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The vacuolar-ATPase (v-ATPase) is a proton transporter found on many intra-cellular organelles and the plasma membrane (PM). The v-ATPase on PMs of cancer cells may contribute to their invasive properties in vitro. Its relevance to human cancer tissues remains unclear. We investigated whether the expression and cellular localization of v-ATPase corresponded to the stage of human pancreatic cancer, and its effect on matrix metalloproteinase (MMP) activation in vitro. The intensity of v-ATPase staining increased significantly across the range of pancreatic histology from normal ducts to pancreatic intra-epithelial neoplasms (PanIN) and finally pancreatic ductal adenocarcinoma (PDAC). Low-grade PanIN lesions displayed polarized staining confined to the basal aspect of the cell in the majority (86%) of fields examined. High-grade PanIN lesions and PDAC demonstrated intense and diffuse v-ATPase localization. In pancreatic cancer cells, PM-associated v-ATPase co-localized with cortactin, a component of the leading edge that helps direct MMP release. Blockade of the v-ATPase with concanamycin or shRNA targeting the V1E subunit reduced MMP-9 activity; this effect was greatest in cells with prominent PM-associated v-ATPase. In cells with detectable MMP-2 activities, however, treatment with concanamycin markedly increased MMP-2’s most activated forms. V-ATPase blockade inhibited functional migration and invasion in those cells with predominantly MMP-9 activity. These results indicate that human PDAC specimens demonstrate loss of v-ATPase polarity and increased expression that correlates with increasing invasive potential. Thus, v-ATPase selectively modulates specific MMPs that may be linked to an invasive cancer phenotype.

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The lymphatic system is part of the circulatory system and plays a key role in normal vascular function. Its failure plays a crucial role in the development and maintenance of various diseases including liver diseases. Lymphangiogenesis (the growth of lymphatic vessels) and changes in the properties of lymphatic vessels are associated with pathogenesis of tumor metastases, ascites formation, liver fibrosis/cirrhosis and portal hypertension. Despite its significant role in liver diseases and its importance as a potential therapeutic target for those diseases, the lymphatic vascular system of the liver is poorly understood. Therefore, how the lymphatic vascular system in general and lymphangiogenesis in particular are mechanistically related to the pathogenesis and maintenance of liver diseases are largely unknown. This article summarizes: 1) the lymphatic vascular system; 2) its role in liver tumors, liver fibrosis/cirrhosis and portal hypertension; and 3) its role in ascites formation.

Nogo-B, also known as Reticulon 4B, plays important roles in vascular injuries. Its function in the liver is not understood. The aim of this study was to characterize Nogo-B in liver fibrosis and cirrhosis. Nogo-B distribution was assessed in normal and cirrhotic human liver sections. We also determined the levels of liver fibrosis in wild-type (WT) and Nogo-A/B knockout (NGB KO) mice after sham operation or bile duct ligation (BDL). To investigate the mechanisms of Nogo-B’s involvement in fibrosis, hepatic stellate cells were isolated from WT and NGB KO mice and transformed into myofibroblasts. Portal pressure was measured to test whether Nogo-B gene deletion could ameliorate portal hypertension. In normal livers, Nogo-B expression was found in nonparenchymal cells, whereas its expression in hepatocytes was minimal. Nogo-B staining was significantly elevated in cirrhotic livers. Fibrosis was significantly increased in WT mice 4 weeks after BDL compared with NGB KO mice. The absence of Nogo-B significantly reduced phosphorylation of Smad2 levels upon transforming growth factor β (TGF-β) stimulation. Reconstitution of the Nogo-B gene into NGB KO fibroblasts restored Smad2 phosphorylation. Four weeks after BDL, portal pressure was significantly increased in WT mice by 47%, compared with sham-operated controls (P = 0.03), whereas such an increase in portal pressure was not observed in NGB KO mice (P = NS). Conclusion Nogo-B regulates liver fibrosis, at least in part, by facilitating the TGFβ/Smad2 signaling pathway in myofibroblasts. Because absence of Nogo-B ameliorates liver fibrosis and portal hypertension, Nogo-B blockade may be a potential therapeutic target in fibrosis/cirrhosis.

These data highlight the importance of lipid metabolism in the tumour microenvironment and identify PEDF as a critical negative regulator of both adiposity and tumour invasion in the pancreas.

Pigment epithelium-derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF-directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator-activated receptor-γ (PPARγ), adiponectin, and other adipocyte markers by nearly 90% compared with control-treated cells (P<0.001). Differentiation to osteoblasts by PEDF resulted in a common pathway that involved PPARγ suppression (P<0.01). Canonical Wnt-β-catenin signaling results in a MSC differentiation pattern analogous to that seen with PEDF. Thus, adding PEDF enhanced Wnt-β-catenin signal transduction in human MSCs, demonstrating a novel Wnt agonist function. In PEDF knockout (KO) mice, total body adiposity was increased by >50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.

Brain edema is a leading cause of death in fulminant hepatic failure (FHF). Patients with FHF who develop brain edema and intracranial hypertension exhibit an increase in cerebral blood flow (CBF). 1-3 Changes in cerebral perfusion have been documented with different techniques, are not related to alterations in systemic hemodynamics, 4 and contribute to intracranial hypertension by increasing cerebral blood volume. Although some subjects with FHF show a reduced CBF, 5,6 the majority of these patients do not exhibit brain edema. 1-2 An increase in cerebral perfusion has been described in experimental models of severe hyperammonemia. 7 Recent studies link elevated plasma ammonia levels to the development of cerebral herniation in FHF. 8,9 Insight into the importance of cerebral perfusion arises from work in an experimental model of brain edema, i.e., rats with portacaval anastomosis (PCA) infused with ammonium acetate. In this preparation, brain swelling and an increase in intracranial pressure (ICP) predictably develop after 3 hours of ammonia infusion in the absence of acute liver injury, allowing the performance of pathophysiologic studies in an otherwise stable model. 10 Using the radioactive microsphere technique, we have shown a selective increase in CBF at 3 hours of infusion, 11 without concomitant changes in systemic hemodynamics or flow to other regional beds. In this model, the increase in cerebral perfusion appears related to an intracerebral signal generated after the detoxification of ammonia to glutamine in astrocytes. Administration of methionine-sulfoximine, an irreversible inhibitor of glutamine synthetase, ameliorated the increase in CBF seen in this model. 12 The nature of the intracerebral signal has not been elucidated. Our initial postulate of increased nitric oxide synthesis in the brain 12,13 led to experiments using nonselective and selective inhibitors of nitric oxide synthase isoforms. Under these conditions, the rise in CBF was still present. 14 We proposed that the development of cerebral hyperemia is necessary for the appearance of cerebral swelling in this model. Mild hypothermia (33°C and 35°C) was shown to prevent the development of ammonia-induced brain edema 11 as well as forestall brain water accumulation in a model of FHF. 15 Clinical studies confirm the protective effect of hypothermia to 32°C in patients with FHF and severe intracranial hypertension. 3 The mechanisms by which hypothermia reduces ICP may be multiple, 16 but an effect on cerebral perfusion has been well documented in our experimental model 11 as well as in humans with FHF. 3 High values of CBF seen in both the experimental and human condition are normalized by hypothermia.We designed an experiment in which indomethacin, a known cerebral vasoconstrictor, 17 was coadministered to PCA rats receiving an ammonia infusion. The drug is a nonselective inhibitor of endothelial cyclooxygenase and has minimal penetration into the brain. 18 If a selective effect on cerebral blood flow influences the development of brain ...