While dysfunctional BMP signaling is linked to the pathogenesis of IPAH, our present findings identified apelin as a secreted protein downstream of BMP signaling that regulates pulmonary vascular homeostasis. We showed that BMP-mediated expression of apelin was regulated by formation of a transcriptional complex between PPARγ and β-catenin and that this complex was induced by endogenous PPARγ ligands, such as NO2
-FAs. BMP-mediated apelin production contributed to EC homeostasis by promoting PAEC survival, proliferation, and migration. These features are functionally significant in the preservation and regeneration of the vasculature in response to injury. In addition to the autocrine functions of apelin, it displays a paracrine effect by attenuating the response of PASMCs to growth factors and by promoting apoptosis. These findings support the beneficial effect of apelin on cardiac function in rats with PAH (28
), underscore the adverse impact of its loss on the severity of PAH (29
), and make apelin an attractive pharmacological target for the treatment of PAH.
Our observation that the PPARγ antagonist GW9662 as well as the agonist rosiglitazone impaired BMP-2–mediated survival of PAECs or PMVECs was consistent with previous studies showing that these reagents can both induce anomalous PPARγ activation (19
). These observations reinforce current doubt as to how well this class of PPARγ-targeted drugs act as physiological regulators of PPARγ. The use of thiazolidinediones (TZDs), such as rosiglitazone, as antidiabetic drugs is under critical scrutiny, since it is becoming apparent that they are manifesting adverse side effects that include weight gain, fluid retention, hepatotoxicity, and risk of adverse cardiovascular events (33
). This motivates consideration of whether the adverse effects of TZDs are related to their disruption of more salutary PPARγ-mediated gene regulation. At least one case report indicates an adverse effect of rosiglitazone on pulmonary hypertension in a patient with diabetes (34
). Our studies suggest a need for more effective PPARγ-targeted drugs that have partial agonist properties that are more consistent with endogenous ligands. Endogenous ligands for PPARγ include NO and NO2
-FAs. These species show a very high affinity for activating PPARγ as partial agonists by covalently adducting the Cys285 of the ligand binding domain (16
). Our present studies demonstrated that NO2
-FAs were also distinguished from TZD PPARγ agonists, such as rosiglitazone, by not disrupting the interaction of PPARγ with β-catenin (9
). Moreover, NO2
-FAs were capable of inducing PPARγ/β-catenin complex formation independent of exogenous BMP stimulation, consistent with their overall promotion of PAEC survival.
Our group previously described BMP-mediated regulation of PPARγ in PASMCs (13
), but in these studies, β-catenin is only transiently transcriptionally active (38
). Moreover, rosiglitazone functioned like BMP-2 in repressing proliferation of SMCs in response to growth factors. Previous cancer cell studies (21
) indicated a physical interaction between PPARγ and β-catenin that was attributed to PPARγ-mediated translocation of β-catenin to the cytosol for proteasomal degradation (22
). Notably, one report indicates that the PPARγ/β-catenin complex can bind a PPRE, and that β-catenin can promote transcription of a PPRE reporter (21
). Association of β-catenin with several other nuclear receptors has been well documented, although the functional significance of these interactions was not well defined (39
). We showed herein that interaction between PPARγ and β-catenin promoted regulation of genes that confer normal function and homeostasis to vascular cells. The mechanism promoting this interaction is not known, but our previous studies suggest that it is not pSmad dependent (9
We carried out ChIP-chip and microarray analyses to determine which of the co-occupied genes are downstream of BMPR2-mediated signaling and have β-catenin–dependent expression. With the criteria used, we may have excluded a large number of gene targets of the PPARγ/β-catenin complex. Although we identified a number of genes related to EC homeostasis, we focused on APLN
among the 18 targets, both because it was among the most highly regulated and because its reduction, like that of BMPR2, was linked to clinical PAH (28
Apelin is highly expressed by systemic ECs, and a previous study showing that it can promote vascular regeneration (31
) is in keeping with the downstream actions of BMPR2 signaling. We showed herein that decreased apelin expression in PAECs increased their susceptibility to apoptosis, an event that can augment microvascular injury and impair recovery. As decreased survival is also observed in ECs deficient in BMPR2 (8
), our observations suggest that this may be related to reduced levels of apelin, and thus might be prevented by apelin administration.
Paracrine effects of apelin were previously related to its activation of cardiac contractility (40
) and vasodilatation, in part by inducing NO production (30
). Our coculture studies demonstrated that the apelin-deficient PAECs also promoted PASMC proliferation, related either to reduced EC survival (42
) or to active production of mediators of PASMC proliferation. We documented direct growth-suppressing effects of apelin on SMCs that were minimized in apelin-deficient EC–conditioned medium. Whereas previous studies suggested that apelin promotes systemic SMC proliferation (43
), the opposite effect occurred in PASMCs. However, our study is consistent with a recent report indicating that apelin protects against atherosclerosis and that apelin treatment can inhibit angiotensin II–mediated neointimal formation (44
Apelin is the only known ligand for the APJ receptor, and the APJ receptor can mediate the effects of apelin on the cardiovascular system. However, the phenotype of the apelin-KO mouse is far less severe that that of the APJ-KO mouse, which dies in embryonic life in association with cardiac defects (45
), suggestive of additional APJ ligands or ligand-independent effects of the receptor. Apelin-deficient mice have decreased myocardial contractility under stress (45
) and retardation of retinal angiogenesis (46
), as well as exaggerated pulmonary hypertension (29
). The latter has been attributed to the induction of eNOS by apelin. In those studies, as in ours, apelin can induce angiogenesis by interacting with VEGFA and serum factors such as FGF2, as well as via eNOS induction.
Although it was previously suggested, using a lacZ reporter transgenic mouse model (47
), that apelin expression is restricted to ECs of capillaries and veins, our observations and those of others noted expression of apelin in precapillary arteries in human lungs and in other tissues (48
). This could be related to additional apelin promoter elements not included in the lacZ reporter construct or to lack of sensitivity of lacZ staining. We attribute the reduced apelin in PAECs from patients with IPAH to their reduced BMPR2 expression.
Since loss of BMPR2 in ECs is lethal in the embryo, we chose to test whether the PAH observed in mice as a consequence of PPARγ deletion in ECs and reduced apelin production could be reversed by adding back exogenous apelin. In the TIE2CrePPARγfl/fl mouse, the manifestation of apelin loss is the absence of its paracrine effect in suppressing SMC proliferation and muscularization of distal arteries. Adding exogenous apelin was sufficient to reverse this morphological change and the associated mild PAH and RVH and was in keeping with the studies in cultured PAECs showing that apelin induced SMC apoptosis. It would be of interest to test whether apelin is reduced in other models of PAH and whether it also promotes regeneration of lost microvessels associated with PAH.
Current experimental approaches to reverse pulmonary vascular remodeling and treat PAH have focused either on promoting SMC apoptosis (49
) or on inducing regeneration of precapillary arteries (50
). Some of these approaches are already finding their way into clinical trials. Our observations in the present study suggest that apelin can do both. Moreover, it represents the replacement of a key downstream gene that is lost when there is dysfunctional BMPR2 signaling. This, coupled with its properties as a vasodilator and as a potent activator of LV and RV contractility (28
), makes apelin a very attractive therapeutic target for treating PAH.