The vascular endothelium is the predominant interface between the hypoxic insult and surrounding tissues. Simultaneously, the endothelium provides the primary determinant of vascular permeability (31
). As such, PMN influx across the protective endothelium, such as occurs during ischemia-reperfusion, creates the potential for endothelial barrier dysfunction, loss of fluid and edema formation (7
). In this study we compared the direct influence of activated PMN on paracellular permeability in normoxic and posthypoxic endothelial cells. The results revealed the existence of PMN-derived mediator(s), which selectively promote posthypoxic endothelia. This mediator was identified as ATP based on its chemical characteristics and chromatographic behavior. Further studies demonstrated that hypoxia promotes the induction of CD39, an endothelial surface apyrase responsible for the initiation of ATP phosphohydrolysis to adenosine (33
). We show here that increased activity of endothelial CD39, as seen after hypoxic exposure, is necessary for the barrier protective influence of ATP. Similarly, these studies revealed that additional metabolic and signaling molecules (CD73 and AdoRA2B
, respectively) are coordinately induced by hypoxia () . In vivo relevance for this response revealed that cd39
-null mice are more susceptible to the development of endothelial barrier dysfunction during hypoxia exposure. Taken together, these studies identify a pathway which results in both elevated tissue levels of adenosine and in amplified endothelial responses to adenosine generated at sites of hypoxia.
Figure 8. Proposed model of coordinated nucleotide metabolism and nucleoside signaling in posthypoxic endothelial cells: in areas of ongoing inflammation, diminished oxygen supply coordinates the induction of CD39, CD73, and AdoRA2B. At such sites, activated PMN (more ...)
It is well documented that adenosine tissue and plasma levels are increased during hypoxia, however, mechanisms of this response are less clear (34
). For example, in human volunteers exposed to ambient hypoxia (SpO2
= 80% over 20 min), plasma adenosine concentrations increased from 21 to 51 nM in the presence of dipyridamole, an inhibitor of adenosine reuptake (36
). Similarly, when measuring adenine nucleotide concentrations in the neurally and vascularly isolated, perfused skeletal muscles of anesthetized dogs, normobaric hypoxia is associated with increases of adenosine in the venous blood, but not of AMP, ADP, or ATP (37
). In conjunction with these studies, we describe here a metabolic pathway, which results in increased tissue adenosine concentrations. While it is well documented that adenosine levels are increased in hypoxic tissue, the source of adenosine remains unclear. We show here, for the first time, that PMN actively release ATP to the extracellular milieu. At present, we do not know the origin of ATP release. Several mechanisms for ATP release have been proposed, including direct transport through ATP-binding cassette (ABC) proteins, transport through connexin hemichannels, as well as vesicular release (38
). As part of the present experiments, some studies were done in an attempt to identify the compartmentalization of ATP (unpublished data). For example, we compared the kinetics of ATP release with activated release of PMN granules (myeloperoxidase, MPO). These studies indicated that while ATP levels were maximal within 1 min, activated degranulation contents such as MPO were maximal at time points >5 min, suggesting that ATP is not granule bound. Moreover, in experiments using isolated granules from unactivated PMN, >95% of MPO activity was associated with granules, <5% of ATP was measurable within this granule pool, suggesting that ATP is unlikely to be granule-bound in PMN. At present, the mechanism by which leukocytes release ATP remains unclear.
As part of these studies, we found that targeted disruption of CD39 in an animal model of ambient hypoxia was associated with barrier dysfunction at baseline and after hypoxic exposure. Such increases in permeability in the cd39
-null mice may be related to decreased concentrations of adenosine and activation of endothelial adenosine receptors. In addition, activation of neutrophil A2
adenosine receptors has been shown to play a critical part for the limitation and termination of PMN-mediated systemic inflammatory responses (39
). Thus, it is reasonable to propose that the presence of CD39/CD73 is important in modulating inflammatory responses during hypoxia. Coordinated induction of both CD39 and CD73 by hypoxia may provide increased tissue adenosine concentrations, thus leading to increased stimulation of PMN adenosine A2
-receptors and decreased leukocyte adherence and transmigration, as has been demonstrated by others (41
). Moreover, recent studies have indicated that adenosine A2A
-receptors may contribute to attenuated vascular leak associated with leukocyte accumulation (43
). As such, direct influences of adenosine via hypoxia-amplified CD39 and CD73 may provide antiinflammatory synergism with the observed protection of endothelial barrier during hypoxia.
As outlined above, leukocyte transmigration associated with hypoxic/ischemic tissues creates the potential for disturbance of the endothelial barrier. We have previously shown that adenosine activation of AdoRA2B
leads to a barrier resealing response after PMN transmigration (11
). Activation of the AdoRA2B
is associated with increases in intracellular cAMP concentration following activation of the adenylate cyclase (44
). By inhibition of cAMP formation, the resealing of the endothelial barrier during PMN transmigration can be obviated (11
). Such increases in cAMP after activation of the AdoRA2B
lead to an activation of protein kinase A (PKA; reference 12
). Further studies revealed a central role of PKA-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a protein responsible for controlling the geometry of actin-filaments (45
). Adenosine-receptor mediated phosphorylation of VASP is responsible for changes in the geometry and distribution of junctional proteins, thereby affecting the characteristics of the junctional complex and promoting increases in barrier function (12
We show here that the barrier-protective response to extracellular ATP is most prevalent in posthypoxic endothelial cells. After suppression of CD39 induction with specific siRNA, this barrier-protective influence to ATP is obviated, providing insight that this molecule may efficiently coordinate permeability responses to PMN during inflammation and hypoxia. In parallel, we showed that the ecto-nucleotidase CD73 is similarly induced by hypoxia. These findings in endothelia coincide with recent work in epithelia identifying a predominant role for hypoxia-inducible factor-1 (HIF-1) in CD73 induction by hypoxia (18
) and recently in enothelial cells (46
). Currently, the mechanism for the hypoxia-inducibility of AdoRA2B
and CD39 remains unclear. Relatively little is known about the transcriptional regulation of CD39 and AdoRA2B
, and while HIF-1 is currently an area of intense investigation in a variety of disorders (47
), including inflammation (5
), it remains to be seen whether the coordinated transcription of CD39, CD73, and AdoRA2B
have common regulatory elements.
Some precedent exists for coordinated, metabolic responses to hypoxia. For example, in the regulation of oxygen hemostasis, HIF-1 has been shown to regulate multiple metabolic and compartmental steps in several metabolic pathways (47
). As such, at least 13 different genes encoding glucose transporters and glycolytic enzymes are coordinately decreased in HIF-1 deficiency (47
). Similarly in the present study, transcriptional and metabolic control-points of extracellular nucleotide metabolism and nucleoside signaling are controlled in a coordinated fashion. Interestingly, recent evidence indicates that the AdoRA2A
and CD73 are coordinately regulated by mitogenic stimuli in human B cells, and that such a pathway drives increased signal transduction (48
). The present results of a coordinated response to decreased oxygen availability of CD39, CD73, and the AdoRA2B
suggest a similar evolutionary adaption to hypoxia.
In summary, these results define a previously unappreciated metabolic pathway prevalent at the vascular interface during inflammation/hypoxia. We show here that PMN-derived ATP selectively promotes barrier function of posthypoxic endothelia. The observed differences were attributable to a coordinated transcriptional response initiated by hypoxia, and resulting in amplified metabolism of extracellular ATP to adenosine as well as enhanced signaling through the AdoRA2B. We propose that this feed-forward mechanism may contribute an endogenous, protective pathway for highly vascular tissues during adaptation to hypoxia.