Pigment Epithelium-derived Factor Concentration Research Articles

Vascular endothelial growth factor and pigment epithelium-derived factor in aqueous humor of patients with choroidal neovascularization

To detect the levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in aqueous humor of patients with active choroidal neovascularization (CNV). Aqueous humor samples were obtained from 32 patients with active CNV. The concentrations of VEGF and PEDF in aqueous humor were measured by enzyme linked immunosorbent assay (ELISA) for quantitative analysis. VEGF and PEDF in 10 samples of aqueous humor from patients with cataract were also detected by the same methods as control. The mean VEGF and PEDF concentrations in aqueous humor of active CNV patients were higher than those in the control group (P=0.000). The patients with active CNV exhibit significantly higher VEGF and PEDF levels than those in control, indicating that VEGF along with PEDF may modulate the formation of CNV.

A phospholipase A2‐linked receptor for PEDF

Pigment epithelium-derived factor (PEDF), a 50-kDa extracellular serpin, is a neurotrophic and antiangiogenic factor for the retina. Its biological functions are independent from its serine protease inhibition potential. Previously we provided evidence for high affinity PEDF-binding sites and proteins in retina cell-surfaces. Recently a novel human retinal pigment epithelial gene was identified as potential PEDF receptor and termed PEDF-R (pseudonyms: PNLP2, TTS-2.1, ATGL, iPLAζ, desnutrin). It codes for a polypeptide (504 amino acids) with a patatin-like phospholipase (PLA2) domain, and 4 transmembrane, 3 intracellular and 2 extracellular regions. RT-PCR and immunohistochemistry confirmed the PEDF-R expression in the retina. The potential PLA2 activity of PEDF-R was investigated. Recombinant epitope-tagged PEDF-R polypeptide was synthesized in cell-free systems and purified. Using dilinoleoyl phosphatidylcholine as substrate, PEDF-R exhibited potent PLA activity, which was optimum at pH 7.5. Surface plasmon resonance revealed high affinity for the PEDF:PEDF-R interactions (KD = ~3 nM), which decreased significantly with other serpins (maspin, ovalbumin). Preincubation of PEDF-R with increasing concentrations of PEDF stimulated the PLA activity of PEDF-R, but the stimulation did not occur when BSA replaced PEDF. PEDF alone did not show activity, and did not stimulate hog pancreas PLA2. These results demonstrate that the recombinant PEDF-R exhibited PLA activity that liberates fatty acids, an activity that PEDF can stimulate, suggesting a molecular pathway by which ligand/receptor interaction on the cell-surface could generate a cellular signal. Supported by the Intramural Research Program of the NIH, NEI.

Detection of PEDF in Subretinal Fluid of Retinal Detachment: Possible Role in the Prevention of Subretinal Neovascularization

PEDF (pigment-epithelium-derived factor) is a member of the serpin family of protease inhibitors. It is considered to be an important regulator of human eye disease and is known to inhibit angiogenesis. We have therefore investigated the presence of PEDF in the subretinal fluid of patients with retinal detachment. Methods: Eighteen samples from SRF were collected from patients during retinal detachment surgery. Specific ELISA analysis was performed with specific IgG against human PEDF. Results: PEDF was detected in the subretinal fluid of all cases. The mean concentration of PEDF was 33.9 ng/ml (SD 23.7 ng/ml; range 5.3–74.7 ng/ml). The majority of samples had however a concentration of more than 22 ng PEDF/ml fluid. Conclusion: PEDF appears to be a constant component of the fluid accumulating in the subretinal space after retinal detachment. The known effects of PEDF, however, suggest that it may be involved in physiological processes of wound healing in the subretinal space.

Vitreous and aqueous concentrations of proangiogenic, antiangiogenic factors and other cytokines in diabetic retinopathy patients with macular edema: Implications for structural differences in macular profiles

The aim of the study was to determine anatomical and growth factor profiles in patients with clinically significant macular oedema (CSMO) undergoing pars plana vitrectomy (PPV). Twenty patients with moderate nonproliferative diabetic retinopathy (NPDR) with persistent CSMO underwent PPV. Patients had baseline and postoperative clinical assessment including Ocular Coherence Tomography (OCT). Baseline vitreous and aqueous and serial postoperative aqueous samples were analysed for vascular endothelial growth factor-A (VEGF-A), pigment epithelium derived Factor (PEDF) and other factors (pg/ml) including hepatocyte growth factor, MMP 9, soluble flt-1 Receptor, and TGF β1 by ELISA. Vitreous from patients with full thickness macular holes (8) and proliferative diabetic retinopathy (22) were collected for comparison as controls. Vitreous VEGF-A concentration in the NPDR group was 957 pg/ml compared to 239 pg/ml in the macula hole (FTMH) control ( p<0.0001) and 596 pg/ml compared to PDR ( p=0.006). The median diabetic vitreous PEDF concentration was 1.36 μg/ml (FTMH 2.6 μg/ml p=0.05). In NPDR, it was higher (1.59 μg/ml) than PDR (1.27 μg/ml) p=0.02. There were changes to the HGF, soluble flt-1 Receptor and TGF b1 concentrations in the NPDR compared to either PDR or the normal state. In CSMO, two OCT profiles were identified: dome-shaped macular elevation (Group 1) ( n=4) and diffuse-low elevation profile (Group 2) ( n=16) which also showed differences in the postoperative median aqueous VEGF concentrations despite macular volume decreasing for both. The results suggest that there is an up-regulation of VEGF in the vitreous of the diabetic eye with a reciprocal decrease in PEDF. The structural and molecular differences between the two OCT macular profiles may explain the varying response to PPV in patients with diffuse CSMO.

Low Levels of Pigment Epithelium-Derived Factor in Highly Myopic Eyes With Chorioretinal Atrophy

To determine the concentration of pigment epithelium-derived factor (PEDF) in the aqueous humor of highly myopic eyes. Observational case series. The PEDF concentration in the aqueous humor of 23 eyes of 17 patients with high myopia (axial length >26 mm) who underwent cataract surgery was measured by enzyme-linked immunosorbent assay. The mean concentration of PEDF in eyes with high myopia (0.54 +/- 0.12 microg/ml) was significantly lower than that in control eyes (0.86 +/- 0.04 microg/ml, P = .0022). The PEDF level in myopic eyes with chorioretinal atrophy (0.32 +/- 0.05 microg/ml) was lower than that in myopic eyes without chorioretinal atrophy (0.71 +/- 0.12 microg/ml; P = .041) and control eyes (P = .0003). The significantly lower concentration of PEDF in eyes with chorioretinal atrophy-associated high myopia probably resulted from degeneration of the retinal pigment epithelial cells and/or the retinal ganglion cells that are the main sources of PEDF in the eye.

Effect of pigment epithelium derived factor on microvascular cells

Objectives: Angiogenesis is a key process of tumor growth and metastasis. Recently, pigment epithelium derived factor (PEDF) is known as a potent antiangiogenic factor. We examined the expression of PEDF in human endothelial cells and pericytes as microvascular cells. Further we examined the effects of exogenous PEDF on endothelial cell and pericyte proliferation. Methods: Pericytes utilized in this study were human retinal capillary pericytes. Endothelial cells were human umbilical vein endothelial cells (HUVEC). The mRNA expression of PDGF and c-fos was examined by RT-PCR. Signal transduction assays were done with Western blot. Cell proliferation was evaluated by DNA synthesis (BrdU assay). Results: Pericytes expressed the high levels of PDGF mRNA while the expression of PEDF in endothelial cells was almost undetectable. PEDF stimulates pericyte proliferation at both low (0.1–1.0 μg/mL) and high (50 μg/mL) doses. The stimulation by PEDF also activated MAPK and CREB and induced early response gene c-fos mRNA expression. In contrast, the effects of exogenous PEDF on endothelial cells were dose-dependent. PEDF increased cell proliferation at the low doses (0.1–1.0 μg/ml) while cell proliferation was significantly decreased with high (50 μg/ml) dose of PEDF. More than 80% of cells treated with the high dose of PEDF were TUNEL positive, suggesting that endothelial cells undergo apoptosis under the high concentration of PEDF. Conclusion: Pericyte is an important source of PEDF. In physiological condition, PEDF enhances pericyte viability and prevent endothelial cell proliferation. In the environment with low levels of PEDF, endothelial cell proliferation can be easily induced.

Suppression of corneal neovascularization by PEDF release from human amniotic membranes.

Human amniotic membrane (HAM) transplantation is commonly used in corneal surface reconstruction and is known to inhibit neovascularization of this tissue. The purpose of the present study is to reveal the molecular basis underlying antiangiogenic activity of HAM. The effects of HAM protein on proliferation of vascular endothelial cells and corneal epithelial cells were determined by quantifying viable cells using the MTT assay. The presence of pigment epithelium-derived factor (PEDF) in HAM was demonstrated at the protein level by Western blot analysis and immunohistochemistry using a monoclonal antibody specific to human PEDF. The PEDF concentration was measured by a specific ELISA. The expression of PEDF in HAM was confirmed at the RNA level by RT-PCR and DNA sequencing. Soluble proteins from HAM inhibited proliferation of human umbilical vein endothelial cells and bovine retinal capillary endothelial cells (BRCECs) while promoting proliferation of bovine cornea epithelial cells. Moreover, the HAM-induced inhibition of BRCECs was neutralized by a specific anti-PEDF antibody. PEDF protein was identified with an abundance of 103.84 +/- 33.21 ng/mg of soluble proteins, which is comparable to that in the retina, a PEDF-rich tissue. PEDF expression was predominantly localized in the basement membrane of HAM. RT-PCR using specific PEDF primers amplified a single product from HAM RNA. The PCR product has a sequence identical with that of human PEDF. HAM specifically inhibits endothelial cell growth and thus suppresses neovascularization in the cornea. PEDF in HAM has a major role in eliciting this antiangiogenic activity.

Decreased levels of pigment Epithelium–derived factor in eyes with neuroretinal dystrophic diseases

PurposeTo determine the concentration of pigment epithelium–derived factor (PEDF) in the aqueous humor of eyes with neuroretinal dystrophy. DesignObservational case series. MethodsAqueous humor was obtained from patients during cataract surgery, and the PEDF concentration in the aqueous humor was measured by enzyme-linked immunosorbent assay. The primary diagnosis was cataract in 162 eyes; of these there were five eyes with retinitis pigmentosa, nine eyes with advanced glaucoma, and 148 eyes with cataract alone. ResultsThe mean levels of PEDF in eyes with retinitis pigmentosa (0.24 ± 0.04 μg/ml, mean ± SE, P = .0004) and advanced glaucoma (0.46 ± 0.08 μg/ml, P = .003) were significantly lower than that in eyes with cataract alone (0.86 ± 0.04 μg/ml). ConclusionThe lower levels of PEDF in eyes with neuroretinal dystrophy may be related to the loss of the retinal ganglion cells or retinal pigment epithelium cells that synthesize PEDF.

Decrease of pigment epithelium-derived factor in aqueous humor with increasing age

Purpose To determine whether the level of pigment epithelium-derived factor (PEDF) in the aqueous humor is altered with age. Design Observational case series. Methods The PEDF concentration in the aqueous humor was measured by enzyme-linked immunosorbent assay in 152 eyes of 121 patients who underwent cataract surgery. Results The mean aqueous level of PEDF was 0.86 ± 0.04 μg/ml (mean ± standard error, 70.7 ± 1.0 years). The PEDF levels decreased with increasing age, and the decrease was significantly correlated with age (Pearson product moment correlation coefficient, r = − 0.22, P = .006). The mean PEDF level in the men (0.76 ± 0.06 μg/ml, 53 eyes, 71.5 ± 2.0 years) was significantly lower than that in women (0.91 ± 0.04 μg/ml, 99 eyes, 70.2 ± 1.1 year, P = .03). Conclusions The negative correlation of PEDF level and age should be considered in age-related eye diseases, especially those associated with angiogenesis.

Antiangiogenic property of pigment epithelium-derived factor in hepatocellular carcinoma.

Pigment epithelium-derived factor (PEDF) is one of the most powerful endogenous antiangiogenic reagents discovered to date. Its antiangiogenic potential in neoplastic disease remains unclear. In this study, we investigated antiangiogenic property of PEDF in hepatocellular carcinoma (HCC), a typical hypervascular tumor. In HCC cell lines, constitutive messenger RNA and protein expression of PEDF varied. Genomic DNA encoding the PEDF gene was the same in the cell lines examined by Southern blotting. In chemically induced hypoxic conditions, secreted PEDF protein was suppressed in contrast to elevation of vascular endothelial growth factor protein. When PEDF was overexpressed by gene transfer, proliferation and migration of endothelial cells were inhibited in conditioned media derived from all HCC cell lines. However, the serum concentration of PEDF, as measured by enzyme-linked immunosorbent assay, was decreased in patients with cirrhosis or HCC complicated by cirrhosis compared to healthy volunteers and patients with chronic hepatitis. According to the endothelial cell proliferation assay, the serum PEDF of patients with HCC had antiangiogenic activity. Moreover, intratumoral injection of a PEDF-expressing plasmid in athymic mouse models caused significant inhibition of preestablished tumor growth. In conclusion, PEDF plays a role in the angiogenic properties of HCC. Reduction of serum PEDF concentration associated with the development of chronic liver diseases may contribute to the progression of HCC. In addition, gene therapy using PEDF may provide an efficient treatment for HCC.

Pigment epithelium-derived factor in the monkey retinal pigment epithelium and interphotoreceptor matrix: apical secretion and distribution

Pigment epithelium-derived factor (PEDF) is an extracellular protein derived from the retinal pigment epithelium (RPE), a tissue formed by polarized cells that release growth and trophic factors in a directional fashion. We have investigated the distribution and directional release of PEDF protein by the monkey RPE. We established primary cultures of monkey RPE cells that expressed the PEDF gene, and that synthesized and secreted the PEDF protein. Northern analysis of RPE cultures and monkey ocular tissues showed that PEDF transcripts were highly expressed in RPE as compared with several other monkey ocular tissues, being even more abundant in cultured cells than they were in the native RPE. The differentiated RPE cells in culture secreted protein that shared the immunological, biochemical and biological characteristics of PEDF. The overall PEDF levels in the RPE conditioned media reached 6·5 mg ml − after 8 days in culture (i.e. 1·1 pg of PEDF per RPE cell). RPE cells were cultivated on permeable supports as monolayers forming a barrier between apical and basal compartments. Apical and basal culture media were sampled at three or four-day intervals for 18 cycles, and the PEDF content was quantified. Most of the PEDF protein was significantly higher in the apical than in the basal medium (>4 times) at the initial recovery intervals, to be detected only in the apical medium at the latter intervals. In the native monkey eye, the concentration of soluble PEDF in the interphotoreceptor matrix (144 n m) was 7-fold and 25-fold greater than in vitreous and aqueous, respectively. PEDF was abundant in the interphotoreceptor matrix surrounding rod and cone outer segments, and was detectable at lower levels in the RPE as visualized by confocal microscopy. We concluded that PEDF synthesized by the RPE is secreted preferentially from the apical surface and is distributed apically to the RPE bordering the outer segments of photoreceptors. PEDF can be a useful marker for RPE polarization and differentiation. The polarization of RPE may be an important mechanism to control PEDF secretion and our results offer interesting possibilities on regulation of PEDF.

Unbalanced vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor in diabetic retinopathy

PURPOSE: To determine the levels of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in the vitreous of patients with diabetic retinopathy (DR). DESIGN: Experimental study of PEDF and VEGF levels in vitreous samples collected during vitrectomy. METHODS: The levels of PEDF and VEGF were measured by enzyme-linked immunosorbent assay in the vitreous of 46 eyes of 43 patients who underwent vitrectomy with diabetic retinopathy (DR) (32 eyes of 29 patients) and an idiopathic macular hole (MH) (14 eyes of 14 patients). RESULTS: The vitreal concentration of PEDF was significantly lower at 1.11 ± 0.14 μg/ml (mean ± standard error) in eyes with DR than in eyes with MH at 1.71 ± 0.22 μg/ml ( P = .021). The VEGF level was 1799 ± 478 pg/ml in eyes with DR and not detectable in MH. The PEDF level in proliferative DR (PDR) (0.94 ± 0.12 μg/ml) was lower than that in nonproliferative DR (NPDR) (2.25 ± 0.32 μg/ml), and that in active DR (0.85 ± 0.14 μg/ml) was significantly lower than that in inactive DR (1.59 ± 0.24 μg/ml; P = .01). The VEGF level was 2025 ± 533 pg/ml in PDR and 215 ± 201 pg/ml in NPDR and that in active DR (2543 ± 673 pg/ml) was significantly higher than that in inactive DR (395 ± 188 pg/ml; P = .0098). CONCLUSIONS: These results suggest that lower levels of PEDF and higher levels of VEGF may be related to the angiogenesis in DR that leads to active PDR.

Inverse levels of pigment epithelium-derived factor and vascular endothelial growth factor in the vitreous of eyes with rhegmatogenous retinal detachment and proliferative vitreoretinopathy

PURPOSE: To determine the vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor in eyes with rhegmatogenous retinal detachment and proliferative vitreoretinopathy. DESIGN: Prospective, noncomparative case series. METHODS: Pigment epithelium-derived factor and vascular endothelial growth factor concentrations were measured by enzyme-linked immunosorbent assay in 26 eyes with retinal detachment, 6 with proliferative vitreoretinopathy, and 14 with an idiopathic macular hole. RESULTS: Pigment epithelium-derived factor concentration in proliferative vitreoretinopathy (0.57 ± 0.05 μg/ml) was lower ( P = .0069), and retinal detachment (2.37 ± 0.34 μg/ml) was higher ( P = .16) than that in macular hole (1.71 ± 0.22 μg/ml). Vascular endothelial growth factor concentration (168 ± 139 μg/ml) in proliferative vitreoretinopathy was significantly higher than that in retinal detachment (11 ± 11 μg/ml, P = .0084) and macular hole (not detectable, P = .0095). CONCLUSION: Lower levels of pigment epithelium-derived factor and higher levels of vascular endothelial growth factor may be related to ocular cell proliferation.

Loss of the antiangiogenic pigment epithelium-derived factor in patients with angiogenic eye disease.

Retinal neovascularization characterizes proliferative diabetic retinopathy (PDR). Pigment epithelium-derived factor (PEDF) has been shown to be a major antiangiogenic growth factor in the mammalian eye. PEDF expression is suppressed by hypoxia, and changes in PEDF have been correlated to the development of retinal neovascularization in animal models of hypoxic eye disease. However, whether this concept of a reduced angiogenesis inhibitor holds true in humans is as yet unclear. In this study, we analyzed the in vivo regulation of PEDF in patients with and without hypoxic eye disease. We used immunoblots to measure PEDF in ocular fluids obtained from 64 nondiabetic and diabetic patients. In addition, immunohistochemistry of PEDF was carried out in specimens of normal human retinas and retinas with various degrees of diabetic retinopathy. The PEDF concentrations in patients with PDR (P < 0.001) or extensive nondiabetic retinal neovascularization caused by retinal-vein occlusion (P < 0.001) were lower than in control patients. Levels of PEDF were replenished in PDR patients with previous retinal scatter photocoagulation compared with PDR patients without previous photocoagulation (P = 0.01). Immunohistochemistry revealed an interstitial staining pattern as expected for a secreted protein, with an intense staining in retinas of patients without proliferative eye disease. However, in patients with PDR, little or no staining was detectable. Our data strongly support the concept that retinal angiogenesis is induced by loss of the major angiogenesis inhibitor in the eye, PEDF, in combination with an increased expression of angiogenic growth factors such as vascular endothelial growth factor. Our findings suggest that substitution of angiogenesis inhibitors may be an effective approach in the treatment of PDR.

Pigment epithelium-derived factor in the vitreous is low in diabetic retinopathy and high in rhegmatogenous retinal detachment

PURPOSE: To report the levels of pigment epithelium-derived factor in the vitreous of patients with diabetic retinopathy, rhegmatogenous retinal detachment, and idiopathic macular hole. METHODS: Using enzyme-linked immunosorbent assay, we measured the levels of pigment epithelium-derived factor in the vitreous of 34 eyes of 33 patients who underwent vitrectomy for the treatment of diabetic retinopathy (17 eyes of 16 patients), rhegmatogenous retinal detachment (10 eyes), and idiopathic macular hole (seven eyes). RESULTS: The vitreal concentration of pigment epithelium-derived factor was 1.15 ± 0.23 μg/ml (mean ± standard error) in eyes with diabetic retinopathy, 3.28 ± 0.69 μg/ml in rhegmatogenous retinal detachment, and 1.71 ± 0.39 μg/ml in idiopathic macular hole. The pigment epithelium-derived factor level in rhegmatogenous retinal detachment was significantly higher than that in diabetic retinopathy ( P = .0008) and idiopathic macular hole ( P = .034). For eyes with diabetic retinopathy, the pigment epithelium-derived factor level was 0.88 ± 0.21 μg/ml in proliferative diabetic retinopathy and 2.43 ± 0.37 μg/ml in nonproliferative diabetic retinopathy ( P = .0083). Additionally, the pigment epithelium-derived factor level in active diabetic retinopathy (0.70 ± 0.22 μg/ml) was significantly lower than the level in inactive diabetic retinopathy (1.79 ± 0.35 μg/ml; P = .018). CONCLUSIONS: These results suggest that pigment epithelium-derived factor inhibits angiogenesis and that lower levels of pigment epithelium-derived factor may be related to the angiogenesis in diabetic retinopathy and result in active proliferative diabetic retinopathy. The results also suggest that higher levels of pigment epithelium-derived factor in the eyes with rhegmatogenous retinal detachment may act as a neuroprotective agent for the detached retina.