Pigment epithelium-derived factor inhibits glioma cell growth in vitro and in vivo

Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that pigment epithelium-derived factor (PEDF) can induce differentiation and inhibit angiogenesis of several tumors. This study was designed to determine whether gliomas angiogenesis and tumor growth could be inhibited by PEDF. We found that PEDF down-regulated expression levels of vascular endothelial growth factor and up-regulated the expression of thrombospondin-2 and augmented apoptosis in a dose-dependent manner in both A172 and U87 glioma cells lines after 48 h of treatment. Analysis of the cell cycle showed arrest in the G₁ phase and block in S phase of the cell cycle. Meanwhile PEDF induced apoptosis was associated with increases of p53 and Bax and inhibition of Bcl-2. Conditioned medium with PEDF showed a significantly reductive effect on migration in vitro accompanied with a significant reduction of matrix metalloproteinase-9 expression. PEDF suppressed glioma cell migration in vitro and tumor burden in athymic nude mice. These results demonstrate for the first time inhibitory effects of PEDF on the growth and migration of human gliomas via induction of apoptosis and blocking of migratory-related factors. PEDF activation can be a novel approach for future therapeutic purposes against gliomas.     

Pigment epithelium-derived factor is elevated in CSF of patients with amyotrophic lateral sclerosis

Pigment epithelium-derived factor (PEDF), a recently defined retinal trophic factor and anti-angiogenic factor for the eye, is also present in the CNS and is a motor neuron protectant. We asked whether PEDF levels in CSF are altered in patients with amyotrophic lateral sclerosis (ALS). Pigment epithelium-derived factor protein was detected by quantitative western blot analysis with a PEDF-specific antiserum. Levels of PEDF in CSF, expressed as a ratio to total CSF protein, were significantly elevated 3.4-fold in 15 patients with ALS compared with neurologic disease controls (p < 0.0003). This increase does not seem likely to reflect up-regulation of PEDF synthesis in muscle in response to denervation, as CSF PEDF was not elevated in severe denervating diseases other than ALS. Nor does the increase represent some non-specific release in neurodegeneration, as CSF PEDF was not elevated in other neurodegenerative diseases. While the mechanism of this presumably reactive increase is not known, the distinctive, surprisingly elevated level of PEDF in the CSF may be an autoprotective reaction in ALS.     

Identification of differentially expressed proteins in Ficedula flycatchers

This is the first report on the large-scale identification and comparison of proteins in non-model organisms, Ficedula flycatchers. It highlights the potential of proteomics approaches in both non-sequenced and non-model organisms for identification of differentially expressed proteins. Not surprisingly, more than 55% of the proteins failed to be identified even though the MS spectra were of high quality. Nevertheless, the protein information obtained in this study will serve as a valuable resource for continued research.     

Isolation of Human Serum Alpha-2 HS Glycoprotein

Alpha-2 HS glycoprotein was obtained in an almost pure state from normal human serum, by ion exchange chromatography followed by curtain electrophoresis, gel filtration and ammonium sulfate precipitation. Alpha-2 HS glycoprotein has a sedimentation coefficient of 3.5 S, and an approximate molecular weight of 49,000 Dal tons. Purified α₂HS glycoprotein enhances phagocytosis and may be the human homologue of rat α₂ opsonic protein.     

Pigment epithelium derived factor regulates human Sost/Sclerostin and other osteocyte gene expression via the receptor and induction of Erk/GSK-3beta/beta-catenin signaling

Mutations in Serpinf1 gene which encodes pigment epithelium derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective mineralization. We reported that PEDF suppressed expression of Sost/Sclerostin and other osteocyte related genes in mineralizing osteoblast cultures and suggested that this could be part of the mechanisms by which PEDF regulates matrix mineralization (Li et al. J Cellular Phys. 2014). We have used a long-term differentiated mineralizing osteoblast culture (LTD) to define mechanisms by which PEDF regulates osteocyte gene expression. LTD cultures were established by culturing human osteoblasts in an osteogenic medium for 4?months followed by analysis of osteocytes related genes and encoded proteins. LTD cells synthesized Sclerostin, matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein (DMP-1) and their synthesis was reduced by treatment with PEDF. Treatment of the cultures with PEDF induced phosphorylation of Erk and glycogen synthase kinase 3-beta (GSK-3β), and accumulation of nonphosphorylated β-catenin. Inhibition of Erk activation and neutralizing antibodies to the pigment epithelium derived receptor (PEDF-R) suppressed GSK-3β phosphorylation and accumulation of nonphosphorylated β-catenin in presence of PEDF. Topflash assays demonstrated that PEDF activated luciferase reporter activity and this activity was inhibited by treatment with Erk inhibitor or neutralizing antibodies to PEDF-R. Dickkopf-related protein 1 treatment of the cells in presence of PEDF had minimal effect suggesting that GSK-3β phosphorylation and accumulation of nonphosphorylayted β-catenin may not involve LRP5/6 in osteocytes. Taken together, the data demonstrate that PEDF regulates osteocyte gene expression through its receptor and possible involvement of Erk/GSK-3β/β-catenin signaling pathway.     

Proteomic analysis of the cerebrospinal fluid of patients with lumbar disk herniation

To better understand the pathophysiologic mechanisms underlying spinal nerve root injury induced by lumbar disk herniation (LDH), comparative proteomic analysis of cerebrospinal fluid (CSF) between patients with LDH (the experiment group) and the otherwise healthy patients who had had implants removed from healed fractures in the lower limbs (the control group) was carried out using 2-DE followed by LC-IT-MS and database searching. Image analysis of silver-stained 2-DE gels revealed that 15 protein spots showed significant differential expression between the two groups of CSF samples (p < 0.05). After searching the database we found that in CSF of LDH patients, the expression of cystatin C, apolipoprotein A-IV, vitamin D-binding protein, neurofilament triplet L protein, IgG, tetranectin, and hemoglobin were elevated. However, ProSAAS, prostagladin D2 synthase, creatine kinase B, superoxide dismutase 1 and peroxiredoxin 2 were decreased. The subsequent ELISA measured the concentration of tetranectin, vitamin D-binding protein and cystatin C and confirmed the results of proteomic analysis. These identified proteins are involved in the pathophysiological process of spinal nerve root injury caused by herniated lumbar disk. The functional implications of the alterations in the levels of these proteins are discussed in this paper.     

钙离子和氯化钴促进成纤维细胞色素上皮衍生因子的表达

目的探讨色素上皮衍生因子(PEDF)在成纤维细胞中的表达以及钙离子和氯化钴对其表达的影响。方法 19例包皮标本根据年龄分为2组:13例40岁以下者为I组,6例40岁以上者为II组。以I型胶原酶分离真皮成纤维细胞,37oC5%CO2培养。以1.0mmol/L氯化钙和100μmol/L的氯化钴作用于培养的成纤维细胞。以免疫荧光法检测PEDF蛋白质的表达,以逆转录-聚合酶链反应(RT-PCR)检测PEDF mRNA的表达情况。结果 PEDF表达于正常成纤维细胞,而且在组I和组II间mRNA相对表达量无差异性(分别为,P>0.05)。1.0mmol/L氯化钙和100μmol/L氯化钴显著增加PEDF mRNA相对表达量,分别为2.3倍和1.4倍,二者差异具有显著性(P<0.05)。结论真皮成纤维细胞表达PEDF,而且受钙离子和氯化钴的调节。     

Optimising ovine cerebrospinal fluid preparation for two-dimensional gel electrophoresis

Biomarkers for neurodegenerative disorders are potentially present in cerebrospinal fluid (CSF) and can be detected using proteomic technologies. Since CSF is high in salt and low in protein, its study by proteomic methods requires appropriate sample preparation. In this study, we applied four different sample treatments to the same ovine CSF sample. Precipitation with acetone or using a 2-D Clean-Up Kit (GE Healthcare BioSciences, Little Chalfont, UK) preserved more proteins, and produced more gel spots than spin columns from Sigma and Bio-Rad. A 53-kDa spot, identified by MS/MS as transthyretin (TTR) tetramer, was not detected in samples treated with the 2-D Clean-Up Kit, though it was always present on all gels prepared using the other three methods. Western immunoblotting confirmed the low recovery of tetrameric TTR by the 2-D Clean-Up Kit and showed that the tetrameric form of TTR predominated in ovine but not in rat CSF. In one ovine CSF sample haemoglobin was found, indicating blood contamination. We conclude that acetone precipitation is a simple and efficient way to prepare ovine CSF for 2-DE. The use of the 2-D Clean-Up Kit leads to the disappearance of tetrameric TTR only from ovine CSF proteome.     

Osteoblasts and osteoclasts express PEDF, VEGF-A isoforms, and VEGF receptors: possible mediators of angiogenesis and matrix remodeling in the bone

Pigment epithelial derived factor (PEDF) is one of the most effective inhibitors of angiogenesis described so far, especially in controlling the growth of blood vessels in the eye. We now describe the localization of PEDF in regions of active bone formation in the mid-gestation mouse embryo and its specific and high levels of secretion by osteoblasts. PEDF is detected to a lesser extent in osteoclasts as well. The proangiogenic factors, VEGF and its receptors VEGF-R1 and VEGF-R2, are also expressed by both osteoblasts and osteoclasts. These findings suggest that bone angiogenesis and matrix remodeling may be mediated both by PEDF and by VEGF.     

Involvement of pigment epithelium-derived factor,docosahexaenoic acid and neuroprotectin D1 in corneal inflammation and nerve integrity after refractive surgery

Alterations in corneal innervations result in impaired corneal sensation, severe dry eye and damage to the epithelium that may in turn lead to corneal ulcers, melting and perforation. These alterations can occur after refractive surgery. We have discovered that pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA or the docosanoid bioactive neuroprotectin D1 (NPD1)) induces nerve regeneration after corneal surgery that damages the stromal nerves. We found that PEDF is released from corneal epithelial cells after injury, and when DHA is provided to the cells it stimulates the biosynthesis of NPD1 by an autocrine mechanism. The combination of PEDF plus DHA also decreased the production of leukotriene B4 (LTB4), a neutrophil chemotactic factor, thereby decreasing the inflammation induced after corneal damage. These studies suggest that PEDF plus DHA and its derivative NPD1 hold promise as a future treatment to restore a healthy cornea after nerve damage.     

Involvement of the collagen I-binding motif in the anti-angiogenic activity of pigment epithelium-derived factor

Pigment epithelium-derived factor (PEDF) is the most potent endogenous inhibitor of angiogenesis in age-related macular degeneration and tumors. However, the molecular mechanism of the anti-angiogenic activity of PEDF is poorly understood. PEDF interacts with the extracellular matrix (ECM) in vitro. Here, we investigated the possible involvement of the motif for ECM interaction in the anti-angiogenic activity of PEDF. The growth rates of HeLa cells in culture were not affected by transfection of PEDF, indicating that PEDF did not suppress tumor cell growth directly. In tumor xenografts, the overexpression of wild-type PEDF significantly suppressed tumor growth, whereas a mutant of the collagen I-binding site of PEDF (Col-mut PEDF) did not inhibit tumor growth. A mutant of the heparin-binding site of PEDF (Hep-mut PEDF) suppressed tumor growth. Histological analysis showed that the density and area of microvasculatures in either PEDF or Hep-mut PEDF were suppressed when compared with those in either vector or Col-mut PEDF. Our data indicate that PEDF inhibits tumor growth via its anti-angiogenic activity, and the collagen I-binding motif of PEDF is involved in the biological activity.     

D2-class dopamine receptor inhibition of NMDA currents in prefrontal cortical neurons is platelet-derived growth factor receptor-dependent

NMDA receptor function is modulated by both G-protein-coupled receptors and receptor tyrosine kinases. In acutely isolated rat hippocampal neurons, direct activation of the platelet-derived growth factor (PDGF) receptor or transactivation of the PDGF receptor by D4 dopamine receptors inhibits NMDA-evoked currents in a phospholipase C (PLC)-dependent manner. We have investigated further the ability of D2-class dopamine receptors to modulate NMDA-evoked currents in isolated rat prefrontal cortex (PFC). We have demonstrated that, similar to isolated hippocampal neurons, the application of PDGF-BB or quinpirole to isolated PFC neurons induces a slow-onset and long-lasting inhibition of NMDA-evoked currents. However, in contrast to hippocampal neurons, the inhibition of NMDA-evoked currents by quinpirole in PFC neurons is dependent upon D2/3, rather than D4, dopamine receptors. In PFC slices, application of both PDGF-BB and quinpirole induced a phosphorylation of the PDGF receptor at the PLCgamma binding and activation site, Tyr1021. The PDGF receptor kinase inhibitor, tyrphostin A9, and the D2/3 dopamine receptor antagonist, raclopride, inhibited quinpirole-induced Tyr1021 phosphorylation. These finding suggest that quinpirole treatment inhibits NMDAR signaling via PDGF receptor transactivation in both the hippocampus and the PFC, and that the effects of quinpirole in these regions are mediated by D4 and D2/3 dopamine receptors, respectively.     

Pigment epithelium-derived factor expression prolongs survival and enhances the cytotoxicity of low-dose chemotherapy in castration-refractory prostate cancer

There is currently no cure for advanced castration-refractory prostate cancer (CRPC) despite the recent approval of several new therapeutic agents. We report here the anti-tumor effect of the angio-inhibitory pigment epithelium-derived factor (PEDF) in the metastatic LNCaP-derivative CRPC CL1 model and explore PEDF anti-neoplasic efficacy in combination with low-dose chemotherapy. Androgen-sensitive LNCaP and CRPC PC3 cell lines were examined as comparison. Using a retroviral expression system, we showed that PEDF limited the proliferation of all prostatic cell lines tested; an effect attributed to interleukin 8 (IL8)-CXCR1/IL8RA inhibition. PEDF also reduced the number and size of 3D tumor spheroids in vitro, but only induced cell differentiation in CRPC spheroids. Similarly, PEDF inhibited the migration of CRPC cells suggesting both anti-proliferative and anti-migratory functions. In vivo, PEDF decreased by 85% and 65% the growth of subcutaneous (s.c.) PC3 and CL1 tumors, respectively. In the CL1 orthotopic model, tumor intake with lethal metastases was found in all animals; nevertheless, PEDF prolonged the median survival of tumor-bearing mice (95% confidence interval: 53±0.001 to 57±1 days). Accordingly, PEDF delayed the emergence of skeletal-related event in intra-tibial xenografts. Next, we evaluated low-dose docetaxel (DTX; 5, 1, 0.5 mg/kg) or cyclophosphamide (CTX; 10–20 mg/kg) on established s.c. PC3 tumors that conditionally express PEDF anti-tumoral epitope/NT3. Although NT3–DTX-5 mg/kg combination was inefficient, NT3–DTX-1 mg/kg and -0.5 mg/kg inhibited by 95% and 87.8%, respectively, tumor growth compared with control and induced tumor stasis. Both NT3–CTX combinations were advantageous. Inversely, PEDF–DTX-5 mg/kg and PEDF–CTX-10 mg/kg delayed the most CL1 tumor growth (15, 11 and 5 days for PEDF–DTX-5 mg/kg, PEDF–CTX-10 mg/kg and single treatments, respectively) with elevated apoptosis and serum thrombospondin-1 as possible mechanism and marker, respectively. As well, both PEDF–CTX-10 mg/kg and PEDF–DTX-5 mg/kg prolonged significantly the survival of tumor-bearing mice compared with single treatments. Metastases were reduced in PEDF–DTX-5 mg/kg compared with other treatments, suggesting that PEDF–DTX delayed metastases formation. Our results advocate that PEDF/low-dose chemotherapy may represent a new therapeutic alternative for CRPC.     

Identification of elicitor‐responsive proteins in rice leaves by a proteomic approach

Experiments were conducted to identify the differentially expressed proteins in rice (Oryza sativa L.) plants after treatment with the glycoprotein elicitor CSB I, purified from ZC₁₃, a race of the rice blast fungus Magnaporthe grisea. The interactions of two near isogenic lines of rice, C101A51 and CO39, with ZC₁₃ resulted in completely incompatible and compatible types, respectively. Proteins were extracted from rice leaves at 12 and 24 h after treatment with CSB I. Temporal changes in total proteins were examined using 2‐DE. Among more than 900 protein spots reproducibly detected on each gel, 11 were up‐regulated, three were down‐regulated and seven were newly induced during, at a minimum, one time point. Twenty‐one differentially expressed proteins were identified by linear ion trap quadrupole (LTQ)‐MS/MS. The identified proteins were classified into six categories based on their putative function reported: (i) defense proteins (PR‐10a, PR‐5 and putative salt‐induced protein), (ii) signal transduction (nucleoside diphosphate kinase and putative profilin), (iii) ROS (Mn‐SOD, Cu/Zn‐SOD, GST and CAT), (iv) programmed cell death (translationally controlled tumor protein), (v) molecule biosynthesis (putative ribosomal protein S5, putative ribosomal protein L12, putative translational elongation factor Tu and putative chaperonin 21 precursor) and (vi) metabolism (putative fructose‐bisphosphate aldolase class‐I, putative malate dehydrogenase, cytoplasmic malate dehydrogenase, putative acid phosphatase, putative transketolase1 and gamma hydroxybutyrate dehydrogenase‐like protein). All of these proteins (except Cu/Zn‐SOD, putative acid phosphatase and translationally controlled tumor protein) were induced faster and to a higher degree in C101A51 than in CO39. These data suggest that the incompatible rice line may possess a more sensitive recognition system that can identify and react to specific chemical, biological or physical triggers in a more efficient manner, thus eliciting an early and fast defense response.