Increased Pigment Epithelium-derived Factor Expression Research Articles

Transcription Factor EGR1 Facilitates Neovascularization in Mice with Retinopathy of Prematurity by Regulating the miR-182-5p/EFNA5 Axis.

Neovascularization is the hallmark of retinopathy of prematurity (ROP). Early growth response 1 (EGR1) has been reported as an angiogenic factor. This study was conducted to probe the regulatory mechanism of EGR1 in neovascularization in ROP model mice. The ROP mouse model was established, followed by determination of EGR1 expression and assessment of neovascularization [vascular endothelial growth factor-A (VEGF-A) and pigment epithelium-derived factor (PEDF)]. Retinal vascular endothelial cells were cultured and treated with hypoxia, followed by the tube formation assay. The state of oxygen induction was assessed by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assay to determine hypoxia-inducible factor 1-alpha (HIF-1A). The levels of microRNA (miRNA)-182-5p and ephrin-A5 (EFNA5) in tissues and cells were determined by RT-qPCR. Chromatin immunoprecipitation and dual-luciferase assay were used to validate gene interaction. EGR1 and EFNA5 were upregulated in the retina of ROP mice while miR-182-5p was downregulated. EGR1 knockdown decreased VEGF-A and HIF-1A expression and increased PEDF expression in the retina of ROP mice. In vitro, EGR1 knockdown also reduced neovascularization. EGR1 binding to the miR-182-5p promoter inhibited miR-182-5p transcription and further promoted EFNA5 transcription. miR-182-5p downregulation or EFNA5 overexpression averted the inhibition of neovascularization caused by EGR1 downregulation. Overall, EGR1 bound to the miR-182-5p promoter to inhibit miR-182-5p transcription and further promoted EFNA5 transcription, thus promoting retinal neovascularization in ROP mice.

Sevoflurane but not propofol enhances ovarian cancer cell biology through regulating cellular metabolic and signaling mechanisms.

Perioperative risk factors, including the choice of anesthetics, may influence ovarian cancer recurrence after surgery. Inhalational anesthetic sevoflurane and intravenous agent propofol might affect cancer cell metabolism and signaling, which, in turn, may influence the malignancy of ovarian cancer cells. The different effects between sevoflurane and propofol on ovarian cancer cell biology and underlying mechanisms were studied. Cultured ovarian cancer cells were exposed to 2.5% sevoflurane, 4μg/mL propofol, or sham condition as the control for 2h followed by 24-h recovery. Glucose transporter 1 (GLUT1), mitochondrial pyruvate carrier 1 (MPC1), glutamate dehydrogenase 1 (GLUD1), pigment epithelium-derived factor (PEDF), p-Erk1/2, and hypoxia-inducible factor 1-alpha (HIF-1α) expressions were determined with immunostaining and/or Western blot. Cultured media were collected for 1H-NMR spectroscopy-based metabolomics analysis. Principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) were used to analyze metabolomics data. Sevoflurane increased the GLUT1, MPC1, GLUD1, p-Erk1/2, and HIF-1α expressions but decreased the PEDF expression relative to the controls. In contrast to sevoflurane, propofol decreased GLUT1, MPC1, GLUD1, p-Erk1/2, and HIF-1α but increased PEDF expression. Sevoflurane increased metabolite isopropanol and decreased glucose and glutamine energy substrates in the media, but the opposite changes were found after propofol treatment. Our data indicated that, unlike the pro-tumor property of sevoflurane, propofol negatively modulated PEDF/Erk/HIF-1α cellular signaling pathway and inhibited ovarian cancer metabolic efficiency and survival, and hence decreased malignancy. The translational value of this work warrants further study. • Sevoflurane promoted but propofol inhibited ovarian cancer cell biology. • Sevoflurane upregulated but propofol downregulated the GLUT1, MPC1, and GLUD1 expressions of ovarian cancer cells. • Sevoflurane enhanced but propofol inhibited ovarian cancer cellular glucose. metabolism and glutaminolysis. • Sevoflurane downregulated PEDF but upregulated the Erk pathway and HIF-1α, while propofol had the adverse effects on ovarian cancer cells.

Pigment Epithelium-Derived Factor Promotes the Angiogenic Activity of VEGF

Introduction: We successfully identified a subset of CD11b+ monocytes expressing CX3CR1, the only receptor of fractalkine (Fkn; CX3CL1), in G-CSF mobilized peripheral blood stem cell collection (PBSC). We found that Fkn-treated CD11b+CX3CR1+ monocytes express pigment epithelium-derived factor (PEDF) once Fkn activates Fkn/CX3CR1 signaling. PEDF is a glycoprotein, which belongs to the serpin family and involves various physiologic processes such as angiogenesis, cell proliferation, and survival. In this study, we investigate the role of PEDF and its mechanism of action in promoting angiogenesis. Materials andmethods: To mobilize mononuclear cells (MNCs) including CD11b+CX3CR1+ monocytes into peripheral blood (PB), heathy donors were subcutaneously injected with G-CSF (10μg/kg) for 5 days. Apheresis MNCs were collected from donor PB using a COBE spectra cell separator (COBE, Lakewood, CO) 5 days after daily G-CSF injection. Then, CD11b+CX3CR1+ cells were isolated with a MoFlo™ XDP Cell Sorter (Beckman Coulter, Brea, CA). Human umbilical vein endothelial cells (HUVECs) were isolated from human cords and cultured at 37 °C in 5% CO2 on top of collagen in four different settings: (1) untreated (HUVECs alone) (2) VEGF (1ng/mL) treated HUVECs; (3) PEDF (3,30 and 300 ng/mL) treated HUVECs; (4) VEGF (1ng/mL)+PEDF (3,30 and 300 ng/mL) treated HUVECs. To assess dose-dependency, PEDF was administered at different dosages (0, 3, 30 and 300 ng/mL) for 7 days in either the presence or absence of VEGF (1ng/mL). HUVEC growth areas were measured by image J. Results: CD11b+CX3CR1+ monocytes were found at 19.6±3.58% of total MNCs in human G-CSF mobilized PBSC. Fractalkine treatment (50ng/mL for 30 minutes) of these monocytes promoted endothelial cell proliferation of HUVECs and increased PEDF expression according to the angiogenic protein array results. Administration of PEDF inhibitor significantly decreased HUVEC proliferation and vascular structure formation compared to the control group (p <0.001). We further investigated pro-angiogenic effects of PEDF on HUVEC proliferation. In PEDF (3,30 and 300 ng/mL) treated HUVECs, PEDF itself has minimal effect on HUVEC proliferation. However, EC sprouting at the edge of vessel-like structures was greater in the 3 and 30 ng/mL. Interestingly, there was no EC sprouting with 300 ng/mL PEDF. PEDF (3 and 30 ng/mL) itself showed little effect on HUVEC proliferation. However, co-culturing with VEGF (1ng/mL) and low PEDF concentrations (3 and 30 ng/mL) significantly increased EC proliferation (EC proliferation area (mm2), 1.7±0.4 (VEGF only) vs 3.0±0.6 (VEGF+3 ng/mL PEDF) and 2.5±1.1 (VEGF+30 ng/mL PEDF); p=0.002 and 0.048, respectively). Higher concentration of PEDF did not induced EC proliferation even in the presence VEGF (1.7±0.4 (VEGF only) vs 1.5±0.9 (VEGF+300 ng/mL PEDF); p=0.192). The results suggest that low concentrations of PEDF (3 and 30 ng/mL) has no effect on HUVEC proliferation, but has a potent effect on HUVEC migration regardless of VEGF activity. PEDF is not pro-angiogenic by itself, but can markedly enhance HUVEC proliferation in PEDF (3 and 30 ng/mL) treated HUVECs with VEGF. Conclusion: Fractalkine-treated CD11b+CX3CR1+ monocytes promote angiogenesis by increasing PEDF expression, which acts as a pro-angiogenic factor only under conditions where VEGF is present. DisclosuresNo relevant conflicts of interest to declare.

Use of antibiotic‐resistance‐free plasmids for non‐viral transfection of pigment epithelium‐derived factor in rat primary cells

AbstractPurpose We aim to assess the pigment epithelium‐derived factor (PEDF) production in cells after transfection with plasmids free of antibiotic resistance markers (pFAR4) in a non‐viral Sleeping Beauty transposon system (SB100X).Methods ARPE‐19 cell line and primary rat iris and retinal pigment epithelial (IPE and RPE) cells were transfected with SB100X transposase and PEDF transposon encoding pFAR4 plasmids (1:16 ratio). Control cells were transfected with SB100X and pFAR4‐Venus plasmids. Supernatants were taken every 24 hours for kinetics. The effect of transfecting different amount of cells was also analyzed. PEDF production was tested by Western blot and ELISA.Results A cumulative PEDF secretion over time in ARPE‐19 and primary cells was shown, reaching almost 3‐fold higher PEDF production in ARPE‐19 cells. Control cells did not express recombinant PEDF, but showed fluorescence. The transfection of 10,000 cells clearly showed higher PEDF expression than cells that were transfected as a group of 70,000 and subsequently divided.Conclusion Rat primary cells are efficiently transfected and produced PEDF as it happens in ARPE‐19 cell line. Merging of the SB100X and pFAR4 technologies led to increased PEDF expression and improved safety by avoiding the transfer and potential integration of antibiotic resistance genes. This study demonstrates the feasibility of our approach based on ex vivo transfection of a low number of primary autologous pigment epithelial cells.

Relationship between pigment epithelium-derived factor expressed in placentas and the pathogenesis of preeclampsia disease

To investigate the effect of pigment epithelium-derived factor (PEDF) on the pathogenesis of preeclampsia disease, by detecting the expression of PEDF in the placentas, as well as the relationship between PEDF and the production of placental vessels. A study was performed in 60 cases of pregnant women with preeclampsia in the obstetrical department of Nanfang Hospital affiliated to southern medical university from October 2011 to January 2013, in which 30 cases were patients with mild preeclampsia(mPE) and other 30 cases were those with severe preeclampsia (sPE).40 normal pregnant women who also been hospitalized and delivered were selected as control group. The expression of PEDF and micro-vessel density (MVD) in placentas were assayed by using western blot and SP immunohistochemical method, then the relationship between PEDF and MVD was analyzed. (1) The pathological changes of placentas:the placental weight were lightened obviously in the mild preeclampsia and severe preeclampsia groups, the reduced blood vessels and luminal stegnosis were found in chorionic villus, basement membrane of trophocytes were thickening. The hyperplasia syneytiotrophoblast were like nodosity, with focus infarction, fibrinoid necrosis, or thrombogenesis.While there was no the above mentioned pathological alteration in normal control group. (2)The levels of PEDF expression in mild and severe preeclampsia group were 0.63 ± 0.09, 0.93 ± 0.07, while 0.47 ± 0.04 in control group, which in mild and sever preeclampsia groups were significantly higher than that in normal group (P < 0.05). Compared to mild preeclampsia group, the expression of PEDF was significantly increased in severe preeclampsia group, there was statistical significance between the difference (P < 0.05).(3) The amount of microvessel density (MVD) in mild and severe preeclampsia group were 106 ± 9, 93 ± 8, while 136 ± 9 in control group, which were significantly reduced in mild and severe preeclampsia group, compared to that in normal control group (P < 0.05). And it was significantly lower in severe preeclampsia group than that in mild preeclampsia group (P < 0.05). (4) The expression of PEDF was negatively correlated with the amount of MVD in mild and severe preeclampsia group (r = -0.426, P < 0.05; and r = -0.646, P < 0.05 respectively), which was also negative in control group (r = -0.589, P < 0.05). Increased PEDF expression in placentas of women with preeclampsia induce the dysfunction of the placental vascular reconstruction and the pathological alteration like ischemic and hypoxia in placentas, which may be involved in pathogenesis and pathogenic progress of preeclampsia.

Abstract LB-161: Metformin increases lipolytic metabolism and PEDF expression in prostate cancer cells

Abstract Obesity increases the risk of prostate cancer (PCa) progression; however, the mechanism remains unclear. Obesity results in metabolic dysfunction, including increases in circulating and tissue lipid levels. A common sequela of obesity is Type II diabetes, and some studies suggest that PCa patients on the antidiabetic drug, metformin, have a reduced PCa risk. The antitumor mechanism of metformin is largely unknown. Metformin exerts its antidiabetic function via AMPK activation-dependent inhibition of hepatic gluconeogenesis, with secondary decrease in serum insulin/IGF-1 levels that may represent an indirect mechanism for its proposed antitumor effects. Previous studies have shown that metformin directly inhibits PCa cell proliferation and cyclin D1 expression in vitro. Expression of pigment epithelium-derived factor (PEDF), a potent angiogenesis inhibitor, is decreased in PCa tissues, and PEDF inhibits PCa cell proliferation. Interestingly, PEDF knockout mice have defects in lipid metabolism and develop prostate hyperplasia, so we hypothesized that metformin's antitumor mechanism could be mediated, in part, through regulation of PEDF expression and lipolytic metabolism. To simulate obesity-induced cellular triglyceride (TG) accumulation, we treated cells with oleic acid (OA, 1.0 mM) ± PEDF (10 nM) or metformin (5 mM). Proliferation (cell count or MTT assay) and lipid content (TG isolation) were quantified, and lipid and protein were assessed by lipid staining and immunocytochemistry or Western blot, respectively. In androgen-insensitive PCa cells (PC-3 and DU145), OA treatment promoted TG accumulation, cyclin D1 expression and proliferation, with increased expression of lipid droplet-associated proteins, adipose triglyceride lipase (ATGL) and CGI-58, and conversely suppressed PEDF expression. Furthermore, PEDF treatment decreased basal TG levels (32.89 ± 1.0 control vs. 17.3 ± 0.6 in PEDF; P&amp;lt;0.0002) and suppressed OA-induced TG accumulation (130.33 ± 1.2 μg/1x106 cells in OA vs. 72.92 ± 3.5 OA + PEDF; P&amp;lt;0.0002) and proliferation (P&amp;lt;0.05), concomitantly decreasing ATGL and CGI-58 expression. Interestingly, metformin blocked OA-induced proliferation (P&amp;lt;0.02), decreased ATGL and CGI-58 expression, and increased PEDF expression. When free glycerol release was quantified as a measure of TG lipolysis, both metformin and PEDF increased PCa cell TG turnover compared to control cells (P&amp;lt;0.05). These data suggest that metformin exerts its anti-proliferative effect on PCa cells, in part, through upregulation of lipolytic metabolism via increased PEDF expression. These data provide a novel pharmacodynamic biomarker for mechanistic studies in patients, and platform for rational design of “personalized” cancer therapies targeting altered lipid metabolism in obesity-induced cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-161. doi:1538-7445.AM2012-LB-161

Pigment Epithelium-Derived Factor Regulates Early Pancreatic Fibrotic Responses and Suppresses the Profibrotic Cytokine Thrombospondin-1

Pigment epithelium-derived factor (PEDF) is important for maintaining the normal extracellular matrix. We hypothesized that the initiation of pancreatic fibrosis is dependent on the loss of PEDF. Pancreatic PEDF expression was assessed in wild-type mice fed either a control or ethanol diet using an intragastric feeding model. Pancreatitis responses were elicited with either a single episode or a repetitive cerulein-induced (50 μg/kg, 6 hourly i.p. injections) protocol in wild-type and PEDF-null mice. Quantitative real-time PCR and immunoblotting were performed to assess fibrogenic responses. In wild-type animals, PEDF expression increased with pancreatitis and was more pronounced in mice fed ethanol. Compared with wild-type mice, α-smooth muscle actin staining and expression levels of fibrogenic markers (eg, transforming growth factor-β1, platelet-derived growth factor, collagen I, and thrombospondin-1) were higher in PEDF-null mice at baseline. Sirius red staining revealed more fibrosis in PEDF-null versus wild-type pancreas 1 week after pancreatitis. Differences in tissue fibrosis resolved with longer recovery periods. PEDF overexpression suppressed thrombospondin-1 levels in vitro. Ethanol feeding and experimental pancreatitis increased PEDF expression in wild-type mice. PEDF-null mice, however, demonstrated enhanced early fibrotic responses compared with wild-type mice with pancreatitis. These findings indicate that PEDF acts as a compensatory antifibrotic cytokine in pancreatitis.

Increased expression of pigment epithelium-derived factor in aged mesenchymal stem cells impairs their therapeutic efficacy for attenuating myocardial infarction injury‡

AimsMesenchymal stem cells (MSCs) can ameliorate myocardial infarction (MI) injury. However, older-donor MSCs seem less efficacious than those from younger donors, and the contributing underlying mechanisms remain unknown. Here, we determine how age-related expression of pigment epithelium-derived factor (PEDF) affects MSC therapeutic efficacy for MI.Methods and resultsReverse transcriptase–polymerized chain reaction and enzyme-linked immunosorbent assay analyses revealed dramatically increased PEDF expression in MSCs from old mice compared to young mice. Morphological and functional experiments demonstrated significantly impaired old MSC therapeutic efficacy compared with young MSCs in treatment of mice subjected to MI. Immunofluorescent staining demonstrated that administration of old MSCs compared with young MSCs resulted in an infarct region containing fewer endothelial cells, vascular smooth muscle cells, and macrophages, but more fibroblasts. Pigment epithelium-derived factor overexpression in young MSCs impaired the beneficial effects against MI injury, and induced cellular profile changes in the infarct region similar to administration of old MSCs. Knocking down PEDF expression in old MSCs improved MSC therapeutic efficacy, and induced a cellular profile similar to young MSCs administration. Studies in vitro showed that PEDF secreted by MSCs regulated the proliferation and migration of cardiac fibroblasts.ConclusionsThis is the first evidence that paracrine factor PEDF plays critical role in the regulatory effects of MSCs against MI injury. Furthermore, the impaired therapeutic ability of aged MSCs is predominantly caused by increased PEDF secretion. These findings indicate PEDF as a promising novel genetic modification target for improving aged MSC therapeutic efficacy.