CD39 Modulates Endothelial Cell Activation and Apoptosis

Molecular Medicine 6(7): 591–603, 2000 Molecular Medicine © 2000 The Picower Institute Press CD39 Modulates Endothelial Cell Activation and Apoptosis Christian Goepfert, Masato Imai, Sophie Brouard, Eva Csizmadia, Elzbieta Kaczmarek, and Simon C. Robson Center for Immunobiology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A. Accepted April 23, 2000 Abstract Background: CD39 is the dominant vascular nucleoside triphosphate diphosphohydrolase (NTPDase) that exerts major effects on platelet reactivity by the regulated hydrolysis of extracellular adenine nucleotides. The effects of NTPDases on endothelial cell (EC) activation and apoptosis remain unexplored. Material and Methods: Recombinant replicationdeficient adenoviruses were constructed with human CD39 cDNA (rAdCD39) or the bacterial -galactosidase (rAdgal). Results: Intact human umbilical vein EC cultures infected with rAdCD39 had substantial and stable increases in NTPDase biochemical activity (14.50  3.50 Pi nmole/well/min), when contrasted with noninfected cells (0.95  0.002) and rAdgal infected cells (1.010.02; p0.005). Increased NTPDase activity efficiently inhibited immediate type 2Y purinergic receptor (P2Y)-mediated EC activation responses viz. von Willebrand factor secretion in response to extracellular ATP. In addition, CD39 up-regulation blocked ATP-induced translocation of the transcription nuclear factor (NF)-B to the cell nucleus, and abrogated transcription of mRNA encoding E-selectin, and consequent protein synthesis. CD39 also decreased the extent of apoptosis triggered by putative type-2X purinergic (P2X7) receptors in response to high concentrations of extracellular ATP in vitro. Conclusion: These properties of CD39 indicate primary vascular protective effects with potential therapeutic applications. Introduction (7–9). These ectonucleotidases and related proteins are wholly responsible for the chain of extracellular nucleotide hydrolysis in the blood (5). Both ATP and ADP are released at high concentrations from activated platelets and injured EC (10,11). These extracellular nucleotides bind to and activate specific purinergic type 2 (P2) receptors expressed on EC (12). P2Y receptors are G-protein-coupled proteins; whereas, P2X family members are ligand-gated channels (7). Following exposure to adenine nucleotides, EC undergo immediate activation responses with release of prestored proteins, such as von Willebrand factor (vWF) (13), with secretion of prostacyclin and nitric oxide (NO) (12). Ligation of P2Y receptors results in the release of vWF stored in Weibel Palade bodies; whereas, adenosine can potentiate this effect via binding to the specific adenosine or P1 receptors (13). These early events are designated The vascular nucleoside triphosphate diphosphohydrolase (NTPDase; EC 3.5.1.5 or CD39) is expressed on the luminal surface and caveolar microdomains of endothelial cells (EC) (1). Vascular CD39 sequentially hydrolyses extracellular plasma ATP and ADP to the monophosphate derivative AMP (2–5). CD73 (ecto-5’-nucleotidase; EC 3.1.3.5) associates with the vasculature, then generates adenosine from the adenosine monophosphate (6). Adenosine reacts with specific receptors in the vasculature, on leukocytes, or on platelets, and is taken up by EC via concentrative transporters Address correspondence and reprint requests to: Dr. Simon C. Robson, Rm. 370 H, Research North, Beth Israel Deaconess Medical Center, 99 Brookline Avenue, Boston, MA 02215, U.S.A. Phone: 617-632-0881; Fax: 617-632-0880; E-mail: 592 Molecular Medicine, Volume 6, Number 7, July 2000 type I activation and do not require protein synthesis (14,15). Delayed or type II EC responses are generally regulated at the transcriptional level and consist of de novo synthesis and expression of a variety of proteins, including adhesion proteins such as E-selectin, proinflammatory cytokines such as IL-1 or monocyte chemoattractant protein-1 (MCP-1), and procoagulant molecules such as tissue factor (16,17). These latter pathways are usually dependent on the nuclear translocation of the transcription nuclear factor NF-B (18,19), that can be induced by distinct P2 receptor activation responses (20,21). Extracellular ATP also induces apoptotic cell death through interactions with the P2X7 purinergic receptor (22). The regulated hydrolysis of adenine nucleotides and generation of nucleosides by the vascular NTPDase has potential implications for proximate ADP-mediated platelet reactivity (23–25). We demonstrated such thromboregulatory effects by the generation of mutant mice deficient in cd39 (26). Somewhat paradoxically, these mice have defects in hemostasis associated with platelet P2Y1-type desensitization that is comparable to that seen in the recently described P2Y1-deficient mice (27). Mechanisms underlying the disordered vascular EC tissue factor expression, and consequent high levels of fibrin sequestration in the cd39-deficient mice, are under further investigation (26,28). CD39-associated NTPDase activity is also rapidly lost with graft rejection (29), ischemiareperfusion injury (30), oxidant perturbation (2,31) and in cytokine-mediated EC activation responses (25). Such inflammatory changes commonly are associated with extensive vascular injury with the involvement of other mechanisms in vivo (32,33). Multiorgan vascular injury is also observed in cd39-null mice, compared with wild-type mice (27). Excessive murine graft rejection reactions and organ failure also appear to be linked to the suppression or deletion of NTPDase expression [(26); unpublished data]. In addition, supplementation of cellular NTPDase activity by the infusion of apyrases effectively blocks platelet sequestration at the sites of vascular injury within cardiac grafts (34). The aim of this study was to investigate any putative benefits of high-level expression of CD39 in the modulation of inflammatory responses in EC exposed to purinergic mediators. We present clear evidence that CD39 can down-regulate adenine nucleotide induced type I and type II EC activation and influence the process of ATP-induced apotosis. These experimental observations have important implications for the pharmacological use of CD39 in vascular inflammatory and thrombotic states. Materials and Methods Cell Culture and Reagents Human umbilical vein endothelial cells (HUVEC) were kindly provided by Dr. Ewenstein, Brigham and Women’s Hospital, Harvard Medical School, and maintained in M199 medium (BioWhittacker, Walkersville, MD) supplemented with 15% fetal calf serum (FCS; Atlanta Biologicals, Norcross, GA), NaH2CO3 (20mM), N-2-hydroxyethylpiperazine-N-2 ethanesulfonic acid (HEPES; 25mM), glutamine (5mM; BioWhittacker), heparin (100 g/mL), gentamicin (50 g/mL), and endothelial growth factor (50 g/ml; Biomedical Technologies, Stoughton, MA). Cells were used up to the third passage, as described before (...truncated)