Abstract-Extracellular ATP liberated during hypoxia and inflammation can either signal directly on purinergic receptors or can activate adenosine receptors following phosphohydrolysis to adenosine. Given the association of polymorphonuclear leukocytes (PMNs) with adenine-nucleotide/nucleoside signaling in the inflammatory milieu, we hypothesized that PMNs are a source of extracellular ATP. Initial studies using high-performance liquid chromatography and luminometric ATP detection assays revealed that PMNs release ATP through activation-dependent pathways.In vitro models of endothelial barrier function and neutrophil/endothelial adhesion indicated that PMN-derived ATP signals through endothelial adenosine receptors, thereby promoting endothelial barrier function and attenuating PMN/endothelial adhesion. Metabolism of extracellular ATP to adenosine required PMNs, and studies addressing these metabolic steps revealed that PMN express surface ecto-apyrase (CD39). In fact, studies with PMNs derived from cd39 Ϫ/Ϫ mice showed significantly increased levels of extracellular ATP and lack of ATP dissipation from their supernatants. After excluding lytic ATP release, we used pharmacological strategies to reveal a potential mechanism involved in PMN-dependent ATP release (eg, verapamil, dipyridamole, brefeldin A, 18-␣-glycyrrhetinic acid, connexin-mimetic peptides). These studies showed that PMN ATP release occurs through connexin 43 (Cx43) hemichannels in a protein/phosphatase-A-dependent manner. Findings in human PMNs were confirmed in PMNs derived from induced Cx43 Ϫ/Ϫ mice, whereby activated PMNs release less than 15% of ATP relative to littermate controls, whereas Cx43 heterozygote PMNs were intermediate in their capacity for ATP release (PϽ0.01). Taken together, our results identify a previously unappreciated role for Cx43 in activated PMN ATP release, therein contributing to the innate metabolic control of the inflammatory milieu. (Circ Res. 2006;99:1100-1108.) Key Words: nucleotide Ⅲ nucleoside Ⅲ adenosine Ⅲ endothelia Ⅲ inflammation Ⅲ ATP Ⅲ connexin Ⅲ inflammation Ⅲ hypoxia P ast studies have revealed a central role of extracellular nucleotide phosphohydrolysis and nucleoside signaling in innate immune responses during conditions of limited oxygen availability (hypoxia) or during acute inflammation. For example, metabolic enzymes and vascular nucleotide levels are consistently increased during hypoxia. 1,2 The contribution of individual nucleotides (ATP, ADP, AMP) to these innate responses remain unclear. Polymorphonuclear granulocytes (PMNs) function as a first line of cellular response during acute inflammatory episodes. 3 Previous reports have suggested that PMNs may release ATP during conditions of inflammation or hypoxia. 1 Such extracellular ATP can either signal directly to vascular ATP receptors 4 or may function as a metabolite following conversion via ecto-apyrase (CD39, conversion of ATP to AMP) and ecto-5Ј-nucleotidase (CD73, conversion of AMP to adenosine).In the present study, we aimed to identify mol...

Mucosal diseases are often characterized by an inflammatory infiltrate that includes polymorphonuclear leukocytes (PMNs), monocytes, lymphocytes, and platelets. A number of studies have suggested that the interaction of platelets with leukocytes has an essential proinflammatory role. Here, we examined whether platelets migrate across mucosal epithelium, as PMNs are known to do, and whether platelets influence epithelial cell function. Initial studies revealed that human platelets did not efficiently transmigrate across human epithelial cell monolayers. However, in the presence of human PMNs, platelet movement across the epithelium was proportional to the extent of PMN transmigration, and strategies that blocked PMN transmigration diminished platelet movement. Furthermore, platelet-PMN comigration was observed in intestinal tissue derived from human patients with inflammatory bowel disease (IBD). The translocated platelets were found to release large quantities of ATP, which was metabolized to adenosine via a 2-step enzymatic reaction mediated by ectonucleotidases, including CD73 and ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases), expressed on the apical membrane of the intestinal epithelial cells. In vitro studies and a mouse model of intestinal inflammation were employed to define a mechanism involving adenosine-mediated induction of electrogenic chloride secretion, with concomitant water movement into the intestinal lumen. These studies demonstrate that ecto-NTPDases are expressed on the apical membrane of epithelial cells and are involved in what we believe to be a previously unappreciated function for platelets in the inflamed intestine, which might promote bacterial clearance under inflammatory conditions.

Interactions between the vascular endothelium and polymorphonuclear leukocytes (PMNs) are central to PMN emigration into inflamed tissues, and to neutrophil-endothelial crosstalk pathways that modulate inflammatory responses and vascular barrier function. For example, during episodes of inflammation, the transendothelial migration (TEM) of PMNs potentially disturbs vascular barrier and gives rise to intravascular fluid extravasation and edema. However, because of the close special relationship between PMNs and the vascular endothelium, TEM creates an ideal situation for neutrophil-endothelial crosstalk. While investigating innate mechanisms to dampen intravascular fluid loss and edema occurring during TEM, we observed that PMNs release adenine nucleotides after activation (adenosine triphosphate [ATP] and adenosine monophosphate [AMP]). ATP and AMP are metabolized by endothelial cell-surface enzymes, the ecto-apyrase (CD39, metabolizes ATP to AMP) and the 5'-ecto-nucleotidase (CD73, metabolizes AMP to adenosine). Adenosine generated in this fashion can activate endothelial adenosine receptors, leading to increases in intracellular cyclic AMP and resealing of the endothelial junctions, thereby promoting vascular barrier function. This crosstalk pathway provides an endogenous mechanism to dampen vascular leak syndrome during neutrophil-endothelial interaction. In other words, during TEM, neutrophils close the door behind them.

Increased tissue permeability is commonly associated with hypoxia of many origins. Since hypoxia-inducible factor (HIF) represents a predominant hypoxia signaling mechanism, we compared hypoxia-elicited changes in tissue barrier function in mice conditionally lacking intestinal epithelial hypoxia-inducible factor-1alpha (hif1a). Somewhat surprisingly, these studies revealed that mutant hif1a mice were protected from hypoxia-induced increases in intestinal permeability in vivo. Guided by microarray analysis of tissues derived from these mutant hif1a mice, we identified HIF-1-dependent repression of vasodilator-stimulated phosphoprotein (VASP), a molecule known to be important in the control of cytoskeletal dynamics, including barrier function. Studies at the mRNA and protein level confirmed hypoxia-elicited repression of VASP in murine tissue, cultured epithelia and endothelia, as well as human saphenous vein ex vivo. Targeted repression of VASP by siRNA recapitulated our findings with hypoxia and directed overexpression of VASP abolished hypoxia-induced barrier dysfunction. Studies in the cloned human VASP promoter revealed hypoxia-dependent transcriptional repression, and functional studies by chromatin immunoprecipitation (ChIP) and site-directed mutagenesis revealed hypoxia-dependent binding of HIF-1alpha to the human VASP promoter. These studies identify HIF-1-dependent repression of VASP as a control point for hypoxia-regulated barrier dysfunction.

Previously unappreciated, the phosphorylation of VASP performs a key function for the formation of PNCs that is crucially important for the extent of myocardial IR injury.

Taken together, these results indicate that hypoxia-induced increases in vascular leakage and acute inflammation are attenuated by hydroxyethyl starch treatment.

During episodes of hypoxia and inflammation, polymorphonuclear leukocytes (PMN) move into underlying tissues by initially passing between endothelial cells that line the inner surface of blood vessels (transendothelial migration, TEM). TEM creates the potential for disturbances in vascular barrier and concomitant loss of extravascular fluid and resultant edema. Recent studies have demonstrated a crucial role for nucleotide metabolism and nucleoside signaling during inflammation. These studies have implicated multiple adenine nucleotides as endogenous tissue protective mechanisms in vivo. Here, we review the functional components of vascular barrier, identify strategies for increasing nucleotide generation and nucleoside signaling, and discuss potential therapeutic targets to regulate the vascular barrier during inflammation.Abbreviations: AMP -5 0 -adenosine monophosphate; APCP -5 0

Hypoxia has been widely implicated in many pathological conditions, including those associated with inflammation and tumorigenesis. A number of recent studies have implicated hypoxia in the control of vasculogenesis and permeability, the basis for which is not fully understood. Here we examine the transcriptional regulation of angiogenesis and permeability by hypoxia in endothelial cells. Guided by a global profiling approach in cultured endothelial cells, these studies revealed the selective induction of human gravin (protein kinase A anchoring protein 12) by hypoxia. Analysis of the cloned gravin promoter identified a functional hypoxia-responsive region including 2 binding sites for hypoxia-inducible factor (HIF). Site-directed mutagenesis identified the most distal HIF-binding site as essential for the induction of gravin by hypoxia. Further studies examining gravin gain and loss of function confirmed strong dependence of gravin in control of microvascular endothelial tube formation, wherein gravin functions as a "braking" system for angiogenesis. Additional studies in confluent endothelia revealed that gravin functionally couples to control endothelial barrier function in response to protein kinase A (PKA) agonists. Taken together, these results demonstrate transcriptional coordination of gravin by HIF-1α and amplified PKA-dependent endothelial responses. These findings provide an important link between hypoxia and metabolic conditions associated with inflammation and angiogenesis.