Role of Pigment Epithelium-Derived Factor (PEDF) in Arsenic-Induced Cell Apoptosis of Liver and Brain in a Rat Model

Although studies have shown that arsenic exposure can induce apoptosis in a variety of cells, the exact molecular mechanism of chronic arsenicosis remains unclear. Based on our previous study on human serum, the present study was to determine whether pigment epithelium-derived factor (PEDF) plays a role in the damage induced by chronic arsenic exposure in a rat model and to explore the possible signaling pathway involved. Thirty male Wistar rats were randomly divided into three groups and the arsenite doses administered were 0, 10, and 50 mg/L, respectively. The experiment lasted for 6 months. Our results showed that level of arsenic increased significantly in serum, liver, brain, and kidney in arsenic-exposed groups. It was indicated that PEDF protein was widely distributed in the cytoplasm of various types of cells in liver, brain, and kidney. PEDF protein level was only changed when the arsenite dose reached 50 mg/L in liver and brain, whereas it was not changed in the kidney. In order to investigate the possible mechanism of PEDF-exerted damages upon arsenite exposure, apoptosis in liver and brain was assessed. The proportion of apoptotic cells gradually increased with increasing arsenic administration. The ratio of Bax/Bcl-2 in the high arsenic group (50 mg/L) was significantly higher than that in the control group. Therefore, we thought PEDF played a role in cell apoptosis of liver and brain which induced by sodium arsenite exposure, and the results also demonstrated that Bax and Bcl-2 might be two key targets in the action of PEDF.     

Pigment Epithelium-derived Factor (PEDF) Prevents Retinal Cell Death via PEDF Receptor (PEDF-R): IDENTIFICATION OF A FUNCTIONAL LIGAND BINDING SITE*

The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu¹⁵⁹–Met³²⁵) with affinity similar to the full-length PEDF-R (Met¹–Leu⁵⁰⁴). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile¹⁹³–Leu²³²) and P1 (Thr²¹⁰–Leu²⁴⁹) peptides. Recombinant C-terminal truncated PEDF-R4 (Met¹–Leu²³²) and internally truncated PEDF-R and PEDF-R4 (ΔHis²⁰³–Leu²³²) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His²⁰³–Leu²³² region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.     

Antagonism of CD11b with Neutrophil Inhibitory Factor (NIF) Inhibits Vascular Lesions in Diabetic Retinopathy

Leukocytes and proteins that govern leukocyte adhesion to endothelial cells play a causal role in retinal abnormalities characteristic of the early stages of diabetic retinopathy, including diabetes-induced degeneration of retinal capillaries. Leukocyte integrin αmβ2 (CD11b/CD18, MAC1), a protein mediating adhesion, has been shown to mediate damage to endothelial cells by activated leukocytes in vitro. We hypothesized that Neutrophil Inhibitory Factor (NIF), a selective antagonist of integrin αmβ2, would inhibit the diabetes-induced degeneration of retinal capillaries by inhibiting the excessive interaction between leukocytes and retinal endothelial cells in diabetes. Wild type animals and transgenic animals expressing NIF were made diabetic with streptozotocin and assessed for diabetes-induced retinal vascular abnormalities and leukocyte activation. To assess if the leukocyte blocking therapy compromised the immune system, animals were challenged with bacteria. Retinal superoxide production, leukostasis and leukocyte superoxide production were increased in wild type mice diabetic for 10 weeks, as was the ability of leukocytes isolated from diabetic animals to kill retinal endothelial cells in vitro. Retinal capillary degeneration was significantly increased in wild type mice diabetic 40 weeks. In contrast, mice expressing NIF did not develop any of these abnormalities, with the exception that non-diabetic and diabetic mice expressing NIF generated greater amounts of superoxide than did similar mice not expressing NIF. Importantly, NIF did not significantly impair the ability of mice to clear an opportunistic bacterial challenge, suggesting that NIF did not compromise immune surveillance. We conclude that antagonism of CD11b (integrin αmβ2) by NIF is sufficient to inhibit early stages of diabetic retinopathy, while not compromising the basic immune response.     

Pigment Epithelial-derived Factor (PEDF)-triggered Lung Cancer Cell Apoptosis Relies on p53 Protein-driven Fas Ligand (Fas-L) Up-regulation and Fas Protein Cell Surface Translocation

Pigment epithelium-derived factor (PEDF), a potent antiangiogenesis agent, has recently attracted attention for targeting tumor cells in several types of tumors. However, less is known about the apoptosis-inducing effect of PEDF on human lung cancer cells and the underlying molecular events. Here we report that PEDF has a growth-suppressive and proapoptotic effect on lung cancer xenografts. Accordingly, in vitro, PEDF apparently induced apoptosis in A549 and Calu-3 cells, predominantly via the Fas-L/Fas death signaling pathway. Interestingly, A549 and Calu-3 cells are insensitive to the Fas-L/Fas apoptosis pathway because of the low level of cell surface Fas. Our results revealed that, in addition to the enhancement of Fas-L expression, PEDF increased the sensitivity of A549 and Calu-3 cells to Fas-L-mediated apoptosis by triggering the translocation of Fas protein to the plasma membrane in a p53- and FAP-1-dependent manner. Similarly, the up-regulation of Fas-L by PEDF was also mediated by p53. Furthermore, peroxisome proliferator-activated receptor γ was determined to be the upstream regulator of p53. Together, these findings uncover a novel mechanism of tumor cell apoptosis induced by PEDF and provide a potential therapeutic strategy for tumors that are insensitive to Fas-L/Fas-dependent apoptosis because of a low level of cell surface Fas.     

Enterostatin (VPDPR) and Its Peptide Fragment DPR Reduce Serum Cholesterol Levels after Oral Administration in Mice

We found that enterostatin (VPDPR), an anorexigenic peptide for a high-fat diet, significantly reduces serum cholesterol levels after oral administration of 100 mg/kg for 3 days in mice fed a high cholesterol-cholic acid diet. DPR, a peptide fragment of VPDPR, also had hypocholesterolemic activity at a dose of 50 mg/kg. Food intake was not suppressed under these dietary conditions. Fecal excretion of cholesterol and bile acids was increased significantly by both VPDPR and DPR. Interestingly, DPR induced hypocholesterolemic effects just two hours after a single oral administration at a dose of 100 mg/kg.     

Reduced Levels of Brain Derived Neurotrophic Factor (BDNF) in the Serum of Diabetic Retinopathy Patients and in the Retina of Diabetic Rats

Diabetic retinopathy (DR) is widely recognized as a neurovascular disease. Retina, being a neuronal tissue of the eye, produces neurotrophic factors for its maintenance. However, diabetes dysregulates their levels and thereby may damage the retina. Among neurotrophins, brain derived neurotrophic factor (BDNF) is the most abundant in the retina. In this study, we investigated the level of BDNF in the serum of patients with DR and also in the serum and retina of streptozotocin-induced diabetic rats. The level of BDNF was significantly decreased in the serum of proliferative diabetic retinopathy patients as compared to that of non-diabetic healthy controls (25.5 ± 8.5–10.0 ± 8.1 ng/ml, p < 0.001) as well as compared to that of diabetic patients with no retinopathy (21.8 ± 4.7–10.0 ± 8.1 ng/ml, p < 0.001), as measured by ELISA techniques. The levels of BDNF in the serum and retina of diabetic rats were also significantly reduced compared to that of non-diabetic controls (p < 0.05). In addition, the expression level of tropomyosin-related kinase B (TrkB) was significantly decreased in diabetic rat retina compared to that of non-diabetic controls as determined by Western blotting technique. Caspase-3 activity was increased in diabetic rat retina after 3 weeks of diabetes and remained elevated until 10 weeks, which negatively correlated with the level of BDNF (r = ?0.544, p = 0.013). Our results indicate that reduced levels of BDNF in diabetes may cause apoptosis and neurodegeneration early in diabetic retina, which may lead to neuro-vascular damage later in DR.     

Pigment epithelium-derived factor (PEDF) interacts with transportin SR2, and active nuclear import is facilitated by a novel nuclear localization motif

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.     

Stem Cell Transplantation Upregulates Sirt1 and Antioxidant Expression,Ameliorating Fatty Liver in Type 2 Diabetic Mice

Nonalcoholic fatty liver disease (NAFLD) is associated with insulin resistance, oxidative stress, and obesity. The db/db mouse model displays increased levels of insulin resistance, obesity, and an over-accumulation of hepatic triglycerides, making it an excellent model for studying NAFLD. In db/db mice, intra-bone marrow-bone marrow transplantation plus thymus transplantation (IBM-BMT+TT) improves type 2 diabetes mellitus (T2 DM) by normalizing the T-cell imbalance. We hypothesized that this approach would improve Sirt1 expression in the liver and benefit liver development.The db/db mice were treated with IBM-BMT+TT, and plasma MCP-1, IL-6, adiponection, LDL, Sirt1, and HO-1 levels were then assessed. Stem cell transplantation decreased the levels of plasma inflammatory cytokines and LDL while it increased the expression of Sirt1 and HO-1, resulting in decreased progression of fatty liver. Moreover, Sirt1 and HO-1 expression were both detected in the thymus and many HO-1-positive cells were observed in the bone marrow.This is the first report of stem cell transplantation improving the antioxidant function in the liver, thymus, and bone marrow of db/db mice by increasing the levels of Sirt1 and HO-1. This approach may prove useful in the treatment of nonalcoholic steatohepatitis and its clinical manifestations.     

B Cell Activating Factor (BAFF) and T Cells Cooperate to Breach B Cell Tolerance in Lupus-Prone New Zealand Black (NZB) Mice

The presence of autoantibodies in New Zealand Black (NZB) mice suggests a B cell tolerance defect however the nature of this defect is unknown. To determine whether defects in B cell anergy contribute to the autoimmune phenotype in NZB mice, soluble hen egg lysozyme (sHEL) and anti-HEL Ig transgenes were bred onto the NZB background to generate double transgenic (dTg) mice. NZB dTg mice had elevated levels of anti-HEL antibodies, despite apparently normal B cell functional anergy in-vitro. NZB dTg B cells also demonstrated increased survival and abnormal entry into the follicular compartment following transfer into sHEL mice. Since this process is dependent on BAFF, BAFF serum and mRNA levels were assessed and were found to be significantly elevated in NZB dTg mice. Treatment of NZB sHEL recipient mice with TACI-Ig reduced NZB dTg B cell survival following adoptive transfer, confirming the role of BAFF in this process. Although NZB mice had modestly elevated BAFF, the enhanced NZB B cell survival response appeared to result from an altered response to BAFF. In contrast, T cell blockade had a minimal effect on B cell survival, but inhibited anti-HEL antibody production. The findings suggest that the modest BAFF elevations in NZB mice are sufficient to perturb B cell tolerance, particularly when acting in concert with B cell functional abnormalities and T cell help.     

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Signaling and Cell Death in the Immature Central Nervous System after Hypoxia-Ischemia and Inflammation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family. The interaction of TRAIL with death receptor 4 (DR4) and DR5 can trigger apoptotic cell death. The aim of this study was to investigate the role of TRAIL signaling in neonatal hypoxia-ischemia (HI). Using a neonatal mouse model of HI, mRNA, and protein expression of TRAIL, DR5 and the TRAIL decoy receptors osteoprotegerin (OPG), mDcTRAILR1, and mDcTRAILR2 were determined. In vitro, mRNA expression of these genes was measured in primary neurons and oligodendrocyte progenitor cells (OPCs) after inflammatory cytokine (TNF-α/IFN-γ) treatment and/or oxygen and glucose deprivation (OGD). The toxicity of these various paradigms was also measured. The expression of TRAIL, DR5, OPG, and mDcTRAILR2 was significantly increased after HI. In vitro, inflammatory cytokines and OGD treatment significantly induced mRNAs for TRAIL, DR5, OPG, and mDcTRAILR2 in primary neurons and of TRAIL and OPG in OPCs. TRAIL protein was expressed primarily in microglia and astroglia, whereas DR5 co-localized with neurons and OPCs in vivo. OGD enhanced TNF-α/IFN-γ toxicity in both neuronal and OPC cultures. Recombinant TRAIL exerted toxicity alone or in combination with OGD and TNF-α/IFN-γ in primary neurons but not in OPC cultures. The marked increases in the expression of TRAIL and its receptors after cytokine exposure and OGD in primary neurons and OPCs were similar to those found in our animal model of neonatal HI. The toxicity of TRAIL in primary neurons suggests that TRAIL signaling participates in neonatal brain injury after inflammation and HI.     

Impairment of Colour Vision in Diabetes with No Retinopathy: Sankara Nethralaya Diabetic Retinopathy Epidemiology and Molecular Genetics Study (SNDREAMS- II,Report 3)

Purpose

To assess impairment of colour vision in type 2 diabetics with no diabetic retinopathy and elucidate associated risk factors in a population-based cross-sectional study.

Methods

This is part of Sankara Nethralaya Diabetic Retinopathy Epidemiology and Molecular-genetics Study (SN-DREAMS II) which was conducted between 2007–2010. FM 100 hue-test was performed in 253 subjects with no clinical evidence of diabetic retinopathy. All subjects underwent detailed ophthalmic evaluation including cataract grading using LOCS III and 45° 4-field stereoscopic fundus photography. Various ocular and systemic risk factors for impairment of colour vision (ICV) were assessed in subjects with diabetes but no retinopathy. P value of < 0.05 was considered statistically significant.

Results

The mean age of the study sample was 57.08 ± 9.21 (range: 44–86 years). Gender adjusted prevalence of ICV among subjects with diabetes with no retinopathy was 39.5% (CI: 33.5–45.5). The mean total error score in the study sample was 197.77 ± 100 (range: 19–583). The risk factors for ICV in the study were women OR: 1.79 (1.00–3.18), increased resting heart rate OR: 1.04 (1.01–1.07) and increased intraocular pressure OR: 1.12 (1.00–1.24). Significant protective factor was serum high-density lipoprotein OR: 0.96 (0.93–0.99).

Conclusions

Acquired ICV is an early indicator of neurodegenerative changes in the retina. ICV found in diabetic subjects without retinopathy may be of non-vascular etiology.     

Susceptibility to Seizure-Induced Excitotoxic Cell Death Is Regulated by an Epistatic Interaction between Chr 18 (Sicd1) and Chr 15 (Sicd2) Loci in Mice

Seizure-induced cell death is believed to be regulated by multiple genetic components in addition to numerous external factors. We previously defined quantitative trait loci that control susceptibility to seizure-induced cell death in FVB/NJ (susceptible) and C57BL/6J (resistant) mice. Two of these quantitative trait loci assigned to chromosomes 18 (Sicd1) and 15 (Sicd2), control seizure-induced cell death resistance. In this study, through the use of a series of novel congenic strains containing the Sicd1 and Sicd2 congenic strains and different combinations of the Sicd1 or Sicd2 sub region(s), respectively, we defined these genetic interactions. We generated a double congenic strain, which contains the two C57BL/6J differential segments from chromosome 18 and 15, to determine how these two segments interact with one another. Phenotypic comparison between FVB-like littermates and the double congenic FVB.B6-Sicd1/Sicd2 strain identified an additive effect with respect to resistance to seizure-induced excitotoxic cell death. It thus appears that C57BL/6J alleles located on chromosomes 18 and 15 interact epistatically in an additive manner to control the extent of seizure-induced excitotoxic cell death. Three interval-specific congenic lines were developed, in which either segments of C57BL/6J Chr 18 or C57BL/6J Chr 15 were introduced in the FVB/NJ genetic background, and progeny were treated with kainate and examined for the extent of seizure-induced cell death. All of the interval-specific congenic lines exhibited reduced cell death in both area CA3 and the dentate hilus, associated with the C57BL/6J phenotype. These experiments demonstrate functional interactions between Sicd1 and Sicd2 that improve resistance to seizure-induced excitotoxic cell death, validating the critical role played by gene-gene interactions in excitotoxic cell death.     

Scorpion Venom Heat-Resistant Peptide (SVHRP) Enhances Neurogenesis and Neurite Outgrowth of Immature Neurons in Adult Mice by Up-Regulating Brain-Derived Neurotrophic Factor (BDNF)

Scorpion venom heat-resistant peptide (SVHRP) is a component purified from Buthus martensii Karsch scorpion venom. Although scorpions and their venom have been used in Traditional Chinese Medicine (TCM) to treat chronic neurological disorders, the underlying mechanisms of these treatments remain unknown. We applied SVHRP in vitro and in vivo to understand its effects on the neurogenesis and maturation of adult immature neurons and explore associated molecular mechanisms. SVHRP administration increased the number of 5-bromo-2’-dexoxyuridine (BrdU)-positive cells, BrdU- positive/neuron-specific nuclear protein (NeuN)-positive neurons, and polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive immature neurons in the subventricular zone (SVZ) and subgranular zone (SGZ) of hippocampus. Furthermore immature neurons incubated with SVHRP-pretreated astrocyte-conditioned medium exhibited significantly increased neurite length compared with those incubated with normal astrocyte-conditioned medium. This neurotrophic effect was further confirmed in vivo by detecting an increased average single area and whole area of immature neurons in the SGZ, SVZ and olfactory bulb (OB) in the adult mouse brain. In contrast to normal astrocyte-conditioned medium, higher concentrations of brain-derived neurotrophic factor (BDNF) but not nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) was detected in the conditioned medium of SVHRP-pretreated astrocytes, and blocking BDNF using anti-BDNF antibodies eliminated these SVHRP-dependent neurotrophic effects. In SVHRP treated mouse brain, more glial fibrillary acidic protein (GFAP)-positive cells were detected. Furthermore, immunohistochemistry revealed increased numbers of GFAP/BDNF double-positive cells, which agrees with the observed changes in the culture system. This paper describes novel effects of scorpion venom-originated peptide on the stem cells and suggests the potential therapeutic values of SVHRP.     

Statocyte and Ocellar Pigment Cell in Embryos and Larvae of the Ascidian, Styela plicata (Lesueur)

The processes of formation of two pigmented cells, the statocyte and the ocellar pigment cell, in the cerebral vesicle of larvae of the ascidian Styela plicata were investigated in whole mount specimens and serial paraffin sections by light microscopy. The pigmentations of the two cells became visible simultaneously in embryos at the stage of tail elongation, 5–6 hr after fertilization. The pigmented cells were at first located side by side in the dorsal wall of the neurocoel. Growth of the pigment mass in the ocellus ceased at about 6.5 hr, while that in the statocyte continued through the hatching period (9–10 hr) up to the swimming stage. The pigment mass in the statocyte consisted of two blocks which joined together during their growth. The statocyte migrated from the dorsal to the ventral wall of the cerebral vesicle by the swimming stage. In swimming larvae, the more ventral of the two pigment blocks of the statocyte formed an inverted pigment cup and a cluster of protuberances projected into it from the ventral wall of the cerebral vesicle. Phylogenetically, the sensory organs in the cerebral vesicle of Styela plicata seem intermediate between those in Pyuridae and Botryllinae with respect of their structure and process of differentiation.     

Cardiac-Specific Over-Expression of Epidermal Growth Factor Receptor 2 (ErbB2) Induces Pro-Survival Pathways and Hypertrophic Cardiomyopathy in Mice

Background

Emerging evidence shows that ErbB2 signaling has a critical role in cardiomyocyte physiology, based mainly on findings that blocking ErbB2 for cancer therapy is toxic to cardiac cells. However, consequences of high levels of ErbB2 activity in the heart have not been previously explored.

Methodology/Principal Findings

We investigated consequences of cardiac-restricted over-expression of ErbB2 in two novel lines of transgenic mice. Both lines develop striking concentric cardiac hypertrophy, without heart failure or decreased life span. ErbB2 transgenic mice display electrocardiographic characteristics similar to those found in patients with Hypertrophic Cardiomyopathy, with susceptibility to adrenergic-induced arrhythmias. The hypertrophic hearts, which are 2–3 times larger than those of control littermates, express increased atrial natriuretic peptide and β-myosin heavy chain mRNA, consistent with a hypertrophic phenotype. Cardiomyocytes in these hearts are significantly larger than wild type cardiomyocytes, with enlarged nuclei and distinctive myocardial disarray. Interestingly, the over-expression of ErbB2 induces a concurrent up-regulation of multiple proteins associated with this signaling pathway, including EGFR, ErbB3, ErbB4, PI3K subunits p110 and p85, bcl-2 and multiple protective heat shock proteins. Additionally, ErbB2 up-regulation leads to an anti-apoptotic shift in the ratio of bcl-xS/xL in the heart. Finally, ErbB2 over-expression results in increased activation of the translation machinery involving S6, 4E-BP1 and eIF4E. The dependence of this hypertrophic phenotype on ErbB family signaling is confirmed by reduction in heart mass and cardiomyocyte size, and inactivation of pro-hypertrophic signaling in transgenic animals treated with the ErbB1/2 inhibitor, lapatinib.

Conclusions/Significance

These studies are the first to demonstrate that increased ErbB2 over-expression in the heart can activate protective signaling pathways and induce a phenotype consistent with Hypertrophic Cardiomyopathy. Furthermore, our work suggests that in the situation where ErbB2 signaling contributes to cardiac hypertrophy, inhibition of this pathway may reverse this process.