Involvement of SSAO-mediated deamination in adipose glucose transport and weight gain in obese diabetic KKAy mice

Semicarbazide-sensitive amine oxidase (SSAO) is located on outer surfaces of adipocytes and endothelial and vascular smooth muscle cells. This enzyme catalyzes deamination of methylamine and aminoacetone, leading to production of toxic formaldehyde and methylglyoxal, respectively, as well as hydrogen peroxide and ammonium. Several lines of evidence suggest that increased SSAO activity is related to chronic inflammation and vascular disorders related to diabetic complications. We found that a highly potent and selective SSAO inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (FPFA), was capable of reducing numbers of atherosclerotic lesions as well as weight gain in obese KKAy mice fed an atherogenic diet. SSAO inhibitors cause a moderate and long-lasting hyperglycemia. Such an increase in serum glucose is a result of reduction of glucose uptake by adipocytes. SSAO-mediated deamination of endogenous methylamine substrates induces adipocyte glucose uptake and lipogenesis. Highly selective SSAO inhibitors can effectively block induced glucose uptake. The results suggest that increased SSAO-mediated deamination may be concomitantly related to obesity and vascular disorders associated with type 2 diabetes.     

内源性甲醛与心血管疾病

内源性甲醛是甲胺由氨基脲敏感性胺氧化酶催化而生成,广泛存在于动物体内多种组织细胞。已经证实,内源性甲醛参与了神经变性病、免疫性疾病以及肿瘤等疾病的发病过程。脂肪细胞、血管内皮细胞和平滑肌细胞富含甲醛生成酶氨基脲敏感性胺氧化酶(semicarbazide-sensitive a-mine oxidase,SSAO)。甲醛具有细胞毒性,易损伤血管内皮并介导多种致病因素诱导的血管损伤过程,在动脉粥样硬化和糖尿病及其并发症的发病中都具有重要作用。     

Studies on semicarbazide-sensitive amine oxidase in patients with diabetes mellitus and in transgenic mice

Patients with diabetes mellitus and with vascular complications in particular, exhibit higher plasma activities of semicarbazide-sensitive amine oxidase (SSAO) compared to control subjects. It has been speculated that production of cytotoxic products of SSAO may cause endothelial damage and thus contribute to the development of diabetic vascular complications such as retino-, nephro-, and neuropathies as a result of SSAO activity.In order to explore the possibility that high SSAO activity contributes to the development of vascular complications in diabetes, we have performed two studies in patients with Type-2 diabetes quantifying plasma SSAO activity, HbA(1c), and urinary levels of the SSAO substrate, methylamine. We also examined the prevalence of retinopathy in these patients. Additionally, we have studied a model of transgenic mice expressing human SSAO in smooth muscle cells. The transgenic mice have an increased SSAO activity as well as mRNA expression. Histological studies revealed a specific aorta phenotype with a condensed and rigid vessel wall in some of the transgenic mice. No wild-type animals displayed this phenotype.In conclusion, we suggest that this transgenic mouse model may be of great value for increasing the knowledge about to what extent human SSAO contributes to vascular complications in diabetes, and also to which extent inhibition of SSAO can prevent the development of such complications.     

p53 phosphorylation is involved in vascular cell death induced by the catalytic activity of membrane-bound SSAO/VAP-1

Semicarbazide sensitive amine oxidase (SSAO) is a multifunctional enzyme present mainly in adipocytes, endothelial and smooth muscle cells. It metabolizes primary aliphatic and aromatic amines generating products able to contribute to cellular oxidative stress. SSAO is expressed in a membrane-bound form and is also present as a soluble enzyme in plasma. Both isoforms are increased in several pathologies, and the catalytic products generated by the soluble enzymatic activity can induce cytotoxicity of vascular cells in culture. We have analyzed whether the transmembrane form of the enzyme is able to produce a cytotoxic effect through methylamine oxidation. Since cells in culture lose the expression of this enzyme, we used an SSAO stably transfected smooth muscle cell line. Herein we report that cell treatment with the substrate methylamine induced a dose and time dependent cytotoxic effect. The tumor suppressor protein p53 played an important role in the molecular pathway involved in this cell death. Moreover, we also observed the induction of PUMA-alpha expression with mitochondrial Bcl-2 family proteins being affected, and final effector caspases being activated.     

A fluorometric high-performance liquid chromatography procedure for simultaneous determination of methylamine and aminoacetone in blood and tissues

Methylamine and aminoacetone are endogenous aliphatic amines found in human blood and urine. They can be oxidized by semicarbazide-sensitive amine oxidase (SSAO), leading to the production of toxic aldehydes such as formaldehyde and methylglyoxal as well as hydrogen peroxide and ammonia. SSAO is localized on the surface of vascular endothelial and smooth muscle cells and of adipocytes. Increases in SSAO activity are linked to vascular disorders associated with pathological conditions such as diabetic complications, heart failure, and vascular dementia. Quantitative assessment of methylamine and acetonitrile in tissues has been hampered due to the volatility and hydrolipophilicity of these amines as well as interference by complex biological constituents. We have overcome this problem and developed an FMOC/HPLC (9-fluorenylmethyl chloroformate-Cl/high-performance liquid chromatography) method for simultaneous assessment of methylamine and aminoacetone. This method has been validated using rodent tissues with a detection limit at the picogram level. Methylamine and aminoacetone distributed unevenly among different tissues ranged from 0.1 to 27 nmol/g. To our knowledge, this is the first report on simultaneous determination of methylamine and aminoacetone in mammal tissues.     

Semicarbazide-sensitive amine oxidase in aortic smooth muscle cells mediates synthesis of a methylglyoxal-AGE: implications for vascular complications in diabetes

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes formation of methylglyoxal (MG) from aminoacetone; MG then reacts with proteins to form advanced glycation end products or AGEs. Because of its potential to generate MG, SSAO may contribute to AGE-associated vascular complications of aging and diabetes. We developed a method to measure SSAO activity in bovine aortic smooth muscle cells (BASMC) based on the oxidation of 2',7'-dichlorofluorescin by hydrogen peroxide and horseradish peroxidase. The SSAO activity was completely inhibited by 10 mM semicarbazide. Argpyrimidine is a readily detectable fluorescent product of the reaction between MG and arginine. Cell lysates incubated with aminoacetone formed argpyrimidine in a reaction that was inhibited by 20 mM semicarbazide. Immunostaining of tissue sections showed that aminoacetone-treated rats (normal as well as diabetic) formed more argpyrimidine in aortic smooth muscle than untreated controls. We believe that SSAO can enhance AGE synthesis in the macrovasculature of diabetic individuals by production of MG.     

Physiological and pathological implications of semicarbazide-sensitive amine oxidase

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the deamination of primary amines. Such deamination has been shown capable of regulating glucose transport in adipose cells. It has been independently discovered that the primary structure of vascular adhesion protein-1 (VAP-1) is identical to SSAO. VAP-1 regulates leukocyte migration and is related to inflammation. Increased serum SSAO activities have been found in patients with diabetic mellitus, vascular disorders and Alzheimer's disease. The SSAO-catalyzed deamination of endogenous substrates, that is, methylamine and aminoacetone, led to production of toxic formaldehyde and methylglyoxal, hydrogen peroxide and ammonia, respectively. These highly reactive aldehydes have been shown to initiate protein cross-linkage, exacerbate advanced glycation of proteins and cause endothelial injury. Hydrogen peroxide contributes to oxidative stress. 14C-methylamine is converted to 14C-formaldehyde, which then forms labeled long-lasting protein adduct in rodents. Chronic methylamine treatment increased the excretion of malondialdehyde and microalbuminuria, and enhanced the formation of fatty streaks in C57BL/6 mice fed with an atherogenic diet. Treatment with selective SSAO inhibitor reduces atherogenesis in KKAy diabetic mice fed with high-cholesterol diet. Aminoguanidine, which blocks advanced glycation and reduces nephropathy in animals, is in fact more potent at inhibiting SSAO than its effect on glycation. It suggests that SSAO is involved in vascular disorders under certain pathological conditions. Although SSAO has been known for several decades, its physiological and pathological implications are just beginning to be recognized.     

A spectrophotometric method for determining the oxidative deamination of methylamine by the amine oxidases

Previously published studies on the oxidative deamination of methylamine by the amine oxidases have determined the formation of radioactively labeled formaldehyde from [(14)C]methylamine. The present work describes a coupled spectrophotometric assay, using formaldehyde dehydrogenase, for the continuous determination of the oxidative deamination of methylamine by semicarbazide-sensitive amine oxidase (SSAO) and its potential use for determining methylamine concentrations in plasma. In this assay, the formaldehyde produced by methylamine deamination is further oxidized to formate, with the reduction of NAD(+), by formaldehyde dehydrogenase. The NADH generated is monitored continuously at 340 nm. Interference from the presence of a rotenone-insensitive NADH oxidase activity in crude tissue homogenates and microsomal fractions can be minimized by pretreating samples with Triton X-100 or substituting NAD(+) by APAD(+) in the coupled assay. This relatively inexpensive and reproducible assay procedure avoids the use of radioactively labeled material.     

The cellular function and molecular mechanism of formaldehyde in cardiovascular disease and heart development

As a common air pollutant, formaldehyde is widely present in nature, industrial production and consumer products. Endogenous formaldehyde is mainly produced through the oxidative deamination of methylamine catalysed by semicarbazide-sensitive amine oxidase (SSAO) and is ubiquitous in human body fluids, tissues and cells. Vascular endothelial cells and smooth muscle cells are rich in this formaldehyde-producing enzyme and are easily damaged owing to consequent cytotoxicity. Consistent with this, increasing evidence suggests that the cardiovascular system and stages of heart development are also susceptible to the harmful effects of formaldehyde. Exposure to formaldehyde from different sources can induce heart disease such as arrhythmia, myocardial infarction (MI), heart failure (HF) and atherosclerosis (AS). In particular, long-term exposure to high concentrations of formaldehyde in pregnant women is more likely to affect embryonic development and cause heart malformations than long-term exposure to low concentrations of formaldehyde. Specifically, the ability of mouse embryos to effect formaldehyde clearance is far lower than that of the rat embryos, more readily allowing its accumulation. Formaldehyde may also exert toxic effects on heart development by inducing oxidative stress and cardiomyocyte apoptosis. This review focuses on the current progress in understanding the influence and underlying mechanisms of formaldehyde on cardiovascular disease and heart development.     

Chronic effect of doxorubicin on vascular endothelium assessed by organ culture study.

We have attempted to determine the chronic effects of doxorubicin, a commonly used anticancer agent, on vascular endothelium using an organ culture system. In rabbit mesenteric arteries treated with 0.3 microM doxorubicin for 7 days, rounding and concentrated nuclei and TUNEL-positive staining were observed in endothelial cells, indicating DNA damage and the induction of apoptosis. However, the endothelium-dependent relaxation induced by substance P and the expression of mRNA encoding endothelial NO synthase (eNOS) did not differ from those in control arteries. In arteries treated with a higher concentration (1 microM) of doxorubicin, apoptosis and damage to nuclei occurred in the endothelial cells at the third day of treatment, and the detachment and excoriation of endothelium from the tunica interna of the vascular wall were also observed. The impairment of endothelium-dependent relaxation was observed at the fifth day of the treatment with 1 microM doxorubicin. Additionally, apoptotic change in the smooth muscle layer was observed at this concentration of doxorubicin. Apoptotic phenomena were further confirmed by DNA fragmentation using isolated bovine aortic endothelial cells (BAECs) and A7r5 vascular smooth muscle cells, and it was revealed that BAECs are more sensitive than A7r5 to the apoptotic effect of doxorubicin. These results suggest that chronic treatment with doxorubicin at therapeutic concentrations induces apoptosis and excoriation of endothelial cells, which diminishes endothelium-dependent relaxation.     

Diabetes and semicarbazide-sensitive amine oxidase (SSAO) activity: a review

The enzyme of semicarbazide-sensitive amine oxidase (SSAO) activity has been reported to be elevated in blood from diabetic patients. SSAO are widely distributed in plasma membranes of various tissues and blood plasma. SSAO-mediated production of toxic aldehydes has been proposed to be related to pathophysiological conditions. Cytotoxic metabolites by SSAO may cause endothelial injury and subsequently induce atherosclerosis. The precise physiological functions of SSAO could play an important role in the control of energy balance in adipose tissue. It is possible that the increased SSAO activity in diabetes may be a result of up-regulation due to increase of SSAO substrates, such as methylamine or aminoacetone. SSAO could play an important role in the regulation of adipocyte homeostasis. Inhibition of SSAO could be of therapeutic value for treatment of diabetic patient.     

Receptor-mediated mitogenic effects of substance P on cultured smooth muscle cells

The neuropeptide substance P, a known mitogen for human blood T-lymphocytes, now is shown to stimulate proliferation of embryonic rat aortic smooth muscle cells of the A7r5 line, at concentrations of 10(-9) M to 10(-6) M. Neurotensin (NT), that has vascular and smooth muscle activity similar to SP, failed to induce proliferation of A7r5 cells. At proliferation-enhancing concentrations, SP increased the concentration of cytosolic Ca2+ in A7r5 cells, suggesting activation of the phosphatidylinositol pathway. Binding of [125I]-substance P to A7r5 cells reached equilibrium rapidly at 4 degrees C, and was saturable, implying that the activation of smooth muscle cells by SP is a receptor-mediated process.     

Vascular metabolic dysfunction and lipotoxicity

The purpose of this study was to determine the role of lipotoxicity in vascular smooth muscle (VSM). C(1)-BODIPY 500/510 C(12) used to assess the ability of VSM A7r5 cells to transport long-chain fatty acids showed that lipid transport did not appear to limit metabolism. Thin layer chromatography revealed that storage of transported fatty acid occurred primarily as mono- and diglycerides and fatty acids but not as triglycerides. We used lipid-induced apoptosis as a measure of lipotoxicity and found that 1.5 mM palmitate (6.8:1) bound to albumin resulted in a 15-fold increase in the number of apoptotic cells compared to the control at 24 hours. This apoptosis did not seem to be due to an increase in reactive oxygen species (ROS) since VSM cells incubated in palmitate showed less ROS production than cells incubated in albumin only. Similar exposure to oleate did not significantly increase the number of apoptotic cells compared to the control. Oleate actually significantly attenuated the apoptosis induced by palmitate, suggesting that unsaturated fatty acids have a protective effect on cells undergoing palmitate-induced apoptosis. These results suggest that vascular smooth muscle is vulnerable to lipotoxicity and that this lipotoxicity may play a role in the development of atherosclerosis.     

Benzylamine antihyperglycemic effect is abolished by AOC3 gene invalidation in mice but not rescued by semicarbazide-sensitive amine oxidase expression under the control of aP2 promoter

Semicarbazide-sensitive amine oxidase (SSAO) is a transmembrane enzyme that metabolizes primary amines from endogenous or dietary origin. SSAO is highly expressed in adipose, smooth muscle and endothelial cells. In each of these cell types, SSAO is implicated in different biological functions, such as glucose transport activation, extracellular matrix maturation and leucocyte extravasation, respectively. However, the physiological functions of SSAO and their involvement in pathogenesis remain uncompletely characterized. To better understand the role of adipose tissue SSAO, we investigated whether it was necessary and/or sufficient to produce the antihyperglycemic effect of the SSAO-substrate benzylamine, already reported in mice. Therefore, we crossed SSAO-deficient mice invalidated for AOC3 gene and transgenic mice expected to express human SSAO in an adipocyte-specific manner, under the control of aP2 promoter. The aP2?Chuman AOC3 construct (aP2?ChAOC3) was equally expressed in the adipose tissue of mice expressing or not the native murine form and almost absent in other tissues. However, the corresponding SSAO activity found in adipose tissue represented only 20?% that of control mice. As a consequence, the benzylamine antihyperglycemic effect observed during glucose tolerance test in control was abolished in AOC3-KO mice but not rescued in mice expressing aP2?ChAOC3. The capacity of benzylamine or methylamine to activate glucose uptake in adipocytes exhibited parallel variations in the corresponding genotypes. Although the aP2?ChAOC3 construct did not allow a total rescue of SSAO activity in adipose tissue, it could be assessed from our observations that adipocyte SSAO plays a pivotal role in the increased glucose tolerance promoted by pharmacological doses of benzylamine.     

Inactivation of Semicarbazide-Sensitive Amine Oxidase Stabilizes the Established Atherosclerotic Lesions via Inducing the Phenotypic Switch of Smooth Muscle Cells

Given that the elevated serum semicarbazide-sensitive amine oxidase (SSAO) activity is associated with the severity of carotid atherosclerosis in clinic, the current study aims to investigate whether SSAO inactivation by semicarbazide is beneficial for established atherosclerotic lesions in LDLr knockout mice on a high-fat/high- cholesterol Western-type diet or after dietary lipid lowering. Despite no impact on plasma total cholesterol levels, the infiltration of circulating monocytes into peripheral tissues, and the size of atherosclerotic lesions, abrogation of SSAO activity resulted in the stabilization of established lesions as evidenced by the increased collagen contents under both conditions. Moreover, SSAO inactivation decreased Ly6C^high monocytosis and lesion macrophage contents in hypercholesterolemic mice, while no effect was observed in mice after normalization of hypercholesterolemia by dietary lipid lowering. Strikingly, abrogation of SSAO activity significantly increased not only the absolute numbers of smooth muscle cells (SMCs), but also the percent of SMCs with a synthetic phenotype in established lesions of mice regardless of plasma cholesterol levels. Overall, our data indicate that SSAO inactivation in vivo stabilizes the established plaques mainly via inducing the switch of SMCs from a contractile to a synthetic phenotype. Targeting SSAO activity thus may represent a potential treatment for patients with atherosclerosis.     

Proteomic profiling and identification of cofilin responding to oxidative stress in vascular smooth muscle

We used 2-DE and MALDI-TOF/TOF to identify proteins of vascular smooth muscle cells whose expression was or was not altered by exposure to 500 microM H2O2 for 30 min. We detected more than 800 proteins on silver-stained gels of whole protein extracts from rat aortic smooth muscle strips. Of these proteins, 135 clearly unaffected and 19 having levels altered by exposure to H2O2 were identified. Protein characterization revealed that the most prominent vascular smooth muscle proteins were those with antioxidant, cytoskeletal structure, or muscle contraction. In addition, cofilin, an isoform of the actin depolymerizing factor family, shifted to its basic site on the 2-DE gel as a result of H2O2 treatment. In Western blot analysis of proteins from A7r5 aortic smooth muscle cells, the phosphorylation, but not the expression, of cofilin was decreased by H2O2 in a dose-dependent manner. The H2O2-induced dephosphorylation of cofilin and apoptosis was inhibited by Na3VO4, an inhibitor of protein tyrosine phosphatase (PTP). These results suggest that cofilin is one of the proteins regulated by H2O2 treatment in vascular smooth muscle, and has an important role in the induction of vascular apoptosis through PTP-dependent mechanisms.     

Polyamine synthesis inhibition induces S phase cell cycle arrest in vascular smooth muscle cells

Polyamines are important for cell growth and proliferation and they are formed from arginine and ornithine via arginase and ornithine decarboxylase (ODC). Arginine may alternatively be metabolised to NO via NO synthase. Here we study if vascular smooth muscle cell proliferation can be reversed by polyamine synthesis inhibitors and investigate their mechanism of action. Cell proliferation was assessed in cultured vascular smooth muscle A7r5 cells and in endothelium-denuded rat arterial rings by measuring [³H]-thymidine incorporation and by cell counting. Cell cycle phase distribution was determined by flow cytometry and polyamines by HPLC. Protein expression was determined by Western blotting. The ODC inhibitor DFMO (1–10 mM) reduced polyamine concentration and attenuated proliferation in A7r5 cells and rat tail artery. DFMO accumulated cells in S phase of the cell cycle and reduced cyclin A expression. DFMO had no effect on cell viability and apoptosis as assessed by fluorescence microscopy. Polyamine concentration and cellular proliferation were not affected by the arginase inhibitor NOHA (100–200 μM) and the NO synthase inhibitor l-NAME (100 μM). Lack of effect of NOHA was reflected by absence of arginase expression. Polyamine synthesis inhibition attenuates vascular smooth muscle cell proliferation by reducing DNA synthesis and accumulation of cells in S phase, and may be a useful approach to prevent vascular smooth muscle cell proliferation in cardiovascular diseases.     

Contamination in HPLC quantified benzaldehyde is from polypropylene microtubes

Semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.6) is a copper-containing enzyme predominantly expressed by vascular smooth muscle cells. SSAO deaminates primary amines to produce aldehydes and oxygen peroxides, and may thus play a role in vascular damage. SSAO activity can be quantified by assaying benzaldehyde production using fluorescent derivatisation and separation by HPLC. We performed the derivatisation step in polypropylene or borosilicate glass tubes over 45 min at 95 degrees C. High and obstructing background levels of benzaldehyde were found in one batch of polypropylene vials, as opposed to its alternatives. Treatment and handling of product shipment into the country did not account for introduction of contaminant into packaged vials nor did any reagent used in the assay. We conclude that the source of contamination was most likely due to variation in the commercial production process. Use of borosilicate vials for assays based on aldehyde production and derivatisation is recommended.     

Culture medium enhances semicarbazide-sensitive amine oxidase activity

Components of fetal calf serum (FCS) are known to contribute to growth and maintenance of cultured cells. Fetal calf serum supplementation of media also may contribute to the cytotoxicity of other substances to cells grown in vitro. Semicarbazide-sensitive amine oxidase (SSAO) enzyme, present in FCS, metabolizes primary amines and contributes to amine cytotoxicity in vascular smooth muscle cells (VSMC). In cell culture experiments, the media used may greatly affect enzymic activities such as SSAO. In these studies, the SSAO activity in FCS, cultured rat aortic VSMC, and rat plasma was determined in the presence and absence of various culture media. Semicarbazide-sensitive amine oxidase activity in FCS (5-20 microl) was significantly enhanced (approximately 1.5- to 2-fold) in the presence of various culture media, with Dulbecco modified Eagle medium (DMEM), causing the greatest enhancement. Dulbecco modified Eagle medium enhanced the SSAO activity of cultured VSMC in two of the four passages but reduced activity in two passages. Activity in rat plasma was reduced by approximately 25% in the presence of DMEM. The concentrations of various media components, such as glucose, sodium pyruvate, pyridoxine.HCl, and L-glutamine, were not correlated with enhancement. This study identifies an important enhancement effect of culture media on the FCS enzyme, SSAO, although the media components responsible for the enhancement are yet to be identified.