Low-affinity nerve growth factor receptor p75NTR immunoreactivity in the myocardium with sympathetic hyperinnervation

INTRODUCTION: We previously demonstrated the relationship between sympathetic nerve density in myocardium and the occurrences of ventricular arrhythmia. Nerve growth factor (NGF) regulates myocardial sympathetic innervation. However, it is unclear whether the NGF high-affinity receptor tyrosine kinase A (TrkA) and the NGF low-affinity receptor p75NTR are altered in the state of sympathetic hyperinnervation in the heart. The aim of this study was to determine the density and location of TrkA and p75NTR in canine ventricles with sympathetic hyperinnervation. METHODS AND RESULTS: Myocardial sympathetic hyperinnervation was induced by local infusion of NGF into myocardium or left stellate ganglia, or chronic subthreshold electric stimulation to the left stellate ganglia. The results showed that TrkA immunoreactivity was absent in the myocardium. Low-affinity receptor p75NTR immunoreactivity was present in axons, Schwann cells, and interstitial cells of sympathetic nerves, as well as in interstitial cells of the myocardium. The density of p75NTR immunolabeled myocardial interstitial cells at the NGF infusion site was lower than that at the site remote from NGF infusion, yet the sympathetic nerve density was higher at the infusion site than the remote area. The density of p75NTR also was lower in the myocardium with high sympathetic nerve density, induced by NGF infusion or chronic electric stimulation of the left stellate ganglia, compared to control groups. CONCLUSION: The data indicate that p75NTR may be the main NGF receptor in the myocardium, and p75NTR immunopositive interstitial cells may have a role in regulating sympathetic nerve growth in canine heart.     

姜黄素对人肝星状细胞中神经生长因子及其低亲和力受体表达的影响

目的观察姜黄素对人肝星状细胞(HSC)中神经生长因子(NGF)及其低亲和力受体P75NTR表达的影响,探讨姜黄素逆转肝纤维化的可能机制。方法体外培养人HSC株LX-2,分别给予0~80μmol/L不同浓度的姜黄素作用于LX-2,MTT法检测细胞增殖;选取30~50μmol/L姜黄素分别作用于LX-2,细胞免疫化学染色检测NGF和P75NTR表达,计算阳性细胞率。结果 10~20μmol/L姜黄素作用下LX-2增殖与对照组相比,差异无统计学意义(P0.05);在30~80μmol/L浓度范围内,LX-2增殖显著降低(P0.05);在30~50μmol/L浓度范围内,NGF蛋白表达率分别为(46.59±8.15)%、(90.75±4.72)%、(83.32±4.78)%,对照组为(27.11±5.50)%;P75NTR为(37.17±4.71)%、(88.74±6.27)%、(83.79±4.70)%,对照组为(24.33±5.17)%,差异均有统计学意义(P0.01)。结论姜黄素可诱导人HSC中NGF和P75NTR的蛋白表达,这可能是姜黄素逆转肝纤维化的机制之一。     

High nerve growth factor receptor (p75NTR) expression is a favourable prognostic factor in paediatric B cell precursor-acute lymphoblastic leukaemia

Nerve growth factor (NGF) plays a pivotal role in cellular survival/death decisions with the low affinity receptor p75NTR predominately transmitting anti-proliferative signals. In spite of its established role in B-cell function and identification as a prognostically favourable marker in a number of malignancies, little is known about the expression pattern and prognostic significance of p75NTR in B cell precursor-acute lymphoblastic leukaemia (BCP-ALL). p75NTR expression was prospectively studied on primary ALL-blasts in a cohort of paediatric patients with common ALL (n = 86) and preB-ALL (n = 34) treated within the Co-operative study group for childhood acute lymphoblastic leukaemia (CoALL) protocol, CoALL06-97. Flow cytometric analysis showed that almost half of the patients expressed no or negligible amounts of p75NTR (<10%). The median expression in patients expressing p75NTR beyond that threshold was 49% (range 11-100%). In patients classified as low-risk at diagnosis, p75NTR expression was significantly higher than in high-risk patients (P = 0.001). Of note, p75NTR expression was lower in the 21 patients who subsequently developed relapse compared with those remaining in remission (P = 0.038). Accordingly, relapse-free survival was significantly better in patients expressing high surface p75NTR (P = 0.041). Thus, in this prospective analysis, high p75NTR expression was a strong prognostic marker that identified a group of paediatric ALL patients with favourable outcome.     

Kinetic changes of liver regeneration and hepatocellular carcinoma cells after partial hepatectomy in rats

We investigated, using rats, the effect of partial hepatectomy (PH) on hepatocellular carcinoma (HCC, KDH-8 and AH-66) cells, and the effect of HCC cells on the regeneration of remaining hepatocytes after PH. Our results showed that PH significantly enhanced the growth of HCC cells in rats. Tumor volume increased more significantly in the partially hepatectomized group (H-group) than in the control group, and the tumor wet weights on the 14th postoperative day were significantly higher in the H-group than in the control group. Such an enhanced growth effect of PH on the injected (s.c) HCC cells was related to an abrupt increase of tumor volume within 24 hours after operation, which was supported by the mitotic indices (MI) of the KDH-8 cells. These phenomena of the enhanced growth of the HCC cells following PH were not observed at all in rats injected with estrogen receptor (ER)-negative mammary carcinoma (SST-2) or nonepithelial fibrosarcoma (KMT-75) cells. The MIs of the remaining hepatocytes after PH increased abruptly at the 30th postoperative hour and reached a maximum at the 36th postoperative hour, and the MIs were significantly higher in the H-group with the KDH-8 cells than in the H-group without them from the 42th to the 60th postoperative hour. In the control group, the MIs of hepatocytes were not regardless of the presence of KDH-8 cells. From these results, we speculate that some growth factor(s) induced by PH may act on injected (s.c.) HCC cells, and that the other growth factor(s) secreted by HCC cells may act on the regenerating hepatocytes after PH. This work was supported in part by a grant from the Japanese Ministry of Education. Science and Culture.     

神经生长因子抑制肝星状细胞增殖的观察

目的 观察神经生长因子(NGF)对大鼠肝星状细胞(HSC)增殖的抑制作用,并探讨其作用机制. 方法 体外培养大鼠肝星状细胞株HSC-T6,将HSC-T6与不同浓度NGF孵育后,用XTT比色法检测NGF对HSC增殖的影响,流式细胞术分析NGF对HSC细胞周期的影响,透射电镜观察经100 ng/ml NGF作用24h后HSC形态学变化.结果 (100、200、400) ng/ml浓度时NGF对HSC的抑制作用经XTT法测得A值分别为0.66±0.03、0.69±0.03和0.66±0.03,与对照组(0.73±0.01)比较,差异具有统计学意义(P＜0.05),但无浓度依赖性(P＞0.05).100、200、400ng/ml NGF作用于HSC 24h后,G2期比例分别为14.83％±5.41％、14.73％±2.50％和14.87％±2.06％,与对照组(7.47％±4.39％)比较,明显增加(P＜0.05),透射电镜可以见到细胞凋亡的形态学变化. 结论 NGF可抑制HSC增殖,通过使HSC细胞周期停滞于G2期而抑制HSC增殖可能为其作用机制之一;经NGF作用的大鼠肝星状细胞可出现明显的增殖受抑、细胞凋亡的形态学改变.     

Effect of serotonin receptor 2 blockage on liver regeneration after partial hepatectomy in the rat liver.

The effect of serotonin receptor 2 blockade (5-HT(2)) on liver regeneration after 30-34% and 60-70% partial hepatectomy in the rat liver was investigated. Materials and methods: Male Wistar rats were subjected to 60-70% (group I) and 30-34% (group II) partial hepatectomy. Serotonin receptor 2 blockade was exerted by intraperitoneal administration of ketanserin at different doses and time points after partial hepatectomy. The rats of all groups were killed at different time points until 96 h after partial hepatectomy. The rate of liver regeneration was evaluated by the mitotic index in hematoxylin and eosin sections, the immunochemical detection of Ki67 and proliferating cell nuclear antigens, the rate of [(3)H]-thymidine incorporation into hepatic DNA and liver thymidine kinase enzymatic activity. Results: Liver regeneration peaked at 24 and 32 h after partial hepatectomy in 60-70% hepatectomized rats. In 30-34% hepatectomized rats liver regeneration peaked at 60 h, whereas low rates of regenerative activity were observed between 24 and 72 h after partial hepatectomy. Ketanserin administration arrested liver regeneration only when administered at 16 h after 60-70% partial hepatectomy. Ketanserin also abrogated the observed peak of regenerative activity at 60 h in 30-34% hepatectomized rats when administered at 52 h after partial hepatectomy. All indices of liver regeneration were affected by ketanserin administration. Conclusions: Serotonin receptor 2 blockade can arrest liver regeneration only when administered close to G1/S transition point, and that while serotonin may be a cofactor for DNA synthesis, it does not play a role in initiation of liver regeneration.     

Rat peripheral mononuclear cell thymidine kinase activity increases during liver regenerative processes after partial hepatectomy

Deoxythymidine kinase (TK; EC 2.7.1.21) activity in the liver has been used as a marker of liver regeneration after partial hepatectomy. In this study we examined TK activity of various organs, plasma and peripheral blood mononuclear cells (PMNC) in 70% partially hepatectomized rats. TK activity of lymph nodes, small intestine, heart, lung, kidney and thymus did not increase significantly during the course of the study, except for spleen at 72 h. On the other hand, PMNC-TK and liver cystolic TK activity increased in a parallel fashion at all times after partial hepatectomy; they began to increase 12 h after surgery and peaked 48 h post-surgery. Fractionation of PMNC into T cells and B cells revealed that both populations increased and peaked 48 h post-surgery. Plasma TK peaked 12–24 h after surgery, then declined at 36, 48 and 72 h after partial hepatectomy. This change paralleled plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). PMNC-TK activity correlated significantly with liver cystolic TK activity 24 h (r = 0.743; P < 0.05) and 48 h (r = 0.708; P < 0.05) after partial hepatectomy. However, it did not correlate with plasma levels of TK, AST and ALT. The results indicate that in the early stage of liver regeneration PMNC-TK may provide a marker of liver regenerative processes.     

Mechanism of impairment on liver regeneration in elderly patients: Role of hepatic stellate cell function

Japan, along with most other countries in the world, is facing an increasingly aging population with a prolonged life expectancy. Concurrently, the need for medical intervention, including hepatectomy, has also increased for the elderly. Although surgical outcomes for older patients are reported to be comparable with those for younger patients, additional care in the selection of older patients for hepatectomy is considered necessary. Although the effect of aging on human liver regeneration is not fully understood, the regeneration of liver tissue after hepatectomy in elderly patients is shown to be generally worse than in younger patients and, to date, the mechanisms involved in the impairment of liver regeneration have not been fully clarified. Hepatic stellate cells (HSCs) are liver‐specific mesenchymal cells that play critical roles in liver physiology and fibrogenesis. Recent studies in liver regeneration have increasingly focused on HSCs rather than on hepatocytes, Kupffer cells, endothelial cells, or infiltrating immune cells and suggest that HSCs might play a critical role in liver regeneration. In this review, we summarize the mechanisms involved in the impairment of liver regeneration in elderly patients, especially focusing on HSCs. We also discuss how HSCs contribute to the impairment of liver regeneration.     

Beneficial effects of Kampo medicine Inchin-ko-to on liver function and regeneration after hepatectomy in rats.

Aim: Inchin-ko-to (ICKT), Kampo medicine, is known to inhibit hepatocyte apoptosis as well as promote the secretion and excretion of bile. The aim of this study is to clarify the effects of ICKT on liver function and hepatic regeneration after massive hepatectomy in rats. Methods: Male Wistar rats received 2 g/kg ICKT from 3 days preoperatively and underwent 90% hepatectomy. Liver sections were stained using immunohistochemistry (hemeoxygenase-1 [HO-1], alpha-smooth muscle actin [SMA], and proliferating cell nuclear antigen [PCNA]). Results: The survival period was significantly prolonged, and the remnant liver/body weight ratio was significantly increased postoperatively in the ICKT group. The values of transaminase, total bile acid, and total bilirubin were significantly improved in the ICKT group. In the ICKT group, PCNA and HO-1 were strongly expressed early postoperatively, but the expression of alpha-SMA was weak. Conclusion: The preoperative administration of ICKT has been suggested to provide beneficial effects in promoting hepatic regeneration and preventing postoperative hepatic failure. The reduced activation of stellate cells may be involved in their mechanisms.     

Segmental cholangitis impairs hepatic regeneration capacity after partial hepatectomy in rats

Background:

Patients with hilar cholangiocarcinoma or hepatolithiasis often develop segmental cholangitis (SC), but it is unclear whether hepatectomy for patients with SC can be performed safely.

Methods:

Rats were subjected to segmental bile duct ligation (SBDL) with LPS (SC group) or a saline (Sham group) infusion into the bile duct of the ligated lobes. The rats were sacrificed at 3, 24 and 48 h after the SBDL. For another experiment, the rats were subjected to partial hepatectomy (PHx) for the ligated lobes. Hepatic regeneration rates and the expression of regeneration-associated genes were evaluated.

Results:

In the SC group, severe parenchymal damage was observed in the acute phase (3 h). Altered gene expression in the liver in response to biliary infection occurred not only in the infected lobes but also in the non-infected lobes. In the rats of the SC group, both the hepatic regeneration rate and serum HGF levels were significantly lower than in the Sham group.

Conclusion:

These results clearly demonstrate that SC impairs the regeneration capacity of the contralateral remnant liver. Therefore, hepatectomy should be avoided for patients with SC even if it occurs in the part of the liver to be resected.     

Modulation of the expression of galactose-specific receptors on Kupffer cells after partial hepatectomy and zymosan stimulation

ABSTRACT— Partial hepatectomy (PH) and a single intravenous injection of zymosan were used to provoke expansion of the Kupffer cell population in rat liver. The number of Kupffer cells per unit of area of liver increased after both stimulations. During these growth phases the number of binding sites for galactose-exposing ligands was studied on Kupffer cell plasma membranes. Both partial hepatectomy and zymosan stimulation affected the binding of lactosylated bovine serum albumin conjugated to gold particles (LacBSA-Au₅). The reduction observed depended on the time after the PH or zymosan injection studied. Low levels of binding sites were found at 24 h from PH and 5 days after zymosan stimulation and represented only 1/4 or 1/10 of the binding sites expressed on the cell surface of Kupffer cells from normal adult rats. In addition, in both experimental situations we observed that the clustered distribution of gold particles typical of adult Kupffer cell was changed.     

Effects of heparin on hepatic regeneration and function after partial hepatectomy in rats

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Somatostatin inhibits pro-inflammatory cytokine secretion from rat hepatic stellate cells.

BACKGROUND/AIMS: Activated hepatic stellate cells (HSCs) have been implicated in hepatic fibrosis. Somatostatin (SOM) has an immunomodulatory role. The aim of this study was to assess the secretion of pro-inflammatory cytokines by HSCs and to determine the effect of SOM on the secretion of these mediators. METHODS: Activated rat HSCs were evaluated for their secretion of IL-1beta, IL-8, and TNF-alpha using ELISA. RNA protection assay was used to determine cytokine mRNA levels. The expression of chemokine and cytokine mRNA and the secretion of these mediators were assessed following incubation with SOM or octreotide. RESULTS: HSCs spontaneously secreted IL-1beta, IL-8, and TNF-alpha. This secretion was augmented following stimulation by IL-1beta and TNF-alpha. SOM inhibited the spontaneous and TNF-alpha-induced secretion of IL-1beta, IL-8, and TNF-alpha and suppressed the expression of IL-1beta and TNF-alpha mRNA. Octreotide suppressed the secretion of IL-1beta and IL-8. CONCLUSIONS: These observations indicate that SOM exerts an inhibitory immunomodulatory effect on HSCs.     

Hepatocarcinoma cells stimulate the growth,migration and expression of pro‐angiogenic genes in human hepatic stellate cells

Background: Activated hepatic stellate cells (HSC) and other fibrogenic cell types are frequently found around hepatocellular carcinoma. It is unknown whether hepatocarcinoma cells regulate the biological functions of HSC. Aims: This study aimed to investigate the paracrine effects of hepatocarcinoma cells on human HSC using a co‐culture system. Methods: Huh7 or HepG2 cells, human hepatocarcinoma cell lines, were co‐cultured with primary human HSC. Intracellular calcium mobilization, proliferation, migration, expression of pro‐angiogenic and fibrogenic genes, smooth muscle α‐actin (α‐SMA) protein expression, inflammatory properties (nuclear factor kappa B activation and interleukin 8 secretion) and intracellular signalling pathways (AKT and ERK) were analysed in HSC. Results: Culture of HSC with Huh7 cells for 24 h stimulated HSC proliferation, migration and expression of pro‐angiogenic genes. The migration effect was corroborated with HepG2 cells. The effects of Huh7 cells on cell proliferation and migration were mediated mainly by PI3K/AKT activation. Moreover, Huh7 cells reduced the expression of genes involved in fibrogenesis, while they did not modify the inflammatory properties of HSC. The expression of α‐SMA was induced by Huh7 cells. Because hepatitis C virus (HCV) infection is a major cause of hepatocarcinoma, we next investigated whether these effects are regulated by the expression of HCV in hepatocarcinoma cells. Expression of a subgenomic replicon expressing HCV nonstructural proteins (NS3–NS5) in Huh7 cells did not affect paracrine actions in HSC (cell proliferation and migration). Conclusions: These results suggested that there is a cross‐talk between hepatocarcinoma cells and HSC. Activated HSC may be stimulated by cancer cells to accumulate and express angiogenic genes.     

Fenretinide stimulates the apoptosis of hepatic stellate cells and ameliorates hepatic fibrosis in mice

Aim: To investigate whether fenretinide, a clinically proved apoptosis‐inducing chemopreventive agent in tumor cells, can induce apoptosis in hepatic stellate cells (HSCs) and resolve hepatic fibrosis. Methods: CCl₄‐induced liver fibrosis in mice and rat activated hepatic stellate cells (HSC‐T6) as well as hepatocytes (BRL‐3A) were studied. Results: The duplex staining of proliferating cell nuclear antigen and α‐ smooth muscle actin or terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling and α‐ smooth muscle actin demonstrated that fenretinide executed its anti‐fibrosis effect in liver by inducing apoptosis rather than inhibiting proliferation of HSCs, while it had no apparently apoptotic effect on hepatocytes. Fenretinide could elicit apoptosis of HSC‐T6 in vitro at the concentration range from 0.5 to 5 µM, but at higher concentrations ≥5 µM was required to induce apoptosis in hepatocytes (BRL‐3A). Conclusion: Further studies using malondialdehyde measurement, Western blot, antioxidant, inhibitors for p53, caspase 8 and 9 – as well as anti‐Fas neutralizing antibody – have shown that in HSC‐T6, fenretinide‐induced apoptosis involves a reactive oxygen species (ROS)‐generated, P53‐independent, mitochondria‐associated intrinsic pathway, whereas in hepatocytes (BRL‐3A), a ROS‐generated, P53‐dependent, Fas‐related extrinsic pathway is triggered only at high concentration.