IntroductionPrevious studies have implicated extracellular nucleotide metabolites, predominantly adenosine, as triggers of endogenous protective mechanisms in a number of acute injury models. [1][2][3][4][5][6][7] Extracellular adenosine is derived primarily through phosphohydrolysis of adenosine 5Ј-monophosphate (AMP). Ecto-5Ј-nucleotidase (CD73), a ubiquitously expressed ectoenzyme, is the pacemaker of this reaction. 8 Studies on the role of CD73 in tissue-injury showed that cd73 Ϫ/Ϫ mice develop profound vascular leakage and pulmonary edema upon hypoxia exposure. 8 Once generated into the extracellular space, adenosine can signal through any of 4 G-protein coupled adenosine-receptors (ARs: A1AR/A2AAR/A2BAR/A3AR). All of these receptors are expressed on vascular endothelia 9 and have been implicated in tissue-protection in different models of injury. [1][2][3]7,[10][11][12][13][14][15][16][17][18] Changes in vascular barrier function closely coincide with tissue injury of many etiologies, and result in fluid loss, edema, and organ dysfunction. [19][20][21] The predominant barrier (ϳ90%) to movement of macromolecules across a blood vessel wall is presented by the vascular endothelium. 20,22 Under physiologic conditions, macromolecules such as albumin (molecular weight ϳ70 kD) can cross the endothelial monolayer via a paracellular route (eg, by passing between adjacent endothelia) with some contribution of transcellular passage. 23,24 Endothelial barrier function correlates inversely with the size of molecules that can gain entry into tissues and differs between tissues of different origins. Endothelial permeability is highly regulated and may increase markedly upon exposure to inflammatory stimuli (eg, lipopolysaccharide, bacteria, bacterial compounds, prostaglandins, reactive oxygen species, leukotrienes) or adverse conditions such as ischemia or hypoxia. 18,20,[25][26][27][28][29] Given that activation of ARs can lead to an elevation of intracellular cAMP, and that elevated cAMP in endothelia promotes barrier function, 20,30 we considered the possibility of endothelial AR-signaling to regulate vascular permeability. In contrast to previous studies that found tissue protection during hypoxia or inflammation through signaling pathways involving the A2AAR, 1,3,7,31,32 the present studies conclude that the A2BAR is central to the control of vascular leak in hypoxia. Methods Cell cultureHuman microvascular endothelial cells (HMEC)-1 were cultured as described previously. 9,18 For preparation of experimental HMEC-1 monolayers, confluent endothelial cells were seeded at approximately less than 10 5 cells/cm 2 onto either permeable polycarbonate inserts or 100-mm Petri dishes. Endothelial cell purity was assessed by phase microscopic "cobblestone" appearance and uptake of fluorescent acetylated low-density lipoprotein. Stable repression of AR expression by siRNAWith the help of the siRNA Wizard (www.sirnawizard.com; InvivoGen, San Diego, CA) the following primer sequences were chosen within the coding region of the g...

Adenosine has been widely associated with hypoxia of many origins, including those associated with inflammation and tumorogenesis. A number of recent studies have implicated metabolic control of adenosine generation at sites of tissue hypoxia. Here, we examine adenosine receptor control and amplification of signaling through transcriptional regulation of endothelial and epithelial adenosine receptors. Initial studies confirmed previous findings indicating selective induction of human adenosine A2B receptor (A2BR) by hypoxia. Analysis of the cloned human A2BR promoter identified a functional hypoxia-responsive region, including a functional binding site for hypoxia-inducible factor (HIF) within the A2BR promoter. Further studies examining HIF-1alpha DNA binding and HIF-1alpha gain and loss of function confirmed strong dependence of A2BR induction by HIF-1alpha in vitro and in vivo mouse models. Additional studies in endothelia overexpressing full-length A2BR revealed functional phenotypes of increased barrier function and enhanced angiogenesis. Taken together, these results demonstrate transcriptional coordination of A2BR by HIF-1alpha and amplified adenosine signaling during hypoxia. These findings may provide an important link between hypoxia and metabolic conditions associated with inflammation and angiogenesis.

Background-Extracellular adenosine, generated from extracellular nucleotides via ectonucleotidases, binds to specific receptors and provides cardioprotection from ischemia and reperfusion. In the present study, we studied ecto-enzymatic ATP/ADP-phosphohydrolysis by select members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family during myocardial ischemia. Methods and Results-As a first step, we used a murine model of myocardial ischemia and in situ preconditioning and performed pharmacological studies with polyoxometalate 1, a potent E-NTPDase inhibitor ( 01). Heightened levels of injury after myocardial ischemia and negligible preconditioning benefits in cd39Ϫ/Ϫ mice were corrected by infusion of the metabolic product (AMP) or apyrase. Moreover, apyrase treatment of wild-type mice resulted in 43Ϯ4.2% infarct size reduction (PϽ0.01). Conclusions-Taken together, these studies reveal E-NTPDase 1 in cardioprotection and suggest apyrase in the treatment of myocardial ischemia.

Staphylococcus aureus is resistant to alpha-defensins, antimicrobial peptides that play an important role in oxygen-independent killing of human neutrophils. The dlt operon mediates d-alanine incorporation into teichoic acids in the staphylococcal cell envelope and is a determinant of defensin resistance. By using S. aureus wild-type (WT) and Dlt- bacteria, the relative contributions of oxygen-dependent and -independent antimicrobial phagocyte components were analyzed. The Dlt- strain was efficiently killed by human neutrophils even in the absence of a functional respiratory burst, whereas the killing of the WT organism was strongly diminished when the respiratory burst was inhibited. Human monocytes, which do not produce defensins, inactivated the WT and Dlt- bacteria with similar efficiencies. In addition, mice injected with the Dlt- strain had significantly lower rates of sepsis and septic arthritis and fewer bacteria in the kidneys, compared with mice infected with the WT strain.

BackgroundExtracellular ATP is an important signaling molecule for vascular adaptation to limited oxygen availability (hypoxia). Here, we pursued the contribution of vascular endothelia to extracellular ATP release under hypoxic conditions.Methodology, Principal FindingsWe gained first insight from studying ATP release from endothelia (HMEC-1) pre-exposed to hypoxia. Surprisingly, we found that ATP release was significantly attenuated following hypoxia exposure (2% oxygen, 22±3% after 48 h). In contrast, intracellular ATP was unchanged. Similarly, lactate-dehydrogenase release into the supernatants was similar between normoxic or hypoxic endothelia, suggesting that differences in lytic ATP release between normoxia or hypoxia are minimal. Next, we used pharmacological strategies to study potential mechanisms for endothelial-dependent ATP release (eg, verapamil, dipyridamole, 18-alpha-glycyrrhetinic acid, anandamide, connexin-mimetic peptides). These studies revealed that endothelial ATP release occurs – at least in part - through connexin 43 (Cx43) hemichannels. A real-time RT-PCR screen of endothelial connexin expression showed selective repression of Cx43 transcript and additional studies confirmed time-dependent Cx43 mRNA, total and surface protein repression during hypoxia. In addition, hypoxia resulted in Cx43-serine368 phosphorylation, which is known to switch Cx43 hemi-channels from an open to a closed state.Conclusions/SignificanceTaken together, these studies implicate endothelial Cx43 in hypoxia-associated repression of endothelial ATP release.

Extracellular levels of adenosine increase during hypoxia. While acute increases in adenosine are important to counterbalance excessive inflammation or vascular leakage, chronically elevated adenosine levels may be toxic. Thus, we reasoned that clearance mechanisms might exist to offset deleterious influences of chronically elevated adenosine. Guided by microarray results revealing induction of endothelial adenosine deaminase (ADA) mRNA in hypoxia, we used in vitro and in vivo models of adenosine signaling, confirming induction of ADA protein and activity. Further studies in human endothelia revealed that ADA-complexing protein CD26 is coordinately induced by hypoxia, effectively localizing ADA activity at the endothelial cell surface. Moreover, ADA surface binding was effectively blocked with glycoprotein 120 (gp120) treatment, a protein known to specifically compete for ADA-CD26 binding. IntroductionPhysiologic adaptation and pathophysiologic response to hypoxia are currently areas of intense investigation, and several reports suggest that both transcriptional and metabolic pathways may contribute to a broad range of diseases. 1 For example, during episodes of hypoxia/ischemia, polymorphonuclear leukocytes (PMNs) are mobilized from the intravascular space to the interstitium, 2,3 and such responses may contribute significantly to tissue damage during subsequent reperfusion. 4 Emigration of PMNs through the endothelial barrier is associated with a disruption of tissue barriers, creating the potential for vascular fluid leakage and subsequent edema formation. 5,6 At the same time, extracellular nucleotide metabolites (particularly adenosine) may function as endogenous anti-inflammatory mediators during hypoxia. 1,3,4,6,7 Vascular adenosine signaling during hypoxia has been implicated, dampening pathophysiologic changes related to increased tissue permeability, accumulation of inflammatory cells, and transcriptional induction of proinflammatory cytokines during hypoxia. 1,8 Several lines of evidence support this assertion: first, adenosine receptors are widely expressed on vascular endothelial cells, and have been studied for their capacity to modulate inflammation; 1,3,5 second, murine models of inflammation and/or hypoxia provide evidence for adenosine receptor signaling as a mechanism for regulating hypoxia responses in vivo. Indeed, mice genetically deficient in surface enzymes necessary for adenosine generation (ecto-apyrase, CD39 [conversion of ATP to AMP] and ecto-5Ј-nucleotidase, CD73 [conversion of AMP to adenosine]) show increased hypoxia-associated tissue damage and vascular leak syndrome during hypoxia. 5,6 Third, hypoxia accompanies the normal inflammatory response [9][10][11] and is associated with significantly increased levels of adenosine. 12,13 The exact source(s) of adenosine are not well defined, but likely include a combination of increased intracellular metabolism and amplified extracellular phosphohydrolysis of adenine nucleotides via surface ectonucleotidases. 5,6 In addition, recent stud...

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During a systemic inflammatory response endothelial-expressed surface molecules have been strongly implicated in orchestrating immune responses. Previous studies have shown enhanced extracellular nucleotide release during acute inflammatory conditions. Therefore, we hypothesized that endothelial nucleotide receptors could play a role in vascular inflammation. To address this hypothesis, we performed screening experiments and exposed human microvascular endothelia to inflammatory stimuli, followed by measurements of P2Y or P2X transcriptional responses. These studies showed a selective induction of the P2Y 6 receptor (> 4-fold at 24 hours). Moreover, studies that used real-time reverse transcription-polymerase chain reaction, Western blot analysis, or immunofluorescence confirmed time-and dosedependent induction of P2Y 6 IntroductionVascular responses contribute significantly to inflammatory disorders such as systemic inflammatory response syndrome, sepsis, or acute lung injury. [1][2][3][4] Because of its large surface area and its anatomic position at the interface between the blood stream and surrounding tissues, the vascular endothelium has an exquisite regulatory function in orchestrating acute inflammatory responses. In fact, inflammatory cells such as phagocytes or lymphocytes can emigrate from the bloodstream in response to molecular changes on the surface of blood vessels that signal injury or infection. For example, ϳ 70 million polymorphonuclear neutrophils exit the vasculature per minute. These inflammatory cells move into underlying tissue by initially passing between endothelial cells that line the inner surface of blood vessels. This process, referred to as transendothelial migration is particularly prevalent in inflamed tissues. Although several studies suggested that specific molecules may establish "bottlenecks" to the control of the inflammatory response of the vasculature, only limited information exists about the biochemical events that initialize and dynamically regulate vascular inflammation.Under pathologic conditions such as acute inflammation, ischemia, or hypoxia extracellular nucleotides are released by multiple cell types. 5,6 For example, vascular endothelia, platelets, erythrocytes, or inflammatory cells can release nucleotides. Moreover, activation of extracellular nucleotide receptors (P2X receptors, ligand-gated ion channels; P2Y receptors, G protein-coupled receptors) has been implicated in driving an inflammatory phenotype in different disease models. 7 As such, studies in human and in mice implicate P2 receptor activation in pulmonary inflammation in the context of asthma 8 or chronic lung injury. 9 However, the role of nucleotide signaling during a systemic inflammatory response syndrome or sepsis remains largely unknown.On the basis of these findings, we hypothesized that vascular inflammation could drive transcriptional alterations of P2 receptors involved in the dynamic regulation of vascular inflammation. Consistent with this hypothesis, we found a selective induction of...