Naoki Uyama

Fascin, a novel marker of human hepatic stellate cells, may regulate their proliferation, migration, and collagen gene expression through the FAK-PI3K-Akt pathway

Fascin is a component of actin bundles and may regulate various cellular events. The expression and function of fascin in human hepatic stellate cells (HSCs) has remained largely uncharacterized. Fascin expression in human liver tissue was studied using immunohistochemistry. To identify cells expressing fascin, double immunofluorescent staining with vimentin, α-smooth muscle actin (α-SMA), or fibulin-2 was performed and analyzed with confocal microscopy. In culture experiments, fascin expression and the phosphorylation of focal adhesion kinase (FAK) and Akt in LX-2 cells, a cell line of human HSCs, were investigated using western blot. Specific siRNAs were used to reduce the expression of fascin in LX-2 cells. Proliferation and migration were assayed with a CyQuant assay kit and a Matrigel-coated culture insert system, respectively. Levels of matrix metalloproteinase (MMP)-2 and collagen mRNAs were examined using quantitative RT-PCR. Immunohistochemistry revealed the expression of fascin along sinusoids and overlapping with vimentin and α-SMA in both non-fibrotic and fibrotic liver tissue, but it was almost absent in periportal myofibroblastic cells and did not colocalize with fibulin-2, a marker of portal myofibroblasts. In addition, fascin immunoreactivity was almost undetectable in septa of fibrotic human liver tissue. The expression of fascin in LX-2 cells was confirmed using western blot. Two different specific siRNAs against fascin significantly reduced the number of viable LX-2 cells to 65% compared with control cultures and downregulated the mRNAs levels of types I and III collagen and MMP-2 to 62%, 65%, and 70% of control levels, respectively. This condition also reduced the migration activity of LX-2 cells to 46% of control cells and the phosphorylation level of both FAK and Akt. Fascin may be an excellent novel marker of human HSCs that distinguishes HSCs from periportal myofibroblasts. Fascin may regulate functions of human HSCs through the FAK-phosphoinositide 3-kinase-Akt pathway.

Angiogenesis is crucial for liver regeneration after partial hepatectomy.

BACKGROUND Recent studies of hepatic regeneration have mainly focused on the growth of parenchymal cells. However, remodeling of liver vessels seems to be crucial during hepatic regeneration. In this study, we investigated the influence of antiangiogenesis on hepatic regeneration using sFlt-1, a soluble receptor for vascular endothelial growth factor that acts as a dominant negative receptor, and the hepatocyte growth factor antagonist NK4. METHODS A sFlt-1-expressing adenoviral vector, an NK4-expressing adenoviral vector, or both combined were infected into C57BL6 mice via the tail vein. A 70% partial hepatectomy was performed on all of the mice 48 hours after infection. The remnants of the liver were removed after the partial hepatectomy, and hepatic regeneration was assessed by measuring the remnant liver weight and hepatocyte mitosis, bromodeoxyuridine staining, immunohistochemical staining with anti-platelet endothelial cell adhesion molecule-1 antibodies, and real-time polymerase chain reaction studies for angiogenic factors. RESULTS The immunohistochemical staining for CD31 showed suppression of sinusoidal endothelial cells growth in sFlt-1-expressing adenoviral vector-and NK4-expressing adenoviral vector-infected mice. Increases in the remnant hepatic weight were significantly lower in the sFlt-1-expressing adenoviral vector-infected mice. The bromodeoxyuridine index and mitotic cell results revealed a significant decrease in hepatic regeneration in the sFlt-1-expressing adenoviral vector-and NK4-expressing adenoviral vector-infected mice. The suppressive effects on hepatic regeneration were significantly enhanced by combined sFlt-1-expressing adenoviral vector and NK4-expressing adenoviral vector infection. Real-time polymerase chain reaction results revealed the significant suppression of angiogenic growth factor receptors Tie-1 and Tie-2. CONCLUSION The angiogenesis inhibitor significantly suppressed hepatic regeneration. These results suggest that hepatic regeneration after hepatectomy closely correlates with angiogenesis.

Splenectomy attenuates murine liver fibrosis with hypersplenism stimulating hepatic accumulation of Ly-6Clo macrophages

BACKGROUND & AIMS Splenectomy in cirrhotic patients has been reported to improve liver function; however the underlying mechanism remains obscure. In the present study, we investigated the mechanism using a murine model, which represents well the compensated liver cirrhosis. METHODS C57BL/6 male mice were allowed to drink water including thioacetamide (TAA: 300 mg/L) ad libitum for 32 weeks. After splenectomy at 32 weeks, mice were sacrificed on days one, seven, and 28, respectively, while TAA-administration was continued. Perioperative changes in peripheral blood and liver tissues were analyzed. RESULTS TAA treatment of mice for 32 weeks reproducibly achieved advanced liver fibrosis with splenomegaly, thrombocytopenia, and leukocytopenia. After splenectomy, liver fibrosis was attenuated, and macrophages/monocytes were significantly increased in peripheral blood, as well as in the liver. Progenitor-like cells expressing CK-19, EpCAM, or CD-133 appeared in the liver after TAA treatment, and gradually disappeared after splenectomy. Macrophages/monocytes accumulated in the liver, most of which were negative for Ly-6C, were adjacent to the hepatic progenitor-like cells, and quantitative RT-PCR indicated increased canonical Wnt and decreased Notch signals. As a result, a significant amount of β-catenin accumulated in the progenitor-like cells. Moreover, relatively small Ki67-positive hepatic cells were significantly increased. Protein expression of MMP-9, to which Ly-6G-positive neutrophils contributed, was also increased in the liver after splenectomy. CONCLUSIONS The hepatic accumulation of macrophages/monocytes, most of which are Ly-6C(lo), the reduction of fibrosis, and the gradual disappearance of hepatic progenitor-like cells possibly play significant roles in the tissue remodeling process in cirrhotic livers after splenectomy.

A Case of Biliary Cystadenocarcinoma with Ovarian Like Stroma

症例は63歳の女性で，約8か月前より肝嚢胞を指摘され経過観察されていた．2009年6月，CA19-9が上昇したため精査目的にて入院となった．腹部超音波,CT，MRIにて肝左葉に嚢胞性病変を認め，嚢胞壁の一部に壁肥厚を認めた．FDG-PETを施行したところ嚢胞内部に異常集積像を認めた．腫瘍マーカーの上昇およびFDG-PETの異常集積像より胆管嚢胞腺癌と診断し，肝左葉切除術を施行した．切除標本では嚢胞の一部に壁肥厚を認め，嚢胞内部に血腫および粘液を認めた．病理組織学的検査では胆管嚢胞腺癌であり，卵巣様間質を伴っていた．術後合併症は認めず，術後12か月の現在，再発なく健在である．卵巣様間質を伴う胆管嚢胞腺癌は極めてまれな疾患であり，若干の文献的考察を加え報告する．

Analysis of unique liver volume restoration after laparoscopic fenestration of liver cysts

Laparoscopic fenestration is a standard procedure for the treatment of non‐parasitic liver cysts. After fenestration, the remnant liver often restores its volume. However, no systematic analysis of the phenomenon exists. In the present study, the pattern of liver volume restoration after laparoscopic fenestration of liver cysts was analyzed, and the mechanism for the unique restoration was investigated.

Immunohistochemical characterization of cancer-associated fibroblasts at the primary sites and in the metastatic lymph nodes of human intrahepatic cholangiocarcinoma

Cancer-associated fibroblasts (CAFs) are an important constituent of the cancer stroma. In intrahepatic cholangiocarcinoma (ICC), the features of CAFs at the primary site and in the metastatic lymph nodes (Met-LNs) and their origin have been unclear. In the present study, we characterized CAFs at the primary site (n = 42) and in the Met-LNs (n = 10) of human ICC by immunohistochemistry using potential molecular markers of CAFs, portal fibroblasts (PFs), hepatic stellate cells (HSCs), and bone marrow-derived fibrocytes (BMDFs). At the primary site, the stroma was strongly positive for α-smooth muscle actin (α-SMA; marker for CAFs), platelet-derived growth factor receptor-β (PDGFR-β) (common marker for HSCs and PFs), fibulin-2, and thymus cell antigen-1 (Thy-1; PF marker), whereas immunoreactivity for fascin (HSC marker) was scarce. Most of the α-SMA-positive cells were found to express PDGFR-β, Thy-1, and fibulin-2 by double immunostaining. A small population of BMDF marker-positive (α-SMA+CD45+CD34+) cells was found by triple immunostaining. In the micro-Met-LNs, α-SMA-positive cells were absent in cancer aggregates of the LN sinus, whereas they were present in the invasion area of cancer cells from the LN sinus to the LN parenchyma. In the macro-Met-LNs, there were abundant α-SMA-positive cells that were also positive for PDGFR-β and Thy-1 but negative for fibulin-2 and fascin. Thus, regarding the expression of molecular markers, CAFs at the primary site of ICC are similar to PFs and different from those of HSCs or CAFs in the Met-LNs. CAFs at the primary sites and in the Met-LN are thought to be derived from PFs/BMDFs and resident cells of LNs, respectively.

AB061. P033. A comparison of delayed gastric emptying and nutritional status after pylorus-preserving versus stomach-preserving pancreaticoduodenectomy

Background: This study was performed to compare the incidence of delayed gastric emptying (DGE), postoperative outcome and long-term nutritional status between pyloruspreserving pancreaticoduodenectomy (PPPD) and subtotal stomach-preserving pancreaticoduodenectomy (SSPPD). Methods: We retrospectively analyzed 133 patients who undergoing PPPD (n=89) or SSPPD (n=44) between March 2011 and December 2015. All cases of duodenojejunostomy in PPPD and gastrojejunostomy in SSPPD were performed antecolically. The postoperative nutritional status was explored by changes ratio in the body weight, serum total protein and serum albumin for 1 year after surgery. Results: The overall incidence of the DGE was 12%. The incidence of DGE was 13.5% (grade A: 5.6%, grade B: 4.5%, grade C: 3.4%) in PPPD group and 9.1% (grade A: 4.5%, grade B: 4.5%, grade C: 0%) in SSPPD group, and was no significant differences in both groups. The mean postoperative hospital stay was 42.8 days in the PPPD group and 37.2 days in the SSPPD group, and was no significant differences in both groups. The body weight ratio was decreased at 6 months after surgery in the SSPPD group, whereas it continued to decrease at 9 months after surgery in the PPPD group. It was gradually increased 9 months later after surgery in SSPPD group, and it was increased 12 months later after surgery in PPPD group. The serum total protein ratio and serum albumin ratio were decreased at 3 months after surgery and were gradually increased 6 months later after surgery in both groups. There were no significant differences with regard to postoperative body weight ratio, serum total protein ratio and serum albumin ratio in both groups for 1 year after surgery. Conclusions: SSPPD is equivalent outcomes in incidence of DGE and in postoperative long-term nutritional status comparing PPPD.