Gennady G. Yegutkin

A Cell Surface Amine Oxidase Directly Controls Lymphocyte Migration

Lymphocytes leave the blood using a sequential adhesion cascade. Vascular adhesion molecule-1 (VAP-1) is a surface-expressed endothelial glycoprotein, which belongs to a distinct subgroup of monoamine oxidases. We show here that catalytic activity of VAP-1 on primary endothelial cells directly regulates lymphocyte rolling under defined laminar shear. VAP-1 seems to bind to a primary amino group presented on the lymphocyte surface and oxidatively deaminate it in a reaction, which results in the formation of a transient covalent bond between the two cell types. Instead, soluble reaction products (aldehydes and hydrogen peroxide) are not needed for the VAP-1-dependent rolling. Enzymatic regulation of lymphocyte adhesion to endothelium provides a previously unrecognized rapid way of controlling the extravasation process.

Effect of shear stress on the release of soluble ecto-enzymes ATPase and 5'-nucleotidase along with endogenous ATP from vascular endothelial cells.

Stimulation of endothelial cells from human umbilical vein by shear stress induced release of endogenous ATP which was accompanied by an extracellular increase in the activity of enzymes degrading both ATP (ATPases) and AMP (5′‐nucleotidases). The activity of soluble ATPase was progressively increased from 1.62±0.27 to 12.7±1.0 pmoles ml−1 h−1 after 60 min of stimulation by shear stress. The rate of [3H]‐ATP hydrolysis in the medium was inhibited by the purinergic agents suramin, Reactive blue 2 and pyridoxalphosphate‐6‐azophenyl‐2′4′‐disulphonic acid, and remained insensitive to the classic inhibitors of ion‐pumping and intracellular ATPases. Shear stress also increased the activity of 5′‐nucleotidase in the medium from 2.0±0.5 to 27.2±2.8 pmoles ml−1 h−1. When shear stress was applied after removal of ecto‐5′‐nucleotidase by phosphatidylinositol‐specific phospholipase C, the release of 5′‐nucleotidase was drastically reduced. These results show that soluble ATPase and 5′‐nucleotidase which are released during shear stress are not released from an intracellular compartment together with ATP but have an extracellular origin.

Origins of Serum Semicarbazide-Sensitive Amine Oxidase

Semicarbazide-sensitive amine oxidases (SSAO) are enzymes that are capable of deaminating primary amines to produce aldehyde, ammonia, and hydrogen peroxide. This activity has been associated with vascular adhesion protein-1 (VAP-1) and is found in the serum, endothelium, adipose, and smooth muscle of mammals. Circulating SSAO activity is increased in congestive heart failure, diabetes, and inflammatory liver diseases. To investigate the origin of circulating SSAO activity, two transgenic mouse models were created with full-length human VAP-1 (hVAP-1) expressed on either endothelial (mTIEhVAP-1) or adipose tissues (aP2hVAP-1), with tie-1 and adipocyte P2 promoters, respectively. Under normal conditions a circulating form of hVAP-1 was found at high levels in the serum of mice with endothelium-specific expression and at low levels in the serum of mice with adipose specific expression. The level of circulating hVAP-1 in the transgenic mice varied with gender, transgene zygosity, diabetes, and fasting. Serum SSAO activity was absent from VAP-1 knockout mice and endothelial cell–specific expression of human VAP-1 restored SSAO activity to the serum of VAP-1 knockout mice. Together, these experiments show that in the mouse VAP-1 is the only source of serum SSAO, that under physiological conditions vascular endothelial cells can be a major source of circulating VAP-1 protein and SSAO, and that serum VAP-1 can originate from both endothelial cells and adipocytes during experimental diabetes. An increased endothelial cell capacity for lymphocyte binding and altered expression of redox-sensitive proteins was also associated with the mTIEhVAP-1 transgene.

Semicarbazide sensitive amine oxidase overexpression has dual consequences: insulin mimicry and diabetes-like complications

Semicarbazide‐sensitive amine oxidases (SSAO) are copper‐containing enzymes that oxidatively deaminate primary amines to produce hydrogen peroxide, ammonium, and specific aldehydes. Vascular adhesion protein‐1 (VAP‐1) is a cell surface and soluble molecule that possesses SSAO activity. VAP‐1 protein, SSAO activity, and SSAO reaction products are elevated in the serum of patients with diabetes, congestive heart failure, and specific inflammatory liver diseases. By expressing human VAP‐1/SSAO on mouse endothelial cells and subsequently in the serum, and by chronically treating the transgenic mice for 15 months with a high‐fat diet and a physiological substrate for SSAO, methylamine, the in vivo roles of SSAO were assessed. The VAP‐1 transgene increased the mouse body mass index and subcutaneous abdominal fat pad weights in a manner independent of food consumption. The transgene together with increased SSAO substrate availability enhanced glucose uptake in an SSAO‐dependent manner. The increased SSAO activity also led to diabetes‐like complications, including advanced glycation end product formation, elevated blood pressure, altered atherosclerosis progression, and nephropathy. These findings suggest that, although manipulation of VAP‐1/SSAO has potential to serve as a therapeutic treatment in insulin‐resistant conditions, care must be taken to fully understand its impact on obesity and vascular damage.

Altered purinergic signaling in CD73 deficient mice inhibits tumor progression

CD73/ecto‐5′‐nucleotidase dephosphorylates extracellular AMP into adenosine, and it is a key enzyme in the regulation of adenosinergic signaling. The contribution of host CD73 to tumor growth and anti‐tumor immunity has not been studied. Here, we show that under physiological conditions CD73‐deficient mice had significantly elevated ATPase and ADPase activities in LN T cells. In a melanoma model, the growth of primary tumors and formation of metastasis were significantly attenuated in mice lacking CD73. Among tumor‐infiltrating leukocytes there were fewer Tregs and mannose receptor‐positive macrophages, and increased IFN‐γ and NOS2 mRNA production in CD73‐deficient mice. Treatment of tumor‐bearing animals with soluble apyrase, an enzyme hydrolyzing ATP and ADP, significantly inhibited tumor growth and accumulation of intratumoral Tregs and mannose receptor‐positive macrophages in the WT C57BL/6 mice but not in the CD73‐deficient mice. Pharmacological inhibition of CD73 with α,β‐methylene‐adenosine‐5′‐diphosphate in WT mice retarded tumor progression similarly to the genetic deletion of CD73. Together these data show that increased pericellular ATP degradation in the absence of CD73 activity in the host cells is a novel mechanism controlling anti‐tumor immunity and tumor progression, and that the purinergic balance can be manipulated therapeutically to inhibit tumor growth.

Human vascular adhesion protein-1 in smooth muscle cells

Human vascular adhesion protein-1 (VAP-1) is a dual-function molecule with adhesive and enzymatic properties. In addition to synthesis in endothelial cells, where it mediates lymphocyte binding, VAP-1 is expressed in smooth muscle cells. Here we studied the expression, biochemical structure, and function of VAP-1 in muscle cells and compared it to those in endothelial cells. VAP-1 is expressed on the plasma membrane of all types of smooth muscle cells, but it is completely absent from cardiac and skeletal muscle cells. In tumors, VAP-1 is retained on all leiomyoma cells, whereas it is lost in half of leiomyosarcoma samples. In smooth muscle VAP-1 predominantly exists as a approximately 165-kd homodimeric glycoprotein, but a trimeric (approximately 250 kd) form of VAP-1 is also found. It contains N-linked oligosaccharide side chains and abundant sialic acid decorations. In comparison, in endothelial cells dimeric VAP-1 is larger, no trimeric forms are found, and VAP-1 does not have N-glycanase-sensitive oligosaccharides. Unlike endothelial VAP-1, VAP-1 localized on smooth muscle cells does not support binding of lymphocytes. Instead, it deaminates exogenous and endogenous primary amines. In conclusion, VAP-1 in smooth muscle cells is structurally and functionally distinct from VAP-1 present on endothelial cells.

Soluble purine-converting enzymes circulate in human blood and regulate extracellular ATP level via counteracting pyrophosphatase and phosphotransfer reactions.

Extracellular ATP and other purines play a crucial role in the vasculature, and their turnover is selectively governed by a network of ectoenzymes expressed both on endothelial and hematopoietic cells. By studying the whole pattern of purine metabolism in human serum, we revealed the existence of soluble enzymes capable of both inactivating and transphosphorylating circulating purines. Evidence for this was obtained by using independent assays, including chromatographic analyses with 3H‐labeled and unlabeled nucleotides and adenosine, direct transfer of γ‐terminal phosphate from [γ‐32P]ATP to NDP/AMP, and bioluminescent measurement of ATP metabolism. Based on substrate‐specificity and competitive studies, we identified three purine‐inactivating enzymes in human serum, nucleotide pyrophosphatase (EC 3.6.1.9), 5′‐nucleotidase (EC 3.1.3.5), and adenosine deaminase (EC 3.5.4.4), whereas an opposite ATP‐generating pathway is represented by adenylate kinase (EC 2.7.4.3) and NDP kinase (EC 2.7.4.6). Comparative kinetic analysis revealed that the Vmax values for soluble nucleotide kinases significantly exceed those of counteracting nucleotidases, whereas the apparent Km values for serum enzymes were fairly comparable and varied within a range of 40–70 µmol/l. Identification of soluble enzymes contributing, along with membrane‐bound ectoenzymes, to the active cycling between circulating ATP and other purines provides a novel insight into the regulatory mechanisms of purine homeostasis in the blood.

Enzymes involved in metabolism of extracellular nucleotides and nucleosides: Functional implications and measurement of activities

Abstract Extracellular nucleotides and nucleosides mediate diverse signaling effects in virtually all organs and tissues. Most models of purinergic signaling depend on functional interactions between distinct processes, including (i) the release of endogenous ATP and other nucleotides, (ii) triggering of signaling events via a series of nucleotide-selective ligand-gated P2X and metabotropic P2Y receptors as well as adenosine receptors and (iii) ectoenzymatic interconversion of purinergic agonists. The duration and magnitude of purinergic signaling is governed by a network of ectoenzymes, including the enzymes of the nucleoside triphosphate diphosphohydrolase (NTPDase) family, the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, ecto-5′-nucleotidase/CD73, tissue-nonspecific alkaline phosphatase (TNAP), prostatic acid phosphatase (PAP) and other alkaline and acid phosphatases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). Along with “classical” inactivating ectoenzymes, recent data provide evidence for the co-existence of a counteracting ATP-regenerating pathway comprising the enzymes of the adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK/NME/NM23) families and ATP synthase. This review describes recent advances in this field, with special emphasis on purine-converting ectoenzymes as a complex and integrated network regulating purinergic signaling in such (patho)physiological states as immunomodulation, inflammation, tumorigenesis, arterial calcification and other diseases. The second part of this review provides a comprehensive overview and basic principles of major approaches employed for studying purinergic activities, including spectrophotometric Pi-liberating assays, high-performance liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) analyses of purine substrates and metabolites, capillary electrophoresis, bioluminescent, fluorometric and electrochemical enzyme-coupled assays, histochemical staining, and further emphasizes their advantages, drawbacks and suitability for assaying a particular catalytic reaction.

IFN-α Induced Adenosine Production on the Endothelium: A Mechanism Mediated by CD73 (Ecto-5′-Nucleotidase) Up-Regulation

CD73 (ecto-5′-nucleotidase; EC 3.1.3.5) participates in lymphocyte binding to endothelial cells and converts extracellular AMP into a potent anti-inflammatory substance adenosine. However, the regulation of expression and function of CD73 has remained largely unknown. In this study, we show that IFN-α produces a time- and dose-dependent long-term up-regulation of CD73 on endothelial cells, but not on lymphocytes both at protein and RNA levels. Moreover, CD73-mediated production of adenosine is increased after IFN-α treatment on endothelial cells, resulting in a decrease in the permeability of these cells. Subsequent to induction with PMA, FMLP, dibutyryl cAMP, thrombin, histamine, IL-1β, TNF-α, and LPS, no marked changes in the level of CD73 expression on endothelial cells are observed. We also show that CD73 is up-regulated in vivo on the vasculature after intravesical treatment of urinary bladder cancers with IFN-α. In conclusion, distinct behavior of lymphocyte and endothelial CD73 subsequent to cytokine treatment further emphasizes the existence of cell type-specific mechanisms in the regulation of CD73 expression and function. Overall, these results suggest that IFN-α is a relevant in vivo regulator of CD73 in the endothelial-leukocyte microenvironment in infections/inflammations, and thus has a fundamental role in controlling the extent of inflammation via CD73-dependent adenosine production.

Insulin-Regulated Increase of Soluble Vascular Adhesion Protein-1 in Diabetes

Vascular adhesion protein-1 (VAP-1) is one of the molecules on the endothelial cell membrane, which may guide inflammatory cells into atherosclerotic lesions. This dual function molecule may also contribute to the pathogenesis of atherosclerosis and other vasculopathies via its enzymatic activity that oxidizes primary amines to produce their corresponding aldehydes, hydrogen peroxide, and ammonium. Because VAP-1 also exists in a soluble form, we analyzed its potential usefulness as a biomarker to monitor and predict the extent of ongoing atherosclerotic processes. Soluble VAP-1 (sVAP-1) levels were determined from the sera of 136 Finnish men with established coronary heart disease and in 275 controls using sandwich enzyme immunoassays and correlated to multiple risk factors for coronary events. Intriguingly, sVAP-1 showed a statistically significant correlation with diabetes in both cohorts. We then collected patients with type 1 diabetes and observed that sVAP-1 levels were highly elevated when the patients were metabolically compromised. On normalization of their blood glucose and ketone body levels by exogenous insulin, their sVAP-1 concentration rapidly decreased to control levels. Intravenous glucose tolerance and hyperinsulinemic clamp tests further showed that elevation of blood glucose per se did not increase sVAP-1 levels, but rather, sVAP-1 was inversely correlated with circulating insulin concentrations. In conclusion insulin appears to regulate shedding or clearance of VAP-1, and an increase in sVAP-1 because of absolute or relative insulin deficiency may be directly involved in the pathogenesis of diabetic angiopathy.