Data reported here show that apelin blocks a spectrum of Ang II–mediated effects on atherosclerosis in the ApoE
-KO model. These endpoints result from a number of pathophysiological processes that are regulated by Ang II in the setting of vascular wall disease, including direct effects on cellular adhesion, proliferation, migration, hypertrophy, and synthesis/degradation of matrix proteins and collagen (31
). Given the number of endpoints evaluated and the consistent findings obtained, it is likely that the apelin-APJ pathway also regulates Ang II signaling in human vascular wall disease. These findings are important, particularly in light of extensive experimental and clinical data regarding the role of Ang II in the initiation and progression of human atherosclerosis. Blocking Ang II atherosclerosis-related effects through angiotensin converting enzyme-1 inhibition and AT1R blockade reduces cardiovascular events, independent of lowering blood pressure (35
). Regulation of Ang II signaling by the endogenous apelin-APJ pathway thus becomes an important variable for consideration. Based on the markedly similar expression patterns of AT1R and APJ receptors, we feel that the apelin-APJ pathway may serve a clinically important role in endogenous regulation of Ang II signaling in vascular disease.
One accepted mechanism for many of the actions of Ang II in vascular wall disease relates to the effect of Ang II on the balance of reactive oxygen species. The related decrease in NO availability and the inflammatory consequences of increased O2•–
signaling in vascular wall cells are thought to be intimately involved in the disease process (36
). Prior studies of apelin’s effects on the vascular wall have focused on the endothelium and the eNOS-dependent production of NO, where apelin potentiates phosphorylation of Akt and eNOS (13
). It thus seemed likely that one mechanism for apelin-mediated amelioration of atherosclerosis might be related to increased NO production, and indeed, experiments reported here show that apelin does increase production of NO, and that l
-NAME blocks apelin-mediated amelioration of Ang II–
exacerbated vascular disease in the ApoE
-KO model. Increased NO is expected to quench O2•–,
and thus mitigate the pathological effects of O2•–
production in the vessel wall, and this is a likely mechanism for some of the in vivo effects of apelin documented in this work. In the models employed in these experiments, there was a decrease in O2•–
that correlated with disease improvement. Evidence against this being the sole mechanism for the disease-inhibiting actions of apelin is the observation that l
-NAME had no effect on O2•–
levels in the group treated with both apelin and Ang II. It thus seems likely that additional actions of apelin are an integral part of its Ang II inhibiting functions. While most of these data are focused on apelin blocking the actions of exogenous Ang II, our findings also support the hypothesis that apelin infusion antagonizes the actions of endogenous Ang II. We found significant increases in NO bioavailability (Figure A), with significant decreases in native atherosclerosis in the aortic root (Supplemental Figure 2) and O2•–
levels in aortas from ApoE
mice treated with apelin (Figure , B and C). In the face of existing data suggesting a prominent role of Ang II in mediating atherosclerosis in hypercholesterolemic mice, it is plausible that the beneficial effects of apelin, in the absence of exogenous Ang II infusion, is at least in part due to antagonism of increased levels of endogenous Ang II in ApoE
-KO mice (38
Cellular studies reported here suggest additional mechanisms for the inhibition of Ang II exacerbation of atherosclerosis. Most significant are the data documenting a direct inhibitory effect of Ang II–mediated increases in gene transcription by concurrent cellular stimulation with apelin. These data establish for what we believe is the first time a direct antagonism at the level of cytoplasmic or nuclear signaling between these 2 peptide ligand pathways. This apelin-mediated antagonism of Ang II signaling correlates with the observed decrease in disease endpoints. These data are intriguing, given the very similar receptor structure and likely similarity between G protein coupling and regulatory proteins employed. Given that a number of Ang II downstream signaling targets were blocked by apelin and that apelin did not block activation of the shared downstream targets of other stimuli such as TNF-α and H2O2, it seemed likely that crosstalk between the Ang II and apelin pathways was occurring at a proximal point in the signaling cascades.
The receptor-receptor interaction studies described here demonstrate one potential molecular mechanism of crosstalk between the apelin and Ang II pathways. Two different experimental approaches employed in these studies have shown that AT1R and APJ physically associate in the cell, presumably on the cell membrane, and this association of APJ appears to inhibit AT1R signaling in a stoichiometric fashion. Ang II–mediated downstream protein phosphorylation and reporter gene activation were both inhibited by expression of APJ in cells also expressing AT1R. We have shown that this association is dynamically regulated by Ang II levels in vitro but did not find evidence that apelin regulates this interaction to a significant degree. These experiments do not exclude the possibility that apelin can modulate receptor-receptor interaction. Further experiments are necessary to define the apelin-mediated modulation of AT1R receptor signaling, which may involve further downstream mechanisms not investigated in the present study. One additional potential mechanism is ligand-mediated variation in receptor expression levels, as relative expression level of the 2 receptors would be expected to have a significant effect on the interaction. Also, in vitro culture conditions and the phenotype of the cultured cells might not accurately reflect the situation in native cells in the diseased tissues.
If apelin does regulate receptor heterodimerization, it could account for some portion of the inhibitory signaling crosstalk that was demonstrated and modulate Ang II signaling in vivo in the disease models. There is now convincing data that such GPCR interactions can promote disease. For instance, interaction of AT1R and the bradykinin B2 receptor is a critical aspect of the preeclampsia syndrome, with AT1R-B2 heterodimerization increasing responsiveness to Ang II (40
). Given the ability of APJ to inhibit AT1R signaling, one important fact that emerges from these studies is the critical importance of disease-related APJ expression level, which is worthy of careful follow-up study in these and other model systems.
While we have provided evidence for crosstalk between the apelin and Ang II signaling pathways inside the cell, it is important to consider which cells in the vessel wall might be the site of such interaction. If the observed effects in the models are due to apelin stimulation of eNOS and resulting increased NO production, the endothelial cell is obviously the cell type primarily involved. Also, in the endothelial cell, blocking Ang II signaling would decrease the well characterized proatherosclerotic effects of Ang II, such as increased adhesion molecule expression, no matter if this occurred at the receptor or intracellular signaling level. Both the APJ and AT1R are known to be expressed by the endothelium, although APJ expression in the adult vasculature has not been thoroughly characterized for either normal or diseased vessel wall. We have employed SMC in some of our experiments, because they are the only cell type that we have found to reliably express both APJ and AT1R in vitro. However, it is unclear whether APJ is expressed on these cells in vivo in atherosclerotic tissues. In fact, radioligand binding studies have shown coronary artery medial receptor expression to be low at baseline and to not be increased in atherosclerotic coronary tissue (41
). There is also very little data regarding APJ expression by macrophages, with one study showing that brain-derived macrophages do not express APJ in culture (42
). Further studies are necessary to elucidate the role of apelin signaling in these cell types.
While this work was being prepared for publication, Hashimoto et al. reported data suggesting conclusions contrary to those derived from studies reported here. In their studies, the ApoE
-KO mice lacking APJ expression had significantly reduced aortic atherosclerosis and O2•–
). This is in contrast to our findings in the infusion models as well as our studies of the apelin-ApoE
double-knockout mice, in which apelin’s effects were clearly antiatherosclerotic. There are a number of differences in the experimental designs of the 2 studies that likely account for the apparent inconsistency of results produced with these different experimental approaches. The genetic background employed in the animal models in the 2 sets of experiments were markedly different, and there is good evidence that modifying loci can significantly influence vascular disease mouse models (44
). The other group employed mice that were fed a very high (1%) cholesterol diet for 15 weeks, while the mice used in our studies were fed a moderate (0.2%) cholesterol diet for shorter durations. Also, Ang II signaling may undergo compensatory downregulation in the APJ-KO mice due to loss of apelin signaling, and this possibility needs to be investigated. Finally, it is possible that alternative genetic loci are developmentally activated in the APJ-KO animals and serve to compensate for loss of apelin-APJ signaling in the disease setting in the adult. Expression of these compensatory loci might account for the phenotype presumed to be due to loss of apelin-APJ. It will be important to repeat these experiments with an inducible recombinase system that allows the functional deletion of the apelin-APJ pathway in the adult animal, which should prevent the confounding activation of compensatory pathways with partially overlapping function, and to perform these studies on the C57BL/6 background that was used in the studies reported here.
In summary, these data establish that apelin can block a number of Ang II–related pathological processes associated with atherosclerosis, including the production of O2•– in the vascular media. This inhibitory activity is likely mediated by increased endothelial NO production and blocking Ang II signaling to the nucleus, with some portion of the latter mediated by physical association of AT1R and APJ. These data suggest a role for the apelin-APJ pathway in atherosclerosis and Ang II–mediated vascular remodeling and provide directions for additional studies further investigating the mechanisms of the observed effects.