EphrinB2 and EphB4 expression has been previously characterized in the developing arteries and veins of the superficial vascular plexus in the neonatal mouse retina (Saint-Geniez et al.,2003; Uemura et al.,2006). The current study extends the prior findings by establishing that ephrinB2 and EphB4 expression was associated with a subset of capillaries in the developing deep retinal vascular plexus and subsets of pathologic preretinal neovascular tufts. Furthermore, ephrinB2 and EphB4 expression helped delineate arteriovenous shunts that were observed in the OIR model.
Expression of EphrinB2 and EphB4 During Deep Capillary Plexus Formation
The deep capillary plexus of the retina begins to develop between approximately P7 and P8 (Dorrell et al.,2002; Davies et al.,2003) by an angiogenic process (Fruttiger,2007). Our results have revealed that the deep retinal vascular plexus consists of subsets of both ephrinB2+ or EphB4+ vessels, as well as a large number of ephrinB2−/EphB4− capillaries. Expression of ephrinB2 has been previously noted to occur at sites of secondary angiogenesis, including normal physiologic and tumor angiogenesis (Gale et al.,2001; Shin et al.,2001). The ephrinB2-labeled tumor vessels arose from previously existing ephrinB2+ arterioles, supporting the notion that ephrinB2+ vessels are characteristic of secondary capillary networks. Similar to our results, EphB4 has also been previously noted to be expressed by angiogenic capillaries, while modulation of EphB4 altered vascular patterning, confirming a functional role beyond venous phenotype marker (Erber et al.,2006; Taylor et al.,2007). It has been noted that symmetric distribution of ephrinB2+ and EphB4+ vessels are essential for normal vascular development (Visconti et al.,2002). This suggests both vessel populations are required for proper formation of the deep retinal plexus, likely providing focal-adhesion and migration clues required for the developing network of vessels (Fuller et al.,2003). However, based on their known repulsive properties it would be unlikely that ephrinB2+ and EphB4+ vessels would directly connect to each other (Marston et al.,2003; Zimmer et al.,2003). As mentioned above, we observed a large number of ephrinB2−/EphB4− deep retinal vessels, which would allow intercalation between ephrinB2+ and EphB4+ vessels permitting formation of a capillary plexus. In support of our data, Oike and colleagues proposed a model for capillary network formation that is regulated by ephrinB2 and EphB4 interactions, in which an ephrinB2− capillary is required to bind with an EphB4+ capillary (Oike et al.,2002). The proposed model was subsequently supported experimentally by lacZ staining experiments in which an ephrinB2−/EphB4− EC population was found situated between the ephrinB2+ and EphB4+ ECs (Fuller et al.,2003).
Altered Expression of EphB4 During Hyperoxia Phase
The genetic pre-determination of arterial-venous identity is under the complex control of several gene pathways (Gridley,2007; Lin et al.,2007), while vessel specification needs to be maintained by local micro-environmental cues and can be altered by changes in blood flow, physiological requirements, and pathologic conditions (Korff et al.,2006; Aitsebaomo et al.,2008; Rocha and Adams,2009). After hyperoxia exposure, ephrinB2 expression is maintained in the developing arteries and arterioles, consistent with a prior study in which ephrinB2 expression in developing retinal arteries was not affected by exposure to 60% hyperoxia (Claxton and Fruttiger,2005). In contrast, EphB4 expression was present in the central veins but attenuated in the peripheral veins after 24 hr of hyperoxia and completely absent in veins after additional hyperoxia exposure. Similarly, EphB4 mRNA has also been previously noted to be reduced in the OIR model during hyperoxia (Zamora et al.,2005).
EphrinB2 expression is dependent upon VEGF induction and peak expression has been noted on P6 during normal retinal vascular development, a period of VEGF expression and normal physiologic angiogenesis in the retina (You et al.,2005; Hong et al.,2006; Salvucci et al.,2009). Thus, ephrinB2 is associated with arteries and small arterioles when the retina is exposed to hyperoxia on P7, and despite the down-regulation of VEGF during the phase of hyperoxia (Pierce et al.,1995), expression of eprhinB2 is maintained, suggesting alternative regulation. In addition to VEGF, endothelial ephrinB2 expression can also be regulated by contact with smooth muscle cells or pericytes, with expression at points of contact (Korff et al.,2006; Salvucci et al.,2009). As shown by the SMA expression pattern, the relationship between ephrinB2+ arterioles and mural cells is maintained during the hyperoxia phase. Although the initial onset of ephrinB2 expression is independent of mechanical forces, shear stress or wall tension could also induce and maintain gene expression of ephrinB2 as a result of arterial blood flow (Othman-Hassan et al.,2001; Obi et al.,2009).
Recently, COUP-TFII has been identified as a regulator of venous identity, down-regulating the Notch signaling pathway and expression of the VEGF co-receptor, neuropilin 1 (NP-1), thereby inhibiting arterial fate (You et al.,2005; Lin et al.,2007). Although we observed down-regulation of EphB4 expression during the hyperoxia exposure, mRNA expression of COUP-TFII is unchanged (unpublished observations). Similar to our results, EphB4 expression has also been shown to be lost in a model of vein graft adaptation, without the vessel acquiring an arterial phenotype (Kudo et al.,2007). In regards to alternative regulation of EphB4 expression, erythropoietin (Epo) is capable of inducing a venous phenotype in EC cultures by up-regulating EphB4 expression (Muller-Ehmsen et al.,2006). During the hyperoxia phase in which EphB4 expression is reduced, local Epo levels are also significantly suppressed (Chen et al.,2008), suggesting that the reduced Epo levels observed in the OIR model could potentially result in suppression of EphB4 expression. PECAM is another molecule with the potential to modulate EphB4 expression in the retina (Dimaio et al.,2008). Functional studies will be required to further evaluate the role of these potential regulators of EphB4 expression in the retina.
Retinal EphB4 expression permitted us to appreciate that a subset of veins was more sensitive to hyperoxia exposure as compared to arteries, regressing after 24 hr of hyperoxia via apoptosis. One molecule with the potential to contribute to vein and capillary survival is Angiopoietin-1 (Ang-1). Ang-1 has been identified as an essential EC survival factor, playing a key role in protecting retinal vessels from apoptosis (Hoffmann et al.,2005; Childs et al.,2008). Additionally, modulation of Ang-1 was observed to specifically alter the number of major blood vessels in both retinal and early vascular development models (Suri et al.,1996; Uemura et al.,2002). In humans, the venous vasculature appears to be more sensitive to defects in the Ang-1/Tie-2 pathway than arterial vessels with the development of venous malformations (Gaengel et al.,2009). In regards to the mouse OIR model, the exposure of mice at P7 to hyperoxia has been shown to significantly suppress the retinal expression of Ang-1, increasing vascular susceptibility to EC apoptosis and subsequent vessel regression (Hoffmann et al.,2005). Interestingly, in addition to promoting a venous phenotype, the Epo signaling pathway is also a major positive regulator of Ang-1 expression, but as previously noted, Epo levels are significantly reduced during the hyperoxia phase, potentially making retinal EC at risk for apoptosis from both reduced Epo and Ang-1 levels (Kertesz et al.,2004; Chen et al.,2008). Finally, perhaps the retinal veins are at higher risk for apoptotic-induced regression as compared to arteries secondary to reduced intrinsic numbers of smooth muscle cells and associated EC survival factors (Transforming Growth Factor-β, Platelet-derived Growth Factor) (Shih et al.,2003; Gaengel et al.,2009). There is also data to suggest that arterial ECs are intrinsically resistant to apoptosis as compared to capillaries and veins by expression of inhibitory Smads (I-smad) (Kiyono and Shibuya,2006).
EphrinB2 and EphB4 Expression During Relative Hypoxia Phase
We initially sought to compare and contrast the deep vascular plexus expression of ephrinB2 and EphB4 after hyperoxia-induced injury to normal development. On P13, the superficial arteries expressed ephrinB2 while the deep vascular plexus starts to recover (Davies et al.,2003). Although initially deep plexus vessels did not express ephrinB2 or EphB4, there was a recovery of ephrinB2+ vessels in the deep network by P15. In contrast, EpB4 was expressed by superficial veins but continued to be absent in the deep plexus vessels on P15. VEGF expression is rapidly increased in the OIR model on days P12.5 to P17 (Pierce et al.,1995) and we have previously demonstrated significant EC cell proliferation in the deep network on P14 (Davies et al.,2006). VEGF also regulates ephrinB2 expression, which correlates with the recovery of ephrinB2 in the deep network by P15. Altered signaling pathways, as a result of the hyperoxia exposure, likely account for the attenuated EphB4 expression (Hong et al.,2006; Lin et al.,2007). As previously noted, Epo is a molecule that can influence EphB4 expression and is suppressed during the hyperoxia phase (Muller-Ehmsen et al.,2006; Chen et al.,2008). In contrast to VEGF, Epo up-regulation is delayed until P15 during the relative hypoxia phase and could be one factor for EphB4 expression being limited to superficial vessels at this time point.
The finding of arteriovenous malformations (AVMs) during vascular recovery has been previously noted in the OIR model (Lobov et al.,2007) but the altered expression patterns of ephrinB2/EphB4 and loss of artery-vein boundaries has not been previously appreciated in the retina. However, AVMs with altered arterial and venous boundaries have been previously described in mouse studies of early vascular development (Urness et al.,2000; Sorensen et al.,2003). In humans with venous malformations, expression of ephrinB2 has been observed in malformed veins with reduced expression in accompanying arteries (Diehl et al.,2005). In addition, in mice in which Delta-like 4 ligand is over-expressed, AVMs also develop with arterialization of veins with associated ectopic ephrinB2 expression, similar to the AVMs noted in our study (Carlson et al.,2005; Trindade et al.,2008). Altered blood flow force and shear stress are also known to regulate ephrinB2 and EphB4 expression and artery-vein differentiation (le Noble et al.,2004). It remains to be determined if the development of AVMs in the oxygen-injured retina is related to altered ephrin/Eph expression or changes in hemodynamic forces. Direct experimental manipulation of ephrin/Eph expression (e.g., gain of function/loss of function studies) will be required to further delineate their involvement in the etiology of AVMs.
Expression of EphrinB2 and EphB4 in Neovascularization Phase
We found that ephrinB2 and/or EphB4 were expressed in a subset of neovascular tufts confirming their involvement in retinal NV. Expression of ephrinB2 and EphB4 has been previously observed at sites of NV in models of cutaneous wound healing and tumor angiogenesis, as well as human pathological specimens of inflammatory and ocular angiogenesis (Yuan et al.,2000; Gale et al.,2001; Shin et al.,2001; Umeda et al.,2004). Co-expression of ephrinB2 and EphB4 was also noted in some neovascular tufts, which is consistent with previous observations by Fuller and colleagues, implying a role for these molecules beyond artery/vein demarcation (Fuller et al.,2003). A biologic role for the ephrinB2/EphB4 pathway in retinal NV has recently been confirmed by immunolocalization of phosphorylated ephrinB in neovascular tufts (Salvucci et al.,2009). The promotion of angiogenesis by ephrinB2 and EphB4 is thought to occur through signaling effectors that modulate cell adhesion, migration of ECs, and interactions with pericytes (Foo et al.,2006; Pasquale,2008; Salvucci et al.,2009). Activation of EphB4 can also enhance endothelial progenitor cell adhesion and targeting to sites of angiogenesis and this cell population has been localized to neovascular tufts in a mouse model of retinal angiogenesis (Grant et al.,2002; Foubert et al.,2007). Our findings of ephrinB2 and EphB4 expression suggest that pre-retinal neovascular tufts are a viable target for anti-angiogenesis therapies that modulate ephrinB2/EphB4 interactions. Additional studies examining ephrinB2 and EphB4 expression after modulation are warranted because of the complex interactions of these multi-functional ligands and receptors with other cell populations (Pasquale,2008).
Expression of EphrinB2 in Activated Müller Cells
Retinal Müller cells are known to be activated in the OIR model as evidenced by increased GFAP expression (Smith et al.,1994). Activated Müller cells are often located at the border between the vascular and avascular ischemic retina, an area of neuronal apoptosis and microglia/macrophage activation (Davies et al.,2003,2006). The expression of ephrinB2 in activated Müller cells suggests a function beyond just angiogenic in the ischemic retina. Expression of ephrinB1 and ephrinB2 ligands is up-regulated in activated Müller cells and astrocytes in glaucomatous optic neuropathy and in a model of spinal cord transection supporting the theme of increased ephrin expression at sites of neuronal injury (Bundesen et al.,2003; Schmidt et al.,2007).
In summary, our data demonstrate that ephrinB2 and EphB4 expression is altered during hyperoxia-induced injury to the retina with subsequent localization to neovascular tufts during the phase of relative hypoxic recovery. These data offer an important foundation for additional mechanistic investigations into the role of ephrinB2 and EphB4 in retinal NV. For example, EphB4 gain-of-function studies, during the hyperoxia phase, are warranted in order to determine the effect upon vein regression and subsequent development of AVMs. In addition, the response of ephrinB2+ and /or EphB4+ neovascular tufts to a specific agonist/antagonist intervention can be monitored to confirm on-target response.