Pigment Epithelium-Derived Factor Binding to VEGFR-1 (Flt-1) Increases the Survival of Retinal Neurons

Abstract

Purpose: The purpose of this study was to examine possible involvement of vascular endothelial growth factor (VEGF) receptor (VEGFR)-1/Flt-1 in pigment epithelium-derived factor (PEDF)-promoted survival of retinal neurons.

Methods: Survival of growth factor-deprived retinal ganglion cells (RGCs) and R28 cells and activation of ERK-1/-2 MAP kinases were assessed in the presence of PEDF, placental growth factor (PlGF), and VEGF using cell cultures, viability assays and quantitation of ERK-1/-2 phosphorylation. VEGFR-1/Flt-1 expression was determined using quantitative PCR (qPCR) and Western blotting. VEGFR-1/Flt-1 was knocked down in R28 cells by small interfering RNA (siRNA). Binding of a PEDF-IgG Fc fusion protein (PEDF-Fc) to retinal neurons, immobilized VEGFR-1/Flt-1 and VEGFR-1/Flt-1-derived peptides was studied using binding assays and peptide scanning.

Results: PEDF in combination with PlGF stimulated increased cell survival and ERK-1/-2 MAP kinase activation compared to effects of either factor alone. VEGFR-1/Flt-1 expression in RGCs and R28 cells was significantly upregulated by hypoxia, VEGF, and PEDF. VEGFR-1/Flt-1 ligands (VEGF and PlGF) or soluble VEGFR-1 (sflt-1) competed with PEDF-Fc for binding to R28 cells. Depleting R28 cells of VEGFR-1/Flt-1 resulted in reduced PEDF-Fc binding when comparing VEGFR-1/Flt-1 siRNA- and control siRNA-treated cells. PEDF-Fc interacted with immobilized sflt-1, which was specifically blocked by VEGF and PlGF. PEDF-Fc binding sites were mapped to VEGFR-1/Flt-1 extracellular domains D3 and D4. Peptides corresponding to D3 and D4 specifically inhibited PEDF-Fc binding to R28 cells. These peptides and sflt-1 significantly inhibited PEDF-promoted survival of R28 cells.

Conclusions: These results suggest that PEDF can target VEGFR-1/Flt-1 and this interaction plays a significant role in PEDF-mediated neuroprotection in the retina.

Figure 1.

PEDF, PlGF, and VEGF stimulate survival of retinal neurons. Cells were deprived of trophic factors and incubated for 24 hours in the presence or absence of PlGF (50 ng/mL), PEDF (500 ng/mL), and VEGF (100 ng/mL). Shown is the treatment of RGCs and R28 cells with single and combined (white bars) factors. (A, B) The numbers of Calcein-AM-metabolizing RGCs were counted and cell survival was expressed in relationship to numbers of DAPI-positive nuclei: treatment with (A) PEDF and PlGF, the panel shows averaged data from two single experiments, error bars depict standard deviation; and (B) PEDF and VEGF (n = 7; means ± SEM, versus medium control culture, **P < 0.01, ***P < 0.001). (C) Survival of stimulated R28 cells was determined by an MTT assay (n = 6). (D) ERK-1/-2 MAP kinase activation is involved in PEDF-, PlGF-, and VEGF-activated intracellular signaling in R28 cells. Cells were stimulated for 30 minutes, lysed and an equal amount of protein was separated by SDS-PAGE. The figure shows one Western blot out of four independent experiments. In bar charts, columns represent the mean ratio of phosphorylated ERK (ERK-P)/ERK signal intensity (n = 5). Results in (C) and (D, bar charts) are expressed as relative values (means ± SEM, versus medium control culture, dashed lines, *P < 0.05, **P < 0.01; versus PEDF-exposed culture, ^◆P < 0.05, ^◆◆◆P < 0.001; versus PlGF-exposed culture, ^◊P < 0.05; versus VEGF-exposed culture, ^◊P < 0.05, ^◊◊◊P < 0.001).

Figure 2.

VEGFR-1/Flt-1 expression in retinal neurons demonstrated by (A) RT-PCR or (B) microscopic imaging following immunofluorescence labeling of RGCs and R28 cells (scale bars = 20 µm) and (C) human retina (scale bar = 50 µm). (A) RGCs and R28 cells express VEGFR-1/Flt-1 mRNA transcripts. Total RNA was extracted and mRNA expression in (1) RGCs and (2) R28 cells was detected by RT-PCR (NC, negative PCR control). Amplified DNA was visualized using agarose gel electrophoresis. (B, C) Labeling of VEGFR-1/Flt-1 with specific antibodies (B, rabbit anti-VEGFR-1/Flt-1, red; C, mAb #MAB321, green) and counterstaining of cell nuclei with DAPI (blue) were performed using fixed/ permeabilized cells or retinal tissue. Negative controls using rabbit IgG or non-immune mouse IgG1, respectively, demonstrated negligible reactivity (not shown). Images are representative of four independent experiments (B) or staining of retinae from two donors (C). (C) Double-labeling of retinal cryosections revealed co-localization of VEGFR-1/Flt-1 and class III β-tubulin (red) in the GCL/NFL (yellow merge signal, arrows). VEGFR-1/Flt-1 was predominantly localized to the ILM. ILM, internal limiting membrane; NFL, nerve fiber layer; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptor layer.

Figure 3.

Hypoxia (0.2% O₂), PEDF, and VEGF upregulate VEGFR-1/Flt-1 expression in retinal neurons. (A) Elevated VEGFR-1/Flt-1 mRNA levels were detected in RGCs (n = 3) and R28 cells (n = 8) and (B) VEGFR-1/Flt-1 protein expression increased in R28 cells (n = 5) after 24 hours of hypoxia. (C) Exposure (24 hours) of cells to PEDF (500 ng/mL) and VEGF (50 ng/mL) induced VEGFR-1/Flt-1 mRNA upregulation in RGCs (n = 3) and R28 cells (n = 6), and (D) caused elevated VEGFR-1/Flt-1 protein levels in R28 cells (n = 4). (A, C) The mRNA transcripts prepared from retinal neurons were reversed transcribed and analyzed by qPCR and (B), (D) VEGFR-1/Flt-1 and β-actin expression in cell lysates of equalized cell numbers was analyzed by Western blotting. Results are expressed as relative values (fold change in VEGFR-1/Flt-1 expression; means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001) compared with values from unstimulated (normoxic, medium) control cell cultures (solid bars).

Figure 4.

PEDF-Fc, a fusion protein consisting of human PEDF and hinge and constant regions of mouse (m)IgG2b heavy chain, binds to retinal neuronal cells. (A) Schematic representation of PEDF-Fc. (B) PEDF-Fc binds to RGCs and R28 cells, and binding is blocked by PEDF or sflt-1. Cells were stained with PEDF-Fc or exposed to a fusion protein control. Immunofluorescence (red) was detected using microscopic imaging (first line, blue fluorescence: cell nuclei labeled with DAPI). RGCs (second line) or R28 cells (third line) were either preincubated with 500 ng/mL PEDF or exposed to PEDF-Fc preincubated with sflt-1 or a human IgG control (each at 1 µg/mL; scale bars = 20 µm). Results are shown from one experiment of two performed with RGCs of different mice or independently cultured R28 cells. (C) Binding of PEDF-Fc was revealed by colorimetric immunodetection. R28 cells were preincubated either in binding buffer (–), with 500 ng/mL PEDF, 100 ng/mL VEGF, or 50 ng/mL PlGF and subsequently exposed to PEDF-Fc. In addition, cells were left in binding buffer (dashed line), incubated with mIgG2b, the fusion protein control, and PEDF-Fc in the presence of sflt-1 or human IgG (control). Significant differences to values from cells exposed to mIgG2b (**P < 0.01) or PEDF-Fc (black bar; ^◆P < 0.05, ^◆◆P < 0.01) are indicated (means ± SEM; n = 4). (D, E) R28 cells were transfected with siRNAs and cultured for 48 hours. Two different VEGFR-1/Flt-1 siRNAs or control (non-transfected cells and control siRNA) conditions were applied in two independent experiments. (D) The effect of siRNAs on VEGFR-1/Flt-1 expression as compared to that of non-targeting control siRNA (–) was determined by qPCR (averaged values from three PCRs; error bars = standard deviation). (E) Knockdown of VEGFR-1/Flt-1 mRNA abolishes PEDF-Fc binding. Binding was revealed by immunofluorescence labeling and microscopic imaging (see panel B) and is shown for R28 cells transfected with VEGFR-1/Flt-1 siRNAs or non-transfected and control siRNA-transfected cells (scale bars = 20 µm). Cells exposed to mIgG2b or the fusion protein control demonstrated negligible reactivity (not shown).

Figure 5.

(A) PEDF competes with PEDF-Fc for binding to immobilized sflt-1. Chimeric Fc-linked human sflt-1 (400 ng per lane) was subjected to Western blotting and strips of the blot were incubated with (1) an mAb directed to human IgG or (2, 3) PEDF-Fc. A strip preincubated with PEDF prior to exposure to PEDF-Fc is shown in lane 3; sflt-1 migrated at approximately 250 kDa under non-reducing conditions. (B) Binding of PEDF-Fc to immobilized sflt-1 was competed by increasing amounts of PlGF or VEGF in a solid-phase binding assay. Biotinylated PEDF-Fc, in the absence (-, black bar) or presence of cytokines, was added to wells coated with sflt-1 and binding was revealed using streptavidin-based detection. PEDF-Fc binding is expressed as relative values (means ± SEM, n = 3, compared with fusion protein control, dashed line, ***P < 0.001; versus PEDF-Fc binding without cytokines, ^◆P < 0.05, ^◆◆P < 0.01, ^◆◆◆P < 0.001). Control values resulting from exposure of wells to biotinylated mIgG2b control or sflt-1-replacing binding buffer are also shown.

Figure 6.

The amino acid sequence of human soluble VEGFR-1/Flt-1 preprotein was fragmented into 13-mer peptides, thereby shifting by two amino acids. (A) Interaction of PEDF-Fc with cellulose-bound peptides was revealed by chemiluminescent immunodetection. Signals in the boxed areas correspond to the amino acid sequences, (1) ²⁴⁵EKNKRASVRRRIDQSNSHANI²⁶⁵, (2) ³⁰³YDKAFITVKHRKQQVLETVAGKR³²⁵, and (3) ⁶²⁹IRGEHCNKKAVFSRISKFKSTRN⁶⁵¹. (B) The amino acid sequence encompassing domains (shaded in grey) D3 (residues 204 – 301) and D4 (residues 309 – 395) of mature human VEGFR-1/Flt-1 (huFlt-1) was aligned with the homologous sequence of mature rat VEGFR-1/Flt-1 (RtFlt-1). Sequences used for alignment were obtained from Genbank (accession numbers NP_002010.2 and NP_062179.2). Amino acid numbering of human VEGFR-1/Flt-1 is related to Ser as the first amino acid of the mature protein. Sequence stretches (1) and (2) that interact with PEDF-Fc (see panel A) are indicated in bold. Underlined amino acids constitute peptides AE10, AF10, DX21, and DW21 used in a competitive binding assay as shown in panel (C): the peptides (each at 10 µM, based on prior findings in RPE cells) competed binding of PEDF-Fc to R28 cells. PEDF-Fc was preincubated with AE10, AF10, DX21, and DW21 or a control peptide (solid bar) and further incubated with R28 cells overnight. As controls, cells were either left in binding buffer (dashed line) or incubated with mIgG2b, a control fusion protein or PEDF-Fc. Binding of PEDF-Fc was revealed by colorimetric immunodetection. Significant differences to cells exposed to mIgG2b (***P < 0.001) or PEDF-Fc in the presence of a control peptide (^◆P < 0.05, ^◆◆P < 0.01, ^◆◆◆P < 0.001) are indicated (means ± SEM; n = 4).

Figure 7.

VEGFR-1/Flt-1 is involved in PEDF-promoted survival of R28 cells. PEDF (500 ng/mL) was preincubated with (A) sflt-1 (see legend to Fig. 5A) or (B) VEGFR-1/Flt-1-derived peptides. Samples were then incubated with growth factor-deprived R28 cells for 24 hours and cell survival was determined by MTT assays. Results are expressed as relative values in relationship to values from medium control cultures (dashed lines). Data are representative of six experiments using independent R28 cell cultures. (A) Sflt-1 (50 ng/mL) attenuates enhanced cell survival induced by PEDF or PlGF (50 ng/mL). Significant differences to control cultures (presence of human IgG, huIgG, 50 ng/mL; ^◆P < 0.05, ^◆◆◆P < 0.001) and the effects of PEDF and PlGF in huIgG control cultures (**P < 0.01, ***P < 0.001) are indicated (means ± SEM; n = 6). (B) VEGFR-1/Flt-1 peptides (10 µM) inhibit R28 cell survival promoted by PEDF. The effect of DX21, AE10, DW21, and AF10 compared to that of a control peptide (^◆◆P < 0.01, ^◆◆◆P < 0.001) and the effect of PEDF in control cultures (***P < 0.001) are shown (means ± SEM; n = 6).