Pigment Epithelium-derived Factor Research Articles

Pigment epithelium-derived factor inhibits adipogenesis in 3T3-L1 adipocytes and protects against high-fat diet-induced obesity and metabolic disorders in mice

Obesity is a major cause of metabolic syndrome and type II diabetes, and it presents with metabolic disorders, such as hyperglycemia, hyperlipidemia, and insulin resistance. Pigment epithelium-derived factor (PEDF), a protein isolated from retinal pigment epithelial cells, has multiple functions, including neuronal protection, antineoplastic effects, and anti-inflammatory activity. The aim of this study is to investigate the antiobesity effects of PEDF. The antiobesity effects of PEDF on fat accumulation, inflammation, energy expenditure, insulin resistance, and obesity-related physiological parameters and protein levels were assessed in high-fat diet (HFD)-induced obese mice in vivo and in 3T3-L1 adipocytes, palmitate (PA)-treated HepG2 cells, and C2C12 myotubes in vitro. In an in vivo assay, PEDF effectively decreased body weight gain, white adipose tissue mass, and inflammation and improved insulin resistance, dyslipidemia, and hyperglycemia in HFD-induced mice. In liver tissue, PEDF decreased lipid accumulation and fibrosis. In an in vitro assay, PEDF diminished the differentiation of 3T3-L1 preadipocytes. We also determined that PEDF promoted lipolysis and prolonged cell cycle progression, through the mTOR-S6K pathway and downstream transcription factors, such as peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein α (CEBP-α), and CEBP-β. In addition, PEDF decreased reactive oxygen species production in PA-induced HepG2 cells and improved glucose uptake ability in PA-induced HepG2 cells and C2C12 myotubes. In the present study, PEDF protected against HFD-induced obesity and metabolic disorders in mice, inhibited adipogenesis, and improved insulin resistance. These results provide a new potential treatment for obesity in the future.

Pigment Epithelium-Derived Factor Promotes Axon Regeneration and Functional Recovery After Spinal Cord Injury

Although neurons in the adult mammalian CNS are inherently incapable of regeneration after injury, we previously showed that exogenous delivery of pigment epithelium-derived factor (PEDF), a 50-kDa neurotrophic factor (NTF), promoted adult retinal ganglion cell neuroprotection and axon regeneration. Here, we show that PEDF and other elements of the PEDF pathway are highly upregulated in dorsal root ganglion neurons (DRGN) from regenerating dorsal column (DC) injury paradigms when compared with non-regenerating DC injury models. Exogenous PEDF was neuroprotective to adult DRGN and disinhibited neurite outgrowth, whilst overexpression of PEDF after DC injury in vivo promoted significant DC axon regeneration with enhanced electrophysiological, sensory, and locomotor function. Our findings reveal that PEDF is a novel NTF for adult DRGN and may represent a therapeutically useful factor to promote functional recovery after spinal cord injury.

Fabrication of resveratrol coated gold nanoparticles and investigation of their effect on diabetic retinopathy in streptozotocin induced diabetic rats

This study includes the fabrication of gold nanoparticles (AuNPs) with the help of a plant polyphenol called Resveratrol through an ecofriendly synthetic process without any use of harmful reductants. In the fabrication of AuNPs, Resveratrol acts as both stabilizing and reducing agent. The prepared AuNPs is tested on streptozotocin (STZ) induced diabetic rats for their amelioration consequence. The images of TEM displayed the development of spherical nanoparticles (NPs) with a median of 20 nm particle size. The STZ injected diabetic rats were administrated orally with calcium dobesilate (CD; 500 mg/kg/day) or AuNPs (200, 300 mg/kg/day) for a period of 3 months. The characteristics displayed by AuNPs were found to be similar with CD in decreasing permeability of blood–retinal barrier in STZ injected diabetic rats. The retinal vessels in the AuNPs administrated diabetic rats were observed to be decreased through the retinal histopathological examination. In the AuNPs administrated diabetic rats, the retinal expression of renal Pigment Epithelium-Derived Factor (PEDF) was observed to be increased and the Vascular Endothelial Growth Factor (VEGF-1), which was increased in diabetic rats was declined on treating with AuNPs. On treating the STZ injected diabetic rats with AuNPs, all the retinal mRNA expressions of VEGF-1, Tumor Necrosis Factor (TNFα), Monocyte Chemotactic Proteins-1 (MCP-1), Intercellular Adhesion Molecule-1 (ICAM-1), and Interleukin (IL)-6, IL-1β were observed to be reduced. Furthermore, AuNPs can reduce phosphorylation of Nuclear Factor Kappa B (NF-κB) p65 and Extracellular signal Regulated Kinase (ERK) 1/2 along with a growth in nuclear translocation of pNF-κB p65 produced by STZ. To conclude, the protective effect of AuNPs on STZ injected diabetic rats could help in redeveloping the balance among the inhibitors and stimulators of angiogenesis. Furthermore, on treating with AuNPs results in inhibiting the signaling pathway of ERK1/2 as well as with amelioration of retinal inflammation through trans repression of NF-κB.

Pedf derived peptides affect colorectal cancer cell lines resistance and tumour re-growth capacity

Relapse after chemotherapy treatment depends on the cancer initiating cells (CICs). PEDF (Pigmented Epithelium Derived Factor) is an anti-angiogenic, neurotrophic and self-renewal regulator molecule, also involved in CICs biology. Acute and chronic exposition of colon cancer cell lines to CT/CTE PEDF-derived peptides decreased drug-resistance to conventional colorectal cancer treatments, such as oxaliplatin or irinotecan. We confirmed a reduction in the irinotecan and oxaliplatin IC50 doses for all tested tumour cell lines. After xenograft transplantation, CT/CTE treatments also produced a reduction in resistance to conventional chemotherapy treatments as in culture-assays. Metastatic capacity of these treated cell lines was also depleted. The PEDF signaling pathway could be a future therapeutic tool for use as an adjuvant therapy that decreases IC50 dosis, adverse effects and treatment costs. This pathway could also be involved in an increase of the time relapse in patients, decreased tumourigenicity, and decreased capacity to produce metastasis.

Triple-threat activity of PEDF in bone tumors: Tumor inhibition, tissue preservation and cardioprotection against doxorubicin.

Pigment epithelium-derived factor (PEDF) is known for its osteogenic properties, but its effects against primary and secondary bone tumors have not comprehensively been demonstrated. We show the ubiquitous expression of PEDF in murine embryonic tissue. Continuous administration of PEDF in pregnant mice for five days did not adversely affect foetal health, despite PEDF's known potent antiangiogenic properties. In the case of the devastating childhood bone cancer osteosarcoma, PEDF has direct anticancer activity per se, and protects against the toxicity of doxorubicin in the heart, small intestine and testes. PEDF demonstrated anti-proliferative and pro-apoptotic effects against human prostate and breast cancer cells, tumors which are known to metastasize to bone as the preferred secondary site. Caspase-2 was activated in both tumor cell types by PEDF. In models of prostate and breast cancer in bone, PEDF significantly reduced tumor volumes. When combined with zoledronic acid, continuously-administered PEDF significantly reduced breast tumor volume at the bone, and was able to preserve the quality of bone better than the combination therapy. These multiple positive findings make PEDF an ideal endogenous and safe biological for possible future clinical testing.

Autophagy: a new mechanism for regulating VEGF and PEDF expression in retinal pigment epithelium cells.

To investigate the regulation of vascular endothelial growth factors (VEGF) and pigment epithelium-derived factor (PEDF) expression by autophagy in retinal pigment epithelium (RPE) cells on exposure to hypoxia. ARPE-19, an RPE cell line, was treated as following: the control group was kept in a normoxic incubator; the hypoxia group was incubated in a hypoxic incubator with 1% O2/5% CO2/94% N2 for 24h; the hypoxia + 3-methyladenine (3-MA) group was pretreated with 10 mmol/L 3-MA for 1h and then in the hypoxic incubator for 24h; and the hypoxia + chloroquine (CQ) group was pretreated with 50 µmol/L CQ for 1h and then in the hypoxic incubator for 24h. The morphology and ultrastructure of the cells was observed by an inverted microscope or a transmission electronic microscope (TEM). Western blot was performed to assay the expression of autophagy-associated markers, including microtubule associated protein 1 light chain 3 B (LC3B), Beclin-1, Atg5 and p62. The concentration of VEGF and PEDF in the culture supernatant was determined by ELISA, and the ratio of VEGF/PEDF was calculated. There were no obvious differences in cell morphology among different groups and autolysosomes could be observed in the cytoplasm in all groups. Compared to the control cells, the LC3B-II/I ratio and levels of Beclin-1 and Atg5 were significantly increased and p62 level was significantly decreased in the hypoxia group. With the increase of VEGF and decrease of PEDF concentration, the VEGF/PEDF ratio was significantly increased in the hypoxia group compared to the control cells. The LC3B-II/I ratio was significantly reduced by 3-MA treatment and increased by CQ treatment. The expressions of Beclin-1 and Atg5 were significantly reduced by 3-MA or CQ treatment, while expression of p62 was increased in the 3-MA or CQ treated cells. The concentration of VEGF was significantly decreased and PEDF increased, thereby the VEGF/PEDF ratio was decreased in the hypoxia + 3-MA group and hypoxia + CQ group compared with that in the hypoxia group. Hypoxia leads to elevated autophagy in RPE cells, and expression of VEGF and PEDF might be regulated by autophagy on exposure to hypoxia to further participate in regulating the formation of retinal neovascularization.

Clinic Case of Rare Type VI Osteogenesis Imperfecta

Osteogenesis imperfect is genetically heterogeneous group of diseases which are characterized by bone brittleness and fractures. It was thought for a long time that this is happening due to mutations in collagen genes. However, within past decade the understanding of osteogenesis imperfecta etiology has changed as a result of genetics development. The majority of all cases is related to mutations in collagen genes whereas rare mostly recessive forms are related to mutations in genes encoding collagen post-translational modification. Mutations in SERPINF1 gene were chosen as molecular cause of osteogenesis imperfecta type VI in 2011. Thus the new pathophysiology of this disease was revealed. Children with osteogenesis imperfecta type VI have high-frequency of fractures despite the management with bisphosphonates because mineralized bone osteoid is considerably reduced.

C1q/TNF-related protein-9 attenuates retinal inflammation and protects blood–retinal barrier in db/db mice

C1q/TNF-related protein-9(CTRP9) is an adipose cytokine, a closest adiponectin paralog, which has anti-inflammatory, antioxidant, vasodilation and anti-atherosclerosis effects. In addition, it can increase insulin sensitivity, decrease blood glucose level and inhibit the apoptosis of endothelial cells. However, it remains unclear whether CTRP9 has beneficial effects on diabetic retinopathy (DR). An adenoviral vector expressing CTRP9 was intravenously injected into db/db mice, aged 12 weeks, at day 15 post injection, and the process was repeated. The transfection efficiency of CTRP9 was assessed by enzyme linked immunosorbent assay. We used RT-PCR, immunofluorescence, and Western blot to determine proinflammatory cytokines, adhesion molecules and tight-junction proteins. The breakdown of blood–retinal barrier (BRB) was evaluated using Evans blue and retinal staining. CTRP9 suppresses the expression of interleukin-1 beta, tumor necrosis factor-alpha, monocyte chemotactic protein-1 and adhesion molecules in the retina of db/db mice. CTRP9 can balance the expression of pigment epithelium-derived factor and vascular endothelial growth factor. CTRP9 can also inhibit the activation of nuclear factor Kappa B in the retina of db/db mouse. In addition, CTRP9 can prevent the breakdown of BRB and downregulation of tight-junction proteins in the retina of db/db mice. Evans blue assay revealed the breakdown of BRB and vascular leakage in the retinas of diabetic mice. CTRP9 can both qualitatively and quantitatively alleviate the vascular leakage in the early stage of diabetic retinas. CTRP9 can inhibit the inflammation of diabetic retinopathy and protect blood–retinal barrier via decreasing proinflammatory cytokines and preventing the downregulation of tight-junction proteins.

Limbal niche cells can reduce the angiogenic potential of cultivated oral mucosal epithelial cells

BackgroundAutologous cultivated oral mucosal epithelial transplantation (COMET) is an important treatment for limbal stem cell deficiency. However, peripheral corneal neovascularization after surgery hinders its application. This study aims to employ a culture system using allogenic limbal niche cells (LNCs) instead of mouse-derived 3T3 cells as a feeder layer that could relieve postoperative neovascularization.MethodsRat oral mucosal epithelial cells (OMECs) were co-cultured with rat LNCs or 3T3 cells. Cultivated oral mucosal epithelial cells (COMECs) of different culture systems were identified by hematoxylin and eosin staining and immunocytochemistry. The expression levels of the angiogenesis-related factors were analyzed by RT-qPCR and western blotting/ELISA. Angiogenic potential was reconfirmed by cell viability and tube formation assays with human umbilical vein endothelial cells (HUVECs).ResultsCOMECs were obtained from both culture systems successfully. Immunocytochemistry showed approximately equal percentages of positive staining cells for p63α (p = 0.9177), ABCG2 (p = 0.526), Ki67 (p = 0.0987), and CK3 (p = 0.4000) in COMECs of different groups. RT-qPCR and western blotting/ELISA showed that COMECs of the LNC group expressed a significantly lower amount of basic fibroblast growth factor (bFGF) (p = 0.0038 for RT-qPCR, p = 0.0026 for western blotting) but more pigment epithelium-derived factor (PEDF) (p = 0.0172 for RT-qPCR, p = 0.0253 for western blotting) and soluble fms-like tyrosine kinase-1 (sFlt-1) (p < 0.0001 for RT-qPCR, p = 0.0064 for ELISA) than the COMECs of the 3T3 group. Furthermore, compared with COMECs of the 3T3 group, COMECs of the LNC group could reduce the viability (p = 0.0002) and tube formation (p = 0.0002) of HUVECs.ConclusionsLNCs could substitute 3T3 cells for expanding OMECs in vitro, and the COMECs obtained in this system are less likely to induce postsurgical neovascularization, which provides an alternative option for an ex vivo culture system and promotes the application of COMET.

Analysis of miR‐21‐5p and ‐3p expression and function during pathological neovascularization

Retinal neovascularization (RNV) is a sight compromising complication of diabetes and prematurity. Previous studies from our laboratory have shown that the small noncoding microRNA, microRNA‐21‐5p (miR‐21‐5p), is an inducer of RNV. Emerging new evidence has shown that miRNA passenger strands, the 3p forms, which were thought to be degraded, possess biological functions. In this study, we have investigated the role of miR‐21‐3p in RNV. In particular, we have assessed the effect of miR‐21‐3p on the same molecular targets of miR‐21‐5p: the tissue inhibitor of matrix metalloproteinases 3 (TIMP3), peroxisome proliferator‐activated receptor alpha (PPAR‐α) and pigment epithelium‐derived factor (PEDF). Real‐time PCR (RT‐PCR) showed that the expression of miR‐21‐3p is significantly upregulated in retinas and blood spot samples of mice subjected to oxygen‐induced retinopathy (OIR), a model of RNV. Furthermore, we noted a significant upregulation of miR‐21‐3p levels, measured by RT‐PCR, in human retinal endothelial cells (HuREC) and pigment epithelial cells (ARPE‐19) when exposed to angiogenic stimuli such as 20ng/mL of vascular endothelial growth factor (VEGF) or to hypoxic conditions (pO2= 2+/−0.5) for different times (1, 3, 6, and 12 hours). Transfection of HuREC and ARPE‐19 with miR‐21‐3p mimic (50nM) for 24 and 48 hours had only marginal inhibition on the miR‐21‐5p target genes (TIMP3, PPARα, and PEDF). Additionally, blockade of miR‐21‐3p achieved by transfecting cells with a specific miR‐21‐3p inhibitor (50nM) did not rescue VEGF‐changes in TIMP3, PPARα, and PEDF expression. Our data demonstrated that the passenger strand, miR‐21‐3p, is co‐expressed with miR‐21‐5p in ischemic conditions, but targets different genes. Although further studies will be necessary to clarify miR‐21‐3p biological function, our studies suggest that, together with miR‐21‐5p, miR‐21‐3p may hold potential diagnostic and therapeutic value for treatment of RNV in ischemic retinopathies.Support or Funding InformationAll support for this research came from the Bartoli laboratory of the Ophthalmology Department of the Medical College of Georgia at Augusta University and the funding sources (EY0246, Knights Templar –GA Chapter).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A "Hibernating-Like" Viable State Induced by Lentiviral Vector-Mediated Pigment Epithelium-Derived Factor Overexpression in Rat Acute Ischemic Myocardium.

The failure to maintain the viability of ischemic myocardium is one of the mechanisms that causes ischemic heart dysfunction after revascularization. Hibernating myocardium is considered to be able to maintain long-term viability during chronic hypoperfusion. Pigment epithelium-derived factor (PEDF) decreases the contractility of hypoxic cardiomyocytes and protects cardiomyocytes against ischemic injury, which is strikingly similar to the pathophysiologic characteristics of hibernating myocardium. It was therefore postulated that PEDF may induce acute ischemic myocardium into a "hibernating-like" state to maintain its viability. Adult Sprague-Dawley rat models of acute myocardial infarction were surgically established. Lentiviral vectors carrying the PEDF gene (PEDF-LVs) were delivered into myocardium with infarction to overexpress PEDF locally. It was found that PEDF local overexpression significantly reduced myocardial infarct size and cardiomyocytes necrosis but did not improve cardiac function at rest. The contractile reserve assessed by low-dose dobutamine stress echocardiography and "perfusion-metabolism mismatch" assessed by positron emission tomography, which are the characteristics of viable myocardium in hibernation, were observed in the PEDF overexpressed ischemic heart. Ultrastructural changes observed by electron microscopy and glycogen deposition explored by Periodic acid-Schiff staining were similar to the histological characteristics of hibernating myocardium. Moreover, PEDF overexpression protected the cardiomyocytes against anoxic injury and retained their functional recovery potential after reoxygenation in vitro. PEDF local overexpression may induce acute ischemic myocardium into a "hibernating-like" state and maintain its viability. This novel effect of PEDF presents an important clinical approach to enhance functional recovery after revascularization therapy in acute myocardial infarction.

PEDF promotes nuclear degradation of ATGL through COP1

Adipose triglyceride lipase (ATGL) plays a compelling role in hepatic lipid turnover and in the pathophysiology of non-alcoholic fatty liver disease. Hepatic ATGL is post-transcriptionally regulated by E3 ubiquitin ligase constitutive photomorphogenic1 (COP1) through polyubiquitylation and proteasomal degradation. However the physiological cue for COP1-mediated hepatocellular degradation of ATGL remained unknown. Here we checked for the role of pigment epithelium-derived factor (PEDF), a moonlighting hepatokine and the so-called ligand of ATGL for its stability in hepatocytes. We show that PEDF diminishes ATGL protein stability by promoting its proteasomal degradation in COP1-dependent manner. Despite being a secretory glycoprotein, PEDF is also sequestered in the nuclear compartment so as COP1. Interestingly, PEDF enhances nuclear import of predominantly cytosolic ATGL protein for its subsequent proteasomal degradation in the nucleus. PEDF also controls cell autonomous hepatocyte lipid accumulation and mobilization through COP1-ATGL axis, thereby unraveling a novel pathway for hepatic lipid metabolism.

Association of pigment epithelium derived factor with von Willebrand factor and plasminogen activator inhibitor 1 in patients with type 2 diabetes.

To compare circulating pigment epithelium derived factor (PEDF) levels in type 2 diabetes patients (T2D) with and without metabolic syndrome (MetS+/-) to healthy controls and assess PEDF association with plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF) as markers of endothelial dysfunction. Fifty T2D individuals and forty healthy controls were included. PEDF, PAI-1, vWF, anthropological parameters, lipids, and markers of insulin resistance were investigated in all subjects. Compared to controls only MetS+ diabetics had higher PEDF levels [14.2 (10.2-16.0) mg/l vs. 11.1 (8.6-14.4) mg/l; p<0.05]. PEDF significantly correlated: positively with body mass index (rho=0.25), smoking (rho=0.21), C-reactive protein (rho=0.22), triglycerides (rho=0.38), non-HDL-cholesterol (rho=0.39), apolipoprotein B (rho=0.38), fasting glucose (rho=0.22), glycated hemoglobin (rho=0.24), C-peptide (rho=0.28), insulin (rho=0.26); and negatively with HDL-cholesterol (rho=-0.42) and apolipoprotein A1 (rho=-0.27). Independent association of PEDF with vWF in T2DMetS- subjects was found. Significantly elevated PEDF in T2DMet+ patients and its association with adverse metabolic profile confirmed PEDF as a marker of insulin resistance. Negative independent association of PEDF with vWF in T2DMetS- patients may reveal its angio-protective role.

Evaluation of Retinal Pigment Epithelial Cell Cytotoxicity of Recombinant Tissue Plasminogen Activator Using Human-Induced Pluripotent Stem Cells.

Purpose The evaluation of drug-induced cytotoxicity is of great importance for the clinical application of pharmaceutical products, and human-induced pluripotent stem cells (hiPSCs) have received considerable scrutiny as a cell source for in vitro cytotoxicity testing. The aim of this study is to validate the concept of cytotoxicity testing using hiPSC-derived retinal pigment epithelium (hiPSC-RPE) by comparing the responsiveness of human fetal RPE (hfRPE) and human RPE cell line (ARPE19) to recombinant tissue plasminogen activator (rtPA). Methods HfRPE, two types of hiPSC-RPE, and ARPE19 were cultured in media with or without rtPA. A lactate dehydrogenase release assay was performed to investigate the dose- and time-dependent effects of rtPA on cell death. RPE function was evaluated by measuring the secretion of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) and RPE-specific gene expression. Results Rates of cell damage in hfRPE and both hiPS-RPE were increased by rtPA supplementation (2000 and 4000 μg/ml) for 1 hour, whereas ARPE19 cell damage was increased by supplementation with rtPA at concentrations higher than 50 μg/ml. Although 100 μg/ml rtPA for 24 hours did not affect RPE cell function, sustained rtPA exposure induced prolonged cytotoxic effects in hfRPE and two hiPSC-RPE, but not ARPE19. Conclusion The responsiveness of hiPSC-RPE to rtPA is similar to that of hfRPE in terms of cell death and cell function. Thus, hiPSC-RPE is a valuable cell source for in vitro cytotoxicity testing.

PEDF decreases cardiomyocyte edema during oxygen‑glucose deprivation and recovery via inhibiting lactate accumulation and expression of AQP1.

Myocardial edema is divided into cellular edema and interstitial edema; however, the dynamic change of cardiomyocyte edema has not been described in detail. Pigment epithelium-derived factor (PEDF) is known for its protective effects on ischemic cardiomyocytes; however, the association between PEDF and cardiomyocyte edema remains to be fully elucidated. In the present study, rat neonatal left ventricular cardiomyocytes were isolated and treated with oxygen-glucose deprivation (OGD) and recovery. During OGD and recovery, the cardiomyocytes exhibited significant edema following 30 min of OGD (OGD 30 min) and OGD 30 min with recovery for 6 h. PEDF significantly decreased the lactate content and extracellular acidification rate of the OGD-treated cardiomyocytes, thereby reducing cellular osmotic gradients and preventing the occurrence of cell edema. In addition, the glycolytic agonist, fructose-1, 6-diphosphate, eliminated the effect of PEDF on inhibiting edema in the OGD-treated cardiomyocytes. Furthermore, PEDF reduced the protein and mRNA expression of aquaporin 1 (AQP1), and thus downregulated cardiomyocyte edema during the OGD/recovery period. The addition of AQP1 agonist, arginine vasopressin, inhibited the inhibitory effect of PEDF on cardiomyocyte edema during OGD/recovery. In conclusion, the present study revealed a novel mechanism for the regulation of cardiomyocyte edema by PEDF involving lactate levels and the expression of AQP1 during OGD/recovery. The reduction of lactate content during OGD was associated with a decrease in the protein level of AQP1 during OGD/recovery; therefore, PEDF decreased cardiomyocyte edema and cellular apoptosis, prolonging the viability of the cells.

Effects of concentration of amyloid \u03b2 (A\u03b2) on viability of cultured retinal pigment epithelial cells

BackgroundAmyloid beta (Aβ) is a constituent of drusen that is a common sign of age-related macular degeneration (AMD). The purpose of this study was to investigate the effect of Aβ on human retinal pigment epithelial (RPE) cells in culture.MethodsCells from a human RPE cell line (ARPE-19) were exposed to 0 to 25 μM of Aβ 1–40 for 48 h, and the number of living cells was determined by WST-8 cleavage. Replicative DNA synthesis was measured by the incorporation of 5′-bromo-2′-deoxyuridine. The cell death pathway was investigated by the WST-8 cleavage assay after the addition of caspase-9 inhibitor, an anti-apoptotic factor. Real-time qRT-PCR was performed using Aβ-exposed cellular RNA to determine the level of vascular endothelial growth factor (VEGF)-A and pigment epithelium derived factor (PEDF). To determine the effect of receptor-for-advanced glycation end products (RAGE), the siRNA for RAGE was inserted into ARPE-19 treated with Aβ, and the levels of expression of VEGF-A and PEDF were determined.ResultsThe number of living ARPE-19 cells was increased by exposure to 5 μM Aβ but was decreased by exposure to 25 μM of Aβ. Replicative DNA synthesis by ARPE-19 cells exposed to 25 μM of Aβ was significantly decreased indicating that 25 μM of Aβ inhibited cell proliferation. Real-time RT-PCR showed that the level of the mRNA of PEDF was increased by exposure to 5 μM Aβ, and the levels of the mRNAs of PEDF and VEGF-A were also increased by exposure to 25 μM Aβ. The addition of an inhibitor of caspase-9 blocked the decrease the number of ARPE-19 cells exposed to 25 μM Aβ. Exposure to si-RAGE attenuated the increase of VEGF-A and PEDF mRNA expression in ARPE-19 exposed to Aβ.ConclusionsExposure of ARPE-19 cells to low concentrations of Aβ increases the level of PEDF which then inhibits the apoptosis of ARPE-19 cells leading to RPE cell proliferation. Exposure to high concentrations of Aβ induces RPE cell death and enhances the expression of the mRNA of VEGF-A in RPE cells. The Aβ-RAGE pathway may lead to the expression VEGF-A and PEDF in RPE cells. These results suggest that Aβ is strongly related to the pathogenesis of choroidal neovascularization.

Decompensation of PEDF Accelerates Atherosclerosis Development via Promoting VEGFB-FATP3/4-Dependent Endothelial FA Uptake in Mice with Hyperlipidemia

Background: Pigment epithelial-derived factor (PEDF) is protectively involved in atherosclerosis induced by hyperlipidemia, but expression characteristics of PEDF and the role of PEDF in the later stage of progressive atherosclerosis remain unclear. Methods: Circulating PEDF were analyzed in patients with atherosclerotic cardiovascular disease (ASCVD) and in murine models of wildtype (WT) mice and ApoE-/- mice under long-term high-fat diet (HFD). PEDF-/-/ ApoE-/- mice and matching PEDF+/+/ ApoE-/- mice fed with HFD for 24, 36 and 48 weeks, combined with injections of adeno-associated virus encoding PEDF (AAV-PEDF) in ApoE-/-/PEDF-/- mice were used to investigate the effects of PEDF knockdown on vascular function and structure. Experiments in vitro and in vivo were applied to identify PEDF regulate vascular endothelial growth factor (VEGFB) signaling pathway mediated endothelial fatty acid (FA) uptake. Findings: Circulating PEDF was increased in ASCVD patients compared to normal individuals, while decreasing trends were confirmed in murine models of WT mice and ApoE-/- mice on long-term HFD. ApoE-/-/PEDF-/- mice placed on HFD were fatter and had accelerated atherosclerotic formation, as evidenced by higher serum lipid levels, increased atherosclerotic plaques, pronounced vascular dysfunction and increased lipid accumulation in peripheral tissues, whereas injections of AAV-PEDF in ApoE-/-/PEDF-/- mice significantly improved. Mechanistically, PEDF deletion activated VEGFB paracrine signaling by upregulating expressions of VEGFB in peripheral tissues and its downstream targets on endothelial cells, including its receptors, namely VEGFR1 and neuropilin-1, and fatty acid transport protein 3 and 4 to enhance FA uptake. Interpretation: Decreasing expression of PEDF may decompensate to aggravate atherosclerosis. Funding: National Nature Science Foundation of China, Science and Technology Project of Guangzhou. Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The study was approved by the ethical committees of Sun Yat-sen Memorial Hospital and Sun Yat-sen University.

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.

Characterization of pigment epithelium-derived factor expression in mechanically expanding skin

Objective To characterize the pigment epithelium-derived factor (PEDF) expression in the mechanically expanding skin tissues. Methods The rat PEDF expression was identified by immunofluorescence staining on skin tissue sections and by quantitative characterization using Western blotting on tissue lysates originated from the mechanically expanding skin samples. To further elucidate the effects of expansion-relevant tissue microenvironment stimuli on the cellular PEDF expression, the PEDF gene transcription levels were determined respectively on epidermal cells that were incubated in vitro under either hypoxia or mechanical stretching conditions. Results For normal oxygen group, epidermal cell line HaCaT cells were cultured and collected at 1, 3, 6 and 9 h, and the PEDF mRNA espression levels were 0.392±0.174, 0.385±0.207, 0.491±0.134, 0.122±0.040 respectively at 1, 3, 6 and 9 h. For hypoxia group, PEDF mRNA expression levels were markedly upregulated correspondingly [2.271±0.781 (P<0.05), 3.419±0.774 (P<0.05), 2.886±0.019 (P<0.05), and 2.673±0.219 (P<0.05) respectively at 1, 3, 6 and 9 h]. For stretch group, HaCaT cells were applied to mechanical stretch for the same period under the normal oxygen. It was found that the PEDF mRNA expression levels were also upregulated correspondingly [1.535±0.199, 1.078±0.167, 1.295±0.012 and 1.500±0.104 respectively at 1, 3, 6 and 9 h]. However, the difference in the mRNA expression levels between stretch group and normal oxygen group was just significant at 1 h after the onset of mechanical stretching. Conclusion The microenvironmental hypoxia and mechanical strain significantly upregulate epidermal cellular PEDF expression respectively. Key words: Pigment epithelium-derived factor; Soft tissue expansion; HaCaT cells

Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

Retinal ganglion cells (RGCs) undergo rapid cell death by apoptosis after injury but can be rescued by suppression of caspase-2 (CASP2) using an siRNA to CASP2 (siCASP2). Pigment epithelium-derived factor (PEDF), has neuroprotective and anti-angiogenic functions and protects RGC from death. The purpose of this study was to investigate if suppression of CASP2 is a possible mechanism of neuroprotection by PEDF in RGC. Adult rat retinal cells were treated in vitro with sub-optimal and optimal concentrations of siCASP2 and PEDF and levels of CASP2 mRNA and RGC survival were then quantified. Optic nerve crush (ONC) injury followed by intravitreal injections of siCASP2 or PEDF and eye drops of PEDF-34 were also used to determine CASP2 mRNA and protein reduction. Results showed that PEDF and PEDF-34 significantly suppressed CASP2 mRNA in culture, by 1.85- and 3.04-fold, respectively, and increased RGC survival by 63.2 ± 3.8% and 81.9 ± 6.6%, respectively compared to cells grown in Neurobasal-A alone. RGC survival was significantly reduced in glial proliferation inhibited and purified RGC cultures suggesting that some of the effects of PEDF were glia-mediated. In addition, intravitreal injection of PEDF and eye drops of PEDF-34 after ONC also suppressed CASP2 mRNA levels by 1.82- and 3.89-fold and cleaved caspase-2 (C-CASP2) protein levels by 4.98- and 8.93-fold compared to ONC + PBS vehicle groups, respectively, without affecting other executioner caspases. Treatment of retinal cultures with PEDF and PEDF-34 promoted the secretion of neurotrophic factors (NTF) into the culture media, of which brain-derived neurotrophic factor (BDNF) caused the greatest reduction in CASP2 mRNA and C-CASP2 protein. The neuroprotective effects of PEDF were blocked by a polyclonal antibody and PEDF suppressed key elements in the apoptotic pathway. In conclusion, this study shows that some of the RGC neuroprotective effects of PEDF is regulated through suppression of CASP2 and downstream apoptotic signalling molecules.