Apelin Signaling: a new GPCR pathway in lymphatic vascular development

The identification of novel factors that orchestrate the development, growth and function of the lymphatic vascular system is a relatively new and coveted goal in the broad field of vascular biology. With the increased incidence of lymphedema and the recent recognition of the important roles that lymphatic vessels play in cancer, obesity and metabolic disorders, there is a strong motivation to identify factors that could serve as pharmacological targets for the therapeutic modulation of lymphatic vessel growth and function¹. Since G protein-coupled receptors (GPCRs) constitute the largest proportion of targets for prescribed pharmaceuticals, there is intense interest in revealing novel receptor pathways that could be targeted with highly specific agonists and antagonists². In this issue of Arteriosclerosis, Thrombosis and Vascular Biology, Kim et al³ discover a new player in lymphatic vessels; Apelin signaling—a broadly-expressed and multifunctional GPCR pathway that is part of the broader family of adipocytokine signaling^{4, 5}.

Much of our knowledge about lymphatic vessel growth has come from developmental studies, whereby the establishment of lymphatic progenitor fate, migration, proliferation and maturation can be elegantly teased apart using genetic approaches in mice and aquatic animal model systems^{6, 7}. Lymphatic progenitors arise from venous endothelial cells, and the process of lymphatic endothelial cell migration away from parental veins can be stereotypically visualized and quantitated in zebrafish. Therefore, identifying key growth factors that are spatiotemporally expressed during this critical point of lymphatic vessel formation provides a strong foundation for the elucidation of new players. This pattern of highly restricted expression is precisely what led Kim et al to explore the possible functions of Apelin signaling in lymphatic development. Previous studies had shown that while the expression of Apelin receptors was broad during early zebrafish development, their expression became restricted to blood vasculature at later stages^{8, 9}. Moreover, by 24 hours post fertilization (24hpf), apelin receptor a (aplnra) is expressed exclusively with venous endothelial cells of the trunk⁹—corresponding with the spatiotemporal onset of lymphangiogenesis.

However, the study of lymphatic vascular development in Apelin- or Apelin receptor-deficient conditions is precluded by the dramatic and early-onset cardiovascular phenotypes in both mice and zebrafish (reviewed in⁴). Therefore in an elegant strategy, Kim et al circumvented the confounding early embryonic defects associated with loss of Apelin signaling by injecting zebrafish with suboptimal and titrated doses of morpholinos targeted against either the Apelin receptor, aplnra or the Apelin ligand, apln. At low dose morpholino injections, this approach allowed for normal development and function of the heart and blood vasculature through 4 days post fertilization (4dpf), and thus the opportunity to evaluate the effects of partial Apelin signaling loss at later developmental time points.

The most striking observation was that aplnra morpholino-injected animals lacked a thoracic duct and had significantly fewer lymphatic endothelial cells compared to control-injected animals. Zebrafish have two Apelin receptor genes, aplnra and aplnrb. Previous studies have shown that morpholino knockdown of aplnrb causes early embryonic lethality associated with abnormal heart field development during early zebrafish gastrulation^{8, 10}. Therefore, it is worth noting that the marked lymphatic phenotypes caused by temporal and suboptimal knockdown of aplnra were not compensated for by the functions of aplnrb.

Similarly, loss of the Apelin ligand by titrated morpholino injection also resulted in a dose-dependent reduction in numbers of lymphatic endothelial cells and complete absence of the parachordal vessels—which give rise to lymphatic structures later in zebrafish development. Moreover, micro-lymphangiography showed dysfunctional lymphatics in Apelin signaling morphants. Importantly, the lymphatic phenotype of apln morpholino embryos could be phenotypically reversed with dosed co-injections of apln mRNA, thereby ruling out any secondary effects due to morpholino injection or cardiovascular defects. Thus, the ability to develop and characterize a range of depleted Apelin signaling during development has revealed novel roles for this multifunctional adipokine system during later stages of embryogenesis.

The next important questions were to determine the precise cellular defects caused by reduced Apelin signaling and to elucidate which downstream effector pathways might be activated by Apelin signaling in lymphatic endothelial cells. Since Apelin is an adipokine, with previously described roles in controlling cell migration, the authors used siRNA approaches to knockdown the Apelin receptor (APLNR) in cultured human lymphatic endothelial cells (hLECs). Using a scratch wound assay, they found significant impairment of migration in APLNR-knockdown hLECs, even in response to exogenously-added Apelin ligand. The knockdown of APLNR in hLECs was also associated with a robust decline in basal levels of phospho-AKT1/2, whereas stimulation of control hLECs with Apelin ligand caused a dose-dependent increase in phospho-AKT1/2. Importantly, the effects of reduced Apelin signaling on levels of phospho-AKT1/2 were recapitulated in the dorsal aorta and cardinal veins of apln morpholino-injected zebrafish during early developmental stages (48hpf). However, this early developmental analysis of apln morphants precluded the ability to address whether disrupted AKT signaling was present in lymphatic progenitors of Apelin signaling mutants. Therefore, the Authors used two well-established chemical antagonists of AKT signaling (LY294002 and Torin1) to inhibit AKT phosphorylation later during development in both control- and morpholino-injected zebrafish. As expected, chemical inhibition of AKT phosphorylation dramatically attenuated the number of lymphatic endothelial cells present in 4dpf embryos. This effect was substantially exacerbated by 40% when apln morpholino-injected animals were also treated with these inhibitors. However, phospho-AKT is unlikely to be the only downstream effector of Apelin signaling in lymphatic endothelial cells, because the converse experiment—to rescue Apelin signaling deficiency with chemical activation of AKT—failed to increase the number of lymphatic endothelial cells in the morpholino-injected mutants.

These findings raised the possibility that Apelin signaling may be influencing the actions of VEGF-C—the key stimulus for lymphatic vascular migration and development, which also potently activates AKT. Although expression levels of vegfc or its receptor, flt4, were not affected by morpholino-induced reduction in Apelin signaling (and vice-versa), co-injection of apln and vegfc morpholinos at suboptimal doses significantly exacerbated the lymphatic phenotypes of Apelin signaling mutants. These results indicate that together, these two potent stimuli of AKT activation play important roles in lymphatic development. However, their functions appear to be non-redundant because ectopic expression of apln mRNA was unable to rescue vegfc morpholino-induced lymphatic defects, and conversely, vegfc mRNA also had no significant effect on the apln-knockdown phenotype.

This raises the interesting possibility that the functions of these two potent AKT activators may be spatially and temporally segregated at different times during lymphatic vascular development. To provide support for this hypothesis, the Authors examined the temporal expression of both Apelin and VEGFC signaling components throughout the first five days of zebrafish development. Consistently, they found that levels of Apelin signaling components were perpetually increased during the first five days of development, while the levels of vegfr3 dropped precipitously at 2dpf—the time at which lymphatic endothelial cells first begin to emerge from intersomitic vessels. Based on this data, the Authors speculate that the functions of VEGFC and Apelin in lymphatic endothelial cells may be temporally distinguished, such that VEGFC plays critical roles in early lymphatic migration from venous endothelium, while Apelin may be required at slightly later stages to maintain AKT levels for normal migration and proliferation of lymphatic endothelial cells. However, additional studies will be required in order to substantiate this model. For example, the relative expression levels of these factors to each other and their spatial distribution within the animal could not be determined from these whole-animal gene expression profiles. Therefore, it will become important to precisely map the spatiotemporal expression of the Apelin and VEGFC receptors in lymphatic endothelial cells throughout development. In addition, recently identified and highly-specific Apelin receptor agonist¹¹ and antagonists¹², will provide useful and beneficial approaches for developing dose-dependent activation and inhibition of Apelin signaling during embryogenesis, in a manner that will circumvent the caveats of short-lived morpholinos. In this way, the distinct roles of Apelin signaling, versus VEGFC, in controlling the temporal aspects of AKT-mediated lymphatic vascular development could be directly addressed.

The dramatic lymphatic phenotypes of Apelin signaling morpholino zebrafish also raise several interesting questions regarding the possible functions of Apelin signaling in the mammalian lymphatic vascular system. Importantly, the Authors of this study found that the number of VEGFR3-positive lymphatic endothelial cells was significantly reduced in the diaphragms of homozygous null Apelin receptor mice (Apj^−/−), suggesting that a conserved function for Apelin signaling in lymphatics may exist across species. However, to date, a thorough phenotypic characterization of lymphatic development in mice lacking either the Apelin^{13, 14} or Apelin receptor^{15, 16} genes has yet been reported. Indeed, since the cardiovascular developmental phenotype of these animals is severe, conditional and temporal deletion of the Apelin receptor in lymphatic endothelial cells should be an extremely interesting and informative next-step.

These developmental studies also raise the important question of whether and how the Apelin signaling pathway might regulate normally quiescent adult lymphatic vessels? While numerous studies have demonstrated an important role for Apelin signaling in blood vessel maturation and stabilization⁴, only a few have begun to explore the effects on adult lymphatic vessels. For example, transgenic mice over-expressing Apelin in dermal keratinocytes are protected from UV-induced edema and display reduced inflammation which is associated with enlarged and stabilized lymphatic vessels¹⁷. Similarly, the effect of Apelin in stabilizing lymphatic vessel permeability was also recently observed in a high fat diet, obesity mouse model¹⁸. It is likely that the effects of Apelin signaling on lymphatic endothelial cells are conserved from development to adulthood. Therefore, this highly conserved G protein-coupled receptor pathway may offer new avenues for pharmacologically targeting the adult lymphatic vascular system in lymphatic-related disease conditions such as lymphedema and tumor lymphangiogenesis.