Vascular Adhesion Protein-1 Activity Research Articles

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

BACKGROUNDNon-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.AIMTo assess the potential for inhibition of the amine oxidase enzyme vascular adhesion protein-1 (VAP-1) to modify hepatic lipid accumulation in NAFLD.METHODSWe 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.RESULTSWe 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.CONCLUSIONOur 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.

Mechanism-based modeling of the effect of a novel inhibitor of vascular adhesion protein-1 on albuminuria and renal function markers in patients with diabetic kidney disease

The vascular adhesion protein-1 (VAP-1) inhibitor ASP8232 reduces albuminuria in patients with type 2 diabetes and chronic kidney disease. A mechanism-based model was developed to quantify the effects of ASP8232 on renal markers from a placebo-controlled Phase 2 study in diabetic kidney disease with 12 weeks of ASP8232 treatment. The model incorporated the available pharmacokinetic, pharmacodynamic (plasma VAP-1 concentration and activity), serum and urine creatinine, serum cystatin C, albumin excretion rate, urinary albumin-to-creatinine ratio, and urine volume information in an integrated manner. Drug-independent time-varying changes and different drug effects could be quantified for these markers using the model. Through simulations, this model provided the opportunity to dissect the relationship and longitudinal association between the estimated glomerular filtration rate and albuminuria and to quantify the pharmacological effects of ASP8232. The developed drug-independent model may be useful as a starting point for other compounds affecting the same biomarkers in a similar time scale.

Primary outcomes of the VIDI study: phase 2, double-masked, randomized, active-controlled study of ASP8232 for diabetic macular edema

BackgroundASP8232 is a potent and specific small molecule vascular adhesion protein-1 (VAP-1) inhibitor. This study evaluated the effect of ASP8232 on excess retinal thickness when given alone or in combination with ranibizumab in patients with center-involved diabetic macular edema (CI-DME).MethodsThis was a phase 2a, placebo and sham-injection controlled, double-masked, randomized, parallel-group clinical trial. Participants were patients with CI-DME and central subfield thickness (CST) ≥ 375 µm in the study eye as assessed by spectral domain optical coherence tomography. Eligible patients were randomized to (1) daily oral ASP8232 40 mg monotherapy; (2) combination therapy of daily oral ASP8232 40 mg and monthly intravitreal ranibizumab 0.3 mg; or (3) monthly intravitreal ranibizumab 0.3 mg monotherapy. The treatment period was 12 weeks. CST and best corrected visual acuity (BCVA) were assessed at baseline and at Weeks 2, 4, 8, 12, 16 and 24. The primary outcome was the mean percent change from baseline in excess CST at Week 12. Secondary outcomes were BCVA, safety and tolerability, and pharmacokinetic and pharmacodynamic characteristics of ASP8232.ResultsAfter 12 weeks, the mean (95% confidence interval) percent change in excess CST was 11.4% (− 15.0%, 37.8%) in the ASP8232 group, − 61.7% (− 86.1%, − 37.2%) in the ASP8232/ranibizumab group, and − 75.3% (− 94.8%, − 55.8%) in the ranibizumab group. The change from baseline in the two ranibizumab arms was statistically significant (P < 0.001) as was the difference between the ranibizumab groups and the ASP8232 group (P < 0.001). Mean (SD) increase in BCVA score from baseline was 3.1 (7.3) in the ASP8232 group, 5.2 (7.1) in the ASP8232/ranibizumab group, and 8.2 (9.5) in the ranibizumab group. The increase from baseline in BCVA score was statistically and clinically significant in the ranibizumab group compared with the ASP8232 group (P = 0.015). ASP8232 resulted in near complete inhibition of plasma VAP-1 activity whilst ranibizumab had no effect.ConclusionsNear complete inhibition of plasma VAP-1 activity with ASP8232 had no effect on CST in patients with CI-DME. Furthermore, combination therapy did not provide additional benefit to treatment with ranibizumab alone, which significantly reduced CST and improved BCVA.Trial registration clinicaltrials.gov; NCT02302079. Registered on November 26, 2014

Oxidative Deamination of Emixustat by Human Vascular Adhesion Protein-1/Semicarbazide-Sensitive Amine Oxidase.

Emixustat potently inhibits the visual cycle isomerase retinal pigment epithelium protein 65 (RPE65) to reduce the accumulation of toxic bisretinoid by-products that lead to various retinopathies. Orally administered emixustat is cleared rapidly from the plasma, with little excreted unchanged. The hydroxypropylamine moiety that is critical in emixustat's inhibition of RPE65 is oxidatively deaminated to three major carboxylic acid metabolites that appear rapidly in plasma. These metabolites greatly exceed the plasma concentrations of emixustat and demonstrate formation-rate-limited metabolite kinetics. This study investigated in vitro deamination of emixustat in human vascular membrane fractions, plasma, and recombinant human vascular adhesion protein-1 (VAP-1), demonstrating single-enzyme kinetics for the formation of a stable aldehyde intermediate (ACU-5201) in all in vitro systems. The in vitro systems used herein established sequential formation of the major metabolites with addition of assay components for aldehyde dehydrogenase and cytochrome P450. Reaction phenotyping experiments using selective chemical inhibitors and recombinant enzymes of monoamine oxidase, VAP-1, and lysyl oxidase showed that only VAP-1 deaminated emixustat. In individually derived human vascular membranes from umbilical cord and aorta, rates of emixustat deamination were highly correlated to VAP-1 marker substrate activity (benzylamine) and VAP-1 levels measured by enzyme-linked immunosorbent assay. In donor-matched plasma samples, soluble VAP-1 activity and levels were lower than in aorta membranes. A variety of potential comedications did not strongly inhibit emixustat deamination in vitro.

Six-Week Endurance Exercise Alters Gut Metagenome That Is not Reflected in Systemic Metabolism in Over-weight Women.

Recent studies suggest that exercise alters the gut microbiome. We determined whether six-weeks endurance exercise, without changing diet, affected the gut metagenome and systemic metabolites of overweight women. Previously sedentary overweight women (n = 19) underwent a six-weeks endurance exercise intervention, but two were excluded due to antibiotic therapy. The gut microbiota composition and functions were analyzed by 16S rRNA gene amplicon sequencing and metagenomics. Body composition was analyzed with DXA X-ray densitometer and serum metabolomics with NMR metabolomics. Total energy and energy-yielding nutrient intakes were analyzed from food records using Micro-Nutrica software. Serum clinical variables were determined with KONELAB instrument. Soluble Vascular Adhesion Protein 1 (VAP-1) was measured with ELISA and its' enzymatic activity as produced hydrogen peroxide. The exercise intervention was effective, as maximal power and maximum rate of oxygen consumption increased while android fat mass decreased. No changes in diet were observed. Metagenomic analysis revealed taxonomic shifts including an increase in Akkermansia and a decrease in Proteobacteria. These changes were independent of age, weight, fat % as well as energy and fiber intake. Training slightly increased Jaccard distance of genus level β-diversity. Training did not alter the enriched metagenomic pathways, which, according to Bray Curtis dissimilarity analysis, may have been due to that only half of the subjects' microbiomes responded considerably to exercise. Nevertheless, tranining decreased the abundance of several genes including those related to fructose and amino acid metabolism. These metagenomic changes, however, were not translated into major systemic metabolic changes as only two metabolites, phospholipids and cholesterol in large VLDL particles, decreased after exercise. Training also decreased the amine oxidase activity of pro-inflammatory VAP-1, whereas no changes in CRP were detected. All clinical blood variables were within normal range, yet exercise slightly increased glucose and decreased LDL and HDL. In conclusion, exercise training modified the gut microbiome without greatly affecting systemic metabolites or body composition. Based on our data and existing literature, we propose that especially Akkermansia and Proteobacteria are exercise-responsive taxa. Our results warrant the need for further studies in larger cohorts to determine whether exercise types other than endurance exercise also modify the gut metagenome.

Leukocyte trafficking-associated vascular adhesion protein 1 is expressed and functionally active in atherosclerotic plaques.

Given the important role of inflammation and the potential association of the leukocyte trafficking-associated adhesion molecule vascular adhesion protein 1 (VAP-1) with atherosclerosis, this study examined whether functional VAP-1 is expressed in atherosclerotic lesions and, if so, whether it could be targeted by positron emission tomography (PET). First, immunohistochemistry revealed that VAP-1 localized to endothelial cells of intra-plaque neovessels in human carotid endarterectomy samples from patients with recent ischemic symptoms. In low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 (LDLR−/−ApoB100/100), VAP-1 was expressed on endothelial cells lining inflamed atherosclerotic lesions; normal vessel walls in aortas of C57BL/6N control mice were VAP-1-negative. Second, we discovered that the focal uptake of VAP-1 targeting sialic acid-binding immunoglobulin-like lectin 9 based PET tracer [68Ga]DOTA-Siglec-9 in atherosclerotic plaques was associated with the density of activated macrophages (r = 0.58, P = 0.022). As a final point, we found that the inhibition of VAP-1 activity with small molecule LJP1586 decreased the density of macrophages in inflamed atherosclerotic plaques in mice. Our results suggest for the first time VAP-1 as a potential imaging target for inflamed atherosclerotic plaques, and corroborate VAP-1 inhibition as a therapeutic approach in the treatment of atherosclerosis.

Dysregulated hepatic expression of glucose transporters in chronic disease: contribution of semicarbazide-sensitive amine oxidase to hepatic glucose uptake.

Insulin resistance is common in patients with chronic liver disease (CLD). Serum levels of soluble vascular adhesion protein-1 (VAP-1) are also increased in these patients. The amine oxidase activity of VAP-1 stimulates glucose uptake via translocation of transporters to the cell membrane in adipocytes and smooth muscle cells. We aimed to document human hepatocellular expression of glucose transporters (GLUTs) and to determine if VAP-1 activity influences receptor expression and hepatic glucose uptake. Quantitative PCR and immunocytochemistry were used to study human liver tissue and cultured cells. We also used tissue slices from humans and VAP-1-deficient mice to assay glucose uptake and measure hepatocellular responses to stimulation. We report upregulation of GLUT1, -3, -5, -6, -7, -8, -9, -10, -11, -12, and -13 in CLD. VAP-1 expression and enzyme activity increased in disease, and provision of substrate to hepatic VAP-1 drives hepatic glucose uptake. This effect was sensitive to inhibition of VAP-1 and could be recapitulated by H2O2. VAP-1 activity also altered expression and subcellular localization of GLUT2, -4, -9, -10, and -13. Therefore, we show, for the first time, alterations in hepatocellular expression of glucose and fructose transporters in CLD and provide evidence that the semicarbazide-sensitive amine oxidase activity of VAP-1 modifies hepatic glucose homeostasis and may contribute to patterns of GLUT expression in chronic disease.

SSAO inhibitors suppress hepatocellular tumor growth in mice

Vascular adhesion protein-1 (VAP-1) is both an endothelial adhesion molecule involved in leukocytes emigration, and an oxidase belonging to the family of semicarbazide-sensitive amine oxidases (SSAOs). The enzyme activity of VAP-1 plays an important role in the migration of myeloid-derived suppressor cells (MDSCs) into tumor site, and SSAO inhibitors can block the function of VAP-1. The effects of SSAO inhibitors on leukocyte infiltration and tumor progression were evaluated in H22 hepatocellular carcinoma-bearing C57BL/6 mice. Tumor weight and volume were measured after SSAO inhibitor treatment. Then, MDSCs recruitment and neo-angiogenesis were determined using immunostaining. SSAO inhibitors significantly blocked the catalytic activity of VAP-1 in tumor, attenuated tumor progression, and reduced neo-angiogenesis. CD11b+ and Gr-1+ MDSCs, which normally infiltrate into tumors, were significantly diminished in tumor-bearing mice treated with SSAO inhibitors. The present study demonstrated that SSAO inhibitors might have an anti-tumor effect on hepatocellular carcinoma by inhibiting recruitment of CD11b+ and Gr-1+ cells and hindering angiogenesis, which could be attributed to impairing the catalytic activity of VAP-1.

Functional modulation of vascular adhesion protein-1 by a novel splice variant.

Vascular Adhesion Protein-1 (VAP-1) is an endothelial adhesion molecule belonging to the primary amine oxidases. Upon inflammation it takes part in the leukocyte extravasation cascade facilitating transmigration of leukocytes into the inflamed tissue. Screening of a human lung cDNA library revealed the presence of an alternatively spliced shorter transcript of VAP-1, VAP-1Δ3. Here, we have studied the functional and structural characteristics of VAP-1Δ3, and show that the mRNA for this splice variant is expressed in most human tissues studied. In comparison to the parent molecule this carboxy-terminally truncated isoform lacks several of the amino acids important in the formation of the enzymatic groove of VAP-1. In addition, the conserved His684, which takes part in coordinating the active site copper, is missing from VAP-1Δ3. Assays using the prototypic amine substrates methylamine and benzylamine demonstrated that VAP-1Δ3 is indeed devoid of the semicarbazide-sensitive amine oxidase (SSAO) activity characteristic to VAP-1. When VAP-1Δ3-cDNA is transfected into cells stably expressing VAP-1, the surface expression of the full-length molecule is reduced. Furthermore, the SSAO activity of the co-transfectants is diminished in comparison to transfectants expressing only VAP-1. The observed down-regulation of both the expression and enzymatic activity of VAP-1 may result from a dominant-negative effect caused by heterodimerization between VAP-1 and VAP-1Δ3, which was detected in co-immunoprecipitation studies. This alternatively spliced transcript adds thus to the repertoire of potential regulatory mechanisms through which the cell-surface expression and enzymatic activity of VAP-1 can be modulated.

Vascular adhesion protein‐1 and syndecan‐1 in septic shock

Constituents of vascular endothelial surface layer (glycocalyx), e.g. an anchor protein syndecan-1 (SDC-1), can be detected in plasma in many inflammatory conditions. In inflammation, vascular adhesion protein-1 (VAP-1) is rapidly translocated to the apical side of the endothelial cells and may be released to plasma in a soluble form. We hypothesized that glycocalyx injury coincides with VAP-1 activation on endothelial cells. To test the hypothesis, we measured SDC-1 and VAP-1 levels in 20 patients with septic shock. A prospective observational study was conducted in two multidisciplinary critical care units in two tertiary academic teaching hospitals with 20 mechanically ventilated adult patients with septic shock, on days 1 and 4 of treatment. Twenty healthy adults were enrolled as a control group. Plasma SDC-1 content, serum VAP-1 activity, platelets, and leukocyte count were measured in septic shock group at baseline and at 72 h and compared with those of healthy controls. VAP-1 activity and SDC-1 content were significantly increased in septic patients' group (P < 0.01) in comparison with controls. VAP-1 activity and SDC-1 content correlated positively to each other, and negatively to platelet count. In the septic shock group SDC-1 correlated on day 1 to SOFA score. We found increased VAP-1 activity and SDC-1 content in critically ill patients with septic shock. Based on our results, the role of VAP-1 in shock pathogenesis should be studied with semicarbazide-sensitive amine oxidase activity blocking agents and substrate affinity testing.

Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 and can be used in PET imaging of inflammation and cancer

AbstractLeukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1–dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

P88 Vascular adhesion protein-1 (VAP-1) modulates glucose and lipid uptake in Non-Alcoholic Fatty Liver Disease (NAFLD)

IntroductionNAFLD is characterised by steatosis, chronic inflammation and fibrosis. The underlying mechanisms include insulin resistance, increased free fatty acid flux from de-novo lipogenesis and decreased lipid oxidation. Vascular Adhesion Protein—1...

Vascular Adhesion Protein-1 Enhances Tumor Growth by Supporting Recruitment of Gr-1+CD11b+ Myeloid Cells into Tumors

Cancer growth is regulated by several nonmalignant cell types, such as leukocytes and endothelial cells, which reside in the stroma of the tumor. Vascular adhesion protein-1 (VAP-1) is an amine oxidase enzyme that is expressed on the surface of endothelial cells. It supports leukocyte traffic into inflamed tissues, but nothing is known about its possible role in cancer biology in vivo. Here, we report that B16 melanoma and EL-4 lymphoma remain smaller in VAP-1-deficient mice than in wild-type controls. We found an unexpected defect in tumor angiogenesis in the absence of VAP-1. VAP-1 also selectively enhanced the recruitment of Gr-1+CD11b+ myeloid cells into the tumors. Generation of mice expressing enzymatically inactive VAP-1 showed that the oxidase activity of VAP-1 was necessary to support neoangiogenesis, myeloid cell recruitment, and tumor growth in vivo. These data describe VAP-1 as the first adhesion molecule known to be involved in the recruitment of Gr-1+CD11b+ myeloid cells into tumors. They also suggest that VAP-1 is a potential new tool for immunotherapy of tumors that could be exploited to reduce tumor burden by controlling the traffic of Gr-1+CD11b+ myeloid cells.

Ischemia‐reperfusion injury is attenuated in VAP‐1‐deficient mice and by VAP‐1 inhibitors

Neutrophils mediate the damage caused by ischemia-reperfusion both at the site of primary injury and in remote organs. Vascular adhesion protein-1 (VAP-1) is an ectoenzyme expressed on endothelial cells and it has been shown to regulate leukocyte extravasation. Here we show for the first time using VAP-1-deficient mice that VAP-1 plays a significant role in the intestinal damage and acute lung injury after ischemia-reperfusion. Separate inhibition of VAP-1 by small molecule enzyme inhibitors and a function-blocking monoclonal antibody in WT mice revealed that the catalytic activity of VAP-1 is responsible for its pro-inflammatory action. The use of transgenic humanized VAP-1 mice also showed that the enzyme inhibitors alleviate both the ischemia-reperfusion injury in the gut and neutrophil accumulation in the lungs. These data thus indicate that VAP-1 regulates the inflammatory response in ischemia-reperfusion injury and suggest that blockade of VAP-1 may have therapeutic value.

Inhibition of Semicarbazide-Sensitive Amine Oxidases Decreases Lymphocyte Infiltration in the Early Phases of Rat Liver Allograft Rejection

Vascular adhesion protein-1 (VAP-1) has been shown to mediate lymphocyte adhesion to endothelia at sites of inflammation in vitro and in vivo. VAP-1 is also an ectoenzyme with semicarbazide-sensitive amine oxidase (SSAO) activity. In this study we investigated whether inhibition of SSAO influences the inflammatory infiltration in acute rat liver allograft rejection. BN recipients of DA liver allografts were treated with 50 mg/kg/d semicarbazide, an inhibitor of SSAO, or similar volumes of saline. 10 rats/group were followed for graft survival, and 10 rats/group were sacrificed on day 7 post-transplantation for histology and T-lymphocyte isolation. The area percentage of portal inflammatory infiltrates in the grafts was assessed from digital photomicrographs. The proportion of CD4-, CD8- and IL2-receptor positive lymphocytes in the graft was quantified with flow cytometry. On day 7, semicarbazide treatment significantly decreased the inflammatory infiltrate area in the grafts. CD4-, CD8- and IL2-receptor positive cells were equally affected. However, animal survival was not affected. Blockade of the enzymatic activity of VAP-1 has a significant effect on lymphocyte infiltration early in acute liver rejection. Later, activation of other adhesion pathways can by-pass the blockade caused by VAP-inhibition.

The oxidase activity of vascular adhesion protein-1 (VAP-1) induces endothelial E- and P-selectins and leukocyte binding

AbstractLeukocyte migration from the blood into tissues is pivotal in immune homeostasis and in inflammation. During the multistep extravasation cascade, endothelial selectins (P- and E-selectin) and vascular adhesion protein-1 (VAP-1), a cell-surface–expressed oxidase, are important in tethering and rolling. Here, we studied the signaling functions of the catalytic activity of VAP-1. Using human endothelial cells transfected with wild-type VAP-1 and an enzymatically inactive VAP-1 point mutant, we show that transcription and translation of E- and P-selectins are induced through the enzymatic activity of VAP-1. Moreover, use of VAP-1–deficient animals and VAP-1–deficient animals carrying the human VAP-1 as a transgene show a VAP-enzyme activity–dependent induction of P-selectin in vivo. Up-regulation of P-selectin was found both in high endothelial venules in lymphoid tissues and in flat-walled vessels in noninflamed tissues. VAP-1 activity in vivo led to increased P-selectin–dependent binding of lymphocytes to endothelial cells. These data show that the oxidase reaction catalyzed by VAP-1 alters the expression of other molecules involved in the leukocyte extravasation cascade. Our findings indicate cross-talk between adhesion molecules involved in the tethering and rolling of leukocytes and show that VAP-1–dependent signaling can prime the vessels for an enhanced inflammatory response.

Vascular Adhesion Protein‐1 as a Potential Therapeutic Target in Liver Disease

Vascular adhesion protein-1 (VAP-1) is a homodimeric, transmembrane sialoglycoprotein, and amine-oxidase enzyme constitutively expressed by hepatic endothelial cells, and as a soluble protein in serum (sVAP-1). VAP-1 mediates leukocyte adhesion and migration in an enzyme activity-dependent manner. We wished to determine whether VAP-1 blockade reduces leukocyte recruitment in inflammatory liver disease, and the mechanism by which this occurs. Our results show that VAP-1 is upregulated in the liver and serum of patients with inflammatory liver disease. Expression is maintained on hepatic sinusoidal endothelial cells (HSECs) isolated from explanted livers. Blockade of VAP-1 activity modestly decreases migration of normal lymphocytes across HSECs but has significant effects on the migration of peripheral blood lymphocytes (PBLs) and liver-derived lymphocytes across HSECs. Engagement of VAP-1 results in PI3-kinase-dependent NF-kappaB activation and increased chemokine and adhesion molecule expression. Thus complex mechanisms regulate VAP-1-mediated recruitment of leukocytes. Direct binding to endothelial VAP-1 protein, indirect enzyme-dependent activation of other endothelial adhesive pathways, and activation of leukocyte by VAP-1 ligand occupancy all contribute to adhesion. The restricted expression of VAP-1 and increased production of sVAP-1 in inflammatory liver disease confirm the validity of this molecule as a therapeutic target.

Developmental regulation of the adhesive and enzymatic activity of vascular adhesion protein-1 (VAP-1) in humans

Vascular adhesion protein-1 (VAP-1) is a homodimeric glycoprotein that belongs to a unique subgroup of cell-surface-expressed oxidases. In adults, endothelial VAP-1 supports leukocyte rolling, firm adhesion, and transmigration in both enzyme activity-dependent and enzyme activity-independent manner. Here we studied the induction and function of VAP-1 during human ontogeny. We show that VAP-1 is already found in the smooth muscle at embryonic week 7. There are marked time-dependent switches in VAP-1 expression in the sinusoids of the liver, in the peritubular capillaries of the kidney, in the capillaries of the heart, and in the venules in the lamina propria of the gut. Fetal VAP-1 is dimerized, and it is enzymatically active. VAP-1 in fetal-type venules is able to bind cord blood lymphocytes. Also, adenovirally transfected VAP-1 on human umbilical vein endothelial cells is involved in rolling and firm adhesion of cord blood lymphocytes under conditions of physiologic shear stress. We conclude that VAP-1 is synthesized from early on in human vessels and it is functionally intact already before birth. Thus, VAP-1 may contribute critically to the oxidase activities in utero, and prove important for lymphocyte trafficking during human ontogeny.

Carbohydrates located on the top of the “cap” contribute to the adhesive and enzymatic functions of vascular adhesion protein‐1

Vascular adhesion protein 1 (VAP-1) is an endothelial adhesion molecule with an enzymatic activity. It deaminates biogenic amines, resulting in the formation of aldehydes and hydrogen peroxide. During the enzymatic reaction a transient Schiff base is formed between endothelial VAP-1 and its leukocytic ligand, and this interaction is important for lymphocyte adhesion. VAP-1 monomer has six potential N-linked, and three putative O-linked glycosylation sites and an SSSS sequence potentially forming an attachment site for an adjacent O-linked site. In this work we modeled the carbohydrate decorations on a structural model of VAP-1, and studied which of those potential glycosylation sites are utilized, and whether those decorations accessible to a lymphocyte ligand are important in lymphocyte adhesion and enzymatic activity of VAP-1. We show that, unlike the O-linked attachment sites, all six N-linked glycosylation sites are in use. Furthermore, mutation of the N-linked attachment sites strategically located on the top of the molecule reduces lymphocyte adhesion in non-static conditions, and enhances the catalytic activity of membrane-bound human VAP-1 in static conditions, suggesting that glycosylation regulates the functional properties of VAP-1.

A peptide inhibitor of vascular adhesion protein-1 (VAP-1) blocks leukocyte-endothelium interactions under shear stress.

Vascular adhesion protein-1 (VAP-1) is an endothelial adhesion molecule mediating leukocyte interactions with blood vessels during leukocyte extravasation. Molecularly VAP-1 is a cell-surface-expressed ecto-enzyme belonging to the group of semicarbazide-sensitive amine oxidases (SSAO; EC 2.4.6.3), which deaminate primary amines. Here we asked whether peptides displaying a suitable free amine group could be a substrate or inhibitor of SSAO and thus regulate VAP-1-mediated leukocyte adhesion. On the basis of a molecular model of VAP-1, we designed synthetic peptides that fit to the substrate channel of VAP-1. One of these lysine-containing peptides effectively inhibits VAP-1-dependent lymphocyte rolling and firm adhesion to primary endothelial cells under physiologically relevant shear conditions. The same peptide inhibits the SSAO activity of endothelial and recombinant VAP-1 in a selective and long-lasting manner. We also show that all enzymatically active VAP-1 is displayed on the cell surface. Our results suggest that, in addition to soluble amines, specific cell-surface-bound molecules containing free NH(2) groups in a suitable position may modulate the enzymatic activity of SSAO. Moreover, the inhibitory peptide diminishes leukocyte interactions with endothelial cells under conditions of shear, and thus it may be useful to treat inflammatory conditions.