Pigment Epithelium-derived Factor Protein Expression Research Articles

Pigment epithelium-derived factor modulates periodontal homeostasis in mice and induces osteogenic differentiation of human periodontal ligament fibroblasts

ABSTRACT Aim The aim of this study was to investigate the influence of pigment epithelium-derived factor (PEDF) on periodontal homeostasis in mice and the osteogenic differentiation of human periodontal ligament fibroblasts (PDLFs). Materials and Methods Micro-computed tomography and histology were performed to compare the alveolar bone volume, density, and bone-related markers between PEDF-deficient (PEDF−/−) and wild-type (WT) mice. Furthermore, after recombinant human PEDF treatment, the PDLF viability and osteogenic differentiation were examined using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) activity assay, Von Kossa staining, Alizarin red staining, real-time quantitative polymerase chain reaction (qRT-PCR), and immunoblotting. Results The alveolar bone volume and density of PEDF−/− mice were significantly lower than those of the WT mice. Higher receptor activator for nuclear factor-κB ligand (RANKL) expression and lower osteoprotegerin (OPG) expression levels were observed in the PEDF−/− group. Moreover, PEDF treatment did not affect the PDLF proliferation. PEDF dose-dependently improved mineral deposition. Compared with the control group, 250 ng/mL PEDF promoted OPG mRNA expression in PDLFs on Day 3 but inhibited RANKL, Wnt5a, GSK3b mRNA, and non-phosphorylated β-catenin protein expression. However, 250 ng/mL PEDF had no significant effect on the expression of Wnt3a. On Day 7, after culture with 250 ng/mL PEDF in osteogenic medium, the ALP and RUNX2 protein levels were upregulated. VEGF protein expression was reduced in a dose-dependent manner after PEDF stimulation. The PEDF protein expression increased as the osteogenic induction time increased. Conclusion PEDF gene knockout suppresses periodontal homeostasis in mice, and PEDF treatment induces PDLF osteogenic differentiation in vitro.

Upregulated pigment epithelium-derived factor (PEDF) promotes trophoblast apoptosis and inhibits invasion in preeclampsia

Preeclampsia (PE) is a severe pregnancy-specific disorder. Previous findings indicated that pigment epithelium-derived factor (PEDF) was upregulated in placentas of women with PE. Here, we investigated the role of PEDF in trophoblast function, especially under hypoxia. The effects of hypoxia on the morphology of extravillous trophoblast (EVT)-derived HTR-8Svneo cells were observed under inverted microscope. Transfections with Lipofectamine LTX were performed according to the manufacturer's protocol. The expression of PEDF protein and mRNA were confirmed by immunofluorescence (IF) and quantitative real-time PCR (qPCR). Apoptosis was detected by transferase-mediated dUTP nick end labeling (TUNEL) assay, and proliferation of trophoblast was detected by CCK-8 method. The invasion capacity of trophoblast was assessed by Transwell assay. PEDF was expressed in HTR-8/SVneo under both normoxia and hypoxic stress. However, cells of hypoxia groups had higher expression level of PEDF, increased apoptosis and decreased invasion capability, as compared with normoxia group. Moreover, after transfection with plasmid expressing PEDF gene, overexpression of PEDF modulated trophoblast activities. In addition, PEDF expression was negatively associated with invasion while positively correlated with apoptosis.Our data suggest that PEDF is an important factor to maintain the biological function of trophoblast cells, thus representing a rational therapeutic target in PE.

PEDF relieves kidney injury in type 2 diabetic nephropathy mice by reducing macrophage infiltration.

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with anti-angiogenic, antioxidant and anti-inflammatory properties. PEDF is involved in the pathogenesis of diabetic retinopathy, but its exact role in diabetic kidneys remains unclear. P78-PEDF is an active peptide sequence consisting of 44 amino acids with biological activity similar to that of PEDF. The present study aimed to investigate whether PEDF can alleviate renal damage in type 2 diabetic nephropathy mice by inhibiting macrophage infiltration. The db/db mice were randomly divided into a diabetes PEDF intervention group (DM-P78-PEDF), a diabetes empty carrier intervention group (DM-Vehicle), and a diabetes mellitus group (DM). Subsequently, they were injected subcutaneously P78-PEDF (0.3 μg/g/d) and PBS for 6 weeks. The ratio of kidney weight to body weight was observed in the mice. An automatic biochemical analyser was used to determine fasting blood glucose (GLU), blood urea nitrogen (UREA), serum creatinine (CREA), and haemoglobin (Hb) content. Histological and ultrastructural pathological changes in the kidneys were examined through H&E and PAS staining. Kidney tissue levels of interleukin-1β (IL-1β), interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) were determined by ELISA. Expression of the macrophage infiltration and typing as well as that of PEDF, NF-kB, and TLR4 was evaluated in the kidneys. PEDF was located in glomeruli, and the expression of PEDF protein and mRNA in the kidney of diabetic mice declined significantly. Compared with diabetic mice treated with vehicle, continuous infusion of P78-PEDF could reduce blood urea nitrogen, serum creatinine (CREA), renal macrophage recruitment, inflammatory cytokines, and histological changes and restore the expression of TLR4/NF-κB signalling pathway-related factors in diabetic mice. These findings highlight the importance of P78-PEDF peptide as a potential treatment in the occurrence and development of diabetic renal injury.

Preclinical Evaluation of a Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System in Choroidal Neovascularization

Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats, and transfected primary cells (transfected RPE [tRPE] and transfected IPE [tIPE] cells) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinases 2 and 9 (MMP-2/9), and microglial/macrophage markers were measured. Injection with tRPE/IPE cells significantly reduced the leakage area at 7 and 14 days and the CNV area at 7 days. There was a significant increase in PEDF and the PEDF/VEGF ratio with tRPE cells and a reduction in the MMP-2 activity. Our data demonstrated that ex vivo non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.

Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System Reduces Neovascularisation in Rats

Background: Age-related macular degeneration (AMD) is a progressive retinal disorder, characterised by imbalanced pro- and anti-angiogenic signals, leading vision loss. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy on primary cells with stable expression of human pigment epithelium derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Methods: Rat retinal pigment epithelial cells (RPEs) and iris pigment epithelial cells (IPEs) were co-transfected with pFAR4-ITRs CMV PEDF BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats and transfected primary cells (tRPE and tIPE) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinase 2 and 9 (MMP-2/9) and microglial/macrophage markers were measured and evaluated. Findings: The animals injected with tRPE/IPE cells significantly showed a reduction in the leakage area at 14 days while at 7 days only tIPE cells showed a significant reduction. The CNV area at 7 days was reduced with tRPE/IPE cells. There was a significant increase in PEDF and PEDF/VEGF ratio with tRPE cells along with a reduction in the MMP-2 activity. Activated microglial fluorescence intensity increased in all groups analyzed. Interpretation: Our data demonstrate that a single administration of SB100X-engineered RPE/IPE cells release enough hPEDF to favour an anti-angiogenic environment that ultimately reduces CNV suggest that ex vivo non-viral gene therapy could be an effective and safe therapeutic option for angiogenic retinal diseases. Funding: This work was supported by the European Union’s Seventh Framework Programme for research, technological development and demonstration, grant agreement no. 305134 and Fundacion Jesus Gangoiti Barrera. LGG received a predoctoral grant from the Asociacion de Amigos de la Universidad de Navarra. Declaration of Interest: A. Garcia-Layana is consultant in Thea, Allergan, Bayer, Novartis and Roche Laboratories. Z. Izsvak and Z. Ivics are co-inventors on several patents on Sleeping Beauty transposon technology. Ethical Approval: The study was performed according to the Association for Research in Vision and Ophthalmology (ARVO) Resolution on the Use of Animals in Ophthalmic and Vision Research and approved by the Ethics Committee for Animal Research of the University of Navarra (protocol approval number 023-13).

Dan Huang Ming Mu Recipe Suppresses the Progression of Streptozotocin-induced Diabetic Retinopathy After Retinal Laser Photocoagulation in Brown Norway Rats via Down-regulating Vascular Endothelial Growth Factor and Up-regulating Pigment Epithelium-derived Factor

ObjectiveTo analyze the effects of Dan Huang Ming Mu Recipe (DHMMR) (a pharmaceutical preparation from herbs and having the function of replenishing vital essence, removing heat, promoting blood circulation and excreting pathogenic water) on diabetic retinopathy (DR) after retinal laser photocoagulation through regulating the expression of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). MethodsForty male Brown Norway (BN) rats were randomly divided into blank group (group A, 10 BN rats) and model group (30 BN rats). DR models were induced by 40 mg/kg streptozotocin (STZ) and the body weight and blood glucose of rats were monitored. After 12-week injection, fluorescein fundus angiography (FFA) and histopathological examination were detected to confirm the successful establishment of DR models. Subsequently, the right eyes of model group rats were conducted retinal laser photocoagulation with the left eyes having no retinal laser photocoagulation and rats of the model group were randomly divided into group B (the model control group), group C (the positive control group), and group D (the DHMMR group), with 10 rats in each group. Rats of the group A and the group B were given vehicle, and the group C were given calcium dobesilate suspension by gavage, and the group D were given DHMMR by gavage. After 4-week gavage, FFA was carried out to observe the fundus microvascular change. Then, the rats were sacrificed to do histopathological examinations and the serum levels of P-selectin, cAMP, cGMP, and the relative expression of the protein of VEGF and PEDF in retina were detected. ResultsAfter 12-week STZ injection, the blood glucose of the model group were pronouncedly higher than the blank group (P < 0.01). Fundus micro-hemangioma changes were observed in the rats of the model group through FFA, and the rats of the blank group did not see any changes. The pictures of HE staining showed that the retinal structure of the model group was more disordered than the blank group. After 4-week gavage, treatments with DHMMR showed dramatic reduction of serum levels of P- selectin, cAMP and cGMP compared with the group B (P < 0.01). FFA and histopathological examinations of DHMMR-treated rats revealed significantly suppression of retinal edema, fundus microvascular destruction and retinal destruction distinguishing from the group B. And the relative expression of VEGF protein of the group D and the group A was markedly lower than that of the group B (P < 0.01). The relative expression of PEDF protein of the group D and the group A was dramatically higher than that of the group B to the contrary (P < 0.01). ConclusionsDHMMR demonstrates pronounced suppressive effects on the progression of DR after retinal laser photocoagulation, through down-regulating VEGF and up- regulating PEDF.

Clinicopathological Features and Prognosis of Papillary Thyroid Microcarcinoma for Surgery and Relationships with the BRAFV600E Mutational Status and Expression of Angiogenic Factors.

ObjectiveTo investigate the clinicopathological characteristics of papillary thyroid microcarcinoma (PTMC) for surgery by comparing the difference between PTMC and larger papillary thyroid carcinoma (LPTC).MethodsWe analyzed the differences in the clinicopathological characteristics, prognosis, B-type RAF kinase (BRAF)V600E mutational status and expression of angiogenic factors, including pigment epithelium-derived factor (PEDF), Vascular Endothelial Growth Factor (VEGF), and hypoxia-inducible factor alpha subunit (HIF-1α), between PTMC and LPTC by retrospectively reviewing the records of 251 patients with papillary thyroid carcinoma, 169 with PTMC, and 82 with LPTC (diameter >1 cm).ResultsThere were no significant differences in the gender, age, multifocality, Hashimoto’s thyroiditis, TNM stage, PEDF protein expression, rate of recurrence, or mean follow-up duration between patients with PTMC or LPTC. The prevalence of extrathyroidal invasion (EI), lymph node metastasis (LNM), and BRAF mutation in patients with PTMC was significantly lower than in patients with LPTC. In addition, in PTMC patients with EI and/or LNM and/or positive BRAF (high-risk PTMC patients), the prevalence of extrathyroidal invasion, Hashimoto's disease, lymph node metastasis, tumor TNM stage, PEDF positive protein expression, the rate of recurrent disease, and the mRNA expression of anti-angiogenic factors was almost as high as in patients with larger PTC, but with no significant difference.ConclusionsExtrathyroid invasion, lymph node metastases, and BRAFV600E mutation were the high risk factors of PTMC. PTMC should be considered for the same treatment strategy as LPTC when any of these factors is found. Particularly, PTMC with BRAFV600E gene mutations needed earlier surgical treatment. In addition, the high cell subtype of PTMC with BRAFV600E gene mutation is recommended for total thyroidectomy in primary surgery to reduce the risk of recurrence.

Chronic intermittent hypoxia increases encoding pigment epithelium-derived factor gene expression, although not that of the protein itself, in the temporal cortex of rats.

Objective: Obstructive sleep apnea syndrome is mainly characterized by intermittent hypoxia (IH) during sleep, being associated with several complications. Exposure to IH is the most widely used animal model of sleep apnea, short-term IH exposure resulting in cognitive and neuronal impairment. Pigment epithelium-derived factor (PEDF) is a hypoxia-sensitive factor acting as a neurotrophic, neuroprotective, and antiangiogenic agent. Our study analyzed performance on learning and cognitive tasks, as well as PEDF gene expression and PEDF protein expression in specific brain structures, in rats exposed to long-term IH. Methods: Male Wistar rats were exposed to IH (oxygen concentrations of 21-5%) for 6 weeks-the chronic IH (CIH) group-or normoxia for 6 weeks-the control group. After CIH exposure, a group of rats were allowed to recover under normoxic conditions for 2 weeks (the CIH+N group). All rats underwent the Morris water maze test for learning and memory, PEDF gene expression and PEDF protein expression in the hippocampus, frontal cortex, and temporal cortex being subsequently assessed. Results: The CIH and CIH+N groups showed increased PEDF gene expression in the temporal cortex, PEDF protein expression remaining unaltered. PEDF gene expression and PEDF protein expression remained unaltered in the frontal cortex and hippocampus. Long-term exposure to IH did not affect cognitive function. Conclusions: Long-term exposure to IH selectively increases PEDF gene expression at the transcriptional level, although only in the temporal cortex. This increase is probably a protective mechanism against IH-induced injury.

Synergistic antitumor effect of recombinant adeno-associated virus-mediated pigment epithelium-derived factor with hyperthermia on solid tumor.

Adeno-associated virus (AAV) is an ideal choice for gene delivery; however, its further development has been limited owing to its low transduction efficiency. DNA-damaging agents can improve AAV-mediated transgene expression. Hyperthermia, as one of the oldest documented tumor treatment modalities, can cause DNA damage as well. However, combined treatment consisting of hyperthermia and AAV-mediated gene therapy has not been reported yet. In this work we investigated whether therapy consisting of AAV-mediated pigment epithelium-derived factor (PEDF) delivery combined with hyperthermia has synergistic antitumor effect on established solid tumors. We produced the recombinant AAV encoding PEDF (rAAV-PEDF). The therapeutic effect of rAAV-PEDF plus hyperthermia was evaluated in a subcutaneous fibrosarcoma mouse model, and the possible mechanism of antitumor effect was investigated. We found that rAAV-PEDF could infect a murine fibrosarcoma cell line (Meth-A) and express PEDF protein with bioactivity in vitro. In addition, in vivo experiments suggested that the combination of rAAV-PEDF with hyperthermia could significantly suppress tumor growth and prolong survival time of treated mice. Immunofluorescence studies indicated that the combination therapy could inhibit angiogenesis and induce apoptosis in tumor tissues. An immunohistochemistry assay of tumor tissue showed that PEDF expression in the combined treatment group was significantly higher than in the rAAV-PEDF group, which implied that hyperthermia could improve the expression of PEDF protein in vivo. No significant differences were observed in each group by hematoxylin-eosin staining of major organs, serum chemistry test, and complete blood assay. These results indicate that the combination of rAAV-PEDF with hyperthermia has synergistic therapeutic effects on established solid tumors, with no side effects. In addition, hyperthermia could improve AAV-mediated transgene expression, which suggests that hyperthermia could serve as a promising adjunctive therapy for AAV-mediated gene therapy.

Abstract 744: PEDF silencing a novel mechanism of antihormone resistance in breast cancer

Abstract Despite the benefits of endocrine therapies in treating estrogen receptor alpha (ERα)-positive breast cancer, many tumors eventually become resistant. Identifying the underlying cellular and molecular mechanisms responsible for endocrine resistance remains a critical and immediate need. Pigment epithelium-derived factor (PEDF) is a multifunctional protein released by adipocytes and increased in metabolic syndrome and insulin-resistance. Moreover, PEDF belongs to the serine protease inhibitor family but does not inhibit proteases. It is a potent endogenous inhibitor of angiogenesis and a promising agent for diabetic retinopathy and potentially cancer. Previous studies have shown that PEDF expression is significantly reduced in breast cancer cells (compared to normal breast epithelium) and its reduction is associated with disease differentiation (lowest levels in poorly differentiated tumors), disease progression and poor patient outcome, however, it is not known whether PEDF plays a role in endocrine resistance. In the present study, we evaluated PEDF expression in endocrine-sensitive MCF-7, T47D, and ZR-75-1 breast cancer cells versus endocrine resistant MCF-7:5C, MCF-7:2A, and BT474 cells and found that PEDF mRNA and protein levels were dramatically reduced in the latter group of cells. In addition, tissue microarray analysis of primary tumors from patients (N=66) who subsequently developed invasive recurrence after adjuvant tamoxifen treatment revealed that expression of PEDF protein was reduced by 45% and loss of PEDF was associated with enhanced expression of phosphoSer167ERα and the receptor tyrosine kinase RET (Rearranged during Transfection). Importantly, we found that silencing endogenous PEDF in tamoxifen-sensitive MCF-7 and T47D breast cancer cells abolished their sensitivity to the inhibitory effects of tamoxifen whereas reexpression of PEDF in endocrine resistant MCF-7:5C and MCF-7:2A cells restored their sensitivity to tamoxifen and raloxifene. Furthermore, we found that reexpression of PEDF in endocrine-resistant cells significantly reduced the levels of RET, cyclin D1 and phospho-AKT, in these cells. Together, these findings suggest that PEDF silencing might be a novel mechanism for the development of endocrine resistance in breast cancer and that PEDF expression could be used as a predictive marker of endocrine sensitivity. Furthermore, these findings suggest that recombinant PEDF might be beneficial in treating patients with antihormone resistant disease with potential application in ER-negative disease. This work was supported by the NIH Career Development Grant K01CA120051 01A2 and the Hollenbach Family Fund (JLW). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 744. doi:10.1158/1538-7445.AM2011-744

OX-LDL Up-Regulates the Vascular Endothelial Growth Factor-to-Pigment Epithelium-Derived Factor Ratio in Human Retinal Pigment Epithelial Cells

Native and oxidized (OX) low-density lipoprotein (LDL) may contribute to the pathogenesis of age related macular degeneration (AMD). In this study, we investigated the effects of lipoproteins, including LDL and ox-LDL, on cell viability, apoptosis, and vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) expression in cultured human retinal pigment epithelial (RPE) cells. ARPE-19 cells were incubated with 10-100 mg/ml n-LDL, ox-LDL for 24 hr. Cell viability was assessed using the Cell Titer 96 Aqueous One Solution cell proliferation assay. The apoptosis of RPE was measured with TUNEL. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the levels of VEGF and PEDF mRNA in RPE cells. The expression of VEGF and PEDF protein was measured by western blotting. To examine the role of MAPK signal transduction in LDL- and OX-LDL-induced VEGF and PEDF protein expression, ARPE-19 cells were pretreated with one of several MAPK inhibitors for 2 hr and then incubated with native LDL or OX-LDL for 24 hr. One-way analysis of variance was used to compare the differences. OX-LDL treatment decreased ARPE-19 cell viability in a dose-dependent manner, whereas native LDL had no effect. Incubation of ARPE-19 cells with 10 mg/mL OX-LDL induced marked apoptosis, compared with untreated control cells. OX-LDL also increased VEGF expression and decreased PEDF expression, whereas native LDL had no significant effect. The VEGF-to-PEDF ratio was elevated after OX-LDL treatment. OX-LDL-induced VEGF protein synthesis was partly abolished by inhibiting p38 and JNK, while inhibiting ERK did not show a significant effect. OX-LDL treatment induced cellular changes in ARPE-19 cells that appeared to reflect pathogenic events in neovascular AMD, providing potential insight into the roles of OX-LDL in human RPE cells and its potential role in the pathogenesis of AMD.

Pigment epithelium-derived factor (PEDF) is one of the most abundant proteins secreted by human adipocytes and induces insulin resistance and inflammatory signaling in muscle and fat cells

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic and anti-angiogenic properties. More recently it became evident that PEDF is upregulated in patients with type 2 diabetes and also contributes to insulin resistance in mice. During characterization of the secretome of in vitro differentiated human adipocytes by two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-MS, we found that PEDF is one of the most abundant proteins released by adipocytes. The aim of this study was to investigate the regulation and autocrine function of PEDF in human adipocytes and to determine its paracrine effects on human skeletal muscle cells (hSkMC) and human smooth muscle cells (hSMC). Human primary adipocytes secrete 130 ng ml(-1) PEDF over 24 h from 1 million cells, which is extremely high as compared with adiponectin, interleukin-6 (IL-6) or IL-8. This release of PEDF is significantly higher than from other primary cells, such as adipose-tissue located macrophages (50-times), hSkMC and hSMC (5-times). PEDF protein expression significantly increases during adipogenesis, which is paralleled by increased PEDF secretion. Furthermore, tumor necrosis factor-α and hypoxia significantly downregulate PEDF protein levels. PEDF secretion was significantly reduced by troglitazone and hypoxia and significantly increased by insulin. Treatment of adipocytes and hSkMC with PEDF induced insulin resistance in adipocytes, skeletal and smooth muscle cells at the level of insulin-stimulated Akt phosphorylation, which was dose dependent and more prominent in adipocytes. Furthermore, inflammatory nuclear factor-κB (NF-κB) signaling was induced by PEDF. In hSMC, PEDF induced proliferation (1.7-fold) and acutely activated proliferative and inflammatory signaling pathways (NF-κB, p38 mitogen-activated protein kinase and mammalian target of rapamycin). PEDF is one of the most abundant adipokines and its secretion is inversely regulated by insulin and hypoxia. PEDF induces insulin resistance in adipocytes and hSkMC and leads to inflammatory signaling in hSMC. Because of these diverse actions, PEDF is a key adipokine, which could have an important role in diabetes and obesity-related disorders.

Pigment Epithelium-Derived Factor Stimulates Tumor Macrophage Recruitment and Is Downregulated by the Prostate Tumor Microenvironment

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis but whether it has additional effects on the tumor microenvironment is largely unexplored. We show that overexpression of PEDF in orthotopic MatLyLu rat prostate tumors increased tumor macrophage recruitment. The fraction of macrophages expressing inducible nitric oxide synthase, a marker of cytotoxic M1 macrophages, was increased, suggesting that PEDF could enhance antitumor immunity. In addition, PEDF overexpression reduced vascular growth both in the tumor and in the surrounding normal tissue, slowed tumor growth, and decreased lymph node metastasis. Contrary, extratumoral lymphangiogenesis was increased. PEDF expression is, for reasons unknown, often decreased or lost during prostate tumor progression. When AT-1 rat prostate tumor cells, expressing high levels of PEDF messenger RNA (mRNA) and protein, were injected into the prostate, PEDF is markedly downregulated, suggesting that factors in the microenvironment suppressed its expression. One such factor could be macrophage-derived tumor necrosis factor α (TNFα). A fraction of the accumulating macrophages expressed TNFα, and TNFα treatment downregulated the expression of PEDF protein and mRNA in prostate AT-1 tumor cells in vitro and in the rat ventral prostate in vivo. PEDF apparently has multiple effects in prostate tumors: it suppresses angiogenesis and metastasis, but it also causes macrophage accumulation. Accumulating macrophages may inhibit tumor growth, but they may also suppress PEDF and enhance lymph angiogenesis and, in this way, eventually enhance tumor growth.

Expression of pigment epithelium-derived factor in human melanocytes and malignant melanoma cells and tissues

Pigment epithelium-derived factor (PEDF) was first isolated from the medium conditioned by human fetal retinal pigment epithelial cells and has been detected in a broad range of human fetal and adult tissues. Recent studies have indicated that PEDF activity is inhibitory to angiogenesis. To study the expression and distribution of pigment epithelium-derived factor (PEDF) in human melanocytes, malignant melanoma cells and tissues. PEDF was expressed in human melanocytes. The expression of PEDF protein diminished in the following orders healthy skin, pigmented nevus and human malignant melanoma (p < 0.001). Both the expression of PEDF mRNA and protein was much lower or almost absent in the malignant melanoma cell line A375 than that in human melanocytes (p < 0.001). The expression and distribution of PEDF in human healthy skin, pigmented nevus and malignant melanoma were studied. The expression of PEDF mRNA in human melanocytes and malignant melanoma cell line A375 was measured by reverse transcription polymerase chain reaction (RT-PCR) and PEDF protein was detected by immunohistochemical method and Western blotting analysis. The lack of PEDF expression may contribute to the pathogenesis of malignant melanoma.

Increased Expression of Pigment Epithelium–Derived Factor in Allergic Rhinitis

To investigate the expression of messenger RNA (mRNA) of the gene for pigment epithelium-derived factor (PEDF) (OMIM *172860) and PEDF protein and to localize the PEDF protein in the nasal mucosa of patients with allergic rhinitis and of control subjects. Investigation of PEDF mRNA and PEDF protein expression in the nasal mucosa using reverse transcription-polymerase chain reaction, Western blotting, and immunohistochemical staining. We used inferior turbinate mucosal samples from 10 patients with allergic rhinitis and 10 matched healthy control subjects. We extracted PEDF mRNA from the inferior turbinate mucosa samples and performed reverse transcription-polymerase chain reaction analysis. We used Western blotting to analyze differences in expression levels of PEDF protein between patients with allergic rhinitis and healthy controls, and the PEDF protein was localized immunohistochemically. The expression levels of PEDF mRNA and PEDF protein in the nasal mucosa were significantly increased in patients with allergic rhinitis compared with those in nonallergic controls. The PEDF protein was expressed in the epithelium and submucosal glands. We found that PEDF protein is expressed in the human nasal mucosa, and its expression is increased in allergic rhinitis. These results suggest a possible contribution of PEDF to the chronic inflammation of the nasal mucosa in allergic rhinitis.

Pigment epithelium-derived factor expression in the retina in response to hypobaric hypoxia

The present study was undertaken to examine the expression of pigment epithelium-derived factor (PEDF) in the retina of adult rat following hypobaric hypoxic exposures. Forty-eight adult Wistar rats were divided into 6 groups randomly (8 rats in each group), of which 5 groups (40 rats) were subjected to hypobaric hypoxia for 2 hours following which expression of PEDF in the retina was investigated from 3 hours to 14 days by real time RT-PCR, western blotting and immunohistochemistry. PEDF mRNA and protein expression in the retina was decreased at 3 hours to 14 days following exposure to hypoxia. Compared with normal control group (1.00+/-0.00), PEDF mRNA expression decreased to 0.83+/-0.05 at 3 hours, 0.72+/-0.04 at 24 hours, 0.56+/-0.04 at 3 days, 0.54+/-0.05 at 7 days and 0.49+/-0.05 at 14 days respectively; Compared with normal control group (1.90+/-0.02), PEDF protein expression decreased to 1.49+/-0.02 at 3 hours, 1.19+/-0.05 at 24 hours, 1.15+/-0.05 at 3 days, 1.09+/-0.06 at 7 days and 1.07+/-0.03 at 14 days respectively. The findings of this study indicate hypoxia can downregulate PEDF in adult retina.

Hydrogen Peroxide Stimulates Pigment Epithelium-derived Factor Gene and Protein Expression in the Human Hepatocyte Cell Line OUMS-29

Pigment epithelium-derived factor (PEDF) may have a protective role in atherosclerosis and is associated with the presence of components of the metabolic syndrome. Since oxidative stress has been postulated to play an important role in the pathogenesis of vascular injury in the metabolic syndrome, this study investigated the effects of hydrogen peroxide (H2O2) on PEDF in the immortalized human hepatocyte cell line OUMS-29. PEDF gene expression was measured using quantitative real-time reverse transcription-polymerase chain reaction and PEDF protein expression was analysed by Western blot. H2O2 upregulated PEDF mRNA levels and increased PEDF protein production in OUMS-29 cells in time- and dose-dependent manners. The anti-oxidant N-acetylcysteine significantly blocked H2O2-induced PEDF overexpression in OUMS-29 cells. These results suggest that hepatic PEDF levels may be elevated to counteract the effects of oxidative stress. H2O2-induced PEDF overproduction in the liver may act as a negative feedback system against vascular damage in the metabolic syndrome.

Pigment Epithelium-Derived Factor Is Estrogen Sensitive and Inhibits the Growth of Human Ovarian Cancer and Ovarian Surface Epithelial Cells

Epithelial ovarian carcinoma is the most lethal gynecological cancer. However, little is known about the molecular mechanisms underlying the disease development and progression. In this study, we found that the expression of pigment epithelium-derived factor (PEDF) was greatly reduced in ovarian tumors and in ovarian cancer cell lines when compared with their normal precursor, ovarian surface epithelium (OSE). In addition, we showed that exogenous PEDF inhibited the growth of cultured human OSE as well as ovarian cancer cell lines, whereas targeted inhibition of endogenous PEDF using small interfering RNA or neutralizing PEDF antibody promoted the growth of these cells, confirming that the growth-inhibitory effect was PEDF specific. We also report for the first time that estrogen is an important upstream regulator of PEDF in human OSE. Treatment of the cultured cells with 17 beta-estradiol (E2) inhibited the expression of PEDF protein and mRNA in a dose- and time-dependent manner, which could be reversed by the specific estrogen receptor antagonist, ICI 182,780, indicating that the regulation was estrogen receptor-mediated. We further showed that this down-regulation of PEDF gene transcription was a direct, primary effect of E2. E2 promoted OSE and ovarian cancer cell growth, whereas simultaneous treatment with E2 and PEDF abrogated the estrogenic growth stimulation of these cells. This study is the first to demonstrate a role of PEDF in OSE biology and ovarian cancer and suggests that the loss of PEDF may e of relevance in carcinogenesis.

Pigment Epithelium-derived Factor as a New Diagnostic Marker for Melanocytic Tumors

Pigment epithelium-derived factor (PEDF), a potent antiangiogenetic factor, has been lately known to correlate well with angiogenic and metastatic potentials of tumor cells. We investigated the expression of PEDF protein in various types of human tumor cells by an immunohistochemical technique using a monoclonal antibody. Consequently, we found the significantly frequent and intense expression of PEDF in human melanocytic tumor cells including malignant melanoma as compared to non-melanocytic ones. We evaluated the diagnostic usefulness of anti-PEDF antibody in melanocytic tumors by comparing its immunoreactive sensitivity with that of other conventional melanocytic markers such as S-100 protein, HMB-45 and Melan-A, and found that PEDF possess the equal ability to others on its sensitivity. We finally concluded that PEDF is a useful diagnostic marker for melanocytic tumors, especially malignant melanomas, by its use in combination with other markers.

Survival Effects of Pigment Epithelium-Derived Factor Expressed by a Lentiviral Vector in Rat Cerebellar Granule Cells

We have previously shown that pigment epithelium-derived factor (PEDF) acts as a survival factor for cerebellar granule cells (CGCs), by blocking apoptotic death, and can also protect these cells against glutamate-induced neurotoxicity. In preparation for gene therapy studies, pseudotyped HIV-1-based lentiviral vectors containing the PEDF gene, as well as either green fluorescent protein or β-galactosidase, were prepared. These bicistronic vectors are unique in that they express two genes efficiently under one promoter. Primary cell cultures of CGCs from postnatal day 8 rats were infected with the vectors encoding PEDF. RT-PCR demonstrated expression of mRNA and Western blot analysis confirmed that infected CGCs secrete PEDF protein to the medium. Assays for cell survival demonstrated that PEDF-infected cells were significantly more protected compared with mock-infected controls for 6–8 days in culture, as well as against induced apoptosis. The PEDF vectors expressing tat (trans-acting transcription factor) provided more protection than the tat(–) vectors. These results demonstrate that while the lentiviral vectors expressing PEDF are as neuroprotective as the protein itself for CGCs, the vectors have the advantage of providing long-lasting expression of PEDF protein, which will be more effective in in vivo studies. The present results suggest that this system may be useful for gene therapy for neurodegenerative disorders.