Pigment epithelium-derived factor (PEDF), a serpin with potent anti-angiogenic and neurite outgrowth-promoting properties

Pigment epithelium-derived factor is a member of the serpin superfamily of proteins, but one that lacks inhibitory properties against either serine or cysteine proteinases. Nevertheless it possesses a number of physiological properties that make it a potentially important protein in regulation of angiogenesis, in neuronal cell survival and in protection of neurons from neurotoxic agents. It is also a protein that is highly up-regulated in the G0 phase of early-passage cells compared with rapidly proliferating cells or senescent cells, and so is also linked to both the cell cycle and cell senescence. The determination of a high resolution X-ray crystal structure of native PEDF provides insight into regions of the protein that may be involved in one or more of these functions.     

Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells.

Pigment epithelium-derived factor (PEDF) is a serpin found in the interphotoreceptor matrix of the eye, which, although not a proteinase inhibitor, possesses a number of important biological properties, including promotion of neurite outgrowth and differential expression in quiescent versus senescent states of certain cell types. The low amounts present in the eye, together with the impracticality of using the eye as a source for isolation of the human protein, make it important to establish a system for overexpression of the recombinant protein for biochemical and biological studies. We describe here the expression and secretion of full-length glycosylated human recombinant PEDF at high levels (> 20 micrograms/ mL) into the growth medium of baby hamster kidney cells and characterization of the purified rPEDF by circular dichroism and fluorescence spectroscopies and neurite outgrowth assay. By these assays, the recombinant protein behaves as expected for a correctly folded full-length human PEDF. The availability of milligram amounts of PEDF has permitted quantitation of its heparin binding properties and of the effect of reactive center cleavage on the stability of PEDF towards thermal and guanidine hydrochloride denaturation.     

Binding of pigment epithelium-derived factor (PEDF) to retinoblastoma cells and cerebellar granule neurons. Evidence for a PEDF receptor.

Pigment epithelium-derived factor (PEDF) has neuronal differentiation and survival activity on retinoblastoma and cerebellar granule (CG) cells. Here, we investigated the presence of PEDF receptors on retinoblastoma Y-79 and CG cells. PEDF radiolabeled with (l25)I remained biologically active and was used for radioligand binding analysis. The binding was saturable and specific to a single class of receptors on both cells and with similar affinities (K(d) = 1.7-3.6 nM, B(max) = 0.5-2.7 x 10(5) sites/Y-79 cell; and K(d) = 3.2 nM, B(max) = 1.1 x 10(3) sites/CG cell). A polyclonal antiserum to PEDF, previously shown to block the PEDF neurotrophic activity, prevented the (125)I-PEDF binding. We designed two peptides from a region previously shown to confer the neurotrophic property to human PEDF, synthetic peptides 34-mer (positions 44-77) and 44-mer (positions 78-121). Only peptide 44-mer competed for the binding to Y-79 cell receptors (EC(50) = 5 nM) and exhibited neuronal differentiating activity. PEDF affinity column chromatography of membrane proteins from both cell types revealed a PEDF-binding protein of approximately 80 kDa. These results are the first demonstration of a PEDF-binding protein with characteristics of a PEDF receptor and suggest that the region comprising amino acid positions 78-121 of PEDF might be involved in ligand-receptor interactions.     

Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris

Pigment epithelium-derived factor (PEDF) combines neurotrophic, neuroprotective, anti-angiogenic, anti-tumor and neural stem cell self-renewal properties in a single molecule, making this protein a valuable potential therapeutic agent. We herein analyzed the expression of human recombinant full-length PEDF, and its N- and C-terminal regions (amino acids 1-243 and 195-418, respectively) in three mammalian cell lines (HEK-293T, COS-1, and 26HCMsv), and in the yeast Pichia pastoris. The highest production of recombinant PEDF was achieved in P. pastoris which secreted approximately 30 microg of full-length rPEDF, and 47 microg of C-terminal/ml of culture medium. Full-length rPEDF was purified by one-step Ni-chelating high-performance liquid chromatography, recovering almost 70% of secreted rPEDF with a purity of 98.6%. The C-terminal region of PEDF was isolated by low-pressure liquid chromatography, recovering around 4% of the recombinant molecule with a purity of 98%. The N-terminal region of PEDF was not secreted by any expression system assayed. The two isolated recombinant PEDF polypeptides inhibited in vitro endothelial cell migration, and full-length rPEDF also increased cerebellar granule cell survival, thus demonstrating their biological activity. These polypeptides can be used to investigate the therapeutic role of PEDF in cancer, neurodegenerative and ocular diseases, and stem cell-based therapies.     

Diabetic nephropathy: serum miR-9 confers a poor prognosis in and is associated with level changes of vascular endothelial growth factor and pigment epithelium-derived factor

Objective

To investigate the relationship between the serum level of miR-9 and the progression of diabetic nephropathy (DN) and related molecular mechanisms.

Results

Thirty-five healthy subjects and 140 DN patients were divided into five groups: control, DN I–II, DN III, DN IV and DN V. Serum level of miR-9 was measured by real-time qPCR. Serum levels of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF) lipids, fasting glucose, insulin, hemoglobin A1c (HBA1c), creatinine, fibrinogen and insulin resistance (HOMA-IR) were also measured. The results show that the levels of miR-9, PEDF and VEGF are increased with DN progression (P < 0.05). miR-9, VEGF and PEDF are independent risk factors of DN (R² = 0.430). Spearman rank correlation analysis showed that miR-9 level is positively related to the levels of VEGF, PEDF, cholesterol, triglyceride, fasting glucose, fasting insulin, HBA1c, creatinine, fibrinogen and HOMA-IR (P < 0.05).

Conclusions

Serum miR-9 is a potential marker for conferring a poor prognosis in DN and associated with the levels of VEGF, PEDF and biochemical indices.

     

Increased levels of vascular endothelial growth factor and advanced glycation end products in aqueous humor of patients with diabetic retinopathy.

Clinical studies have shown a relationship between diabetic retinopathy and vascular endothelial growth factor (VEGF) levels in ocular fluid. Advanced glycation end products (AGEs) have been implicated in diabetes complications, including diabetic retinopathy. Nepsilon-(carboxymethyl) lysine (CML) is a glycoxidation product that may be a marker of oxidative stress. In this study, we used enzyme-linked immunosorbent assays to determine the levels of VEGF, non-CML AGE and CML in the aqueous humor and serum of 82 Japanese patients with type 2 diabetes and 60 non-diabetic subjects. VEGF, non-CML AGE, and CML concentrations in aqueous humor and serum were then compared with the severity of diabetic retinopathy. Immunohistochemical detection analysis of non-CML AGE and CML was also performed using retinal tissues from patients with progressive diabetic retinopathy. Aqueous levels of VEGF, non-CML AGE and CML increased along with the progression of diabetic retinopathy compared to age-matched controls. After coagulation therapy, the VEGF, non-CML AGE, and CML levels were significantly reduced. Immunostaining showed diffuse co-localization of non-CML AGE and CML around microvessels and in the glial cells of proliferative membranes from patients with progressive diabetic retinopathy. These findings suggest that glycation and glycoxidation reactions (or oxidation, as revealed by CML) may contribute to both the onset and progression of diabetic retinopathy.     

Down-regulation of vascular endothelial growth factor and up-regulation of pigment epithelium-derived factor: a possible mechanism for the anti-angiogenic activity of plasminogen kringle 5.

We have previously shown that intravitreal injection of plasminogen kringle 5 (K5), a potent angiogenic inhibitor, inhibits ischemia-induced retinal neovascularization in a rat model. Here we report that K5 down-regulates an endogenous angiogenic stimulator, vascular endothelial growth factor (VEGF) and up-regulates an angiogenic inhibitor, pigment epithelium-derived factor (PEDF) in a dose-dependent manner in vascular cells and in the retina. The regulation of VEGF and PEDF by K5 in the retina correlates with its anti-angiogenic effect in a rat model of ischemia-induced retinopathy. Retinal RNA levels of VEGF and PEDF are also changed by K5. K5 inhibits the p42/p44 MAP kinase activation and nuclear translocation of hypoxia-inducible factor-1alpha, which may be responsible for the down-regulation of VEGF. Down-regulation of endogenous angiogenic stimulators and up-regulation of endogenous angiogenic inhibitors, thus leading toward restoration of the balance in angiogenic control, may represent a mechanism for the anti-angiogenic activity of K5.     

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.     

Low lipoprotein(a) concentration is associated with cancer and all-cause deaths: a population-based cohort study (the JMS cohort study)

Background

Experimental studies support the anti-neoplastic effect of apo(a), but several clinical studies have reported contradictory results. The purpose of this study was to determine whether a low lipoprotein(a) [Lp(a)] concentration is related to mortality from major causes of death, especially cancer.

Methods

The subjects were 10,413 participants (4,005 men and 6,408 women) from a multi-center population-based cohort study in Japan (The Jichi Medical School cohort study). The average age at registration was 55.0 years, and the median observation period was 4,559 days. As the estimated hazard ratio was high for both the low and very high Lp(a) levels, we defined two Lp(a) groups: a low Lp(a) group [Lp(a)<80 mg/L] and an intermediate-to-high Lp(a) group [Lp(a)≥80]. Participants who died from malignant neoplasms (n = 316), cardiovascular disease (202), or other causes (312) during the observation period were examined.

Results

Cumulative incidence plots showed higher cumulative death rates for the low Lp(a) group than for the intermediate-to-high Lp(a) group for all-cause, cancer, and miscellaneous-cause deaths (p<0.001, p = 0.03, and p = 0.03, respectively). Cox proportional hazards analyses with the sex and age of the participants, body mass index, and smoking and drinking histories as covariates showed that a low Lp(a) level was a significant risk for all-cause, cancer, and miscellaneous-cause deaths (p<0.001, p = 0.003, and p = 0.01, respectively). The hazard ratio (95% CI) [1.48, 1.15–1.92] of a low Lp(a) level for cancer deaths was almost the same as that for a male sex (1.46, 1.00–2.13).

Conclusions

This is the first report to describe the association between a low Lp(a) level and all-cause or cancer death, supporting the anti-neoplastic effect of Lp(a). Further epidemiological studies are needed to confirm the present results.     

Candidate gene case-control and functional study shows macrophage inhibitory factor (MIF) polymorphism is associated with cutaneous leishmaniasis

American tegumentary leishmaniasis (ATL) is an infectious disease caused mostly by Leishmania (Viannia) braziliensis in Southeast Brazil. The clinical manifestations are vast, ranging from asymptomatic to severe mucosal leishmaniasis (ML). It has been suggested that variation of the pathogen does not fully explain the response spectrum and the variability of clinical manifestations. Previous data have shown that host genetics also play a role in disease outcome. Herein, we have tested the association of TNF, IL10, IL12 and MIF single nucleotide polymorphisms (SNPs) using a case-control study design including 110 cutaneous leishmaniasis (CL) patients and 682 healthy subjects. The genotype–phenotype correlation was also assessed using leishmania antigens to stimulate peripheral blood mononuclear cells obtained from cured CL patients. Results demonstrated that the MIF ?173C allele is associated with leishmaniasis outcome and also with lower levels of MIF in culture supernatants. Also, the TNF ?308AA genotype was statistically increased among leishmaniasis patients. The results showed here suggest that the lower levels of MIF produced by MIF ?173C carriers could influence the host–Leishmania interaction, favoring infection and disease progression. On the other hand, high TNF levels can contribute to tissue damage, consequently leading to skin lesions.     

High plasma level of long pentraxin 3 (PTX3) is associated with fatal disease in bacteremic patients: a prospective cohort study

Introduction

Long pentraxin 3 (PTX3) is an acute-phase protein secreted by various cells, including leukocytes and endothelial cells. Like C-reactive protein (CRP), it belongs to the pentraxin superfamily. Recent studies indicate that high levels of PTX3 may be associated with mortality in sepsis. The prognostic value of plasma PTX3 in bacteremic patients is unknown.

Methods

Plasma PTX3 levels were measured in 132 patients with bacteremia caused by Staphylococcus aureus, Streptococcus pneumoniae, β-hemolytic streptococcae and Escherichia coli, using a commercial solid-phase enzyme-linked immunosorbent assay (ELISA). Values were measured on days 1–4 after positive blood culture, on day 13–18 and on recovery.

Results

The maximum PTX3 values on days 1–4 were markedly higher in nonsurvivors compared to survivors (44.8 vs 6.4 ng/ml, p<0.001) and the AUC^ROC in the prediction of case fatality was 0.82 (95% CI 0.73–0.91). PTX3 at a cut-off level of 15 ng/ml showed 72% sensitivity and 81% specificity for fatal disease. High PTX3 (>15 ng/ml) was associated with hypotension (MAP <70 mmHg)(OR 7.9;95% CI 3.3–19.0) and high SOFA score (≥4)(OR 13.2; 95% CI 4.9–35.4). The CRP level (maximum value on days 1 to 4) did not predict case fatality at any cut-off level in the ROC curve (p = 0.132). High PTX3 (>15 ng/ml) remained an independent risk factor for case fatality in a logistic regression model adjusted for potential confounders.

Conclusions

PTX3 proved to be a specific independent prognostic biomarker in bacteremia. PTX3 during the first days after diagnosis showed better prognostic value as compared to CRP, a widely used biomarker in clinical settings. PTX3 measurement offers a novel opportunity for the prognostic stratification of bacteremia patients.     

Decreased steady-state insulin-like growth factor binding protein-3 (IGFBP-3) mRNA level is associated with differentiation of cultured porcine myogenic cells

Insulin-like growth factor binding proteins (IGFBPs) affect the biological activity of IGF-I in several cell types, including cultured muscle cells. Additionally, at least one of the IGFBPs, IGFBP-3, has been shown to have IGF-independent effects on cell proliferation. Numerous studies have shown that immortalized muscle cell lines produce various IGFBPs, but to date no muscle cell line has been reported to produce IGFBP-3 protein or mRNA. Unlike muscle cell lines, primary cultures of porcine embryonic myogenic cells express IGFBP-3 mRNA and secrete a protein that is immunologically identifiable as IGFBP-3. Additionally, steady-state IGFBP-3 levels change significantly during differentiation. Here we report that differentiation of porcine myogenic cells in an IGFBP-3-free medium is accompanied by reduced steady-state IGFBP-3 mRNA levels. Steady-state levels of IGFBP-3 mRNA decreased approximately sevenfold (P < .05) during differentiation and then increased to predifferentiation levels once differentiation was complete. Addition of TGF-beta1 (0.5 ng/ml) to porcine myogenic cultures suppressed fusion and resulted in a sevenfold increase in steady-state IGFBP-3 mRNA and a 1.8-fold increase in IGFBP-3 protein levels as compared to untreated control cultures (P < .05). Results suggest that alterations in IGFBP-3 mRNA and protein may play a role in differentiation of porcine embryonic muscle cells.     

Newly diagnosed latent autoimmune diabetes in adults (LADA) is associated with low level glutamate decarboxylase (GAD65) and IA-2 autoantibodies. Diabetes Incidence Study in Sweden (DISS).

A quantitative assay with microSepharose was used to determine GAD65Ab and IA-2Ab levels in 771 population-based patients diagnosed with diabetes mellitus at 15 to 34 years of age, and in 828 matched controls. Among the patients, 587 (76%) were classified with type I, 108 (14%) with type II, and 76 (10%) with unclassifiable diabetes. The levels above normal demonstrated a prevalence of GAD65Ab in 66% of type I diabetes, 50% of type II diabetes and 54% of unclassifiable patients and for IA-2Ab in 40%, 17% and 21%, respectively. Among the autoantibody-positive sera, the LADA patients had a lower GAD65Ab index (median 0.19, p < 0.0001) and IA-2Ab index (median 0.28, p < 0.0001) than the type I patients (median 0.37 and 0.66). Patients with unclassifiable diabetes had a GAD65Ab (median 0.43) or IA-2Ab (median 0.63) index which was not different from the type I diabetes patients. Our data demonstrate that young adult new-onset LADA patients have low level GAD65Ab and IA-2Ab. The low-level autoantibodies may signify a less aggressive beta-cell autoimmunity, which may explain why these patients are often classified with type II or non-insulin-dependent diabetes.     

Association of a low molecular weight helper factor(s) with thymocyte proliferative activity.

Human mixed leukocyte supernatants contain thymocyte proliferative activity (TPA) and a low m.w. helper factor, designated HP-1, which is capable of partially restoring the antibody response of T-cell-deficient adherent murine spleen cells to the thymic-dependent antigen, SRC. TPA and HP-1 appear to have a comparable m.w. (14,000 to 14,500 daltons) by Sephadex gel filtration column chromatography. Furthermore, HP-1 and TPA exhibit similar patterns of heterogeneity on DEAE-cellulose chromatography, elute together on CM-cellulose chromatography, and manifest identical patterns of migration on polyacrylamide gel electrophoresis. These data suggest that the TPA and HP-1 activities reside in either the same molecule(s) or in different molecules with identical charge/mass ratios. Furthermore, the results support the hypothesis that the helper activity of HP-1 is derived from its capacity to activate T and/or pre-T cells.     

Receptor for advanced glycation end-product (RAGE) gene polymorphism 2245G/A is associated with pro-inflammatory,oxidative-glycation markers and sRAGE in diabetic retinopathy

Background

Receptor for advanced glycation end-product (RAGE) gene polymorphism 2245G/A is associated with diabetic retinopathy (DR). However, the mechanism on how it affects the disease development is still unclear.

Aim

This study aims to investigate the relationship between 2245G/A RAGE gene polymorphism and selected pro-inflammatory, oxidative-glycation markers in DR patients.

Methods

A total of 371 unrelated type 2 diabetic patients [200 with retinopathy, 171 without retinopathy (DNR)] and 235 healthy subjects were recruited. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism method followed by DNA sequencing. The nuclear and cytosolic extracts from peripheral blood mononuclear cells were used for nuclear factor kappa B (NF-κB) p65 and superoxide dismutase activity measurement respectively. Plasma was used for glutathione peroxidase activity, advanced oxidation protein product (AOPP), monocyte chemoattractant protein (MCP)-1, pentosidine and soluble RAGE (sRAGE) measurements.

Results

DR patients with 2245GA genotype had significantly elevated levels of activated NF-κB p65, plasma MCP-1, AOPP and pentosidine but lower level of sRAGE when compared to DR patients with wild-type 2245GG.

Conclusion

The RAGE gene polymorphism 2245G/A is associated with pro-inflammatory, oxidative-glycation markers and circulating sRAGE in DR patients. Patients with 2245GA RAGE genotype could aggravate DR possibly via NF-κB mediated inflammatory pathway.     

A PvuII polymorphism of the low density lipoprotein receptor gene is not associated with plasma concentrations of low density lipoproteins including LP(a)

Lipoprotein(a) [Lp(a)] is a low density lipoprotein (LDL), in which apolipoprotein B-100 (apo B-100) is attached to apolipoprotein(a) [apo(a)], a glycoprotein of variable size. Lp(a) may be as atherogenic as LDL. In normal populations, Lp(a) concentrations in plasma are largely determined by the apo(a) gene locus on chromosome 6, but regulation of synthesis and catabolism of Lp(a) is poorly understood. In some studies, a PvuII restriction fragment length polymorphism (RFLP) in the LDL receptor gene seems to affect concentrations of LDL in plasma, and other studies have indicated that Lp(a) catabolism could be mediated by the LDL receptor. We therefore expected that the PvuII polymorphism in the LDL receptor gene might be associated with Lp(a) levels in 170 Caucasian men aged 40 years, selected to have a high representation of low molecular weight apo(a) phenotypes. However, plasma concentrations of cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides and Lp(a) were all unrelated to the LDL receptor gene PvuII polymorphism both in the group as a whole and when it was subgrouped by apo(a) phenotype. Therefore our data do not support the concept that this particular LDL receptor gene polymorphism is associated with LDL receptor function, and our data therefore neither support nor rule out a role for the LDL receptor in Lp(a) catabolism.