Neurons and guidance cues: Semaphorin 3A in degenerative retinal vascular diseases
Retinal vasculopathies such as diabetic retinopathy (DR), retinopathy of prematurity (ROP), and age-related macular degeneration (AMD) are the most common causes of vision loss in the industrialized world. According to the National Eye Institute, 7.7 million Americans are affected by DR, an additional 10 million have AMD, and of the 3.9 million infants born annually in the U.S., ~15 000 are affected to some degree by ROP. In wet AMD, unchecked proliferation of choroidal vessels leads to photoreceptor compromise and vision loss. In ischemic retinopathies such as ROP and DR, degeneration of retinal vessels is followed by misguided pathological neovascularization. Recent studies on DR, ROP and AMD have revealed the paramount role for neurons and neuronal guidance cues in disease progression. During retinal embryogenesis, coordinated interplay between neurons, blood vessels and immune cells is critical for proper retinal development. Although neurovascular and neuroimmune crosstalk shapes vascular development in the retina, it has received limited attention in disease etiology. A better understanding of this crosstalk is now providing novel druggable targets to counter vasodegenerative and vasoproliferative eye diseases.
New evidence suggests that CNS neurons and guidance cues secreted by neurons such as retinal ganglion cells (RGCs) and photoreceptors have an inherent ability to influence vascular and immune responses in retinopathies. Photoreceptors were likely the first neurons suggested to influence the progression of pathological retinal neovascularization. For instance, a negative correlation was drawn between incidences of retinitis pigmentosa, which causes photoreceptor degeneration, and proliferative DR. Further investigation demonstrated that retinal neovascularization associated with long-standing diabetes mellitus spontaneously regressed with the onset of retinitis pigmentosa. This observation held true in animal models where mice with genetically ablated photoreceptors failed to mount reactive retinal neovascularization in a model of oxygen-induced retinopathy (OIR), which mimics the vasodegenerative phase of ROP and loosely represents the vasoproliferative phases of ROP and DR. These studies draw a link between neuronal energy demand and retinal neovascularization.
A mechanism via which central neurons communicate with their environment is through production of classical neuronal guidance cues such as semaphorins. These proteins are now widely regarded to have angiogenic and inflammatory functions, underscoring their pleiotropic nature. While these signaling proteins were initially thought to exclusively pattern the nervous system, it is now clear they also play a critical role in blood vessel development and influence the immune response during embryogenesis, and tissue homeostasis in adulthood.
In addition to photoreceptors, RGCs have the propensity to significantly influence their retinal angiogenesis either positively or negatively and are directly apposed to the retinal vasculature that degenerates in DR and ROP. For example, in the mouse model of OIR, the guidance cue SEMA3A is secreted by hypoxic neurons, repelling regenerating vessels from the most ischemic regions of the retina. Silencing the Sema3A gene enhances physiological vascular regeneration of the hypoxic retina. In non-proliferative DR, SEMA3A is produced by RGCs in hyperglycemic conditions and contributes to inner blood-retinal barrier disruption and consequent macular edema. Therapeutic neutralization of SEMA3A reduces pathological retinal vascular permeability.
In addition to its influence on blood vessels, SEMA3A also affects immune cells such as mononuclear phagocytes (microglia/macrophages) that are central to the progression of proliferative retinopathies. While cytokine signatures responsible for retinal inflammation in DR and ROP are well characterized (notably IL-4, IL-6 and TNFα), the pro-inflammatory properties of neuronally expressed SEMA3A in retinopathies highlight its unique value as therapeutic targets. Hypoxic retinal neurons secrete SEMA3A, attracting pro-angiogenic mononuclear phagocytes to sites of pathological neovascularization. These mononuclear phagocytes infiltrate the retina, take on a microglial phenotype and actively partake in vascular degeneration and later pathological angiogenesis. Therapeutic inhibition of SEMA3A consequently reduces ischemia-driven retinal inflammation.
Pharmacological modulation of guidance cues such as semaphorins offers an alternative and novel strategy to restore healthy functional retinal vasculature to ischemic zones of the retina and consequently reduce the destructive inflammation associated with these diseases.
Semaphorins are a family of proteins initially identified as axonal guidance molecules that direct neuronal growth cones during embryogenesis. The Semaphorin family counts at least 21 vertebrate genes and 8 additional genes in invertebrates. All Semaphorins contain a ~500 amino acid SEMA domain that is required for signaling. Class 3 Semaphorins (such as Semaphorin3A) are secreted members of the family and hence capable of acting at a distance. While Semaphorins were initially thought to exclusively pattern the nervous system, it is now clear they also play a critical role in blood vessel development, regulate the immune response, influence tumour microenvironments and play a role in maintaining bone homeostasis. While physiological functions for Semaphorins have been described in adults, they are thought to predominantly play roles during embryogenesis, given that their levels, at least in the central nervous system, decline after birth.
Molecular Surface of Sema3A (Antipenko et al. Neuron 2003).
Levels of SEMA3A
Patients with either diabetic macular edema (upper pannel) or proliferative diabetic retinopathy (below) have high levels of SEMA3A in their vitreous (Cerani et al. Cell Metabolism, 2013 & Dejda et al, The Journal of Clinical Investigation, 2014).
SEMA3A is synthesized as a disulphide-linked homodimer and dimerization is essential for signaling. SEMA3A binds to its cognate receptor Neuropilin-1 (NRP1). Neuropilin-1 has the particular ability to bind at least two structurally dissimilar ligands via distinct sites on its extracellular domain. It binds not only SEMA3A provoking cytoskeletal collapse but also VEGF165 enhancing binding to VEGFR2 and thus increasing its angiogenic potential. Crystallographic evidence revealed that VEGF165 and SEMA3A do not directly compete for NRP1 but rather can simultaneously bind to NRP1 at distinct, non-overlapping sites. Moreover, genetic studies show that NRP1 distinctly regulates the effects of VEGF and SEMA3A on neuronal and vascular development.
Neuropilin-1 is a single-pass transmembrane receptor with a large 860 amino acid extracellular domain subdivided into 3 sub-domains (a1a2, b1b2 and c) and a short 40 amino acid intracellular domain. In neurons, binding of SEMA3A to NRP-1 recruits Plexins which transduce their intracellular signal23 and provoke cytoskeletal collapse; the transduction mechanism in endothelial cells remains ill-defined. NRP1 binds Semaphorin3A primarily via its a1a2 (but also b1) domains (provoking cytoskeletal collapse) and VEGF165 via its b1b2 domain (enhancing binding to VEGFR2 and thus increasing its angiogenic potential).
Semaphorins and Diabetes
While tissue and circulating levels of Semaphorins are typically low in healthy adults, they have been shown to rise in the retinas, kidneys and blood of patients with diabetes. Production of secreted Semaphorins such as SEMA3A is triggered by tissue ischemia, inflammatory factors such as IL-1b and overall hyperglycemia.
During diabetes, elevated levels of SEMA3A activate cellular signaling pathways that compromise vascular barrier function and lead to increased vascular permeability. In the retina, this causes macular edema (Cerani et al. Cell Metabolism, 2013)
The deterioration of the inner blood-retinal barrier and consequent macular edema is a cardinal manifestation of diabetic retinopathy (DR) and the clinical feature most closely associated with loss of sight. We have discovered evidence from both human and animal studies for the critical role of the classical neuronal guidance cue, semaphorin 3A, in instigating pathological vascular permeability in diabetic retinas via its cognate receptor neuropilin-1. In doing so, we have shown that semaphorin 3A is induced in early hyperglycemic phases of diabetes within the neuronal retina and precipitates initial breakdown of endothelial barrier function. We have also demonstrated that neutralization of semaphorin 3A efficiently prevents diabetes-induced retinal vascular leakage in a stage of the disease when vascular endothelial growth factor neutralization is inefficient. These observations were corroborated in TgCre-Esr1/Nrp1flox/flox conditional knockout mice.
In non-proliferative DR, semaphorin 3A is produced by retinal ganglion neurons (directly adjacent inner retinal blood vessels) early in disease during hyperglycemic conditions and contributes to inner blood-retinal barrier disruption and consequent macular edema. Through activation of its cognate receptor NRP1, SEMA3A triggers signalling cascades that ultimately provoke phosphorylation of Vascular-Endothelial Cadherin junctions and compromise of endothelial cell barrier function. Importantly, patients with diabetic macular edema or proliferative diabetic retinopathy have significantly elevated levels of SEMA3A in both their vitreous and plasma.
Our findings have confirmed Sema3A as a therapeutic target for macular edema and provide further evidence for neurovascular crosstalk in the pathogenesis of DR.
Aggressive inflammatory cells (white arrows) are recruted by SEMA3A into the retina during retinal vascular diseases. (Dejda et al, The Journal of Clinical Investigation, 2014)
The SEMA3A receptor NRP1 is robustly expressed on a subset of resident retinal microglia as well as a population of highly pro-angiogenic infiltrating mononuclear phagocytes (MNPs). These pro-angiogenic NRP1-expressing MNPs are attracted by high levels of SEMA3A in diabetic retinas. Ultimately, they contribute to pathological angiogenesis. Neutralizing SEMA3A prevents this detrimental inflammation associated with diabetic retinopathy.
Retinal neurons in zones where blood vessels have died out produce SEMA3A which acts as a chemical wall that prevents vascular regeneration. Pharmacological inhibition of SEMA3A with SemaThera’s proprietary traps provokes vascular regeneration and repairs the retina. (editorial from Duh, Blood, 2011 for Joyal et al. Blood 2011)
The generation of blood vessels is a highly synchronized process requiring the coordinated efforts of several vascular and nonvascular cell populations as well as a stringent orchestration by the tissue being vascularized. Stereotyped angiogenesis is vital for both developmental growth and to restore tissue metabolic supply after ischemic events. Central neurons such as those found in the brain, spinal cord, and retina are vast consumers of oxygen and nutrients and therefore require high rates of perfusion by functional vascular networks to ensure proper sensory transmission. During a metabolic mismatch, such as that occurring during a cerebrovascular infarct or in ischemic retinopathies, there is increasing evidence that central neurons have an inherent ability to influence the vascular response to injury. Data focused on retina and retinal ischemic disorders, confirms the ever-growing evidence suggesting that central neurons have the propensity to impact tissue vascularization and reparative angiogenesis. Moreover, it addresses the paradoxical ability of severely ischemic neurons to hinder vascular regrowth and thus segregate the most severely injured zones of nervous tissue.
SEMA3A is produced in ischemic zones of the retina such as that seen in patients with late stage proliferative diabetic retinopathy or retinopathy of prematurity. Production of SEMA3A by retinal ganglion neurons within the ischemic retina deviates nascent blood vessels away from the inner retina towards the physiologically avascular vitreous. SEMA3A thus creates a chemical wall that hinders desirable vascular regeneration of the avascular retina. Antagonism of SEMA3A relieves the vasorepulsive force and allows for desirable revascularization and thus repair of the ischemic retina.