QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zelzer, E. Articles by Olsen, B. R. Search for Related Content PubMed PubMed Citation Articles by Zelzer, E. Articles by Olsen, B. R. Social Bookmarking                         What's this?

Skeletal defects in VEGF^120/120 mice reveal multiple roles for VEGF in skeletogenesis

¹ Harvard Medical School, Department of Cell Biology, 240 Longwood Avenue, Boston, MA, USA
² Schepens Eye Research Institute, Department of Ophthalmology, Boston, MA

View larger version (50K): [in a new window]   Fig. 1. A comparison of the skeletons of wild-type and VEGF120/120 mice reveals reduced size of areas stained for Alizarin Red, suggesting a reduction in mineralization of mutant bones. Regions significantly affected include the long bones in both forelimbs and hind limbs (arrows), and calvarial bones (arrowheads).

View larger version (107K): [in a new window]   Fig. 2. Histological study of wild-type and VEGF120/120 mice identifies marked differences in skeletal elements during development. At E14.5, the hypertrophic zone is larger in wild-type (A) than in VEGF120/120 (B) tibia and fibula. At E15.5, blood vessel invasion into the hypertrophic cartilage can be observed in wild-type mice (C, arrow) but not in VEGF120/120 mice (D, arrow). (E-H) At E16.5, bone marrow and bone trabeculae are present in wild-type mice (E,G) but there is a significant delay at this stage in VEGF120/120 mice (F,H), with blood vessel invasion occurring only in the most central region of the diaphysis. t, tibia; r, radius; f, fibula; u, ulna. (I,J) At E17.5, the tibial VEGF120/120 growth plate (J) displays a much longer hypertrophic zone than the wild-type growth plate (I). (K,L) At E16.5, wild-type calvarial bones (K) appear thicker than those in VEGF120/120 mice (L) (b, brain; arrow indicates calvarial bone). In Fig. 2A,B,E-J the extent of the hypertrophic zone is indicated by a bracket.

View larger version (138K): [in a new window]   Fig. 3. Comparison of vascularization of wild-type and VEGF120/120 skeletal elements by CD31 immunostaining. At E14.5, blood vessels surround the tibia in wild-type mice (A, arrows) but remain far from the surface of the tibia and fibula in the VEGF120/120 mice (B; arrows indicate surfaces of tibia and tibula). At E15.5, blood vessels have fully penetrated into the primary ossification center of wild-type (C) tibia (arrow). At E15.5, vessels are only now surrounding the hypertrophic zone in tibia and fibula of VEGF120/120 mice (D, arrow), whereas they are invading the diaphysis at E16.5 (E). At E16.5, large numbers of blood vessels surround the developing calvarial bones in the wild-type (F) mice (arrow), but in the VEGF120/120 (G) mice there are fewer vessels (arrow), and they appear large in diameter (arrowheads).

View larger version (126K): [in a new window]   Fig. 4. VEGF-lacZ expression in developing limb and calvarium. At E13.5, there is robust expression of VEGF in the periochondrium and surrounding tissues (arrow) of the radius (r) and ulna (u) as observed in whole-mount (A) and histological sections (B). In the calvarium at E13.5, expression of VEGF can be seen (C, arrows; b, brain), and this expression is stronger at E14.5 in the region of mesenchyme that is destined to differentiate into osteoblasts (D, area of expression defined by bracket; b, brain). At E14.5, VEGF expression is associated with cells covering the surface of calvarial bone (E, arrows; c, cranial base cartilage) in regions where osteoid and mineral are being deposited.

View larger version (155K): [in a new window]   Fig. 5. Expression analysis of VEGF and its receptors, VEGFR1 (vR1), VEGFR2 (vR2) and neuropilin 2 (np2) in the developing tibia. No significant differences are observed in VEGF expression between wild-type (A) and VEGF120/120 (B) mice at E15.5 (arrowheads define the regions of VEGF expression). At E15.5, there is significantly higher level of expression of VEGFR1 and VEGFR2 in wild-type tibia (C,E, arrowheads) than in VEGF120/120 tibia (D,F, arrowheads). No significant differences are observed in neuropilin 2 expression between wild-type (G) and VEGF120/120 (H) mice at E15.5. At E16.5, there is a significantly stronger signal for VEGFR-1 and VEGFR-2 in wild-type tibia (I,K, arrowheads) than in VEGF120/120 tibia (J,L, arrowheads). At E16.5, there is a dramatic difference in the expression of neuropilin 2 in wild-type mice (M, arrows) when compared with VEGF120/120 mice (N, arrows).

View larger version (71K): [in a new window]   Fig. 6. Analysis of osteoclast markers demonstrates reduced numbers of osteoclasts in the perichondrium of VEGF120/120 mice. MMP9 expression in wild-type (A) radius and ulna (arrowheads) is much more extensive than in VEGF120/120 (B) radius (arrowheads). This is consistent with the results of TRAP staining. Numerous TRAP-positive cells are seen in the perichondrium of wild-type radius (C, arrowheads), in contrast to the almost complete absence of TRAP-positive cells in VEGF120/120 radius (D). Inserts show TRAP-positive cells at a higher magnification.

View larger version (136K): [in a new window]   Fig. 7. Expression of chondrocyte differentiation markers. At E14.5, Col2a1 expression is seen throughout the cartilage anlagen in both wild-type (A) and VEGF120/120 (B) mice. In the wild-type cartilage there is a region of lower Col2a1 expression in the center of the diaphysis, but this is not as apparent in VEGF120/120 mice. In both wild-type (C) and VEGF120/120 (D) mice at E14.5, Col10a1 expression is seen in the region of chondrocyte differentiation to hypertrophy. By E15.5, the differences observed between wild-type and VEGF120/120 mice in Col2a1 (E,F) and Col10a1 (G,H) expression are small.

View larger version (98K): [in a new window]   Fig. 8. Comparative analysis of mineralization and expression of osteoblastic markers in long bones and calvaria. At E16.5, there are dramatic differences in the mineralization of wild-type (A, Alizarin Red; C, von Kossa) and VEGF120/120 (B, Alizarin Red; D, von Kossa) tibias (A,B) and radii (C,D). Most of the mineralization that is observed in the tibia and radius of VEGF120/120 mice is not associated with osteoblastic activity, but is hypertrophic zone mineralization (hypertrophic zone identified by bold line, A and B). At E16.5, expression of Col1a1 in wild-type tibia (E) is much more extensive than in VEGF120/120 tibia (F). At E16.5, expression of osteocalcin in wild type (G) is much stronger than that in VEGF120/120 radius and ulna (H) (arrowheads identify regions of expression). At both E15.5 and E16.5, reduced mineralization is observed in VEGF120/120 mouse calvaria (J,L,N) when compared with wild-type calvaria (I,K,M). At E16.5, expression of Col1a1 is significantly stronger in the wild-type calvarium (O) than in the VEGF120/120 calvarium (P).

View larger version (71K): [in a new window]   Fig. 9. Mineral deposition in calvarial bone cell cultures. Incubation of primary calvarial cells under conditions that allow mineral deposition in the absence (cont; 20,000 pixels/well) and presence of 25 ng/ml VEGF164 (57,000 pixels/well) or VEGF120 (100,000 pixels/well) shows increased mineralization when recombinant VEGF is present.

View larger version (46K): [in a new window]   Fig. 10. Calvarial organ culture demonstrates stimulatory effect of VEGF on bone formation. Calvarial explants were cultured without or with VEGF164 or with Flt-fc. The thickness of the parietal bone was measured at three different points (P1, P2 and P3) determined by analysis of images taken of the whole calvaria prior to processing the sample (A). The thickness was measured as a distance between inner and outer surfaces of the bone based upon alkaline phosphatase staining of the osteoblasts (see high magnification insets) (B). VEGF164 treatment led to a significant increase in calvarial bone thickness (average thicknesses determined at points P1, P2 and P3) when compared with the control, while treatment with Flt-fc prevented all growth during the culture period (C).

View larger version (17K): [in a new window]   Fig. 11. Two-step model of cartilage vascularization. At E13.5, high levels of VEGF are expressed in the perichondrium and surrounding tissues; this stimulates vascular ingrowth into this area. At E14.5, VEGF expression is reduced in the perichondrial regions as expression in the hypertrophic region begins. At E15.5, high-level expression of VEGF in the hypertrophic zone leads to chondroclast/osteoclast recruitment and vascular invasion. At E17.5, VEGF expression is maintained in the hypertrophic zone after a marrow cavity is established.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter