Pigment epithelium-derived factor (PEDF) peptide eye drops reduce inflammation, cell death and vascular leakage in diabetic retinopathy in Ins2(Akita) mice

Abstract

Inflammation, neurodegeneration and microvascular irregularities are included in the spectrum of defects associated with diabetic retinopathy. Here, we evaluated intraocular deliverability features of two pigment epithelium-derived factor (PEDF) derivatives given as eye drops and their efficacy in modulating diabetes-induced retinal complications. The antiangiogenic PEDF60-77 (P60) and neuroprotective PEDF78-121 (P78) derivatives were applied to Ins2(Akita) mouse eyes once a week for 15 wks at the onset of hyperglycemia. Peptides, labeled with Alexa Fluor 488, were observed penetrating the cornea by 1-4 h and gained access to the ciliary body, retinal pigment epithelium (RPE)-choroid complex, retina microvasculature and vitreous. Peak vitreous levels were 0.2 μg/mL for P60 and 0.9 μg/mL for P78 after 0.5 and 4 h, respectively. Both peptides reduced vascular leakage by ~60% and increased zona occludens 1 (ZO1) and occludin expression in the microvasculature to nondiabetic levels. P60 induced pERK1/2 and P78 promoted pAKT in Muller glia, two signals that were dampened in diabetic conditions. Pharmacologically inhibiting AKT signaling in the retina blocked effects of the peptides on ZO1 and occludin expression. P78 reduced levels of 9/20 cytokines in diabetic vitreous including interferon (IFN)-γ, interleukin (IL)-6, IL-3 and tumor necrosis factor (TNF)-α. P60 lowered levels of 6/20 cytokines but was less effective than P78. Neuroprotective P78 prevented diabetes-induced microglia activation by ~60%, retinal ganglion cell (RGC) death by ~22% and inner plexiform layer thinning by ~13%. In summary, we provide evidence that PEDF bioactive derivatives gained access to the retina by topical delivery and validated their efficacy in reducing diabetic retinopathy complications. Our findings argue for glia regulation of microvascular leakage and an early root cause for RGC degeneration embedded in microglia activation.

Figure 1

Expression and purification of the antiangiogenic (P60) and neuroprotective (P78) PEDF derivatives. (A) The N-terminal locations of P60 at residues 60–77 and P78 at residues 78–121 of the PEDF protein are indicated in the diagram. (B) Schematic showing cloning of the peptide derivatives into pET32a. Bands on the PCR gels represent the amplified P60 and P78 fragments. (C) On the SDS-PAGE gradient gel (4–20%) expression of P60 and P78 (U, uncleaved from the upstream thioredoxin vector sequence) and purity of the peptides after cleavage (C, cleaved) at 2.1 and 4.7 kDa, respectively, are shown (M, Bio-Rad Precision Plus Protein™ Dual Xtra Standards). (D) The Western blot confirms identity of the two fragments by using a PEDF polyclonal antibody. Calculated yields of the expressed (U) and purified (C) peptides are given in Table 1.

Figure 2

Penetration of topically applied Alexa Fluor 488–labeled P60 (5 μg) and P78 in ATs across a diabetic (>300 mg/dL glucose) Ins2^Akita mouse eye. (A) Cross-section of a mouse eye 2 h after receiving the eye drops. The fluorescently labeled peptides were visible on the corneal surface and in the ciliary body (CB), RPE/Choroid (RPE/ch) and retina (n = 8). (B) Cross-sections of the corneal epithelium 1–4 h after treatment with eye drops containing dialyzed unconjugated Alexa Fluor 488 label (control), labeled P60, P78 or PEDF. Controls receiving dialyzed unconjugated Alexa Fluor 488 alone indicate that most of the free label was removed during dialysis. Labeled P60 penetrated the cornea earlier than P78, and most of the larger full-length PEDF (50 kDa) remained at the corneal epithelium (n = 5) (scale bar = 50 μmol/L). (C) Distribution of labeled P60 and P78 in the retina, 2 h after eye drops were given. The fluorescently labeled peptides were visible in the RPE/choroid, vitreous and vasculature of the inner retina (arrows) and in some retinal cells (arrowheads). RPE/Ch: RPE-choroidal complex (n = 4) (scale bar = 50 μmol/L).

Figure 3

Detection of Alexa Fluor 488–labeled P60 and P78 in rat vitreous after corneal application. (A) Fluorimetric detection of peptide levels in the vitreous. Peak levels of 0.2 ng/mL for P60 and 0.9 ng/mL for P78 were calculated at 0.5 and 4 h, respectively. Ratios of the peptide levels are given at the top of the graph bars (n = 15). Values are presented as means ± SEM. (B) Western blot (4–20% gradient SDS-PAGE) using an N-terminal PEDF polyclonal antibody confirms the presence of P60 and P78 in vitreous samples (20 μL) at 0.5 and 4 h, respectively, after the eye drops were given. Endogenous PEDF was also detected in the vitreous as the usual doublet migrating at ~50 kDa (M, molecular markers). (C) Densitometric analyses of Western blots indicate the difference in levels of the P60 and P78 peptides reaching the vitreous at the time points when their highest levels are seen by fluorometric analysis (A).

Figure 4

Reduction of vascular leakage in the diabetic Ins2^Akita mouse retina by PEDF derivatives. Retinas were evaluated 30 min after animals received tail-vein injections of BSA-FITC. (A) Left: Cross-section of a diabetic retina (NT) showing diffusion of fluorescently labeled BSA into the adjacent parenchyma of a blood vessel. The arrow points to a large vessel in the inner retina (OS, outer segment; ONL, outer nuclear layer; INL, inner nuclear layer). Middle: Control: Retina flat mount of a diabetic retina treated with vehicle alone (AT). The arrows point to several areas of BSA-FITC accumulation in the retinal parenchyma indicative of hemorrhaging. The vascular bed contains vessels that are thin and discontinuous. Right: Retina flat mount from a diabetic animal treated with the peptide eye drops. Fewer focal leakage areas and a thicker more continuous vascular bed are seen after treatment. (B) Quantitative measurements confirm that the number of hemorrhaging areas in the retina is reduced by ~55% after treatment, with either peptide or a combination of the two when compared with controls (AT) (p < 0.05). Differences were statistically insignificant between P60, P78 and combination treatments (n = 15). (C) Fluorometric measurements confirm a reduction in total BSA-FITC content in retina homogenates by ~40% after treatment with the PEDF derivatives compared with controls (FU, fluorescence units; p = 0.004). (D) Reduced levels of leaked albumin in the diabetic retina after treatment with either peptide was verified by Western blots of retina lysates (M, molecular weight standard; +, positive control – purified albumin protein). (E) Densitometry measurements of Western blots confirm a significant decrease in leaked albumin content in the peptide-treated group compared with AT controls (p = 0.002). Difference between P60 and P78 was statistically insignificant (n = 5). Values are means ± standard deviation (SD).

Figure 5

Regulation of occludin expression in the diabetic retina by P60 and P78. (A) The confocal images show a decrease in occludin expression in diabetic Ins2^Akita retinas (AT, vehicle treated) compared with ND controls (ND). Treatment with a combination of PEDF derivatives increased occludin expression in the retinal vasculature (arrow) of the diabetic animals (scale bar = 50 μmol/L) (n = 5). Western blot (B), densitometry measurements (C) and mRNA expression studies (D) confirm that the PEDF peptides significantly increased occludin levels in the diabetic retinas. P78 had a stronger effect than P60 in boosting both protein and mRNA expression (p ≤ 0.05). Differences between P78 and the combination peptide treatment were statistically insignificant. p values are indicated in the histograms where statistical significance was observed. All values are means ± SD.

Figure 6

Regulation of ZO1 expression in diabetic retinas by P60 and P78. (A) The confocal images indicate that ZO1 expression is decreased in the retinal vasculature (arrowheads) in diabetic conditions (AT) compared with the ND retinas (ND). Increased ZO1 expression was seen in the vasculature (arrows) of the diabetic retinas after treatment with a combination of the PEDF derivatives (scale bar = 50 μmol/L) (n = 6). Western blot analysis (B) and densitometry measurements (C, D) confirm a decrease in two ZO1 isoforms (220 and 214 kDa) in the diabetic groups and an increase in their expression after treatment with P60 or P78. (E) PCR analysis provides supporting evidence that both derivatives increased ZO1 mRNA expression. Differences between P60, P78 and the combination treatment were statistically insignificant (n = 4). p values are indicated in the histograms where statistical significances were observed among the treatment groups. Values are means ± SD.

Figure 7

Modulation of ERK1/2 and AKT signaling in the diabetic retina after peptide treatment. (A) The confocal micrographs show that ERK1/2 and AKT signaling were decreased in diabetic retinas (AT) compared with ND controls (ND). Treatment with the PEDF derivatives induced activation of both ERK and AKT in diabetic retinas. Similar to the nondisease controls, ERK signaling was confined to Muller glia processes in the outer retina and AKT activation in Muller glia cell processes of the inner retina. (B) Higher magnification of the micrographs in (A) showing pERK (outer retina) and pAKT (inner retina) immunolocalization in Muller processes in animals receiving the peptide eye drops (n = 5) (scale bar = 50 μmol/L). (C) The Western blots show that ERK and AKT activation were also evident in retinal explants cultured for 24 h in high glucose (HG) (25 mmol/L) and were then treated with P60 or P78 for 15–120 min. (D, E) Densitometric analyses of the Western blots indicate that P60 induced rapid activation of ERK1/2. P78 reduced ERK1 phosphorylation below the HG controls but had no effect on ERK2 activation, and P78 (F) promoted and sustained activation of AKT throughout the treatment period (n = 3; p < 0.05) (NG, normal glucose). Statistically significant comparisons for P60 and P78 are indicated in the figures. All values are means ± SD.

Figure 8

Regulation of ZO1 and occludin expression by the PEDF derivatives through the PI3K/AKT pathway. Quantitative PCR measurements indicate that the effects of P60 and P78 on ZO1 and occludin expression are mediated through the PI3K/AKT pathway in a retinal explant model of diabetes. Explants were cultured in high glucose (25 mmol/L) for 24 h in the presence/absence of the pharmacological inhibitors of PI3K (iPI3K; LY294,002), MAPK (iMAPK; U0126) and one or the other of the PEDF derivatives (n = 4). iPI3K blocked P78 effects on both ZO1 and occludin expression. It also inhibited the effects of P60 on ZO1 expression but not on occludin levels. p values are indicated for statistically significant comparisons. All values are means ± SD.

Figure 9

Reduction in glia activation in diabetic retinas by P78. (A) The confocal images show an increase in numbers of IBA1-labeled activated microglia in the Ins2^Akita diabetic retinas (AT) compared with retinas from ND mice (ND). P78 or the combination peptide treatment reduced numbers of reactive microglia in the diabetic retina. P60 had no effect on the activation of these cells. The arrow in the control (AT) indicates large amoeboid-like microglia expressing the IBA1 marker in the inner retina. (B) Higher magnification of the control from (A) showing ramifications of large microglia in the inner retina. (C) GFAP expression was detected in astrocytes and not in Muller glia cells in controls and treated and untreated samples (scale bar = 50 μmol/L). (D) Cell counts confirm that P78 reduced microglia activation in the diabetic retina by ~60% (p = 0.001). The difference between AT and P60 treatment was statistically insignificant (n = 8). Western blot (E) and densitometry measurements (F) confirm an increased in IBA1 levels and a reduction after treatment in diabetic retinas. (G) mRNA levels in the retina provide supporting evidence that P78 reduced microglia activation in diabetic conditions (p = 0.001) (n = 3). Densitometry measurements of Western blots (H) and quantitative PCR (I) indicate that GFAP protein and mRNA levels were not altered with diabetes. Comparisons among the peptide- and vehicle-treated groups where p values are not indicated in the graphs are statistically insignificant. All values are means ± SD.

Figure 10

Reduction in inflammatory cytokine levels in the diabetic vitreous after treatment with P60 and P78 peptide eye drops. The histograms represent Affymetrix Luminex bead measurements of cytokine levels in the vitreous. An increase in 8/20 inflammatory cytokines was detected in the diabetic condition compared with the normal controls. Both of the PEDF derivatives reduced vitreous levels of these cytokines, but maximal effect was seen after P78 treatment. Only P78 reduced TNFα (p = 0.003) and IL2 (p = 0.006) levels (n = 5). All values are means ± SD.

Figure 11

Neuronal protection in diabetic retinas by P78. (A) Nuclear staining of fixed retinas with PI showing the various nuclear layers in the control vehicle (AT) and peptide-treated diabetic retinas. A loss of cells in the RGL (arrows) was noted in the diabetic condition and a reduction in cell drop out in the peptide-treated retinas. (B, C) Higher magnification of the RGL in the diabetic control (*) and treated (**) samples in (A) showing cell dropout (arrows) in the RGL in controls compared with the treated groups (scale bar = 50 μmol/L). (D) Cell counts confirm a loss of cells in the RGL in diabetic retinas (p = 0.04) (ND versus NT) and cell rescue after treatment with P78 (p = 0.03). (E) Measurements of the mean thickness of the IPL also indicate that P78 was effective in reducing IPL thinning (p = 0.04). The difference between AT and P60 was insignificant (n = 12). All values are means ± SD.

Figure 12

The schematic represents key events regulated by P60 and P78 that may reduce complications in diabetic retinopathy. The antiangiogenic P60 may reduce vascular leakage by increasing tight junction proteins in retina vessels through Muller glia signaling and by lowering levels of inflammatory cytokines that promote vessel abnormalities. Inhibition of vascular pathology could in turn have downstream effects on retina cell survival. The neuroprotective P78 derivative may reduce RGC loss in the diabetic retina by reducing early microglia activation and levels of inflammatory cytokines such as TNFα. It may block late-stage vascular leakage through a Muller glia-mediated upregulation of tight junction proteins in the retina microvasculature. P78 could also have direct effects on RGC survival by yet undefined mechanisms.