Hepatocyte Growth Factor Activator Research Articles

Hepatocyte growth factor activator inhibitor type 2 (HAI-2) modulates hepcidin expression by inhibiting the cell surface protease matriptase-2

Matriptase-2, a recently identified cell surface protease, is the key enzyme of iron homoeostasis modulating the expression of the liver peptide hormone hepcidin. HAI (hepatocyte growth factor activator inhibitor) types 1 and 2 (HAI-1 and HAI-2 respectively) have been shown to inhibit the close homologue, i.e. matriptase. By co-expressing matriptase-2 and the inhibitor HAI-2 we have identified HAI-2 displaying high inhibitory potential against matriptase-2 at the cell surface as well as in conditioned medium. Accordingly, complex formation between matriptase-2 and HAI-2 was demonstrated by isolation of the complex via immobilizing either HAI-2 or matriptase-2 from lysates and conditioned medium of co-expressing cells. Furthermore, HAI-2 indirectly influences the expression of the hepcidin-encoding gene HAMP. The inhibitor abrogates the matriptase-2-mediated suppression of HAMP expression, presumably by inhibiting the supposed potential of matriptase-2 to cleave membrane-bound HJV (haemojuvelin). Taken together, the results of the present study have characterized HAI-2 as an inhibitor of matriptase-2 that modulates the synthesis of hepcidin and provides new insights into the regulatory mechanism of iron homoeostasis, with clinical importance for a treatment of iron overload diseases.

The novel phosphoinositide 3‐kinase–mammalian target of rapamycin inhibitor, BEZ235, circumvents erlotinib resistance of epidermal growth factor receptor mutant lung cancer cells triggered by hepatocyte growth factor

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, is a critical problem in the management of patients with EGFR mutant lung cancer. Several mechanisms have been reported involved in this acquired resistance, including hepatocyte growth factor (HGF) activation of an alternative pathway. PI3K and mTOR are downstream molecules of receptor tyrosine kinases, such as EGFR and Met, and are thought to be ideal targets for controlling various tumor types. We assessed whether BEZ235, a dual inhibitor of PI3K and mTOR, could overcome the EGFR-TKI resistance induced by HGF in an EGFR mutant lung cancer model. Exogenous and endogenous HGF triggered resistance to erlotinib in the PC-9 and HCC827, EGFR mutant lung cancer cell lines. BEZ235 alone inhibited the viability of PC-9 and HCC827 cells in vitro, irrespective of the presence or the absence of HGF. Using a xenograft model of severe combined immunodeficient mice with HGF-gene-transfected PC-9 cells (PC-9/HGF), we found that BEZ235 inhibited tumor growth, whereas erlotinib did not. BEZ235 monotherapy also inhibited the phosphorylation of Akt and p70S6K/S6RP, downstream molecules of PI3K and mTOR, respectively, as well as suppressing tumor-cell proliferation and angiogenesis of PC-9/HGF tumors. These results suggest that BEZ235, even as monotherapy, may be useful in managing HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

Hepatocyte Growth Factor Activator Inhibitor-1 Is Induced by Bone Morphogenetic Proteins and Regulates Proliferation and Cell Fate of Neural Progenitor Cells

BackgroundNeural progenitor cells (NPCs) in the developing neuroepithelium are regulated by intrinsic and extrinsic factors. There is evidence that NPCs form a self-supporting niche for cell maintenance and proliferation. However, molecular interactions and cell-cell contacts and the microenvironment within the neuroepithelium are largely unknown. We hypothesized that cellular proteases especially those associated with the cell surface of NPCs play a role in regulation of progenitor cells in the brain.Methodology/Principal FindingsIn this work, we show that NPCs, isolated from striatal anlage of developing rat brain, express hepatocyte growth factor activator inhibitor-1 and -2 (HAI-1 and HAI-2) that are cell surface-linked serine protease inhibitors. In addition, radial glia cells derived from mouse embryonic stem cells also express HAI-1 and HAI-2. To study the functional significance of HAI-1 and HAI-2 in progenitor cells, we modulated their levels using expression plasmids or silencing RNA (siRNA) transfected into the NPCs. Data showed that overexpression of HAI-1 or HAI-2 decreased cell proliferation of cultured NPCs, whilst their siRNAs had opposite effects. HAI-1 also influenced NPC differentiation by increasing the number of glial fibrillary acidic protein (GFAP) expressing cells in the culture. Expression of HAI-1 in vivo decreased cell proliferation in developing neuroepithelium in E15 old animals and promoted astrocyte cell differentiation in neonatal animals. Studying the regulation of HAI-1, we observed that Bone morphogenetic protein-2 (BMP-2) and BMP-4 increased HAI-1 levels in the NPCs. Experiments using HAI-1-siRNA showed that these BMPs act on the NPCs partly in a HAI-1-dependent manner.ConclusionsThis study shows that the cell-surface serine protease inhibitors, HAI-1 and HAI-2 influence proliferation and cell fate of NPCs and their expression levels are linked to BMP signaling. Modulation of the levels and actions of HAI-1 in NPCs may be of a potential value in stem cell therapies in various brain diseases.

Abstract B24: Hepatocyte growth factor activator inhibitor type 1 (HAI-1) suppresses tumor growth and metastasis of pancreatic adenocarcinoma cells

Abstract Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound serine proteinase inhibitor expressed on epithelial and carcinoma cell surface. HAI-1 regulates hepatocyte growth factor activator, kallikrein 1-related peptidase 4 and 5 and several membrane-anchored serine proteinases such as matriptase, prostasin, hepsin, TMPRSS13 and human airway trypsin-like protease. These proteases are reported to be involved in tumor progression, and among them, matriptase and prostasin are known to activate protease-activated receptor-2 (PAR-2). Previously, we reported that S2-CP8 cells, a metastatic variant of SUIT-2 pancreatic adenocarcinoma cells, showed decreased HAI-1 expression compared to SUIT-2 and acquired EMT phenotype. To assess the HAI-1 function in metastasis of S2-CP8 cells, we established a stable S2-CP8 subline that expressed HAI-1 under control of tetracycline-regulated promoter and analyzed in a nude mouse orthotopic xenograft model. S2-CP8 cells expressed matriptase, TMPRSS13, TMPRSS4, prostasin and PAR-2. Matrigel invasion assay revealed that S2-CP8 cells showed enhanced invasion compared to SUIT-2 cells. The enhanced invasiveness was significantly suppressed by forced HAI-1 expression. Moreover, the HAI-1 loss-induced invasiveness was partly alleviated by knockdown of matriptase or PAR-2. Then, we examined the effect of HAI-1 expression on metastasis in vivo using an orthotopic xenograft model. Four weeks after the transplantation, mice were sacrificed and prepared for histological evaluation. We found that 50% of control mice developed distant metastasis. However, mice treated with tetracycline to express HAI-1 did not develop the metastasis. Though there was no difference in cell proliferation rates in vitro, tumor size was decreased by the expression of HAI-1. These results suggested that, in SUIT-2 pancreatic adenocarcinoma cell line, loss of HAI-1 enhances cell invasiveness, at least in part, through PAR-2/matriptase system in vitro, and HAI-1 plays a suppressive role in metastasis of the cells in vivo. Citation Format: Jing-Jia Ye, Makiko Kawaguchi, Yukihiro Haruyama, Ai Kanemaru, Tsuyoshi Fukushima, Hiroaki Kataoka. Hepatocyte growth factor activator inhibitor type 1 (HAI-1) suppresses tumor growth and metastasis of pancreatic adenocarcinoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B24.

Hepatocyte growth factor activator inhibitor-2 prevents shedding of matriptase

Hepatocyte growth factor activator inhibitor-2 (HAI-2) is an inhibitor of many proteases in vitro, including the membrane-bound serine protease, matriptase. Studies of knock-out mice have shown that HAI-2 is essential for placental development only in mice expressing matriptase, suggesting that HAI-2 is important for regulation of matriptase. Previous studies have shown that recombinant expression of matriptase was unsuccessful unless co-expressed with another HAI, HAI-1. In the present study we show that when human matriptase is recombinantly expressed alone in the canine cell line MDCK, then human matriptase mRNA can be detected and the human matriptase ectodomain is shed to the media, suggesting that matriptase expressed alone is rapidly transported through the secretory pathway and shed. Whereas matriptase expressed together with HAI-1 or HAI-2 accumulates on the plasma membrane where it is activated, as judged by cleavage at Arg614 and increased peptidolytic activity of the cell extracts. Mutagenesis of Kunitz domain 1 but not Kunitz domain 2 abolished this function of HAI-2. HAI-2 seems to carry out its function intracellularly as this is where the vast majority of HAI-2 is located and since HAI-2 could not be detected on the basolateral plasma membrane where matriptase resides. However, minor amounts of HAI-2 not undergoing endocytosis could be detected on the apical plasma membrane. Our results suggest that Kunitz domain 1 of HAI-2 cause matriptase to accumulate in a membrane-bound form on the basolateral plasma membrane.

Pathologic Effects of Hepatocyte Growth Factor, Hepatocyte Growth Factor Activator, and Arginine in a Pilot Rodent Model of Inflammatory Bowel Disease

Pathologic Effects of Hepatocyte Growth Factor, Hepatocyte Growth Factor Activator, and Arginine in a Pilot Rodent Model of Inflammatory Bowel Disease

Hepatocyte growth factor as an indicator of neonatal maturity

Hepatocyte growth factor (HGF) is a potent mitogen and morphogen expressed in fetal organs. It requires activation by specific serine proteases. The role of activated HGF during fetal development has not been evaluated. We hypothesized that the serum levels of activated HGF would be increased in preterm neonates. Cord blood total HGF and activated HGF levels were measured in 19 preterm (gestational age, 28.04±2.39 weeks) and 24 term (gestational age, 39.37±0.95 weeks) newborns. Anthropometric parameters and metabolic indices were evaluated. Activated HGF was higher in preterm than in term neonates (0.81±0.05 vs. 0.61±0.06 ng/mL, p<0.05), whereas total HGF levels did not differ significantly between groups. In addition, total and activated HGF further increased by ∼40% in preterm neonates 12 h after birth. Finally, activated HGF correlated inversely with gestational age (r=-0.369; p=0.015) and birth weight (r=-0.440; p=0.003). Our study demonstrates that regulation of HGF activity and circulating HGF levels differ between term and preterm neonates along fetal development.

Imaging a functional tumorigenic biomarker in the transformed epithelium

Proteases responsible for the increased peritumoral proteolysis associated with cancer represent functional biomarkers for monitoring tumorigenesis. One attractive extracellular biomarker is the transmembrane serine protease matriptase. Found on the surface of epithelial cells, the activity of matriptase is regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Quantitative mass spectrometry allowed us to show that, in selected cancers, HAI-1 expression decreases, leading to active matriptase. A preclinical probe specific for the measurement of emergent active matriptase was developed. Using an active-site-specific, recombinant human antibody for matriptase, we found that the selective targeting of active matriptase can be used to visualize the tumorigenic epithelium. Live-cell fluorescence imaging validated the selectivity of the antibody in vitro by showing that the probe localized only to cancer cell lines with active matriptase on the surface. Immunofluorescence with the antibody documented significant levels of active matriptase in 68% of primary and metastatic colon cancer sections from tissue microarrays. Labeling of the active form of matriptase in vivo was measured in human colon cancer xenografts and in a patient-derived xenograft model using near-infrared and single-photon emission computed tomography imaging. Tumor uptake of the radiolabeled antibody, (111)In-A11, by active matriptase was high in xenografts (28% injected dose per gram) and was blocked in vivo by the addition of a matriptase-specific variant of ecotin. These findings suggest, through a HAI-1-dependent mechanism, that emergent active matriptase is a functional biomarker of the transformed epithelium and that its proteolytic activity can be exploited to noninvasively evaluate tumorigenesis in vivo.

Furin-cleaved Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Is Active and Modulates Low Density Lipoprotein Receptor and Serum Cholesterol Levels

Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg(218)-Gln(219) in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg(218)-Gln(219) more efficiently than furin but also cleaves at Arg(215)-Phe(216). Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser(153)-Arg(218)), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.

Regulation of pericellular proteolysis by hepatocyte growth factor activator inhibitor type 1 (HAI-1) in trophoblast cells

Hepatocyte growth factor activator inhibitor type 1/serine protease inhibitor Kunitz type 1 (HAI-1/SPINT1) is a membrane-bound Kunitz-type serine protease inhibitor that is abundantly expressed on the surface of cytotrophoblasts, and is critically required for the formation of the placenta labyrinth in mice. HAI-1/SPINT1 regulates several membrane-associated cell surface serine proteases, with matriptase being the most cognate target. Matriptase degrades extracellular matrix protein such as laminin and activates other cell surface proteases including prostasin. This study aimed to analyze the role of HAI-1/SPINT1 in pericellular proteolysis of trophoblasts. In HAI-1/SPINT1-deficient mouse placenta, laminin immunoreactivity around trophoblasts was irregular and occasionally showed an intense punctate pattern, which differed significantly from the linear distribution along the basement membrane observed in wild-type placenta. To explore the molecular mechanism underlying this observation, we analyzed the effect of HAI-1/SPINT1 knock down (KD) on pericellular proteolysis in the human trophoblast cell line, BeWo. HAI-1/SPINT1-KD BeWo cells had increased amounts of cellular laminin protein and decreased laminin degradation activity in the culture supernatant. Subsequent analysis indicated that cell-associated matriptase was significantly decreased in KD cells whereas its mRNA level was not altered, suggesting an enhanced release and/or dislocation of matriptase in the absence of HAI-1/SPINT1. Moreover, prostasin activation and pericellular total serine protease activities were significantly suppressed by HAI-1/SPINT1 KD. These observations suggest that HAI-1/SPINT1 is critically required for the cell surface localization of matriptase in trophoblasts, and, in the absence of HAI-1/SPINT1, physiological activation of prostasin and other protease(s) initiated by cell surface matriptase may be impaired.

Expression of Factors in the Hepatocyte Growth Factor (HGF) Pathway in Whole Bone Marrow Biopsies in Association to the Osteolytic Bone Disease of Multiple Myeloma

AbstractAbstract 3977Osteolytic bone disease (OBD) in multiple myeloma (MM) is known to be caused by a combination of osteoclast hyperactivation and osteoblast inhibition. One of the pathways known to be involved in osteoblast inhibition from in vitro studies is the HGF pathway consisting of HGF, its receptor MET, the co-receptor Syndecan-1 (SDC-1), the partial MET antagonist Decorin and HGF activator responsible for HGF processing to its active form. So far, gene expression studies in MM have been performed on isolated MM plasma cells or bone marrow (BM) aspirates, which are not completely representative of the cell composition in the BM micro-environment. We used a novel strategy, whereby gene expression of factors associated with the HGF pathway was evaluated in snap-frozen BM biopsies, and moreover we determined the protein levels in matched BM plasma samples.An additional BM core biopsy obtained during the diagnostic procedure of MM patients was snap-frozen. Biopsies were cut, homogenized and RNA was purified and analyzed by qRT-PCR using low density arrays (Applied Biosystems). The relative quantitative gene expression was calculated using 3 internal reference genes (ABL, GAPDH and GUS). OBD was evaluated using standard radiographs. All patients were untreated and did not receive medicine that could influence bone remodeling. We examined 10 healthy volunteers (HV), 35 monoclonal gammopathy of unknown significance (MGUS) and 65 untreated MM patients, which according to radiographic findings were divided into NO/LOW and advanced OBD, i.e. OBD in ≥2 regions.ELISA was performed on a total of 31 matched BM plasma samples of HV, MGUS and MM obtained at the same time point as the biopsies. In addition, extra samples without gene data (N=52) were analyzed. Commercial kits for SDC-1 (Diaclone), HGF (RnD, Quantikine) and Decorin (RnD, Duoset) were run in duplicates according to manufacturer's instructions.Gene expression of HGF, SDC1, and MET were significantly different comparing HV, MGUS, no/low and advanced OBD (p<0.05) (For HGF, see figure 1). Decorin was not associated to OBD. HGF activator was not expressed in any of our samples, but only in the positive control.A significant correlation between gene and protein expression levels measured by ELISA was found for SDC-1 (Spearman's rho= 0.463, p=0.0058) and HGF (Spearman's rho=0.45, p=0.01). No correlation was found between Decorin gene levels and BM plasma levels (Spearman's rho =-0.24, p=0.22).The protein level of SDC-1 and HGF in BM plasma were both upregulated in MM and associated significantly to OBD level (p<0.05), while Decorin were significantly downregulated in MGUS and MM samples compared to HVs (p<0.05). A significant difference in HGF BM plasma levels were found between patients with no/limited OBD (median: 1.7ng/mL) and advanced OBD (median: 6.2ng/mL) in BM plasma.In our expression study reflecting the in vivo situation in MM patients, genes in the HGF pathway and proteins were significantly associated to OBD. The use of whole snap-frozen BM biopsies is a novel strategy for evaluation of gene expression in MM making it possible to investigate patients independent of degree of MM plasma cell infiltration. In addition to the dys-regulated gene expression levels alteration of SDC-1 and HGF was also observed at protein level, supporting the gene expression findings, and underscoring the usefulness of BM biopsies for gene expression studies in MM. Furthermore, our study for the first time shows up regulation of HGF in association with OBD at both gene and protein level in a large clinical material. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Phase II Study of the c-Met Inhibitor ARQ 197 (Tivantinib) in Patients with Relapsed Multiple Myeloma.

Abstract 2976 Background:c-Met receptor tyrosine kinase (RTK) activity has been implicated in establishing the oncogenic phenotype across several human cancers with high levels of the activating c-Met ligand, hepatocyte growth factor (HGF). Malignant plasma cells secrete HGF-activator (HGFA), which converts HGF to its active form, and high HGF levels are correlated with a poor prognosis in multiple myeloma (MM). Syndecan 1 (CD138) on malignant plasma cells binds HGF and potentiates interleukin-6-induced growth and migration. HGF stimulation of myeloma cells also activates autophosphorylation of c-Met and other critical downstream signaling pathways promoting oncogenesis. Finally, pre-clinical studies have shown that suppression of c-Met signaling with a number of small molecules, including ARQ 197, induced myeloma cell apoptosis. Tevantinib-mediated cytotoxic response was observed at concentrations of less than 5 μM, which are achievable in the clinic. These findings supported the hypothesis that suppression of the HGF/c-Met signaling axis could be a rational strategy against relapsed multiple myeloma. Methods:In this phase II study, the efficacy and safety of ARQ 197, a non-competitive and highly selective inhibitor of the c-Met RTK, is being studied in patients with relapsed multiple myeloma. Primary objectives were to determine the overall response rate (ORR) to single-agent tivantinib in patients with relapsed multiple myeloma who had received one to four prior lines of therapy, and to define the toxicities in this population. ARQ 197 was administered at a starting oral dose of 360 mg twice daily with meals for each day of every 4-week treatment cycle. This dose was selected from prior phase I investigations in solid tumors, and at this dose level, steady-state plasma level sof ARQ-197 were 7 μM. Treatment could continue providing that patients did not experience undue toxicities, or disease progression. Tivantinib is provided through the Cancer Therapy Evaluation Program (CTEP), and this study is supported by CTEP, as well as the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results:A total of 10 patients have been enrolled and treated to date, all of whom were evaluable for toxicity, with 8 evaluable for response based on having completed two treatment cycles. Patients had received from 1–3 prior lines of therapy for their disease, and 7/10 (70%) had presented with International Staging System stage I disease at diagnosis. All patients on study had an ECOG performance status of 1 or better, and received a median of 3.5 cycles of tivantinib (range 1–7). The most common adverse events (AEs) of any grade seen in at least 30% of patients included diarrhea (30%), dizziness (30%), dry eyes (30%), shortness of breath (30%), memory change (30%), myalgias (40%), fatigue (60%), and neutropenia (60%). Serious AEs (SAEs) occurred in 2 patients, including one patient with grade 3 syncope, and another with grade 4 neutropenia and a grade 3 anal fissure. Stable disease (SD) has been seen as the best response in 5/7 (71%) evaluable patients, which was maintained for up to 7 cycles, while the remaining patients showed evidence of disease progression. Conclusion:Enrollment is continuing to this first study of any c-Met inhibitor in patients with relapsed multiple myeloma to better define the role of single-agent tivantinib in this setting. To date, tivantinib has been tolerated well, and some evidence of activity has been seen, with stable disease in 63% of patients, all of whom were progressing at the time of enrollment. Updated toxicity and efficacy data will be presented at the time of the Annual Meeting. Correlative studies are also underway with the goal of identifying potential predictive biomarkers. Disclosures:Off Label Use: Tivantinib is being evaluated for patients with relapsed myeloma, but is not yet approved in this setting.

Antibodies to Human Factor XII with Antithrombotic Properties

AbstractAbstract 1106The plasma protease factor XIIa (FXIIa) contributes to vascular occlusion in murine thrombosis models, at least partly through activation of factor XI (FXI). While there is good correlation between plasma FXI levels and thrombotic events in humans, the situation is not as clear for FXII (the precursor of FXIIa), suggesting fundamental differences in thrombus formation in mice and humans. To facilitate studies on the effects of FXII/XIIa on thrombus formation, we developed novel inhibitory antibodies to human FXII, designated 9A2 and 15H8, by immunizing FXII-deficient mice with human FXII. Using recombinant human FXII molecules that lack various domains, and chimeras in which specific domains in FXII are replaced with those from the related protein hepatocyte growth factor activator, we determined that 9A2 and 15H8 bind to the FXII/XIIa non-catalytic heavy chain at different sites. 9A2 binds on or near the EGF2 domain, while 15H8 binds to the fibronectin type I and/or kringle domain. These areas have been implicated in FXII binding to polyanionic surfaces. Saturating concentrations of 9A2 or 15H8 reduced FXII activity by 50% and 90%, respectively, in an aPTT assay using normal plasma, while combining the antibodies resulted in >95% inhibition. However, in assays in which clot formation was triggered by adding FXIIa directly to plasma, preincubation of FXIIa with either antibody did not prolong the clotting time. Furthermore, neither antibody had a strong effect in a chromogenic assay of FXI activation by FXIIa, indicating the antibodies interfere with the aPTT assay primarily by inhibiting FXII activation. FXII activation in the aPTT assay is initiated by addition of a polyanion such as silica to the plasma to induce contact activation. In vivo, polymers of inorganic phosphate (polyP) may serve a similar function. Contact activation is triggered in plasma when FXII bound to the polyanion is activated, probably by trace amounts of FXIIa or another protease present in the plasma. Once formed, FXIIa converts the zymogens prekallikrein and FXI to the proteases kallikrein and FXIa, both of which can activate additional FXIIa to amplify the process. In the presence of 9A2 or 15H8, activation of pure FXII in the presence of either silica or polyP was significantly reduced. Interestingly, the antibodies actually potentiated FXII activation by kallikrein or FXIa in the absence of a polyanion. Taken as a whole, these results suggest that binding of 9A2 or 15H8 to FXII results in conformational changes that make FXII a better substrate for kallikrein and FXIa, possibly by mimicking the effect of FXII binding to a polyanion, but that prevent activation of FXII by FXIIa (autoactivation), blunting the overall rate of activation. We tested the effects of 9A2 and 15H8 in a mouse model in which thrombotic occlusion of the carotid artery is induced by exposing the vessel to a 3.5% solution of ferric chloride. Wild type C57Bl/6 mice develop arterial occlusion within 5 to 10 minutes, while FXII-deficient mice are resistant to arterial occlusion. Infusion of human FXII into FXII-deficient mice restores the wild type phenotype. 15H8 prevented thrombus formation in mice reconstituted with human FXII, while 9A2 reduced the rate of thrombotic occlusion by 50%. In an ex vivo flow model, perfusion of human blood through collagen-coated tubes at a shear rate of 300 sec−1 results in tube occlusion by platelet and fibrin rich clot in ∼15 minutes. 15H8 effectively blocked fibrin formation and reduced platelet accumulation, preventing tube occlusion. 9A2 was also effective at preventing clot formation, but there was evidence of some fibrin accumulation over time. In summary, the monoclonal anti-human FXII IgGs 9A2 and 15H8 prevent thrombus formation in whole blood in vivo and ex vivo by interfering with FXII activation. Our data support the hypothesis that pharmacologic inhibition of FXII activation may have therapeutic utility in disorders that are driven or aggravated by the blood contact system. Disclosures:No relevant conflicts of interest to declare.

Matriptase Is Highly up-Regulated in Chronic Lymphocytic Leukemia and Promotes Cancer Cell Invasion

AbstractAbstract 4612Proteases are known to play a critical role in cancer development and progression. Matriptase is an epithelial transmembrane serine protease essential for skin development and barrier function. Matriptase overexpression has been reported in many human solid tumors including prostate, breast, ovarian, kidney and lung cancers, indicating that matriptase may play an important role in cancer. In mice, dysregulated matriptase activity promotes malignant transformation and carcinogenesis.In human blood, low levels of matriptase mRNA expression were detected in monocytes and B-lymphocytes but the biological significance is unclear. In this study, we examined matriptase mRNA and protein expression in 15 hematological cancer cell lines and 223 bone marrow samples from patients with leukemia by reverse transcription PCR (RT-PCR), quantitative real-time PCR (qRT-PCR) and Western blotting. Flow cytometry and immunostaining were used to determine the cell membrane expression of matriptase in B cell-derived Namalwa cells and bone marrow cells from leukemia patients. Small hairpin RNA (shRNA) interference and a recombinant matriptase inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1, were used to inhibit matriptase expression and activity in Namalwa, Raji and chronic lymphocytic leukemia (CLL) cells to test the effect of matriptase inhibition on cancer cell invasion in matrigel-based assays.Our results showed that matriptase mRNA was highly expressed in Namalwa and Raji cells and in bone marrow cells from patients with CLL. Such an expression profile was not found in bone marrow cells from patients with acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). By Western blotting, matriptase protein expression was confirmed in bone marrow samples from the majority (79/81, 97.5%) of CLL patients. By immunostaining and flow cytometry, matriptase protein was localized on the surface of Namalwa and CLL cells. In these cells, the matriptase inhibitors, HAI-1 and HAI-2, were not detected, indicating that the upregulated matriptase activity may not be inhibited in CLL cells. In functional studies, silencing matriptase gene expression by specific shRNAs markedly reduced Namalwa and Raji cells and bone marrow cells from CLL patients in invading matrigels. Similarly, a recombinant HAI-1 dose-dependently inhibited matrigel invasion of these cancer cells.In conclusion, our results showed that matriptase was highly expressed on the surface of CLL cells. Inhibition of matriptase by gene silencing or specific inhibitor prevented CLL cells in invading matrigels, indicating that matriptase contributes to the invasiveness of the cancer cells. Our results suggest that matriptase may represent a new cell surface marker for CLL and that blocking matriptase expression or activity may be used as a therapeutic strategy to inhibit CLL progression. Disclosures:No relevant conflicts of interest to declare.

Improvement of metabolic syndrome by irbesartan via the PPARγ/HGF pathway in apolipoprotein E knockout mice

Irbesartan, a partial agonist of peroxisome proliferators activated receptor-γ (PPARγ), has been reported to improve insulin resistance and lipid profile in patients with diabetes mellitus or metabolic syndrome (MS). However, the down effectors of PPARγ have yet to be elucidated. Thus, in this study, we focused on the role of the hepatocyte growth factor (HGF) in the anti-metabolic effects of irbesartan, using apolipoprotein E (ApoE) knockout (KO) mice. ApoE KO mice placed on a high-fat diet (HFD) for 12 weeks were divided into four groups: i) the control (HFD only), ii) the HFD + irbesartan (5 mg/kg/day), iii) the HFD + irbesartan + GW9662, a PPARγ antagonist (0.5 mg/kg/day) and iv) the HFD + irbesartan + anti-HGF neutralizing antibody (200 μg/week). The liver and epididymal adipose tissues were evaluated histologically. Serum adiponectin and HGF levels were also measured by ELISA. Fatty liver (as detected by oil-red O staining) and macrophage infiltration were markedly reduced by irbesartan. Irbesartan treatment also reduced macrophage infiltration into epididymal adipose tissue and hypertrophy of adipocytes. However, these effects of irbesartan were attenuated by GW9662 as well as by anti-HGF neutralizing antibody. Serum and hepatic HGF levels were also markedly increased by irbesartan, whereas GW9662 decreased the HGF level. In conclusion, irbesartan, an angiotensin (Ang) receptor blocker (ARB) and partial agonist of PPARγ (metabosartan), demonstrated a reduction in fatty liver and chronic inflammation, such as macrophage infiltration, beyond its blood pressure-lowering effect. These favorable characteristics of irbesartan might be due to local HGF activation through its partial PPARγ agonistic action, in addition to Ang II blockade. Upregulation of local HGF by irbesartan might provide a novel advantage in a strategy for the prevention and treatment of cardiovascular diseases (CVDs).

Loss of HGF activator inhibits foveolar hyperplasia induced by oxyntic atrophy without altering gastrin levels

Spasmolytic polypeptide/trefoil family factor 2 expressing metaplasia (SPEM) is induced by oxyntic atrophy and is known as a precancerous or paracancerous lesion. We now have sought to determine whether hepatocyte growth factor (HGF) influences the development of SPEM and oxyntic atrophy. DMP-777, a parietal cell ablating reagent, was administered to HGF activator (HGFA)-deficient mice and wild-type mice. Gastric mucosal lineage changes were analyzed in the DMP-777 treatment phase and recovery phase. Both wild-type and HGFA knockout mice showed SPEM, and there was no difference in SPEM development. However, after cessation of DMP-777, HGFA-deficient mice showed delayed recovery from SPEM compared with wild-type mice. Foveolar cell hyperplasia and the increase in proliferating cells after parietal cell loss were reduced in HGFA-deficient mice. The HGFA does not affect emergence of SPEM. However, the absence of HGFA signaling causes a delay in the recovery from SPEM to normal glandular composition. HGFA also promotes foveolar cell hyperplasia and mucosal cell proliferation in acute oxyntic injury.

Roles of CUB and LDL receptor class A domain repeats of a transmembrane serine protease matriptase in its zymogen activation

Matriptase is a type II transmembrane serine protease containing two complement proteases C1r/C1s-urchin embryonic growth factor-bone morphogenetic protein domains (CUB repeat) and four low-density lipoprotein receptor class A domains (LDLRA repeat). The single-chain zymogen of matriptase has been found to exhibit substantial protease activity, possibly causing its own activation (i.e. conversion to a disulfide-linked two-chain fully active form), although the activation seems to be mediated predominantly by two-chain molecules. Our aim was to assess the roles of CUB and LDLRA repeats in zymogen activation. Transient expression studies of soluble truncated constructs of recombinant matriptase in COS-1 cells showed that the CUB repeat had an inhibitory effect on zymogen activation, possibly because it facilitated the interaction of two-chain molecules with a matriptase inhibitor, hepatocyte growth factor activator inhibitor type-1. By contrast, the LDLRA repeat had a promoting effect on zymogen activation. The effect of the LDLRA repeat seems to reflect its ability to increase zymogen activity. The proteolytic activities were higher in pseudozymogen forms of recombinant matriptase containing the LDLRA repeat than in a pseudozymogen without the repeat. Our findings provide new insights into the roles of these non-catalytic domains in the generation of active matriptase.

Activation of hepatocyte growth factor-induced apoptosis in hepatic stellate cells

To determine whether apoptosis is induced in rat hepatic stellate cells (HSCs) in response to activation of the hepatocyte growth factor (HGF) by hepatocyte growth factor activator (HGFA) by using a co-culture system of bone marrow mesenchymal stem cells (BMSCs) and HSCs. In this study, cells were divided into the following five groups: HSC control group: HSCs co-cultured with fibroblast cells; HSCs blank group: HSCs cultured alone; BMSCs blank group: BMSCs cultured alone; Experimental group: BMSCs + HSCs; HGFA intervention group: HSCs treated with 70 ng/mL of HGFA. The culture systems were established in culture plates with transwell inserts, and cells were assessed at 24, 48, and 72 h of growth. Dynamic changes in cell morphology were observed under an inverted phase contrast microscope. The surface markers of BMSCs and the apoptosis rate of HSCs were detected by Annexin-V-FITC/propidium iodide (PI). Expression of a-smooth muscle actin (SMA) in HSCs was evaluated by immunohistochemistry. The presence of activated HGF (HGF-a chain) was determined by immunofluorescent staining. HSC proliferation was measured by MTT assay, and the concentrations of HGF and HGFA were quantified by enzyme-linked immunosorbent assay (ELISA). MTT results indicated that treatment with HGF alone had no effect on HSC proliferation rate (vs. HSC blank group, P more than 0.05), but that 24 h treatment with HGFA significantly inhibited the proliferation rate (0.26 ± 0.00 vs. blank group: 0.13 ± 0.04, P = 0.02); moreover, this effect was concentration-dependent. Expression of HGF-a was lower in the experimental group than in the HGFA intervention group at 72 h (37.24 ± 1.03 vs. 40.44 ± 0.77, P = 0.04), and both of these groups had higher expression than the control group at all time points examined (P less than 0.05). The apoptosis rate was consistently higher in the experimental group than in the HGFA intervention group, but most robustly at 72 h (40.77 ± 1.16% vs. 33.35 ± 2.04%, P = 0.00); moreover, the apoptosis rate was significantly higher than that in the control group at all time points examined (P less than 0.01). The concentration of HGF in the experimental group and the HGFA intervention group showed a time-dependent reduction, and was consistently lower than that in the HSCs control group (P less than 0.05). Finally, the concentration of HGFA was higher in the experimental group than in the blank group at all time points examined (P less than 0.05). The BMSC-HSC co-culture system can promote secretion of HGFA from HSCs and HGF activation, thereby inducing apoptosis of HSCs.

Reduced Prostasin (CAP1/PRSS8) Activity Eliminates HAI-1 and HAI-2 Deficiency–Associated Developmental Defects by Preventing Matriptase Activation

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1–deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2–deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.

Time‐coordinated prevalence of extracellular HGF, FGF2 and TGF‐β3 in crush‐injured skeletal muscle

Successful regeneration and remodeling of neuromuscular junctions are critical for restoring functional capacities and properties of skeletal muscle after damage, and axon-guidance molecules may be involved in the signaling that regulates such restoration. Recently, we found that early-differentiated satellite cells up-regulate a secreted neural chemorepellent Sema3A upon in vivo muscle-crush injury. The study also revealed that Sema3A expression is up-regulated in primary satellite-cell cultures in response to hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) and is prevented by transforming growth factor (TGF)-β2, 3. In order to verify the physiological significance of this regulation in vitro, the present study was designed to estimate the time-course of extracellular HGF, FGF2 and TGF-β3 concentrations after crush-injury of Gastrocnemius muscle in the rat lower hind-limb, using a combination of a non-homogenization/non-spin extraction of extracellular wound fluids and enhanced chemiluminescence-Western blotting analyses. Results clearly demonstrated that active HGF and FGF2 are prevalent in 2-8 days post-crush, whereas active TGF-β3 increases after 12 days, providing a better understanding of the time-coordinated levels of HGF, FGF2 and TGF-β3 that drive regulation of Sema3A expression during regenerative intramuscular moto-neuritogenesis.