Short-term experience with aspiration embolectomy using a ACE68 reperfusion catheter to treat embolic superior mesenteric artery occlusion.

We report the short-term results with aspiration embolectomy using an ACE68 reperfusion catheter to treat patients with acute embolic superior mesenteric artery (SMA) occlusion. Our study included 4 consecutive male patients ranging in age from 72 to 86 years (mean age 79 years). In all patients, the main trunk of the SMA was occluded. The technical success rate was 100% for all procedures. There were no major procedure-related complications. One patient underwent laparotomy with intestinal resection after successful recanalization. No patient reported clinical symptoms of abdominal ischemia at follow-up. Our short-term experience shows that percutaneous aspiration embolectomy using an ACE68 reperfusion catheter is an effective treatment for acute mesenteric ischemia.

Role of hepatocyte growth factor activator (HGF activator) in invasive growth of human glioblastoma cells in vivo.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that is involved in invasive growth of tumor cells via its receptor MET, a protein product of c-met proto-oncogene. HGF activator (HGFA) is a serine proteinase responsible for the activation of proform of HGF/SF (proHGF/SF). In our study, we examined the effects of engineered expression of HGFA on 2 human glioblastoma cell lines (YKG-1 and U251). Both cells expressed MET, while only YKG-1 expressed endogenous proHGF/SF. Enhanced MET phosphorylation and increased migratory activity were induced by the expression of HGFA in YKG-1 cells in vitro in the presence of thrombin, which is a known activator of proHGFA. In contrast, MET phosphorylation was consistently observed in U251 that lacked endogenous HGF/SF, suggesting ligand-independent activation of MET in this cell line. Consequently, the expression of HGFA in U251 did not enhance the MET phosphorylation and following cellular response even with the thrombin treatment. However, addition of exogenous proHGF/SF resulted in enhanced migratory activity of HGFA-expressing U251 cells in the presence of thrombin in vitro. The engineered HGFA expression resulted in significantly enhanced tumor growth with increased vascular density in vivo when YKG-1 cells were implanted in nude mouse brain. This effect was not observed in U251 lacking endogenous proHGF/SF. These results indicate the possible existence of multiple mechanisms of MET activation in glioblastomas and that the activation system of proHGF/SF is important in progression of glioblastomas that express endogenous proHGF/SF and require ligand-dependent MET activation.

Diverse roles of hepatocyte growth factor activator inhibitor type 1 (HAI-1) in the growth of glioblastoma cells in vivo.

Hepatocyte growth factor activator inhibitor type-1 (HAI-1) is an integral-membrane proteinase inhibitor. In this study, we examined the effects of HAI-1 on human glioblastoma cells. Two glioblastoma cell lines (YKG-1, U251) were stably transfected with expression plasmid harboring mature membrane-form or truncated secreted-form HAI-1. Culture characteristics were not altered by the expression of HAI-1, whereas in vitro invasiveness of U251 was suppressed. On the other hand, the expression of membrane-form HAI-1 resulted in significantly enhanced tumorigenicity of both cell lines in vivo. In contrast, secreted-form HAI-1 did not promote the tumorigenicity. These results suggest that HAI-1 may play complex roles in progression of glioblastoma cells, and membrane-form HAI-1 may mediate an undefined important signaling in the cells.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is required for branching morphogenesis in the chorioallantoic placenta.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-associated Kunitz-type serine proteinase inhibitor that was initially identified as a potent inhibitor of hepatocyte growth factor activator. HAI-1 is also a cognate inhibitor of matriptase, a membrane-associated serine proteinase. HAI-1 is expressed predominantly in epithelial cells in the human body. Its mRNA is also abundant in human placenta, with HAI-1 specifically expressed by villous cytotrophoblasts. In order to address the precise roles of HAI-1 in vivo, we generated HAI-1 mutant mice by homozygous recombination. Heterozygous HAI-1+/- mice underwent normal organ development. However, homozygous HAI-1-/- mice experienced embryonic lethality which became evident at embryonic day 10.5 postcoitum (E10.5). As early as E9.5, HAI-1-/- embryos showed growth retardation that did not reflect impaired cell proliferation but resulted instead from failed placental development and function. Histological analysis revealed severely impaired formation of the labyrinth layer, in contrast all other placental layers, such as the spongiotrophoblast layer and giant cell layer, which were formed. Our results indicate that mouse HAI-1 is essential for branching morphogenesis in the chorioallantoic placenta and lack of HAI-1 function may result in placental failure.

Regeneration of injured intestinal mucosa is impaired in hepatocyte growth factor activator-deficient mice.

BACKGROUND AND AIMS: Hepatocyte growth factor activator (HGFA) is a serum proteinase that specifically converts an inactive single-chain form of hepatocyte growth factor (HGF) into an active 2-chain form. HGFA is produced in its precursor form and then activated in injured tissues. To address the precise role of HGFA and to investigate the mechanisms of HGF activation in injured tissues, we generated mice deficient in HGFA. METHODS: HGFA-deficient mice were generated using targeted gene disruption. The regenerating process of intestinal mucosa damaged by oral administration of dextran sodium sulfate (DSS) or by rectal administration of acetic acid was examined in both HGFA-deficient and control mice. HGF processing activity was analyzed using Western blotting and an HGF activation assay. RESULTS: Homozygous mutant mice were viable and fertile without obvious abnormalities. When mice were treated with 3% DSS in drinking water for 6 days followed by distilled water without DSS, 72% of HGFA-deficient mice died through day 12 while 75% of control mice survived injury. Similar results were also observed in the acetic acid-induced intestinal injury; the survival rate was 36.6% in HGFA-deficient mice and 84.2% in control mice. In HGFA-deficient mice, the injured mucosa was not sufficiently covered by regenerated epithelium and the activation of HGF was impaired in the injured colon. CONCLUSIONS: These results indicate that HGFA is required for repair of injured intestinal mucosa but is not essential for normal development during embryogenesis or after birth.

Establishment and characterization of a new human glioblastoma cell line, NYGM.

A cell line designated NYGM was established from a human cerebral glioblastoma multiforme (GBM) obtained from a 75-year-old Japanese woman. The cell line has grown slowly without interruption and has been propagated continuously by serial passages (more than 80 passage) during the past 3 years. The cultured cells were fusiform or polyhedral in shape. The population doubling time was 24 hours. The chromosomal number varied between 77 and 88, with modal chromosomal number of 84. NYGM cells concomitantly expressed MET receptor tyrosine kinase (a product of c-met protooncogene) and its ligand HGF/SF (hepatocyte growth factor/scatter factor), as well as HGF activator and HGF activator inhibitors. The cells might be useful for the study of pericellular regulation of HGF/SF-MET signaling and HGF activation of GBM cells.

Roles of hepatocyte growth factor (HGF) activator and HGF activator inhibitor in the pericellular activation of HGF/scatter factor.

The activation of hepatocyte growth factor (HGF)/scatter factor (SF) in an extracellular milieu is a critical limiting step in HGF/SF-induced signaling that is believed to have important roles in invasive growth of tumor cells and regeneration of injured tissue. This activation is caused by a proteolytic cleavage at the bond between Arg494-Val495 in the single-chain HGF/SF precursor, generating an active two-chain heterodimeric form. The HGF activator (HGFA) is a coagulation factor XII-like serine proteinase critically involved in this process in injured tissues including tumor tissues. In the past several years, the identification of endogenous HGFA inhibitors (HAIs) has provided detailed knowledge of the regulation of HGFA activity. Currently, two types of HAIs, namely HAI-1 and HAI-2, have been reported. Both are Kunitz-type serine proteinase inhibitors and inhibit not only HGFA but also other serine proteinases, such as membrane-type serine protease 1 (matriptase), plasmin, trypsin and kallikreins. HAIs are of particular interest because they are synthesized as type-I transmembrane proteins. Therefore, HAIs must have important regulatory roles in a cell surface proteolytic reaction, which has emerged as an important mechanism for the generation of biologically active proteins mediating a diverse range of cellular functions. This review is a summary and interpretation of recent data regarding the regulation of pericellular HGF/SF activation mediated by HGFA and HAIs and includes a discussion of the possible role of the type I transmembrane Kunitz-type inhibitor in pericellular proteolysis.

Characterization of transcripts generated from mouse hepatocyte growth factor activator inhibitor type 2 (HAI-2) and HAI-2-related small peptide (H2RSP) genes: chimeric mRNA transcribed from both HAI-2 and H2RSP genes is detected in human but not in mouse.

We previously reported a novel small gene, designated hepatocyte growth factor activator inhibitor type 2 (HAI-2) related small peptide (H2RSP), in the process of the search for splicing variant forms of HAI-2 by 3(')-rapid amplification of cDNA ends method [Biochem. Biophys. Res. Commun. 288 (2001) 390]. Human H2RSP gene consisted of four exons spanning approximately 1kbp and was located in 11kbp downstream of HAI-2 gene. In this study, we cloned and characterized the mouse counterpart of H2RSP gene, which was located in 6.6kbp downstream of mouse HAI-2 gene, and analyzed the transcripts generated from both genes. Similar to human, mouse H2RSP mRNA (0.5kb) was detected abundantly in various tissues including the gastrointestinal tract, and has nuclear localization signal (NLS) in the lysine-rich region (exon 4), which was well-conserved between human and mouse genes. However, chimeric mRNA transcribed from both HAI-2 (exons 1-7) and H2RSP (exons 2-4) genes, which was found in the kidney, prostate, and placenta of human by Northern blot analysis, was not detected in mouse tissue even by a reverse transcription-polymerase chain reaction (RT-PCR). Instead of the chimeric mRNA, a novel splicing variant lacking putative transmembrane domain of HAI-2 was found in mouse but not in human as a putative secrete form of HAI-2. These results suggest that the organization of H2RSP and HAI-2 gene complex is well-conserved, but the usage of these genes was quite different between human and mouse.