Vascular Endothelial Growth Factor-D Activates VEGFR-3 Expressed in Osteoblasts Inducing Their Differentiation

Abstract

Vascular endothelial growth factor (VEGF)-D is a member of the VEGF family of angiogenic growth factors that recognizes and activates the vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 on blood and/or lymphatic vessels. We show that in the long bones of newborn mice, VEGF-D and VEGFR-3 are expressed in the osteoblasts of the growing plate. The treatment of primary human osteoblasts with recombinant VEGF-D induces the expression of osteocalcin and the formation of mineralized nodules in a dose-dependent manner. A monoclonal neutralizing antibody, anti-VEGF-D, or silencing of VEGFR-3 by lentiviral-mediated expression of VEGFR-3 small hairpin RNA affects VEGF-D-dependent osteocalcin expression and nodule formation. Moreover, in primary human osteoblasts, VEGF-D expression is under the control of VEGF, and inhibition of VEGF-D/VEGFR-3 signaling, by monoclonal antibodies or VEGFR-3 silencing, affects VEGF-dependent osteoblast differentiation. These experiments establish that VEGF-D/VEGFR-3 signaling plays a critical role in osteoblast maturation and suggest that VEGF-D is a downstream effector of VEGF in osteogenesis. Vascular endothelial growth factor (VEGF)-D is a member of the VEGF family of angiogenic growth factors that recognizes and activates the vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 on blood and/or lymphatic vessels. We show that in the long bones of newborn mice, VEGF-D and VEGFR-3 are expressed in the osteoblasts of the growing plate. The treatment of primary human osteoblasts with recombinant VEGF-D induces the expression of osteocalcin and the formation of mineralized nodules in a dose-dependent manner. A monoclonal neutralizing antibody, anti-VEGF-D, or silencing of VEGFR-3 by lentiviral-mediated expression of VEGFR-3 small hairpin RNA affects VEGF-D-dependent osteocalcin expression and nodule formation. Moreover, in primary human osteoblasts, VEGF-D expression is under the control of VEGF, and inhibition of VEGF-D/VEGFR-3 signaling, by monoclonal antibodies or VEGFR-3 silencing, affects VEGF-dependent osteoblast differentiation. These experiments establish that VEGF-D/VEGFR-3 signaling plays a critical role in osteoblast maturation and suggest that VEGF-D is a downstream effector of VEGF in osteogenesis. During endochondral bone formation, mesenchymal cells differentiate into chondrocytes, which secrete a cartilage template. Chondrocytes in the centers of the cartilage templates become hypertrophic and produce vascular endothelial growth factor (VEGF) 2The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; shRNA, small hairpin RNA; mAb, monoclonal antibody; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; RT, reverse transcription. 2The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; shRNA, small hairpin RNA; mAb, monoclonal antibody; PBS, phosphate-buffered saline; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; RT, reverse transcription. that stimulates vascular invasion of the cartilage template. Upon this process, the hypertrophic chondrocytes die through apoptosis and are replaced by osteoblasts brought in from the bone collar (1Karsenty G. Genes Dev. 1999; 13: 3037-3051Crossref PubMed Scopus (271) Google Scholar, 2Wagner E.F. Karsenty G. Curr. Opin. Genet. Dev. 2001; 11: 527-532Crossref PubMed Scopus (188) Google Scholar). The interplay between chondrocytes and osteoblasts at the growth plate determines the longitudinal growth of long bones. Osteoblasts are responsible for matrix deposition and bone mineralization. Early during their differentiation, osteoblasts express RUNX2 (also known as CBFA1), which is held inactive by Twist proteins (3Bialek P. Kern B. Yang X. Schrock M. Sosic D. Hong N. Wu H. Yu K. Ornitz D.M. Olson E.N. Justice M.J. Karsenty G. Dev. Cell. 2004; 6: 423-435Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). Later, osteoblasts express the specific marker osteocalcin, which is required for bone mineralization (4Ducy P. Schinke T. Karsenty G. Science. 2000; 289: 1501-1504Crossref PubMed Scopus (887) Google Scholar). Several growth factors expressed in the growth plate including epidermal growth factor, members of fibroblast growth factor family, insulin growth factor-1, platelet growth factor, members of the transforming growth factor-β family, Indian hedgehog, and VEGF are involved in cell proliferation and differentiation during bone formation (1Karsenty G. Genes Dev. 1999; 13: 3037-3051Crossref PubMed Scopus (271) Google Scholar, 2Wagner E.F. Karsenty G. Curr. Opin. Genet. Dev. 2001; 11: 527-532Crossref PubMed Scopus (188) Google Scholar, 5Gerber H.P. Vu T.H. Ryan A.M. Kowalski J. Werb Z. Ferrara N. Nat. Med. 1999; 5: 623-628Crossref PubMed Scopus (1673) Google Scholar). Among these, VEGF has been shown to play a critical role. VEGF was first identified as an angiogenic factor that is expressed in different splicing forms. It binds and activates VEGFR-1 (also known as Flt-1) and VEGFR-2 (also known as KDR or Flk1) on endothelial cells, and their expression is required for vascular development and adult angiogenesis (6Ferrara N. Gerber H.P. LeCouter J. Nat. Med. 2003; 9: 669-676Crossref PubMed Scopus (7667) Google Scholar, 7Carmeliet P. Nat. Med. 2003; 9: 653-660Crossref PubMed Scopus (3394) Google Scholar). During skeletal development, VEGF has been shown to play a role in blood vessel invasion that is essential for coupling resorption of cartilage with mineralization of the extracellular matrix and bone formation (8Gerber H.P. Ferrara N. Trends Cardiovasc. Med. 2000; 10: 223-228Crossref PubMed Scopus (311) Google Scholar, 9Carano R.A.D. Filvaroff E.H. Drug Discov. Today. 2003; 8: 980-989Crossref PubMed Scopus (593) Google Scholar, 10Aoyama J. Tanaka E. Miyauchi M. Takata T. Hanaoka K. Hattori Y. Sasaki A. Watanabe M. Tanne K. Histochem. Cell Biol. 2004; 122: 35-40Crossref PubMed Scopus (29) Google Scholar) and synergizes with BMP2 to promote bone formation and bone healing via modulation of angiogenesis (11Peng H. Usas A. Olshanski A. Ho A.M. Gearhart B. Cooper G.M. Huard J. J. Bone Miner Res. 2005; 20: 2017-2027Crossref PubMed Scopus (336) Google Scholar). VEGF is essential not only for normal angiogenesis but also to allow normal differentiation of hypertrophic chondrocytes, osteoblasts, and osteoclasts (5Gerber H.P. Vu T.H. Ryan A.M. Kowalski J. Werb Z. Ferrara N. Nat. Med. 1999; 5: 623-628Crossref PubMed Scopus (1673) Google Scholar, 12Midy V. Plouet J. Biochem. Biophys. Res. Commun. 1994; 199: 380-386Crossref PubMed Scopus (269) Google Scholar, 13Maes C. Carmeliet P. Moermans K. Stockmans I. Smets N. Collen D. Bouillon R. Carmeliet G. Mech. Dev. 2002; 111: 61-73Crossref PubMed Scopus (353) Google Scholar, 14Mayr-Wohlfart U. Waltenberger J. Hausser H. Kessler S. Gunther K.-P. Dehio C. Puhl W. Brenner R.E. Bone. 2002; 30: 472-477Crossref PubMed Scopus (374) Google Scholar, 15Maes C. Stockmans I. Moermans K. Van Looveren R. Smets N. Carmeliet P. Bouillon R. Carmeliet G. J. Clin. Investig. 2004; 113: 188-199Crossref PubMed Scopus (222) Google Scholar, 16Zelzer E. McLean W. Ng Y.S. Fukai N. Reginato A.M. Lovejoy S. D'Amore P.A. Olsen B.R. Development (Camb.). 2002; 129: 1893-1904Crossref PubMed Google Scholar, 17Zelzer E. Glotzer D.J. Hartmann C. Thomas D. Fukai N. Soker S. Olsen B.R. Mech. Dev. 2001; 106: 97-106Crossref PubMed Scopus (293) Google Scholar, 18Aldridge S.E. Lennard T.W. Williams J.R. Birch M.A. Biochem. Biophys. Res. Commun. 2005; 335: 793-798Crossref PubMed Scopus (82) Google Scholar). VEGF plays a role in bone repair. In mouse femur fractures, treatment with exogenous VEGF enhanced not only blood vessel formation but also ossification and new bone maturation, whereas VEGF inhibition decreased bone formation and callus mineralization (19Street J. Bao M. deGuzman L. Bunting S. Peale Jr., F.V. Ferrara N. Steinmetz Hoeffel H. Cleland J. Daugherty J.L. van Bruggen A. Redmond N. Carano H.P. Filvaroff R. A.E.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9656-9661Crossref PubMed Scopus (1137) Google Scholar). Although experiments in osteoblasts demonstrated an increase of cell migration in response to VEGF (14Mayr-Wohlfart U. Waltenberger J. Hausser H. Kessler S. Gunther K.-P. Dehio C. Puhl W. Brenner R.E. Bone. 2002; 30: 472-477Crossref PubMed Scopus (374) Google Scholar), no functional data of the activity of VEGFRs in osteoblasts has been reported. During mouse development, the expression of another member of the VEGF family, VEGF-D, was detected in the periosteum/osteoblast layer of the developing vertebral column, the limb buds, and the dental mesenchyme close to the enamel epithelium (20Avantaggiato V. Acampora D. Orlandini M. Oliviero S. Simeone A. Mech. Dev. 1998; 73: 221-224Crossref PubMed Scopus (52) Google Scholar). Because in mouse VEGF-D only recognizes murine VEGFR-3 (21Baldwin M.E. Catimel B. Nice E.C. Roufail S. Hall N.E. Stenvers K.L. Karkkainen M.J. Alitalo K. Stacker S.A. Achen M.G. J. Biol. Chem. 2001; 276: 19166-19171Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), its pattern of expression suggests that VEGF-D/VEGFR-3 signaling plays a role in bone development. VEGFR-3 has been previously shown to be involved in vascular development, lymphatic maintenance, and tumor angiogenesis (22Dumont D.J. Jussila L. Taipale J. Lymboussaki A. Mustonen T. Pajusola K. Breitman M. Alitalo K. Science. 1998; 282: 946-949Crossref PubMed Scopus (683) Google Scholar, 23Veikkola T. Jussila L. Makinen T. Karpanen T. Jeltsch M. Petrova T.V. Kubo H. Thurston G. McDonald D.M. Achen M.G. Stacker S.A. Alitalo K. EMBO J. 2001; 20: 1223-1231Crossref PubMed Scopus (558) Google Scholar, 24Makinen T. Jussila L. Veikkola T. Karpanen T. Kettunen M.I. Pulkkanen K.J. Kauppinen R. Jackson D.G. Kubo H. Nishikawa S. Yla-Herttuala S. Alitalo K. Nat. Med. 2001; 7: 199-205Crossref PubMed Scopus (625) Google Scholar, 25Partanen T.A. Alitalo K. Miettinen M. Cancer. 1999; 86: 2406-2412Crossref PubMed Scopus (231) Google Scholar, 26Valtola R. Salven P. Heikkila P. Taipale J. Joensuu H. Rehn M. Pihlajaniemi T. Weich H. deWaal R. Alitalo K. Am. J. Pathol. 1999; 154: 1381-1390Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 27Kubo H. Fujiwara T. Jussila L. Hashi H. Ogawa M. Shimizu K. Awane M. Sakai Y. Takabayashi A. Alitalo K. Yamaoka Y. Nishikawa S.I. Blood. 2000; 96: 546-553Crossref PubMed Google Scholar, 28Achen M.G. Williams R.A. Baldwin M.E. Lai P. Roufail S. Alitalo K. Stacker S.A. Growth Factors. 2002; 20: 99-107Crossref PubMed Scopus (51) Google Scholar). We investigated the involvement of the angiogenic growth factor VEGF-D and its receptor VEGFR-3 in osteoblasts. We show that osteoblasts of the long bones of newborn mice and primary human osteoblasts express VEGFR-3. Osteoblasts treated with recombinant VEGF-D respond with VEGFR-3 autophosphorylation, osteocalcin expression, and nodule formation. Moreover, VEGF treatment induces VEGF-D expression in these cells. Accordingly, the inactivation of VEGF-D activity by neutralizing antibodies or VEGFR-3 silencing inhibited both VEGF- and VEGF-D-dependent nodule formation in osteoblasts. Our data demonstrate the involvement of VEGF-D in maturation and regulation of osteoblastic activity via VEGFR-3. Primary Human Osteoblast Cultures—Bone samples were obtained from 10 women and 4 men (aged 56–78 years, with a mean age of 66 years) who underwent total hip replacement surgery for degenerative joint disease. After extensive washes of trabecular bone explants, small bone chips were placed in flasks with Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 2 mm l-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. Cultures were incubated at 37 °C in a humidified, 5% CO2 atmosphere. For differentiation assays, osteoblasts were seeded in 6-well plates. Upon confluence, recombinant human VEGF-D (0–50 ng/ml) with or without the mouse monoclonal anti-human VEGF-D-neutralizing antibody (monoclonal antibody (mAb) 3.11A25) at a concentration of 5–36 μg/ml was added. Medium was changed every 2 days. The mineralized nodule formation was determined after 15 days by using Alizarin Red S staining as described previously (29Frediani B. Spreafico A. Capperucci C. Chellini F. Gambera D. Ferrata P. Baldi F. Falsetti P. Santucci A. Bocchi L. Marcolongo R. Bone. 2004; 35: 859-869Crossref PubMed Scopus (42) Google Scholar). Immunofluorescent Microscopy—For histological analysis, newborn Swiss mice were sacrificed, and limbs were dissected and embedded in Tissue-Tek® OCT™ compound. 10-μm cryostat sections were cut and fixed in 3% paraformaldehyde for 20 min at room temperature. Human osteoblasts were grown on glass coverslips and, after treatment, fixed in 3% paraformaldehyde for 15 min at room temperature. For permeabilization, cryostat sections were incubated with 0.5% Triton X-100 in PBS for 5 min at 4 °C. For the staining of human osteoblasts, cells were not were in PBS, with bovine serum in PBS for at room and incubated for at 37 °C with the primary L. S. L. E. A. M. F. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar), monoclonal and in bovine serum After were incubated for at with or antibodies and in were using a and of Human the human VEGF-D a the of the VEGF-D M. M. A. Orlandini M. B. E. A. Oliviero S. 1998; PubMed Scopus Google Scholar) was with a an and a reverse a The was into the of the expression The was by cells were grown for at after and in VEGF-D was by under in the of mm using an a VEGF-D the was 10 mm 5 mm 50 mm 5 mm 5 mm mm 2 in the as without 2 was for VEGF-D was a in and with mm was on a column, and the formation was by using a the were from used in and VEGF-D in in a new and human recombinant VEGF-D were using a D. Am. J. Med. PubMed Scopus Google Scholar). were by were and into mice for of The were determined using the mouse monoclonal antibody were by and by and cell proliferation Cell and endothelial cells were grown in medium fetal bovine serum and growth 5 cells were in a plate and for in Dulbecco's modified Eagle's medium fetal bovine serum and bovine serum were treated with VEGF-D in the of different of an and After 20 cell proliferation was as by a The for cell were by using to and osteoblast analysis, were in Dulbecco's modified Eagle's medium bovine serum for cells were incubated with mm for 10 min to were for min with VEGF-D at 37 with PBS mm and in of mm mm Triton mm mm 10 mm mm 10 mm mm Cell were incubated on for 10 min and at for 15 and the were incubated with For of VEGF family growth human and VEGF-D were of from cells were with 2 of in 50 mm mm 0.5% were by were as described previously M. S. Oliviero S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The primary antibodies were mouse monoclonal and and and The were incubated with and by using the enhanced VEGFR-3 RNA hairpin was and the recombinant were as described previously A. A. M. F. Oliviero S. Blood. 2004; PubMed Scopus Google Scholar). for human VEGFR-3 and were to a and by a and their reverse by as an RNA The were and into the cells were using to the The were and and through were by for 2 at was by cells with a protein and by of a with the protein RNA was from cells by the and was by The expression was using a and data were with the The were in using the RNA the For were and the expression was to the of on the The used are in of VEGF-D and VEGFR-3 in staining for murine VEGF-D on the expression of VEGF-D to osteoblasts to hypertrophic chondrocytes VEGF-D expression was with a role in bone formation, the expression of its receptor as in mouse VEGF-D only recognizes VEGFR-3 (21Baldwin M.E. Catimel B. Nice E.C. Roufail S. Hall N.E. Stenvers K.L. Karkkainen M.J. Alitalo K. Stacker S.A. Achen M.G. J. Biol. Chem. 2001; 276: 19166-19171Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). VEGFR-3 expression in the growth plate at the between the bone and the hypertrophic chondrocytes is a by new blood vessel formation and osteoblasts. VEGFR-3 is expressed in endothelial cells during angiogenesis (22Dumont D.J. Jussila L. Taipale J. Lymboussaki A. Mustonen T. Pajusola K. Breitman M. Alitalo K. Science. 1998; 282: 946-949Crossref PubMed Scopus (683) Google Scholar, 27Kubo H. Fujiwara T. Jussila L. Hashi H. Ogawa M. Shimizu K. Awane M. Sakai Y. Takabayashi A. Alitalo K. Yamaoka Y. Nishikawa S.I. Blood. 2000; 96: 546-553Crossref PubMed Google Scholar, van J. P. T.A. J. Pathol. 2001; PubMed Scopus Google Scholar). with of VEGFR-3 expression in endothelial cells in the of new mice, the that a of angiogenesis in the growing bone staining between VEGFR-3 and a marker of endothelial cells, a of these that VEGFR-3 is expressed in endothelial cells at this A VEGFR-3 was in osteocalcin cells These data demonstrate that VEGFR-3 is expressed in mouse osteoblasts. VEGF-D VEGFR-3 on Primary Human VEGF-D/VEGFR-3 signaling plays a functional role in osteoblasts, obtained from trabecular bone primary human osteoblasts and VEGF-D response on these cells. We detected VEGFR-3 expression in primary human osteoblasts by and by whereas not VEGFR-2 in these cells or VEGFR-3 in not VEGFR-3 autophosphorylation, human osteoblasts were treated with recombinant VEGF-D, and cell were with an antibody VEGFR-3 and with that VEGF-D treatment VEGFR-3 in osteoblasts VEGFR-3 was to an signaling as that VEGF-D treatment was by an increase of the of the protein These experiments demonstrate that VEGF-D recognizes and activates VEGFR-3 expressed in primary human osteoblasts. VEGF-D and VEGF-D affects osteoblast differentiation, primary human osteoblasts were grown in medium for days in the of different of shown in VEGF-D the of mineralized nodules in a dose-dependent In the nodules and mineralized of the control that and mineralized nodules the of VEGF-D activity affects osteoblast differentiation, used the to VEGF-D and to VEGF-D-dependent proliferation of endothelial cells The treatment of osteoblast with decreased nodule formation to of cells, whereas a antibody not nodule formation at the treatment of osteoblasts for days with of not osteoblast these data demonstrated that VEGF-D induces osteoblasts to mineralization of the expression of a marker of differentiation of and osteocalcin, a marker of osteoblast differentiation, that VEGF-D treatment not RUNX2 expression, whereas osteocalcin was in a dose-dependent Moreover, osteocalcin was inhibited by the antibody also that VEGF-D treatment osteocalcin with to cells that VEGF-D plays a role in osteoblast treatment stimulates osteocalcin a to osteoblast differentiation. of the osteocalcin and RUNX2 by RNA was from primary human osteoblasts treated for days with of of the osteocalcin by RNA was from osteoblasts treated for days with VEGF-D and different of the antibodies and human osteoblasts or treated for days with VEGF-D and by staining using and of the shown in and D. the S.E. of experiments in of VEGFR-3 VEGF-D-dependent osteoblast differentiation via VEGFR-3 signaling, and to VEGFR-3. Primary human osteoblasts with VEGFR-3 shRNA, but not an shRNA, a VEGFR-3 protein expression and VEGF-D-dependent Osteoblast for VEGFR-3 of nodule formation and osteocalcin VEGF-D treatment and VEGF plays a role in osteoblast via the of VEGFR-1 (14Mayr-Wohlfart U. Waltenberger J. Hausser H. Kessler S. Gunther K.-P. Dehio C. Puhl W. Brenner R.E. Bone. 2002; 30: 472-477Crossref PubMed Scopus (374) Google Scholar), to a functional between VEGF- and VEGF-D-dependent osteoblast differentiation, VEGF-D expression in osteoblasts on VEGF that VEGF treatment, osteoblasts with an expression of VEGF-D, that in osteoblasts, VEGF-D expression is under the control of VEGF VEGF-D plays a role in VEGF-dependent osteoblast differentiation, treated osteoblasts with VEGF in the of the monoclonal antibody VEGF-D activity and the nodule formation by VEGF treatment of primary human osteoblasts a of mineralization The of these with VEGF-D-neutralizing antibodies inhibited VEGF-dependent nodule formation inhibition the VEGFR-3 signaling as also inhibition of VEGF-dependent nodules formation in cells for VEGFR-3 and these demonstrate that VEGF induces nodule formation in osteoblasts via the of the VEGF-D/VEGFR-3 that VEGF-D/VEGFR-3 signaling induces primary human osteoblast differentiation. of long bones of newborn mice that VEGF-D is expressed with VEGFR-3 in the osteoblasts of the growth that VEGF-D/VEGFR-3 signaling to the bone formation in by an of osteoblasts during their Accordingly, that VEGFR-3 is expressed in primary human osteoblasts and VEGF-D stimulates their differentiation as osteocalcin and mineralized nodule formation. by VEGFR-3 silencing, that VEGFR-3 signaling plays a functional role in VEGF-D-dependent osteoblast Because in mouse VEGFR-3 is expressed in osteoblasts and VEGF-D binds only this receptor (21Baldwin M.E. Catimel B. Nice E.C. Roufail S. Hall N.E. Stenvers K.L. Karkkainen M.J. Alitalo K. Stacker S.A. Achen M.G. J. Biol. Chem. 2001; 276: 19166-19171Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), these suggest that VEGF-D/VEGFR-3 signaling in bone formation is a between mouse and VEGF-D induces as as of via lymphatic VEGFR-3 on vascular and lymphatic endothelial cells L. S. L. E. A. M. F. Oliviero S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar, M.G. Jeltsch M. E. Makinen T. A. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, S.A. C. Baldwin M.E. Williams R.A. R. Jackson D.G. Nishikawa S. Kubo H. Achen M.G. Nat. Med. 2001; 7: PubMed Scopus Google Scholar). a role in lymphatic vessel VEGFR-3 is in the of the primary vascular and in the of endothelial during angiogenesis (22Dumont D.J. Jussila L. Taipale J. Lymboussaki A. Mustonen T. Pajusola K. Breitman M. Alitalo K. Science. 1998; 282: 946-949Crossref PubMed Scopus (683) Google Scholar, 25Partanen T.A. Alitalo K. Miettinen M. Cancer. 1999; 86: 2406-2412Crossref PubMed Scopus (231) Google Scholar, 26Valtola R. Salven P. Heikkila P. Taipale J. Joensuu H. Rehn M. Pihlajaniemi T. Weich H. deWaal R. Alitalo K. Am. J. Pathol. 1999; 154: 1381-1390Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar, 27Kubo H. Fujiwara T. Jussila L. Hashi H. Ogawa M. Shimizu K. Awane M. Sakai Y. Takabayashi A. Alitalo K. Yamaoka Y. Nishikawa S.I. Blood. 2000; 96: 546-553Crossref PubMed Google Scholar). endothelial cells, VEGFR-3 signaling activates and A. F. M. F. Oliviero S. Blood. 2005; 106: PubMed Scopus Google Scholar). The data in this demonstrate in to a in endothelial cells, VEGF-D/VEGFR-3 signaling is also in osteoblast differentiation. on primary human osteoblasts demonstrated that VEGF-D induces the expression of osteocalcin, a marker of differentiation, whereas has no on the marker in these cells that the of Twist and Twist expressed during osteoblast differentiation (3Bialek P. Kern B. Yang X. Schrock M. Sosic D. Hong N. Wu H. Yu K. Ornitz D.M. Olson E.N. Justice M.J. Karsenty G. Dev. Cell. 2004; 6: 423-435Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar), are in osteoblasts VEGF-D These data suggest that VEGF-D/VEGFR-3 signaling is not involved in the of RUNX2 but be involved in the of of osteoblast differentiation that at a of osteoblast is also by the that VEGF-D mineralization in these cells. chondrocytes secrete a of growth factors including VEGF, which blood vessel formation, maturation, the differentiation of osteoblasts the mineralized bone as as the of osteoclasts into hypertrophic cartilage 16Zelzer E. McLean W. Ng Y.S. Fukai N. Reginato A.M. Lovejoy S. D'Amore P.A. Olsen B.R. Development (Camb.). 2002; 129: 1893-1904Crossref PubMed Google and In with these are with a in which VEGF on bone differentiation by vessel formation, osteoblast to the growth and also VEGF-D in osteoblasts. VEGF-D in as a downstream effector of VEGF with activity on osteoblasts. these factors to the of osteoblast differentiation. These also that VEGF, on stimulates a different response VEGF-D on VEGFR-3. A was previously by the of endothelial cells in which VEGFR-1 these cells to produce the growth factor, whereas VEGFR-2 their proliferation J. B. Gerber H.P. K.J. Ferrara N. Science. 2003; PubMed Scopus Google Scholar). of the downstream signaling from these the between these in osteoblasts. Our data suggest that VEGF-D plays a role in the development of long bones. During mouse development, VEGF-D expression was also detected in the layer of the developing vertebral and in the dental mesenchyme close to the enamel and enamel are (20Avantaggiato V. Acampora D. Orlandini M. Oliviero S. Simeone A. Mech. Dev. 1998; 73: 221-224Crossref PubMed Scopus (52) Google Scholar), that VEGF-D also play a role in bone formation. We for