Mitsuhiko Abe

Copper incorporation into ceruloplasmin is regulated by Niemann–Pick C1 protein

Aim:  Wilson disease is a genetic disorder of copper metabolism characterized by impaired biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. Our previous study showed the late endosome localization of ATP7B and described the copper transport pathway from the late endosome to trans‐Golgi network (TGN). However, the cellular localization of ATP7B and copper metabolism in hepatocytes remains controversial. The present study was performed to evaluate the role of Niemann–Pick type C (NPC) gene product NPC1 on intracellular copper transport in hepatocytes.

Hepatitis C virus core protein upregulates the expression of vascular endothelial growth factor via the nuclear factor‐κB/hypoxia‐inducible factor‐1α axis under hypoxic conditions

Aim:  Hepatitis C virus (HCV) core protein critically contributes to hepatocarcinogenesis, which is often observed in liver cirrhosis. Since the liver cirrhosis microenvironment is affected by hypoxia, we focused on the possible driving force of HCV core protein on signal relay from hypoxia‐inducible factor (HIF)‐1α to vascular endothelial growth factor (VEGF).

Human peripheral blood CD34-positive cells enhance therapeutic regeneration of chronically injured liver in nude rats.

We investigated whether transplantation of purified human peripheral blood CD34+ cells could reduce established liver fibrosis and up‐regulate therapeutic regeneration. Human peripheral blood CD34+ cells were isolated from total mononuclear cells of healthy volunteers by magnetic cell sorting. Recipient nude rats were injected intraperitoneally with carbon tetrachloride (CCl4) twice weekly for 3 weeks before single administration of CD34+ cells. CCl4 was then re‐administered twice weekly for 3 more weeks, and the nude rats were sacrificed. Saline (control group), 1 × 105 (low‐dose group), 5 × 105 (middle‐dose group), or 2 × 106 (high‐dose group) CD34+ cells/kg body weight were intrasplenically transplanted after CCl4 treatment for 3 weeks. Reverse transcriptase‐polymerase chain reaction analysis of the freshly isolated CD34+ cells revealed the expression of CD31, keratin19, α‐smooth muscle actin (α‐SMA), and epithelial growth factor, but not other liver related markers. The transplanted cells differentiated into vascular and sinusoidal endothelial cells, and vascular smooth muscle cells. CD34+ cell transplantation reduced liver fibrosis in a dose‐dependent fashion, with decreased collagen type‐I and α‐SMA‐positive cells after 6 weeks of CCl4 treatment by Mallorys Azan and immunohistochemical staining. Gelatin zymography showed that the expression levels of active matrix metalloproteinase‐2 and ‐9 in CD34+ cell transplanted livers were significantly stronger than those in saline‐infused livers. In recipients of high‐doses of CD34+ cells, the number of PCNA‐positive hepatocyte increased 6 weeks after CCl4 treatment compared with saline‐infused livers. We conclude that human peripheral blood CD34+ cell transplantation halts established liver fibrosis and promotes hepatic regeneration in CCl4‐induced chronic liver injury. J. Cell. Physiol. 227: 1538–1552, 2012.

Vandetanib, an Inhibitor of VEGF Receptor-2 and EGF Receptor, Suppresses Tumor Development and Improves Prognosis of Liver Cancer in Mice

Purpose: VEGF, EGF, and TGF-α are expressed in hepatocellular carcinomas (HCC) and play a role in its growth. Vandetanib, a multikinase inhibitor, suppresses the phosphorylation of VEGF receptor 2 (VEGFR-2) and EGF receptor (EGFR). The aim of this study was to clarify the antitumor effect of vandetanib in mouse HCCs. Experimental Design: We evaluated the effects of vandetanib on proliferation of human umbilical vein endothelial cells (HUVEC) and three hepatoma cell lines, as well as the phosphorylation of VEGFR-2 and EGFR in these cells. Mice were implanted with hepatoma cells subcutaneously or orthotopically in the liver and treated with 50 or 75 mg/kg vandetanib. We analyzed the effects of treatment on tumor cell proliferation and apoptosis, vessel density, phosphorylation of VEGFR-2 and EGFR, and production of VEGF, TGF-α, and EGF in tumor tissues. Adverse events on vandetanib administration were also investigated. Results: Vandetanib suppressed phosphorylation of VEGFR-2 in HUVECs and EGFR in hepatoma cells and inhibited cell proliferation. In tumor-bearing mice, vandetanib suppressed phosphorylation of VEGFR-2 and EGFR in tumor tissues, significantly reduced tumor vessel density, enhanced tumor cell apoptosis, suppressed tumor growth, improved survival, reduced number of intrahepatic metastases, and upregulated VEGF, TGF-α, and EGF in tumor tissues. Treatment with vandetanib was not associated with serious adverse events, including alanine aminotransferase abnormality, bone marrow suppression, or body weight loss. Conclusions: The antitumor effects of vandetanib in mice suggest that it is a potentially suitable and safe chemotherapeutic agent for HCCs. Clin Cancer Res; 18(14); 3924–33. ©2012 AACR.

Transplantation of endothelial progenitor cells ameliorates vascular dysfunction and portal hypertension in carbon tetrachloride‐induced rat liver cirrhotic model

In cirrhosis, sinusoidal endothelial cell injury results in increased endothelin‐1 (ET‐1) and decreased nitric oxide synthase (NOS) activity, leading to portal hypertension. However, the effects of transplanted endothelial progenitor cells (EPCs) on the cirrhotic liver have not yet been clarified. We investigated whether EPC transplantation reduces portal hypertension.

Pigment Epithelium-Derived Factor Inhibits Lysosomal Degradation of Bcl-xL and Apoptosis in HepG2 cells

Pigment epithelium-derived factor (PEDF) has several biological actions on tumor cells, but its effects are cell-type dependent. The aim of this study was to examine the pathophysiological role of PEDF in hepatocellular carcinoma (HCC). PEDF expression was examined in various hepatoma cell lines and human HCC tissues, and was seen in various hepatoma cell lines including HepG2 cells. In human HCC tissues, PEDF expression was higher than in adjacent non-HCC tissues. In addition, serum PEDF levels were higher in HCC patients than in non-HCC patients, and curative treatment of HCC caused significant reductions in serum PEDF levels compared with pretreatment levels. In vitro experiments, camptothecin (CPT) was used to induce apoptosis and the effect of PEDF was investigated by knockdown of the PEDF gene in CPT-treated HepG2 cells. Knockdown of the PEDF gene enhanced CPT-induced apoptosis, simultaneously down-regulating Bcl-xL expression in HepG2 cells. Expression of apoptosis-related molecules and effects of bafilomycin A1 on CPT-induced apoptosis were also examined in PEDF gene knockdown HepG2 cells. Treatment with bafilomycin A1 suppressed CPT-induced decreases in Bcl-xL expression and increases in apoptosis in PEDF gene knockdown HepG2 cells. PEDF may, therefore, exert anti-apoptotic effects through inhibition of lysosomal degradation of Bcl-xL in CPT-treated HepG2 cells.

Prevention of liver fibrosis and liver reconstitution of DMN‐treated rat liver by transplanted EPCs

Eur J Clin Invest 2012; 42 (7): 717–728

CD34+ cell therapy is safe and effective in slowing the decline of hepatic reserve function in patients with decompensated liver cirrhosis

Preclinical studies in rodent models of chronic liver fibrosis have shown that transplantation of peripheral blood (PB) CD34+ cells leads to hepatic regeneration and a reduction of liver fibrosis by suppressing hepatic stellate cell activity and increasing matrix metalloproteinase activity. The aim of this study was to examine the safety and clinical efficacy of intrahepatic transplantation of autologous granulocyte colony‐stimulating factor (G‐CSF)‐mobilized PB‐CD34+ cells in patients with decompensated liver cirrhosis.

Ablation of endothelial VEGFR1 improves metabolic dysfunction by inducing adipose tissue browning

Angiogenesis plays an instrumental role in the modulation of adipose tissue mass and metabolism. Targeting adipose vasculature provides an outstanding opportunity for treatment of obesity and metabolic disorders. Here, we report the physiological functions of VEGFR1 in the modulation of adipose angiogenesis, obesity, and global metabolism. Pharmacological inhibition and genetic deletion of endothelial VEGFR1 augmented adipose angiogenesis and browning of subcutaneous white adipose tissue, leading to elevated thermogenesis. In a diet-induced obesity model, endothelial-VEGFR1 deficiency demonstrated a potent anti-obesity effect by improving global metabolism. Along with metabolic changes, fatty liver and insulin sensitivity were also markedly improved in VEGFR1-deficient high fat diet (HFD)–fed mice. Together, our data indicate that targeting of VEGFR1 provides an exciting new opportunity for treatment of obesity and metabolic diseases, such as liver steatosis and type 2 diabetes.

High expression of the putative cancer stem cell marker, DCLK1, in rectal neuroendocrine tumors.

Doublecortin-like kinase 1 (DCLK1), a microtubule-associated protein, is known to regulate neuronal differentiation, migration and neurogenesis. Recent evidence suggests that the protein is a putative marker for intestinal and pancreatic stem cells, including their cancer stem cell counterparts. The present study conducted immunohistochemical analyses for DCLK1 and the stemness marker, NANOG, in human intestinal neuroendocrine tumors (NETs), as their expression had not been previously investigated in these tumors. Eighteen patients with endoscopically resected rectal NETs were enrolled in the study. The mean age of the patients was 51 years old. The mean diameter of the resected tumors was 5.2 mm, and a histological diagnosis of NET grade G1 was formed for all tumors. Immunohistochemical analysis was performed not only for DCLK1, but also for the known NET markers, synaptophysin, chromogranin A and cluster of differentiation (CD)56. The intensity and distribution of staining were scored on a scale of 0–3 and 0–2, respectively. The sum of the scores was calculated for each specimen. Co-expression of DCLK1 and NANOG was also examined. The mean scores for DCLK1 and synaptophysin were significantly higher than those for chromogranin A (P<0.0001) and CD56 (P<0.01). There were no significant differences in the scores between DCLK1 and synaptophysin or between chromogranin A and CD56. Notably, NANOG was expressed in high quantities in all the tumor tissues studied, showing clear co-expression with DCLK1. In conclusion, DCLK1 may be a novel marker for rectal NET, potentially indicating the presence of the stemness gene product, NANOG.