Selective induction of endothelial P2Y6 nucleotide receptor promotes vascular inflammation.

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 dose-dependent induction of P2Y(6) with tumor necrosis factor α or Lipopolysaccharide (LPS) stimulation in vitro and in vivo. Studies that used MRS 2578 as P2Y(6) receptor antagonist showed attenuated nuclear factor κB reporter activity and proinflammatory gene expression in human microvascular endothelial cells in vitro. Moreover, pharmacologic or genetic in vivo studies showed attenuated inflammatory responses in P2Y(6)(-/-) mice or after P2Y(6) antagonist treatment during LPS-induced vascular inflammation. These studies show an important contribution of P2Y(6) signaling in enhancing vascular inflammation during systemic LPS challenge and implicate the P2Y(6) receptor as a therapeutic target during systemic inflammatory responses.

Signaling through the A2B adenosine receptor dampens endotoxin-induced acute lung injury.

Sepsis and septic acute lung injury are among the leading causes for morbidity and mortality of critical illness. Extracellular adenosine is a signaling molecule implicated in the cellular adaptation to hypoxia, ischemia, or inflammation. Therefore, we pursued the role of the A2B adenosine receptor (AR) as potential therapeutic target in endotoxin-induced acute lung injury. We gained initial insight from in vitro studies of cultured endothelia or epithelia exposed to inflammatory mediators showing time-dependent induction of the A2BAR (up to 12.9 + or - 3.4-fold, p < 0.05). Similarly, murine studies of endotoxin-induced lung injury identified an almost 4.6-fold induction of A2BAR transcript and corresponding protein induction with LPS exposure. Studies utilizing A2BAR promoter constructs and RNA protection assays indicated that A2BAR induction involved mRNA stability. Functional studies of LPS-induced lung injury revealed that pharmacological inhibition or genetic deletion of the A2BAR was associated with dramatic increases in lung inflammation and histologic tissue injury. Studies of A2BAR bone marrow chimeric mice suggested pulmonary A2BAR signaling in lung protection. Finally, studies with a specific A2BAR agonist (BAY 60-6583) demonstrated attenuation of lung inflammation and pulmonary edema in wild-type but not in gene-targeted mice for the A2BAR. These studies suggest the A2BAR as potential therapeutic target in the treatment of endotoxin-induced forms of acute lung injury.

Upregulation of the water channel aquaporin-4 as a potential cause of postischemic cell swelling in a murine model of myocardial infarction.

Ischemia of the myocardium is generally accepted to be characterized by swelling of myocytes resulting in cardiac dysfunction. However, data are limited concerning the molecular mechanisms of fast water fluxes across cell membranes in ischemic hearts. Since aquaporin-4 (AQP4) is a water channel with an enormous water flux capacity, we investigated in this study whether this water channel protein might play a role in myocyte swelling following myocardial infarction. For this purpose, we studied the expression of AQP4 mRNA at different time points of ischemia in a murine model of myocardial infarction. We observed a significant correlation between the upregulation of AQP4 mRNA and the size of the infarction. In situ hybridization experiments showed comparably higher expression levels of AQP4 mRNA in ischemic myocytes, and anti-AQP4 immunoreactivity was found to be stronger in the sarcolemma of ischemic myocytes. Our findings imply a role of AQP4 in the formation of myocardial edema and this might be important for future prevention and treatment strategies of this distressing situation in order to minimize cardiac dysfunction and mortality in a variety of cardiac diseases in which cell swelling is prevalent.

ATP release from vascular endothelia occurs across Cx43 hemichannels and is attenuated during hypoxia.

BACKGROUND: Extracellular 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 FINDINGS: We 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/SIGNIFICANCE: Taken together, these studies implicate endothelial Cx43 in hypoxia-associated repression of endothelial ATP release.