REVIEW

Curtin Biosciences Research Precinct1; School of Pharmacy2, Curtin University, Bentley, Australia

Potential of pigment epithelium-derived factor (PEDF) as a regenera- tive biopharmaceutical

P. X. LEE1, C. R. DASS1, 2

Received October 21, accepted November 20, 2015 Professor Crispin R. Dass, School of Pharmacy, Curtin University, GPO Box U1987, Perth 6845, Australia [email protected] Pharmazie 71: 171–174 (2016) doi: 10.1691/ph.2016.5829

Bone is very much a dynamic tissue, capable of various functions not limited to protection of the marrow, serving as a reservoir for calcium, maintaining posture and facilitating mobility. It is also a tissue that is fully capable of regenerating itself at most stages of life, with a diminishing capacity with increasing age. Bone defects can arise from a variety of factors not limited to bone tumours and fractures. At present, clinically, most diseased bone is removed and the patient fitted with prosthetics, with use of certain factors such as bone morphogenetic proteins (BMPs) to aid healing. Recently, the protein pigment epithelium-derived factor (PEDF) has been found to have favourable effects on bone regeneration, which is reviewed here. Numerous studies have shown the potential of PEDF in vitro, with increasing reports of success in small animal models of bone trauma. This review puts forward the advantages, and some disadvantages, in the use of PEDF as a biopharmaceutical for bone regeneration.

1. Introduction magnesium and carbonate are transported to the bone and stored. Bone is a vital connective tissue comprising minerals, water, If the blood mineral level decreases, minerals contained within the collagen, and non-collagenous proteins (Burr and Akus 2014). bone will be released into the blood (Burr and Akkus 2014). The Most of the composite for are extracellular matrix rather blood concentration of these minerals is therefore maintained at than cells (Klein 2014; Buckwalter et al. 1995). The properties of a steady level in normal conditions under the influence of para- bones consist of tensile strength and balance of stiffness. Bone is . Hence, storage of these minerals is crucial for constantly changing throughout one’s life to a greater extent in the performance of various cellular functions when cells face dire comparison to other organs such as the brain and (Morgan conditions. et al. 2013). This change in nature is due to the response of the Two structural types of bones exist and they are the trabecular/ bones to the environment which include loading in the form of cancellous and cortical bones (Jähn and Bonewald 2012). Trabec- exercise and unloading such as immobilization which occurs ular bones consist of larger surface area as well as lower density with patients in nursing homes. The cells within the bone that compared to cortical bones. Trabecular bones serve to support the are responsible for these changes include , load and transfer the stress to the cortical bones. The cortical bones and osteocytes. function to carry most of the load due to a denser consistency (Burr Osteoblasts are the bone-forming cells responsible for laying down and Akkus 2014). Bones are organised as multiscale materials, new bones (Tombran-Tink and Barnstable 2004; Jähn and Bone- therefore, they are adapted to avoid fractures caused by repetitive wald 2012). Osteoblasts have the tendency to secrete , loading. a hormone that affects the homeostasis of glucose, male fertility as well as energy expenditure (Karsenty 2014; Kousteni 2013). 2. Bone regeneration It regulates homeostasis by promoting hyperinsulinemia and improve sensitivity of insulin, therefore resulting in improved Bone is one of the few tissues that has the ability to regenerate glucose tolerance and metabolism. It also promotes male fertility (Shrivats et al. 2014; Neffe et al. 2014). However, there is a poten- by favouring the production of as well as promoting tial for defects to occur and the regeneration process is usually germ cell survival Kousteni 2013). Osteoclasts involve the breaking compromised if the bone defects lead to abnormalities. These crit- down of bones as well as removing the bone during growth and ical bone defects can be due to the presence of diseases, tumour repair (Tombran-Tink and Barnstable 2004). Osteocytes are and trauma, or in cases such as atrophic non-unions, avascular matured osteoblasts surrounded by bone matrix or by osteoid as a necrosis and osteoporosis (Neffe et al. 2014; Elbackly et al. 2014). result of minerals entering during calcification (Tombran-Tink and If these critical defects occur, the capability of the regeneration of Barnstable 2004; Clarke 2008). bones intrinsically cannot occur and therefore, it is one of the most Bone plays multiple roles in supporting and protecting vital organs difficult regenerative therapies to achieve. such as the brain, heart and lungs (Burr and Akkus 2014). Bones Bone regeneration is a complex physiological process of bone are also responsible for haematopoiesis, the formation of blood formation that involves the continuous remodelling of bones cellular components, due to the bones having red bone marrow (Dimitriou et al. 2011). It involves a number of intracellular and filled deep within the cavities, giving rise to the formation of blood extracellular molecular signalling pathways in order to improve cells and platelets (Morgan et al. 2013). Other than that, due to the function and repair of bones. The regeneration processes can be their composite structure, they can resist gravity as well as permit enhanced by treatment with bone grafting allogeneically or autol- motility (Klein 2014). Furthermore, bone is involved in metabolic ogously, demineralised bone matrix, bone , exercise processes associated with mineral homeostasis. For instance, and electrical fields. One of the more popular treatments for bone major minerals in the blood which include calcium, phosphorus, regeneration is biomaterial implants. Pharmazie 71 (2016) 171 REVIEW

One of the common bone regeneration processes is fracture healing 3. Pigment epithelium-derived factor (PEDF) – involve- (Jiliang and Stocum 2014). Unlike in other tissues, fractures heal ment in bone without the formation of scar tissues and the bones regenerated are largely stored and the newly formed bone having indistinguishable PEDF is an endogenous glycoprotein of 418 amino acid residues properties in comparison to the adjacent uninjured bones. There with a molecular weight of 50kDa, having a strong role in the are four stages occurring during the healing of bone fractures inhibition of angiogenesis due to the suppression of endothelial which includes an inflammatory response, formation of cartilage, cell migration, proliferation as well as induction of endothelial primary bone formation and the remodelling of bones (Jiliang cell apoptosis (Wang et al. 2003; Quan et al. 2015). It belongs to and Stocum 2014). However, in some cases, fracture healing is one of the members of the serpin superfamily of serine protease not successful due to impaired bone regeneration (Dimitriou et al. inhibitors, however, it does not inhibit proteases (Stratikos et al. 2011). 1996). Other non- protease inhibitors that are also part of the Fracture healing can be classified as primary fracture healing serpin family include ovalbumin and angiotensinogen. PEDF was and secondary fracture healing (Jiliang and Stocum 2014). The first discovered as a protein secreted by cultured pigment epithe- difference between the two is based on the differences in the local lium cells from human retina and was identified as an extracellular motion between the fragments of the fractures. Primary healing component of an adult bovine eye in the retinal interphotoreceptor involves the reestablishment continuity of the cortex after its inter- matrix as well as in the vitreous and aqueous humors (Shao et al. ruption, however, the formation of a fracture callus is not present 2003). (Sathyendra and Darowish 2013). This process takes place when PEDF is expressed mainly by osteoblasts that line the bone spic- there is an establishment of a decrease in interfragmentary motion ules in the ossification zone of the metaphyseal bone as well as and presence of alignment stability by the rigid internal fixation. the osteoblasts that line the cortical periosteum (Broadhead et For instance, osteoblasts lay down osteoid on the exposed surfaces al. 2010). It is expressed to a lesser extent by osteoclasts. During of bones. Healing involves the four classical stages which include endochondral bone formation, PEDF is expressed within the carti- haemorrhagic inflammation, soft callus formation, mineraliza- lage and bone cells. The expression was demonstrated in chondro- tion and remodelling (Jiliang and Stocum 2014). It takes place cytes within the resting, proliferative and upper hypertrophic zones at the fracture site, surrounding the soft tissues and periosteum. of the epiphyseal growth plate (Williams et al. 2007). Its functional Secondary bone healing is contributed by mesenchymal and osteo- role involves the promotion of neuronal development, survival and progenitor cells by endochondral ossification. maintenance. In addition to that, it plays an important role in regu-

Table: Shortcomings of growth factors currently used for bone healing

Other growth factors Shortcomings References

Bone morphogenetic protein-2 and -4 – Expensive (BMP) – Found in proliferating chondrocytes but at low levels in hypertrophic Oryan et al. (2013); Canalis mature chondrocytes (2012) – Proangiogenic – Promote osteoclastogenesis – Overexpression of BMP-4 causes osteopenia and increased number of

Platelet-derived growth factor (PDGF) – Released by the degranulation of platelets – Upregulates expression of VEGF and promote angiogenesis Fries and Colins (1992); Han- – mRNA has a very short half-life, therefore, transcription level is low in kenson et al. (2011) vivo

Transforming growth factor–β (TGF–β) – Weakly expressed in proliferating mesenchymal cells and endothelial Oryan et al. (2013); Canalis cells in inflammatory phase (2012) – Proangiogenic – Promotes osteoclastogenesis – Inhibits alkaline phosphatase activity and synthesis of osteocalcin -> inhibit differentiated function of osteoblasts – Not direct maturation of undifferentiated cells to osteoblasts – Concentration-dependent – High concentration- inhibits osteoclast formation – Low concentration- promote osteoclast formation

Insulin-like growth factor-1 – Produced by bone marrow stromal cells, lineage cells of osteoblasts that Dimitriu et al. (2011); Meinel et express IGF-1 receptor as well as endothelial cells of bone al. (2003; Guvakova (2007) -– Regulates role in inflammation via nuclear factor kappa-light-chain-en- hancer of activated B cells (NF-κB) pathway – Not expressed in the inflammatory phase of repair – Systemic administration is not appropriate due to the poor bioavailability at the injury site

Fibroblast growth factor-1 – Expressed only in osteoblasts Fries and Colins (1992), Han- (FGF-1) – Expressed during the development of bones kenson et al. (2011); Hurley et – Actions are dependent on the interaction with FGF receptors al. (2008)

Fibroblast growth factor –2 – Expression is similar throughout repair, without any peaks Dimitriou et al. (2011); Canalis (FGF-2) – Inhibits type I collagen, alkaline phosphatase activity, synthesis of osteo- (2013); Hankenson et al. (2011); pontin and osteocalcin Hurley et al. (2008) – Increases osteoclastogenesis, and thus increases bone resorption – Transcription of FGF-2 is induced by

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Cancer Diabetic retinopathy ● direct suppression of tumours17 ● loss of anti-angiogenic PEDF18 – proliferation and pro- differentiation ● overexpression of VEGF18 ● indirect suppression of tumours17 ● abnormal growth of new blood vessel18 – anti-angiogenesis ● visual loss18 ● affects bone, prostate, breast, lung and pancreatic carcinoma 17

PEDF

Insulin resistance Angiogenesis ● present in adipocytes and HSkMC19,20 ● formation of new blood vessels21 Fig. 1: Effects of PEDF on cancer, diabetic retino- (human skeletal muscle cell) ● downregulated by PEDF22 pathy, insulin resistance and angiogene sis ● short-term use → reduce ● upregulated by VEGF 22 The diagram shows the various associa- 20 tions of PEDF with diseases. Most of the insulin signal transduction data has been acquired preclinically in vi- ● long-term use → stimulate tro and relevant animal models of disease. lipolysis in adipose tissues20 VEGF, vascular endothelial growth factor. lating cell cycle and growth arrest. The presence of PEDF in bone bone tumours, as well as osteoarthritis (Takenaka et al. 2005). The tissue makes it a promising candidate for bone therapy in light of diseases associated with PEDF are summarised in Fig. 1. the drawbacks of other growth factors currently used (Table). PEDF is expressed throughout the body (Ek et al. 2006). PEDF is 4. Diseases associated with PEDF responsible for the differentiation of precursor cells such as mesen- chymal cells into osteoblasts, leading to the formation of bones. In PEDF was initially thought to be a neurotrophic factor that can be addition to that, it is also responsible for the inhibition of adipo- differentiated into non-proliferating neurons from Y79 retinoblas- genesis, the differentiation of cells from pre-adipocytes into adipo- toma (Crow et al. 2009). However, it was later discovered that it has far greater anti-angiogenic activity compared to other known cytes, and by doing so suppresses peroxisome proliferator- acti- endogenously-produced factors. Pathological conditions which vated receptor- γ (PPARγ), as well as other adipocyte include atherosclerosis, diabetic complications, chronic inflamma- markers (Gattu et al. 2013). Moreover, the differentiation of cells tory diseases as well as cancers were investigated with a focus on into osteoblasts by PEDF resulted in a common pathway that also the role of PEDF in these disorders (Crowe et al. 2009; Zhang et γ involves the suppression of PPAR . However, PEDF is expressed al. 2005). to a lesser extent by osteoclasts, which are cells responsible for PEDF has therapeutic potential in cancer treatment as it is asso- resorption of bones (Tombran-Tink and Barnstable 2004). ciated with direct and indirect suppression of tumours (Ek et al. Angiogenesis/neovascularization is a process that involves the 2006). PEDF expresses its anti-tumour effects directly in two formation of new blood vessels from pre-existing blood vessels ways, proliferation and pro-differentiation (Takenaka et al. 2005). (Tombran-Tink and Barnstable 2004; Hankenson et al. 2011). On the other hand, PEDF exhibits its anti-tumour effects indirectly This process is stimulated by the upregulation of vascular endo- via anti-angiogenesis (Tombran-Tink and Barnstable 2004; Take- thelial growth factor (VEGF), and the downregulation of PEDF naka et al. 2005). Tumour cells undergo apoptosis by inducing the (Tombran-Tink and Barnstable 2004; Takenaka et al. 2005). In Fas/Fas ligand pathway when PEDF interacts with the tumour cells other words, it is the counterbalance between the actions of pro-an- directly via anti-proliferation (Ek et al. 2006). It also decreases giogenic and the actions of anti-angiogenic factors (Hankenson et the number of cells that enter the S-phase, hence, increasing the al. 2011; Takenaka et al. 2005). PEDF was shown to be expressed number of cells that enter the G0 phase which is the resting phase during early bone development by osteoblasts and therefore PEDF by the anti-proliferative pathway. Through the indirect anti-tumour plays a role in balancing the actions of pro-angiogenic factor pathway which involves the decrease in the expression of pro-an- VEGF and its receptors (Hankenson et al. 2011). Nonetheless, if giogenic factors, as well as the direct anti-proliferative effects, bone injuries occur, angiogenesis will be triggered for the repair PEDF inhibits the source of oxygen and nutrients by inhibiting of bones, however, it can promote the onset and progression of the development of new blood supply, which results in tumour cell

Fracture healing Growth plate cartilage (GPC) ● bone regeneration24 ● cartilage that is present within the ● more studies required for PEDF metaphysis at the end of each long bone25 ● site for bone elongation

Differentiation Bone ● preosteoblast → osteoblast17 ● mesenchymal stem cells → osteoblast17

Osteogenesis imperfecta Reduction in osteosarcoma ● brittle bone disease26 ● reduce proliferation of tumour cells27 Fig. 2: Association of PEDF with bone physiology ● fragile bones and reduced ● PEDF reduce new blood supply to and pathophysiology skeletal mass26 tumour cells27 Links between PEDF and bone normal and ● contains unmineralised matrix26 disease biology. The diagram summarises ● due to loss of PEDF26 data from in vitro to clinical studies. Pharmazie 71 (2016) 173 REVIEW death (Ek et al. 2006; Takenaka et al. 2005). Hence, overexpression Crowe S, Wu LE, Economou C, Turpin SM, Matzaris M, Hoehn KL, Hevener AL, of PEDF will significantly reduce the proliferation of tumour cells, James DE, Duh EJ, Watt MJ (2009) Pigment epithelium-derived factor contributes to insulin resistance in obesity. Cell Metabol 10: 40-47. for instance, suppressing osteosarcoma growth and the develop- Dimitriou R, Jones E, McGonagle D, Giannoudis PV (2011) Bone regeneration: ment of pulmonary metastases (Ek et al. 2006). Other than that, current concepts and future directions. BMC Med 9: 66. it is also involved in the development and progression of various Ek E, Dass CR, Choong P (2006) PEDF: a potential molecular therapeutic target with tumours which include breast, prostate, lung and pancreatic carci- multiple anti-cancer activities. Trends Mol Med 12: 497-502. Elbackly RM, Mastrogiacomo M, Cancedda R (2014) Bone regeneration and bioen- noma (Tombran-Tink and Barnstable 2004; Takenaka et al. 2005). gineering. In: Orlando G et al. (eds) Regenerative Medicine Applications in Organ PEDF is an important inhibitor of angiogenesis in the mamma- Transplantation. Amsterdam, pp. 783-797. lian eye regulated by oxygen (Crowe et al. 2009). Therefore, Famulla S, Lamers D, Hartwig S, Passlack W, Horrighs A, Cramer A, Lehr S, Sell loss of anti-angiogenic PEDF will result in patients developing H, Eckel J. (2011) Pigment epithelium-derived factor (PEDF) is one of the most abundant proteins secreted by human adipocytes and induces insulin resistance and sight-threatening eye diseases such as retinopathy, especially in inflammatory signalling in muscle and fat cells. Int J Obes 35: 762-772. premature infants and diabetic patients (Stellmach et al. 2001). The Fedarko NS (2014) /osteoclast development and function. In: Shapiro JR abnormal growth of new blood vessels in the retina are responsible et al. (eds) Osteogenesis Imperfecta. A Translational Approach to Brittle Bone for visual loss (Takenaka et al. 2005; Stellmach et al. 2001). Isch- Disease. London, pp. 45-56. Fries J, Colins T (1992) Platelet-derived growth factor expression in a transgenic aemic retinopathies are normally driven by the downregulation of model. Int 41: 584-589. PEDF and an overexpression of VEGF, an inducer of angiogenesis Gattu AK, Swenson ES, Iwakiri Y, Samuel VT, Troiano N, Berry R, Church CD, and leakiness of vessels, which occurs due to inadequate oxygen Rodeheffer MS, Carpenter TO, Chung C (2013) Determination of mesenchymal (Stellmach et al. 2001). stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content. FASEB J 27: 4384-4394. PEDF reduces the progression of retinopathy; however, it contrib- Guvakova MA (2007) Insulin-like growth factors control cell migration in health and utes to insulin resistance in adipocytes and human muscle skel- disease. Int J Biochem Cell Biol 39: 890-909. etal muscle cells (HSkMCs) (Crowe et al. 2009; Famulla et al. Hankenson KD, Dishowitz M, Gray C, Schenker M (2011) Angiogenesis in bone 2011). The short term use of PEDF will result in a reduction in regeneration. Injury 42: 556-561. 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