Obesity of mice lacking VAP-1/SSAO by Aoc3 gene deletion is reproduced in mice expressing a mutated vascular adhesion protein-1 (VAP-1) devoid of amine oxidase activity.

The product of Aoc3 gene is known as vascular adhesion protein-1 (VAP-1), a glycoprotein contributing to leukocyte extravasation and exhibiting semicarbazide-sensitive amine oxidase activity (SSAO). Regarding the immune functions of VAP-1/SSAO, it is known that mice bearing Aoc3 gene knock-out (AOC3KO) exhibit defects in leukocyte migration similar to those of mice expressing a mutated VAP-1 lacking functional SSAO activity (knock-in, AOC3KI). However, it has not been reported whether these models differ regarding other disturbances. Thus, we further compared endocrine-metabolic phenotypes of AOC3KO and AOC3KI mice to their respective control. Special attention was paid on adiposity, glucose and lipid handling, since VAP-1/SSAO is highly expressed in adipose tissue (AT). In both mouse lines, no tissue SSAO activity was found, while Aoc3 mRNA was absent in AOC3KO only. Although food consumption was unchanged, both AOC3KO and AOC3KI mice were heavier and fatter than their respective controls. Other alterations commonly found in adipocytes from both lines were loss of benzylamine insulin-like action with unchanged insulin lipogenic responsiveness and adiponectin expression. A similar downregulation of inflammatory markers (CD45, IL6) was found in AT. Glucose handling and liver mass remained unchanged, while circulating lipid profile was distinctly altered, with increased cholesterol in AOC3KO only. These results suggest that the lack of oxidase activity found in AOC3KI is sufficient to reproduce the metabolic disturbances observed in AOC3KO mice, save those related with cholesterol transport. Modulation of SSAO activity therefore constitutes a potential target for the treatment of cardiometabolic diseases, especially obesity when complicated by low-grade inflammation.

Metabolic strategies for the degradation of the neuromodulator agmatine in mammals.

Agmatine (1-amino-4-guanidinobutane), a precursor for polyamine biosynthesis, has been identified as an important neuromodulator with anticonvulsant, antineurotoxic and antidepressant actions in the brain. In this context it has emerged as an important mediator of addiction/satiety pathways associated with alcohol misuse. Consequently, the regulation of the activity of key enzymes in agmatine metabolism is an attractive strategy to combat alcoholism and related addiction disorders. Agmatine results from the decarboxylation of L-arginine in a reaction catalyzed by arginine decarboxylase (ADC), and can be converted to either guanidine butyraldehyde by diamine oxidase (DAO) or putrescine and urea by the enzyme agmatinase (AGM) or the more recently identified AGM-like protein (ALP). In rat brain, agmatine, AGM and ALP are predominantly localised in areas associated with roles in appetitive and craving (drug-reinstatement) behaviors. Thus, inhibitors of AGM or ALP are promising agents for the treatment of addictions. In this review, the properties of DAO, AGM and ALP are discussed with a view to their role in the agmatine metabolism in mammals.

Soluble Vascular Adhesion Protein-1 Mediates Spermine Oxidation as Semicarbazide-Sensitive Amine Oxidase: Possible Role in Proliferative Diabetic Retinopathy.

Purpose/Aim of the study: To explore the possible role of vascular adhesion protein-1 (VAP-1) via its enzymatic function as a semicarbazide-sensitive amine oxidase (SSAO) in the pathogenesis of proliferative diabetic retinopathy (PDR). MATERIALS AND METHODS: The levels of soluble VAP-1/SSAO and the unsaturated aldehyde acrolein (ACR)-conjugated protein, Nε-(3-formyl-3, 4-dehydropiperidino) lysine adduct (FDP-Lys), were measured in vitreous fluid samples of PDR and non-diabetic patients using ELISA. Recombinant human VAP-1/SSAO (rhVAP-1/SSAO) was incubated with spermine, with or without semicarbazide or RTU-1096 (a specific inhibitor for VAP-1/SSAO). Immunofluorescence assays were performed to assess the localization of VAP-1/SSAO and FDP-Lys in fibrovascular tissues from patients with PDR. The impact of ACR on cultured retinal capillary endothelial cells was assessed using a cell viability assay and total glutathione (GSH) measurements. RESULTS: The levels of sVAP-1/SSAO and FDP-Lys were elevated in the vitreous fluid of patients with PDR. Incubation of rhVAP-1 with spermine resulted in the generation of hydrogen peroxide and FDP-Lys and the production was inhibited by semicarbazide and RTU-1096. In fibrovascular tissues, FDP-Lys and VAP-1/SSAO were present in endothelial cells. ACR stimulation reduced GSH levels in the cultured endothelial cells in a dose-dependent manner and caused cellular toxicity. CONCLUSIONS: Our results indicate the pathological role of sVAP-1/SSAO to generate hydrogen peroxide and toxic aldehyde ACR, both of which are associated with oxidative stress, as a consequence of spermine oxidation in eyes with PDR.

Cross-talk between Aß and endothelial SSAO/VAP-1 accelerates vascular damage and Aß aggregation related to CAA-AD.

An association between semicarbazide-sensitive amine oxidase (SSAO) and cerebral amyloid angiopathy (CAA) related to Alzheimer's disease (AD) has been largely postulated. Increased SSAO activity and expression have been detected in cerebrovascular tissue and plasma of AD patients, colocalizing with cerebrovascular amyloid-beta (Aß) deposits. As an enzyme, SSAO metabolizes primary amines generating hydrogen peroxide, ammonia, and aldehydes. The ability of these products to generate oxidative stress, to enhance the advanced glycation end-product generation, to promote the Aß aggregation in vitro, and to induce apoptosis supports its role in CAA-related vascular pathology. However, whether the SSAO increase constitutes a cause or it is a consequence of the pathologic process has not been elucidated so far. To set up the nature of this relationship, vascular cell models expressing SSAO were treated with different Aß forms, simulating the CAA conditions in vitro. It was found that the presence of the vasculotropic Dutch-mutated Aß1-40 increases (Aß1-40 D) the SSAO-dependent toxicity, which is accompanied by an increase of SSAO protein availability in endothelial cell membranes. In addition, SSAO enhances Aß1-40 D and Aß1-42 deposition on vascular cells by both activity-dependent and -independent mechanisms. Thus, we provide evidences indicating that Aß itself could be one of the factors inducing SSAO increase in AD, enhancing its toxic effect, and inducing the vascular dysfunction and, in turn, that SSAO stimulates Aß deposition on the vascular walls, thereby contributing to the CAA-AD progression. Therefore, molecules inhibiting SSAO could provide an alternative treatment for preventing/delaying the progress of CAA-AD-associated vasculopathy.

Involvement of SSAO/VAP-1 in oxygen-glucose deprivation-mediated damage using the endothelial hSSAO/VAP-1-expressing cells as experimental model of cerebral ischemia.

BACKGROUND: In the acute phase of ischemic stroke, endothelial cells are activated and induce the expression of adhesion molecules. Vascular adhesion protein 1 (VAP-1) is a proinflammatory protein that mediates leukocyte recruitment through its semicarbazide-sensitive amine oxidase (SSAO) activity (EC 1.4.3.21). Plasmatic SSAO activity predicts the appearance of parenchymal hemorrhages after tissue plasminogen activator treatment in ischemic stroke patients, and it is increased as well in hemorrhagic stroke patients. The aim of this study has been to elucidate the role of SSAO/VAP-1 present in endothelial cells during ischemic stroke conditions. METHODS: Based on the use of endothelial cells expressing, or not expressing, the human SSAO/VAP-1 protein, we have set up an easy ischemic model using oxygen-glucose deprivation (OGD) as an experimental approach to the stroke process. Different OGD and reoxygenation conditions have been analyzed. Western blotting has been used to analyze the activated apoptotic pathways. Several metalloproteinase inhibitors were also used to determine their role in the SSAO/VAP-1 release from the membrane of endothelial cells to the culture media, as a soluble form. Adhesion assays were also performed in order to assess the SSAO/VAP-1-dependent leukocyte adhesion to the endothelia under different OGD and reoxygenation conditions. RESULTS: Our results show that SSAO/VAP-1 expression increases the susceptibility of endothelial cells to OGD, and that its enzymatic activity, through specific substrate oxidation, increases vascular cell damage under these experimental conditions. Caspase-3 and caspase-8 are activated during the death process. In addition, OGD constitutes a stimulus for soluble SSAO/VAP-1 release, partly mediated by metalloproteinase-2-dependent shedding. Short-time OGD induces SSAO/VAP-1-dependent leukocyte binding on endothelial cells, which is partly dependent on its enzymatic activity. CONCLUSIONS: Our results show that SSAO/VAP-1 could participate in some of the processes occurring during stroke. Its expression in endothelial cells increases the OGD-associated cell damage. SSAO/VAP-1 mediates also part of the tissue damage during the reoxygenation process by oxidizing its known enzymatic substrate, methylamine. Also, OGD constitutes a stimulus for its soluble-form release, found elevated in many pathological conditions including stroke. OGD induces SSAO-dependent leukocyte-binding activity, which may have consequences in disease progression, since leukocyte infiltration has shown a determinant role in cerebral ischemia. For all the stroke-related processes in which SSAO/VAP-1 participates, it would be an interesting therapeutic target. Therefore, this model will be a very useful tool for the screening of new molecules as therapeutic agents for cerebral ischemia.

Semicarbazide-sensitive amine oxidase and kidney disease.

With better understanding of the molecular mechanisms underpinning chronic kidney disease, the roles of inflammation and fibrosis are becoming increasingly inseparable. The progression of renal disease is characterized by pathomorphological changes that consist of early inflammatory responses followed by tubulointerstitial fibrosis, tubular atrophy, and glomerular and vascular sclerosis. Currently available therapies that reduce hypertension, proteinuria, hyperglycemia, and interruption of the renin-angiotensin-aldosterone system are at best only partially effective. Hence, there remains a need to explore agents targeting nonrenin-angiotensin-aldosterone system pathways. In this review, we discuss mechanistic aspects in the physiological and pathological role of semicarbazide-sensitive amine oxidase, a protein enzyme involved in cellular trafficking and inflammation, with respect to the kidney. We explore the evidence for the use of semicarbazide-sensitive amine oxidase inhibitors as potential agents in renal fibrosis to delay the onset and progression of chronic kidney disease.

Adhesion molecules in CNS disorders: biomarker and therapeutic targets.

Mounting evidence has been provided regarding the crucial role of leukocyte extravasation and subsequent inflammatory response in several central nervous system (CNS) disorders. The infiltrated leukocytes release proinflammatory mediators and activate resident cells, leading to tissue injury. Leukocyte-endothelia interaction is critical for leukocyte extravasation and migration from the intravascular space into the tissue during inflammation. The basic physiology of leukocyte-endothelia interaction has been investigated extensively. Traditionally, three kinds of adhesion molecules, selectin, integrin, and immunoglobulin families, are responsible for this multiple-step interaction. Furthermore, blocking adhesion molecule function by genetic knockout, antagonizing antibodies, or inhibitory pharmacological drugs provides neuroprotection, which is associated with a reduction in leukocyte accumulation within the tissue. Detection of the soluble form of adhesion molecules has also been proven to predict outcomes in CNS disorders. Lately, vascular adhesion protein-1, a novel adhesion molecule and endothelial cell surface enzyme, has been implicated as a brake in the rolling step of the adhesion cascade, and also a regulator of leukocyte transmigration step. In this review, we summarize the functions of traditional adhesion molecules as well as vascular adhesion protein-1in the leukocyte adhesion cascade. We also discuss the diagnostic and therapeutic potential of adhesion molecules in CNS disorders.

Implication for functions of the ectopic adipocyte copper amine oxidase (AOC3) from purified enzyme and cell-based kinetic studies.

AOC3 is highly expressed in adipocytes and smooth muscle cells, but its function in these cells is currently unknown. The in vivo substrate(s) of AOC3 is/are also unknown, but could provide an invaluable clue to the enzyme's function. Expression of untagged, soluble human AOC3 in insect cells provides a relatively simple means of obtaining pure enzyme. Characterization of enzyme indicates a 6% titer for the active site 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor and corrected k(cat) values as high as 7 s(-1). Substrate kinetic profiling shows that the enzyme accepts a variety of primary amines with different chemical features, including nonphysiological branched-chain and aliphatic amines, with measured k(cat)/K(m) values between 10(2) and 10(4) M(-1) s(-1). K(m)(O(2)) approximates the partial pressure of oxygen found in the interstitial space. Comparison of the properties of purified murine to human enzyme indicates k(cat)/K(m) values that are within 3 to 4-fold, with the exception of methylamine and aminoacetone that are ca. 10-fold more active with human AOC3. With drug development efforts investigating AOC3 as an anti-inflammatory target, these studies suggest that caution is called for when screening the efficacy of inhibitors designed against human enzymes in non-transgenic mouse models. Differentiated murine 3T3-L1 adipocytes show a uniform distribution of AOC3 on the cell surface and whole cell K(m) values that are reasonably close to values measured using purified enzymes. The latter studies support a relevance of the kinetic parameters measured with isolated AOC3 variants to adipocyte function. From our studies, a number of possible substrates with relatively high k(cat)/K(m) have been discovered, including dopamine and cysteamine, which may implicate a role for adipocyte AOC3 in insulin-signaling and fatty acid metabolism, respectively. Finally, the demonstrated AOC3 turnover of primary amines that are non-native to human tissue suggests possible roles for the adipocyte enzyme in subcutaneous bacterial infiltration and obesity.

Tyrosine 381 in E. coli copper amine oxidase influences substrate specificity.

Copper amine oxidases are important for the metabolism of a range of biogenic amines. Here, we focus on substrate specificity in the E. coli copper amine oxidase (ECAO) and specifically the role of Tyr 381. This residue, and its equivalent, in other copper amine oxidases has been referred to as a "gating" residue able to move position depending upon the presence or absence of amine substrate. The position of this residue suggests a role in substrate selectivity. We have compared the properties of two variant forms of ECAO, Y381F and Y381A, with wild-type enzyme by steady-state kinetics of oxidation of a number of amine substrates, modes of inhibitor interactions and X-ray structure determination. Y381F displays a similar catalytic efficiency to wild type against the preferred substrate ß-phenylethylamine. In both cases oxidation of the alternative aromatic amine substrate benzylamine is relatively poor, although Y381F represents an efficient benzylamine oxidase. By contrast, Y381A performed poorly against both aromatic substrates predominantly due to an increased K (M) which we propose is due to the lack of an aromatic residue to orient substrate towards the TPQ and active site base. These results are supported by different behaviour of Y381A to inhibition with 2-hydrazinopyridine. We also report on methylamine turnover by the three enzymes. We propose that Y381, together with another residue Y387, may be considered of critical importance for the substrate selectivity of ECAO, through stacking or hydrophobic interactions with substrate.

Effect of aminoguanidine on the conjunctival histamine and nitrite levels in experimental conjunctivitis.

PURPOSE: Histamine and nitric oxide (NO) play pivotal roles in ocular surface hypersensitivity reactions, whereas the activity of their metabolic enzymes diamine oxidase (DAO) and NO synthase (NOS) may affect their function. This study aimed at investigating the effects of ocular administration of aminoguanidine (AMG), a multiple action DAO and NOS inhibitor, on the conjunctival histamine and nitrite levels in a model of experimental conjunctivitis. METHODS: AMG, at 0.81, 81 or 81×10(3) µM, was instilled into the lower conjunctival fornix of normal and compound 48/80 (C48/80)-challenged eyes of male Wistar rats in the absence or presence of 40 mg/mL disodium cromoglycate. Histamine and nitrite were quantified in the conjunctival homogenate and lavage fluid 45 min and 6 h postchallenge, respectively. RESULTS: AMG induced no significant alterations in basal histamine and nitrite levels in the normal rat eye. In experimental conjunctivitis, AMG failed to modify the reduction in histamine content and partially circumvented the increases in nitrite levels observed during the early and late phase reactions, respectively. In the presence of disodium cromoglycate, AMG significantly increased the levels of both proinflammatory mediators in the normal rat eye. CONCLUSIONS: The data suggested that DAO may not be the main route of in situ histamine catabolism in the normal and C48/80-challenged rat conjunctiva, whereas NOS contributes to the phenotypic alterations observed in mast cell-dependent conjunctivitis. Mast cell stabilizing agents and AMG-modulated systems seem to interact through yet undefined mechanisms in the different phases of ocular hypersensitivity reactions.

Hepatic consequences of vascular adhesion protein-1 expression.

The liver is constantly exposed to antigens present in the blood and to particulate antigens delivered from the gut. To maintain effective levels of immune surveillance and yet tolerate food antigens, the hepatic environment has become highly specialised. A low flow environment exists within the hepatic sinusoids that not only facilitates the exchange of toxins and nutrients within the liver parenchyma, but also provides an ideal niche for the recruitment of leukocytes. One such adhesion molecule involved in this process, the vascular adhesion protein-1 (VAP-1), is unusual in the context of the leukocyte adhesion cascade in that it is both an adhesion molecule and a primary amine oxidase. In this review, we examine the biological functions of VAP-1 and examine what role this molecule might play in the establishment and progression of chronic liver disease.

Preliminary studies of the effects of vascular adhesion protein-1 inhibitors on experimental corneal neovascularization.

Vascular adhesion protein-1 (VAP-1) controls the adhesion of lymphocytes to endothelial cells and is upregulated at sites of inflammation. Moreover, it expresses amine oxidase activity, due to the sequence identity with semicarbazide-sensitive amine oxidase. Recent studies indicate a significant role for VAP-1 in neovascularization, besides its contribution to inflammation. Pathological blood vessel development in severe ocular diseases (such as diabetes, age-related macula degeneration, trauma and infections) might lead to decreased visual acuity and finally to blindness, yet there is no clear consensus as to its appropriate treatment. In the present case study, the effects of two VAP-1 inhibitors on experimentally induced corneal neovascularization in rabbits were compared with the effects of a known inhibitor of angiogenesis, bevacizumab, an anti-vascular endothelial growth factor antibody. In accordance with recent literature data, the results of the preliminary study reported here indicate that the administration of VAP-1 inhibitors is a potentially valuable therapeutic option in the treatment of corneal neovascularization.

Inhibition of semicarbazide-sensitive amine oxidase attenuates myocardial ischemia-reperfusion injury in an in vivo rat model.

AIMS: This study tested the hypothesis that the inhibition of semicarbazide-sensitive amine oxidase (SSAO) after ischemia could attenuate myocardial ischemia-reperfusion (I/R) injury. MAIN METHODS: Anesthetized male Sprague-Dawley rats underwent myocardial I/R injury. Saline, semicarbazide (SCZ, 30 mg/kg), hydralazine (HYD, 10mg/kg), or LJP 1207 (30 mg/kg) was administered intraperitoneally 3 min before reperfusion. After 30 min of ischemia and 180 min of reperfusion, the myocardial infarct size was determined using nitroblue tetrazolium staining. Myocardial myeloperoxidase activity was determined through biochemical assay. HE staining was used for histopathological evaluation. Myocardial SSAO activity was assayed with high performance liquid chromatography analysis. Additionally, the endothelial expression of P-selectin was evaluated using immunohistochemistry after 30 min of ischemia and 20 min of reperfusion. KEY FINDINGS: Myocardial SSAO activity was increased in myocardial I/R injury. Administration of SCZ, HYD, or LJP 1207 reduced the myocardial infarct size and decreased leukocyte infiltration and endothelial P-selectin expression in myocardial I/R injury in vivo. SIGNIFICANCE: These data suggest that myocardial I/R injury up-regulates myocardial SSAO activity, and the inhibition of SSAO prior to reperfusion is able to attenuate acute myocardial I/R injury.

Estrogen regulates amiloride-binding protein 1 through CCAAT/enhancer-binding protein-beta in mouse uterus during embryo implantation and decidualization.

Embryo implantation is an intricate interaction between receptive uterus and active blastocyst. The mechanism underlying embryo implantation is still unknown. Although histamine and putrescine are important for embryo implantation and decidualization, excess amount of histamine and putrescine is harmful. Amiloride binding protein 1 (Abp1) is a membrane-associated amine oxidase and mainly metabolizes histamine and putrescine. In this study, we first showed that Abp1 is strongly expressed in the decidua on d 5-8 of pregnancy. Abp1 expression is not detected during pseudopregnancy and under delayed implantation but is detected after estrogen activation. Because Abp1 is mainly localized in the decidua and also strongly expressed during in vitro decidualization, Abp1 might play a role during mouse decidualization. The regulation of estrogen on Abp1 is mediated by transcription factor CCAAT/enhancer-binding protein-ß. Abp1 expression is also regulated by cAMP, bone morphogenetic protein 2, and ERK1/2. Abp1 may be essential for mouse embryo implantation and decidualization.

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.

The unique substrate specificity of human AOC2, a semicarbazide-sensitive amine oxidase.

Semicarbazide-sensitive amine oxidases (SSAOs) catalyze oxidative deamination of primary amines, but the true physiological function of these enzymes is still poorly understood. Here, we have studied the functional and structural characteristics of a human cell-surface SSAO, AOC2, which is homologous to the better characterized family member, AOC3. The preferred in vitro substrates of AOC2 were found to be 2-phenylethylamine, tryptamine and p-tyramine instead of methylamine and benzylamine, the favored substrates of AOC3. Molecular modeling suggested structural differences between AOC2 and AOC3, which provide AOC2 with the capability to use the larger monoamines as substrates. Even though AOC2 mRNA was expressed in many tissues, the only tissues with detectable AOC2-like enzyme activity were found in the eye. Characterization of AOC2 will help in evaluating the contribution of this enzyme to the pathological processes attributed to the SSAO activity and in designing specific inhibitors for the individual members of the SSAO family.

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.