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. Author manuscript; available in PMC: 2018 Apr 1.
Published in final edited form as: Dev Dyn. 2016 Dec 29;246(4):227–234. doi: 10.1002/dvdy.24463

Vascular Endothelial Growth Factor Control Mechanisms in Skeletal Growth and Repair

Kai Hu 1, Bjorn R Olsen 1
PMCID: PMC5354946  NIHMSID: NIHMS824633  PMID: 27750398

Abstract

Vascular Endothelial Growth factor A (VEGF) is a critical regulator of vascular development, postnatal angiogenesis and homeostasis, and it is essential for bone development and repair. Blood vessels serve both as structural templates for bone formation and they provide essential cells, growth factors and minerals needed for synthesis and mineralization, as well as turnover, of the extracellular matrix in bone. Through its regulation of angiogenesis, VEGF contributes to coupling of osteogenesis to angiogenesis, and it directly controls the differentiation and function of osteoblasts and osteoclasts. In this review we summarize the properties of VEGF and its receptors that are relevant to bone formation and repair; the roles of VEGF during development of endochondral and membranous bones; and the contributions of VEGF to bone healing during different phases of bone repair. Finally, we discuss contributions of altered VEGF function in inherited disorders with bone defects as part of their phenotypes, and we speculate on what will be required before therapeutic strategies based on VEGF modulation can be developed for clinical use to treat patients with bone growth disorders and/or compromised bone repair.

Introduction

Bones are highly vascularized organs. Blood vessel cells and bone cells communicate in many ways during development, fracture healing and bone regeneration (Clarkin and Gerstenfeld, 2013, Carano and Filvaroff, 2003). For example, osteoblasts are major sources of angiogenic factors, including VEGF, that stimulates angiogenesis (Wang et al., 2007, Hu and Olsen, 2016), and blood vessels provide oxygen, nutrients and minerals as well as secreted factors that are necessary for bone formation (Kusumbe et al., 2014). Within the bone marrow space, stem cell niches contain osteoblastic progenitor cells residing around blood vessels (Worthley et al., 2015), and periosteal osteoblast precursors, in a pericyte-like manner, migrate into bone-forming sites along with invading blood vessels during development and fracture healing (Maes et al., 2010). Vascular morphogenesis also provides a spatial and functional template for skeletal morphogenesis in development and postnatal growth. In early development, generation of hypoxic regions by loss of blood vessels is an essential step that allows mesenchymal condensations and cartilage models of endochondral bones to form (Amarilio et al., 2007); in turn, the condensations produce VEGF that regulates vascular morphogenesis in the tissues surrounding the cartilage-forming regions (Eshkar-Oren et al., 2009). During postnatal growth and homeostasis, lamellae of cortical bone are patterned around blood vessels and nerves in Haversian canals (Bogonatov and Gonchar-Zaikina, 1976), and metaphyseal trabecular bone is patterned around the vasculature that invades hypertrophic cartilage in developing and postnatal growth plates (Maes, 2013). Thus, osteogenesis and angiogenesis are coupled processes (Clarkin and Gerstenfeld, 2013; Ramasamy et al., 2015).

Since vascular control is essential for bone development and repair, impairment of this control affects the skeletal system. Deficiencies in vascular supply can lead to osteonecrosis (Childs, 2005), often involving mandibles or the ends of long bones. Reduction in the number of capillaries in trabecular bone is associated with decreased bone formation and bone mass in osteoporosis (Burkhardt et al., 1987). Lack of blood supply after bone injury is considered to be the major reason for compromised fracture healing, affecting about 10% of patients with bone fracture (Gomez-Barrena et al., 2015, Bishop et al., 2012). VEGF is one of the most important regulators of vascular development and angiogenesis (Coultas et al., 2005, Hoeben et al., 2004), and it is therefore also critical for bone health. In addition, VEGF has direct effects on osteoblast and osteoclast differentiation and function. In this review, we summarize the roles of VEGF in bone growth and repair, and we speculate on how VEGF-based therapeutic strategies may improve bone health.

VEGF

The VEGF family includes at least 6 members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (Cross et al., 2003, Ferrara et al., 2003). VEGF-A, usually referred to as VEGF, was discovered first and plays critical roles in angiogenesis and promotion of vessel permeability (Ferrara et al., 2003). Through the utilization of two transcriptional start sites and alternative splicing, the two VEGF-A primary transcripts generate several mRNAs (Arcondéguy et al., 2013). Consequently, several protein isoforms containing different numbers of amino acid residues exist. These include VEGF121, freely diffusing through the extracellular matrix (ECM) because it lacks heparin-binding sites, VEGF189 and VEGF206, both containing two heparin-binding sites and therefore sequestered in the ECM, and VEGF165, the most abundant isoform containing one heparin-binding site (Conn et al., 1990, Pepper et al., 1994). A variety of cell types within or around blood vessels express VEGF, such as smooth muscle cells, pericytes, osteoblasts, pneumocytes, podocytes and hepatocytes (Maharaj et al., 2006, Saint-Geniez et al., 2008). Although they are major targets of VEGF, endothelial cells (ECs) do not generally secrete much VEGF (Couffinhal et al., 1997). Studies using Vegf–lacZ mice, in which the location of VEGF expression can be identified by β-galactosidase staining, demonstrate that VEGF expression occurs in arterial ECs, but not in veins and capillaries (Maharaj et al., 2006). High levels of VEGF is found in ECs lining the aorta (Maharaj et al., 2006). Through binding to receptors on the endothelial surface, VEGF stimulates proliferation and migration in a paracrine manner; in contrast, the ability of VEGF in arterial ECs to maintain postnatal vascular homeostasis is likely based on an autocrine mechanism (Lee et al., 2007).

VEGF binds to the extracellular domains of two tyrosine kinase receptors, VEGF receptor 1 (VEGFR1/Flt-1) and VEGF receptor 2 (VEGFR2/KDR/Flk-1) (Ferrara et al., 2003; Koch and Claesson-Welsh, 2012). VEGFR1 is expressed as both secreted and membrane-bound forms, depending on alternative splicing of the primary transcript. Secreted (soluble) VEGFR1 (sVEGFR1) serves as a decoy receptor by binding to extracellular VEGF, preventing its binding to VEGFR2, the major receptor transducing VEGF signaling. Membrane-bound VEGFR1 serves either as a decoy receptor for VEGF in endothelial cells, particularly in tumor endothelium (Ferrara et al., 2003, Jinnin et al., 2008), or as a signaling receptor like VEGFR2, such as in VEGF-induced monocyte migration (Barleon et al., 1996, Maru et al., 1998). As a signaling receptor, VEGFR2 mediates angiogenesis and promotion of vessel permeability in response to VEGF (Ferrara et al., 2003). It is also expressed and functions in other cell types, such as pericytes, lymphocytes and osteoclasts. The levels of VEGFR expression in osteoblastic cells depend on animal species and methods used for cell isolation. VEGF receptors have been detected in cultures of primary human osteoblasts, and the migration, proliferation and differentiation of the cells could be stimulated by recombinant VEGF (rVEGF) (Mayr-Wohlfart et al., 2002). In other studies, primary murine mesenchymal progenitors and osteoblasts failed to respond to rVEGF, raising questions about VEGF receptor function in mouse osteoblastic cells (Liu et al., 2012, Hu and Olsen, 2016). However, mice with conditional deletion of Vegfr1 or Vegfr2 in osteoblastic cells exhibited reduced bone density two weeks after birth (Liu et al., 2012), and their bone marrow had reduced numbers of osteoprogenitors (Liu et al., 2012). These findings suggest that both VEGFR1 and VEGFR2 are positive regulators of skeletal development. However, during bone repair it is possible that VEGFR2 may also act as a negative regulator of intramembranous bone formation, since mice with VEGFR2-deficient osteoblasts exhibited increased intramembranous bone formation at surgically induced cortical repair sites (Hu and Olsen, 2016). Thus, further studies of cellular mechanisms by which VEGFR2 affects bone formation are clearly needed to resolve such seemingly conflicting data.

VEGF in skeletal development

VEGF in endochondral bone growth

Bone is formed by endochondral ossification and intramembranous bone formation. Endochondral ossification is responsible for development and growth of most of the vertebrate skeleton, including the limb bones, vertebral column and skull base. During endochondral bone formation, osteochondroprogenitor cells form cartilage templates of the future bones within avascular regions. This is followed by hypertrophy of chondrocytes within the templates and invasion of osteoblastic precursor cells, endothelial cells, hematopoietic cells and osteoclasts from the perichondrial region into the hypertrophic cartilage. Within these primary ossification centers (POCs), the cartilage is degraded and replaced by bone marrow and trabecular bone (Kronenberg, 2003, Zelzer and Olsen, 2003).

At several points in this process, VEGF is critical (Figure 1). Expression of VEGF by mesenchymal cells and chondrocytes occurs in response to increased protein levels of hypoxia-induced factor 1µ (HIF-1µ) and the chondrocyte differentiation factor Sox9 during mesenchymal cell condensation and cartilage template formation (Amarilio et al. 2007). Later, when chondrocytes mature to hypertrophy in the central regions of the cartilage templates, their expression of VEGF increases dramatically, and this induces osteoblast precursors in the perichondrium to migrate into the POCs together with blood vessels, osteoclasts and hematopoietic cells (Maes et al., 2010, Zelzer et al., 2002). Mice with Vegfa deleted in Col2-expressing cells exhibit a delay in osteoclast and blood vessel invasion into POCs and cartilage removal (Haigh et al., 2000, Zelzer et al., 2002). Osterix (Osx), a transcription factor expressed by osteoblastic precursor cells in the perichondrium and hypertrophic chondrocytes in POCs and metaphyseal growth plates, is essential for osteoblast differentiation and positively regulates VEGF expression by binding to VEGF promoters (Nakashima et al., 2002; Tang et al., 2012). Mice with deletion of Vegfa in Osx-positive osteolineage cells show decreased numbers of blood vessels in perichondrium and impaired differentiation of osteoblast precursors during development of long bones (Duan et al., 2015).

Figure 1.

Figure 1

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Diagrams illustrating stages in which VEGF has a critical role during endochondral bone formation. Left: Mesenchymal condensations occur in blood vessel-free, hypoxic regions with elevated levels of HIF-1µ, SOX9 and VEGF. This VEGF is required for the morphogenesis of vessels surrounding the cartilage-forming area. Middle: VEGF expressed by chondrocytes serves as a survival factor for the cells. Right: Increased levels of VEGF expression by hypertrophic chondrocytes stimulate invasion of osteoclasts, blood vessels, osteoblast precursors and hematopoietic stem cells into the hypertrophic region (the primary ossification center).

In addition to the effects of chondrocyte-derived VEGF in generation of POCs, a moderate level of VEGF expression in the central region of epiphyseal chondrocytes, controlled by HIF-1µ, is necessary for chondrocyte survival. Maes et al. observed cell death in chondrocytes within epiphyseal regions of long bones from VEGF188/188 mice (mice only expressing the VEGF 188 isoform) (Maes et al., 2004). In mice with Vegfa deleted in Col2-positive chondrocytes, massive chondrocyte apoptosis was also observed in the central epiphyseal/growth plate region of developing skeletal elements (Zelzer et al., 2004). Hypoxia is a major driver of VEGF expression. The protein levels of HIF-1µ are greatly elevated under low oxygen stress in osteoblasts, and this promotes transcription of various angiogenic factors, including VEGF (Spector et al., 2001, Steinbrech et al., 2000). In mice with HIF-1µ deficiency, chondrocyte apoptosis in central epiphyseal regions of developing cartilage is greatly increased (Schipani et al., 2001). Overexpression of VEGF in mice lacking HIF-1µ partially rescues chondrocyte apoptosis, suggesting that VEGF is a critical downstream effector of HIF-1µ in the support of chondrocyte survival (Maes et al., 2012).

VEGF in intramembranous bone growth

Most craniofacial bones are formed by intramembranous ossification, in which cranial neural crest cell (CNC)-derived progenitors directly differentiate into osteoblasts (Figure 2) (Bronner and LeDouarin, 2012, Chai and Maxson, 2006). VEGF is widely expressed in these cells and their offspring (Stalmans et al., 2003). Mice lacking the VEGF isoform VEGF164 exhibit multiple craniofacial defects including cleft palate, unfused cranial sutures, and shorter jaws (Stalmans et al., 2003). Mice with Vegfa deleted in CNC-derived cells exhibit similar craniofacial phenotypes as VEGF164-deficient mice, including hypoplasia of the cranium and mandible, cleft lip and palate, and changes in size and shape of the Meckel's cartilage, resulting in shorter and misshapen mandibles (Hill et al., 2015, Wiszniak et al., 2015).

Figure 2.

Figure 2

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Diagrams illustrating the role of osteoblast-generated VEGF in stimulating angiogenesis and membranous bone formation by cranial neural crest-derived precursors in the palatal shelves and around the Meckel's cartilage in the developing mandible.

Cleft palate and mandibular hypoplasia are among the most common craniofacial defects, and compromised VEGF expression contributes to the pathogenesis of these disorders. Conditional deletion of Vegf in CNC-derived cells results in mice with cleft palate; the palatal shelves show decreased cellular proliferation and abnormal elongation and elevation, leading to failure of fusion. Decreased VEGF levels in CNC-derived cells also leads to reduced vascularization of the palate, and this is coupled with deficient intramembranous ossification of the maxillary and palatal mesenchyme (Hill et al., 2015). These findings demonstrate that CNC-derived VEGF stimulates proliferation, vascularization and ossification during development of maxilla and palate.

Mandibular hypoplasia is often associated with genetic disorders affecting craniofacial development, such as Treacher Collins, DiGeorge and Goldenhar syndromes, based on developmental deficiencies in the first pharyngeal arch (Wiszniak et al., 2015). Mandibular development is a complex process. CNC cells delaminate from the neural tube and migrate into mandibular primordia, where they interact with resident epithelium and mesoderm to initiate differentiation programs leading to formation of skeletal jaw components (Thomas et al., 1998). CNC cells first differentiate into pre-chondrocytes to form Meckel’s cartilage (MC). The MC provides a scaffold for the differentiating mandibular osteoblasts and serves also as a driving force for the outgrowth of the jaw. Mesenchymal cells surrounding the MC differentiate into osteoblasts and deposit bone matrix for the future mandible (Parada and Chai, 2015, Ramaesh and Bard, 2003). VEGF from CNC cells and their derivatives supports MC extension, enhances vascularization of the jaw, and stabilizes major mandibular arteries. Mandibular hypoplasia and blood vessel defects within developing mandibles in mice with Vegfa deleted in CNC-derived cells resemble defects in patients with hemifacial microsomia. To elucidate the role of VEGF derived from specific subpopulations of CNC during craniofacial skeletal development, Duan et al. used mice with conditional deletion of Vegf in Osx-positive osteoblast precursor cells and found that loss of VEGF in these cells led to reduced ossification of calvarial and mandibular bones without affecting MC (Duan et al., 2016). This study further demonstrated that VEGF, derived from Osx-positive osteoblast precursors, is required for optimal ossification of the developing mandible.

The role of VEGF in bone repair

Unlike many other organs, bones can regenerate. Small bone defects are healed by synthesis of new bone that is indistinguishable from the bone that was injured. However, large bone defects do not heal without surgical intervention to stimulate bone healing, such as bone grafting or mechanical stimulation. Fracture healing is the most common form of bone repair. Delayed union or nonunion of bone fragments occurs in about 5–10% of patients with bone fracture (Gomez-Barrena et al., 2015). Possible causes include, but are not limited to, impaired blood supply, periosteal damage, reduced numbers of osteoprogenitor cells as a result of aging, inadequate immobilization and infection. Among potential causes, decreased vascular supply has been considered a major factor, and various strategies, developed to promote vascularization, have been tested in preclinical and clinical models. Fracture healing includes several overlapping phases; inflammation, soft callus formation, cartilage turnover (replacement by bony callus) and bone remodeling (Marsell and Einhorn, 2011). Due to disruption of blood vessels, a hematoma is formed at the injury site after fracture, and neutrophils are recruited into the hematoma. Influx of macrophages follows to remove dead neutrophils, promote angiogenic responses and initiate the repair cascade (Claes et al., 2012). Along with invasion of blood vessels, osteochondroprogenitors migrate to the injury site, where they proliferate and differentiate to either osteoblasts or chondrocytes, depending on the stability of fracture fixation and supply of blood vessels (Dimitriou et al., 2005). Finally, newly formed immature woven bone is gradually remodeled to mature lamellar bone. VEGF plays critical roles in almost all the phases of bone repair.

Roles of VEGF during the inflammation phase

After bone trauma, VEGF is concentrated in the hematoma. Hypoxia, a major inducer of VEGF expression (Krock et al., 2011, Wang et al., 2007), induces VEGF expression in surrounding bone cells or inflammatory cells. Release of neutrophils from bone marrow into the circulation and subsequent recruitment into the hematoma are early signs of acute inflammation. Neutrophils help remove bone debris and microbial pathogens. Osteoblastic lineage cells are thought to play a role in regulating neutrophil release through a CXCL12-CXCR4-dependent mechanism (Eash et al., 2010). Mice with deletion of Vegf in osteoblastic cells exhibit decreased neutrophil release into the circulation during the acute inflammatory phase after bone injury (Hu and Olsen, 2016). VEGF induces neutrophil chemotaxis and increases permeability of sinusoidal endothelial cells (Ancelin et al., 2004, Lim et al., 2014); thus, VEGF may facilitate entry of neutrophils into the circulation during this phase. Osteoblastic precursors and hematopoietic cells reside close to bone marrow sinusoids, and osteoprogenitors and immune cells, including monocytes and macrophages, may migrate to the injury site along with newly formed blood vessels. During the resolution phase (3–7 days after fracture), macrophages are recruited. Uptake of apoptotic neutrophils causes macrophages to change the phenotype from activated M1 to reparative M2 states, and release of mediators, such as TGF-β1, suppress the pro-inflammatory response and initiate the repair process (Brancato and Albina, 2011). In mice with Vegfa deleted in osteoblastic cells, VEGF levels are reduced at injury sites. As a result, recruitment of macrophages and stimulation of angiogenesis are decreased. Consequently the density of mesenchymal precursor cells is reduced at the injury site; in turn, this leads to a delay in the repair process (Hu and Olsen, 2016). There is a strong association between the density of macrophages and blood vessels during the inflammation phase of bone repair (Hu and Olsen, 2016), but what comes first is not entirely clear. In fact, it is likely that angiogenesis and macrophage recruitment are coupled processes. Newly formed blood vessels may recruit macrophages, and these in turn may generate angiogenic factors, including VEGF, to further promote angiogenesis.

Roles of VEGF in cartilage turnover

Both endochondral and intramembranous ossification occur in bone repair, depending on the stability of the fracture, blood vessel supply and the location of bone formation. Stable fractures heal primarily via intramembranous ossification, but moderate amounts of cartilage may form in the injured periosteum. Endochondral ossification predominates in unstable fractures and large amounts of cartilage may be formed, facilitated by lack of blood supply (Dimitriou et al., 2005). Osteochondral progenitor cells in the periosteum may differentiate into chondrocytes or osteoblasts while osteoblastic precursors in endosteum and bone marrow are prone to osteoblastic differentiation. The endochondral ossification repair process recapitulates the stages in developmental endochondral bone formation; cartilage formation, vascular and osteoclast invasion, cartilage resorption and replacement by bone. This process occurs during callus formation as a result of the periosteal response to injury. Inhibition of VEGF signaling in skeletal progenitor cells facilitates cartilage formation at the expense of bone formation (Chan et al., 2015). In mice with a surgically induced cortical bone defect in tibia, knock-down of Vegf in hypertrophic chondrocytes and osteoblastic precursors causes strong induction of chondrogenesis in the injured periosteum (Hu and Olsen, 2016), consistent with the conclusion that VEGF stimulates differentiation of periosteal progenitor cells to osteoblasts.

In the later phases of bone repair by endochondral ossification, chondrocytes stop proliferating, mature to hypertrophy and synthesize collagen type X. Hypertrophic chondrocytes also express Osx, a strong inducer of VEGF expression (Carlevaro et al., 2000, Zelzer et al., 2001). VEGF stimulates vessel invasion and recruitment of osteoclasts/chondroclasts into the hypertrophic cartilage (Carlevaro et al., 2000, Zelzer and Olsen, 2005). In mice with reduced expression of Vegf in hypertrophic chondrocytes, infiltration of vessels and osteoclasts is delayed in the periosteal callus during healing of a cortical defect, consistent with the phenotype of such mice during skeletal development (Hu and Olsen, 2016, Liu et al., 2012). Likewise, inhibiting VEGF signaling by sVEGFR1, delays cartilage turnover, disrupts conversion of the soft cartilaginous callus to a hard bony callus, and impairs healing in mice with femoral fractures (Street et al., 2002). These results are consistent with the findings that VEGF120/120 mice(mice expressing only the VEGF120 isoform) and mice treated with sVEGFR1 during bone development exhibit delayed endochondral bone formation (Gerber et al., 1999, Zelzer et al., 2002).

Roles of VEGF in bone repair by intramembranous ossification

Mesenchymal progenitor cells in endosteum and bone marrow differentiate to either chondrocytes or osteoblasts in vitro depending on the composition of the culture medium. However, the cells differentiate only to osteoblasts in vivo during bone repair. The callus that forms in the intramedullary space during the repair process is the product of osteoblasts derived from mesenchymal progenitor cells in the endosteum and bone marrow. VEGF is critical for this process of intramembranous bone formation.

Stimulated by hypoxia during the inflammation phase, osteoblasts synthesize and secrete VEGF in response to elevated levels of HIF-1µ. This stimulates proliferation and migration of endothelial cells and increases vessel permeability (Wang et al., 2007). Increased angiogenesis brings osteoblastic progenitor cells into the repair site in addition to nutrition, oxygen and minerals necessary for mineralization. In addition, blood vessel cells, including smooth muscle cells, endothelial cells and pericytes, release osteogenic factors, such as TGF-β and BMP2, further stimulating osteoblast differentiation and bone mineralization (Matsubara et al., 2012). Maturating osteoblasts also generate angiogenic factors, including VEGF, that induce angiogenesis via a positive feedback mechanism (Hu and Olsen, 2016). Deletion of Vegf in osteoblasts interrupts this coupling of osteogenesis and angiogenesis, and delays the intramembranous ossification-mediated repair during cortical bone defect healing (Hu and Olsen, 2016). Administration of neutralizing antibodies against VEGF receptors to mice, undergoing distraction osteogenesis following a cut across the tibial diaphysis, significantly decreases the amount of blood vessel formation and intramembranous bone formation in the distraction gap (Carvalho et al., 2004). Overexpression of HIF-1µ in osteoblasts of similar distraction mice results in a VEGF-dependent increase in blood vessels and mineralized bone in the distraction gap (Wan et al., 2008).

In addition to the indirect effects on bone formation via stimulation of blood vessel cells, osteoblast-derived VEGF may also affect osteoblast function directly through autocrine and intracrine mechanisms. Although expression of VEGFRs in murine osteoblasts has been reported to vary in previous studies, deletion of Vegfr2 in Osx-positive cells enhances osteoblast maturation and mineralization in defect repair (Hu and Olsen, 2016). VEGFR2 has been reported to recruit inhibitory Smad7 and decrease activation of Smad1 in endothelial cells. A similar mechanism may also be utilized by osteoblasts; osteoblasts with reduced VEGFR2 levels exhibit increased mineralization in vitro in the presence of BMP2, for which Smad1/5 are downstream transducers (Hu and Olsen, 2016). Inhibition of BMP pathways by VEGFR2 may also partially explain why high levels of VEGF inhibit BMP2- or BMP4-induced bone repair (Peng et al., 2002, Peng et al., 2005). In addition, failure of exogenous VEGF to enhance intramembranous bone formation in vivo and osteoblast differentiation and mineralization in vitro (Liu et al., 2012, Hu and Olsen, 2016), may be due to the fact that osteoblast-generated VEGF is, in part, intracellular and participates in transcriptional regulation of osteoblast differentiation genes (Gerber et al., 2002, Lee et al., 2007, Liu et al., 2012).

Perspectives

VEGF has a critical role in skeletal development and bone repair following injury. Too little or too much VEGF beyond physiological needs may cause defects in bone development or compromise bone repair. Germline mutations in VEGF are rarely observed in genetic disorders affecting bone development; however, in some genetic disorders, causative mutations may affect factors that regulate or interact with VEGF. Thus, some aspects of phenotypes in patients carrying mutations in genes causing bone growth defects may be due to dysregulation of VEGF. In DiGeorge syndrome, TBX1, a disease-causing factor, appears to interact (indirectly) with VEGF. The defects in mice lacking the VEGF164 isoform resemble the phenotypes found in DiGeorge patients. In addition, Tbx1 expression is reduced in embryos from mice with VEGF164 deficiency, and knocking down Vegf levels enhance the pharyngeal arch artery defects induced by Tbx1 knockdown (Stalmans et al., 2003). The functional connection with VEGF and TBX1 suggests that VEGF can modify functions of other genes with mutations in genetic disorders, including phenotypes of bone development. In the growth retardation, alopecia, pseudoanodontia and progressive optic atrophy (GAPO) syndrome, causative loss-of-function mutations have been identified in the extracellular matrix-binding receptor Anthrax toxin receptor 1 (ANTXR1) (Stranecky et al., 2013). ANTXR1 interacts with β1-integrin and VEGFR2 to form a regulatory protein complex for the control of VEGFR1 transcription in endothelial cells (Jinnin et al., 2008). In endothelial cells from infantile hemangiomas, the function of this complex is compromised. As a result, expression levels of VEGFR1 and sVEGFR1, acting as decoy receptors for VEGF, are very low and VEGFR2 signaling is constitutively activated. The increased activation of VEGFR2 further promotes VEGF expression in a positive feedback manner (Chatterjee et al., 2013). In GAPO syndrome, loss of ANTXR1 causes elevated levels of VEGF in several cell types, including endothelial cells, skin fibroblasts and chondrocytes. This is associated with a phenotype of leaky vessels, accumulation of extracellular matrix proteins and chondrocyte maturation defects (Besschetnova et al., 2015 and unpublished data).

The importance of VEGF in normal bone healing is highlighted by the compromised bone repair in mice treated with VEGF inhibitors, and genetically modified mice with Vegf deleted in osteoblastic cells. In humans, associations between VEGF gene polymorphisms that affect VEGF levels and osteoporotic vertebral fractures in postmenopausal women have been reported (Chung et al., 2010). In animal studies, VEGF levels have been found to be reduced in mesenchymal stem cells from aged mice as well as in the tibial metaphysis following ovariectomy-induced osteoporosis (Maharaj and D'Amore, 2007, Wilson et al., 2010). However, evidence that VEGF levels are reduced in bone cells with age or associated with a systemic disease, such as osteoporosis, is still lacking. Therapeutic strategies, based on VEGF modulation to improve bone repair and regeneration, are limited to preclinical studies. The use of targeted supplements of VEGF to treat, or prevent age-related osteoporosis or defective bone healing in clinical settings, will require clinical evidence for a strong association between VEGF levels and bone health as well as a better understanding of how VEGF functions in the context of other growth factors, skeletal stem cells and bone matrix protein synthesis in humans.

Acknowledgments

Research in the authors' laboratory was supported by grants AR36819 and AR36820 (to B.R. Olsen) from NIH-NIAMS.

Footnotes

The authors have declared that no conflict of interest exists.

References

  1. Amarilio R, Viukov SV, Sharir A, Eshkar-oren I, Johnson RS, Zelzer E. HIF1alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis. Development. 2007;134:3917–3928. doi: 10.1242/dev.008441. [DOI] [PubMed] [Google Scholar]
  2. Ancelin M, Chollet-martin S, Herve MA, Legrand C, El Benna J, Perrot-Applanat M. Vascular endothelial growth factor VEGF189 induces human neutrophil chemotaxis in extravascular tissue via an autocrine amplification mechanism. Lab Invest. 2004;84:502–512. doi: 10.1038/labinvest.3700053. [DOI] [PubMed] [Google Scholar]
  3. Arcondeguy T, Lacazette E, Millevoi S, Prats H, Touriol C. VEGF-A mRNA processing, stability and translation: a paradigm for intricate regulation of gene expression at the post-transcriptional level. Nucleic Acids Res. 2013;41:7997–8010. doi: 10.1093/nar/gkt539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood. 1996;87:3336–3343. [PubMed] [Google Scholar]
  5. Besschetnova TY, Ichimura T, Katebi N, St Croix B, Bonventre JV, OLSEN BR. Regulatory mechanisms of anthrax toxin receptor 1-dependent vascular and connective tissue homeostasis. Matrix Biol. 2015;42:56–73. doi: 10.1016/j.matbio.2014.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bishop JA, Palanca AA, Bellino MJ, Lowenberg DW. Assessment of compromised fracture healing. J Am Acad Orthop Surg. 2012;20:273–282. doi: 10.5435/JAAOS-20-05-273. [DOI] [PubMed] [Google Scholar]
  7. Bogonatov BN, Gonchar-Zaikina NG. The system of bony canals as the basis for the angioarchitectonics of bones. Arkh Anat Gistol Embriol. 1976;70:53–61. [PubMed] [Google Scholar]
  8. Brancato SK, Albina JE. Wound macrophages as key regulators of repair: origin, phenotype, and function. Am J Pathol. 2011;178:19–25. doi: 10.1016/j.ajpath.2010.08.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bronner ME, Ledouarin NM. Development and evolution of the neural crest: an overview. Dev Biol. 2012;366:2–9. doi: 10.1016/j.ydbio.2011.12.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Burkhardt R, Kettner G, Bohm W, Schmidmeier M, Schlag R, Frisch B, Mallmann B, Eisenmenger W, Gilg T. Changes in trabecular bone, hematopoiesis and bone marrow vessels in aplastic anemia, primary osteoporosis, and old age: a comparative histomorphometric study. Bone. 1987;8:157–164. doi: 10.1016/8756-3282(87)90015-9. [DOI] [PubMed] [Google Scholar]
  11. Carano RA, Filvaroff EH. Angiogenesis and bone repair. Drug Discov Today. 2003;8:980–989. doi: 10.1016/s1359-6446(03)02866-6. [DOI] [PubMed] [Google Scholar]
  12. Carlevaro MF, Cermelli S, Cancedda R, Descalzi Cancedda F. Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation. J Cell Sci. 2000;113(1):59–69. doi: 10.1242/jcs.113.1.59. [DOI] [PubMed] [Google Scholar]
  13. Carvalho RS, Einhorn TA, Lehmann W, Edgar C, Al-Yamani A, Apazidis A, Pacicca D, Clemens TL, Gerstenfeld LC. The role of angiogenesis in a murine tibial model of distraction osteogenesis. Bone. 2004;34:849–861. doi: 10.1016/j.bone.2003.12.027. [DOI] [PubMed] [Google Scholar]
  14. Chai Y, Maxson RE., Jr Recent advances in craniofacial morphogenesis. Dev Dyn. 2006;235:2353–2375. doi: 10.1002/dvdy.20833. [DOI] [PubMed] [Google Scholar]
  15. Chan CK, Seo EY, Chen JY, Lo D, Mcardle A, Sinha R, Tevlin R, Seita J, Vincent-Tompkins J, Wearda T, Lu WJ, Senarath-Yapa K, Chung MT, Marecic O, Tran M, Yan KS, Upton R, Walmsley GG, Lee AS, Sahoo D, Kuo CJ, Weissman IL, Longaker MT. Identification and specification of the mouse skeletal stem cell. Cell. 2015;160:285–298. doi: 10.1016/j.cell.2014.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Chatterjee S, Heukamp LC, Siobal M, Schottle J, Wieczorek C, Peifer M, Frasca D, Koker M, Konig K, Meder L, Rauh D, Buettner R, Wolf J, Brekken RA, Neumaier B, Christofori G, Thomas RK, Ullrich RT. Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer. J Clin Invest. 2013;123:1732–1740. doi: 10.1172/JCI65385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Childs SG. Osteonecrosis: death of bone cells. Orthop Nurs. 2005;24:295–301. doi: 10.1097/00006416-200507000-00012. quiz 302-3. [DOI] [PubMed] [Google Scholar]
  18. Chung YS, Hong SH, Min KT, Shin DE, Lee JH, Shim YS, Ahn JY, Kim NK. Association of vascular endothelial growth factor gene polymorphisms with osteoporotic vertebral compression fractures in postmenopausal women. Genes & Genomics. 2010;32:499–505. [Google Scholar]
  19. Claes L, Recknagel S, Ignatius A. Fracture healing under healthy and inflammatory conditions. Nat Rev Rheumatol. 2012;8:133–143. doi: 10.1038/nrrheum.2012.1. [DOI] [PubMed] [Google Scholar]
  20. Clarkin CE, Gerstenfeld LC. VEGF and bone cell signalling: an essential vessel for communication? Cell Biochem Funct. 2013;31:1–11. doi: 10.1002/cbf.2911. [DOI] [PubMed] [Google Scholar]
  21. Conn G, Bayne ML, Soderman DD, Kwok PW, Sullivan KA, Palisi TM, Hope DA, Thomas KA. Amino acid and cDNA sequences of a vascular endothelial cell mitogen that is homologous to platelet-derived growth factor. Proc Natl Acad Sci U S A. 1990;87:2628–2632. doi: 10.1073/pnas.87.7.2628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Couffinhal T, Kearney M, Witzenbichler B, Chen D, Murohara T, Losordo DW, Symes J, Isner JM. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and atherosclerotic human arteries. Am J Pathol. 1997;150:1673–1685. [PMC free article] [PubMed] [Google Scholar]
  23. Coultas L, Chawengsaksophak K, Rossant J. Endothelial cells and VEGF in vascular development. Nature. 2005;438:937–945. doi: 10.1038/nature04479. [DOI] [PubMed] [Google Scholar]
  24. Cross MJ, Dixelius J, Matsumoto T, Claesson-Welsh L. VEGF-receptor signal transduction. Trends Biochem Sci. 2003;28:488–494. doi: 10.1016/S0968-0004(03)00193-2. [DOI] [PubMed] [Google Scholar]
  25. Dimitriou R, Tsiridis E, Giannoudis PV. Current concepts of molecular aspects of bone healing. Injury. 2005;36:1392–1404. doi: 10.1016/j.injury.2005.07.019. [DOI] [PubMed] [Google Scholar]
  26. Duan X, Bradbury SR, Olsen BR, Berendsen AD. VEGF stimulates intramembranous bone formation during craniofacial skeletal development. Matrix Biol. 2016;52–54:127–140. doi: 10.1016/j.matbio.2016.02.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Duan X, Murata Y, Liu Y, Nicolae C, Olsen BR, Berendsen AD. Vegfa regulates perichondrial vascularity and osteoblast differentiation in bone development. Development. 2015;142:1984–1991. doi: 10.1242/dev.117952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Eash KJ, Greenbaum AM, Gopalan PK, Link DC. CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow. J Clin Invest. 2010;120:2423–2431. doi: 10.1172/JCI41649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Eshkar-oren I, Viukov SV, Salameh S, Krief S, Oh CD, Akiyama H, Gerber HP, Ferrara N, Zelzer E. The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation of Vegf. Development. 2009;136:1263–1272. doi: 10.1242/dev.034199. [DOI] [PubMed] [Google Scholar]
  30. Ferrara N, Gerber HP, Lecouter J. The biology of VEGF and its receptors. Nat Med. 2003;9:669–676. doi: 10.1038/nm0603-669. [DOI] [PubMed] [Google Scholar]
  31. Gerber HP, Malik AK, Solar GP, Sherman D, Liang XH, Meng G, Hong K, Marsters JC, Ferrara N. VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism. Nature. 2002;417:954–958. doi: 10.1038/nature00821. [DOI] [PubMed] [Google Scholar]
  32. Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med. 1999;5:623–628. doi: 10.1038/9467. [DOI] [PubMed] [Google Scholar]
  33. Gomez-Barrena E, Rosset P, Lozano D, Stanovici J, Ermthaller C, Gerbhard F. Bone fracture healing: cell therapy in delayed unions and nonunions. Bone. 2015;70:93–101. doi: 10.1016/j.bone.2014.07.033. [DOI] [PubMed] [Google Scholar]
  34. Haigh JJ, Gerber HP, Ferrara N, Wagner EF. Conditional inactivation of VEGF-A in areas of collagen2a1 expression results in embryonic lethality in the heterozygous state. Development. 2000;127:1445–1453. doi: 10.1242/dev.127.7.1445. [DOI] [PubMed] [Google Scholar]
  35. Hill C, Jacobs B, Kennedy L, Rohde S, Zhou B, Baldwin S, Goudy S. Cranial neural crest deletion of VEGFa causes cleft palate with aberrant vascular and bone development. Cell Tissue Res. 2015;361:711–722. doi: 10.1007/s00441-015-2150-7. [DOI] [PubMed] [Google Scholar]
  36. Hoeben A, Landuyt B, Highley MS, Wildiers H, Van Oosterom AT, De Bruijn EA. Vascular endothelial growth factor and angiogenesis. Pharmacol Rev. 2004;56:549–580. doi: 10.1124/pr.56.4.3. [DOI] [PubMed] [Google Scholar]
  37. Hu K, Olsen BR. Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair. J Clin Invest. 2016;126:509–526. doi: 10.1172/JCI82585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Jinnin M, Medici D, Park L, Limaye N, Liu Y, Boscolo E, Bischoff J, Vikkula M, Boye E, Olsen BR. Suppressed NFAT-dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile hemangioma. Nat Med. 2008;14:1236–1246. doi: 10.1038/nm.1877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Koch S, Claesson-Welsh L. Signal transduction by vascular endothelial growth factor receptors. Cold Spring Harb Perspect Med. 2012;2:a006502. doi: 10.1101/cshperspect.a006502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Krock BL, Skuli N, Simon MC. Hypoxia-induced angiogenesis: good and evil. Genes Cancer. 2011;2:1117–1133. doi: 10.1177/1947601911423654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kronenberg HM. Developmental regulation of the growth plate. Nature. 2003;423:332–336. doi: 10.1038/nature01657. [DOI] [PubMed] [Google Scholar]
  42. Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature. 2014;507:323–328. doi: 10.1038/nature13145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Lee S, Chen TT, Barber CL, Jordan MC, Murdock J, Desai S, Ferrara N, Nagy A, Roos KP, Iruela-Arispe ML. Autocrine VEGF signaling is required for vascular homeostasis. Cell. 2007;130:691–703. doi: 10.1016/j.cell.2007.06.054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Lim S, Zhang Y, Zhang D, Chen F, Hosaka K, Feng N, Seki T, Andersson P, Li J, Zang J, Sun B, Cao Y. VEGFR2-mediated vascular dilation as a mechanism of VEGF-induced anemia and bone marrow cell mobilization. Cell Rep. 2014;9:569–580. doi: 10.1016/j.celrep.2014.09.003. [DOI] [PubMed] [Google Scholar]
  45. Liu Y, Berendsen AD, Jia S, Lotinun S, Baron R, Ferrara N, Olsen BR. Intracellular VEGF regulates the balance between osteoblast and adipocyte differentiation. J Clin Invest. 2012;122:3101–3113. doi: 10.1172/JCI61209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Maes C. Role and regulation of vascularization processes in endochondral bones. Calcif Tissue Int. 2013;92:307–323. doi: 10.1007/s00223-012-9689-z. [DOI] [PubMed] [Google Scholar]
  47. Maes C, Araldi E, Haigh K, Khatri R, Van Looveren R, Giaccia AJ, Haigh JJ, Carmeliet G, Schipani E. VEGF-independent cell-autonomous functions of HIF-1alpha regulating oxygen consumption in fetal cartilage are critical for chondrocyte survival. J Bone Miner Res. 2012;27:596–609. doi: 10.1002/jbmr.1487. [DOI] [PubMed] [Google Scholar]
  48. Maes C, Kobayashi T, Selig MK, Torrekens S, Roth SI, Mackem S, Carmeliet G, Kronenberg HM. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev Cell. 2010;19:329–344. doi: 10.1016/j.devcel.2010.07.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Maes C, Stockmans I, Moermans K, Van Looveren R, Smets N, Carmeliet P, Bouillon R, Carmeliet G. Soluble VEGF isoforms are essential for establishing epiphyseal vascularization and regulating chondrocyte development and survival. J Clin Invest. 2004;113:188–199. doi: 10.1172/JCI19383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Maharaj AS, D'amore PA. Roles for VEGF in the adult. Microvasc Res. 2007;74:100–113. doi: 10.1016/j.mvr.2007.03.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Maharaj AS, Saint-Geniez M, Maldonado AE, D'amore PA. Vascular endothelial growth factor localization in the adult. Am J Pathol. 2006;168:639–648. doi: 10.2353/ajpath.2006.050834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Marsell R, Einhorn TA. The biology of fracture healing. Injury. 2011;42:551–555. doi: 10.1016/j.injury.2011.03.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Maru Y, Yamaguchi S, Shibuya M. Flt-1, a receptor for vascular endothelial growth factor, has transforming and morphogenic potentials. Oncogene. 1998;16:2585–2595. doi: 10.1038/sj.onc.1201786. [DOI] [PubMed] [Google Scholar]
  54. Matsubara H, Hogan DE, Morgan EF, Mortlock DP, Einhorn TA, Gerstenfeld LC. Vascular tissues are a primary source of BMP2 expression during bone formation induced by distraction osteogenesis. Bone. 2012;51:168–180. doi: 10.1016/j.bone.2012.02.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Mayr-Wohlfart U, Waltenberger J, Hausser H, Kessler S, Gunther KP, Dehio C, Puhl W, Brenner RE. Vascular endothelial growth factor stimulates chemotactic migration of primary human osteoblasts. Bone. 2002;30:472–477. doi: 10.1016/s8756-3282(01)00690-1. [DOI] [PubMed] [Google Scholar]
  56. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, De Crombrugghe B. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell. 2002;108:17–29. doi: 10.1016/s0092-8674(01)00622-5. [DOI] [PubMed] [Google Scholar]
  57. Parada C, Chai Y. Mandible and Tongue Development. Curr Top Dev Biol. 2015;115:31–58. doi: 10.1016/bs.ctdb.2015.07.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Peng H, Usas A, Olshanski A, Ho AM, Gearhart B, Cooper GM, Huard J. VEGF improves, whereas sFlt1 inhibits, BMP2-induced bone formation and bone healing through modulation of angiogenesis. J Bone Miner Res. 2005;20:2017–2027. doi: 10.1359/JBMR.050708. [DOI] [PubMed] [Google Scholar]
  59. Peng H, Wright V, Usas A, Gearhart B, Shen HC, Cummins J, Huard J. Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J Clin Invest. 2002;110:751–759. doi: 10.1172/JCI15153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Pepper MS, Wasi S, Ferrara N, Orci L, Montesano R. In vitro angiogenic and proteolytic properties of bovine lymphatic endothelial cells. Exp Cell Res. 1994;210:298–305. doi: 10.1006/excr.1994.1042. [DOI] [PubMed] [Google Scholar]
  61. Ramaesh T, Bard JB. The growth and morphogenesis of the early mouse mandible: a quantitative analysis. J Anat. 2003;203:213–222. doi: 10.1046/j.1469-7580.2003.00210.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Saint-Geniez M, Maharaj AS, Walshe TE, Tucker BA, Sekiyama E, Kurihara T, Darland DC, Young MJ, D'amore PA. Endogenous VEGF is required for visual function: evidence for a survival role on muller cells and photoreceptors. PLoS One. 2008;3:e3554. doi: 10.1371/journal.pone.0003554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Schipani E, Ryan HE, Didrickson S, Kobayashi T, Knight M, Johnson RS. Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival. Genes Dev. 2001;15:2865–2876. doi: 10.1101/gad.934301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Spector JA, Mehrara BJ, Greenwald JA, Saadeh PB, Steinbrech DS, Bouletreau PJ, Smith LP, Longaker MT. Osteoblast expression of vascular endothelial growth factor is modulated by the extracellular microenvironment. Am J Physiol Cell Physiol. 2001;280:C72–C80. doi: 10.1152/ajpcell.2001.280.1.C72. [DOI] [PubMed] [Google Scholar]
  65. Stalmans I, Lambrechts D, De Smet F, Jansen S, Wang J, Maity S, Kneer P, Von Der Ohe M, Swillen A, Maes C, Gewillig M, Molin DG, Hellings P, Boetel T, Haardt M, Compernolle V, Dewerchin M, Plaisance S, Vlietinck R, Emanuel B, Gittenberger-De Groot AC, Scambler P, Morrow B, Driscol DA, Moons L, Esguerra CV, Carmeliet G, Behn-Krappa A, Devriendt K, Collen D, Conway SJ, Carmeliet P. VEGF: a modifier of the del22q11 (DiGeorge) syndrome? Nat Med. 2003;9:173–182. doi: 10.1038/nm819. [DOI] [PubMed] [Google Scholar]
  66. Steinbrech DS, Mehrara BJ, Saadeh PB, Greenwald JA, Spector JA, Gittes GK, Longaker MT. VEGF expression in an osteoblast-like cell line is regulated by a hypoxia response mechanism. Am J Physiol Cell Physiol. 2000;278:C853–C860. doi: 10.1152/ajpcell.2000.278.4.C853. [DOI] [PubMed] [Google Scholar]
  67. Stranecky V, Hoischen A, Hartmannova H, Zaki MS, Chaudhary A, Zudaire E, Noskova L, Baresova V, Pristoupilova A, Hodanova K, Sovova J, Hulkova H, Piherova L, Hehir-Kwa JY, De Silva D, Senanayake MP, Farrag S, Zeman J, Martasek P, Baxova A, Afifi HH, St Croix B, Brunner HG, Temtamy S, Kmoch S. Mutations in ANTXR1 cause GAPO syndrome. Am J Hum Genet. 2013;92:792–799. doi: 10.1016/j.ajhg.2013.03.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Street J, Bao M, Deguzman L, Bunting S, Peale FV, Jr, Ferrara N, Steinmetz H, Hoeffel J, Cleland JL, Daugherty A, Van Bruggen N, Redmond HP, Carano RA, Filvaroff EH. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci U S A. 2002;99:9656–9661. doi: 10.1073/pnas.152324099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Tang W, Yang F, Li Y, De Crombrugghe B, Jiao H, Xiao G, Zhang C. Transcriptional regulation of Vascular Endothelial Growth Factor (VEGF) by osteoblast-specific transcription factor Osterix (Osx) in osteoblasts. J Biol Chem. 2012;287:1671–1678. doi: 10.1074/jbc.M111.288472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Thomas T, Kurihara H, Yamagishi H, Kurihara Y, Yazaki Y, Olson EN, Srivastava D. A signaling cascade involving endothelin-1, dHAND and msx1 regulates development of neural-crest-derived branchial arch mesenchyme. Development. 1998;125:3005–3014. doi: 10.1242/dev.125.16.3005. [DOI] [PubMed] [Google Scholar]
  71. Wan C, Gilbert SR, Wang Y, Cao X, Shen X, Ramaswamy G, Jacobsen KA, Alaql ZS, Eberhardt AW, Gerstenfeld LC, Einhorn TA, Deng L, Clemens TL. Activation of the hypoxia-inducible factor-1alpha pathway accelerates bone regeneration. Proc Natl Acad Sci U S A. 2008;105:686–691. doi: 10.1073/pnas.0708474105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Wang Y, Wan C, Deng L, Liu X, Cao X, Gilbert SR, Bouxsein ML, Faugere MC, Guldberg RE, Gerstenfeld LC, Haase VH, Johnson RS, Schipani E, Clemens TL. The hypoxia-inducible factor alpha pathway couples angiogenesis to osteogenesis during skeletal development. J Clin Invest. 2007;117:1616–1626. doi: 10.1172/JCI31581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Wilson A, Shehadeh LA, Yu H, Webster KA. Age-related molecular genetic changes of murine bone marrow mesenchymal stem cells. BMC Genomics. 2010;11:229. doi: 10.1186/1471-2164-11-229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Wiszniak S, Mackenzie FE, Anderson P, Kabbara S, Ruhrberg C, Schwarz Q. Neural crest cell-derived VEGF promotes embryonic jaw extension. Proc Natl Acad Sci U S A. 2015;112:6086–6091. doi: 10.1073/pnas.1419368112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Worthley DL, Churchill M, Compton JT, Tailor Y, Rao M, Si Y, Levin D, Schwartz MG, Uygur A, Hayakawa Y, Gross S, Renz BW, Setlik W, Martinez AN, Chen X, Nizami S, Lee HG, Kang HP, Caldwell JM, Asfaha S, Westphalen CB, Graham T, Jin G, Nagar K, Wang H, Kheirbek MA, Kolhe A, Carpenter J, Glaire M, Nair A, Renders S, Manieri N, Muthupalani S, Fox JG, Reichert M, Giraud AS, Schwabe RF, Pradere JP, Walton K, Prakash A, Gumucio D, Rustgi AK, Stappenbeck TS, Friedman RA, Gershon MD, Sims P, Grikscheit T, Lee FY, Karsenty G, Mukherjee S, Wang TC. Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential. Cell. 2015;160:269–284. doi: 10.1016/j.cell.2014.11.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Zelzer E, Glotzer DJ, Hartmann C, Thomas D, Fukai N, Soker S, Olsen BR. Tissue specific regulation of VEGF expression during bone development requires Cbfa1/Runx2. Mech Dev. 2001;106:97–106. doi: 10.1016/s0925-4773(01)00428-2. [DOI] [PubMed] [Google Scholar]
  77. Zelzer E, Mamluk R, Ferrara N, Johnson RS, Schipani E, Olsen BR. VEGFA is necessary for chondrocyte survival during bone development. Development. 2004;131:2161–2171. doi: 10.1242/dev.01053. [DOI] [PubMed] [Google Scholar]
  78. Zelzer E, Mclean W, Ng YS, Fukai N, Reginato AM, Lovejoy S, D'amore PA, Olsen BR. Skeletal defects in VEGF(120/120) mice reveal multiple roles for VEGF in skeletogenesis. Development. 2002;129:1893–1904. doi: 10.1242/dev.129.8.1893. [DOI] [PubMed] [Google Scholar]
  79. Zelzer E, Olsen BR. The genetic basis for skeletal diseases. Nature. 2003;423:343–348. doi: 10.1038/nature01659. [DOI] [PubMed] [Google Scholar]
  80. Zelzer E, Olsen BR. Multiple roles of vascular endothelial growth factor (VEGF) in skeletal development, growth, and repair. Curr Top Dev Biol. 2005;65:169–187. doi: 10.1016/S0070-2153(04)65006-X. [DOI] [PubMed] [Google Scholar]

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