Chronic hypoxia impairs extracellular nucleotide metabolism and barrier function in pulmonary artery vasa vasorum endothelial cells

MediCity Research Laboratory, University of Turku and National Institute of Health and Welfare, Tykistökatu 6A, 20520, Turku, Finland.
Angiogenesis (Impact Factor: 4.88). 09/2011; 14(4):503-13. DOI: 10.1007/s10456-011-9234-0
Source: PubMed


Vascular remodeling plays a pivotal role in a variety of pathophysiological conditions where hypoxia and inflammation are prominent features. Intravascular ATP, ADP and adenosine are known as important regulators of vascular tone, permeability and homeostasis, however contribution of purinergic signalling to endothelial cell growth and angiogenesis remains poorly understood. By using vasa vasorum endothelial cells (VVEC) isolated from pulmonary artery adventitia of control and chronically hypoxic neonatal calves, these studies were aimed to evaluate the effect of hypoxia on biochemical and functional properties of microvascular endothelial network at the sites of angiogenesis. In comparison with normoxic controls, VVEC from hypoxic animals are characterized by (1) drastically impaired nucleoside triphosphate diphosphohydrolase-1 (NTPDase-1/CD39) and ecto-5'-nucleotidase/CD73 activities with respective increases in basal extracellular ATP and ADP levels (2) higher proliferative responses to low micromolar concentrations of ATP and ADP; and (3) enhanced permeability and disordered adenosinergic control of vascular barrier function (measured as a paracellular flux of 70 kDa fluorescein isothiocyanate-dextran). Together, these results suggest that unique pattern of purine-mediated angiogenic activation and enhanced leakiness of VVEC from chronically hypoxic vessels may be defined by disordered endothelial nucleotide homeostasis at sites of active neovascularization.

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    • "yruvate kinase and phosphoenol pyruvate ( Sorensen et al . , 2003 ) or myokinase ( adenylate kinase ) preparation ( Strehler & Totter , 1952 ) , followed by bioluminescent detection of ADP - derived ATP . Using these techniques , extracellular ADP levels were determined in cultured endothelial , lymphoid and tumor cells ( Helenius et al . , 2012 ; Yegutkin et al . , 2011a ) , rat pancreatic juice ( Sorensen et al . , 2003 ) , keratino - cytes ( Mizumoto et al . , 2002 ) , murine plasma ( Enjyoji et al . , 1999 ; Mercier et al . , 2012 ) , and human vitreous fluids ( Loukovaara et al . , 2014 ) . Likewise , using another mixture of enzymes and substrates , another important ATP metabolite , PP i , can be "
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    ABSTRACT: 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.
    Critical Reviews in Biochemistry and Molecular Biology 11/2014; 49(6):473-97. DOI:10.3109/10409238.2014.953627 · 7.71 Impact Factor
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    • "By regulating extracellular nucleotide levels, NTPDase1 affects haemostasis [12, 13, 37], leukocyte migration [24, 25], immune responses [6, 49], angiogenesis, vascular permeability [17, 50], and vasoconstriction [8, 15]. Therefore the identification of selective NTPDase1 inhibitors would be valuable tools to study the function and pathological consequence of dysregulation of NTPDase1 activity. "
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    ABSTRACT: Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), like other ectonucleotidases, controls extracellular nucleotide levels and consequently their (patho)physiological responses such as in thrombosis, inflammation, and cancer. Selective NTPDase1 inhibitors would therefore be very useful. We previously observed that ticlopidine in its prodrug form, which does not affect P2 receptor activity, inhibited the recombinant form of human NTPDase1 (K i = 14 μM). Here we tested whether ticlopidine can be used as a selective inhibitor of NTPDase1. We confirmed that ticlopidine inhibits NTPDase1 in different forms and in different assays. The ADPase activity of intact HUVEC as well as of COS-7 cells transfected with human NTPDase1 was strongly inhibited by 100 µM ticlopidine, 99 and 86%, respectively. Ticlopidine (100 µM) completely inhibited the ATPase activity of NTPDase1 in situ as shown by enzyme histochemistry with human liver and pancreas sections. Ticlopidine also inhibited the activity of rat and mouse NTPDase1 and of potato apyrase. At 100 µM ticlopidine did not affect the activity of human NTPDase2, NTPDase3, and NTPDase8, nor of NPP1 and NPP3. Weak inhibition (10-20%) of NTPDase3 and -8 was observed at 1 mM ticlopidine. These results show that ticlopidine is a specific inhibitor of NTPDase1 that can be used in enzymatic and histochemistry assays.
    Mediators of Inflammation 08/2014; 2014:547480. DOI:10.1155/2014/547480 · 3.24 Impact Factor
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    • "In the coronary vascular bed, ENTPD1 (CD39) [7,8] and CD73 [9] are thought to be involved in the conversion of ATP to Ado. Recent studies suggested that ectonucleotidase activity is altered under pathophysiological conditions of the heart, such as myocardial ischemia and chronic heart failure [10-13]. Activation of CD73 was found in the preconditioned heart, which was induced by brief periods of myocardial ischemia [11]. "
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    ABSTRACT: Ectonucleotidase plays an important role in the regulation of cardiac function by controlling extracellular levels of adenine nucleotides and adenosine. To determine the influence of ischemia-reperfusion injury on ectonucleotidase activity in coronary vascular bed, we compared the metabolic profile of adenine nucleotides during the coronary circulation in pre- and post-ischemic heart. Langendorff-perfused rat hearts were used to assess the intracoronary metabolism of adenine nucleotides. The effects of ischemia on the adenine nucleotide metabolism were examined after 30 min of ischemia and 30 min of reperfusion. Adenine nucleotide metabolites were measured by high performance liquid chromatography. ATP, ADP and AMP were rapidly metabolized to adenosine and inosine during the coronary circulation. After ischemia, ectonucleotidase activity of the coronary vascular bed was significantly decreased. In addition, the perfusate from the ischemic heart contained a considerable amount of enzymes degrading ATP, AMP and adenosine. Immunoblot analysis revealed that the perfusate from the ischemic heart dominantly contained ectonucleoside triphosphate diphosphohydrolase 1, and, to a lesser extent, ecto-5'-nucleotidase. The leakage of nucleotide metabolizing enzymes from the coronary vascular bed by ischemia-reperfusion was more remarkable in aged rats, in which post-ischemic cardiac dysfunction was more serious. Ectonucleotidases were liberated from the coronary vascular bed by ischemia-reperfusion, resulting in an overall decrease in ectonucleotidase activity in the post-ischemic coronary vascular bed. These results suggest that decreased ectonucleotidase activity by ischemia may exacerbate subsequent reperfusion injury, and that levels of circulating ectonucleotidase may reflect the severity of ischemic vascular injury.
    BMC Cardiovascular Disorders 07/2013; 13(1):53. DOI:10.1186/1471-2261-13-53 · 1.88 Impact Factor
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