Full-length Pigment Epithelium-derived Factor Research Articles

The contrary intracellular and extracellular functions of PEDF in HCC development

Pigment epithelium-derived factor (PEDF), a classic angiogenic inhibitor, has been reported to function as a tumor suppression protein and to downregulate in many types of solid tumors. However, the expression level of PEDF and its role in hepatocellular carcinoma (HCC) are contradictory. The present study investigates the expression and different activities of secreted and intracellular PEDF during HCC development, as well as the underlying mechanism of PEDF on HCC lipid disorders. We found that PEDF had no association with patients’ prognosis, although PEDF was highly expressed and inhibited angiogenesis in HCC tumor tissues. The animal experiments indicated that full-length PEDF exhibited equalizing effects on tumor growth activation and tumor angiogenesis inhibition in the late stage of HCC progression. Importantly, the pro-tumor activity was mediated by the intracellular PEDF, which causes accumulation of free fatty acids (FFAs) in vivo and in vitro. Based on the correlation analysis of PEDF and lipid metabolic indexes in human HCC tissues, we demonstrated that the intracellular PEDF led to the accumulation of FFA and eventually promoted HCC cell growth by inhibiting the activation of AMPK via ubiquitin–proteasome-mediated degradation, which causes increased de novo fatty acid synthesis and decreased FFA oxidation. Our findings revealed why elevated PEDF did not improve the patients’ prognosis as the offsetting intracellular and extracellular activities. This study will lead to a comprehensive understanding of the diverse role of PEDF in HCC and provide a new selective strategy by supplement of extracellular PEDF and downregulation of intracellular PEDF for the prevention and treatment of liver cancer.

Abnormal pigment epithelium-derived factor processing in progressive myopia

Pigment Epithelium-Derived Factor (PEDF) is a secreted glycoprotein belonging to the family of non-inhibitory serpins. It is known, that in cases of complicated myopia, the content of PEDF in aqueous humor of the anterior chamber is significantly reduced. Here we examined a bulk of Tenon's capsule samples obtained from various groups of myopes, to examine PEDF processing in progressive myopia. We have analyzed the distribution of full length PEDF50 and its truncated form PEDF45 in the soluble and insoluble fractions extracted from Tenon's capsule of myopic and control (non-myopic) patients using SDS-polyacrylamide gel electrophoresis, as well as monitored the proteolytic degradation of PEDF ex vivo by enzyme-linked immunosorbent assay. These results were complemented by PEDF mRNA analysis in correspondent tissues by using qPCR and immunohistochemistry analysis of PEDF distribution in normal and myopic specimens. We found that in the Tenon's capsule of patients suffering from a high myopia the level of “soluble” 45 kDa PEDF reduced by 2-fold, while the content of “insoluble” 50 kDa form of PEDF was increased by 4-fold compared to controls. Excessive amount of PEDF50 in myopic specimens have been shown to correlate with the abrogated PEDF processing rather than with an increase of its expression. Moreover, immunohistochemical staining of the myopic Tenon's capsule tissue sections revealed the halo of deposited PEDF50 in the fibroblast extracellular space. These findings suggest that in myopia limited proteolysis of PEDF is altered or abrogated. Accumulation of full-length PEDF insoluble aggregates in the fibroblast intercellular space may affect cell survival and consequently causes the destructive changes in the extracellular matrix of the eye connective tissues. As a result, the abrogation of full-length PEDF normal processing can be an important mechanism leading to biomechanical destabilization of the scleral capsule and myopia progression.

Pigment epithelium-derived factor 34-mer peptide prevents liver fibrosis and hepatic stellate cell activation through down-regulation of the PDGF receptor.

Pigment epithelium-derived factor (PEDF) has been shown previously to prevent liver fibrosis and hepatic stellate cell (HSC) activation. By investigating the functional domains in PEDF, we identified a 34-mer peptide (residues Asp44-Asn77) that harbors the same function as the full-length PEDF protein. Not only did the 34-mer suppress the development of fibrosis in carbon tetrachloride (CCl4)-treated mouse liver but it also upregulated peroxisome proliferator-activated receptor-gamma (PPARγ) expression in HSCs in vivo. Platelet-derived growth factor (PDGF) plays a crucial role on the process of HSC activation in response to liver damage. The 34-mer suppressed PDGF-induced cell proliferation and expression of myofibroblastic marker proteins in primary rat HSC culture, increased the levels of PPARγ mRNA and protein in a dose-dependent manner and markedly reduced the level of active β-catenin protein, an HSC activating factor, in HSC-T6 cells. Similarly, IWR-1, an inhibitor of the Wnt response, displayed the same effect as the 34-mer in preventing HSC-T6 activation. The Wnt signaling-mediated PPARγ suppression was abolished by both the IWR-1 inhibitor and a small interfering RNA (siRNA) targeting β-catenin and the Wnt coreceptor, LRP6. Both PEDF and the 34-mer down-regulated PDGF receptor-α/β expression and blocked the PDGF-induced phosphorylation of Akt and ERK. Moreover, the inhibitory effect on PDGF receptor expression was abolished by PPARγ antagonists and PPARγ siRNA. Our observations indicate that the PEDF-derived 34-mer peptide can mimic PEDF in attenuating HSC activation. Investigation of this 34-mer peptide led to the identification of a signaling mechanism involving PPARγ induction, suppression of Wnt/β-catenin signaling and down-regulation of the PDGF receptor-α/β.

Protective role of small pigment epithelium-derived factor (PEDF) peptide in diabetic renal injury

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with antiangiogenic, antioxidative, and anti-inflammatory properties. PEDF is involved in the pathogenesis of diabetic retinopathy, but its direct role in the kidneys remains unclear. We hypothesize that a PEDF fragment (P78-PEDF) confers kidney protection in diabetic nephropathy (DN). The localization of the full-length PEDF protein were determined in DBA mice following multiple low doses of streptozotocin. Using immunohistochemistry, PEDF was localized in the kidney vasculature, interstitial space, glomeruli, tubules, and renal medulla. Kidney PEDF protein and mRNA expression were significantly reduced in diabetic mice. Continuous infusion of P78-PEDF for 6 wk resulted in protection from diabetic neuropathy as indicated by reduced albuminuria and blood urea nitrogen, increased nephrin expression, decreased kidney macrophage recruitment and inflammatory cytokines, and reduced histological changes compared with vehicle-treated diabetic mice. In vitro, P78-PEDF blocked the increase in podocyte permeability to albumin and disruption of the actin cytoskeleton induced by puromycin aminonucleoside treatment. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in early phase diabetic renal injury.

Formation of amyloid-like fibrillar structures and destruction of fibroblasts of the Tenon’s capsule in progressive myopia due to resistance of the pigment epithelium-derived factor to restricted proteolysis

We have shown previously the presence of full length (50 kD) and truncated proteolytic form (45 kD) of pigment epithelium derived factor (PEDF) in the eye Tenon's capsule in progressive myopia. The full length PEDF is prevalent in myopia that correlates with breach in collagen fibrils forming. Immunohistochemical analysis of Tenon's capsule with polyclonal antibodies to PEDF revealed PEDF in control group being exclusively inside fibroblasts, whereas in myopia, PEDF was distributed extracellularly as halo around blasted fibroblasts. By means of atomic force microscopy and immunodot analysis with anti amyloid fibrils antibodies the ability was studied of recombinant PEDF fragments to form fibrils. Only full length PEDF was shown to form amyloid like fibril structures, but not the truncated form. Accumulation offibrils results in fibroblasts destruction and might be the cause of changes in biochemical and morphological structure of Tenon's capsule observed in myopia.

Inhibition of tumor cell surface ATP synthesis by pigment epithelium-derived factor: Implications for antitumor activity

Recently, we have shown that the antiangiogenic pigment epithelium-derived factor (PEDF) can bind the catalytic β-subunit of F1-ATP synthase and inhibit endothelial cell surface ATP synthase activity. This factor can additionally restrict tumor growth, invasion and metastasis, and can directly induce death on several tumor cell types. Active cell surface ATP synthase is also present in certain tumor cells and its ATP product is considered a stimulus for tumor growth. The present study aimed to elucidate the biological implications of the interactions between the extracellular PEDF and tumor cell surface ATP synthase. Incubation of T24 human urinary bladder carcinoma cells in media containing human recombinant PEDF protein for 48-96h dramatically decreased cell viability in a concentration-dependent fashion as monitored by real-time cell impedance with a microelectronic system, microscopic imaging and biomarkers of live cells. Intact tumor cells exhibited cell surface ATP synthesis activity, which was inhibited by piceatannol, a specific inhibitor of F1/F0-ATP synthase. Immunoblotting revealed that the β subunit of F1-ATP synthase was present in plasma membrane fractions of these cells. Interestingly, pre-incubation of tumor cells with PEDF inhibited the activity of cell surface ATP synthase in a concentration-dependent fashion. The PEDF-derived peptide 34-mer decreased tumor cell viability and inhibited extracellular ATP synthesis to the same extent as full-length PEDF. Moreover, ATP additions attenuated both the PEDF-mediated decrease in tumor cell viability and the inhibition of endothelial cell tube formation. The results lead to conclude that PEDF is a novel inhibitor of tumor cell surface ATP synthase activity that exhibits a cytotoxic effect on tumor cells, and that the structural determinants for these properties are within the peptide region 34-mer of the PEDF polypeptide. The data strongly suggest a role for the interaction between the 34-mer region of PEDF and tumor cell-surface ATP synthase in promoting tumor cell death.

PEDF-derived peptide inhibits corneal angiogenesis by suppressing VEGF expression

Pigment epithelium-derived factor (PEDF) a glycoprotein that belongs to the superfamily of serine protease inhibitors, has been recently shown to be the most potent inhibitor of angiogenesis in the mammalian eye. However, which active domain of PEDF protein could be involved in its anti-angiogenic properties remains unknown. Therefore, in this study, we examined which PEDF-derived synthetic peptides could inhibit corneal neovascularization induced by chemical cauterization in vivo. Rats treated with topical application of PEDF protein had 31% less corneal neovascularization at day 7 after the injury than phosphate-buffered saline (PBS)-treated rats. P5-2 and P5-3 peptides (residues 388–393 and 394–400 of PEDF protein, respectively) significantly suppressed the corneal neovascularization after chemical cauterization at day 7, and its anti-angiogenic potential was almost equal to that of full-length PEDF protein. Further, full-length PEDF protein and P5-3 peptide significantly decreased 8-hydroxy-2′-deoxyguanosine and vascular endothelial growth factor (VEGF) levels in the corneal. Our present study suggests that PEDF-derived synthetic peptide, P5-3 could inhibit the corneal neovascularization induced by chemical cauterization in rats by suppressing VEGF expression via its anti-oxidative properties.

Laminin Receptor Involvement in the Anti-angiogenic Activity of Pigment Epithelium-derived Factor

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic, anti-oxidative, and anti-inflammatory properties. It is also one of the most potent endogenous inhibitors of angiogenesis, playing an important role in restricting tumor growth, invasion, and metastasis. Studies show that PEDF binds to cell surface proteins, but little is known about how it exerts its effects. Recently, research identified phospholipase A(2)/nutrin/patatin-like phospholipase domain-containing 2 as one PEDF receptor. To identify other receptors, we performed yeast two-hybrid screening using PEDF as bait and discovered that the non-integrin 37/67-kDa laminin receptor (LR) is another PEDF receptor. Co-immunoprecipitation, His tag pulldown, and surface plasmon resonance assays confirmed the interaction between PEDF and LR. Using the yeast two-hybrid method, we further restricted the LR-interacting domain on PEDF to a 34-amino acid (aa) peptide (aa 44-77) and the PEDF-interacting domain on LR to a 91-aa fragment (aa 120-210). A 25-mer peptide named P46 (aa 46-70), derived from 34-mer, interacts with LR in surface plasmon resonance assays and binds to endothelial cell (EC) membranes. This peptide induces EC apoptosis and inhibits EC migration, tube-like network formation in vitro, and retinal angiogenesis ex vivo, like PEDF. Our results suggest that LR is a real PEDF receptor that mediates PEDF angiogenesis inhibition.

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 μg of full-length rPEDF, and 47 μg 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.

A PEDF N-terminal peptide protects the retina from ischemic injury when delivered in PLGA nanospheres

The neuroprotective effects of small pigment epithelium-derived factor (PEDF) peptides injected intravitreally as free peptides or delivered in poly(lactide-co-glycolide) (PLGA) nanospheres, were tested in retinal ischemic injury. We induced transient ischemia in C57BL/6 mice by elevating the intraocular pressure to the equivalent of 120 mmHg for 60 min, then injected these eyes with one of the following: PBS, full-length native PEDF, N-terminal peptides—PEDF 136–155 and PEDF 82–121, blank PLGA nanospheres or PLGA loaded with PEDF 82–121 (PLGA-PEDF 82–121). Morphometric analysis and TUNEL assays were used to determine the extent of retinal damage. Transient ischemia caused a rapid reduction in the number of viable cells in the retinal ganglion cell (RGC) layer over 48 h as compared to non-ischemic retinas. About 76% surviving cells in the RGC layer were observed in the full-length PEDF protein treated group, whereas only 32% of cells survived in the PBS group. Thus, PEDF prevented approximately 44% of the cell death in the RGC layer resulting from transient ischemia. PEDF 82–121 peptide was as effective as full-length PEDF when injected as either a free peptide or delivered in PLGA nanospheres. PLGA-PEDF 82–121 showed longer-term protection of the RGC layer with no noticeable side effects at 7 days. PEDF and PEDF 82–121 lessened damage to the IPL as measured by layer thickness. PEDF and PEDF 82–121 also delayed retinal responses to ischemic injury as measured by GFAP immunolabeling in Müller cells. PEDF 82–121 is an effective neuroprotective peptide in retinal ischemia. PLGA-PEDF 82–121 offers greater protection to the retina suggesting that this peptide and the method of delivering therapeutically active drugs have potential clinical advantages for longer-term treatments of retinal diseases.

Mapping the Type I Collagen-binding Site on Pigment Epithelium-derived Factor: IMPLICATIONS FOR ITS ANTIANGIOGENIC ACTIVITY

Pigment epithelium-derived factor (PEDF), a neurotrophic and antiangiogenic serpin, is identified in tissues rich in collagen, e.g. cornea, vitreous, bone, and cartilage. We show that recombinant human PEDF formed complexes with collagens from the bovine cornea and vitreous. We have examined the direct binding of PEDF to collagen I and found that interactions were ionic in nature and occurred when PEDF and collagen I were both in solution, when either one was immobilized, or even when collagen I was denatured under reducing conditions. (125)I-PEDF bound to immobilized collagen I in a saturable fashion (K(D) = 123 nm). Compared with neurotrophic PEDF-derived peptides, ovalbumin and angiogenic inhibitors, only full-length PEDF competed efficiently with (125)I-PEDF for the binding to immobilized collagen I (EC(50) = 3 microg/ml). The collagen-binding region was analyzed using controlled proteolysis and chemically modified PEDF. Cleavage of the serpin exposed loop did not prevent binding to collagen I. Conjugation of lysines with fluorescein increased the collagen binding affinity. However, treatment of PEDF with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide abolished it, implicating the PEDF aspartic and/or glutamic acid residues in its interaction with collagen I. A negatively charged region on the surface of the PEDF molecule is rich in acidic residues (Glu(41), Glu(42), Glu(43), Asp(44), Asp(64), Asp(256), Asp(258), Glu(290), Glu(291), Glu(296), Asp(300), Glu(304)) available to interact directly with positively charged areas of collagen. This represents the first collagen-binding site described for a serpin, which in PEDF, is distinct from its heparin-binding region, neurotrophic active site, and its serpin exposed loop. The collagen-binding property of PEDF may play a role in surface localization and modulation of its antiangiogenic effects in the eye and bone.