Vascular and Nonvascular Roles of VEGF in Bone Development

Chondrocytes

Chondrocytes in the cartilage template and later in the growth plate first proliferate, and then progressively differentiate into mature hypertrophic cells. Several autocrine and/or paracrine factors have been implicated in chondrocyte development, including parathyroid hormone related protein (PTHRP; now known as parathyroid hormone like peptide, PTHLH), indian hedgehog (IHH), bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). PTHRP and IHH form a negative feedback signaling pathway to control the pace of chondrocyte development in the growth plate. IHH also coordinates chondrocyte and osteoblast differentiation, together with the transcription factor RUNX2 (runt related transcription factor; also known as core binding factor 1, CBFA1).^7,8

Hypertrophic chondrocytes, but not immature chondrocytes, consistently express high levels of VEGF in vivo. One factor that may control this VEGF expression is RUNX2.⁹ By expressing VEGF and other angiogenic stimulators, hypertrophic cartilage becomes a target for capillary invasion and angiogenesis. In contrast, immature cartilage remains avascular due to the production of angiogenic inhibitors. As a result, the center of the developing epiphyseal growth plate becomes hypoxic,¹⁰ but chondrocytes are well capable of surviving this challenge. For example, bovine articular chondrocytes are able to survive under oxygen tensions ranging from <0.1% to 20% for at least 7 days in vitro, with no evident differences in cell division or differentiation.¹¹ In response to this physiological hypoxia, immature chondrocytes in the center of the epiphysis upregulate VEGF. Accordingly, VEGF mRNA and protein levels are increased by hypoxia in cultured embryonic limbs and primary chondrocytes in vitro.^5,12 In general, hypoxia induces the expression of VEGF and other genes involved in angiogenesis and glucose metabolism via two transcriptional regulators, the hypoxia-inducible factors HIF1A and HIF2A (previously known as HIF1 alpha and HIF2 alpha; see Chapter 3 by M. Fruttiger). In agreement, inactivation of HIF1A in epiphyseal chondrocytes abolished the upregulation of VEGF in response to hypoxia in vitro.¹² Mice with inactivation of HIF1A in cartilage nevertheless showed increased VEGF expression in the epiphysis, suggesting that HIF2A and/or other factors may compensate for HIF1A loss or contribute to VEGF upregulation in vivo.¹⁰ Studies on the VEGF receptor profile in chondrocytes showed that immature epiphyseal chondrocytes in vivo express the two VEGF isoform-specific receptors NRP1 and NRP2, but no detectable levels of FLT1 or KDR were found.^3,5 Expression of KDR has however been reported in some other cartilage types, such as the permanent thyroid cartilage of humans and cultured hypertrophic chondrocytes of chicken.^13,14

Osteoblasts

Osteoblasts share a common mesenchymal precursor with chondrocytes, and specific regulatory factors direct the osteo-chondroprogenitors to either one of these lineages. As such, RUNX2 dominates the control of osteoblast differentiation. Mature osteoblasts produce bone matrix and abundantly express type I collagen. In a later differentiation stage, osteoblasts mineralize the osteoid and are typified by expression of osteocalcin.

Several groups reported that osteoblastic cells of mouse, rat or human origin express VEGF and its receptors, with highest expression levels being found at the late differentiation stages.^15,16 As observed in chondrocytes, VEGF production in osteoblasts is stimulated by hypoxia in a process that involves HIF1A and HIF2A.^17-19 Furthermore, VEGF expression in osteoblasts is also induced by several osteotropic factors, including BMPs, transforming growth factor beta (TGFB), prostaglandins, insulin-like growth factor 1 (IGF1), platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF2) and 1alpha,25-dihydroxyvitamin D₃, but it is inhibited by bone catabolic factors such as glucocorticoids.

Osteoclasts

Osteoclasts are large, multinucleated cells that are endowed with the unique capacity to degrade mineralized tissues, a process in which secreted MMPs play an important role.²⁰ The development of osteoclasts is a complex multi-step process that involves at least two crucial signaling molecules expressed by osteoblasts and osteoblast progenitors: macrophage-colony stimulating factor (CSF1; previously known as M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL; now also known as tumor necrosis factor superfamily member 11, TNFSF11).²¹

Osteoclasts share a common hematopoietic precursor with monocytes and macrophages, and like them express FLT1 as their main VEGF receptor.^22,23 However, expression of KDR by cultured osteoclasts has also been reported.^24,25 Primary cultures of osteoclasts prepared from murine bone marrow were found to express VEGF by RT-PCR,²⁵ but these results have to be confirmed by additional experimental approaches, as these cultures also contained other bone marrow derived cells.

VEGF Controls the Initial Vascular Invasion during Formation of the Primary Ossification Center

At the time when the primary ossification center develops, VEGF is produced by perichondrial cells, possibly osteoblasts, and by diaphyseal hypertrophic chondrocytes (see below).^4,27 Vegfa transcription is thought to be induced by RUNX2, given that expression of VEGF and its receptors is impaired in the bones of RUNX2-deficient mice. Moreover, these mice show no vascular invasion into any skeletal element, consistent with the idea that VEGF is a critical vascular growth factor during bone formation.⁹ This idea is particularly supported by the observation that the formation of the primary ossification center is delayed in cartilage explants cultured with a VEGF-inhibiting soluble chimeric FLT1 protein.²⁶ The initial vascular invasion and the formation of the primary ossification center are also delayed in Vegfa120/120 and Vegfa188/188 mice, but not in Vegfa164/164 mice, suggesting that specifically the VEGF164 isoform is needed for this process.^4,5 This could be due to its interaction with NRP1. Alternatively, not the VEGF164 isoform in particular, but rather a combination of soluble and bound VEGF molecules may be needed to coordinate the initial capillary invasion into the cartilage anlagen, perhaps to form a chemoattractive gradient for ingrowing vessels. A similar mechanism has been proposed for angiogenesis in other organs (see Chapter 6 by H. Gerhardt). VEGF is also a chemoattractant for osteoclasts invading into developing bones. This process involves MMPs, raising the possibility that release of matrix-bound VEGF from the hypertrophic cartilage matrix by the action of MMPs may account for the close association of vascular and osteoclastic invasion.^28-30

Nonvascular Roles of VEGF during the Formation of the Primary Ossification Center

Table 1

VEGF effects during the 3 key stages of endochondral (more...)

Table 1

VEGF effects during the 3 key stages of endochondral bone development

VEGF Effects	POC Development	Longitudinal Bone Growth	SOC Development
Vascular	Vascular invasion	Metaphyseal vascularization	Epiphyseal vascularization
Nonvascular	HC differentiation (*)	HC apoptosis and resorption (*)	Chondrocyte proliferation
	OB development/activity	OB and OC activity	and differentiation
	OC recruitment/activity		Chondrocyte survival

(*) The direct effect of VEGF in these processes has not unambiguously been shown in vitro. Abbreviations: POC: primary ossification center; SOC: secondary ossification center; HC: hypertrophic chondrocyte; OB: osteoblast; OC: osteoclast.

The Role of VEGF in Metaphyseal Vascularization

During longitudinal bone growth, it is of utmost importance that the key mechanisms of endochondral ossification are rigorously coordinated, and the analysis of several different mouse models has demonstrated that VEGF-mediated metaphyseal angiogenesis plays a critical role in this process. Disruption of VEGF function in developing bones has been achieved by injection of a soluble truncated chimeric VEGF receptor,¹ by targeted inactivation of the VEGF-isoforms VEGF164 and VEGF188 leaving only expression of the soluble isoform VEGF120,^4,6 and by conditional deletion of a single Vegfa allele in cells expressing type II collagen.² In all three instances, (partial) loss of VEGF function impaired metaphyseal bone vascularization. Specifically, vascularization was decreased and disorganized near the growth plate and, concomitantly, trabecular bone formation and bone growth were impaired. Typically, the hypertrophic chondrocyte zone of the growth plate was enlarged, due to reduced resorption and/or apoptosis. Thus, in metaphyseal bone development, VEGF functions to attract vessels to the growth plate, which is accompanied by hypertrophic chondrocyte apoptosis, cartilage resorption by osteoclasts/chondroclasts, and trabecular bone formation by osteoblasts.

Fig. 1H

Fig. 1H

Nonvascular Roles of VEGF during Longitudinal Bone Growth

Recent studies have suggested that VEGF may influence bone formation by directly affecting osteoblasts, as VEGF stimulates osteoblast differentiation and migration in vitro.^15,31,37 Moreover, adenovirus-mediated VEGF gene transfer induces bone formation by increasing osteoblast number and osteoid forming activity in vivo.³⁸ VEGF signaling has also been implicated in osteoclastogenesis and subsequent cartilage/bone resorption: Firstly, VEGF directly enhances the resorption activity and survival of mature osteoclasts in vitro.²⁴ Secondly, RANKL induces osteoclast differentiation from spleen- or bone marrow-derived precursors in culture when provided in combination with either VEGF or CSF1. Thirdly, VEGF, like CSF1, rescues osteoclast recruitment, survival and activity in osteopetrotic op/op mice, which carry an inactivating point mutation in the Csf1 gene that results in low numbers of macrophages and a complete lack of mature osteoclasts.²³ Like monocytes and macrophages, osteoclasts predominantly express FLT1 rather than KDR (see above).^22,23 Moreover, FLT1 ligands are chemoattractive for both monocytes and osteoclasts.^29,39

The Role of VEGF in Epiphyseal Vascularization and the Initiation of Secondary Ossification

Fig. 1I

Nonvascular Roles of VEGF in Epiphyseal Chondrocyte Development and Survival

Fig. 1I

Novel Mouse Models to Understand VEGF Signaling in Bone Development

Whilst it is now evident that VEGF is essential to drive vascularization during endochondral bone development, the in vivo studies performed have also underscored our limited knowledge of the bone's vascular system itself and of its role in regulating the behavior of bone cells. For example, we don't know much about the types of blood vessels involved (capillaries, venous sinusoids, arteries), nor about the presence and role of pericytes or other peri-vascular cells. It is also still largely unclear how epiphyseal vascularization and the formation of vascular canals relate to secondary ossification. Moreover, we need to understand better the resorption processes that accompany vascular invasion. For instance, the contribution of specific proteases that release VEGF from the matrix at particular stages of endochondral bone development could be further addressed by the analysis of mice with specific mutations in MMP genes. The role of the VEGF-responsive cell types involved, such as endothelial cells and (other) resorbing cells, could be addressed by specifically targeting VEGF receptors.

Importantly, it has become clear that VEGF also exerts direct effects on several key bone cell types during endochondral bone development. These direct effects are still incompletely understood, for two reasons: Firstly, in vivo models have often been difficult to analyze, as the alteration of the VEGF expression levels or the VEGF isoform balance almost inevitably causes angiogenic defects, which in turn affect bone development. Secondly, in vitro models are limited in their capacity to accurately reproduce the complex differentiation processes that occur in vivo. However, the combination of both approaches, together with the use of transgenic mice carrying cell type-specific, temporally restricted or even cellular differentiation stage-specific knockout alleles for VEGF and its receptors, including the isoform-specific VEGF receptors, will provide this critical information. Aspects to be addressed include the precise effects, mechanisms of action and regulation of VEGF in osteoblasts, osteoclasts, and chondrocytes. Particularly regarding the regulation of VEGF in cartilage, much remains to be learned about the role of the hypoxia regulatory pathway. Inactivation of von Hippel Lindau (VHL), a mediator of HIF degradation, in murine cartilage was recently shown to alter chondrocyte proliferation, further underscoring that components of the hypoxia regulatory pathway play important physiological roles in cartilage, either directly and/or by affecting VEGF levels.⁴⁵ Furthermore, it will be interesting to see whether this pathway also plays a role in other bone cells. Finally, it will be exciting to explore if VEGF contributes to the very early stages of skeletal development, prior to vascular invasion and ossification, and whether VEGF regulates the development of joints.

From Mice to Men: A Role for VEGF Misexpression in Growth Disorders?

Animal studies have shown that the precise level of VEGF is critically important for embryonic development. Interestingly, the phenotypes of Vegfa188/188 and Vegfa120/120 mice suggest that altering the relative levels of the VEGF isoforms—without affecting the total level of VEGF—impairs developmental processes such as vascular network formation and skeletal development and growth. In particular, normal levels of the VEGF164 isoform appear to be critical for normal bone development in mice. However, it has not yet been examined if subtle variations in VEGF or VEGF isoform expression levels affect the development of the human skeleton. For example, it is conceivable that allelic variations in the human VEGFA gene promoter or abnormal VEGFA mRNA splicing affect either VEGF expression or the production of the VEGF165 isoform (the human ortholog of murine VEGF164). Hypothetically, such changes might cause or increase the risk of growth defects, or add to the severity of skeletal disorders caused by mutation in other genes (e.g., FGF receptor 3 or PTH/PTHRP receptor). It will be particularly important to examine if VEGFA gene polymorphisms are linked to the pathogenesis of human dwarfing syndromes or other skeletal pathologies, because low VEGF levels are already known to predispose humans and mice to motor neuron degeneration (see Chapter 8 by J. Krum, J. Rosenstein and C. Ruhrberg), and loss of VEGF164 expression acts as a modifier of DiGeorge syndrome, a disease with vascular and craniofacial abnormalities.^46,47 Studying the role of VEGF in bone development may also provide the basis for new therapies aimed at treating debilitating and/or unmanageable bone diseases, such as osteoporosis, bone metastases, and nonhealing fractures.