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Apelin signaling plays an important role during embryo development and regulates angiogenesis, cardiovascular activity, and energy metabolism in adulthood. Overexpression and hyperactivity of this signaling pathway is observed in various pathologic states, such as cardiovascular diseases and cancer, which highlights the importance of inhibiting apelin receptor (APJ); therefore, we developed a cell-based screening assay that uses fluorescence microscopy to identify APJ antagonists. This approach led us to identify the U.S. Food and Drug Administration-approved compound protamine-already used clinically after cardiac surgery-as an agent to bind to heparin and thereby reverse its anticlotting activity. Protamine displays a 390-nM affinity for APJ and behaves as a full antagonist with regard to G protein and β-arrestin-dependent intracellular signaling. Ex vivo and in vivo , protamine abolishes well-known apelin effects, such as angiogenesis, glucose tolerance, and vasodilatation. Remarkably, protamine antagonist activity is fully reversed by heparin treatment both in vitro and in vivo Thus, our results demonstrate a new pharmacologic property of protamine-blockade of APJ-that could explain some adverse effects observed in protamine-treated patients. Moreover, our data reveal that the established antiangiogenic activity of protamine would rely on APJ antagonism.-Le Gonidec, S., Chaves-Almagro, C., Bai, Y., Kang, H. J., Smith, A., Wanecq, E., Huang, X.-P., Prats, H., Knibiehler, B., Roth, B. L., Barak, L. S., Caron, M. G., Valet, P., Audigier, Y., Masri, B. Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin., (© FASEB.)

The formation of the vascular system is an early step in organogenesis that involves the participation of various signalling pathways. Integration of the extracellular signals decoded by their cognate membrane receptors orchestrate the cell events, which act at different stages, from the primitive network formed by vasculogenesis to the arborescent network remodeled by angiogenesis. Our laboratory showed the participation of a new signalling pathway in physiological angiogenesis and tumour neovascularisation. This signalling pathway named apelin comprises a G protein-coupled receptor and a peptide ligand. Expression of apelin receptors is observed during the embryonic formation of blood vessels where it is localized in the endothelium. In HUVECs, which endogenously express apelin receptors, apelin promotes the phosphorylation of ERKs, Akt and p70 S6 Kinase. In addition, apelin increases in vitro the proliferation of these endothelial cells. Finally, injection of apelin in the vitreous induces in vivo the sprouting and the proliferation of endothelial cells from the retinal vascular network. Accordingly, all these results led us to study the role of apelin signalling in tumour neovascularisation. In two tumoral cell lines, we showed that hypoxia induces the expression of apelin gene. In addition, the overexpression of apelin gene resulting from stable transfection of these cell lines clearly accelerates in vivo tumour growth, as a consequence of an increased number of vessels irrigating these tumours. The pathological relevance of these data has been validated by the characterization of an overexpression of apelin gene in one third of human tumours. Taken together, apelin signalling is both involved in physiological angiogenesis and pathological neoangiogenesis, and therefore represents an interesting pharmacological target for anti-angiogenic therapies.

The apelin receptor is a G protein-coupled receptor activated by several apelin fragments. Its tissue distribution suggests that apelin signalling is involved in a broad range of physiological functions. Endothelial cells, which express high levels of apelin receptors, respond to apelin through the phosphorylation of key intracellular effectors associated with cell proliferation and migration. In addition, apelin is a mitogen for endothelial cells and exhibits angiogenic properties in matrigel experiments. This review focuses on the therapeutic potential of apelin signalling, which is associated with pathologies that result from decreased vascularisation (ischemias) or neovascularisation (retinopathies and solid tumors).

The apelin receptor is a G protein-coupled receptor to which two ligand fragments, apelin-(65-77) and apelin-(42-77), can bind. To address the physiological significance of the existence of dual ligands for a single receptor, we first compared the ability of the apelin fragments to regulate intracellular effectors, to promote G protein coupling, and to desensitize the response in Chinese hamster ovary cells expressing the murine apelin receptor. We found that both apelin fragments inhibited adenylyl cyclase and increased the phosphorylation of ERK or Akt. Using stably transfected cells expressing a pertussis toxin-insensitive alpha(i) subunit, we demonstrated that each apelin fragment promoted coupling of the apelin receptor to either Galpha(i1) or Galpha(i2) but not to Galpha(i3). Although preincubation with each apelin fragment induced a desensitization at the level of the three effectors, preincubation with apelin-(42-77) also increased basal effector activity. In addition, a C-terminal deletion of the apelin receptor decreased the desensitization induced by apelin-(65-77) but did not alter the desensitization pattern induced by apelin-(42-77). Finally, in umbilical endothelial cells, which we have recently shown to express the apelin receptor, the Galpha(i1) and Galpha(i2) subunits are also expressed, ERK and Akt phosphorylation is desensitized after preincubation with apelin-(65-77), and basal levels of Akt phosphorylation are increased after preincubation with apelin-(42-77). In summary, apelin fragments regulate the same effectors, via the preferential coupling of the apelin receptor to G(i1) or G(i2), but they promote a differential desensitization pattern that may be central to their respective physiological roles.

The discovery of new signalling pathways is always followed by the development of pharmacological agents as drugs that can be used in the treatment of diseases resulting from a dysfunction of the signalling pathway in question. Apelin signalling plays a role in the central and peripheral regulation of the cardiovascular system, in water and food intake, and possibly in immune function. Up-regulation of ligand and receptor is also associated with pathophysiological states such as cardiac dysfunction and neovascularisation. Finally, the apelin receptor is a coreceptor for the entry of several HIV-1 and SIV strains. In view of these features, the apelin receptor constitutes a very interesting target for the design of new drugs for treating the prime causes of human mortality.

Expression of the new G protein-coupled msr/apj receptor in the mouse embryo is restricted to the endothelial layer of the primary blood vessels and the newly forming heart (Mech. Dev. 84 (1999) 199). During development of the retinal vasculature, the msr/apj gene is not expressed throughout the vascular network, indicating a possible relationship between the localization of expression and the acquisition of arterial or venous identity (Mech. Dev. 110 (2002) 183). Here we first established that retinal expression of ephrin-B2 and its putative receptor EphB4 correlates with arterial and venous phenotype, respectively. Then we analyzed the expression pattern of msr/apj in the retinal vessels at various stages of postnatal development by in situ hybridization. In contrast to the expression of ephrin-B2 or EphB4, msr/apj transcripts can be detected as early as postnatal day P3. From P3 to P12, msr/apj expression in the vascular network is restricted to the venules and the associated capillaries. The msr/apj gene is thus an early and specific marker of the venous phenotype in the retinal vasculature.

We report here that apelin (65-77) induces activation of extracellular-regulated kinases (ERKs) in Chinese hamster ovary (CHO) cells expressing the msr/apj receptor. This concentration-dependent activation was transient, peaking at 5 min. Pretreatment of CHO cells with pertussis toxin fully abrogated ERK phosphorylation, whereas overexpression of the beta-adrenergic receptor kinase-1 C-terminal fragment did not alter ERK activation. Transfection with a dominant-negative mutant of Ras was without effect on ERK activation, whereas an inhibitor of many protein kinase C isoforms, GF109203X, strongly decreased it. These results demonstrate that stimulation of the murine msr/apj receptor promotes ERK activation via the alpha subunit of a pertussis toxin-sensitive protein in a Ras-independent pathway., ((c)2002 Elsevier Science.)

We have recently identified a new G protein-coupled receptor, msr/apj, whose transcripts are detected in the endothelium of the primary blood vessels and the newly forming heart (Mech. Dev. 1999;84:199). Its expression during formation of the embryonic vasculature incited us to investigate whether expression of the receptor also increased during the formation of retinal vessels, which occurs postnatally in the mouse. Interestingly, msr/apj transcripts are indeed associated with the forming vessels and trace the centrifugal extension of the superficial vasculature. We also show that expression of the endogenous ligand apelin is upregulated at the leading edge of vessel formation.

We have recently identified a new G protein-coupled receptor, X-msr, whose expression is associated with the endothelial lineage in Xenopus laevis (Devic, E., Paquereau, L., Vernier, P., Knibiehler, B., Audigier, Y., 1996. Expression of a new G protein-coupled receptor X-msr is associated with an endothelial lineage in Xenopus laevis. Mech. Dev. 59, 129-140). Based on its structural analogy to the human orphan receptor APJ, we cloned the murine msr/apj receptor and analyzed its expression in developing tissues. As observed for X-msr, msr/apj transcripts are detected in the endothelium of the primary blood vessels and the forming heart. In addition, they are expressed in somites, limb bud and branchial arches. This expression pattern is distinct from that of the Flk1 gene and suggests that the msr/apj gene is expressed in a subpopulation of endothelial precursors and a mesenchymal population derived from paraaxial mesoderm.

Characterization of a new family of G protein-coupled receptors is reported. Expression of these receptors is associated with endothelial lineage. Cloning of the Xenope X-msr receptor allowed to show that embryonic expression of this receptor occurred in the heart and developing primary blood vessels. Furthermore, within these cardiovascular structures, expression was restricted to the endothelial layer. Because structural similarities with the human orphan receptor h-APJ were found, the msr/apj receptor was cloned in mice. This showed that embryonic expression of this receptor was also confirmed to endothelial precursors. Thus, this receptor is the orthological equivalent in mice to the amphibian receptor X-msr. Molecular phylogenesis studies showed that the X-msr, msr/apj, and h-APJ receptors shared considerable homology with two CXC chemokine receptors, namely LCR1, whose name was recently changed to CXCR4, and RDC1, which is structurally similar to the CXCR2 receptor. The human h-APJ receptor is a co-receptor for entry of the HIV into T cells, a property associated only with CXC chemokine receptors in the lymphocyte population. These data suggest that this new signaling system may participate in endothelial precursor migration during developmental angiogenesis and in endothelial cell migration and proliferation during neoangiogenesis in adults.

In order to determine whether G protein-coupled receptors play a role in early embryogenesis, we looked for cDNA fragments amplified between primers located in consensus sequences of transmembrane segments. Using one such amplified fragment as a probe, we cloned a novel member of the G protein-coupled receptor superfamily in Xenopus. Alignment of the deduced protein sequence with that of other receptors discloses some homology with angiotensin receptors. A single transcript of 2.5 kb is detected at the late blastula stage and its expression increases during gastrulation. In situ hybridization reveals transcripts initially in the ventrolateral involuting marginal zone and later in the lateral plate mesoderm. At larval stages, the transcript is expressed in procardiac tube and forming blood vessels, where it is localized in the inner endothelial layer. Thus, this gene traces an endothelial lineage and represents a very early and unique marker in Xenopus of the specification of cardiac and vascular endothelia. We propose the name of X-msr for mesenchyme-associated serpentine receptor.