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Sphingosine 1-phosphate, a potential target in neovascular retinal disease
  1. Rasha A Alshaikh1,2,
  2. Katie B Ryan1,3,
  3. Christian Waeber1,4
  1. 1School of Pharmacy, University College Cork, Cork, Ireland
  2. 2Department of Pharmaceutical Technology, Tanta University, Tanta, Egypt
  3. 3SSPC The SFI Research Centre for Pharmaceuticals, School of Pharmacy, University College Cork, Cork, Ireland
  4. 4Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland
  1. Correspondence to Dr Christian Waeber, School of Pharmacy, University College Cork, Cork, Ireland; c.waeber{at}ucc.ie

Abstract

Neovascular ocular diseases (such as age-related macular degeneration, diabetic retinopathy and retinal vein occlusion) are characterised by common pathological processes that contribute to disease progression. These include angiogenesis, oedema, inflammation, cell death and fibrosis. Currently available therapies target the effects of vascular endothelial growth factor (VEGF), the main mediator of pathological angiogenesis. Unfortunately, VEGF blockers are expensive biological therapeutics that necessitate frequent intravitreal administration and are associated with multiple adverse effects. Thus, alternative treatment options associated with fewer side effects are required for disease management. This review introduces sphingosine 1-phosphate (S1P) as a potential pharmacological target for the treatment of neovascular ocular pathologies. S1P is a sphingolipid mediator that controls cellular growth, differentiation, survival and death. S1P actions are mediated by five G protein-coupled receptors (S1P1–5 receptors) which are abundantly expressed in all retinal and subretinal structures. The action of S1P on S1P1 receptors can reduce angiogenesis, increase endothelium integrity, reduce photoreceptor apoptosis and protect the retina against neurodegeneration. Conversely, S1P2 receptor signalling can increase neovascularisation, disrupt endothelial junctions, stimulate VEGF release, and induce retinal cell apoptosis and degeneration of neural retina. The aim of this review is to thoroughly discuss the role of S1P and its different receptor subtypes in angiogenesis, inflammation, apoptosis and fibrosis in order to determine which of these S1P-mediated processes may be targeted therapeutically.

  • angiogenesis
  • degeneration
  • inflammation
  • neovascularisation
  • pharmacology

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Introduction

At least 2.2 billion people suffer from vision impairment, which in 1 billion cases can be attributed to a preventable or treatable cause. Diabetic retinopathy (DR) and age-related macular degeneration (AMD) account for more than one-third of these cases.1 Neovascular ocular diseases including DR, wet-AMD and retinal vein occlusion (RVO) have different aetiologies but result in a similar cascade of pathophysiological events (table 1). Pathological angiogenesis is key among these, and a hallmark of disorders that occurs in response to vascular endothelial growth factor (VEGF), a potent hypoxia-induced angiogenic mediator that triggers the formation of new permeable and unstable blood vessels.2 Pathological angiogenesis can originate from retinal vasculature which supplies the inner retina and/or choroidal vasculature which supplies the outer retinal and retinal pigmented epithelium (RPE). RPE is a monolayer of epithelial cells that represents the main structure of outer blood–retinal barrier3 4 and prevents retinal invasion of neovascular tissue from choroidal blood vessels.5 In neovascular ocular disease, angiogenesis is accompanied by disruption of RPE physical and metabolic barrier function (table 1). This results in continuous leakage of blood or blood components to the surrounding tissues, leading to oedema and/or haemorrhage, with possible progression to retinal detachment and irreversible apoptosis of photoreceptors and other retinal cells (table 1). In addition to angiogenesis, chronic hypoxia causes chronic inflammation and overproduction of reactive oxygen species in the retina, triggering cell death and fibrotic cascades (table 1). Fibrosis in the posterior chamber of the eye has unique characteristics, being characterised by the occurrence of gliosis and epithelial to mesenchymal transition (EMT). Later sections describe the role of sphingosine 1-phosphate (S1P) in both gliosis and EMT.

Table 1

Common pathological events associated with different neovascular ocular diseases; most of these have been shown to be affected by S1P signalling

Angiogenesis, oedema, inflammation, apoptosis and fibrosis contribute to the pathophysiology of DR, wet-AMD and RVO (table 1). Current therapeutic options largely rely on the blockade of VEGF signalling with biological therapeutics (antibodies, recombinant fusion proteins or pegylated RNA aptamers). They are expensive, with limited stability and are administered by invasive intravitreal injections that can be associated with retinal detachment, subconjunctival haemorrhage, uveitis and endophthalmitis.6 It would therefore be beneficial to develop novel therapeutic agents with fewer limitations. The lipid mediator S1P is involved in hypoxia-induced angiogenesis. Unlike VEGF, S1P can promote stable blood vessel formation, increase endothelial barrier integrity and positively impact the subsequent pathophysiological steps leading to neovascular ocular diseases.

This review will provide a brief description of S1P metabolism, the distribution and function of S1P receptor subtypes in different retinal tissues, and will then more specifically focus on the documented effects of S1P and the sometimes opposing roles of various S1P receptor subtypes in the processes that contribute to neovascular disease pathogenesis.

S1P production, metabolism and receptor expression in ocular tissues

Sphingolipids are lipid-based cell membrane components. In addition to their structural role, they modulate cellular proliferation, migration, differentiation and survival.7 8 They are synthesised by serine palmitoyl transferase (SPT) from palmitoyl CoA and serine as summarised in figure 1. S1P is produced by phosphorylation of sphingosine by one of two sphingosine kinase isoforms, SphK1 and SphK2.9 S1P can be de-phosphorylated by the action of two phosphatases or degraded by S1P lyase to produce inactive metabolites (figure 1).10

Figure 1

Schematic representation of synthesis and metabolism of sphingolipids, with special emphasis on S1P and the five receptor subtypes it can activate (S1P1–5 receptors) as well as the G proteins they are coupled to. Specific S1P receptor modulators discussed in this review are listed at the bottom of the figure (blue and red circles indicate agonist and antagonist activities, respectively). This figure was created by the authors, using the software Biorender.

The regulation of S1P production and release in different body tissues is not yet completely understood. In plasma, S1P is mainly produced by red blood cells, endothelial cells and platelets. Once produced intracellularly, S1P is transported to the extracellular space leading to significantly higher plasma concentrations of the mediator (~1 µM) compared with interstitial fluid levels.11 Most circulating S1P is not free, but bound to high-density lipoproteins (HDL), albumin and to a lower extent low-density lipoproteins (LDL).

Under normal conditions, SphK2 is the main S1P-producing kinase in rat and mouse retina.12 13 Under hypoxic or light-induced stress conditions, SphK1 but not SphK2 is upregulated leading to increased intracellular S1P levels in murine retina.12 14 Little is known about the levels and role of potential S1P carriers in ocular tissues. Albumin can be found in foetal vitreous, the retina and lens.15 LDL and HDL can be synthesised locally in the retina16 or diffuse from the systemic circulation through the RPE, although HDL diffusion is significantly lower than LDL in rat retina.17 Apolipoprotein E is synthesised by Müller glial cells in neural retina and transported to vitreous humour,18 with no information describing retinal expression of apolipoprotein A4 or M.

While S1P can act as an intracellular second messenger and as an extracellular mediator, the latter effects, mediated by five G protein-coupled receptors (S1P1–5), predominate.14 S1P1–3 receptors are expressed in almost every body tissue, while S1P4 and S1P5 expression is largely restricted to the lymphatic and nervous systems.19 20 S1P receptor expression in retina varies depending on cell type and pathophysiological status (see table 2 for a summary). Under healthy conditions, S1P1 receptors predominate in retina, while photoreceptors mainly express S1P2 receptors (table 2).12 14 RPE cells show robust S1P1–3 receptor expression, with different subtypes predominating in different cell lines.21 22 Retinal vasculature endothelial cells, isolated from human donor tissues, predominantly express S1P2 and S1P3 receptors (table 2).23 New single-cell RNA sequencing (scRNA-seq) data show that S1P1 and S1P3 genes are the most strongly expressed in retinal endothelial cells (RECs),24 while choriocapillaris endothelial cells mostly express S1P1, followed by S1P3.25 Müller glial cells express S1P1 and S1P3 receptors, with S1P3 receptor expression is most evident in peripheral rather than foveal Müller glial cells.26 S1P receptor densities are altered under pathological conditions. S1P2 and S1P3, but not S1P1 receptors, are upregulated following light-induced retinal damage.12 Likewise, a fivefold increase in the retinal S1P2 receptor expression is seen in response to hypoxia.27 Recent reports also highlight the role of S1P2 receptors in laser-induced choroidal neovascular (CNV) lesions.28 Additionally, scRNA-seq reveals noticeable downregulation of both S1P1 and S1P3 receptor expression in the choroidal endothelial cells pooled from a patient with AMD.25

Table 2

S1P receptor expression in different tissues of the ocular posterior segment

Growing evidence suggests that S1P plays a significant role in normal retinal development. S1P1–3 receptor loss leads to significant defects in the retinal vascular network of postnatal mice.29 Additionally, S1P has essential functions in photoreceptor development, proliferation, differentiation and survival.30 A link between sphingolipids and ocular disease was first suspected following the observation of ocular abnormalities in sphingolipidoses, a group of lysosomal storage disorders characterised by a build-up of certain sphingolipids in which retinal degeneration, neovascularisation and blindness are common manifestations.31 32 Hereditary and sensory autonomic neuropathy type 1 (HSAN1) is characterised by mutated SPT. The defective enzyme synthesises deoxysphingolipids lacking the hydroxyl group at C1, which is essential for synthesis of other sphingolipids. Deoxysphingolipids are cytotoxic, suggesting the involvement of these mediators in the neuropathy of HSAN1.33 Deoxysphingolipids are also seen in macular telangiectasia. Patients with this condition have normal SPT but significantly lower serum serine levels, resulting in the use of alanine instead of serine as a substrate for SPT, ending in the formation of deoxysphingolipids. Deoxysphingolipid serum levels in these patients are positively correlated to the disease severity.34 35 However, a role for S1P and its receptors in these diseases has not been reported and they will not be discussed further in this review.

The altered expression and differential effects of various S1P receptor subtypes under stress conditions offer unique opportunities to target these receptors with selective agonists or antagonists in ocular disorders. The following sections describe the role of individual S1P receptor subtypes in the relevant pathological processes. In this context, it is worth bearing in mind the uncertain specificity of the pharmacological agents used in the studies described in this review.36 Better characterised clinical candidates, or clinically used S1P receptor drugs, have been developed primarily for the management of multiple sclerosis (MS).37 38 These include approved S1P receptor modulators fingolimod (FTY720), siponimod (BAF312) and ozanimod (RPC1063). Fingolimod binds to all S1P receptors except S1P2, siponimod and ozanimod are selective S1P1 and S1P5 modulators.39 To the best of our knowledge, these agents have not been used to characterise the role of S1P signalling in ocular pathophysiology. Ocular pathologies and macular oedema are associated with fingolimod therapy in patients with MS,40 but may result from MS progression rather than fingolimod treatment.41

Angiogenesis

Angiogenesis is the process by which new blood vessels develop from pre-existing ones. The process is carefully regulated by a balance of stimulatory (eg, VEGF, hypoxia-inducible factor, transforming growth factor-β (TGF-β)) and inhibitory signalling, which maintains a minimal turnover of endothelial cells. In adults, physiological angiogenesis is transient and only occurs during the menstrual cycle and wound healing.42 Under specific conditions (eg, hypoxia, inflammation, acidosis), quiescent endothelial cells show massive proliferation and migration in a phenomenon called ‘the angiogenic switch’, where the influence of angiogenesis activators exceeds that of the inhibitors.42 The vessel formation is then initiated by the release of pro-angiogenic mediators and growth factors.43 These factors trigger transcriptional responses in the endothelium, with specialised endothelial cells becoming ‘tip cells’ which guide vessel branching toward the angiogenic stimulus. This is followed by enzymatic (eg, metalloproteineases) lysis of the basement membrane and extracellular matrix, while other endothelial cells proliferate and follow the lead of the tip cells. These cells differentiate into stalk cells responsible for lumen formation, basement membrane deposition, growth and quiescence of new endothelial cell, and expression of intercellular junction proteins. This is accompanied by migration of pericytes and vascular smooth muscle cells to support the young blood vessels. Once blood is flowing in the new vessel, fluid shear forces act as an inhibitor of angiogenesis, a process in which S1P1 receptors play a key role.44

Under pathological conditions (eg, DR), persistent tissue hypoxia results in sustained release of angiogenic stimulators and exhaustive endothelium activation.45 This triggers erosion of the basement membrane in multiple locations, substantial tip cell formation and continuous endothelial proliferation and migration. Due to the high concentration of angiogenic mediators, endothelial activation occurs even in newly formed vessels. This leads to the formation of fragile endothelium with no time for maturation and subsequently no ability to restore blood flow,42 further exacerbating hypoxia, leakage of blood components to the surrounding tissues and release of more angiogenic mediators in a vicious cycle that contributes to disease progression in many pathologies including neovascular ocular disease.46

Role of S1P in retinal and choroidal angiogenesis and blood vessel integrity

While S1P can be described as a hypoxia-induced pro-angiogenic mediator, this oversimplifies its complex role in vascular growth and stability.47 48 The effect of S1P in angiogenesis is mediated mainly via S1P1 and S1P2 receptors, which are both highly expressed in retinal endothelium. The number and integrity of the vessels formed in response to this lipid depend on which receptor subtype is principally involved. To add further complexity, the nature of the endothelial response to S1P depends on which carrier protein (albumin, apolipoprotein A4 or M) presents the lipid to its target receptor.49 Similar observations were made in RPE cells.28 Finally, it is important to note that S1P1 receptors are an essential component of fluid shear stress sensing and can be activated in the absence of S1P.44

S1P1 receptors

S1P1 receptors trigger a distinctive and controlled angiogenic pattern where only a limited number of blood vessel sprouts are formed.44 50–52 These sprouts undergo full maturation and acquire the characteristic of mature endothelium forming a competent vascular network.44 53 54 S1P-mediated enhancement of endothelial integrity is abolished in S1P1-knockdown endothelium,54 while S1P1 gene deletion increases tip cell formation in the retinal vasculature, leading to hypersprouting, which is associated with disrupted intercellular junctions, reduced capillary perfusion and hypoxia in surrounding retinal tissues, with VEGF overexpression in retinal endothelium.44 Similar results are obtained in postnatal mice retinas by administering the S1P1 antagonist W146.52 Conversely, S1P1 receptor overexpression reduces the number of tip cells and vessel branch points in retinal vasculature of mouse embryos,44 while S1P1 receptor activation with the S1P1 agonist SEW2871 results in angiogenesis characterised by fewer but longer blood vessel branches and reduces the VEGF angiogenic effects on human umbilical vein endothelial cells (HUVECs) or mouse microvascular endothelial cells.52 As pathological neovascularisation is characterised by higher VEGF levels, the ability of S1P1 receptor activation to antagonise the VEGF angiogenic effect is of pathological relevance.52 Finally, the levels of cell junction proteins are reduced following S1pr1 silencing by small interfering RNA, targeted S1pr1 gene deletion in endothelial cells or VEGF stimulation.52

In conclusion, while S1P1 receptor stimulation is generally pro-angiogenic, it results in the formation of fewer blood vessel sprouts and these develop a competent endothelium. This can restore blood flow to the hypoxic retina without resulting in oedema or haemorrhage (figure 2).

Figure 2

Summary of S1P-mediated effects on angiogenesis. Top: S1P1 receptor activation by S1P reduces pro-angiogenic factor release in response to hypoxia (most importantly VEGF) leading to fewer tip cells being formed and fewer branching points per unit area. This leads to the formation of fewer vessel branches. S1P1 receptors also increase intercellular junction protein expression and perivascular cell coverage of newly formed sprouts. This leads to the formation of competent blood vessels with normal blood flow that restores tissue perfusion (red areas) and downregulates the angiogenic signal. Bottom: hypoxia results in overexpression of SphK1 and S1P2 receptors. This receptor subtype increases VEGF release and tip cell number resulting in increased vessel branching per unit area. The formed branches have defective expression of intercellular junction proteins and irregular perivascular cell coverage. This leads to the formation of leaky endothelium with diminished blood flow which further exacerbates tissue hypoxia (blue areas). Due to sustained angiogenesis, further branching of the new sprouts occurs leading to further leaking and haemorrhage. The net effect of these two opposite signals depends on relative receptor densities and specific receptor upregulation in response to hypoxia. Although their abundance in the eye is not known, the balance of S1P-carrier proteins may also play a role, as ApoM-bound S1P and albumin-bound S1P preferentially activate S1P1 and S1P2-mediated cascades, respectively. This figure was created by the authors using elements from the Servier Medical Arts Database. ApoM, apolipoprotein M; S1P, sphingosine 1-phosphate; VEGF, vascular endothelial growth factor; VSMCs, vascular smooth muscle cells .

S1P2 receptors

S1P2 receptors activate an angiogenetic response similar to that of VEGF,55 in which continuous, uncontrolled blood vessel sprouting occurs in response to hypoxia. Consequently, vascular maturation is defective due to the sustained endothelial cell proliferation and migration. This results in leaky vascular architecture, with interrupted adherens junctions and inadequate blood flow (figure 2). Indeed, hypoxia upregulates S1P2 receptors in retinal endothelium and leads to the formation of blood vessel sprouts with leaky basement membranes and limited perfusion.27 In S1P2-knockout mice, hypoxia-induced angiogenesis is characterised by the formation of competent blood vessel sprouts that have comparable blood flow with the mature vasculature.27 Similar results are seen with an S1P2 receptor antagonist, as laser-induced choroidal neovascularisation is reduced after intravitreal injection of JTE013.28 The effect of S1P2 receptors on angiogenesis may vary in different endothelial cell types, as JTE013 administration increases S1P-mediated abdominal subcutaneous angiogenesis in mice.56 In addition to angiogenic effects on retinal vasculature, S1P2 activation with albumin-bound S1P results in disruption of barrier integrity and increased RPE permeability. Preincubation of RPE with JTE013 results in the repair of disrupted epithelium and reduced vascular leakage. Additionally, RPE shows increased VEGF release in response to S1P, an effect which is inhibited by JTE013, whereas the S1P1/3 receptor antagonist VPC23019 has no effect.28

The net effect of S1P on vascular endothelium depends on the balance between S1P1 and S1P2 receptors. In vivo evidence shows predominant expression of S1P2 receptors in mouse CNV lesions,28 upregulation of S1P2 and S1P3 receptors in light-induced damage in rat retinas12 and upregulation of S1P2 receptors in a mouse model of retinal ischaemia.27 Additionally, S1P2-knockout mice show enhanced retinal vascularisation with normal vascular morphology following ischaemic insult compared with S1P2+/+ mice.27 Likewise, intravitreal injection of the S1P2 antagonist JTE013 significantly reduced CNV lesion areas in mice.28 This evidence explains why administration of anti-S1P antibodies reduces choroidal neovascularisation and vessel leakage,23 57 an action thought to be mediated by S1P2 receptors. The type of S1P-carrying molecules may also matter, as apolipoprotein M-bound S1P elicits a favourable activation of S1P1 receptors resulting in reduced vascular leakage and increased expression of junction proteins in RPE, while albumin-bound S1P results in S1P2-mediated disruption of cellular junctions, increased vascular leakage and reduced endothelial integrity (figure 2).28

Relationship between S1P and VEGF signalling

VEGF affects S1P signalling in different ways. It increases S1P production by upregulating SphK expression in endothelial cells50 and also increases SphK activity in RECs.58 While both actions raise intracellular S1P concentration, most of the intracellular S1P is transported to the extracellular space to act on S1P1–5 receptors in autocrine and paracrine manners. VEGF specifically upregulates S1P1 receptor expression in aortic endothelial cells, potentiating nitric oxide/Akt signalling, but has no effect on S1P2 or S1P3 receptor expression, suggesting that under hypoxic conditions, these endothelial cells might be more sensitive to S1P1 signalling.59 Yet, S1P1 activation with SEW2871 blocks VEGF-induced sprouting in HUVECs and mouse microvascular endothelial cells, while the S1P1 antagonist W146 increases VEGF-induced angiogenesis.52 This suggests that upregulation of S1P1 receptors in response to VEGF can lessen the overall angiogenic response to VEGF under pathological conditions. S1P results in increased VEGF expression in RPE cells, a response that is diminished after S1P2 blockade with JTE013.28 Additionally, S1P can transiently activate VEGFR2 receptors in bovine aortic endothelial cells in a tyrosine kinase inhibitor-sensitive manner, resulting in endothelial nitric-oxide synthase phosphorylation and activation.60 SphK inhibition decreases VEGF-mediated REC proliferation, migration and vascular leakage, indicating that S1P release is involved in VEGF-mediated angiogenesis.58

S1P role in ocular inflammation and release of inflammatory mediators

Inflammatory processes also play a role in AMD, DR and RVO pathophysiology. Indeed, the lower incidence of retinopathy among patients with diabetes on salicylate therapy for rheumatoid arthritis, and the significant effect of corticosteroid therapy on reducing macular oedema and neovascularisation in DR highlight the therapeutic relevance of anti-inflammatory drugs in the progression of DR.61 Retinal ischaemia is known to induce the expression of potent inflammatory cytokines including monocyte chemotactic protein‐1 (MCP‐1) and macrophage inflammatory protein‐1α,62 resulting in leucocyte infiltration and macrophage recruitment. Activated macrophages and microglia secrete inflammatory molecules such as tumour necrosis factor-α and interleukins (ILs),63 which subsequently trigger a complex chain of cellular and vascular responses, the details of which are outside the scope of this review. Levels of MCP-1 are markedly increased in the vitreous of patients with DR64 65 and RVO.65 Furthermore, markedly elevated IL-8 levels are detected in the vitreous fluid of patients with DR and RVO,65 66 higher IL-6 levels are also detected in the vitreous of patients with DR64 and complement system activation is reported in AMD,67 which highlight the significant role of the inflammatory response in neovascular ocular diseases.

In addition to its role in vascular integrity, S1P signalling can modulate inflammation. For instance, S1P reduces vascular leakage, neutrophil infiltration and lung oedema after intratracheal administration of lipopolysaccharide.68 But S1P also increases the production of inflammatory cytokines such as IL-8 and IL-6, among others.22 S1P increases cyclo-oxygenase-2 expression and prostaglandin production via S1P2 receptors in renal mesangial cells.69 Fingolimod suppresses inflammation in an uveoretinitis model70 and inhibits leucocyte infiltration when administered as a single dose before induction of ocular inflammation.71 Patients with fingolimod-treated MS show a lower incidence of ocular inflammation compared with other patients with MS.72 It is unclear whether the ocular anti-inflammatory effects of fingolimod are due to its agonist or functional antagonist activity.39 73 S1P increases IL-8, but not IL-6, production by RPE cells in a pertussis toxin-sensitive manner, suggesting S1P1 receptor involvement.22 However, another study suggests a role for S1P2 receptors, as S1P-induced production of IL-8 and CCL2 in RPE cells is decreased by JTE013, but not by S1P1 or S1P3 antagonists.28 These apparently contradictory reports suggest that further work is needed to assess the role of different S1P receptors in retinal inflammation.

S1P role in photoreceptor apoptosis and neurodegeneration

Retina, being a part of the central nervous system (CNS), comprises full neuronal circuits to acquire, convert and transfer electrical activity of photoreceptors to the brain, which are known as neural retina. The neural retina is a multilayered interconnected structure composed of five cell types; these are photoreceptors, bipolar cells, ganglion cells, horizontal cells and amacrine cells.74 Neurodegeneration in retinal diseases usually refers to apoptosis of retinal ganglionic cells and photoreceptors which leads to significant and progressive loss in visual function.75 Retinal neurodegeneration is evident in DR76 and neovascular AMD,77 although the exact mechanism of neurodegeneration is not fully elucidated. Nevertheless, hypoxia-associated disturbed retinal blood flow in these diseases is suggested to be the main trigger of neuronal death.75 The role of S1P signalling in normal development of CNS is thoroughly reported, as sphingosine kinase-null mice embryos suffered from neuronal tube defects with massive apoptosis in neuroepithelium.78 Additionally, S1P signalling is involved in nerve growth factor-mediated neurite extension79 and neuronal excitability.80 S1P is required for development, differentiation and proliferation of photoreceptors in rat retinas.30

Under stress-induced photoreceptor and retinal ganglionic cell apoptosis, S1P can elicit different responses.81 82 On the one hand, S1P promotes cellular proliferation and reduces photoreceptor apoptosis.83 Decosahexanoic acid (DHA, a mediator of photoreceptor survival and differentiation) increases intracellular S1P levels by upregulating SphK, and the protective effects of DHA are blocked after SphK inhibition.30 Similarly, the action of S1P on S1P1 receptors increases the survival of and mitigates the damage to retinal ganglionic cells following optic nerve injury,84 and the selective S1P1 receptor agonist CYM-5442 reduces retinal ganglionic cell damage after endothelin-1-induced vasoconstriction.85 On the other hand, S1P acts as a pro-apoptotic mediator that can intensify the degenerative response in photoreceptors,86 an action that is suggested to be mainly mediated by S1P2 receptor activation.14

Under pathological conditions in the retina such as hypoxic and oxidative stress, or optic nerve injury, S1P2 receptors are upregulated while S1P1 receptors are downregulated.12 84 This makes the role of S1P1 receptors in ganglionic cell and photoreceptor survival less obvious under pathological conditions.84 At variance with the trophic effect of S1P1 receptors, S1P/S1P2 signalling elicits a detrimental effect on neuronal cells.84

S1P role in fibrosis, gliosis and EMT

Fibrosis is a reparative process that occurs in response to tissue injury, where the injured tissue is replaced by non-functional, collagen-rich fibrous matrix. Outside the CNS, fibroblasts are the main players in fibrosis, as they migrate to the injured location, proliferate, synthesise and deposit extracellular matrix proteins.87 As retina is considered a part of the CNS, the fibrotic response in the posterior chamber uses different mechanisms and cellular incorporation compared with that seen in non-CNS tissues.88 Retina has a scarce fibroblast population; instead the fibrotic response is mainly mediated by RPE and Müller glial cells. RPE and glial cells are quiescent and non-migratory under normal conditions. Under inflammatory conditions or tissue injury, these cells undergo specific transdifferentiation to acquire a fibroblast-like phenotype in processes known as EMT or gliosis. During EMT, RPE cells lose their epithelial traits and acquire a mesenchymal/fibroblast-like phenotype, becoming invasive, migratory, lacking tight junction proteins and expressing mesenchymal markers.89 The fibrocontractile nature of transformed RPE can result in retinal detachment and severe vision impairment ending in further disease exacerbation. Similar transitional events occur in Müller cell gliosis, where Müller glial cells transdifferentiate to a fibroblast-like phenotype, release trophic mediators, and acquire proliferative and migratory properties.90 91 Again, this transition to a fibrocontractile structure results in gliotic scar tissue formation, which further exacerbates retinal damage.92 While multiple cytokines and several proinflammatory signals can trigger a fibrotic response, there is growing evidence that TGF-β is one of the most important cytokines that contribute to EMT93 94 and gliosis,95 as it is detected at higher levels in the vitreous of patients with DR.96

S1P signalling generally triggers fibrotic events in neovascular ocular disease. Administration of an anti-S1P antibody reduces collagen precipitation in subretinal structures after rupture of Bruch’s membrane in a mouse model of CNV.23 Likewise, locally injected anti-S1P monoclonal antibodies can alleviate conjunctival scarring following glaucoma-filtering surgery.97 S1P increases the production of contractile actin fibres and facilitates collagen deposition by RPE in vitro, one of the mesenchymal characteristics of EMT,21 but the exact mechanism and S1P receptor subtype involved have not been reported. Migration of Müller glial cells in vitro is significantly increased by exogenously added S1P. Additionally, inhibition of SphK1, the main isoform in Müller glial cells, abolishes filopodia formation and cellular migration, suggesting that both endogenous and exogenous S1P amplify glial cell migration. S1P-mediated actions are reduced by pretreatment of Müller glial cells with the S1P3 antagonist BML-241, suggesting that the effects are primarily mediated by S1P3 receptors.91 Likewise, SphK1-null mice show diminished gliosis and slower progression of Sandhoff disease, a central neurodegenerative disease. Similar results are obtained by S1P3 receptor gene deletion.98

Although the relationship between S1P and TGF-β signalling in neovascular ocular disease is not yet reported, there is established evidence of crosstalk between S1P and TGF-β in renal mesangial cells.99 TGF-β increases SphK1 in endometriotic stromal cells,100 human fibroblasts101 and in human kidney podocytes.102 SphK1 upregulation in kidney is associated with protective rather than detrimental effects, as SphK1-deficient mice develop more drastic streptozocin-induced nephropathy.102

Conclusion

S1P is a promising therapeutic target that modulates angiogenesis, inflammation, apoptosis and fibrosis associated with neovascular ocular diseases. S1P/S1P1 signalling can induce formation of competent blood vessel sprouts, increase retinal perfusion, and reduce cell apoptosis and neurodegeneration. Blocking S1P2 receptors achieves similar beneficial outcomes, while S1P3 receptor antagonism or S1P1 activation can inhibit gliosis. Under hypoxic conditions, SphK1 and S1P2 are upregulated; this can be accompanied by S1P1 downregulation, resulting in increased S1P production and predominant signalling through S1P2 receptors. Therefore, inhibition of SphK1, S1P1 activation or S1P2/S1P3 antagonism might be used to attenuate retinal damage in ocular neovascular disease. Recent clinically approved S1P receptor modulators include siponimod and ozanimod; both can selectively activate S1P1 receptors (along with S1P5 receptors, which are less prevalent in ocular tissue). Evidence to date suggests that S1P receptor modulation plays important roles in the pathogenesis and treatment of neovascular ocular diseases. However, the role of these agents in the progression of neovascular ocular disease should be elucidated in preclinical models to inform future clinical trials involving S1P receptor modulators already approved for other conditions.

Acknowledgments

Figure 1 was created in part using BioRender. Elements of figure 2 are from the Servier Medical Arts Database.

References

Footnotes

  • Contributors RAA, KBR and CW contributed to the drafting of the manuscript. RAA and CW contributed to the interpretation of the data in the literature. All authors contributed to the critical appraisal and final approval of the manuscript. CW provided the overall supervision of this work.

  • Funding This work was funded by the Irish Research Council (IRC).

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.