How PEDF prevents angiogenesis: a hypothesized pathway

Medical Hypotheses (2005) 64, 74–78

http://intl.elsevierhealth.com/journals/mehy

How PEDF prevents angiogenesis: a hypothesized pathway Jian-Guo Rena,*, Chunfa Jieb, Conover Talbotb a

Department of Pathology, Brighman and Women’s Hospital and Harvard Medical School, Thorn 530, 75 Francis Street, Boston, MA 02115, USA b JHMI Microarray Core, Johns Hopkins University School of Medicine, 733 W Broadway, BRB 527, Baltimore, MD 21205, USA Received 12 March 2004; accepted 18 May 2004

Summary Pigment epithelium-derived factor (PEDF) is a multiple functional protein, coded by the serine proteinase inhibitor, clade F, member 1 (SERPINF1) gene, which has both anti-angiogenic activity and neurotrophic activity at the same time. Its antiangiogenic activity in the mammalian eye is the most potent known at this time. However, the mechanism(s) by which PEDF works in vivo is still uncertain. Some observations suggest that PEDF can simultaneously inhibit the migration and proliferation induced by vascular endothelial growth factor (VEGF), and then further inhibits angiogenesis by interacting with specific cell surface receptors, but no such receptor has been reported to date. Here we propose a hypothesis that PEDF exerts its function by binding with intergrins. Intergrin can therefore serve as the receptor of PEDF. c 2004 Elsevier Ltd. All rights reserved.



Introduction Angiogenesis, the process in which new capillaries form by sprouting from pre-existing ones, occurs normally during embryogenesis and pathologically in tumor growth, diabetic retinopathy, wound healing, psoriasis and rheumatoid arthritis [1–8]. Generally, angiogenesis is exquisitely controlled in most healthy tissues through a finely tuned balance between pro- and anti-angiogenic factors. Pathogenic angiogenesis plays an important role in disease progression. It is well known that tumors

*

Corresponding author. Tel.: +617 732 6839. E-mail address: [email protected] (J.-G. Ren).



cannot grow beyond 2–3 mm in diameter without forming new blood vessels. Not restricted to tumors, the neovascularization occurring in diabetic retinopathy threatens the visions of more than 7 million people in the USA alone [1]. Actually, tumor and diabetes are the two most threatening diseases for human health. The ability to control neovascularization would offer a unique opportunity to impact a wide variety of physiological and pathological functions. Thus, preventing neovascularization becomes a fascinating goal for both tumor and diabetic therapy [8–14]. Developing antiangiogenic factors is becoming a more and more attractive approach for the therapy of tumor, diabetic retinopathies, and other neovascularization-related diseases.

0306-9877/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2004.05.016

How PEDF prevents angiogenesis Pigment epithelium-derived factor (PEDF), a 418-amino acid 50 KDa glycoprotein first identified in 1987 by Tombran-Tink and Johnson in conditioned medium from fetal human retinal pigment epithelium (RPE) cell culture, is among the most potent natural antiangiogenic factors, as it specifically inhibits both the migration and proliferation of endothelial cells [15]. The antiangiogenic activity of PEDF is selective because it is effective against newly forming vessels but spares existing ones, and it is reversible. In specific, as an effective anti-angiogenic factor for clinic purposes, PEDF has the following advantages:first, it is not expected to activate drug-resistance genes, and thus offers the potential for effective long-term anti-angiogenic therapy. Second, due to its tolerance in the body, it is unlikely to produce the toxic side effects of synthetic inhibitors. Finally, given its neurotrophic activity, PEDF has the additional advantage of preserving neurons from the damage often caused by vascular disease of the nervous system. In light of PEDFs great promise for antiangiogenesis, understanding its fundamental mechanism holds the promise of effective therapeutic approaches for treating neovascularization-related diseases. Some researchers have proposed that maintaining a balance between the pro and anti-angiogenic factors is critical for the regulation of angiogenesis [16]. The expression and the role of angiogenic factors in retinal neovascularization have been well established. Normal angiogenesis requires the coordination of growth factor receptors and integrins, leading to the activation of down stream signals in endothelial cells [17]. Although integrins and growth factor receptors can independently propagate intracellular signals [18– 20], it appears that a synergy between signals initiated by the extracellular matrix (ECM) and growth factors enhances their separately induced angiogenesis [20]. In addition, protein phosphatases such as protein tyrosine phosphatase (PTP) and protein kinases like FAK, Akt, Src, and MAPK play key roles in the cross-talk between the growth factor and integrin pathways [20]. Thus, there are two possible means to inhibit angiogenesis: one is to target the receptors of growth factors; the other is to disrupt the activation of integrin. PEDF is the most potential natural antiangiogenic factor found up to now [15]. However, how it is that PEDF inhibits new blood vessels formation has remained uncertain. Some evidence has indicated that PEDF can inhibit the migration, proliferation, and even permeability induced by VEGF, in vitro or in vivo, and thereby inhibit angiogenesis by interacting with specific cell surface

75 receptors [21,22], however, no such receptor has been reported to date.

PEDF exerts its function via integrins Here we hypothesize that integrin can serve as the receptor of PEDF. Collagen is an ideal angiogenic scaffold since angiogenesis depends on proper collagen biosynthesis and cross-linking. Collagen can itself trigger angiogenesis by binding with some integrins, which then serve as collagen receptors [23]. In vivo, PEDF interacts with the ECM proteins such as collagen [24] and heparin [25]. Therefore, it is reasonable to hypothesize that PEDF can regulate the angiogenesis induced by VEGF or other angiogenic factor(s) through its binding with integrin directly and/or indirectly. In vivo PEDF is first concentrated in the ECM through its recruitment by proteins such as collagen or heparin; subsequently, the enriched PEDF interacts further with integrin. In general, the binding of angiogenic factors like VEGF with their receptors on endothelial cells leads to the self-phosphorylation (P) of the receptors, which then triggers the key first-step of angiogenesis by causing the migration and proliferation of endothelial cells. This step sets in motion a complex cascade of inter-related events between cells, soluble factors, second messengers and extracellular matrix components as well. This entire process is regulated by such protein phosphatases as PTP and/or protein kinases like FAK, MAPK and Src. Under normal conditions the binding of PTP to integrins prevents it from reducing the selfphosphorylation of VEGF receptors, thereby permitting the signals initiated by VEGF to be transmitted downstream. At the same time, the autophosphorylation of FAK at Y397, mediated by ECM and integrin action, causes the recruitment of Src through binding its SH2 and SH3 domains and thereby activating Src catalytic activity. Src then phosphorylates additional sites on FAK and its associated proteins such as Raf [26]. FAK and Src form a distinct signaling unit in adherent cells which regulates cell spreading and mobility upon integrin engagement through the recruitment of additional signaling and adapter proteins. The activation of protein phosphatase or the inactivation of protein kinase stops the phosphorylation of VEGFR or its down stream protein, and thereby prevents the receptors from transmitting further signals (for a review see [27]). When PEDF binds with one or more of the possible integrin dimers (i.e avb5, avb3, a3b1, a2b1 and a1b1) directly and/or indirectly via ECM, the complex may somehow

76 facilitate the interaction between PEDF and its receptors, e.g., by inducing a conformational change in PEDF, which accelerates the ligand– receptor interaction. Following the binding of PEDF

Ren et al. and integrin, either the phosphatases may dissociate and then reassociate with VEGF receptors, or the PEDF–integrin complex may inhibit the activation of a kinase such as FAK or Src; Both alterna-

Figure 1 How does PEDF block blood-vessel growth? PEDF is the most potent natural inhibitor of angiogenesis in the mammalian eye. However, its mechanisms still remain unclear. The following is a model, modified from Aplin et al. [27] and Conway and Carmeliet [28], on how PEDF works in vivo. (a) On endothelia cells, when the vascular endothelial growth factor (VEGF) binds with its receptors, the receptors self-phosphorylate (P). This leads to a series of intracellular events that trigger angiogenisis. Here, integrin is associated with protein tyrosine phosphatase (PTP). At the same time, the activation of integrin in adhering cells may activate protein kinases like FAK, thereby triggering a series of down stream events to enhance the angiogenesis induced by VEGF. (b) When PEDF binds with integrins, directly and/or indirectly via ECM, this leads to conformation changes of integrin or collagen–integrin complexes, causing phosphatases to dissociate, and then reassociate with VEGF receptors. This PEDF–integrin complex may also inactivate the protein kinase. The dephosphorylation of receptors or their downstream factors stops signal transduction, thus blocking angiogenesis.

How PEDF prevents angiogenesis tives block phosphorylation, and thus stop signal transduction, thereby blocking angiogenesis. Here, collagen and/or heparin might work as co-receptors of PEDF; Integrin serves as the receptor of PEDF (see Fig. 1). Some evidences provide indirect support to this hypothesis. A number of endogenous angiogenic inhibitors mediate their antiangiogenic effects by direct or indirect interactions with integrins [29– 34]. Recently, Seo et al. [35] found that TIMP-2, tissue inhibitors of metalloproteinases 2, can bind with a3b1 integrin, which binding results in the movement of phosphatase from a a3b1 integrin to the VEGF and FGF-2 receptors. Thus the cells are conferred resistance to VEGF and FGF-2 induced angiogenesis. Similar to TIMP-2, PEDF is also a natural antiangiogenic factor, potentially the most potent natural antiangiogenic factor found to date, which can also inhibit the migration, proliferation and even permeability of VEGF induced endothelial cells. It is quite possible, therefore, that PEDF shares the same mechanism(s) with TIMP-2. If, as we expected, PEDF exerts its angiogenesis and neurotrophic activity through binding to integrins, this will be useful in basic research to further elucidate the role of PEDF and its receptor in signal transduction pathways; futhermore, this will enable the development of drug screening assays to identify agonists and antiagonists of PEDF activity. Since one function of PEDF is antiangiogenesis, then blocking integrins will be contribute greatly to the therapy of tumor and diabetic retinopahies.

Suggestions for experimentation In this hypothesis we put forward a possible mechanism by which PEDF exerts its function in vivo via interaction with integrins. To test this hypothesis, the following experiments will be helpful. First, do an in vitro PEDF–integrin binding experiment to observe whether PEDF can directly bind to integrins or not. Second, determine in an endothelial cell system analysis whether PEDF still can bind to the cell surface after integrins are blocked with integrin antibody. Third, analyse whether PEDF can still exert its angiogenesis and neruotrophic functions after blocking integrins using their antibody.

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