50 E. B. Pasquale

primary lymphatic plexus in the skin (which expresses both ephrin-B2 and EphB4) forms normally but there are defects in subsequent sprouting of new capillaries (expressing only EphB4) and in vascular remodeling. In addition, collecting lymphatic vessels are hyperplastic and lack the luminal valves that allow proper lymphatic drainage. In contrast, knock-in of a mutated form of ephrin-B2 lacking all the cytoplasmic tyrosine phosphorylation sites almost fully compensates for the lack of wild type ephrin-B2. These data indicate that ephrin-B2 interaction with PDZ domain-containing proteins is required for normal development of the lymphatic vasculature, whereas ephrin-B2 tyrosine phosphorylation and interaction with SH2 domain-containing proteins are dispensable. Consistent with this idea, several known ephrin-B2 binding proteins that contain PDZ domains were found to have altered subcellular distribution in lymphatic vessels expressing the mutant ephrinB2 without the PDZ domain-binding site. Ephrin-B2 reverse signaling therefore seems to play a more important role in lymphatic vascular morphogenesis than in blood vessel morphogenesis.

6. Adult Vasculature

Given the importance of Eph receptors and ephrins in the formation of the embryonic vasculature, it is not surprising that these molecules have also been implicated in physiological and pathological forms of postnatal angiogenesis.

6.1. Quiescent vasculature

Ephrin-A1 is downregulated during embryonic development and is not detectable in adult quiescent vasculature,60 but some EphA receptors are expressed in normal adult blood vessels. For example, EphA7 has been detected in the blood vessels of the liver septa and in blood vessels of the renal parenchyma.77 However, the function of these receptors in the adult vasculature and the identity of their ephrin-A ligand counterpart are not known.

A stable molecular difference between arteries and veins persists in the adult quiescent vasculature, where ephrin-B2 remains expressed in arterial endothelial cells.51,78 EphB4 remains expressed in small diameter

EPH Receptors and Ephrins 51

venous microvessels and capillaries as well as in large veins such as the vena cava, where this receptor exhibits a patchy heterogeneous expression. In addition, however, low levels of ephrin-B2 have been detected in some cells of adult veins, such as the vena cava, and EphB4 has been detected in some arteries. Another receptor for ephrin-B2 in the adult vasculature is EphB1, which in adult kidney glomeruli is expressed at higher levels than during development.17 Thus, interactions between ephrin-B2 and EphB receptors likely continue to play a role in the maintenance of a mature vessel configuration.

An increasingly important role of ephrin-B2 at later stages of vascular development may be to regulate vascular smooth muscle cells. The endothelial expression domain of ephrin-B2 expands as development progresses to include the vascular smooth muscle cells of many arteries where this ligand continues to be expressed in the adult.51,78 Interestingly, ephrin-B2 is upregulated in vascular smooth muscle cells only after they have already lined blood vessels and expression starts from the smooth muscle cells directly in contact with endothelial cells and gradually progresses towards more external regions.51

6.2. Physiological angiogenesis

EphA2 is one of the key Eph receptors that play a role in postnatal angiogenesis, even though it does not seem to be involved in angiogenesis during embryonic development (Sec. 4.1). With regard to the B class, ephrin-B2 continues to be expressed in the arterial vasculature at sites of secondary angiogenesis in the embryo, such as the heart, neural tube, kidney and lung. Ephrin-B2 expression also persists at sites of adult angiogenesis, such as the ovarian follicles and the corpus luteum in the female reproductive system.78 Here, ephrin-B2 presumably plays an important role in the continuous vascular remodeling that occurs during the estrous cycle. It has also been recently proposed that the ephrin-B/EphB system plays a role in connecting the blood vessels of the human placenta to the maternal circulation, a process mediated by fetal cytotrophoblast cells that invade the uterine wall to reach arterioles and remodel them.79 During their differentiation, the cytotrophoblast cells lose EphB4 expression and acquire ephrin-B1 and ephrin-B2 expression, which results in repulsive signals and reduced