Absence of CD39 leads to a secondary imbalance between extracellular nucleotides and nucleosides.15
This disordered purinergic signaling results in increased immediate vascular injury, impaired hepatocyte turnover, and later increments in endothelial cell apoptosis in CD39 null mice, after partial hepatectomy. CD39 deletion in mice alters both the early paracrine stimulation of hepatocyte turnover by LSEC and the crucial later proliferative responses of LSEC during liver regeneration. There is also decreased survival in CD39-null mice that is associated with increased liver injury and heightened LSEC apoptosis.
The expression of CD39 in the normal liver is typically confined to Kupffer cells and endothelial cells of muscularized vessels. Interestingly, vascular and sinusoidal expression of CD39 postpartial hepatectomy is up-regulated. Ecto-NTPDase activity is already boosted on LSEC in wild-type mice, prior to the angiogenic responses noted at days 3–5. Such early growth responses of LSEC postpartial hepatectomy are significantly perturbed in mice null for CD39.
We also show that CD39 deletion can alter proliferation of hepatocytes that do not intrinsically express this ecto-enzyme. We suggest that CD39 expression by LSEC might further coordinate liver remodeling by initially facilitating VEGF-induced paracrine release of HGF to regulate hepatocellular proliferation and liver regeneration. Therefore, CD39 and extracellular nucleotide/nucleoside fluxes appear to be implicated in growth factor responses involving the signaling pathways induced by tyrosine receptor kinases in addition to their previously proven roles in angiogenesis.8,15
Postpartial hepatectomy, CD39 appears to boost angiogenesis directly by controlling pericellular nucleotide/nucleoside boluses that regulate endothelial cell proliferation and may also regulate endothelial cell apoptosis. Persistent elevations in pericellular ATP levels in mice null for CD39 also result in P2Y2R receptor desensitization, the receptor that has been previously implicated in the regulation of vascular growth responses.14
In contrast, continuous stimulation of P2X7R, a receptor that is resistant to desensitization, by ATP may differentially promote cell death.16
Similar to our findings in vivo, increased levels of apoptotic LSEC were found in vitro in response to continuous stimulation with extracellular nucleotides in addition to cycloheximide and TNF-α
. We suggest that this is related to unopposed activation of the P2X7R, triggering cellular apoptosis.17–19
The presence of CD39 seems to be crucial in protecting from increased levels of extracellular nucleotides. These mediators are released during inflammatory or surgical stress, as seen in other models of immune liver injury.13
VEGF has been shown to impact liver regeneration in both angiogenesis-dependent and angiogenesis-independent manners, following on the coordinated activation of both VEGFR1 and VEGFR2.4,20
As VEGFR1 and VEGFR2 form heterodimers,21,22
concurrent VEGFR2 signaling by VEGFR1 (and flt-1) may be considered highly probable. In response to soluble flt-1, LSEC can be shown to modulate both HGF and IL-6 expression and functions on hepatocytes.4
In our study, failure of VEGF signaling by CD39-null LSEC is associated with decreased levels of HGF secretion (). Transactivation of VEGFR2 signaling by extracellular nucleotides (UTP/ATP) via P2Y2R seems to impact on postoperative outcome post-partial hepatectomy.
Figure 6 Impact of purinergic signaling upon interactions between LSEC and hepatocytes during early liver regeneration. (1) Absence of CD39 leads to elevated nucleotide levels. These changes in fluxes of extracellular nucleotides (ATP, UTP, and ADP) impact P2 (more ...)
This finding is in complete agreement with observations in other systems in that activation of VEGFR2 is tightly modulated by extracellular nucleotides.14,23
P2Y2R and VEGFR2 are also shown to colocalize on vascular cell membranes, and activation of the P2Y2R thereby induces rapid tyrosine phosphorylation of VEGFR2 in endothelial cells.14,23
Desensitization of P2Y2R can occur in response to heightened and prolonged levels of extracellular nucleotides, following on deletion of CD39.7
This process then leads to failure of VEGFR2 transactivation.8
We propose that the effects of disordered purinergic signaling in vivo are related to vascular expression of CD39 with secondary persistent impacts on P2 nucleotide receptor responses on LSEC and hepatocytes.7,8
Changes in fluxes of extracellular nucleotides impact P2Y receptor signaling (inclusive of desensitization responses) in nonvascular tissues,7
shown here to include liver. This hypothesis was further tested in mice null for P2Y2R. These mice had defects that are comparable with the CD39 null mice with respect to readouts of hepatocellular proliferation in vivo and in vitro and disordered phosphorylation of VEGFR2 in LSEC. Thus, as expected, mice null for P2Y2R also show altered cross talk between endothelial cells and hepatocytes.
Injecting apyrase, a soluble ENTPD, boosted levels of replicating hepatocytes postpartial hepatectomy. In previous studies, short-term bolus injection of ATP into normal livers was noted to result in entry of hepatocytes into cell cycle and increased replication.9
In our study, however, injection was continuous and nucleotide infusion was employed, further perturbing the elevated levels expected with tissue injury secondary to obligatory surgical injury. In this setting, extracellular nucleotide levels are likely to be increased over normal levels. Therefore, the importance of tight regulation including clearance of increased levels of extracellular nucleotides seems to be crucial in this setting.
We conclude that CD39 modulates liver regeneration in both angiogenesis-independent and -dependent manners. We have identified extracellular nucleotides and purinergic pathways as potential targets for therapeutic intervention in liver injury and regeneration. Pharmacologic strategies to enhance liver regeneration with the infusion of soluble CD39 derivatives might result in better clinical outcomes in liver failure and after surgical resection.