Transfected clones were screened for VEGF121 mRNA expression by Northern blot analysis and for VEGF121 protein expression by Western blot analysis. Consequently, we obtained S17 cells that expressed a high level of both VEGF mRNA and VEGF protein. A vector-transfected clone (V7 cell) was used as a control. The experiment with the dorsal air sac method revealed that S17 cells elicited a stronger directional out-growth of capillaries than V7 cells. S17 cells formed faster-growing tumors than did V7 cells when xenografted s.c. into nude mice, although there was no significant difference in their in vitro proliferation. Tumors derived from S17 cells had more vascularity, as assessed by counting capillary vessels after staining with factor VIII, than did tumors derived from V7 cells (P < 0.05). With regard to the metastatic potential, S17 cells exhibited a higher capacity for both hepatic metastasis after the splenic portal inoculation and peritoneal dissemination after i.p. injection than did V7 cells. However, S17 cells showed no apparent metastasis, despite their rapid growth after orthotopic implantation. In conclusion, the present study showed clearly that VEGF plays an important role in cancer growth due to stimulation of angiogenesis by accelerating cell growth after reaching the target organs.

Mouse macrophage metalloelastase gene transfer into a murine melanoma suppresses primary tumor growth by halting angiogenesis.

Mouse macrophage metalloelastase (MME) has been associated with the generation of angiostatin, an internal fragment of plasminogen, which inhibits angiogenesis. To clarify whether tumor cells that consistently generate MME can suppress angiogenesis and, therefore, inhibit the growth of primary tumors in vivo, we transfected a cDNA coding for MME into murine B16-BL6 melanoma cells that grow rapidly and are MME deficient. The generation of active MME in MME-transfected clones was confirmed by immunoprecipitation followed by in vitro cleavage of plasminogen. Subcutaneous implantation of these stable clones in C57BL/6 mice inhibited primary tumor growth by an average of 73% (P = 0.00002), which directly correlated with a significant reduction of blood vessel formation (approximately 76%) in such tumors. Microangiography revealed massive angiogenesis in control tumors (mock and vector); however, in MME-transfected primary tumors it demonstrated a decreased and disrupted vascular network. Western blot analysis using a specific anti-mouse angiostatin antibody demonstrated a strong 38-kDa immunoreactive band in MME-transfected tumors and in the serum of mice bearing those tumor cells. These results show that placing MME gene directly into B16-BL6 melanoma cells is an effective approach to suppress primary tumor growth in vivo because it halts angiogenesis. Our data provide a feasible and promising strategy for gene therapy of cancer by targeting tumor vasculature.

Expression of METH-1 and METH-2 in pancreatic cancer.

PURPOSE: METH-1/hADAMTS-1 and METH-2/hADAMTS-8 are recently identified genes that inhibit angiogenesis, and the murine homologue, ADAMTS-1, shows metalloproteinase function. Because the significance of METH-1 and METH-2 has not been determined in solid tumors, we examined the mRNA expressions of these molecules in pancreatic cancer and hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: METH-1 and METH-2 mRNA expressions were identified in six pancreatic cancer cell lines and were quantified by TaqMan reverse transcription-PCR in 18 paired samples of pancreatic cancer and surrounding noncancerous pancreas, and in 14 samples of pancreatic cancer. METH-1 mRNA expression was also examined in 16 pairs of HCC and cirrhotic liver. Vascularity was estimated by CD34 staining. The correlation between clinicopathological factors and METH-1 expression was additionally analyzed. RESULTS: Four of six pancreatic cancer cell lines expressed METH-1, and 1/6 expressed METH-2. METH-1 was substantially expressed in both pancreatic cancer and noncancerous pancreas, but METH-2 was not. METH-1 expression in pancreatic cancer tissue was significantly lower than that in noncancerous pancreas (P = 0.002), and a similar result was obtained between HCC and cirrhotic liver (P = 0.003). METH-1 expression did not show a significant correlation with vascularity in pancreatic cancer or in HCC. However, pancreatic cancer with higher expression of METH-1 showed significantly severe lymph node metastasis or retroperitoneal invasion (P = 0.033 and P = 0.018, respectively) and worse prognosis (P = 0.038). CONCLUSIONS: METH-1, which was initially reported to have a potent antiangiogenic effect, does not seem to be a predominant determinant of tumor vascularity in pancreatic cancer. Rather, METH-1 seems to be involved in progression of pancreatic cancer through local invasion and lymph node metastasis.

Immunosuppressive effect of long-term drainage of thoracic duct on immunological memory in adult thymectomized rats.

Profound reduction of the recirculating lymphocyte pool using thoracic duct drainage (TDD), a method developed by Gowans et al, has been shown to be of limited immunosuppressive value when applied in experimental as well as in clinical settings across major histocompatibility antigen complex (MHC) differences. This limitation is due to the observation that animals, in particular mice, are normally not able to have the drainage last longer than 8 to 10 days. However, using a simple modification of TDD, we have established a long-term TDD method, ie, more than 20 days. Combining this long-term TDD with adult thymectomy, we have examined the life span of naive and memory T cells specific for the minor histocompatibility antigen H-Y in female lewis rats. Furthermore, we demonstrated that memory T cells specific for the H-Y antigen do not appear to be recirculating lymphocytes.

Comparison of the vector systems for gene transduction into rat dendritic cells and peritoneal exudate cells.

Specialized antigen-presenting cells (APC), known as dendritic cells (DC), play a pivotal role in initiating primary immune responses. It has been reported that several vector systems, including adenoviral vectors, retroviral vectors, Hemagglutinating Virus of Japan (HVJ)-related vectors, and electroporation, are able to transduce genes into mouse and human DC. This has not been achieved for rat DC. To our knowledge, there is no direct evidence to support the view that the currently used vector systems are able to transduce genes into mature DC. Because most, if not all, gene transfer studies investigating DC or DC-related cell populations are carried out using heterogeneous groups of cells, it is therefore very important to determine to what extent gene transduction occurs in rat DC, and also selected mature DC (CD161a+ fully mature DC). In this study, we provide evidence that none of 4 vector systems are able to transfer genes into fully mature rat DC, which are derived from bone marrow cells (BMC), driven by Flt3/Flk2 ligand and interleukin (IL)-6, and purified by CD161a. Nevertheless, the most efficient gene transduction was observed in the developing DC progenitor cells during the long-term culture of rat BMC, and its gene expression was successfully achieved after 2 weeks of culture only with a human immunodeficiency virus (HIV)-based lentiviral vector system. The most critical time point for lentiviral gene transduction was around the 7th day from the beginning of culture with lentiviral vectors. Rat peritoneal exudate cells (PEC) and another cell line (K562) were easily transducted by adenoviral vectors and lentiviral vectors.

Isolation of hepatocyte-like cells from mouse embryoid body cells.

We previously reported that embryoid body (EB) cells derived from embryonic stem (ES) cells are capable of differentiating into functional hepatocyte-like cells both in vitro and in vivo. Because transplantation of EB-derived cells into the liver via the spleen resulted in a low incidence of teratoma formation, purification of hepatocyte-like cells is required to prevent teratoma formation. The aim of this study was to purify hepatocyte-like cells from cultured EBs. For the isolation of hepatocyte-like cells, EBs cultured for 15 days were treated with trypsin-EDTA. The disaggregated cells were plated on a gelatin-coated dish as a monolayer. These cells were separated by Percoll gradient centrifugation, enriched by magnetic cell sorting, and purified by FACS. The purified hepatocyte-like cells in monolayer cultures were positive for immunostaining for albumin and expressed albumin mRNA, but not Oct3/4 mRNA. Transplantation of the purified hepatocyte-like cells derived from mouse ES cells might be an effective treatment for liver failure.

Beneficial effect of tetrahydrobiopterin on the survival of rats exposed to hepatic ischemia-reperfusion injury.

INTRODUCTION: The protective role of nitric oxide (NO) against hepatic ischemia-reperfusion (I/R) injury remains controversial. In this study we investigated the effect of tetrahydrobiopterin (BH4) on the survival of rats exposed to an hepatic I/R injury. METHODS: The rats were subjected to 100 minutes of 70% hepatic ischemia 30 minutes after administration of BH4 or saline. A specific inducible NO synthase (iNOS) blocker, 1400W, was used to evaluate endogenous iNOS. NOS protein measured the histological appearance of the liver by Western blotting, and survival was evaluated after reperfusion. RESULTS: The 1-week survival rate was 60% among the BH4 group and 10% for the saline group. The serum ALT and bilirubin values in the BH4 group were significantly lower than the saline group. Histological examination of the liver revealed only a small necrotic area in the BH4 group as opposed to massive necrosis and cell infiltration in the saline group. Injection of 1400W significantly decreased the prolongation of survival produced by BH4. CONCLUSIONS: BH4 significantly improved the survival rate, the histological findings, and the liver function, thereby reducing liver failure. Western blotting showed a higher level of iNOS protein in the BH4 group than the saline group, 1400W suppressed this effect of BH4. Taken together, these observations suggest that NO derived from reactions driven by BH4-induced iNOS exerts a protective effect against reperfusion injury.

Phenotype and functional identity of GM-CSF-independent dendritic cells generated by long-term propagation of DC progenitor cells in bone marrow cells and skin Langerhans cells.

Evidence is provided that dendritic cells (DC) generated by either long-term bone marrow cell (BMC) culture with Flt3L and interleukin-6 (IL-6), or after short-term BMC culture with granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), contain heterogeneous cell populations of admixed DC and Mphi, regardless of the cytokine source. By employing GM-CSF-independent culture systems with the aid of Flt3/Flk-2 ligand and IL-6 and phenotypic characterization of BMC-derived DC and skin Langerhans cells (LC), revealed similar phenotypes. Furthermore, CD103 (OX62), which is widely used for rat DC separation, was found to be insufficient to enrich DC, due to downregulation of the marker. In this regard, the most efficient selection of rat DC, was obtained by CD161a (NKR-P1A), a member of the C-type lectin family. Despite the phenotypic similarity with BMC-derived DC, the nucleus of LC showed a distinct morphology. A large population of DC generated by Flt3L/IL-6 from GM-CSF receptor-deficient mice by do not express NK1.1 (NKR-P1B and NKR-P1C). The profiles for BMC-derived DC were the same as for skin Langerhans cells.

Studies on the most efficient vector systems for gene transduction into dendritic cells.

Specialized antigen-presenting cells (APC), known as dendritic cells (DC), play a pivotal role in initiating primary immune responses. Several vector systems, including adenoviral vectors, retroviral vectors, hemagglutinating virus of Japan-related vectors, and the electroporation, have been shown to transduce genes into mouse and human but not rat DC. However, there is no direct evidence to support the view that the currently used vector systems are able to transduce genes into mature DC. Inasmuch as most, if not all, gene transfer studies investigating DC or DC-related cell populations are performed employing heterogeneous-groups of cells, it is therefore important to determine the extent to which gene transduction occurs in bona fide DC. In this study, we provide evidence that none of these vector systems are able to transfer genes into mature rat DC, which are derived from bone marrow cells (BMC), driven by Flt3/Flk2 ligand and IL-6, and purified with CD161a. Nevertheless, the most efficient gene transduction was observed with developing DC progenitor cells during long-term culture of rat BMC. Successful gene transfer was achieved after 2-week culture with an HIV-based lentiviral vector system.

Teratoma formation and hepatocyte differentiation in mouse liver transplanted with mouse embryonic stem cell-derived embryoid bodies.

We previously reported that mouse embryonic stem (ES) cells are capable of differentiating into hepatocytes in cultured embryoid bodies (EBs) and that hepatocytes generate in the recipient liver injected with cultured day-9 EB cells via spleen without the formation of a teratoma. Because ES cells frequently form teratomas in recipient mice, we investigated incidence of teratoma formation when day-9 EBs derived from ES cells were transplanted directly into the subcapsule of mouse liver. In contrast to injection of day-9 EB cells through the portal vein via the spleen, direct subcapsular injection of cultured day-9 EB cells into liver, and even of cultured day-15 EBs, resulted in an high incidence of teratoma in the liver. In teratomas of livers injected directly with day-15 EBs, hepatocytes were detected singly and in clusters. These results imply that undifferentiated cells capable of developing into teratomas exist in cultured EBs, and even in cultured day-15 EBs containing differentiated hepatocytes.

Protection by vascular endothelial growth factor against sinusoidal endothelial damage and apoptosis induced by cold preservation.

BACKGROUND: It is well known that sinusoidal endothelial cell (SEC) damage during cold preservation of liver tissue is closely involved in early graft failure. The objective of this study was to investigate the involvement of apoptosis in the SEC damage induced by cold preservation and to demonstrate the protective effect of vascular endothelial growth factor (VEGF) on SEC injury, including apoptotic changes. METHODS: Isolated SECs and liver tissue of Wistar rats were cold-preserved in University of Wisconsin (UW) solution, and the protective effect of VEGF was then investigated. Isolated SECs were cultured for 24 hr, and divided into the following 3 groups: Group A, in which the cells were cultured for an additional 27 hr, Group B, in which the cells were cold-preserved in UW solution for 3 hr, and then recultured for 24 hr, and Group C, in which 20 ng/ml of VEGF was added to both the culture medium and the UW solution of cells cultured according to the Group B protocol. Each group of SECs was morphologically examined using the phase contrast microscopic method and the transmission electron microscopic method (TEM), and quantitatively analyzed using the WST-1 assay. Rat livers were cold-preserved in UW solution and divided into the VEGF(+) group and the VEGF(-) group, depending on whether VEGF was added or not. Each group of livers were analyzed by scanning electron microscopic method (SEM) after 24 hr of preservation. The hyaluronic acid uptake rate (HUR) was also determined after 6 hr of preservation. After 24 hr of preservation and 6 hr of reperfusion, tissues were examined by TEM and by the terminal deoxynucleotidyl transferase d-uridine triphosphate nick end labeling (TUNEL) assay. RESULTS: The phase contrast microscopic method and the WST-1 assay showed a protective effect of VEGF against the injury to isolated SECs during cold preservation and subsequent reculturing. Apoptosis was detected immediately by TEM after isolation of SECs, and the number of apoptotic cells increased with the incubation time. This increase was accelerated after cold preservation. The scanning electron microscopic method and the hyaluronic acid uptake rate showed a protective effect of VEGF against SEC damage in the cold-preserved livers. In the liver tissue, the TEM and the TUNEL assay detected apoptosis of SECs only after cold preservation and subsequent reperfusion. VEGF suppressed the apoptosis of SECs induced by cold preservation in both isolated cells and liver tissue. CONCLUSIONS: We demonstrated that SEC damage in the cold preservation of liver tissue was caused mainly by apoptosis, which required subsequent reperfusion. Moreover, isolated SECs showed spontaneous occurrence of apoptotic changes during culture, and these changes were accelerated by the preceding cold preservation. This is the first report to demonstrate the apoptotic changes of SECs seen here were inhibited by VEGF.

Pathogenic role of Kupffer cell activation in the reperfusion injury of cold-preserved liver.

The present study was designed to investigate the possible participation of Kupffer cells in the development of reperfusion injury of the cold-stored liver graft. In the cold preservation of Kupffer cells with Euro-Collins solution, the proportion of asialo-GM1-positive cells was significantly increased at 12 and 24 hr of storage, and the TNF alpha-producing activity in these cells was approximately fivefold greater than control. Northern blot analysis demonstrated that TNF alpha mRNA was remarkably elevated in the reperfusion of the cold-preserved liver, although that of the prereperfused graft was only slightly induced. The reperfusion experiments of the cold-stored liver graft showed that addition of anti-TNF alpha antibody to the perfusate suppressed the elevation of the effluent levels of GOT and LDH significantly, and that pretreatment with a Kupffer cell inhibitor, gadolinium chloride, inhibited the increase of these enzymes in the effluents almost completely. Histological study revealed deposition of a fibrinlike substance in the sinusoid and the central veins extensively in the reperfused liver graft, whereas no apparent deposition was observed in the gadolinium-pretreated liver. Thus, the present study showed that Kupffer cells were primed by cold preservation with Euro-Collins solution, and then activated when the reperfusion was done. It seems likely that the Kupffer cell activation induced by cold preservation/reperfusion plays a major role in reperfusion injury with sinusoidal microcirculatory disturbance, and that TNF alpha is responsible for the impairment of the reperfused liver graft.

Involvement of thromboxane A2-thromboxane A2 receptor system of the hepatic sinusoid in pathogenesis of cold preservation/reperfusion injury in the rat liver graft.

This study was designed to investigate the possible involvement of the thromboxane A2 (TXA2)-TXA2 receptor (TXA2R) system of the hepatic sinusoid in cold preservation/reperfusion injury in liver grafts. Rat livers were preserved in cold University of Wisconsin solution for either 6 or 24 hr. The number of TXA2Rs in sinusoidal endothelial cells isolated from 0-, 6-, and 24-hr preserved liver specimens was 22.50 +/- 1.80 x 10(3)/cell, 12.66 +/- 1.00 x 10(3)/cell, and 4.17 +/- 0.65 x 10(3)/cell, respectively. Kd and Bmax at 0 hr, 6 hr, and 24 hr of preservation were 8.54 +/- 1.26 nM and 37.34 +/- 3.01 fmol/10(6) cells, 7.08 +/- 1.14 nM and 12.66 +/- 1.00 fmol/10(6) cells, and 1.91 +/- 0.10 nM and 3.88 +/- 0.59 fmol/10(6) cells, respectively. The administration of OKY-046 (inhibitor of TXA2 synthesis) to the University of Wisconsin solution suppressed this reduction in TXA2R number. Furthermore, the concentration of TXA2 in hepatic sinusoid was decreased by OKY-046. In a reperfusion experiment, liver tissue preserved for 24 hr exhibited a higher reperfusion pressure, and effluent levels of both aspartate aminotransferase and lactate dehydrogenase were markedly elevated. The addition of OKY-046 to the preservation solution, however, prevented the rise in reperfusion pressure almost completely and the increase in effluent enzyme levels. This study showed that the TXA2Rs in sinusoidal endothelial cells were internalized through binding with TXA2 during cold preservation, causing activation of the TXA2-TXA2R system. This activation apparently induces an increase in reperfusion pressure, possibly due to sinusoidal contraction, resulting in microcirculatory disturbances.(ABSTRACT TRUNCATED AT 250 WORDS)