Defining a mechanistic link between pigment epithelium-derived factor, docosahexaenoic acid, and corneal nerve regeneration

Abstract

The cornea is densely innervated to sustain the integrity of the ocular surface. Corneal nerve damage produced by aging, diabetes, refractive surgeries, and viral or bacterial infections impairs tear production, the blinking reflex, and epithelial wound healing, resulting in loss of transparency and vision. A combination of the known neuroprotective molecule, pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA), has been shown to stimulate corneal nerve regeneration, but the mechanisms involved are unclear. Here, we sought to define the molecular events of this effect in an in vivo mouse injury model. We first confirmed that PEDF + DHA increased nerve regeneration in the mouse cornea. Treatment with PEDF activates the phospholipase A₂ activity of the PEDF-receptor (PEDF-R) leading to the release of DHA; this free DHA led to enhanced docosanoid synthesis and induction of bdnf, ngf, and the axon growth promoter semaphorin 7a (sema7a), and as a consequence, their products appeared in the mouse tears. Surprisingly, corneal injury and treatment with PEDF + DHA induced transcription of neuropeptide y (npy), small proline-rich protein 1a (sprr1a), and vasoactive intestinal peptide (vip) in the trigeminal ganglia (TG). The PEDF-R inhibitor, atglistatin, blocked all of these changes in the cornea and TG. In conclusion, we uncovered here an active cornea-TG axis, driven by PEDF-R activation, that fosters axon outgrowth in the cornea.

Keywords: DHA; NGF; PEDF; Sema7A; adipose triglyceride lipase; brain-derived neurotrophic factor (BDNF); cornea; lipid signaling; neuropeptides; phospholipase A.

Figure 1.

PEDF + DHA enhances corneal nerve regeneration in the mouse. A, representative image of a frozen section of a mouse cornea immunostained with anti-PEDR-R antibody (green) and DAPI (blue). B, expression of PEDF-R in mouse corneas (pool of six) by Western blotting. C, whole-mount images of corneal nerves were stained with anti-PGP9.5; D, anti-SP after injury and topical treatment with PEDF + DHA and vehicle three times a day for 7 days (Table 1 shows treatment concentrations). The insets in C and D, which are marked by a dashed box in the whole-mount images, show the amplified vortex area inverted to a white background in the treated corneas under ×10 objective lens. Data were normalized to the baseline (uninjured corneas). Bars represent average nerve density of four corneas ± S.D. *, p < 0.05 with t test statistical analysis to compare two groups at 95% of the confidence level. The experiment was repeated three times with similar results.

Figure 2.

Gene induction in the mouse cornea after injury and treatment with PEDF + DHA. A, experimental design (top) and the induction of genes by DHA, PEDF, and PEDF + DHA, with the cutoff at a 2- and 5-fold increase (p < 0.05). In the lower chart, results are shown with three levels of gene induction: <2, between 2 and 5, and >5. Red boxes label three genes with higher expression by PEDF + DHA: bdnf, ngf, and sema7a. B, gene induction of bdnf, ngf, and sema7a by PEDF + DHA as a function of time. Mice were treated as described under “Experimental procedures” (top). Levels of transcripts were normalized to actb, gapdh, and tbp (primer sequences in Table 2). The bars represent the mean of three samples ± S.D. A pool of six corneas/sample was used for the gene expression study.

Figure 3.

PEDF + DHA treatment increases secretion of NGF, BDNF, and Sema7A in tears and the phosphorylation of TrkB and ERK1/2 in TG. A, corneas were injured and treated with PEDF + DHA and tears collected as shown in the experimental design (top). Seven micrograms of protein from the mouse tear film (pool of six eyes/sample) were used for Western blot analysis of BDNF and NGF (bottom). B, Western blot analysis of TrkA, TrkB, p75, Tyr-phosphorylated Trks, and GAPDH in the TG (pool of six TGs, 50 μg of protein per well). Bars in A and B represent the mean of two experiments (two different pooled sample sets for each experiment, four samples in total) ± S.D. *, p < 0.05 with the t test analysis in comparison with the vehicle at the same time point. C, Western blot of Sema7A secreted to tears after PEDF + DHA treatment. Seven micrograms of protein collected from tears (pool of six eyes/sample) were used. D, Western blot analysis of intracellular integrin β1, total ERK, p44/p42 ERK1/2, and GAPDH in the TG (pool of six TGs, 50 μg of protein per well) of corneas treated with PEDF + DHA or vehicle. Details about the antibodies used are in Table 3. Bars in C and D represent the mean of two experiments (two different pooled sample sets for each experiment, four samples in total) ± S.D. of p-ERK/total ERK ratio. *, p < 0.05 with t test analysis in comparison with the vehicle at the same time points.

Figure 4.

Stimulation of gene and protein expression of neurotrophins and Sema7A by 44-mer PEDF + DHA, NPD1, and PEDF + DHA. A, qPCR gene expression analysis of mouse corneas at 3 h after injury and treatments. The concentrations of the compounds are described in Table 1. All genes were significantly induced in comparison with the vehicle-treated groups (six corneas/sample, p < 0.05). Levels of transcripts were normalized to gapdh, tubb3, and tbp (primer sequences in Table 4). B, Western blot analysis of secreted BDNF, NGF, and Sema7A into the media of injured corneas in organ culture treated with the compounds or vehicle for 24 h (six corneas/sample). The data were normalized to GAPDH as an internal standard. *, p < 0.05 with t test analysis in comparison with the vehicle. The bars represent the mean of three samples ± S.D. The experiment was repeated once with similar results.

Figure 5.

DHA incorporation in corneal PC and PE molecular species, from basal to treated conditions. Effect of atglistatin on gene expression, release of DHA and AA, and synthesis of hydroxy-derivatives was examined. A and B, comparison of different molecular species of PC and PE in injured corneas in mice after 1 h of topical DHA treatment in vivo analyzed by LC-MS/MS. Each plot shows the percentage of specific PC/PE species in the vehicle- (left) and DHA (right)-treated corneas. Data were collected from eight corneas individually (one cornea/sample). *, p < 0.05 with the t test statistical analysis to compare two groups at 95% of the confidence level. C, quantification of 22:6/22:6-containing PCs and PEs in the presence of PEDF, DHA, and PEDF + DHA. Single data point represents one treated cornea. *, p < 0.05 with ANOVA analysis plus Fisher post hoc test at 95% of the confidence level. D, experimental design of mice treated with atglistatin injected (i.p.) before injury and topically treated with PEDF + DHA, PEDF + DHA + atglistatin, or vehicle after injury. E and F, gene expression analysis in corneas (E) and TG (F) at 3 h after injury and treatment. G, mass spectrometry-based lipidomic analysis of mouse corneas as explained under “Experimental procedures.” *, p < 0.05 with ANOVA analysis plus Fisher post hoc test at 95% of the confidence level. For lipid analysis, a pool of six corneas/sample was used. Bars represent the mean of three experiments ± S.D.

Figure 6.

Inhibition of PEDF-R decreases corneal nerve regeneration. A, experimental design and Schirmer's test analysis. Volume of mouse tears was measured on days 2, 4, and 6 after injury and treatment. *, p < 0.05 with ANOVA plus Fisher post hoc test at 95% of confidence level. B, representative whole-mount images of corneal nerves stained with anti PGP9.5 and percent of nerve density compared with normal corneas after 7 days of treatment. *, p < 0.05 with ANOVA plus Fisher post hoc test at 95% of confidence level. C, effect of atglistatin on the levels of Sema7A and BDNF in tears analyzed by Western blotting (pool sample of six eyes, 7 μg of protein per well). The intensity of immunoreactive bands was normalized to the baseline (uninjured corneas). The bars represent the mean of three experiments ± S.D. *, p < 0.05 with ANOVA plus Fisher post hoc test at 95% of confidence level.

Figure 7.

Working model of the action of PEDF + DHA in enhancing corneal nerve regeneration. PEDF via its 44-mer neuroprotective domain (in red) activates PEDF-R in the cornea (amplify for clarification). This transmembrane receptor with iPLA₂ activity released DHA, enriched in the sn-2 position of membrane phospholipids, by DHA supplementation. Mouse corneas express a 12- and 15-lipoxygenase (LOX) (55); DHA is converted to 17-HpDHA on the pathway to NPD1 by 15-LOX-1, and DHA is also converted to 14-HpDHA on the pathway to maresin-1 by 12-LOX-1. Docosanoids such as NPD1 (and possibly others not yet identified) induce the gene expression and protein levels of the neurotrophins NGF, BDNF, and Sema7A (all of which are secreted to tears), and of RAGs vip, npy, and sprr1a in the TG. Phosphorylation of TrkB and ERK 1/2 occurs in the TG as a result of BDNF and Sema7A secretion in the tear film. Inhibition of the phospholipase activity of the PEDF-R abolishes this signaling mechanism and corneal nerve regeneration.