Anti-VEGF drugs have been reported to temporarily halt disease progression in some refractory cases of ischemic retinopathies (5
). This pharmacological approach has offered a breakthrough that obviates destructive therapies such as laser photocoagulation and surgical interventions. However, as shown in our study, comprehensive inhibition of angiogenesis may sustain retinal ischemia, which will consequently exacerbate disordered angiogenesis unless the anti-VEGF drugs are repeatedly administered. To overcome this limitation, we postulated that normalization of angiogenic orientation will offer an alternative, but effective treatment to fundamentally resolve retinal ischemia. Starting from this premise, we explored a therapeutic approach that can selectively suppress disoriented angiogenesis by exploiting the molecular mechanisms underlying intraretinal angiogenesis during development.
In postnatal mouse retinas, we observed an intimate association between PlexinD1-expressing ECs and VEGF-expressing astrocytes, which was corroborated by Plxnd1
gene downregulation after blocking VEGF signaling. While blocking PlexinD1 signaling rapidly elongated endothelial filopodia, the disruption of Sema3e
, which is expressed in neurons in the GCL, caused defective patterning of the retinal vasculature. Based on these findings, we propose that Sema3E-PlexinD1 signaling acts as a negative feedback system against VEGF signaling in terms of the filopodia projections as follows: Under hypoxia, VEGF evokes a series of proangiogenic signaling cascades for proliferation, survival, and migration (36
) but simultaneously upregulates PlexinD1 expression. Sema3E diffusing from the underlying neurons constantly signals to endothelial PlexinD1. ECs can project filopodia when VEGF signaling rises above the threshold level defined by the PlexinD1 signaling.
In the present study, we showed for the first time to our knowledge that Sema3E-induced activation of RhoJ mediates endothelial contraction. This contrasts with VEGF-induced activation of Cdc42 (37
), which plays a crucial role in filopodia projections (26
). Interestingly, RhoJ and Cdc42 were inactivated by VEGF and Sema3E, respectively (Supplemental Figure 10A). Thus, it seems likely that VEGF and Sema3E signals inversely activate or inactivate Cdc42 and RhoJ. Upon activation, both RhoJ and Cdc42 bind to the Cdc42/Rac-interactive binding (CRIB) domains of their effector proteins, such as p21-activated kinase (PAK) and neural Wiskott-Aldrich syndrome protein (N-WASP), which regulate intracellular actin polymerization (24
). Given the opposite effects of RhoJ and Cdc42 on endothelial filopodia formation, we suggest that activated RhoJ and Cdc42 competitively bind to their effector proteins, whereby the endothelial filopodia project or retract (Supplemental Figure 10B). Considering the expression of Cdc42 in various types of cells, the endothelial expression of RhoJ indicates the possibility that this molecule could serve as a therapeutic target to specifically manipulate endothelial filopodia projections.
Given the molecular machinery described above, one may ask how such a fine-tuning of endothelial filopodia projections contributes to the establishment of the retinal vascular architecture. A key to answering this question may be the contradictory consequences of filopodia elongation after PlexinD1-Fc injections at different time points. PlexinD1-Fc injection at P3 led to the formation of an excessive capillary network around veins, whereas its injection at P1 resulted in a sparse capillary network with less apparent morphological distinction between arteries and veins. This discrepancy may partly be explained by the extent of preformed vessels at the time of PlexinD1-Fc injection. That is, elongated filopodia may fuse extensively with neighboring vessels after P3, when the retinal vascular network has been considerably formed. By contrast, when PlexinD1 signaling is blocked just after the onset of the retinal vascular development, the aberrantly projected filopodia may fail to anastomose with each other, leading to the formation of a discontinuous capillary network. In line with this idea, the overall vascular density was decreased in Sema3e
-deficient retinas, despite the increased length of endothelial filopodia. Of importance, the Sema3e
-deficient retinas demonstrated a wide variety of vascular defects in their extent as well as in their morphological phenotypes, which is ascribable to the functional redundancy of endogenous ligands for PlexinD1. While Sema3E is the only ligand that can directly bind to PlexinD1, other soluble Sema3 ligands may signal to PlexinD1 in the co-presence of neuropilins (38
), to compensate for the loss of Sema3E or to exert their distinct functions.
In postnatal mouse retinas, developing vessels grow without protruding out of the retinal surface, even after pharmacological blockade of PlexinD1 or genetic disruption of Sema3e
. This excludes the possibility that a deficiency in Sema3E-PlexinD1 signaling alone is sufficient for extraretinal angiogenesis. By contrast, overexpression of soluble VEGF can cause extraretinal projections of endothelial filopodia in developing retinal vasculature (9
). As we showed the complete suppression of extraretinal angiogenesis by pharmacological or genetic disruption of VEGF signaling in the OIR model, it seems likely that aberrant VEGF distribution is primarily responsible for extraretinal angiogenesis. Indeed, the predominant VEGF-expressing cells in the OIR model are neurons rather than astrocytes. In addition, astrocytes failed to deposit extracellular fibronectin matrices (Supplemental Figure 11), which may have led to the loss of physical scaffolds for migrating ECs and the inability to retain heparin-binding VEGF proteins within the retina. The resulting diffusion and accumulation of VEGF in the preretinal space, as documented in human ischemic retinopathies (39
), may induce disoriented extraretinal vascular outgrowth. In this context, a high VEGF concentration accounts for the elevated PlexinD1 expression in extraretinal vessels.
By exploiting the differential distribution of PlexinD1 in the OIR model, we showed that intravitreal Sema3E injection selectively prevented extraretinal vascular outgrowth. Because of the absence of PlexinD1 expression in intraretinal vessels, this treatment did not interfere with vascular regeneration in ischemic retinas. To date, experimental manipulation of various signaling molecules, such as inducible nitric oxide synthase (40
) and sphingosine 1–phosphatase (41
), has been demonstrated to successfully prevent or repair vascular abnormalities in OIR models. Nonetheless, it is intriguing that diffuse administration of soluble Sema3E proteins can correct the imbalance in the complex signaling machineries determining retinal angiogenic orientation. This simplicity may allow clinical adaptation of this treatment for vascular regeneration therapy in ischemic retinopathies.
The feasibility of intravitreal Sema3E therapy is further supported by the specific involvement of PlexinD1 signaling in angiogenic sprouting, which will lessen the undesirable effects on normal vascular functions. However, the expression of PlexinD1 in neurons, although only limited to a small population in the GCL, suggests that caution must be exercised regarding the direct effects of Sema3E on retinal neurons, especially in clinical use for premature infants, whose retinal neural networks may not have been fully established (42
). Indeed, in Sema3e–/–
mice, we occasionally observed disordered axonal fibers in small retinal areas (Supplemental Figure 12), implying a role for Sema3E in axonal pathfinding in specific populations of retinal ganglion cells. While we demonstrated dose-dependent efficacy of intravitreal Sema3E injection in suppressing disoriented angiogenesis, its appropriate dosage remains to be determined to minimize the potential undesirable effects.
Further studies are needed to evaluate whether intravitreal Sema3E administration alone can offer vascular regeneration therapy in ischemic retinopathies, because this treatment did not reduce the unvascularized retinal areas in the OIR model. In some cases of human ROP, in which physiological retinal angiogenesis spontaneously recurs, it has been clinically reported that intravitreal anti-VEGF therapy paradoxically promotes physiological angiogenesis (6
). Nevertheless, our experimental results demonstrated that VEGFR1-Fc injection, irrespective of Sema3E coinjection, comprehensively inhibited vascular regrowth in the OIR model (Supplemental Figure 8), indicating the clinical requirements for restricted use of anti-VEGF drugs in severe ROP. Also, in human PDR, which often lacks spontaneous vascular regeneration in ischemic retinas, selective suppression of extraretinal angiogenesis by Sema3E injection will be more beneficial when conjugated with additional modalities that promote intraretinal vascular growth, such as restoration of proangiogenic properties in retinal astrocytes (43
In conclusion, we have presented a paradigm of vascular regeneration therapy in ischemic retinas by exploiting the antiangiogenic action of Sema3E on PlexinD1-expressing extraretinal vessels. Sema3E administration can also be useful to precisely guide angiogenesis to ischemic tissues in other organs if PlexinD1 expression is restricted to disoriented vessels. Otherwise, in diseases with broad PlexinD1 expression in abnormal vessels, such as cancer (44
), Sema3E administration may be a potent antiangiogenic therapy. Thus, the outcomes of Sema3E therapy may depend on the distribution of endothelial PlexinD1 in distinct disease settings.