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VEGF: an Essential Mediator of Both Angiogenesis and Endochondral Ossification

The Biomedical and Tissue Engineering Group, Department of Orthodontics, Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR, China

Correspondence: ^* corresponding author, rabie{at}hkusua.hku.hk

	ABSTRACT

TOP ABSTRACT INTRODUCTION THE VEGF FAMILY AND... VEGF RECEPTORS REGULATION OF VEGF-A GENE... ANGIOGENESIS AND ENDOCHONDRAL... THERAPEUTIC IMPLICATIONS IN... REFERENCES

 
During bone growth, development, and remodeling, angiogenesis as well as osteogenesis are closely associated processes, sharing some essential mediators. Vascular endothelial growth factor (VEGF) was initially recognized as the best-characterized endothelial-specific growth factor, which increased vascular permeability and angiogenesis, and it is now apparent that this cytokine regulates multiple biological functions in the endochondral ossification of mandibular condylar growth, as well as long bone formation. The complexity of VEGF biology is paralleled by the emerging complexity of interactions between VEGF ligands and their receptors. This narrative review summarizes the family of VEGF-related molecules, including 7 mammalian members, namely, VEGF, placenta growth factor (PLGF), and VEGF-B, -C, -D, -E, and -F. The biological functions of VEGF are mediated by at least 3 corresponding receptors: VEGFR-1/Flt-1, VEGFR-2/Flk-1, VEGFR-3/Flt-4 and 2 co-receptors of neuropilin (NRP) and heparan sulfate proteoglycans (HSPGs). Current findings on endochondral ossification are also discussed, with emphasis on VEGF-A action in osteoblasts, chondroblasts, and chondroclasts/osteoclasts and regulatory mechanisms involving oxygen tension, and some growth factors and hormones. Furthermore, the therapeutic implications of recombinant VEGF-A protein therapy and VEGF-A gene therapy are evaluated. Abbreviations used: VEGF, Vascular endothelial growth factor; PLGF, placenta growth factor; NRP, neuropilin; HSPGs, heparan sulfate proteoglycans; FGF, fibroblast growth factor; TGF, transforming growth factor; HGF, hepatocyte growth factor; TNF, tumor necrosis factor; ECM, extracellular matrix; RTKs, receptor tyrosine kinases; ERK, extracellular signal kinases; HIF, hypoxia-inducible factor

Key Words: angiogenesis • bone, cartilage, endochondral ossification • neovascularization, vascularity, recombinant protein • gene therapy • VEGF • VEGF receptor

	INTRODUCTION

TOP ABSTRACT INTRODUCTION THE VEGF FAMILY AND... VEGF RECEPTORS REGULATION OF VEGF-A GENE... ANGIOGENESIS AND ENDOCHONDRAL... THERAPEUTIC IMPLICATIONS IN... REFERENCES

 
During embryogenesis, blood vessels are formed by two processes, vasculogenesis and angiogenesis. Vasculogenesis is the formation of new blood vessels involving de novo differentiation of endothelial cells from mesoderm-derived precursor cells (angioblasts) (Gonzalez-Crussi, 1971). In contrast, angiogenesis, termed ’neovascularization’, is the formation of new blood vessels from a pre-existing vascular network, with continued expansion of a vascular tree in response to an increase in tissue mass (Risau, 1997; Carmeliet, 2005). Almost all tissues develop a functional circulatory system by sprouting or non-sprouting angiogenesis to form a primary capillary plexus. The metabolism and survival of normal tissue depend on the adequate supply of oxygen and nutrients from the vasculature. During adulthood, however, cells of most blood vessels are quiescent, except for those which respond to physiological processes such as wound healing, tissue remodeling, and the female reproductive cycle and pathological processes such as tumor expansion and metastasis (Folkman and Shing, 1992; Folkman, 1995). However, dysregulated and excessive vessel growth has a significant impact on health, and contributes to various diseases. Insufficient vessel growth or abnormal vessel regression causes stroke, Alzheimer’s disease, amyotrophic lateral sclerosis, hypertension, osteoporosis, respiratory distress, and other disorders. In contrast, cancer, rheumatoid arthritis, psoriasis, and diabetic retinopathy are the best-known diseases resulting from excessive or abnormal angiogenesis (Carmeliet, 2005).

The final event in endochondral ossification of bone growth and development is the replacement of the avascular cartilage template by highly vascularized bone tissue (Fig. 1). In this process, chondrocytes become hypertrophic and then produce a calcified cartilaginous extracellular matrix (ECM) and angiogenic stimulators, thus providing a target for capillary invasion and angiogenesis (Alini et al., 1996). The new vasculature supplies a conduit for the recruitment of the cell types involved in cartilage resorption and bone deposition, while providing the signals necessary for normal morphogenesis (Harper and Klagsbrun, 1999). Histological findings suggest that osteoblasts and osteoprogenitor cells always develop concomitantly with endothelial cells in the newly formed blood vessels at sites where new bone is formed (Deckers et al., 2000). Capillary endothelial cells provide microvasculature during bone remodeling (Erlebacher et al., 1995), and this vascular invasion is a prerequisite for bone formation (Guenther et al., 1986). Moreover, in the mandibular condyle, the terminal ends of the penetrating capillaries closely follow the outline of the previously eroded chondrocytic capsule, and the process of erosion is closely related to and dependent on osteoclastic activity (Durkin et al., 1973). Certainly, angiogenesis is of critical importance for the growth and development of long bones and the mandibular condyle, and these two biological events share a common mediator.

View larger version (54K): [in this window] [in a new window]   Figure 1. Schematic representation of the role of VEGF in mandibular condylar growth. The mandibular condyle is histologically distinguishable as 5 different layers: the fibrus, proliferative, chondroblast, hypertrophic, and erosive layers. Mesenchymal stem cells differentiate into chondroblasts, which form the framework of the cartilage matrix. VEGF, released from hypertrophic chondrocytes, induces endothelial cell invasion, which in turn induces vascular channel formation in the terminal layer of hypertrophic cartilage. The invading blood vessels bring progenitor mesenchymal cells, which later differentiate into the osteoblasts and chondroclasts/osteoclasts involved in endochondral ossification. Some growth factors regulate VEGF expression and participate in different stages of endochondral ossification.

This review summarizes the ongoing efforts made in: (1) characterizing VEGF family members, especially VEGF-A, and their receptors; (2) investigating the regulatory system of VEGF-A expression; (3) exploring the multiple roles of VEGF-A in endochondral ossification of long bones, as well as in mandibular condylar growth; and (4) developing therapeutic applications for recombinant protein and gene therapy.

	THE VEGF FAMILY AND ITS CHARACTERISTICS

TOP ABSTRACT INTRODUCTION THE VEGF FAMILY AND... VEGF RECEPTORS REGULATION OF VEGF-A GENE... ANGIOGENESIS AND ENDOCHONDRAL... THERAPEUTIC IMPLICATIONS IN... REFERENCES

 
VEGF is now understood to be one of a family of VEGFs that includes VEGF, placenta growth factor (PLGF), and VEGF-B, -C, -D, -E, and -F (Veikkola and Alitalo, 1999; Ferrara et al., 2003; Roy et al., 2006). They all share a common structure of 8 characteristically spaced cysteine residues in a VEGF homology domain.

VEGF-A
VEGF, generally referred to as VEGF-A, is a fundamental mediator of physiologic and pathophysiologic angiogenesis. It is also called vascular permeability factor (VPF) and is produced by cultured vascular smooth-muscle cells (Tischer et al., 1991). Structurally, VEGF-A is an antiparallel dimer that has receptor-binding sites at each pole of the dimer. The VEGF-A gene has been mapped to human chromosomes 6p21.3 (Vincenti et al., 1996), which can be alternatively spliced to generate at least 6 distinct isoforms of 121, 145, 165, 189, and 206 amino acids (termed VEGF-A₁₂₁, VEGF-A₁₄₅, VEGF-A₁₆₅, VEGF-A₁₈₉, and VEGF-A₂₀₆, respectively); a variant of VEGF-A₁₆₅, namely, VEGF-A_165b, has also been identified (Tischer et al., 1991) (Fig. 2, Table 1). Murine VEGF-As are shorter than the corresponding human isoforms by 1 amino acid, and they are denoted VEGF-A₁₂₀, VEGF-A₁₄₄, VEGF-A₁₆₄, and VEGF-A₁₈₈ (Tamayose et al., 1996). The VEGF-A isoforms have distinct biological activities, such as differential interaction with heparan sulfate proteoglycans (HSPGs) and neuropilin (NRP). The amino acids that are encoded by exons 1–5 are conserved in all isoforms, but alternative splicing can occur in exons 6 and 7 (Houck et al., 1991).

View larger version (37K): [in this window] [in a new window]   Figure 2. VEGF-A isoforms. There are at least 6 different isoforms of VEGF-A that arise by alternative exon splicing. VEGF165 is the predominant molecular species among the isoforms.

VEGF-A₁₄₅, an isoform lacking exons 6b and 7, appears in an intermediary manner, since it is secreted, but a significant segment remains bound to the cell surface and ECM (Ferrara and Davis-Smyth, 1997). VEGF-A₁₄₅ binds to VEGFR-1, VEGFR-2, HSPG, and NRP-2, but not to NRP-1 (Poltorak et al., 1997; Gluzman-Poltorak et al., 2000). Binding to HSPG, VEGF-A₁₄₅ was found to induce endothelial cell proliferation and in vivo angiogenesis (Poltorak et al., 1997). However, the actual physiological roles of VEGF-A₁₄₅’s affinity to NRP-2 remain unclear.

VEGF-A₁₆₅, lacking exons 6a and 6b, is physiologically the most abundant isoform of VEGF-A (Tischer et al., 1991). It binds to all of the receptors, including VEGFR-1, VEGFR-2, HSPGs, NRP-1, and NRP-2. Moreover, the affinity to VEGFR-1 is 10-fold higher than that to VEGFR-2 (Waltenberger et al., 1994). VEGF-A₁₆₅ and VEGF-A₁₂₁ are highly expressed in muscle, brain, kidney, eye, and chondrocytes (Gengrinovitch et al. , 1995). However, mice expressing only the VEGF-A₁₂₀ isoform (VEGF-A^120/120) presented with delayed recruitment of blood vessels into the perichondrium and delayed invasion of vessels into the primary ossification center (Zelzer et al., 2002), whereas VEGF-A^164/164 mice showed normal vascular development. In contrast, VEGF-A₁₂₁ was reported to have 10-to 100-fold less endothelial cell mitogenic activity than VEGF-A₁₆₅, due to lack of a HSPG-binding basic region (Gitay-Goren et al., 1996). Moreover, when chondrocytes are cultured in low oxygen conditions, they secrete VEGF-A₁₆₅. Both VEGF-A₁₆₅ and VEGF-A₁₂₁ might promote angiogenesis contributing to long bone development, cause permeabilization of blood vessels, induce proliferation of vascular endothelial cells, and stimulate osteoclastogenesis for tooth eruption (Ng et al., 2001; Wise and Yao, 2003; Cramer et al., 2004). The splice variant VEGF-A_165b contains the same quantity of amino acids as VEGF-A₁₆₅, except for 6 amino acids in the C-terminal region, denoted exon 9 (Bates et al., 2002; Woolard et al., 2004). The C-terminal is critical for mitogenic signaling; hence, the differentiation in the VEGF-A_165b variant is likely to affect its biological activity (Cui et al., 2004; Woolard et al., 2004; Byrne et al. , 2005). Although VEGF-A_165b can bind VEGFR-2, its binding leads to neither phosphorylation of the receptor nor activation of the downstream signal pathway. VEGF-A_165b is an endogenously inhibitory form of VEGF-A and reduces VEGF-A-activated proliferation and migration of endothelial cells (Cui et al., 2004; Woolard et al., 2004).

VEGF-A₁₈₉ and VEGF-A₂₀₆ contain all the exons. VEGF-A₁₈₉ has an insertion of 24 amino acids that are highly enriched in basic residues, and VEGF-A₂₀₆ has an additional insertion of 17 amino acids. These features mean that VEGF-A₁₈₉ and VEGF-A₂₀₆ are not secreted from cell surfaces, but are tightly bound to HSPGs and NRP, allowing for the sequestration of VEGF-A₁₈₉ and VEGF-A₂₀₆ in the ECM and on cell surfaces (Houck et al., 1992; Park et al. , 2003). VEGF-A₁₈₉ is usually present in low amounts, and VEGF-A₂₀₆ expression is restricted to embryonic tissues (Ferrara and Davis-Smyth, 1997). Hence, they are less active than other isoforms secreted in vivo , such as VEGF-A₁₂₁, VEGF-A₁₄₅, and VEGF-A₁₆₅. Protease cleavage of matrix-bound VEGF-A₁₈₉ allows for the release of an active, freely diffusible 110-amino-acid fragment (Lee et al., 2005). A recent study demonstrated that VEGF-A₁₈₉ could promote endothelial cell proliferation and migration in vitro, and angiogenesis in the Matrigel plug assay in vivo (Herve et al., 2005). However, VEGF-A^188/188 mice showed dwarfism, impaired development of growth plates and secondary ossification centers, and knee joint dysplasia. This phenotype was at least partly due to defective vascularization surrounding the epiphysis, leading to ectopically increased hypoxia and massive chondrocyte apoptosis in the interior of the epiphyseal cartilage. In addition, an in vitro study showed that the VEGF-A₁₈₈ isoform alone is also insufficient to regulate chondrocyte proliferation and survival responses to hypoxia (Maes et al., 2004).

PLGF
The first member of the VEGF family to be discovered, PLGF, shares 53% identity with the platelet-derived growth factor (PDGF)-like region of VEGF (Nissen et al., 1998). The human PLGF gene has been mapped to chromosome 14q24 (Mattei et al., 1996). PLGF is anticipated to have 149 amino acids and is encoded by 7 exons that span 800 kb (Roy et al., 2006). By alternative splicing, 4 forms of PLGF protein are generated: PLGF-1, PLGF-2, PLGF-3, and PLGF-4 (Maglione et al., 2000). Only PLGF-2 is capable of binding heparin (Hauser and Weich, 1993). Overexpression of PLGF-2 in perivascular tissue raises VEGF-A₁₆₅ and VEGF-A₁₂₁ levels and produces significant angiogenesis (Roy et al., 2006). PLGF has a powerful chemotactic effect on monocytes, since injection of PLGF protein and adenovirus-mediated PLGF gene transfer increases macrophage accumulation (Roy et al., 2005). Furthermore, PLGF deficiency impairs proliferation and differentiation of osteochondroprogenitors during bone repair (Maes et al., 2006).

VEGF-B
VEGF-B, which is also called VEGF-related factor (VRF), encodes 188 amino acids, consists of 8 exons and 6 introns, and is located on chromosome 11q13 (Paavonen et al., 1996). VEGF-B transcripts can be alternatively spliced into two different variants encoding proteins VEGF-B₁₆₇ and VEGF-B₁₈₆ (Li et al., 2001). The promoter region of VEGF-B is similar to that of VEGF-A, in that both promoters are associated with a CpG island and contain transcription-factor-binding sites for Sp1 and AP-2 (Silins et al., 1997). However, the VEGF-B promoter includes Egr-1 sites, not hypoxia-inducible factor-1 and AP-1 sites. Consequently, stimuli such as hypoxia, which can regulate VEGF-A expression, appear to have no effect on the expression of VEGF-B (Enholm et al., 1997; Roy et al., 2006). VEGF-B is highly abundant in the heart, skeletal muscle, and pancreas and is required in vascularization of skeletal muscle (Enholm et al., 1997). Silvestre et al.(2003) demonstrated that VEGF-B, partly through its receptor VEGFR-1, induces angiogenesis associated with an activation of Akt and eNOS-related pathways.

VEGF-C
VEGF-C, which is also referred to as VEGF-related protein (VRP), is located on chromosome 4q34 (Paavonen et al., 1996). The VEGF-C gene contains more than 40 kb of genomic DNA and consists of 7 exons (Roy et al., 2006). VEGF-C is a secreted protein that consists of 399 amino acids and was identified as a ligand for the tyrosine kinase receptor VEGFR-3, which is associated with the lymphatic vasculature (Karkkainen et al., 2004). Structurally, VEGF-C shares approximately 30% sequence homology with the central core of VEGF-A. In vivo, VEGF-C can stimulate angiogenesis in a rabbit ischemic hind-limb model, rabbit cornea assay, and early chick chorioallantoic membrane. In vitro, VEGF-C can stimulate endothelial cell proliferation and migration, but it is less potent than VEGF-A. Gene transfer of VEGF-C produced moderate angiogenesis in rabbit skeletal as well as perivascular tissue (Bhardwaj et al., 2003). VEGF-C is overexpressed in rheumatoid arthritis (RA) synovial tissues, when compared with osteoarthritis (OA) or normal synovial tissues (Wauke et al., 2002). In addition, tumor necrosis factor (TNF)- has been recently identified to induce VEGF-C expression significantly in rheumatoid synoviocytes in a dose-dependent manner. It suggests that VEGF-C may be indispensable for the pathogenesis of RA through contributing to local lymphangiogenesis and tumor angiogenesis (Cha et al., 2007).

VEGF-D
VEGF-D, also called c-fos-induced growth factor (FIGF), can activate the receptors VEGFR-2 and VEGFR-3 in humans, but only VEGFR-3 in mice (Baldwin et al., 2001). It is expressed in many adult tissues, including the limb buds, teeth, liver, and heart as well as the lung and kidney mesenchyme and periosteum of the vertebral column (Avantaggiato et al., 1998). The human VEGF-D gene is 2.0 kb in size and is located on chromosome Xp22.31 (Yamada et al., 1997). VEGF-D is mitogenic for endothelial cells in vitro (Bhardwaj et al., 2003), angiogenic in vivo and in vitro (Marconcini et al., 1999), and lymphangiogenic in tumors (Miyata et al., 2006). Compared with other human VEGFs, VEGF-D is thought to be the most potent angiogenic and lymphangiogenic factor when delivered into rabbit hindlimb skeletal muscle through adenoviral vector-mediated gene transfer (Rissanen et al., 2003).

VEGF-E
VEGF-E was first identified in the genome of NZ-7, NZ-2, and D1701 strains of the Orf virus, a parapoxvirus that infects goats, sheep, and occasionally humans (Ferrara and Davis-Smyth, 1997; Ogawa et al., 1998). VEGF-E exclusively binds to VEGFR-1 with high affinity, similar to VEGF-A₁₆₅, resulting in receptor autophosphorylation and a biphasic rise in the intracellular concentration of free calcium ions (Meyer et al., 1999). Although VEGF-E does not bear a heparin-binding basic region, structurally similar to VEGF-A₁₂₁, its biological activities for endothelial cell growth and vascular permeability are almost equal to those of the potent angiogenesis and vascular permeability factor, VEGF-A₁₆₅ (Ogawa et al., 1998). This VEGF homolog is a potent angiogenesis stimulator and can induce the proliferation, migration, sprouting, and mitotic activity of vascular endothelial cells. More recently, Inoue et al.(2006) reported that plasmid-mediated VEGF-E/PLGF chimera gene therapy significantly promoted angiogenesis in a rat model of hindlimb ischemia without stimulating inflammatory cell infiltration.

VEGF-F
The seventh member of the VEGF family, VEGF-F, was recently identified from snake (viper) venom (Suto et al., 2005). VEGF-F consists of 2 VEGF-related proteins, designated vammin (110 residues) and VR-1 (109 residues), both of which have a 50% primary structural identity with VEGF-A₁₆₅ and bind selectively to VEGFR-2 (Suto et al., 2005). VEGF-F contains a short C-terminal heparin-binding region; the C-terminus of VEGF-F specifically blocks VEGF-A₁₆₅ activity both in vitro and in vivo (Yamazaki et al., 2005).

	VEGF RECEPTORS

TOP ABSTRACT INTRODUCTION THE VEGF FAMILY AND... VEGF RECEPTORS REGULATION OF VEGF-A GENE... ANGIOGENESIS AND ENDOCHONDRAL... THERAPEUTIC IMPLICATIONS IN... REFERENCES

 
The main receptors involved in VEGF signal transduction are the PDGF receptor subfamily of receptor tyrosine kinases (RTKs)—namely, VEGFR-1, VEGFR-2, VEGFR-3—as well as co-receptors, such as NRP-1 and -2 and HSPGs (Ferrara et al., 2003) (Fig. 3). More than one type of VEGF receptor contributes to the biological responses to VEGF, thereby ensuring balanced signaling.

View larger version (29K): [in this window] [in a new window]   Figure 3. Schematic representation of the VEGF receptors and their ligands. The tyrosine kinase receptors (VEGFR-1/Flt-1, VEGFR-2/KDR/Flk-1, VEGFR-3/Flt-4) are organized into 7 extracellular immunoglobulin (Ig)-like domains. VEGF binding has been localized to the second and third Ig-like domains. The fourth Ig-like domains are thought to interact directly with each other in a ligand-induced receptor dimer. In VEGFR-3, the fifth Ig domain is replaced by a disulfide bridge. The extracellular domain is followed by a single transmembrane region, a split tyrosine-kinase domain. The interaction of VEGFR-1, -2 with either NRP-1, -2, or HSPG may facilitate the binding of VEGF to its receptor.

VEGFR-2
VEGFR-2, or kinase insert domain-containing receptor (KDR)/fetal liver kinase 1 (flk-1), is a 230-kDa glycoprotein that is thought to mediate almost all of the observed endothelial cell responses to VEGF (Neufeld et al., 1999). It binds to all the isoforms of VEGF-A, VEGF-C, VEGF-D, and VEGF-F, but not VEGF-B₁₆₇ and PLGF (Ferrara and Davis-Smyth, 1997). VEGFR-2 is predominantly located on the surfaces of endothelial cells and is thought to initiate intracellular signal transduction, which is associated with the integrin-dependent migration of endothelial cells, since it can form a complex with integrin Vβ₃ and the induction of endothelial cell proliferation, migration, and in vivo angiogenesis (Hutchings et al., 2003). It has been reported that VEGFR-2-mediated cell migration induced by VEGF-A₁₆₅, but not by VEGF-A₁₂₁, is strongly enhanced in the presence of NRP-1 receptors (Shraga-Heled et al., 2007). VEGFR-2 knockout mice showed severely impaired vasculogenesis and hematopoiesis in the embryo, leading to embryonic death (Shalaby et al., 1995). Moreover, expression of VEGFR-2 occurs in a differentiation-dependent manner and declines post-natally (Millauer et al., 1993). In contrast, the binding of VEGF to VEGFR-1 does not seem to result in the induction of cell proliferation, and it is believed that its role is mainly inhibitory (Shraga-Heled et al., 2007).

VEGFR-3
VEGFR-3 or flt-4, a 170-kDa glycosylated protein, is a receptor for VEGF-C and VEGF-D, but not for VEGF-A. It is required for the development of blood as well as lymphatic vessels and is thought to have a general function in blood vascularization during early development (Lohela et al., 2003); later, its role becomes restricted mostly to the development of the lymphatic vascular system (Dayoub et al., 2003). Furthermore, expression of VEGFR-3 has been reported to be higher in human osteoarthritic chondrocytes than in persons without arthritis (Shakibaei et al., 2003).

NRPs
NRPs were recently identified to be VEGF co-receptors and belong to a family of non-tyrosine kinase transmembrane receptors that have a small cytoplasmic domain and multiple extracellular domains (Soker et al., 1998). NRP-1 lacks an intracellular tyrosine kinase domain and therefore must act in conjunction with other receptors to mediate VEGF signaling. It can associate with both VEGFR-1 and VEGFR-2 by specifically binding VEGF-A₁₆₅ and PLGF, VEGF-B, and VEGF-E, but not VEGF-A₁₂₁ (Zelzer et al., 2001). NRP-2 also lacks a cytoplasmic signaling domain and can bind to VEGF-A₁₆₅, VEGF-A₁₄₅, and PLGF and can interact with VEGFR-1 (Gluzman-Poltorak et al., 2000, 2001). NRPs act as active mediators in immune responses, neuronal development, and angiogenesis (Klagsbrun et al., 2002). Deletion of the NRP-2 gene impairs formation of small lymphatic vessels, suggesting that NRP-2 may act as a co-receptor for VEGFR-3 (Yuan et al., 2002). Both NRP-1 and NRP-2 can enhance VEGF-A₁₂₁-induced phosphorylation of VEGFR-2 and VEGF-A₁₂₁-induced proliferation of endothelial cells. The enhancement of VEGF-A121 activity by NRP-1 has been reported to be accompanied by a significant increase in the binding affinity of VEGFR-2 to VEGF-A₁₂₁ and was not associated with the formation of new VEGFR-2/NRP-1 complexes (Shraga-Heled et al., 2007). NRP-1 expression in cultured MC3T3-E1 osteoblasts was down-regulated in a differentiation-dependent manner and bound to VEGF-A₁₆₅ (Deckers et al., 2000). Consistent with the in vitro studies, NRP1 is expressed in vivo by osteoblasts, but not osteocytes, in the metaphysis and trabeculae of growing mouse bones and embryonic chick bones (Harper et al., 2001). The overexpression of NRP-1 showed extra digits, suggesting that limb morphogenesis is very sensitive to exogenous neuropilin expression (Kitsukawa et al., 1995). In addition, the binding of semaphorin-3A (an axonal chemorepellent) to NRP-1 may compete with VEGF-A₁₆₅ for an overlapping binding site on NRP-1, and may inhibit the migration of endothelial cells and the outgrowth of capillary tubes from rat aortic segments (Narazaki and Tosato, 2006). Thus, NRP-1 appears to be a novel regulator of osteoblast activity in development of the skeletal system, and semaphorin-3A might affect its affinity to VEGF-A₁₆₅. However, the contribution of NRP-2 to bone development is still unclear.

HSPGs
HSPGs are transmembrane, glycosylphosphatidylinositol-anchored or secreted proteins that contain covalently linked heparan sulfate chains that modulate the activity of a large number of secreted signaling molecules. HSPGs might regulate the binding of VEGF-A₁₆₅ to its receptors via an accessory low-affinity binding site; in this way, HSPGs promote VEGF-A₁₆₅ signaling in transit from the surface of an adjacent cell (Houck et al., 1992; Selleck, 2006). Such ligand-receptor interactions stimulate VEGFR-2 turnover and cause a marked increase in duration and amplitude of the VEGFR signal (Jakobsson et al., 2006; Selleck, 2006). HSPGs also have profound effects on the bioactivity of VEGF-A₁₆₅, but not VEGF-A₁₂₁: The high-affinity binding to VEGF-A₁₆₅ affects its diffusion, half-life, and interaction through VEGFR-2 (Larrivee and Karsan, 2000; Stringer, 2006). HSPGs are considered to be both important co-receptors and important regulators of VEGF-A gradients. Exogenous heparin is known to inhibit both binding ability between VEGFR-2 and VEGF-A₁₆₅ and autophosphorylation of VEGFR-2 induced by VEGF-A₁₆₅, but it has no effect on the interaction of VEGFR-2 with VEGF-A₁₂₁ (Tessler et al., 1994). In contrast, exogenous heparin significantly inhibits the affinity for VEGFR-1 with both VEGF-A₁₆₅- and VEGF-A₁₂₁ (Cohen et al., 1995). These observations suggest that the C-terminal region of VEGF-A₁₆₅ is important in the interaction of cell-associated VEGFR-2, while heparin affects the activity role of VEGFR-1 independently of the heparin-binding ability of the ligands (Yamazaki and Morita, 2006).

	REGULATION OF VEGF-A GENE EXPRESSION

TOP ABSTRACT INTRODUCTION THE VEGF FAMILY AND... VEGF RECEPTORS REGULATION OF VEGF-A GENE... ANGIOGENESIS AND ENDOCHONDRAL... THERAPEUTIC IMPLICATIONS IN... REFERENCES

 
Several mechanisms have been demonstrated to be involved in the regulation of VEGF expression, including hypoxia and several growth factors and hormones. An overview of the regulation factors appears in Table 2, and these will be discussed in the following sections.

Cbfa1
Cbfa1 (core-binding factor a1, also called runx2, Osf2, or AML3), a homolog of the Drosophila Runt protein, is a transcription factor that is required for endochondral and intramembranous bone formation. It is expressed in osteoblasts, pre-chondrogenic mesenchymal condensations, and hypertrophic chondrocytes, where it serves as the earliest transcriptional regulator of osteoblast differentiation (Rabie et al., 2004; Tang and Rabie, 2005). Cbfa1 is sufficient to induce premature and ectopic chondrocyte hypertrophy (Rabie et al., 2004). In Cbfa1-deficient mice, VEGF-A cannot be up-regulated, chondrocyte differentiation to hypertrophy is impaired, and cartilage angiogenesis does not occur. These findings demonstrate that Cbfa1 is an essential factor for mediating VEGF-A functions during endochondral ossification (Zelzer et al., 2001). Whether Cbfa1 acts as a direct transcriptional regulator of the VEGF-A gene is unclear. However, the VEGF-A promoter contains Cbfa1 binding sites, and over-expression of Cbfa1 in cultured fibroblasts increases both mRNA and protein expression levels of VEGF (Zelzer et al., 2001).

BMPs
Bone morphogenetic proteins (BMP) are multi-functional growth factors that belong to the TGF-β superfamily. The addition of BMP-2, -4, and -6 to the murine osteoblast-like cell line KS483 led to an augmentation of calcium deposition and VEGF-A production in a dose-dependent manner (Deckers et al., 2000). Osteogenic protein-1 (OP-1 or BMP-7) increased the steady-state level of VEGF-A mRNA by about three-fold in an OP-1 concentration- and time-dependent manner in primary cultures of fetal rat calvaria cells. The increase in VEGF-A mRNA level depended on transcription and was sensitive to cell replication (Yeh and Lee, 1999). The mRNA levels for VEGFR-1 and VEGFR-2 in the fetal rat calvaria cells were low but detectable by RT-PCR, and were not changed by OP-1 (Yeh and Lee, 1999). Kakudo et al.(2006) investigated whether recombinant human BMP-2 could cause undifferentiated mesenchymal cells to differentiate into chondrocytes and osteoblasts, which then expressed VEGF, thereby creating an advantageous environment for vascularization in bony tissue. In addition to increased VEGF-A expression, BMP-2 is able to up-regulate VEGF-B and VEGF-C expression and synthesis, as well as increase VEGFR-2 levels (Bluteau et al., 2007).

TGF-β1
At least part of the osteogenic activity of TGF-β1 may be attributed to the production of VEGF-A (Chang et al., 2003). The addition of TGF-β1 to MC3T3-E1 cells induced VEGF-A mRNA production, and this induced expression was superinduced by cycloheximide and blocked by actinomycin D and Ro 31-8220, a protein kinase C (PKC) inhibitor. Curcumin, an inhibitor for transcription factor AP-1, also blocked the induction (Spector et al., 2000). Moreover, phorbolmyristate acetate (PMA), a PKC activator, can enhance VEGF expression in cultured dental follicle cells (Wise and Yao, 2003). Further research has shown that both p44/p42 MAP kinase and p38 MAP kinase contribute to TGF-β-stimulated VEGF-A synthesis in osteoblasts, which suggests a positive feedback loop for osteoblast differentiation (Tokuda et al., 2003a).

CTGF
Connective tissue growth factor (CTGF) is a crucial regulator of cartilage ECM (Perbal, 2004). Ctgf deficiency in Ctgf(–/–) mice leads to skeletal dysmorphisms, as a result of impaired endochondral ossification (Ivkovic et al., 2003). These defects are linked to decreased expression of VEGF-A mRNA and protein levels in the expanded hypertrophic zone in newborn Ctgf mutants. Thus, VEGF-A is probably a target of CTGF action in hypertrophic cartilage (Ivkovic et al., 2003). Moreover, this decrease is not the result of reduced Cbfa1 expression, suggesting that CTGF is either downstream of, or acts in parallel with, Cbfa1 in regulating VEGF-A expression (Ivkovic et al. , 2003). VEGF-A₁₆₅-induced angiogenesis was reported to be inhibited selectively and strongly by complexing with CTGF. This complex formation interrupts the binding of VEGF-A₁₆₅ to VEGFR-2 (Inoki et al., 2002).

MMP9
Reduced expression of the osteoclastic protease MMP9 affects angiogenesis-inducing activity in growth plates (Vu et al., 1998), and MMP9-null growth plates share several characteristics with mouse growth plates in which VEGF-A activities have been blocked, such as expansion of the hypertrophic layer and reduction in both vascularization and ossification (Vu et al., 1998). Hence, it has been suggested that MMP9 regulates vascular invasion by releasing the VEGF-A that is bound to the hypertrophic cartilage matrix. Once released, VEGF-A can bind to its receptors on endothelial cells, osteoclasts, and osteoblasts, and, in so doing, can stimulate their migration and activity at the fracture site (Nakagawa et al., 2000).

TNF-
TNF- is a pro-inflammatory cytokine that enhances VEGF-A expression in follicle cells and periodontal ligament cells (Oyama et al., 2000; Wise and Yao, 2003). The enhanced expression of VEGF-A in the dental follicle cells by TNF- is considerable and could be the main avenue by which TNF- expression leads to the recruitment and differentiation of osteoclasts (Wise and Yao, 2003).

Hormones
It is widely recognized that the role of thyroid hormone in bone metabolism is mainly ascribed to the regulation of osteoblast activities. Triiodothyronine (T₃) alone could stimulate VEGF-A release in MC3T3-E1 cells, so it is likely that thyroid hormone stimulates bone metabolism partially via the up-regulation of VEGF-A release (Tokuda et al., 2003b). In human osteoblast-like cells, a steady expression of VEGF-A is induced by 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] (Wang et al., 1996). Furthermore, when alkaline phosphatase(ALP)-positive human osteoblast-like cells are co-cultured with ALP-negative human umbilical vein endothelial cells, the addition of 1,25-(OH)₂D₃ stimulates ALP activity in the osteoblast-like cells, and increases the expression of VEGF mRNA, followed by increased secretion of VEGF-A (Petit et al., 1997). More recently, ED-71, a novel vitamin D[1,25-(OH)₂D₃] analog, has been demonstrated to improve angiogenesis within the bone marrow cavity through the increased expression of VEGF-A₁₂₀ (Okuda et al., 2006).

	ANGIOGENESIS AND ENDOCHONDRAL OSSIFICATION

TOP ABSTRACT INTRODUCTION THE VEGF FAMILY AND... VEGF RECEPTORS REGULATION OF VEGF-A GENE... ANGIOGENESIS AND ENDOCHONDRAL... THERAPEUTIC IMPLICATIONS IN... REFERENCES

 
VEGF-A is predominantly produced in tissues that acquire new capillary networks (Shweiki et al., 1993), and the importance of VEGF-A during angiogenesis has been demonstrated in gene knockout studies. Homozygous Vegfa knockout mice die at embryonic days E8-E9, and mice lacking a single Vegfa allele die at days E11-E12, due to deficient endothelial cell development and lack of blood vessels (Carmeliet et al., 1996; Tammela et al., 2005). Moreover, inducible deletion of VEGF-A in neonatal mice results in stunted growth and increased apoptosis of endothelial cells (Gerber et al., 1999). The knockout of one allele of VEGFR-2 in mice also resulted in lack of normal blood vessel development and embryonic lethality (Shalaby et al., 1995). The failure of vasculogenesis leads to growth retardation, developmental anomalies, and, ultimately, embryonic lethality (Carmeliet et al., 1996). Using a VEGF-A120/120 mouse model created by Cre-loxP-mediated removal of VEGF-A exons 6 and 7, researchers showed that VEGF-A₁₆₄ and/or VEGF-A₁₈₈ is important in both mediating vascularization and ensuring the normal differentiation of progenitors into hypertrophic chondrocytes, osteoblasts, endothelial cells, and osteoclasts (Maes et al., 2002, 2004).

Hence, VEGF-A is not only essential for normal angiogenesis, but it is also involved in endochondral ossification (Midy and Plouet, 1994; Gerber et al., 1999; Rabie et al., 2002a; Aldridge et al., 2005) (Fig. 1). Cells in the upper zone of the hypertrophic cartilage, especially in the mandibular condyle, secrete VEGF-A, which then regulates the invasion of new blood vessels from the perichondrium and influences the removal of the cartilage matrix (Rabie and Hagg, 2002; Rabie et al., 2002a). The invading blood vessels bring progenitor mesenchymal cells to the mineralization front, and the cells later differentiate into osteoblasts and chondroclasts/osteoclasts involved in endochondral ossification (Rabie and Hagg, 2002; Leung et al., 2004). In 24-day-old mice, inactivation of VEGF-A through the systemic administration of a soluble VEGF-A receptor protein, Flt-(1–3)-IgG, suppresses blood vessel invasion, impairs trabecular bone formation, and expands the hypertrophic zone in the growth plate (Gerber et al., 1999). The chondrocyte-specific inactivation of VEGF-A results in aberrant endochondral bone formation, lack of normal blood vessel development, and embryonic lethality in transgenic mice (Haigh et al., 2000). Therefore, VEGF-A seems to play a key role in the highly regulated process of endochondral ossification, which depends on the interplay of chondrocytes, osteoblasts, and osteoclasts/chondroclasts (Fig. 4).

View larger version (31K): [in this window] [in a new window]   Figure 4. Schematic representation of the role of VEGF and the corresponding receptors on osteoblasts, chondrocytes, and chondroclasts/osteoclasts.

VEGF-A Regulates Chondroclast/Osteoclast Activity
It seems that VEGF-A is involved in controlling endothelial cell activities, as well as in osteoclastic differentiation, migration, and activity (Zelzer and Olsen, 2005). In addition, cartilage and bone resorption by chondroclasts and osteoclasts regulates vascular invasion and relies on degradation of the cartilaginous matrix, which occurs mainly via the release of VEGF-A (Rabie et al., 2002a,b). In fact, osteoclasts express VEGF receptor 1, which can bind VEGF-A to induce osteoclast recruitment and bone-resorption activity (Niida et al., 2005). A single injection of recombinant human VEGF-A (rhVEGF-A) into osteopetrotic op/op mice (lack of CSF-1) induced osteoclast recruitment and survival, while stimulating osteoclastic bone resorption (Niida et al., 1999). In contrast, the inhibition of VEGF-A resulted in impaired angiogenesis and a decrease of chondroclast and osteoblast numbers in growth plates (Dass et al., 2007). It has been suggested that VEGF-A is involved not only in osteoclastic recruitment and differentiation, but also in enhancing osteoclastic bone-resorbing activity in cultured rabbit mature osteoclasts (Nakagawa et al., 2000). A recent study demonstrated that VEGF-A-elicited pathways are involved in the induction of receptor of NF-B ligand (RANKL) expression, suggesting that VEGF-A plays an important role in modulating the angiogenic action of RANKL under physiological or pathological conditions (Henriksen et al., 2003). In the process of monocytic precursor cell differentiation, Niida et al.(1999) demonstrated that VEGF-A can substitute for CSF-1 in the presence of RANKL to promote osteoclastogenesis. Semi-quantitative RT-PCR and fluorescence-activated cell-sorter analysis revealed that VEGF-A can significantly increase both mRNA expression and surface protein expression of receptor activator of NF-B (RANK) in human endothelial cells, thereby increasing the angiogenic responses of endothelial cells to RANKL (Min et al., 2003). This up-regulation takes place mainly through the Flk-1/KDR-PKC-ERK signaling pathway (Min et al., 2003). VEGF-A can also up-regulate RANKL expression in osteoclast precursors, but it cannot fully substitute for CSF-1 to promote proliferation and osteoclastogenesis (Yao et al., 2006). Furthermore, according to a review by Zelzer and Olsen (2005), VEGF-A is produced by hypertrophic chondrocytes, induces osteoclastogenesis in the perichondrium, and stimulates migration of the osteoclasts into hypertrophic cartilage through the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway (Henriksen et al., 2003; Min et al., 2003).

VEGF-A Regulates Osteoblastic Activity
Although the exact mechanism of VEGF-A production and secretion in osteoblasts has not yet been fully clarified, VEGFs have been implicated in various aspects of osteoblast function. In vitro, VEGF-A mRNA was found to be expressed in rat calvaria-derived osteoblast-enriched cells (Harada et al., 1994). During calvaria organ culture, treatment with VEGF-A₁₆₄ led to a significant increase in the thickness of parietal bone, demonstrating a stimulatory effect of VEGF-A on bone formation (Zelzer et al., 2002). Several mechanisms for controlling osteoblast activity have been proposed. First, VEGF-A could couple angiogenesis and osteogenesis by manipulating the angiogenic response to osteoblastic activity. Second, VEGF-A could act as an autocrine regulator of osteoblastic differentiation and activity. Third, by expressing VEGF-A, osteoblasts could induce cells in the vicinity to express factors that, in turn, regulate osteoblastic activity (Zelzer and Olsen, 2005). The following studies support all three possibilities.

In 24-day-old mice, the inhibition of VEGF-A by soluble Flt-1 decreased angiogenesis, reduced cartilage formation, and delayed cartilage resorption (Gerber et al., 1999). VEGF-A can also directly promote differentiation of human primary cultured osteoblasts (Street et al., 2002). Deckers et al.(2000) showed that a low level of VEGF-A expression occurs at the early stage of osteoblast differentiation, before the detection of mRNAs encoding bone sialoprotein and osteocalcin. Only during the terminal differentiation of osteoblasts is their expression greatly increased, achieving a maximum level during the period of mineralization (Deckers et al., 2000). This finding suggests that VEGFs are indispensable in the regulation of bone remodeling, because they stimulate osteoblast differentiation. Moreover, the amount of increase in VEGF expression during osteoblastogenesis depends on the maturity of the osteoblastic cells (Furumatsu et al., 2003). Finally, VEGF-A acts as a potent chemoattractant for rat and human osteoblasts, as well as bone-marrow-derived mesenchymal progenitor cells (Nakagawa et al., 2000; Mayr-Wohlfart et al., 2002; Fiedler et al., 2005). VEGF treatment induces VEGF-D expression in osteoblasts, and the inactivation of VEGF-D activity by neutralizing antibodies or VEGFR-3 silencing inhibits both VEGF-A- and VEGF-D-dependent nodule formation in osteoblasts, suggesting that VEGF-D is a downstream effector of VEGF-A in osteogenesis (Orlandini et al., 2006).

	THERAPEUTIC IMPLICATIONS IN ENDOCHONDRAL OSSIFICATION AND PERSPECTIVES

TOP ABSTRACT INTRODUCTION THE VEGF FAMILY AND... VEGF RECEPTORS REGULATION OF VEGF-A GENE... ANGIOGENESIS AND ENDOCHONDRAL... THERAPEUTIC IMPLICATIONS IN... REFERENCES

 
Several experiments with recombinant VEGF proteins or genes have demonstrated that these treatment regimes seem to be safe, and the results have been encouraging for the treatment of coronary and limb ischemia (Street et al., 2002; Kastrup, 2003) and burn wounds (Galeano et al., 2003). However, the VEGF-A that was locally applied to rabbit tibia during distraction osteogenesis increased the blood flow in the distracted limb, but failed to influence bone mineral content and histomorphometric indices of bone regeneration (Eckardt et al., 2003). A possible explanation is that, during distraction osteogenesis, a high level of endogenous VEGF-A has already been secreted, and therefore the additional delivery of VEGF-A has little or no effect, owing to optimal endogenous VEGF-A signaling. Similar results were obtained in ex vivo gene therapy based on retrovirus-transferred muscle-derived stem cells (MDSCs): Supplying VEGF-A alone was not sufficient to initiate the cascade of bone regeneration in the critical-sized calvarial defects (Peng et al., 2002). However, VEGF-A acts synergistically with BMP₄ to recruit mesenchymal stem cells and induce cartilage formation and remodeling during endochondral ossification (Peng et al., 2002).

Gain-of-function studies with recombinant VEGF-A proteins or genes have increasingly found significant increases in vascularization and bone regeneration in defects (Table 3). For example, when poly(lactic-co-glycolic acid) (PLGA) scaffolds that have been incorporated with rhVEGF-A were loaded into radiated calvarial defects of Fisher rats, there were significant increases in blood vessel formation, bone coverage, and bone mineral density when compared with defects loaded with only PLGA scaffolds (Orlandini et al., 2006). Scaffolds coated with a rhVEGF-A-releasing layer (bioactive glass) also demonstrated significant improvements in blood vessel density and bone mineral density in rat cranial bone defects (Leach et al., 2006). The combination of rhVEGF-A and demineralized bone matrix grafts (Rabie, 1997) significantly increased new bone formation in rabbit parietal bone defects (Bassem et al., 2006). Local administration of rhVEGF-A exhibited an increased number of osteoclasts during experimental tooth movement, as well as increases in the amount of tooth movement (Kaku et al., 2001; Kohno et al., 2003). The use of rhVEGF-A in bone defect models has shown that new blood vessel formation preceded the osteogenic front, and that increased angiogenesis corresponded to increased bone formation (Kleinheinz et al., 2005). Although these findings are promising, the possible applications of these growth factors are limited by the need for repeated and expensive dosages, and by their short biological half-life (Zhang et al., 2002).

	ACKNOWLEDGMENTS

 
Work from the authors’ laboratory cited in this article was supported by grant No. 10206152.11222.21700.302.01, awarded by The University Strategic Research Theme: Genomics, Proteomics & Bioinformatics, The University of Hong Kong, as well as by CERG grant No. 10206968.22311.08003.324.01, awarded to Professor Rabie. We thank Mr. Ting Tung Yuen for preparing the illustration and Dr. Trevor Lane for editing assistance.

Received for publication December 8, 2006. Revision received May 22, 2007. Accepted for publication May 28, 2007.

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Journal of Dental Research, Vol. 86, No. 10, 937-950 (2007)
DOI: 10.1177/154405910708601006


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