Regenerative medicine is an encompassing term used to describe methods to revive and/or replace dead or diseased tissue. This is an important field in medicine because in contrast to nonmammalian vertebrates, mammals and humans display limited capacity for the self-regeneration of cells, tissues, and organ systems.¹ Methods used to enhance regeneration include tissue transplantation, biomaterials, tissue scaffolds, cellular supportive (trophic) factors, drug therapies, gene-based therapies and, notably, the use of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).² The field of vitreoretinal surgery is pioneering advances in regenerative medicine, from the first implantation of any ESC-derived cell into humans in 2002³ to numerous stem cell clinical trials occurring today.⁴ In addition, clinicians and scientists are becoming increasingly aware of the presence of specific populations of cells that have the capacity for self-regeneration.⁵ In the central nervous system (CNS), conventional wisdom holds that endogenous stem cells and native tissue regeneration do not exist. However, the dynamic role of neuronal support cells (glia)⁶ and the identification of specialized cellular environments⁷ (microenvironments) are challenging this dogma.

In humans, the process of cellular regeneration is limited in its resting state and often requires initiation by significant external events, such as inflammation, response to injury, and activation of cell-signaling pathways such as the Wnt (Wingless, Int-1) pathway. The fundamental concept of in situ retinal regeneration is that retinal tissue growth may occur by taking advantage of these initiating factors without the application of cells from other sources. Activation of the Wnt signaling pathway is a particularly promising mechanism for promoting retinal anatomic and functional regeneration.

WNT SIGNALING PATHWAY, NORRIN, AND RELATED DISEASE

The Wnt signaling pathway is a cellular signaling pathway that guides tissue differentiation in the developing fetus and plays several roles in adults, including angiogenesis; maintenance of the blood-brain barrier (BBB) and blood-retinal barrier (BRB), which is an essential feature of neurovascular tissue; and promotion of tissue regeneration.⁸ The Wnt gene family consists of 19 genes encoding proteins that act as extracellular signaling factors^2,9 primarily impacting vascular and neural tissues. There are 2 Wnt pathways: (1) The canonical/ β-catenin pathway and (2) the noncanonical pathway, with the canonical pathway primarily controlling cell proliferation, stem-cell self-renewal, and tissue regeneration.^10,11 Norrin is a protein encoded by the NDP gene on the X chromosome that is the strongest known activator of the canonical Wnt signaling pathway in the retina and retinal vascular cells. In the retina, Müller glial cells express NDP and produce norrin.¹² Activation of the canonical Wnt pathway ultimately culminates in the accumulation of β-catenin, a transcription factor that guides gene expression. In the case of vascular endothelial cells, norrin binds to the retinal endothelial cell Frizzled-4 cell surface receptor (FZD4) in conjunction with low-density lipoprotein receptor-related protein-5 (LRP5) and tetraspanin family member-12 (TSPAN12), producing a myriad of proteins that promote healthy cells.¹⁰ While essential in fetal and neonatal tissue development, these key actuators of the Canonical Wnt signaling pathway (norrin, FZD4, LRP5, and TSPAN12) remain expressed in the adult (post-natal) retina,¹³ indicating that driving the canonical Wnt pathway with exogenous norrin might shift cells into a developing or embryologic state and aid in vascular and neural tissue regeneration.

In the eye and retina, a functional Wnt signaling system plays a key fundamental role in the development of a sufficient vascular and neural network to support vision. This is evidenced by patients with Wnt mutations who develop many neurovascular disease, such as familial exudative vitreoretinopathy (FEVR), retinopathy of prematurity (ROP), Coats disease, and Norrie disease.¹⁴ In Norrie disease, the developing fetus is unable to produce a functional norrin protein and thereby unable to produce appropriate vascular or neuronal structures for the brain, ear, and particularly the retina. Because norrin-driven Wnt signaling is the primary Wnt system for the eye,¹² 100% of Norrie disease patients are bilaterally blind, yet only 40% are deaf or developmentally delayed, highlighting that other Wnt signaling pathways are available in the ear and central nervous system.¹⁰

It is also a common observation that young children can recover from injury faster than older adults. In particular, our clinical practice has observed that pediatric patients with traumatic macular holes may heal these injuries with clinically repaired photoreceptors and retinal pigment epithelial (RPE) cells. Could it be that children still possess the ability to activate a norrin-driven Wnt signaling wound repair/regenerative system? It was thought that these conditions could only be seen in fetal and infant cells; however our group has shown that these Wnt functions can be activated in adult human endothelial and neuronal cells.¹⁵ We believe that through the exogenous application of norrin, we can drive the norrin-driven WNT pathway to repair and regrow these retinal vascular and neuronal elements. Herein we describe cellular mechanisms by which the external application of the norrin protein promotes the health, maintenance, and regeneration of retinal vascular and neural cells.

NORRIN-DRIVEN RETINAL VASCULAR REGENERATION

The fundamental theme in norrin-driven retinal vascular regeneration is that the exogenous application of norrin repairs and regenerates vascular endothelial cells by modifying the gene expression of a myriad of proteins that improve endothelial cell tight junctions, strengthen the BRB, inhibit the formation of significant tissue edema, and increase angiogenesis. When blood vessels in the retina are injured in the setting of retinal vascular disease (including diabetic retinopathy [DR], retinal vein occlusion [RVO], FEVR, ROP, Coats, Norrie disease, and others), numerous growth factors, including vascular endothelial growth factor (VEGF), are upregulated in an attempt to promote the growth of blood vessels and increase cellular oxygen delivery.¹⁵ However, VEGF also directs the growth of disorganized and pathologic immature vessels along the retinal surface and into the vitreous cavity (retinal neovascularization) and increases the permeability of the BRB, leading to leakage of plasma and red blood cells through endothelial cells into the retina (retinal edema). Vascular leakage in the retina is clinically observed as macular edema or late-angiographic posterior and peripheral endothelial leakage (LAPPEL).¹⁶ A primary mechanism of vascular leakage and retinal edema is VEGF-induced expression of plasmalemma vesicle-associated protein (PLVAP). Plasmalemma vesicle-associated protein is a cell-specific protein that plays a pivotal role in transendothelial transport and pathologically increases within retinal vascular endothelial cells in the setting of VEGF-driven disease.¹⁷

Contemporary medical therapies for treatment-requiring retinal vascular disease involve tissue destruction with ablative retinal laser therapy and/or intravitreal injection of anti-VEGF agents (bevacizumab, ranibizumab, aflibercept).¹⁸ Intravitreal injection of anti-VEGF agents is the most commonly performed procedure in ophthalmology and possibly all of medicine.¹⁹ Through targeting VEGF, these therapies block an upstream effector of retinal edema and retinal neovascularization. However, VEGF is upregulated for an intended purpose (response to vascular injury), and numerous reports have shown that the presence of VEGF plays a pivotal role in wound healing, neuroprotection, and vascular protection.²⁰ Therefore, long-term anti-VEGF therapy and VEGF suppression may impart unintended consequences on cells.²¹ Targeting a more specific effector of retinal vascular leakage, such as PLVAP, may serve as a more precise approach. This may be accomplished with the activation of norrin, as the norrin protein has been shown to directly downregulate endothelial PLVAP,²² thereby decreasing retinal edema. Our group has shown that the exogenous application of norrin significantly decreases PLVAP levels alone and in the presence of VEGF (Figure 1). The exogenous application of norrin also mobilizes claudin-5, translocating it to the cell membrane, which helps restore the BRB and facilitates modulated angiogenesis.¹⁰ Other proteins favorably driven by the exogenous application of norrin include vascular endothelial cadherin, BMP2, and SOX proteins.¹⁰

With the application of norrin, the goal is to promote the health of the retinal vascular network to decrease tissue ischemia. Our group has realized this goal by showing that Norrin increases vascularization of the retina and decreases retinal neovascularization in animal models of disease.²³ A well perfused retina fails to drive the pathologic upregulation of VEGF, thereby avoiding VEGF-related consequences, including retinal edema and retinal neovascularization, and obviating the need for anti-VEGF therapy.

NORRIN-DRIVEN RETINAL NEURAL CELL REGENERATION

Exogenous application of the norrin protein has also been shown to promote the health, maintenance, and regeneration of retinal neural cells (retinal ganglion cells [RGCs], bipolar cells, photoreceptors). This occurs by norrin either improving the vascular perfusion to neural cells, acting directly on retinal neural cells to increase their health and repair them, or acting on neuronal support cells (Müller glial cells).¹⁵

As described above, the exogenous application of norrin can improve retinal vascularization. Improved tissue perfusion results in many positive effects on cells, including increased oxygen delivery to diseased tissue and the protection and repair of neural cells. To this end, our group^15,21 and others²⁴ have shown that norrin-induced retinal vascularization increases the survival of RGCs in an animal models of retinal disease (Figure 2).

Alternatively, norrin may also act directly and indirectly (through trophic factors) on retinal neural cells to improve their health. The Wnt receptor family of leucine-rich repeat-containing, G-protein-coupled receptors (LGRs), specifically LGR4, is present within RGCs.²⁵ Norrin binding to LGR4 activates Wnt signaling and may have a positive effect on RGC health. Norrin has also been shown to mediate neuroprotection to RGCs by activating Wnt signaling within Müller cells and promoting them to secrete neuroprotective trophic factors.²⁶ Additionally, in an animal model where RPE cells were made to continuously produce the norrin protein, retinal photoreceptors were protected against light-induced cell damage through protective effects of brain-derived neurotrophic factor.²⁷ These studies remind us of the complex relationships between retinal cell types, and show that the norrin protein can promote retinal neural and support cells to act together for cellular health and repair.

In the presence of norrin, retinal neuronal support cells (Müller glial cells) may actually turn into functioning retinal neural cells. This is an evolutionarily conserved pathway still active in fish and birds where Müller cells promote continuous and ongoing retinal regeneration.²⁸ In mammals, it is well known that trauma can induce glial to neural differentiation in the central nervous system to a small degree, mediated by inflammation and related pathways, but there is more evidence pointing toward canonical Wnt signaling being able to promote this even in the absence of injury.²⁹ Retinal Müller cells are the last cell type to form from retinal progenitor cells, indicating that they retain some embryologic memory of turning into other cell types.³⁰ Essentially, activation of the canonical Wnt signaling pathway may revert Müller cells back to a progenitor retinal cell type, which subsequently undergoes differentiation to mature retinal cell types including functioning photoreceptors.³¹ This process seems to be more prominent in youth and decreases with age,³² indicating that it may play a role in the striking ability of children to heal as discussed above. In essence, Müller glial cells are a source of retinal stem cells, and norrin-driven Wnt signaling may promote them to participate in the process of retinal regeneration.

CONCLUSION

The concept of in situ retinal regeneration is complex and seems to involve multiple components acting together in concert to result in meaningful anatomic and functional gains. Through the exogenous application of norrin, we have shown that activating the canonical Wnt signaling pathway may promote retinal vascular and neural regeneration. RP

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