Cellular localization and trafficking of vascular adhesion protein-1 as revealed by an N-terminal GFP fusion protein

Recent studies of vascular adhesion protein-1 (VAP-1) have greatly advanced our understanding of the important role this protein plays in the establishment and progression of inflammatory disease. To facilitate more detailed studies on the function of VAP-1, we developed a GFP-fusion protein that enabled us to monitor the trafficking of the protein in three selected cell types: hepatic sinusoidal endothelial cells, liver myofibroblasts and an hepatic stellate cell line (LX-2). The fusion protein was detected as punctate cytoplasmic GFP staining, but was present only at low levels at the cell surface in all cell types studied. The subcellular distribution of the protein was not altered in a catalytically inactive mutant form of the protein (Tyr471Phe) or in the presence of exogenous VAP-1 substrate (methylamine) or inhibitor (semicarbazide). The GFP-VAP-1 protein was localized to the Golgi apparatus (GM-130), endoplasmic reticulum (GRP94) and early endosomes (EEA-1). Additional staining for VAP-1 revealed that the overexpressed protein was also present in vesicles that were negative for GFP fluorescent signal and did not express EEA-1. We propose that these vesicles are responsible for recycling the fusion protein and that the fluorescence of the GFP moiety is quenched at the low pH within these vesicles. This feature of the protein makes it well suited for live cell imaging studies where we wish to track protein that is being actively trafficked within the cell in preference to that which is being recycled. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00702-013-1003-3) contains supplementary material, which is available to authorized users

A Novel and Cost-Effective Monitoring Approach for Outcomes in an Australian Biodiversity Conservation Incentive Program

We report on the design and implementation of ecological monitoring for an Australian biodiversity conservation incentive scheme - the Environmental Stewardship Program. The Program uses competitive auctions to contract individual land managers for up to 15 years to conserve matters of National Environmental Significance (with an initial priority on nationally threatened ecological communities). The ecological monitoring was explicitly aligned with the Program's policy objective and desired outcomes and was applied to the Program's initial Project which targeted the critically endangered White Box-Yellow Box-Blakely's Red Gum Grassy Woodland and Derived Native Grassland ecological community in south eastern Australia. These woodlands have been reduced to <3% of their original extent and persist mostly as small remnants of variable condition on private farmland. We established monitoring sites on 153 farms located over 172,232 sq km. On each farm we established a monitoring site within the woodland patch funded for management and, wherever possible, a matched control site. The monitoring has entailed gathering data on vegetation condition, reptiles and birds. We also gathered data on the costs of experimental design, site establishment, field survey, and data analysis. The costs of monitoring are approximately 8.5% of the Program's investment in the first four years and hence are in broad accord with the general rule of thumb that 5-10% of a program's funding should be invested in monitoring. Once initial monitoring and site benchmarking are completed we propose to implement a novel rotating sampling approach that will maintain scientific integrity while achieving an annual cost-efficiency of up to 23%. We discuss useful lessons relevant to other monitoring programs where there is a need to provide managers with reliable early evidence of program effectiveness and to demonstrate opportunities for cost-efficiencies.Specialist staff within SEWPaC assisted in the design of the monitoring program to ensure its policy relevance. The funders contributed to development of the monitoring program but this did not prejudice the content of the paper in any way. The funders had no role in data collection, analysis and interpretation of results. The funders read and suggested improvements to the manuscript and agreed to publication

Inhibition of vascular adhesion protein-1 modifies hepatic steatosis in vitro and in vivo

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and dyslipidaemia and currently is estimated to affect up to a third of all individuals in developed countries. Current standard of care for patients varies according to disease stage, but includes lifestyle interventions common insulin sensitizers, antioxidants and lipid modifiers. However, to date specific therapies have shown little histological or fibrosis stage improvement in large clinical trials, and there is still no licensed therapy for NAFLD. Given the high prevalence, limited treatment options and significant screening costs for the general population, new treatments are urgently required. AIM: To assess the potential for inhibition of the amine oxidase enzyme vascular adhesion protein-1 (VAP-1) to modify hepatic lipid accumulation in NAFLD. METHODS: We have used immunochemical and qPCR analysis to document expression of VAP-1 and key functional proteins and transporters across the NAFLD spectrum. We then utilised hepatocytes in culture and human precision cut liver slices in concert with selective enzyme activity inhibitors to test the effects of activating the semicarbazide-sensitive amine oxidase activity of VAP-1 on hepatic lipid uptake and triglyceride export. A murine model of NAFLD was also used to determine the consequences of VAP-1 knockout and gene expression arrays were used to quantify the effects of VAP-1 activity on key lipid modifying and proinflammatory gene expression. RESULTS: We confirmed that increasing severity of NAFLD and progression to cirrhosis was associated with a significant increase in hepatocellular VAP-1 expression. Hepatocytes in vitro exposed to recombinant VAP-1 and its substrate methylamine showed increased lipid accumulation as determined by quantification of Oil Red O uptake. This was recapitulated using hydrogen peroxide, and lipid accumulation was accompanied by changes in expression of the lipid transporter molecules FABP3, FATP6, insulin receptor subunits and PPARα. Human liver tissue exposed to recombinant VAP-1 or substrates for endo/exogenous VAP-1 produced less triglyceride than untreated tissue and demonstrated an increase in steatosis. This response could be inhibited by using bromoethylamine to inhibit the SSAO activity of VAP-1, and mice deficient in VAP-1/AOC3 also demonstrated reduced steatosis on high fat diet. Exposure of human liver tissue to methylamine to activate VAP-1 resulted in increased expression of FABP2 and 4, FATP3-5, caveolin-1, VLDLR, PPARGC1 and genes associated with the inflammatory response. CONCLUSION: Our data confirm that the elevations in hepatic VAP-1 expression reported in nonalcoholic steatohepatitis can contribute to steatosis, metabolic disturbance and inflammation. This suggests that targeting the semicarbazide sensitive amine oxidase capacity of VAP-1 may represent a useful adjunct to other therapeutic strategies in NAFLD

The senescent secretome drives PLVAP expression in cultured human hepatic endothelia to promote monocyte transmigration

Liver sinusoidal endothelial cells (LSEC) undergo significant phenotypic change in chronic liver disease (CLD), and yet the factors that drive this process and the impact on their function as a vascular barrier and gatekeeper for immune cell recruitment are poorly understood. Plasmalemma-vesicle-associated protein (PLVAP) has been characterized as a marker of LSEC in CLD; notably we found that PLVAP upregulation strongly correlated with markers of tissue senescence. Furthermore, exposure of human LSEC to the senescence-associated secretory phenotype (SASP) led to a significant upregulation of PLVAP. Flow-based assays demonstrated that SASP-driven leukocyte recruitment was characterized by paracellular transmigration of monocytes while the majority of lymphocytes migrated transcellularly. Knockdown studies confirmed that PLVAP selectively supported monocyte transmigration mediated through PLVAP's impact on LSEC permeability by regulating phospho-VE-cadherin expression and endothelial gap formation. PLVAP may therefore represent an endothelial target that selectively shapes the senescence-mediated immune microenvironment in liver disease. [Abstract copyright: © 2023 The Author(s).

SCARF-1 promotes adhesion of CD4+ T cells to human hepatic sinusoidal endothelium under conditions of shear stress

Abstract Liver-resident cells are constantly exposed to gut-derived antigens via portal blood and, as a consequence, they express a unique repertoire of scavenger receptors. Whilst there is increasing evidence that the gut contributes to chronic inflammatory liver disease, the role of scavenger receptors in regulating liver inflammation remains limited. Here, we describe for the first time the expression of scavenger receptor class F, member 1 (SCARF-1) on hepatic sinusoidal endothelial cells (HSEC). We report that SCARF-1 shows a highly localised expression pattern and co-localised with endothelial markers on sinusoidal endothelium. Analysis of chronically inflamed liver tissue demonstrated accumulation of SCARF-1 at sites of CD4+ T cell aggregation. We then studied the regulation and functional role of SCARF-1 in HSEC and showed that SCARF-1 expression by HSEC is regulated by proinflammatory cytokines and bacterial lipopolysaccharide (LPS). Furthermore, SCARF-1 expression by HSEC, induced by proinflammatory and gut-derived factors acts as a novel adhesion molecule, present in adhesive cup structures, that specifically supports CD4+ T cells under conditions of physiological shear stress. In conclusion, we show that SCARF-1 contributes to lymphocyte subset adhesion to primary human HSEC and could play an important role in regulating the inflammatory response during chronic liver disease

CD151 supports VCAM-1 mediated lymphocyte adhesion to liver endothelium and is upregulated in chronic liver diseases and hepatocellular carcinoma

CD151, a member of the tetraspanin family of receptors, is a lateral organizer and modulator of activity of several families of transmembrane proteins. It has been implicated in the development and progression of several cancers, but its role in chronic inflammatory disease is less well understood. Here we show that CD151 is upregulated by distinct microenvironmental signals in a range of chronic inflammatory liver diseases and in primary liver cancer, in which it supports lymphocyte recruitment. CD151 was highly expressed in endothelial cells of the hepatic sinusoids and neovessels developing in fibrotic septa and tumor margins. Primary cultures of human hepatic sinusoidal endothelial cells (HSECs) expressed CD151 at the cell membrane and in intracellular vesicles. CD151 was upregulated by VEGF and HepG2 conditioned media but not by proinflammatory cytokines. Confocal microscopy confirmed that CD151 colocalized with the endothelial adhesion molecule/immunoglobulin superfamily member, VCAM-1. Functional flow-based adhesion assays with primary human lymphocytes and HSECs demonstrated a 40% reduction of lymphocyte adhesion with CD151 blockade. Inhibition of lymphocyte adhesion was similar between VCAM-1 blockade and a combination of CD151/VCAM-1 blockade, suggesting a collaborative role between the two receptors. These studies demonstrate that CD151 is upregulated within the liver during chronic inflammation, where it supports lymphocyte recruitment via liver endothelium. We propose that CD151 regulates the activity of VCAM-1 during lymphocyte recruitment to the human liver and could be a novel anti-inflammatory target in chronic liver disease and hepatocellular cancer prevention.NEW & NOTEWORTHY Chronic hepatitis is characterized by lymphocyte accumulation in liver tissue, which drives fibrosis and carcinogenesis. Here, we demonstrate for the first time that the tetraspanin CD151 supports lymphocyte adhesion to liver endothelium. We show that CD151 is upregulated in chronic liver disease and hepatocellular carcinoma (HCC) and is regulated on endothelium by tissue remodeling and procarcinogenic factors. These regulatory and functional studies identify CD151 as a potential therapeutic target to treat liver fibrosis and HCC

Chemogenomics identifies acetyl-coenzyme A synthetase as a target for malaria treatment and prevention

We identify the Plasmodium falciparum acetyl-coenzyme A synthetase (PfAcAS) as a druggable target, using genetic and chemical validation. In vitro evolution of resistance with two antiplasmodial drug-like compounds (MMV019721 and MMV084978) selects for mutations in PfAcAS. Metabolic profiling of compound-treated parasites reveals changes in acetyl-CoA levels for both compounds. Genome editing confirms that mutations in PfAcAS are sufficient to confer resistance. Knockdown studies demonstrate that PfAcAS is essential for asexual growth, and partial knockdown induces hypersensitivity to both compounds. In vitro biochemical assays using recombinantly expressed PfAcAS validates that MMV019721 and MMV084978 directly inhibit the enzyme by preventing CoA and acetate binding, respectively. Immunolocalization studies reveal that PfAcAS is primarily localized to the nucleus. Functional studies demonstrate inhibition of histone acetylation in compound-treated wild-type, but not in resistant parasites. Our findings identify and validate PfAcAS as an essential, druggable target involved in the epigenetic regulation of gene expression