Fingolimod (FTY720), a Sphinogosine-1-Phosphate Receptor Agonist, Mitigates Choroidal Endothelial Proangiogenic Properties and Choroidal Neovascularization

Abstract

Neovascular or wet age-related macular degeneration (nAMD) causes vision loss due to inflammatory and vascular endothelial growth factor (VEGF)-driven neovascularization processes in the choroid. Due to the excess in VEGF levels associated with nAMD, anti-VEGF therapies are utilized for treatment. Unfortunately, not all patients have a sufficient response to such therapies, leaving few if any other treatment options for these patients. Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator found in endothelial cells that participates in modulating barrier function, angiogenesis, and inflammation. S1P, through its receptor (S1PR1) in endothelial cells, prevents illegitimate sprouting angiogenesis during vascular development. In the present paper, we show that, in choroidal endothelial cells, S1PR1 is the most abundantly expressed S1P receptor and agonism of S1PR1-prevented choroidal endothelial cell capillary morphogenesis in culture. Given that nAMD pathogenesis draws from enhanced inflammation and angiogenesis as well as a loss of barrier function, we assessed the impact of S1PR agonism on choroidal neovascularization in vivo. Using laser photocoagulation rupture of Bruch's membrane to induce choroidal neovascularization, we show that S1PR non-selective (FTY720) and S1PR1 selective (CYM5442) agonists significantly inhibit choroidal neovascularization in this model. Thus, utilizing S1PR agonists to temper choroidal neovascularization presents an additional novel use for these agonists presently in clinical use for multiple sclerosis as well as other inflammatory diseases.

Keywords: age-related macular degeneration; choroid; inflammation; microglia; retinal pigment epithelium; retinal vasculature.

Figure 1

Expression of S1PR in various ocular cells. (A) Total RNA was extracted from murine ChEC, ChPC, RPE, REC, RPC, RAC, MGC, ChMC, and BMMC or (B) retina and choroid/RPE tissue, as outlined in Methods. A total of 1 µg of total RNA was used for cDNA synthesis. cDNA was used as the template for qPCR performed in triplicates of three biological replicates using specific gene primers (Table 1). The RpL13A was used as a control. The relative amounts of transcripts were determined, as detailed in Methods. * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001. (n = 3).

Figure 2

The S1PR non-selective agonist FTY720 and the S1PR1 selective agonist CYM5442 impact ChEC viability. (A) ChECs were grown in 96-well plates and incubated with medium containing varying concentrations of FTY720 or CYM5442 (in DMSO) or DMSO for 4 days. (B,C) ChECs were also incubated with different concentrations of active (FTY720(S)-P) or inactive (FTY720(R)-P) FTY720 enantiomers for 2 days. The percent cell viability relative to control samples was determined as described in Methods. * p < 0.05; ** p < 0.01, **** p < 0.0001. (n = 3).

Figure 3

The S1PR agonist FTY720 inhibits ChEC migration. A suspension of ChECs was prepared in serum-free medium and added to the top of Transwell inserts and incubated for 4 h with medium containing agonist (in DMSO) or DMSO. Cells were fixed, stained with H&E, and the number of cells that migrated through the 8 μm pore size membrane was counted, as described in Methods. *** p < 0.001. (n = 3).

Figure 4

Enantiomers of the phosphorylated S1PR agonist FTY720 inhibit ChEC migration. ChECs in suspension were added to the top of Transwell inserts and incubated for 4 h with medium containing varying concentrations of enantiomer (in DMSO) or DMSO. Cells were fixed, stained with H&E, and the number of cells that migrated through the 8 μm pore size membrane was counted as described in Methods. **** p < 0.0001. (n = 3).

Figure 5

The S1PR agonist FTY720 inhibits ChEC capillary morphogenesis. Detached ChECs were incubated with varying concentrations of FTY720 (in DMSO) or DMSO on ice for 20 min. Incubated cells were then applied to Matrigel-coated plates, incubated, and imaged 14 h later as described in Methods. The mean number of branch points was determined as detailed in Methods. **** p < 0.0001. (n = 3).

Figure 6

An enantiomer of the phosphorylated S1PR agonist FTY720 inhibits ChEC capillary morphogenesis. Detached ChECs were incubated with varying concentrations of phosphorylated S1PR agonist (in DMSO) or DMSO on ice for 20 min. Incubated cells were then applied to Matrigel-coated plates, incubated, and photographed after 14 h as described in Methods. The mean number of branch points was determined. *** p < 0.001, **** p < 0.0001. (n = 3).

Figure 7

The S1PR1 agonist CYM5442 inhibits ChEC capillary morphogenesis. ChECs were detached and incubated with varying concentrations of CYM5442 (in DMSO) or DMSO on ice for 20 min. Incubated cells were then applied to Matrigel-coated plates, incubated, and photographed after 14 h as described in Methods. The mean number of branch points was determined. **** p < 0.0001. (n = 3).

Figure 8

Oral delivery of the S1PR agonist FTY720 inhibits CNV in a mouse model of nAMD. Vehicle control (A) or FTY720 (B) was administered orally (0.3 mg/kg body weight) to C57BL/6j male and female mice (2 months old), 2 days before laser photocoagulation and for 2 weeks (5 times per week), as described in Methods. The concentration was chosen to mimic the safe and effective dose frequently used to treat other disease entities in mouse models. Representative images of new choroidal blood vessels stained with anti-ICAM-2 antibodies are shown in Panels A and B, and the quantification of the findings is shown in (C). *** p < 0.001. (n = 10 mice per group).

Figure 9

A single intravitreal injection of the S1PR agonist FTY720 inhibits CNV in a mouse model of nAMD. C57BL/6j male and female mice (2 months old) were injected intravitreally with (A) vehicle control or (B) FTY720, 1 μL of a 10 µM stock, following a laser rupture of Bruch’s membrane as described in Methods. After 14 days, the eyes were processed for staining with anti-ICAM-2 antibodies and quantification of new choroidal blood vessels (C). ** p < 0.01. (n = 10 mice per group).

Figure 10

A single intravitreal injection of the S1PR1 agonist CYM5442 inhibits CNV in a mouse model of nAMD. C57BL/6j male and female mice, 2 months old, received a single intravitreal injection of (A) vehicle control or (B) CYM5442, 1 μL of a 10 µM stock, following the laser rupture of Bruch’s membrane as described in Methods. After 14 days, the eyes were processed for staining with anti-ICAM-2 antibodies and quantification of new choroidal blood vessels (C). *** p < 0.001. (n = 10 mice per group).