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Review of the Effects of Anti-Angiogenic Compounds on the Epiphyseal Growth PlateAstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, England Correspondence: Address correspondence to: Anthony Peter Hall, AstraZeneca R&D, Alderley Park, Safety Assessment UK, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, England; e-mail:Peter.a.hall{at}astrazeneca.com
The formation of new blood vessels from a pre-existing vascular bed, termed "angiogenesis," is of critical importance for the growth and development of the animal since it is required for the growth of the skeleton during endochondral ossification, development and cycling of the corpus luteum and uterus, and for the repair of tissues during wound healing. "Vasculogenesis," the de novo formation of blood vessels is also important for the proper function and development of the vascular system in the embryo. New blood vessel formation is a prominent feature and permissive factor in the relentless progression of many human diseases, one of the most important examples of which is neoplasia. It is for this reason that angiogenesis is considered to be one of the hallmarks of cancer. The development of new classes of drugs that inhibit the growth and proper functioning of new blood vessels in vivo is likely to provide significant therapeutic benefit in the treatment of cancer, as well as other conditions where angiogenesis is a strong driver to the disease process. During the preclinical safety testing of these drugs, it is becoming increasingly clear that their in vivo efficacy is reflected in the profile of "expected toxicity" (resulting from pharmacology) observed in laboratory animals, so much so, that this profile of "desired" toxicity may act as a signature for their anti-angiogenic effect. In this article we review the major mechanisms controlling angiogenesis and its role during endochondral ossification. We also review the effects of perturbation of endochondral ossification through four mechanisms—inhibition of vascular endothelial growth factor (VEGF), pp60 c-Src kinase and matrix metalloproteinases as well as disruption of the blood supply with vascular targeting agents. Inhibition through each of these mechanisms appears to have broadly similar effects on the epiphyseal growth plate characterised by thickening due to the retention of hypertrophic chondrocytes resulting from the inhibition of angiogenesis. In contrast, in the metaphysis there are differing effects reflecting the specific role of these targets at this site.
Key Words: Review • angiogenesis • epiphyseal growth plate • endochondral ossification • corpus luteum • VEGF • VEGFR • pp60 c-Src kinase inhibitor • matrix metalloproteinase inhibitor • vascular targeting/tubulin binding agent Abbreviations: ADEPT, antibody-directed enzyme prodrug therapy strategy •
Angiogenesis, the growth of new capillaries from a pre-existing vascular bed, is a rare event in most adult tissues, with a turnover rate of approximately 0.01%. However it is required for tissue growth and repair, and is a prerequisite for endochondral ossification (Gerber and Ferrara, 2000), wound healing (Howdieshell et al., 2001), corpus luteum formation (Ferrara et al., 1998), uterine endometrial development (Ferrara et al., 1998), and neonatal growth (Gerber et al., 1999). Vasculogenesis, the de novo growth of new blood vessels, is required for the development of the embryo. Pathological angiogenesis, on the other hand represents a non-physiological form of angiogenesis that is associated with, or even drives the disease process. It is involved in a plethora of diseases (reviewed by Carmeliet, 2003) the most important of which is, arguably, neoplasia. Human neoplasia and metastasis is absolutely dependent upon angiogenesis one of the six "hallmarks of cancer" (Hahn and Weinberg, 2002; Hanahan and Weinberg, 2000) since in its absence solid masses are limited to a maximum diameter of 2–3 mm—the diffusion limit for nutrients and oxygen (Folkman, 2002). This dependency is illustrated by the observations that tumour "hotspots" (areas of increased vascular density and mitotic activity) carry considerable clinical relevance, correlating with poorer survival in many types of human cancer (Jannink et al., 1995; Belien et al., 1999; Ahn et al., 2001; Mineo et al., 2004). The degree of vascularisation in turn correlates with the likelihood of progression and metastasis (Clayton, 1991; Weidner et al., 1991; Biesterfeld et al., 1995; Hasan et al, 2002). The discoveries of the dependency of neoplasia (and other diseases) upon angiogenesis, and the elucidation of many of the important molecules controlling angiogenesis have prompted pharmaceutical and biotechnology companies to develop novel treatments that inhibit this process. The results of this effort have propelled the field of angiogenesis, and in particular, inhibition of VEGF into a key area of clinical importance (Cristofanilli et al., 2002; Ferrara and Alitalo et al., 1999; Kerbel and Folkman, 2002) resulting in the development of new compounds to treat human neoplasia. The generation of these novel treatments has of course resulted in significant toxicity to a number of target organs relying on angiogenesis for maintenance and development. This review describes the effects of some of these new compounds on endochondral ossification.
Activation of the Angiogenic Switch Cells readily upregulate pro-angiogenic molecules in response to metabolic stress (hypoxia, decreased pH, and hypocalcaemia), physical stress (pressure), inflammation, and oncogene activation (reviewed by Carmeliet and Jain, 2000). Hypoxia is a potent stimulator of angiogenesis by signalling through a family of hypoxia inducible transcription factors (HIFs) that can upregulate VEGF by up to 30-fold within a few minutes (Pugh and Ratcliffe, 2003). In tumours, this change to the pro-angiogenic phenotype in response to positive and negative regulators of angiogenesis ("the angiogenic switch") is accompanied by the acquisition of activated oncogenes (Rak et al., 2000; Hanahan and Folkman, 1996). The dual roles that activated oncogenes, such as EGF (epidermal growth factor) (Hirata et al., 2002) and Ras (Thompson et al., 1989) play in the initiation and development of cancer, provides a mechanistic rational for the coupling of continued tumour growth with increased angiogenesis.
Activation of the angiogenic switch, either through mutation and oncogene activation or due to physiological angiogenesis in the growing animal causes vessel destabilisation, endothelial basement membrane/matrix dissolution, and finally sprouting/intussusception, migration and proliferation of endothelial cells. Eventually strings of endothelial cells organise themselves into hollow vascular tubes. Many of these effects can be achieved by vascular endothelial growth factor (VEGF) alone (Figure 1) since it not only promotes endothelial cell activation, migration, proliferation and survival, but also promotes MMP-2 (matrix metalloproteinase 2), MMP-9 and MT1-MMP (membrane type-1 MMP) secretion, and increased vascular permeability (reviewed in Rundhaug, 2003). Increased vascular permeability itself promotes the formation of a provisional matrix scaffold via leakage of fibrin, fibronectin and other plasma proteins (Dvorak, 2002). The provisional stroma is able to bind integrin receptors (e.g.,
VEGF and the Angiopoietins The recent discovery of organ specific activators (Ferrara et al., 2004; Pisani et al., 2004; LeCouter et al., 2002) and inhibitors (Albig and Schiemann, 2004; Kee et al., 2002) indicate that there are additional levels of control over angiogenesis. The functional significance that many of these factors contribute to the angiogenic process; however, is still not fully understood. Two key molecules that have received considerable interest and have fairly well defined roles are VEGF and the angiopoietins. The receptors for both these ligands are predominantly expressed upon endothelial cells and regulate the proliferation and survival of endothelial cells. The discovery of VEGF in 1989 (Leung et al., 1989) (also known as vascular permeability factor (Senger et al., 1983)) catalysed research into angiogenesis. Deficiencies in either vascular endothelial growth factor receptor (VEGFR) or its ligand, VEGF, result in embryonic lethality. VEGFR-1/2 null mice die at embryonic day 8.5 (reviewed in Hanahan, 1997) whereas VEGF+/– hemizygous mouse embryos die at E11 to 12 due to ubiquitous defects in their vasculature. Histologically, defects in yolk sac and embryo vessel morphology are seen with accompanying tissue necrosis (Patan, 2004). The VEGFs are dimeric endothelial cell mitogens encoded by 5 genes in mammals designated VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF) (Huang and Bao, 2004). Variations in mRNA splicing generate isoforms of several of the VEGFs, adding to the complexity of the family (McColl et al., 2004). They bind to their cognate receptors, VEGFR-1 (also known as Flt-1) and VEGFR-2 (also known as Flk-1, KDR). The Angiopoietin-Tie receptor system consists of angiopoietin (Ang) –1, which binds to its cognate receptor tyrosine kinase, Tie-2 (Huang and Bao, 2004). Other angiopoietins have been found (Ang2, Ang3, Ang4), which also bind to Tie-2 but do not necessarily induce phosphorylation (reviewed by Thurston, 2003). No known ligand has been found for Tie-1. Tie-1 and Tie-2 receptor null mice, like VEGF/VEGFR deficient mice die in utero, between E9.5-10.5 (Tie-2) and E13.5-(E18.5 depending on strain) (Tie-1) (Dumont et al., 1994; Sato et al., 1995; Puri et al., 1995). Both Tie-1 and Tie-2 receptor knockout mice develop vascular anomalies, with the Tie-2 mutant mice developing abnormally dilated vessels that lack mural cell support (pericytes and vascular smooth muscle cells (vSMCs)) (Patan, 2004). Recently, the Tie-2 receptor has been shown to be expressed on three distinct cell populations involved in tumour neovascularisation: endothelial cells, proangiogenic bone marrow derived monocyte/macrophages and pericyte precursors of mesenchymal origin (De Palma et al., 2005). Angiopoietin-2 (Ang-2) binds to the Tie-2 receptor tyrosine kinase and induces a loosening of the attachment between endothelial cells and supporting stromal cells (pericytes and vSMCs) (Maisonpierre et al., 1997). In the absence of VEGF, Ang-2 induces regression of blood vessels (Sato et al., 1995; Dumont et al., 1994). In the presence of VEGF, Ang-2 facilitates vessel sprouting (the formation of blind ending capillary tubes) (Asahara et al., 1998) and intussusception (the folding of slender tissue pillars or posts into the vessel lumen (Patan et al., 1992)). Vessel sprouting is accompanied by basement membrane/matrix dissolution and the loss of endothelial/matrix attachments. VEGF promotes endothelial vascular permeability and vasodilatation as well as survival, migration and proliferation. Under its influence, a provisional scaffold of plasma proteins and fibrin forms.
Fibroblast Growth Factor and Angiogenesis
The Extracellular Matrix—Role in Angiogenesis Degradation of the extracellular matrix is a necessary prerequisite for angiogenic sprouting (Figure 2). A plethora of intricately balanced proteases and protease inhibitors are involved in this process. The matrix metalloproteinases are considered essential for matrix remodelling and consist of a family of zinc endopeptidases that cleave peptide bonds in extracellular matrix proteins (reviewed by Sottile, 2004). Mice lacking "RECK," a membrane bound inhibitor of MMP-2, -9 and MT1-MMP (membrane type I-matrix metalloproteinase), die in utero at E10.5, due to defects in collagens, basement membrane and vascular development (Oh et al., 2001). Restoration of RECK function in cell lines suppresses invasion and metastasis (Takahashi et al., 1998). Their differential expression is regulated, at least in part, by growth factor stimulation (e.g., FGF and VEGF (Burbridge et al., 2002)).
Matrix degradation promotes vascular sprouting not only by removing a physical barrier, but by stimulating endothelial cell migration by revealing cryptic binding sites on collagens (Hangai et al., 2002) and virtually all other matrix molecules (Schenk and Quaranta, 2003), releasing latent matrix-bound pro-angiogenic factors e.g., VEGF, FGF (Saksela et al., 1990) transforming growth factor-β (TGF-β) (Rifkin et al., 1999; Egeblad and Werb, 2002), and activating angiogenic chemokines (egIL-1β). Matrix degradation appears to be balanced by protease inhibitors e.g., tissue inhibitors of metalloproteinases (TIMPs), and urokinase plasminogen activator inhibitor (PAI-1) that limit excess degradation, but also, by the action of the proteases themselves, liberate matrix fragments with anti-angiogenic activity e.g., endorepellin (Mongiat et al., 2003), endostatin (O’Reilly et al., 1997), a thrombospondin fragment (Good et al., 1990), angiostatin (O’Reilly et al., 1994) and tumstatin (Maeshima et al., 2000; Petitclerc et al., 2000). This is necessary since excess matrix degradation results in impaired angiogenesis (Bajou et al., 1998) possibly due to removal of critical support and guidance cues for migrating endothelial cells (Anderson et al., 2004).
Mural Cell Support
Analysis of the desmoplastic stromal response induced by invasive carcinomas suggests that myofibroblasts express a variety of markers, most commonly vimentin (V) and alpha smooth muscle actin ( Recruitment of PDGFRβ+ (platelet derived growth factor receptor β) mesenchymal pericyte precursors, appears to be critically dependent upon PDGF-B (platelet derived growth factor-B) which is elaborated by the sprouting vascular tip. PDGF-B induced migration is not straight forward, but relies upon receptor "crosstalk," since it requires PDGFRβ regulated activation of the EDG-1/ sphingosine-1-phosphate (S1P1) receptor system ( and Hla, 2002; Rosenfeldt et al., 2003). In return, pericytes synthesise and release VEGF, which is a potent endothelial mitogen, as well as a possible pericyte mitogen in areas of hypoxia, (Yamagishi et al., 1999) creating a local circuit promoting endothelial/pericyte co-migration. PDGF-B ligand stimulates pericyte expansion from mesenchymal precursors and subsequent population of nascent blood vessels (Hellstrom et al., 1999). Genetic ablation of PDGF-B or PDGFRβ results in perinatal lethality due to microaneurysm formation and widespread vascular leakage (Leveen et al., 1994; Soriano, 1994; Lindahl et al., 1997) as a result of increased vascular permeability and lack of structural support from pericyte loss. Attachment of pericytes is promoted by Ang 1, which appears to enhance vessel stabilisation and reduce vascular permeability (Hawighorst et al., 2002; Asahara et al., 1998). TGF-β signalling, occurring mainly during the later stages of angiogenesis, further modifies the angiogenic response. At low concentrations, TGF-β upregulates the angiogenic switch whereas at high concentrations, it stimulates matrix synthesis, induces mesenchymal cell differentiation into (myo)fibroblasts (Pepper, 1997; Chambers et al., 2003) and pericytes ( and D’Amore, 2001) and promotes smooth muscle differentiation (Hirschi et al., 1998). This is mediated, at least in part, through TGF-Rβ type I receptor/Smad signal transduction pathways that can either induce endothelial proliferation and migration via the ALK1 (activin receptor-like kinase) pathway or inhibit it via the ALK5 pathway (Goumans et al., 2002). Coincident with mural cell recruitment, endothelial cells re-establish cell-cell and cell-matrix junctional communication via, for example, vascular endothelial (VE) cadherins (Breviario et al., 1995), occludins (Wu et al., 2000), JAM-1 in tight junctions ( and Dejana, 2001), CD31 (PECAM-1) (Feng et al., 2004), connexins in gap junctions (Yeh et al., 1998) and integrins. Maturation of the nascent capillary network usually returns the tissue to homeostasis. Pathological angiogenesis however is marked by an absence of resolution. Xenograft and transgenic mouse tumours demonstrate abnormalities in angiogenesis due to dysregulated synthesis and release of angiogenic factors, which in turn promote continuous capillary proliferation and an immature vascular phenotype. Nascent microvessels show aberrations in mural/pericyte cell recruitment and attachment (Morikawa et al., 2002) and abnormally dilated and tortuous microvessels with increased permeability (Carmeliet and Jain, 2002). It is hoped and believed that these differences will help distinguish tumour vessels from quiescent vessels, and so make them amenable to therapeutic intervention.
Endochondral Bone Ossification
Immature and proliferating chondrocytes secrete and express angiogenic inhibitors (Moses et al., 1999) and an extracellular matrix (ECM) rich in aggrecan and type II collagen (reviewed in Orth, 1999). Enlargement and terminal differentiation into hypertrophic chondrocytes now allows these cells to act as regulators of the process and engage a genetic programme characterised by expression of type X collagen (Alini et al., 1994), VEGF (Gerber et al., 1999; Horner et al., 1999; Carlevaro et al., 2000; Maes et al., 2002), connective tissue growth factor (Ivkovic et al., 2003), acidic and basic FGF (Baron et al., 1994; Nagai and Aoki, 2002), MMP-13 (Nagai and Aoki, 2002) and the enzyme alkaline phosphatase (Vaananen, 1980). Hypertrophic chondrocytes also deposit mineral and partially degrade the ECM before undergoing programme cell death. Cells migrating inwards from the marrow space also contribute to ECM degradation by secreting MMPs—the most important of which being MMP-2, MMP-9 and MMP-13 that degrade non mineralised matrix (reviewed in Blair et al., 2002) resulting in loss of the "last transverse septum." This septum marks the boundary between apoptotic hypertrophic chondrocytes and the migrating vascular invasion front. Hypertrophic chondrocytes express a number of VEGF isoforms (Maes et al., 2002; Petersen et al., 2002). Long splice forms, such as murine VEGF164 and VEGF188 show increased binding to heparin sulphate containing proteoglycans present on the cell surface and extracellular matrix (Ferrara and Davis-Smyth, 1997), whereas the short murine VEGF120 splice form is completely soluble and unable to bind heparin (Ferrara and Davis-Smyth, 1997). The long VEGF splice forms (Maes et al., 2002) appear to be the most important isoforms mediating endochondral bone ossification (Maes et al., 2002). During ECM degradation, MMPs release the matrix bound long VEGF isoforms, which together with other processed angiogenic growth factors, further encourage the inwards migration of vascular endothelium (Petersen et al., 2002). Capillary invasion into the empty lacunae of terminal hypertrophic chondrocytes now allows access and functional differentiation of other VEGFR expressing nonvascular cells—notably osteoblasts (Midy and Plouet, 1994; Deckers et al., 2000) and osteoclasts (Engsig et al., 2000; Maes et al., 2002). Apoptotic chondrocytes leave behind a mineralised cartilage matrix that becomes the scaffold for invading osteoblasts and osteoclasts. Osteoblasts differentiate on the plates of mineralised cartilage between columns of hypertrophic chondrocytes and lay down delicate spicules of woven bone (primary trabeculae), whereas osteoclasts remove mineralised matrix. Analysis of MMP-9–/– and MMP-13–/– deficient mice indicate that these are the two most active MMPs involved in epiphyseal growth plate endochondral ossification and operate in concert to degrade the unmineralised hypertrophic chondrocyte septae. They are expressed in two different compartments at the chondro-osseous interface. MMP-9 appears to be the most important, being expressed at the invasion front by endothelial cells, monocytes, preosteoclasts, osteoclasts and chondroclasts (reviewed in Ortega et al., 2004). MMP-9 is not only necessary for ECM degradation, a prerequisite for angiogenesis, but also appears to play a role in the processing of kit ligand (stem cell factor) for the recruitment and mobilisation of bone marrow derived stem, haemopoietic and endothelial precursors (Hessig et al., 2002). MMP-13, derived from hypertrophic chondrocytes and osteoblasts at the chondro-osseous border, synergises with MMP-9 during ECM degradation. Primary trabeculae are modelled into fewer and thicker secondary and tertiary trabeculae located more distally in the diaphysis (Porter et al., 1997). The modelling of bone consists of a "coupled" process of osteoclastic resorption and osteoblastic formation. Osteoblasts are thought to provide osteoclastic help by removing the organic phase to expose bone mineral (Chambers and Fuller, 1985). Osteoclasts remove mineralised matrix. They are polarised cells with a ruffled border that enables them to create an acidic microenvironment into which they release lysosomal enzymes, resulting in the decalcification and degradation of bone (Baron, 1989). The formation of the ruffled border, necessary for bone resorption, is dependent upon pp60 c-Src (Boyce et al., 1992; Soriano et al., 1991) and the micropthalmia transcription factor, Mitf (Hodgkinson et al., 1993). The uncoupling of these two processes—osteoblastic bone formation and osteoclastic resorption—is the basis of skeletal imbalances characterised by increased (osteopetrosis) or decreased (osteoporosis) bone density.
Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitors This work has been continued in human patients, where the recombinant humanised anti-VEGF-A antibody (Avastin; Genetech) during phase II and phase III clinical trials showed synergy with standard chemotherapy in a number of malignancies, including colorectal (Midgley and Kerr, 2005) and advanced metastatic non-small cell lung carcinoma (Kabbinavar et al., 2003; Ferrara et al., 2004; Sandler et al., 2004). Avastin has now been approved in the United States as a first-line therapy for metastatic colorectal cancer (reviewed by Ferrara et al., 2004). In addition, several small molecule inhibitors of the VEGFR-1 and/or 2 tyrosine kinase, have now entered clinical trials and are expected to show clinical benefit. The effect of inhibition of VEGF signalling on bone growth has been well described (Gerber et al., 1999; Wedge et al., 2000; Beebe et al., 2003) and results in thickening of the epiphyseal growth plate due to expansion of the hypertrophic zone (Figure 4b). These effects have been attributed to delayed vascular invasion of the epiphyseal growth plate resulting in a reduced rate of hypertrophic chondrocyte apoptosis. Similar effects have also been noted with a small molecule inhibitor of the FGF receptor (Brown et al., 2005). The changes in growth plate thickness can be morphmetrically quantified revealing a dose-dependent increase in the epiphyseal area of up to 481% (Wedge et al., 2005). These changes appear to be restricted to the hypertrophic zone characterised by the accumulation of relatively parallel columns of hypertrophic chondrocytes (Wedge et al., 2000). The reserve, proliferative and maturing zones of the growth plate appear normal. Minimal retention of hypertrophic chondrocytes also occurs in the articular-epiphyseal complex (epiphyseal growth cartilage) of the femoral and tibial condyles (Figure 4f). Since the normal rat femur would be expected to grow approximately 5 mm (female) to 5.5 mm (male) in length between 6 and 10 weeks of age (Hannson et al., 1972) it is likely that despite VEGFR inhibition, some endochondral ossification occurs at a basal level.
Inhibition of VEGF not only interferes with vascular invasion, resulting in growth plate thickening, but also impairs trabecular bone formation. Mice treated with a soluble VEGF receptor chimeric protein, mFlt(1-3)-IgG showed reduced length and number of primary trabeculae with thicker secondary trabeculae (Gerber et al., 1999) (Figure 5b). This observation can be accounted for by both reduced ossification of apoptotic hypertrophic chondrocytes (Gerber et al., 1999) and impaired recruitment and migration of monocyte/macrophage-derived chondroclasts and osteoblasts. These specialised cells are necessary for the formation of bone trabeculae, express VEGF-R1, and migrate in response to VEGF ligand (Midy and Plouet, 1994; Hiratsuka et al., 1998; Mayr-Wohlfart et al., 2002). Restoration of normal angiogenesis by discontinuation of mFlt(1-3)-IgG treatment allows rapid reversal of all growth plate changes within 2 weeks (Gerber et al., 1999). Cessation of treatment reverses femoral shortening and restores the growth plate to a normal histological appearance. This is attributed to renewed vascular invasion inducing resorption of hypertrophic chondrocytes, and modelling of newly formed primary trabeculae into secondary trabeculae.
In addition to changes in the growth plate induced by VEGFR antagonists, we have also observed dental dysplasia in the growing rodent incisor (unpublished observations), and ovarian atrophy (reduced numbers of corpora lutea) (Wedge et al., 2005). Ovarian atrophy is an expected consequence of VEGF inhibition since VEGF expression is essential for the capillary in-growth into the normally avascular Graafian follicle and conversion to the highly angiogenic corpus luteum (Phillips et al., 1990; Ferrara et al., 1998).
Vascular Disrupting/Targeting Agents Recently, low molecular weight vascular disrupting agents have also been developed that target tumour vasculature, such as combretastatin A4 phosphate (Griggs et al., 2001) and ZD6126 (Blakey et al., 2002a). These compounds disrupt the tubulin cytoskeleton of immature tumour endothelium (Davis et al., 2002) and destabilise microtubules resulting in mitotic cell arrest/death (Kanthou et al., 2004) and rapid endothelial cell retraction/exfoliation. This culminates in exposure of vascular basement membrane, thrombus formation and, usually within 24 hours, large areas of ischaemic necrosis of the tumour core (Blakey et al., 2002b). These compounds have now entered clinical trials (Thorpe et al., 2003). Consistent with an anti-angiogenic effect, we have observed mild, often focal thickening of the epiphyseal growth plate together with necrosis of sub-physeal osteocytes (Figure 4c and 5f) (unpublished observations). The local nature of both these lesions, metaphyseal osteocyte necrosis and growth plate thickening, may indicate relative insensitivity of the peripheral areas of the growth plate vasculature to vascular disruption by these agents. This would not be unexpected since treatment with combretastatin A4 phosphate and other tubulin-binding agents produces a characteristic pattern of necrosis in xenograft models, limited to the central core (West and Price, 2004). The rim is usually viable, reflecting relative vascular resistance.
pp60 c-Src Kinase Inhibition
pp60 c-Src kinase is thought to form part of the VEGFR-2 (KDR) downstream signal transduction pathway necessary for the hypoxic induction of VEGF (Mukhopadhyay et al., 1995), and coupling of focal adhesion kinase to pp60 c-Src forms part of a multimeric signalling protein involving gelsolin, pp60 c-Src and phosphatidylinositol 3'-kinase (Chellaiah et al., 1998, 2001). Gelsolin null mice show abnormalities in osteoclastic resorption causing thickening of metaphyseal trabeculae with abnormal trabecular architecture (Chellaiah et al., 2000). Homozygous c-Src–/–-deficient mice develop osteopetrosis (Soriano et al., 1991). Histologically, the bone marrow space becomes occupied with increased bone volume that architecturally appears to be an extension of abnormally modelled primary trabeculae into the diaphysis (Figure 5c). This phenotype is thought to be mediated through an osteoclast defect that renders them inactive and unable to adhere to bone and/or form a bone resorbing ruffled border (Lowe et al., 1993). c-Src–/–-deficient mice also show enhanced osteoblast differentiation and bone formation that contributes to the trabecular changes (Marzi et al., 2000). The precise role that pp60 c-Src Kinase plays in osteoclastic bone resorption however is still unclear. Some authors suggest that only the adaptor function of pp60 c-Src is necessary for bone resorption (Schwartberg et al., 1997; Felsenfeld et al., 1999). Others (Miyazaki et al., 2004) have suggested that both Src kinase activity and Src SH2-dependent formation of the Pyk2/Src complex are necessary for bone resorption.
Broad Spectrum Matrix Metalloproteinase Inhibitors Genetic disruption of the FGFR-3 (Deng et al., 1996), treatment with a FGF receptor small molecule tyrosine kinase inhibitor (Brown et al., 2005) or supplementation with FGF-2 (Nagai and Aoki, 2002) all produce a similar effect on the growth plate. Growth plate thickening is thought to be mediated through one or more mechanisms-directly through inhibition of angiogenesis, or indirectly through inhibition of cartilage degradation, chondrocyte differentiation and vascular invasion (MMP-9 and FGF-2/MMP-13) preventing the exit of hypertrophic chondrocytes from the growth plate (Stickens et al., 2004) or through relief of chondrocyte growth suppression (FGFR-3). Recent studies with MMP-13 deficient mice indicate that MMP-13 expression is upstream of growth plate angiogenesis and that degradation of cartilage extracellular matrix is a prerequisite for vascular invasion (Stickens et al., 2004). This view is further supported by the observation that treatment of wild-type mice with galactin-3 (a matricellular protein) phenocopies the growth plate defect seen in MMP-9-deficient mice, indicating that this protein may be a physiologically relevant substrate for MMP-9 during endochondral bone ossification (Ortega et al., 2005). The role of a number of MMPs has also been documented in the turnover and remodelling of bone in the metaphysis (Kusano et al., 1998; Pelletier et al., 2004). MMP-1, MMP-9, MMP-12 and MMP-13 expression has been immunolocalised under the ruffled borders of osteoclasts and on bone surfaces in rodents (Delaisse et al., 1993; Nakamura et al., 2004) and in osteoclasts in humans (Okada et al., 1995; Hou et al., 2004). MMP-13-deficient mice show metaphyseal changes characterised by increased trabecular bone (Figure 5d). This effect was thought to be due to MMP-13 deficiency in the osteoblasts and therefore independent of the effect in the growth plate cartilage (Stickens et al., 2004). Systemic treatment of growing rats with FGF-2 causes metaphyseal changes which appeared morphologically as a disruption between the cartilage columns and trabecular bone, possibly mediated through diminished expression of MMP-13 in the terminal hypertrophic chondrocytes (Nagai and Aoki, 2002). These changes were characterised by a discontinuity between the metaphyseal trabecular bone and the hypertrophic chondrocyte columns (Figure 5e) resulting in separation and the appearance of horizontally laid vascular channels (Nagai and Aoki, 2002). Disruption of the chondro-osseous junction also been shown in a human case of achondroplasia (Briner et al., 1991) and the skeletal phenotype of FGFR-3 (G380) transgenic mice (Segev et al., 2000).
The requirement for angiogenesis during the normal development of vascular tissues such as the growth plate and the Graafian follicle is well established. The key molecules that regulate this process during normal physiology also play crucial roles in the control of the aberrant angiogenesis found in tumours. It is not surprising therefore that inhibitors of tumour angiogenesis produce a recognisable anti-angiogenic signature in growing animals that can be assessed during preclinical safety studies. The growth plate (and other organs such as the ovary) appear to be a very sensitive biomarker of this effect and thus can contribute to the overall data set supporting the mechanism of action of these compounds in vivo. It is likely as more experience is gathered in this area that other toxicities will be reported, specific for the intended target. These target organs will provide other opportunities to determine the efficacy and mechanism of action of novel angiogenic inhibitors, in the context of essentially normally regulated angiogenesis. It will be interesting therefore to speculate how predictive these biomarkers will be for human toxicity, and for the efficacy of anti-angiogenic compounds in human clinical trials when compared to efficacy data collected in vitro and in the standard xenograft model.
The authors would like to thank Steve Wedge for reviewing this manuscript. The work reviewed in this document complies with the standards of animal care and ethics described in "Guidance on the Operations of the Animals (Scientific Procedures) Act 1986" issued by the UK Home Office and was conducted so that any clinical expression of toxicity remained within a moderate severity limit as described in guidelines agreed with the UK Home Office Inspector.
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Toxicologic Pathology, Vol. 34, No. 2,
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Abstract
Introduction
SMA, alpha smooth muscle actin • Ang, angiopoietins • bFGF, basic fibroblast growth factor • D, desmin • EDB, extra domain B • EDG1, endothelial differentiation/sphingolipid G-protein-coupled receptor, 1 • EG-VEGF, endocrine derived vascular endothelial growth factor • FGF, fibroblast growth factor • FGFR, fibroblast growth factor receptor • HIF, hypoxia inducible factor • IFN-
, interferon gamma • JAM-1, junctional adhesion molecule • Mitf, micropthalmia transcription factor • MMP, matrix metalloproteinase • MT1-MMP, membrane type 1 matrix metalloproteinase • MHC II, major histocompatibility class II • PA, plasminogen activator • PAI-1, plasminiogen activator inhibitor I • PDGF-B, platelet derived growth factor-B • PDGFRβ, platelet derived growth factor receptor beta • PECAM-1, platelet/endothelial cell adhesion molecule • PlGF, placental growth factor • S1P1, sphingosine-1-phosphate-1 • TGF-β, transforming growth factor beta • TGF-Rβ, transforming growth factor receptor beta • TIMPs, tissue inhibitor of metalloproteinases • vSMC, vascular smooth muscle cell • VE, vascular endothelial • VEGFR, vascular endothelial growth factor receptor • VEGF, vascular endothelial growth factor • V, vimentin
