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PEDF and VEGF-A Output from Human Retinal Pigment Epithelial Cells Grown on Novel Microcarriers
Torsten Falk,Nicole R. Congrove,Shiling Zhang,Alexander D. McCourt,Scott J. Sherman,Brian S. McKay
Journal of Biomedicine and Biotechnology , 2012, DOI: 10.1155/2012/278932
Abstract: Human retinal pigment epithelial (hRPE) cells have been tested as a cell-based therapy for Parkinson’s disease but will require additional study before further clinical trials can be planned. We now show that the long-term survival and neurotrophic potential of hRPE cells can be enhanced by the use of FDA-approved plastic-based microcarriers compared to a gelatin-based microcarrier as used in failed clinical trials. The hRPE cells grown on these plastic-based microcarriers display several important characteristics of hRPE found in vivo: (1) characteristic morphological features, (2) accumulation of melanin pigment, and (3) high levels of production of the neurotrophic factors pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor-A (VEGF-A). Growth of hRPE cells on plastic-based microcarriers led to sustained levels (>1 ng/ml) of PEDF and VEGF-A in conditioned media for two months. We also show that the expression of VEGF-A and PEDF is reciprocally regulated by activation of the GPR143 pathway. GPR143 is activated by L-DOPA (1 μM) which decreased VEGF-A secretion as opposed to the previously reported increase in PEDF secretion. The hRPE microcarriers are therefore novel candidate delivery systems for achieving long-term delivery of the neuroprotective factors PEDF and VEGF-A, which could have a value in neurodegenerative conditions such as Parkinson’s disease.
Pigment Epithelium-Derived Factor (PEDF) Interacts with Transportin SR2, and Active Nuclear Import Is Facilitated by a Novel Nuclear Localization Motif  [PDF]
Sergio Anguissola, William J. McCormack, Michelle A. Morrin, Wayne J. Higgins, Denise M. Fox, D. Margaret Worrall
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0026234
Abstract: PEDF (Pigment epithelium-derived factor) is a non-inhibitory member of the serpin gene family (serpinF1) that displays neurotrophic and anti-angiogenic properties. PEDF contains a secretion signal sequence, but although originally regarded as a secreted extracellular protein, endogenous PEDF is found in the cytoplasm and nucleus of several mammalian cell types. In this study we employed a yeast two-hybrid interaction trap screen to identify transportin-SR2, a member of the importin-β family of nuclear transport karyopherins, as a putative PEDF binding partner. The interaction was supported in vitro by GST-pulldown and co-immunoprecipitation. Following transfection of HEK293 cells with GFP-tagged PEDF the protein was predominantly localised to the nucleus, suggesting that active import of PEDF occurs. A motif (YxxYRVRS) shared by PEDF and the unrelated transportin-SR2 substrate, RNA binding motif protein 4b, was identified and we investigated its potential as a nuclear localization signal (NLS) sequence. Site-directed mutagenesis of this helix A motif in PEDF resulted in a GFP-tagged mutant protein being excluded from the nucleus, and mutation of two arginine residues (R67, R69) was sufficient to abolish nuclear import and PEDF interaction with transportin-SR2. These results suggest a novel NLS and mechanism for serpinF1 nuclear import, which may be critical for anti-angiogenic and neurotrophic function.
Pigment Epithelium-Derived Factor (PEDF) Expression Induced by EGFRvIII Promotes Self-renewal and Tumor Progression of Glioma Stem Cells  [PDF]
Jinlong Yin?,Gunwoo Park?,Tae Hoon Kim?,Jun Hee Hong?,Youn-Jae Kim?,Xiong Jin?,Sangjo Kang?,Ji-Eun Jung?,Jeong-Yub Kim?,Hyeongsun Yun
PLOS Biology , 2015, DOI: 10.1371/journal.pbio.1002152
Abstract: Epidermal growth factor receptor variant III (EGFRvIII) has been associated with glioma stemness, but the direct molecular mechanism linking the two is largely unknown. Here, we show that EGFRvIII induces the expression and secretion of pigment epithelium-derived factor (PEDF) via activation of signal transducer and activator of transcription 3 (STAT3), thereby promoting self-renewal and tumor progression of glioma stem cells (GSCs). Mechanistically, PEDF sustained GSC self-renewal by Notch1 cleavage, and the generated intracellular domain of Notch1 (NICD) induced the expression of Sox2 through interaction with its promoter region. Furthermore, a subpopulation with high levels of PEDF was capable of infiltration along corpus callosum. Inhibition of PEDF diminished GSC self-renewal and increased survival of orthotopic tumor-bearing mice. Together, these data indicate the novel role of PEDF as a key regulator of GSC and suggest clinical implications.
Inhibition of Corneal Neovascularization with the Combination of Bevacizumab and Plasmid Pigment Epithelium-Derived Factor-Synthetic Amphiphile INTeraction-18 (p-PEDF-SAINT-18) Vector in a Rat Corneal Experimental Angiogenesis Model  [PDF]
Chien-Neng Kuo,Chung-Yi Chen,San-Ni Chen,Lin-Cheng Yang,Li-Ju Lai,Chien-Hsiung Lai,Miao-Fen Chen,Chia-Hui Hung,Ching-Hsein Chen
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms14048291
Abstract: Bevacizumab, a 149-kDa protein, is a recombinant humanized monoclonal antibody to VEGF. PEDF, a 50-kDa glycoprotein, has demonstrated anti-vasopermeability properties. In this study, we demonstrated that the combination of bevacizumab and plasmid pigment epithelium-derived factor-synthetic amphiphile INTeraction-18 (p-PEDF-SAINT-18) has a favorable antiangiogenic effect on corneal NV. Four groups (Group A: 0 μg + 0 μg, B: 0.1 μg + 0.1 μg, C: 1 μg + 1 μg, and D: 10 μg + 10 μg) of bevacizumab + p-PEDF-SAINT-18 were prepared and implanted into the rat subconjunctival substantia propria 1.5 mm from the limbus on the temporal side. Then, 1 μg of p-bFGF-SAINT-18 was prepared and implanted into the rat corneal stroma 1.5 mm from the limbus on the same side. The inhibition of NV was observed and quantified from days 1 to 60. Biomicroscopic examination, western blot analysis and immunohistochemistry were used to analyze the 18-kDa bFGF, 50-kDa PEDF and VEGF protein expression. No inhibition activity for normal limbal vessels was noted. Subconjunctival injection with the combination of bevacizumab and p-PEDF-SAINT-18 successfully inhibited corneal NV. The bFGF and PEDF genes were successfully expressed as shown by western blot analysis, and a mild immune response to HLA-DR was shown by immunohistochemistry. We concluded that the combination of bevacizumab and p-PEDF-SAINT-18 may have more potent and prolonged antiangiogenic effects, making it possible to reduce the frequency of subconjunctival.Bevacizumab, a 149-kDa protein, is a recombinant humanized monoclonalantibody to VEGF. PEDF, a 50-kDa glycoprotein, has demonstrated anti-vasopermeabilityproperties. In this study, we demonstrated that the combination of bevacizumaband plasmid pigment epithelium-derived factor-synthetic amphiphile INTeraction-18(p-PEDF-SAINT-18) has a favorable antiangiogenic effect on corneal NV. Four groups(Group A: 0 μg + 0 μg, B: 0.1 μg + 0.1 μg, C: 1 μg + 1 μg, and D: 10 μg + 10 μg) ofbevacizumab + p-PEDF-SAINT-18 were prepared and implanted into the ratsubconjunctival substantia propria 1.5 mm from the limbus on the temporal side. Then, 1 μgof p-bFGF-SAINT-18 was prepared and implanted into the rat corneal stroma 1.5 mm fromthe limbus on the same side. The inhibition of NV was observed and quantified from days1 to 60. Biomicroscopic examination, western blot analysis and immunohistochemistry wereused to analyze the 18-kDa bFGF, 50-kDa PEDF and VEGF protein expression. Noinhibition activity for normal limbal vessels was noted. Subconjunctival injection with thecombination of
Aqueous Humor Levels of Pigment Epithelium-Derived Factor and Oscillatory Potentials in Diabetic Retinopathy Patients

臧晶, 管国奇, 王文娟, 鲍炯琳, 陈立伦
Hans Journal of Ophthalmology (HJO) , 2016, DOI: 10.12677/HJO.2016.51005
目的:探讨房水中色素上皮衍生因子(pigment epithelium derived factor, PEDF)与视网膜电图振荡电位(oscillatory potentials, OPs)在各期糖尿病性视网膜病变中的相关性研究分析,为糖尿病视网膜病变(diabetic retinopathy, DR)的早期诊断及病程进展预测提供客观的检测依据。方法:房水来自2型糖尿病患者40例40眼。单纯2型糖尿病患者组19例19只眼,非增殖期糖尿病性视网膜病变(non-proliferative diabetic retinopathy, NPDR)组患者11例11只眼,增殖期糖尿病视网膜病变(proliferative diabetic retinopathy, PDR)组患者10例10只眼,单纯老年性白内障患者20例20只眼作为对照组。用酶联免疫吸附法检測房水中PEDF的水平,用德国 ROLAND CONSULT公司生产的罗兰视觉电生理检查仪检测全视网膜电图中OPs的总振幅值。结果:单纯2型糖尿病患者组、NPDR组及PDR组的房水中PEDF的浓度分别为(9.13 ± 4.43) ng/ml、(7.00 ± 2.04) ng/ml、(6.39 ± 1.66) ng/ml与对照组(13.87 ± 0.36) ng/ml相比,房水中PEDF浓度均明显降低(P < 0.01),单纯2型糖尿病患者组与NPDR组及PDR组相比,房水中PEDF浓度亦均明显降低(P = 0.04, P = 0.02),而NPDR组与PDR组相比无明显差异(P = 0.34);单纯2型糖尿病患者组、NPDR组及PDR组的OPs的总振幅值分别为(164.56 ± 37.41) μV、(79.80 ± 19.89) μV、(28.55 ± 17.47) μV与对照组(237.96 ± 35.96) μV相比,OPs的总振幅值均明显降低(P < 0.01),而且组间两两比较亦有明显差异(P < 0.01);各期糖尿病性视网膜病变房水中PEDF浓度变化与OPs的总振幅值呈典型的正相关(r = 0.905, P < 0.01)。结论:房水中PEDF的浓度与OPs总波幅在各期糖尿病性视网膜病变中呈正相关,可为糖尿病视网膜病变的早期诊断及病程进展预测提供客观的检测依据。
Objective: To investigate the relationship between pigment epithelium derived factor (PEDF) and oscillatory potentials (OPs) in various diabetic retinopathy and to provide an objective basis for the early diagnosis of diabetic retinopathy (diabetic retinopathy, DR) and the prediction of the progression of the disease. Methods: Aqueous humor samples were collected from 40 eyes of 40 type 2 diabetes patients, including 19 eyes without diabetic retinopathy and 21 eyes with diabetic (11 eyes with NPDR and 10 eyes with PDR), twenty aqueous samples from 20 cataract patients without other ocular or systemic diseases served as controls. The PEDF levels in aqueous humor were measured by enzyme-linked immunosorbent assay (ELISA). The total amplitude of OPs in the whole retina was detected by the Roland visual electrophysiological test instrument of the German CONSULT ROLAND company. Results: The PEDF and OPs level in eyes without diabetic retinopathy [(9.13 ± 4.43) ng/ml, 164.56 ± 37.41) μV, P < 0.01] was significantly lower than that in controls [(13.87 ± 0.36) ng/ml, (237.96 ± 35.96) μV]; the PEDF Ievel in eyes with NPDR [(7.00 ± 2.04) ng/ml, (79.80 ± 19.89) μV, P < 0.01] and PDR [(6.39 ± 1.66) ng/ml, (28.55 ± 17.47) μV, P < 0.01] was sig-nificantly lower than that in controls [(13.87 ± 0.36) ng/ml, (237.96 ± 35.96) μV]; the concentration of PEDF in the patients with type 2 diabetes mellitus was significantly lower than that in the NPDR and PDR group (P = 0.04, P = 0.02), while there was no significant
The Emerging Role of PEDF in Stem Cell Biology
Mina Elahy,Swati Baindur-Hudson,Crispin R. Dass
Journal of Biomedicine and Biotechnology , 2012, DOI: 10.1155/2012/239091
Abstract: Encoded by a single gene, PEDF is a 50 kDa glycoprotein that is highly conserved and is widely expressed among many tissues. Most secreted PEDF deposits within the extracellular matrix, with cell-type-specific functions. While traditionally PEDF is known as a strong antiangiogenic factor, more recently, as this paper highlights, PEDF has been linked with stem cell biology, and there is now accumulating evidence demonstrating the effects of PEDF in a variety of stem cells, mainly in supporting stem cell survival and maintaining multipotency.
Suppressive Effect of Hydroquinone, a Benzene Metabolite, on In Vitro Inflammatory Responses Mediated by Macrophages, Monocytes, and Lymphocytes  [PDF]
Jae Youl Cho
Mediators of Inflammation , 2008, DOI: 10.1155/2008/298010
Abstract: We investigated the inhibitory effects of hydroquinone on cytokine release, phagocytosis, NO production, ROS generation, cell-cell/cell fibronectin adhesion, and lymphocyte proliferation. We found that hydroquinone suppressed the production of proinflammatory cytokines [tumor necrosis factor (TNF)-, interleukin (IL)-1, and IL-6], secretion of toxic molecules [nitric oxide (NO) and reactive oxygen species (ROS)], phagocytic uptake of FITC-labeled dextran, upregulation of costimulatory molecules, U937 cell-cell adhesion induced by CD18 and CD29, and the proliferation of lymphocytes from the bone marrow and spleen. Considering that (1) environmental chemical stressors reduce the immune response of chronic cigarette smokers and children against bacterial and viral infections and that (2) workers in petroleum factories are at higher risk for cancer, our data suggest that hydroquinone might pathologically inhibit inflammatory responses mediated by monocytes, macrophages, and lymphocytes.
Nicotine Promotes Proliferation of Human Nasopharyngeal Carcinoma Cells by Regulating α7AChR, ERK, HIF-1α and VEGF/PEDF Signaling  [PDF]
Dingbo Shi, Wei Guo, Wangbin Chen, Lingyi Fu, Jingshu Wang, Yung Tian, Xiangsheng Xiao, Tiebang Kang, Wenlin Huang, Wuguo Deng
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043898
Abstract: Nicotine, the major component in cigarette smoke, can promote tumor growth and angiogenesis, but the precise mechanisms involved remain largely unknown. Here, we investigated the mechanism of action of nicotine in human nasopharyngeal carcinoma (NPC) cells. Nicotine significantly promoted cell proliferation in a dose and time-dependent manner in human NPC cells. The mechanism studies showed that the observed stimulation of proliferation was accompanied by the nicotine-mediated simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling. Treatment of NPC cells with nicotine markedly upregulated the expression of α7AChR and HIF-1α proteins. Transfection with a α7AChR or HIF-1α-specific siRNA or a α7AChR-selective inhibitor significantly attenuated the nicotine-mediated promotion of NPC cell proliferation. Nicotine also promoted the phosphorylation of ERK1/2 but not JNK and p38 proteins, thereby induced the activation of ERK/MAPK signaling pathway. Pretreatment with an ERK-selective inhibitor effectively reduced the nicotine-induced proliferation of NPC cells. Moreover, nicotine upregulated the expression of VEGF but suppressed the expression of PEDF at mRNA and protein levels, leading to a significant increase of the ratio of VEGF/PEDF in NPC cells. Pretreatment with a α7AChR or ERK-selective inhibitor or transfection with a HIF-1α-specific siRNA in NPC cells significantly inhibited the nicotine-induced HIF-1α expression and VEGF/PEDF ratio. These results therefore indicate that nicotine promotes proliferation of human NPC cells in vitro through simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling and suggest that the related molecules such as HIF-1α might be the potential therapeutic targets for tobacco-associated diseases such as nasopharyngeal carcinomas.
Codon Preference Optimization Increases Heterologous PEDF Expression  [PDF]
Anzor G. Gvritishvili,Kar Wah Leung,Joyce Tombran-Tink
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015056
Abstract: Pigment epithelium-derived factor (PEDF) is widely known for its neurotrophic and antiangiogenic functions. Efficacy studies of PEDF in animal models are limited because of poor heterologous protein yields. Here, we redesigned the human PEDF gene to preferentially match codon frequencies of E coli without altering the amino acid sequence. Following de novo synthesis, codon optimized PEDF (coPEDF) and the wtPEDF genes were cloned into pET32a containing a 5′ thioredoxin sequence (Trx) and the recombinant Trx-coPEDF or Trx-wtPEDF fusion constructs expressed in native and two tRNA augmented E coli hosts - BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP, carrying extra copies of tRNAarg,ile,leu and tRNAarg,pro genes , respectively. Trx-PEDF fusion proteins were isolated using Ni-NTA metal affinity chromatography and PEDF purified after cleavage with factor Xα. Protein purity and identity were confirmed by western blot, MALDI-TOF, and UV/CD spectral analyses. Expression of the synthetic gene was ~3.4 fold greater (212.7 mg/g; 62.1 mg/g wet cells) and purified yields ~4 fold greater (41.1 mg/g; 11.3 mg/g wet cell) than wtPEDF in the native host. A small increase in expression of both genes was observed in hosts supplemented with rare tRNA genes compared to the native host but expression of coPEDF was ~3 fold greater than wtPEDF in both native and codon-bias-adjusted E coli strains. ΔGs at ?3 to +50 of the Trx site of both fusion genes were ?3.9 kcal/mol. Functionally, coPEDF was equally as effective as wtPEDF in reducing oxidative stress, promoting neurite outgrowth, and blocking endothelial tube formation. These findings suggest that while rare tRNA augmentation and mRNA folding energies can significantly contribute to increased protein expression, preferred codon usage, in this case, is advantageous to translational efficiency of biologically active PEDF in E coli. This strategy will undoubtedly fast forward studies to validate therapeutic utility of PEDF in vivo.
PEDF in Diabetic Retinopathy: A Protective Effect of Oxidative Stress
Xiao-feng Zhu,Hai-dong Zou
Journal of Biomedicine and Biotechnology , 2012, DOI: 10.1155/2012/580687
Abstract: Diabetic retinopathy (DR) is a major cause of blindness in working age adults, and oxidative stress plays a vital role in the pathogenesis of DR. Pigment-epithelium-derived factor (PEDF), a multifunctional protein, has shown to inhibit the development of DR by accumulating evidence. This paper highlights the current understanding of probable mechanism about how PEDF blocks the deterioration of DR through its antioxidative properties and application prospects of PEDF as a novel therapeutic target in DR. Gene therapy of PEDF is becoming more and more acceptable and will widely be applied to the actual treatment in the near future.
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