Increased skeletal VEGF enhances β-catenin activity and results in excessively ossified bones
2009; Springer Nature; Volume: 29; Issue: 2 Linguagem: Inglês
10.1038/emboj.2009.361
ISSN1460-2075
AutoresChrista Maes, Steven Goossens, Sonia Bartunkova, Benjamin Drogat, Lieve Coenegrachts, Ingrid Stockmans, Karen Moermans, Omar Nyabi, Katharina Haigh, Michael Naessens, Lieven Haenebalcke, Jan Tuckermann, Marc Tjwa, Peter Carmeliet, Vice Mandic, Jean‐Pierre David, Axel Behrens, András Nagy, Geert Carmeliet, Jody J. Haigh,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoArticle10 December 2009free access Increased skeletal VEGF enhances β-catenin activity and results in excessively ossified bones Christa Maes Christa Maes Laboratory of Experimental Medicine and Endocrinology, Department of Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Steven Goossens Steven Goossens Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Sonia Bartunkova Sonia Bartunkova Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Benjamin Drogat Benjamin Drogat Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Lieve Coenegrachts Lieve Coenegrachts Laboratory of Experimental Medicine and Endocrinology, Department of Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Ingrid Stockmans Ingrid Stockmans Laboratory of Experimental Medicine and Endocrinology, Department of Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Karen Moermans Karen Moermans Laboratory of Experimental Medicine and Endocrinology, Department of Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Omar Nyabi Omar Nyabi Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Katharina Haigh Katharina Haigh Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Michael Naessens Michael Naessens Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Lieven Haenebalcke Lieven Haenebalcke Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Jan P Tuckermann Jan P Tuckermann Division of Tissue-Specific Hormone Action, Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Marc Tjwa Marc Tjwa Vesalius Research Center, KU Leuven, Leuven, Belgium Vesalius Research Center, VIB, Leuven, Belgium Search for more papers by this author Peter Carmeliet Peter Carmeliet Vesalius Research Center, KU Leuven, Leuven, Belgium Vesalius Research Center, VIB, Leuven, Belgium Search for more papers by this author Vice Mandic Vice Mandic Department of Internal Medicine 3, Rheumatology and Immunology, University of Erlangen-Nuremberg, Erlangen, Germany Search for more papers by this author Jean-Pierre David Jean-Pierre David Department of Internal Medicine 3, Rheumatology and Immunology, University of Erlangen-Nuremberg, Erlangen, Germany Search for more papers by this author Axel Behrens Axel Behrens Lincoln's Inn Fields Laboratories, London Research Institute, Cancer Research UK, London, UK Search for more papers by this author Andras Nagy Andras Nagy Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada Search for more papers by this author Geert Carmeliet Geert Carmeliet Laboratory of Experimental Medicine and Endocrinology, Department of Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Jody J Haigh Corresponding Author Jody J Haigh Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Christa Maes Christa Maes Laboratory of Experimental Medicine and Endocrinology, Department of Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Steven Goossens Steven Goossens Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Sonia Bartunkova Sonia Bartunkova Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Benjamin Drogat Benjamin Drogat Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Lieve Coenegrachts Lieve Coenegrachts Laboratory of Experimental Medicine and Endocrinology, Department of Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Ingrid Stockmans Ingrid Stockmans Laboratory of Experimental Medicine and Endocrinology, Department of Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Karen Moermans Karen Moermans Laboratory of Experimental Medicine and Endocrinology, Department of Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Omar Nyabi Omar Nyabi Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Katharina Haigh Katharina Haigh Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Michael Naessens Michael Naessens Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Lieven Haenebalcke Lieven Haenebalcke Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Jan P Tuckermann Jan P Tuckermann Division of Tissue-Specific Hormone Action, Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Marc Tjwa Marc Tjwa Vesalius Research Center, KU Leuven, Leuven, Belgium Vesalius Research Center, VIB, Leuven, Belgium Search for more papers by this author Peter Carmeliet Peter Carmeliet Vesalius Research Center, KU Leuven, Leuven, Belgium Vesalius Research Center, VIB, Leuven, Belgium Search for more papers by this author Vice Mandic Vice Mandic Department of Internal Medicine 3, Rheumatology and Immunology, University of Erlangen-Nuremberg, Erlangen, Germany Search for more papers by this author Jean-Pierre David Jean-Pierre David Department of Internal Medicine 3, Rheumatology and Immunology, University of Erlangen-Nuremberg, Erlangen, Germany Search for more papers by this author Axel Behrens Axel Behrens Lincoln's Inn Fields Laboratories, London Research Institute, Cancer Research UK, London, UK Search for more papers by this author Andras Nagy Andras Nagy Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada Search for more papers by this author Geert Carmeliet Geert Carmeliet Laboratory of Experimental Medicine and Endocrinology, Department of Experimental Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Jody J Haigh Corresponding Author Jody J Haigh Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium Search for more papers by this author Author Information Christa Maes1,‡, Steven Goossens2,3,‡, Sonia Bartunkova2,3,‡, Benjamin Drogat2,3, Lieve Coenegrachts1, Ingrid Stockmans1, Karen Moermans1, Omar Nyabi2,3, Katharina Haigh2,3, Michael Naessens2,3, Lieven Haenebalcke2,3, Jan P Tuckermann4, Marc Tjwa5,6, Peter Carmeliet5,6, Vice Mandic7, Jean-Pierre David7, Axel Behrens8, Andras Nagy9,10, Geert Carmeliet1,‡ and Jody J Haigh 2,3,‡ 1Laboratory of Experimental Medicine and Endocrinology, Department of Experimental Medicine, KU Leuven, Leuven, Belgium 2Vascular Cell Biology Unit, Department of Molecular Biology, Ghent University, Ghent, Belgium 3Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium 4Division of Tissue-Specific Hormone Action, Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany 5Vesalius Research Center, KU Leuven, Leuven, Belgium 6Vesalius Research Center, VIB, Leuven, Belgium 7Department of Internal Medicine 3, Rheumatology and Immunology, University of Erlangen-Nuremberg, Erlangen, Germany 8Lincoln's Inn Fields Laboratories, London Research Institute, Cancer Research UK, London, UK 9Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada 10Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada ‡These authors contributed equally to this work *Corresponding author. Vascular Cell Biology Unit, VIB Department for Molecular Biomedical Research, UGent, Technologiepark 927, Ghent, Zwijnaarde 9052, Belgium. Tel.: +32 9 33 13 730; Fax: +32 9 33 13 609; E-mail: [email protected] The EMBO Journal (2010)29:424-441https://doi.org/10.1038/emboj.2009.361 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Vascular endothelial growth factor (VEGF) and β-catenin both act broadly in embryogenesis and adulthood, including in the skeletal and vascular systems. Increased or deregulated activity of these molecules has been linked to cancer and bone-related pathologies. By using novel mouse models to locally increase VEGF levels in the skeleton, we found that embryonic VEGF over-expression in osteo-chondroprogenitors and their progeny largely pheno-copied constitutive β-catenin activation. Adult induction of VEGF in these cell populations dramatically increased bone mass, associated with aberrant vascularization, bone marrow fibrosis and haematological anomalies. Genetic and pharmacological interventions showed that VEGF increased bone mass through a VEGF receptor 2- and phosphatidyl inositol 3-kinase-mediated pathway inducing β-catenin transcriptional activity in endothelial and osteoblastic cells, likely through modulation of glycogen synthase kinase 3-β phosphorylation. These insights into the actions of VEGF in the bone and marrow environment underscore its power as pleiotropic bone anabolic agent but also warn for caution in its therapeutic use. Moreover, the finding that VEGF can modulate β-catenin activity may have widespread physiological and clinical ramifications. Introduction Vascular endothelial growth factor (VEGF) is a positive regulator of bone development (Haigh et al, 2000; Maes et al, 2002, 2004; Zelzer et al, 2002), skeletal growth (Gerber et al, 1999) and fracture repair (Jacobsen et al, 2008). VEGF may couple angiogenesis to osteogenesis both indirectly through its effects on endothelial cells and directly by modulating chondrocytes, osteoblasts and osteoclasts that all express VEGF receptors (Dai and Rabie, 2007; Maes and Carmeliet, 2008). Accordingly, VEGF is currently tested in preclinical models as a potential therapy for stimulating fracture healing (Carano and Filvaroff, 2003). On the other hand, emerging data suggest that a disturbance in the interplay between angiogenesis and osteogenesis might be causal and/or consequential to progression of many bone and haematological pathologies (Calvi et al, 2003; Visnjic et al, 2004; Walkley et al, 2007; Larsson et al, 2008). Future therapeutic use of VEGF in bone pathology therefore warrants in-depth analyses of both the therapeutic merits as well as the potential side effects. A prime goal is to understand better the mechanisms of action of VEGF in the bone environment, including its cellular targets and downstream effectors. A crucial regulator of bone formation is β-catenin, the main effector of canonical signalling by Wnts. In the absence of Wnts, cytoplasmic β-catenin is constitutively degraded through its phosphorylation by glycogen synthase kinase 3-β (GSK3-β) that targets it to the ubiquitin-proteasome pathway. On signalling by Wnts, β-catenin is stabilized and translocates to the nucleus, where it interacts with T-cell factor (TCF)/lymphoid enhancer factor family transcription factors to regulate the expression of its target genes (Clevers, 2006; Grigoryan et al, 2008). In addition, other pathways affecting GSK3-β (e.g. phosphatidyl inositol 3 (PI3)-kinase pathways) can also modulate β-catenin transcriptional activity; Wnt and growth factor signalling can act through convergent pathways and possibly synergistically on GSK3-β and β-catenin (Jin et al, 2008). The importance of β-catenin in skeletal biology was proven recently by studies elucidating its role as a crucial transcription factor in (i) determining osteoblast lineage commitment of early osteo-chondroprogenitors (Day et al, 2005; Hill et al, 2005; Hu et al, 2005; Rodda and McMahon, 2006) and (ii) coupling osteoblast to osteoclast activity by stimulating osteoblastic production of osteoprotegerin (OPG or TNFRSF11B), an inhibitor of osteoclast formation (Glass et al, 2005). β-catenin signalling is also important in vascular biology. In quiescent endothelial cells of established vessels, β-catenin is concentrated at the plasmamembrane where it interacts with vascular endothelial (VE)-cadherin and mediates its linkage to the actin cytoskeleton (Dejana et al, 2008). VEGF promotes endothelial cell survival by stimulating the formation of a multi-protein transmembrane complex including VEGF receptor 2 (VEGFR-2, also known as Flk-1 or KDR), VE-cadherin and β-catenin, activating PI3-kinase/Akt (Dejana et al, 2008). In angiogenic cells, during embryogenesis, pathological angiogenesis or vascular remodelling, β-catenin may translocate to the nucleus and activate cell-cycle gene transcription (such as CyclinD1), contributing to endothelial cell proliferation. Pro-angiogenic effects of the GSK3-β/β-catenin pathway in endothelial cells have been described in vitro (Kim et al, 2002; Skurk et al, 2005) and very recently canonical Wnt signalling in endothelial cells was shown to be critical for vascularization of the developing central nervous system (Liebner et al, 2008; Stenman et al, 2008). Thus, similar to VEGF, β-catenin acts broadly in embryogenesis and adulthood. This suggests that molecular communication involving both molecules may possibly contribute to the coupled osteogenic and angiogenic responses that are systematically seen in bone biology and pathology. In this study, using two independent conditional and/or inducible approaches to over-express VEGF164 in embryonic development, during skeletal growth and in adult bone, we provide evidence for novel mechanistic links between the VEGF and β-catenin signalling pathways. Even short-term gain-of-function of VEGF in the bone microenvironment not only stimulated vascularization and ossification, but also induced dramatic pathological changes. This phenotype correlated with VEGF-induced activation of VEGFR-2 and a PI3-kinase/GSK3-β/β-catenin pathway in both endothelial and osteoblast lineage cells, mediating its downstream responses in the bone and marrow microenvironment. These data underscore the potential of VEGF as a bone anabolic agent but also warn for caution in all therapeutic uses of this powerful molecule, pointing out the bone microenvironment as a critical locus for monitoring potential side effects. Results Locally increased VEGF during skeletal development leads to deformed bones To determine the effects of local VEGF over-expression in the skeleton we targeted a conditional construct encoding the major isoform VEGF164 to the genomic ROSA26 locus (for details see Supplementary data). The expression of the VEGF164 transgene was prevented by a floxed upstream transcriptional stop cassette (Figure 1A). Only in cells expressing Cre, recombination led to the deletion of the stop cassette and activation of constitutive VEGF over-expression. Here, we used collagen type II (Col2)-Cre mice (Ovchinnikov et al, 2000) because Cre expression was reported to be activated early and broadly in the mesenchyme-derived cartilaginous condensations (including the perichondrium and postulated osteo-chondroprogenitors) that give rise to both the cartilage and the bone of the endochondral skeleton (Hilton et al, 2005; Rodda and McMahon, 2006; Ford-Hutchinson et al, 2007). As such, targeted cells and their descendants would encompass the physiological sites of VEGF production in endochondral bone (Maes et al, 2002; Zelzer et al, 2002). ROSA26R β-galactosidase (LacZ) reporter analysis (Soriano, 1999) indeed confirmed evidence of present or past Col2-Cre activity in chondrocyte and osteoblast lineage cells (Figure 1B). In agreement, compared with control littermates, mutant Col2-Cre+;ROSA26-VEGF164 (designated ‘+VEGF164’) embryos had two-fold increased VEGF mRNA levels (P<0.05; real-time quantitative RT–PCR (qRT–PCR) data not shown) and three- to five-fold higher VEGF protein levels in their tibias, both in the cartilaginous epiphyses and the bony diaphyses (Figure 1C). Figure 1.VEGF over-expression in the developing skeleton leads to aberrant ossification and deformed bones. (A) Strategy used to conditionally target VEGF164 to the ROSA26 locus. (B) ROSA26R LacZ reporter analysis visualizing Col2-Cre targeted cells at E17.5 (femur). Blue X-Gal staining shows present or past Col2-Cre activity in cartilage and bone. PC, proliferating chondrocytes; HC, hypertrophic chondrocytes; arrows, osteoblasts; double arrows, osteocytes; arrowheads, perichondrium/periosteum. (C) VEGF protein levels measured by ELISA in E16.5 whole tibias or dissected growth cartilages (epiphyses) and bone shafts (diaphyses). Bars represent mean±s.e.m.; **P<0.01; ***P<0.001. (D) Whole-mount alcian blue (cartilage) and alizarin red (bone) skeletal stains of E16.5 control and +VEGF164 embryos. (E) Histological analysis of E16.5 hindlimbs by Safranin O, Von Kossa and PECAM-1 staining. Download figure Download PowerPoint Mutant +VEGF164 mice died at birth by yet unknown causes and displayed marked deformities of the long bones. In particular, skeletal preparations of E16.5 embryos showed that the red stained ossified diaphyses of the limbs and ribs were abnormally short and thick, associated with kyphosis (bending) in the limbs (Figure 1D). The overall skeletal patterning and growth of the mutants were not impaired. Accordingly, histological analysis showed no striking disruption in the growth cartilage of +VEGF164 limbs other than a mild reduction of the hypertrophic chondrocyte zone (202 μm±5 in controls versus 158 μm±15 in +VEGF164 mice; P=0.047; n=3) (Supplementary Figure S1). In contrast, the primary ossification centre was drastically malformed (Figure 1E, top). As evidenced by Von Kossa staining for mineralized bone, +VEGF164 mutants had laterally expanded ossified centres filled with excessive, disorganized bone. In contrast to control mice, a proper cortex was lacking and instead aberrantly orientated trabecular-like structures extended from the widened periosteal area to the inside of the bone, obliterating the developing marrow cavity (Figure 1E, middle). In line with the strong angiogenic activity of VEGF, staining of vascularization by PECAM-1 immunohistochemistry (IHC) showed a vast increase in blood vessels throughout the mutant bones (Figure 1E, bottom). Thus, skeletal VEGF over-expression was characterized by aberrantly ossified, hypervascularized bones. A similar phenotype in the long bones was obtained using the more osteoprogenitor/osteoblast-directed gene promoters Runx2 (active in subsets of hypertrophic chondrocytes and osteoblasts (J Tuckermann, unpublished data); Supplementary Figure S2) or Osterix (Rodda and McMahon, 2006) (not shown) to drive Cre-mediated VEGF over-expression. Generation of mice with inducible, skeletal-specific transgenic VEGF expression As improved angiogenesis and osteogenesis is much sought after in postnatal conditions such as fracture healing and osteoporosis, we wanted to assess whether VEGF would exert bone anabolic effects later in life as well. Given the neonatal lethality associated with constitutive conditional VEGF gain-of-function, we designed a doxycycline (dox)-inducible transgenic strategy (Figure 2A). The reverse tetracycline transactivator (rtTA) was targeted to the conditional ROSA26-locus described above (as a bicistronic transcript also encoding EGFP), rendering the activation of its expression dependent on Cre activity (Belteki et al, 2005). The corresponding tetracycline-responsive element (tet(o)), induced by rtTA only in the presence of dox, regulated the expression of a VEGF164 transgene (Akeson et al, 2003). As described above, the crossing with Col2-Cre mice mediated the recombination of the conditional ROSA26-locus in embryonic osteo-chondroprogenitor cells and all progeny thereof. Consequently, the rtTA:EGFP transgene became constitutively expressed in the endochondral bones. Widespread expression of EGFP throughout the adult growth plate and metaphyseal and cortical bone areas was accordingly documented by IHC (Supplementary Figure S3A). The presence of dox would enable the produced rtTA to bind to tet(o) and switch on transgenic VEGF164 expression. Thus, VEGF over-expression was controlled both spatially (by tissue-specific Cre expression) and temporally (by dox administration). Administration of dox to triple transgenic mice (Col2-Cre+; ROSA26-Flox/Stop-rtTA-IRES-EGFP; tet(o)-VEGF164), further designated ‘+VEGF164dox mice’, through the drinking water was indeed effective at inducing abundantly increased VEGF expression throughout the bone, both in chondrocytes and in osteoblast lineage cells, as detected by IHC and in situ hybridization (ISH) (Supplementary Figure S3B and C). While leaky transgene expression was not observed in the absence of dox (Supplementary Figure S3D), 2 weeks of dox supplementation, as applied throughout these studies, caused a 10-fold increase in VEGF mRNA levels in long bones of adult +VEGF164dox mice as compared with dox-treated control littermates, associated with a more moderate two- to three-fold induction at the protein level (Supplementary Figure S3E and F). Of note, the serum VEGF levels were not significantly altered in +VEGF164dox mice (Supplementary Figure S3G). Figure 2.VEGF-induction in adult bone causes excessive trabecular bone, cortical porosity, hypervascularization and bone marrow fibrosis. (A) Breeding scheme to generate +VEGF164dox mice expressing the inducible transcriptional activator rtTA and EGFP constitutively in chondrocyte and osteoblast cell lineages. Only on dox administration VEGF over-expression is induced in these cells through the activation of tet(o) by rtTA. Control mice were littermates lacking the tet(o)-VEGF164 or the Col2-Cre transgene, receiving dox. (B) H&E stained tibias showing excessive trabecular bone in the metaphysis (boxed areas magnified in middle) surrounded by abundant stromal cells (magnification in bottom panels) in +VEGF164dox mice after 2 weeks on dox. (C, D) Von Kossa staining of control and +VEGF164dox tibias (C) and histomorphometric analysis (D) performed in three fixed areas (red boxes). Arrows indicate cortical remodelling. BV/TV, bone volume relative to total volume. (E) PECAM-1 immunohistochemistry (IHC) visualizing vascularization. gp, growth plate. (F) Reticulin staining revealing massive fibrosis in +VEGF164dox bones. Boxes, magnified on the right. Download figure Download PowerPoint Short-term increases of VEGF levels disrupt the architecture of juvenile and adult bone Induction of VEGF over-expression in the endochondral skeleton of juvenile mice, by supplying dox to the nursing mother during the first 2 weeks of life, profoundly affected the shape and morphology of the growing long bones. The aberrant bone was associated with abundant stromal cells and blood vessels surrounding the numerous trabeculae (Supplementary Figure S4). In adult mice with normally developed bones, dox supplementation for 14 days also completely disrupted the bone architecture, as seen in +VEGF164dox mice at the age of 3–4 months (Figure 2B). The induced VEGF over-expression characteristically led to excessive, aberrant bone structures and abundant peritrabecular mesenchymal stromal cells in the metaphyseal and epiphyseal regions (primary and secondary centres of ossification) (Figure 2B). Von Kossa staining and histomorphometry indeed showed a dramatically increased metaphyseal trabecular bone mass in +VEGF164dox mice (Figure 2C and D), as did pQCT analysis (70% increased trabecular density, n=12, P<0.05). The adjacent lamellar cortical bone, however, was replaced by trabecular-like porous bone structures (red arrows in Figure 2C). This manifest porosity of the cortex was characterized by abundant intercalating mesenchymal tissue components and osteoclast-rich remodelling units (Supplementary Figure S5A and B). As during development, the morphology of the cartilage was not manifestly disrupted by VEGF over-expression in the adult, but a mild decrease in the growth plate thickness was documented (95 μm±5 in controls versus 79 μm±5 in +VEGF164dox mice; P<0.05; n=7). As well, the growth plate showed increased mineralization as quantified on Von Kossa stained sections (percent calcification: 14.3%±0.8 in controls versus 18.8%±1.7 in +VEGF164dox mice; P<0.05; n=5). As anticipated, the increased VEGF levels again caused hypervascularization of the bones. PECAM-1 IHC visualized the strongly increased microvascular density throughout the altered metaphyseal (Figure 2E) and cortical (Supplementary Figure S5C) bone areas in +VEGF164dox mice. Adjacent to the growth plate, haemangioma-like blood vessels were often observed, indicative of localized supra-physiological VEGF levels (Ozawa et al, 2004) (Figure 2E) (also see below). These data indicate that even short-term induction of transgenic VEGF expression in adult bone dramatically stimulated osteogenesis and angiogenesis. VEGF over-expression in the bone micro-environment causes vascular anomalies, bone marrow fibrosis and haematological alterations In addition to the increased bone mass and hypervascularization caused by increased VEGF at all stages examined in this study, adult +VEGF164dox mice displayed several marked pathological features. Reticulin staining showed increased amounts of collagen IV-positive fibres in the regions of increased osteogenesis, indicative of massive fibrosis (Figure 2F). Furthermore, vascularity in the bone was consistently increased but in severely pathological bones several bone marrow blood vessels exhibited additionally a haemangioma-like morphology filled with erythrocytes (Figure 3A). Notably, these pathological manifestations locally replaced the haematopoietic marrow and consequently, the areas heavily occupied by new bone, marrow fibrosis and aberrant vessels displayed a drastic paucity of myeloid cells (Figure 3A). Moreover, increased spleen size was observed in 25% of the +VEGF164dox mice (n=11/43), suggesting extramedullary haematopoiesis (Figure 3B). As well, we documented evidence of extramedullary haematopoiesis in the liver in +VEGF164dox mice with haematopoietic colonies developing near some central vein regions (data not shown). These observations led us to assess whether +VEGF164dox mice suffered haematological alterations. The bone marrow of +VEGF164dox mice showed excessive PECAM-1+ megakaryocytes displaying abundant extended cellular processes, characteristic of megakaryocyte activation (Figure 3C). The number of megakaryocytes and progenitors in the spleen was also dramatically increased in the mutant mice, as shown by morphological detection, colony forming units (CFU)-MK analysis, and FACS analysis using the megakaryocyte lineage antigen CD41 (Figure 3D). In addition, we detected a three-fold increase in the numbers of haematopoietic progenitor cells (HPCs) in the peripheral blood of mutant mice by analysing the CFU-C, as well as a significant increase of CFU-Cs in the spleen (Figure 3E). No significant changes in CFU-C were documented in flushed bone marrow cultures (Figure 3E). In addition, temporal haematocrit profiling of the peripheral blood did not document significant changes in the number of circulating red or white blood cells but a small significant drop in platelet numbers at the end of the 2 weeks of induction (Supplemen
Referência(s)