Osteoblasts and osteoclasts express PEDF, VEGF-A isoforms, and VEGF receptors: possible mediators of angiogenesis and matrix remodeling in the bone

Pigment epithelial derived factor (PEDF) is one of the most effective inhibitors of angiogenesis described so far, especially in controlling the growth of blood vessels in the eye. We now describe the localization of PEDF in regions of active bone formation in the mid-gestation mouse embryo and its specific and high levels of secretion by osteoblasts. PEDF is detected to a lesser extent in osteoclasts as well. The proangiogenic factors, VEGF and its receptors VEGF-R1 and VEGF-R2, are also expressed by both osteoblasts and osteoclasts. These findings suggest that bone angiogenesis and matrix remodeling may be mediated both by PEDF and by VEGF.     

Pigment epithelium-derived factor (PEDF) prevents advanced glycation end products (AGEs)-elicited endothelial nitric oxide synthase (eNOS) reduction through its anti-oxidative properties

PEDF is an effective inhibitor of angiogenesis in the eye with neuronal differentiating activity. Here, we show that PEDF prevents the AGEs-elicited eNOS reduction through its anti-oxidative properties. Our present study suggests that PEDF may also play a protective role against atherosclerosis.     

Poster Sessions BP02: Oligodendrocytes and Schwann Cells. RAS activation regulates angiogenic and anti‐angiogenic factor expression in NF1‐derived Schwann cells

Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro, the NF1‐derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1‐derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1‐derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1‐derived Schwann cells have increased RAS activity and a three‐fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1‐derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP‐related domain of neurofibromin into the NF1‐derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1‐derived Schwann cells the Schwann cells expressing the GAP‐related domain expressed about one‐half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1‐drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.     

Pigment epithelium-derived factor (PEDF) inhibits angiotensin II-induced smooth muscle cell proliferation through its anti-oxidative properties

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis, suggesting that loss of this factor may be involved in angiogenic eye disease. Here, we show that PEDF inhibits angiotensin II-induced smooth muscle cell proliferation through its anti-oxidative properties. Our present study suggests that PEDF may play a protective role against atherosclerosis.     

Heparin binding induces a conformational change in pigment epithelium-derived factor

Pigment epithelium-derived factor (PEDF) is a noninhibitory serpin found in plasma and in the extracellular space. The protein is involved in different biological processes including cell differentiation and survival. In addition, it is a potent inhibitor of angiogenesis. The function is likely associated with binding to cell surface receptors in a heparin-dependent way (Alberdi, E. M., Weldon, J. E., and Becerra, S. P. (2003) BMC Biochem. 4, 1). We have investigated the structural basis for this observation and show that heparin induces a conformational change in the vicinity of Lys(178). This structural change was evident both when binding to intact heparin and specific heparin-derived oligosaccharides at physiological conditions or simply when exposing PEDF to low ionic strength. Binding to other glycosaminoglycans, heparin-derived oligosaccharides smaller than hexadecasaccharides (dp16), or type I collagen did not affect the structure of PEDF. The conformational change is likely to expose the epitope involved in binding to the receptor and thus regulates the interactions with cell surface receptors.     

Pigment epithelium-derived factor inhibits glioma cell growth in vitro and in vivo

Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that pigment epithelium-derived factor (PEDF) can induce differentiation and inhibit angiogenesis of several tumors. This study was designed to determine whether gliomas angiogenesis and tumor growth could be inhibited by PEDF. We found that PEDF down-regulated expression levels of vascular endothelial growth factor and up-regulated the expression of thrombospondin-2 and augmented apoptosis in a dose-dependent manner in both A172 and U87 glioma cells lines after 48 h of treatment. Analysis of the cell cycle showed arrest in the G₁ phase and block in S phase of the cell cycle. Meanwhile PEDF induced apoptosis was associated with increases of p53 and Bax and inhibition of Bcl-2. Conditioned medium with PEDF showed a significantly reductive effect on migration in vitro accompanied with a significant reduction of matrix metalloproteinase-9 expression. PEDF suppressed glioma cell migration in vitro and tumor burden in athymic nude mice. These results demonstrate for the first time inhibitory effects of PEDF on the growth and migration of human gliomas via induction of apoptosis and blocking of migratory-related factors. PEDF activation can be a novel approach for future therapeutic purposes against gliomas.     

Pigment epithelium-derived factor protects cultured retinal pericytes from advanced glycation end product-induced injury through its antioxidative properties

Pigment epithelium-derived factor (PEDF) has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, suggesting that loss of PEDF is involved in the pathogenesis of proliferative diabetic retinopathy. However, a protective role for PEDF in pericyte loss in early diabetic retinopathy remains to be elucidated. In this study, we investigated whether PEDF proteins could protect against advanced glycation end product (AGE)-induced injury in retinal pericytes. Ligand blot analysis revealed that pericytes possessed a membrane protein with binding affinity for PEDF. PEDF proteins were found to significantly inhibit AGE-induced reactive oxygen species (ROS) generation and the subsequent decrease in DNA synthesis and apoptotic cell death in pericytes. Further, PEDF proteins completely restored the down-regulation of bcl-2 gene expression in AGE-exposed pericytes. The results demonstrated that PEDF proteins protected cultured pericytes from AGE-induced cytotoxicity through its anti-oxidative properties. Our present study suggests that substitution of PEDF proteins may be a promising strategy in treatment of patients with early diabetic retinopathy.     

PEDF derived from glial Müller cells: a possible regulator of retinal angiogenesis

A precise balance between stimulators and inhibitors of angiogenesis, such as vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), respectively, is essential for angiogenic homeostasis in ocular tissues. Retinal hypoxia is accompanied by some pathological conditions that may promote intraocular neovascularization. Here we demonstrate that retinal glial (Müller) cells express and release pigment epithelium-derived factor (PEDF). Decreasing oxygen concentrations cause strong attenuation of PEDF release resulting in enhanced VEGF/PEDF ratios. Exposure of Müller cells to VEGF suppressed PEDF release in a dose-dependent manner. This may represent a novel mechanism of ocular angiogenic homeostasis sufficient in the control of PEDF levels during normoxia or mild hypoxia but supplemented by other (hitherto unknown) mechanisms in cases of strong hypoxia. In spite of the enhanced VEGF/PEDF ratios resulting from hypoxia, conditioned media of Müller cells failed to stimulate additional proliferation of retinal endothelial cells. These findings suggest that in the ischemic retina, Müller cells generate a permissive condition for angiogenesis by secreting more VEGF and less PEDF, but the onset of retinal endothelial cell proliferation requires another triggering signal that remains to be identified.     

Pigment epithelium-derived factor inhibits angiogenesis via regulated intracellular proteolysis of vascular endothelial growth factor receptor 1

Pigment epithelium-derived factor (PEDF) has been identified as one of the most potent of endogenous negative regulators of blood vessel growth in the body. Here we report that PEDF is able to inhibit growth factor-induced angiogenesis in microvascular endothelial cells through a novel pathway requiring cleavage and intracellular translocation of the transmembrane domain of the VEGFR-1. Analysis of the subcellular distribution of VEGFR-1 revealed the appearance of an 80-kDa C-terminal domain in the cytosol of cells treated with VEGF and PEDF that correlated with a decrease of the full-length receptor in the nuclear and cytoskeletal fractions. This regulated intramembrane proteolysis is dependent on gamma-secretase because inhibition of gamma-secretase abolished the inhibitory effect of PEDF on VEGF-induced angiogenesis as well as VEGFR-1 cleavage. The addition of PEDF to microvascular endothelial cells significantly increases gamma-secretase activity even in the absence of VEGF, showing that VEGF binding to VEGF-R1 is essential for substrate availability. This increase in activity was associated with translocation of presenilin 1 from the perinuclear region to the cell membrane. PEDF was also able to inhibit VEGF-induced phosphorylation of VEGFR-1. Taken together we have identified two novel pathways by which PEDF inhibits VEGF-induced angiogenesis: regulated intramembrane proteolysis and inhibition of phosphorylation. This confirms the importance of PEDF and VEGFR-1 in the negative regulation of angiogenesis.     

Recombinant fragment 44–77 of the pigment epithelium-derived factor prevents the development of the pathological cornea neovascularization

The pigment epithelium-derived factor (PEDF), a secreted 50 kDa glycoprotein, is one of the most potent endogenous inhibitors of angiogenesis. The fragment 44–77 of PEDF possesses the antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of diseases of visual organs accompanied by pathological neovascularization. An effective biotechnological method for the large-scale production of the PEDF (44–77) fragment as part of the fusion protein with the SspDnaB intein has been developed. The hybrid protein was produced in Escherichia coli bacterial cells in the form of inclusion bodies, solubilized, and subjected to autocatalytic cleavage with the release of the PEDF (44–77) fragment (reaction yield 77%). The target peptide was separated from the intein by tangential ultrafiltration. The final purification of PEDF (44–77) was performed by reversed-phase HPLC. The yield of the target peptide (purity 99%) was 65 mg per 1 l of culture. The antiangiogenic activity of the peptide was confirmed in vitro using mouse endothelial cells SVEC-4-10. It was found that the preparation at a concentration of 1 nM suppresses the proliferation of endothelial cells by 53% and inhibits the formation of tube-like structures by endothelial cells. The ability of the recombinant PEDF (44–77) to block the initial stages of angiogenesis was shown using an experimental model of rabbit corneal neovascularization.     

The anti-angiogenic factor PEDF is present in the human heart and is regulated by anoxia in cardiac myocytes and fibroblasts

Cardiac diseases such as myocardial infarction and heart failure are among the leading causes of death in western societies. Therapeutic angiogenesis has been suggested as a concept to combat these diseases. The biology of angiogenic factors expressed in the heart such as vascular endothelial growth factor (VEGF) is well studied, whereas data on anti-angiogenic mediators in the heart are scarce. Here we study the expression of the anti-angiogenic factor pigment epithelium-derived factor (PEDF) in the human heart and in human cardiac cells. PEDF expression could be detected in human cardiac tissue on the protein and mRNA levels. PEDF mRNA levels were significantly lower in explanted human ischemic hearts as compared to healthy hearts. Our in vitro experiments showed that human adult cardiac myocytes and fibroblasts constitutively secrete PEDF. In addition to anoxic conditions, cobalt chloride, 2,2'dipyridyl and dimethoxally glycine, which stabilize hypoxia inducible factor-α decreased PEDF expression. Furthermore we show that PEDF inhibits VEGF-induced sprouting. We have identified PEDF in healthy and ischemic human hearts and we show that PEDF expression is down-regulated by low oxygen levels. Therefore, we suggest a role for PEDF in the regulation of angiogenesis in the heart and propose PEDF as a possible therapeutic target in heart disease.     

Total chemical synthesis of the D2 domain of human VEGF receptor 1

The interaction of the vascular endothelial growth factor (VEGF) with its cellular receptors exerts a central role in the regulation of angiogenesis. Among these receptors, the VEGF receptor 1 may be implicated in pathological angiogenesis. Here, we report the first total chemical synthesis of the VEGF‐binding domain of the VEGF receptor 1. Aggregation issues were overcome by the use of a low‐substituted resin and the stepwise introduction of pseudoproline dipeptides and Dmb‐glycines. The folding of the protein was achieved by air oxidation and its biological activity was verified on ELISA‐based assays. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Pigment Epithelium Derived Factor Inhibits the Growth of Human Endometrial Implants in Nude Mice and of Ovarian Endometriotic Stromal Cells In Vitro

Angiogenesis is a prerequisite for the formation and development of endometriosis. Pigment epithelium derived factor (PEDF) is a natural inhibitor of angiogenesis. We previously demonstrated a reduction of PEDF in the peritoneal fluid, serum and endometriotic lesions from women with endometriosis compared with women without endometriosis. Here, we aim to investigate the inhibitory effect of PEDF on human endometriotic cells in vivo and in vitro. We found that PEDF markedly inhibited the growth of human endometrial implants in nude mice and of ovarian endometriotic stromal cells in vitro by up-regulating PEDF expression and down-regulating vascular endothelial growth factor (VEGF) expression. Moreover, apoptotic index was significantly increased in endometriotic lesions in vivo and endometriotic stromal cells in vitro when treated with PEDF. In mice treated with PEDF, decreased microvessel density labeled by Von Willebrand factor but not by α-Smooth Muscle Actin was observed in endometriotic lesions. And it showed no increase in PEDF expression of the ovary and uterus tissues. These findings suggest that PEDF gene therapy may be a new treatment for endometriosis.     

Therapeutic prospects for PEDF: more than a promising angiogenesis inhibitor

Blood vessel growth and stability are under the exquisite control of a network of pro- and anti-angiogenic factors. Disruption of the balance between these factors is a characteristic of tumor growth and many vascular diseases. Endogenous angiogenesis inhibitors, particularly those that act broadly at the earliest stages, could be excellent pharmacological tools in combating pathogenic vessel growth. Pigment-epithelium-derived factor (PEDF) is a natural angiogenesis inhibitor that (1) targets only new vessel growth, (2) can be administered therapeutically as a soluble protein or by viral-mediated gene transfer, (3) is stable and nontoxic when injected, and (4) is more potent than other well-characterized angiogenesis inhibitors. Because PEDF also has differentiating and neuroprotective activities, it has additional benefits for use in the nervous system. The production of PEDF by many tissues suggests its therapeutic potential should be explored in a much wider range of diseases, including tumor proliferation and metastasis.     

Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human osteosarcoma cells

Pigment epithelium-derived factor (PEDF) has been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, thus suggesting that loss of PEDF is involved in angiogenic eye diseases such as proliferative diabetic retinopathy. Angiogenesis is required for tumor growth and progression as well. We, along with others, have recently found that PEDF could inhibit growth of melanoma and hepatocellular carcinoma in nude mice through its anti-angiogenic effects on tumor endothelial cells. However, the possibility of the direct effect of PEDF on tumor cells has remained. In this study, we investigated the effects of PEDF on growth and vascular endothelial growth factor (VEGF) expression in MG63 human cultured osteosarcoma cells. PEDF decreased viable cell number as well as DNA synthesis in MG63 cells in a dose-dependent manner. Furthermore, PEDF was found to increase caspase-3/7 activity and to subsequently induce apoptotic cell death in MG63 cells. PEDF also inhibited VEGF expression in MG63 cells at both mRNA and protein levels. Our present study provides novel beneficial aspects of PEDF on osteosarcoma cells; one is induction of apoptotic cell death of tumor cells, and the other is the suppression of VEGF expression, which would lead to inhibition of tumor angiogenesis. PEDF therefore might be a promising therapeutic agent for treatment of patients with osteosarcoma.     

PEDF inhibits lymphatic metastasis of nasopharyngeal carcinoma as a new lymphangiogenesis inhibitor

Nasopharyngeal carcinoma (NPC) is one of the most malignant tumors in southern China and Asia, and lymph node metastasis is an important cause for treatment failure. Lymphangiogenesis is a crucial step in lymphatic metastasis of NPC, while little is known about lymphangiogenesis in NPC. Similar to angiogenesis, lymphangitic neovascularization is a process of balance between pro-lymphangiogenesis and anti-lymphangiogenesis factors, but there are few studies on endogenous lymphangiogenesis inhibitors. Pigment epithelium-derived factor (PEDF) is a well-known effective endogenous angiogenesis inhibitor. However, the relationship between PEDF and lymphangiogenesis remains unknown. Our present study reveals that PEDF is lowly expressed in human NPC tissues with poor prognosis and is negatively correlated with lymphatic vessel density (LVD). Consistently, PEDF inhibits lymphangiogenesis and lymphatic metastasis of NPC in vivo experiments. Mechanistically, PEDF inhibits the proliferation, migration, and tube formation of lymphatic endothelial cells and promotes cell apoptosis. On the other hand, PEDF reduces the expression and secretion of vascular endothelial growth factor C (VEGF-C) of NPC cells through the nuclear factor-κB (NF-κB) signaling pathway. Our findings indicate that PEDF plays a vital role in lymphatic metastasis by targeting both lymphatic endothelial cells and NPC cells, and PEDF may represent a novel therapeutic target for NPC.Subject terms: Metastasis, Head and neck cancer     

Mesenchymal stem cells overexpressing PEDF decrease the angiogenesis of gliomas

The present study is an exploration of a novel strategy to target a therapeutic gene to brain tumour tissues. In the present study, we evaluated the feasibility of using hMSCs (human mesenchymal stem cells) to deliver PEDF (pigment epithelium-derived factor), a potent inhibitor of tumour angiogenesis, in a model of intracranial gliomas. To assess its potential of tracking gliomas, MSCs (mesenchymal stem cells) were injected into the cerebral hemisphere and it showed that MSCs infiltrated into the vessel beds and scattered throughout the tumour. In vitro migration assay showed that the VEGF (vascular endothelial growth factor) enhanced MSC migration. In contrast, the migratory activity of MSCs was significantly inhibited with the presence of PEDF. Systematic delivery of AAV (adeno-associated virus)–PEDF to established glioma xenografts resulted in increased apoptosis of gliomas. In addition, MSC–PEDF treatment prolonged the survival of mice bearing U87 gliomas. Taken together, these data validate that MSCs–PEDF can migrate and deliver PEDF to target glioma cells, which may be a novel and promising therapeutic approach for refractory brain tumour.     

Recombinant fragment of pigment epithelium-derived factor (44-77) prevents pathological corneal neovascularization

Pigment epithelium-derived factor (PEDF), a 50 kDa secreted glycoprotein, is among the most potent endogenous inhibitors of angiogenesis. PEDF-derived fragment (44-77) possesses antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of ocular neovascular diseases. In this study we propose an efficient scalable biotechnological method for the production of PEDF (44-77) as part of a fusion protein with SspDnaB intein. The fusion protein was obtained in bacterial E. coli cells in the form of inclusion bodies, solubilized and subjected to autocatalytic cleavage with the release of PEDF (44-77) (yield, 77%). The target peptide was separated from the intein using tangential ultrafiltration. The final purification of PEDF (44-77) was performed by reversed-phase HPLC. The yield of the target peptide (purity, 99%) was 65 mg per 1 liter of culture. Antiangiogenic activity of the obtained peptide was studied in vitro using murine endothelial cells SVEC-4-10. PEDF (44-77) suppressed proliferation of endothelial cells by 53% and inhibited endothelial cell tube formation at the concentration of 1 nM. The ability of the recombinant PEDF (44-77) to block initial stages of angiogenesis was demonstrated using the model of rabbit corneal neovascularization.     

Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

Poly(ADP-ribose)polymerase (PARP) inhibitors decrease angiogenesis through reducing vascular endothelium growth factor (VEGF) induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). In contrast to VEGF, pigment epithelium-derived factor (PEDF) has been demonstrated to act as a strong endogenous inhibitor of angiogenesis. Here, we show that PARP inhibition with a specific inhibitor PJ-34 or specific PARP antisense oligonucleotide upregulates hyperglycemia-induced PEDF expression in HUVECs in a dose-dependent manner. This results in the retard of activation of p38 MAP kinase and the concomitant decrease in cell apoptosis. These results give the first direct demonstration that PEDF might represent a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent.