Soluble VEGF isoforms are essential for establishingepiphyseal vascularization and regulating chondrocyte development and survival
Christa Maes, … , Roger Bouillon, Geert Carmeliet
Christa Maes, … , Roger Bouillon, Geert Carmeliet
Published January 15, 2004
Citation Information: J Clin Invest. 2004;113(2):188-199. https://doi.org/10.1172/JCI19383.
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Article Bone biology

Soluble VEGF isoforms are essential for establishingepiphyseal vascularization and regulating chondrocyte development and survival

Abstract

VEGF is crucial for metaphyseal bone vascularization. In contrast, the angiogenic factors required for vascularization of epiphyseal cartilage are unknown, although this represents a developmentally and clinically important aspect of bone growth. The VEGF gene is alternatively transcribed into VEGF120, VEGF164, and VEGF188 isoforms that differ in matrix association and receptor binding. Their role in bone development was studied in mice expressing single isoforms. Here we report that expression of only VEGF164 or only VEGF188 (in VEGF188/188 mice) was sufficient for metaphyseal development. VEGF188/188 mice, however, showed dwarfism, disrupted development of growth plates and secondary ossification centers, and knee joint dysplasia. This phenotype was at least partly due to impaired vascularization surrounding the epiphysis, resulting in ectopically increased hypoxia and massive chondrocyte apoptosis in the interior of the epiphyseal cartilage. In addition to the vascular defect, we provide in vitro evidence that the VEGF188 isoform alone is also insufficient to regulate chondrocyte proliferation and survival responses to hypoxia. Consistent herewith, chondrocytes in or close to the hypoxic zone in VEGF188/188 mice showed increased proliferation and decreased differentiation. These findings indicate that the insoluble VEGF188 isoform is insufficient for establishing epiphyseal vascularization and regulating cartilage development during endochondral bone formation.

Authors

Christa Maes, Ingrid Stockmans, Karen Moermans, Riet Van Looveren, Nico Smets, Peter Carmeliet, Roger Bouillon, Geert Carmeliet

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Central epiphyseal cartilage defects in VEGF188/188 mice. (a–c) Central ...
Central epiphyseal cartilage defects in VEGF188/188 mice. (a–c) Central sections through WT and VEGF188/188 (188/188) tibia/femur at P1.5. (a) Collagen II immunostaining shows its accumulation centrally in VEGF188/188 cartilage. (b) TUNEL analysis detecting centrally localized apoptotic cells only in VEGF188/188 epiphyses. (c) In situ hybridization fails to detect collagen II and collagen X mRNA in the center of mutant bones. (d and e) Hypoxia imaging by EF5 binding. (d) E18.5 distal femurs, showing strongly increased and ectopic hypoxia in mutant cartilage. (e) E16.5 knee joint and adjacent epiphyses (top panel) and magnification of the boxed area (lower panel). Note highly increased EF5 staining in the mutant bones, especially in resting/periarticular cartilage (arrowhead) and extending into the joint (arrow). (f) TUNEL staining detects an apoptotic cell cluster in the region of increased articular EF5 staining (arrow) in the mutants, whereas the epiphyseal cartilage itself is viable (arrowhead). (g and h) Angiography. (g) Lateral and anterior views of distal femur (skeletal staining or angiography), localizing the area of investigation and quantification (boxed area). (h) Low (left) and high (right) magnification of angiographies, showing the epiphyseal vessel network. Vessels appear thinner and more randomly orientated along the condylar rim in VEGF188/188 mice compared with WT, and vascular density on the medial and lateral condyles is reduced. (i) (n = 3; *P < 0.05; **P < 0.01). Scale bars: (a–c) 200 μm; (d–f) 100 μm; (h) 50 μm. Kj, knee joint; rc, resting/periarticular cartilage; pc, proliferating/columnar chondrocytes; hc, hypertrophic cartilage; pe, perichondrium; tb, trabecular bone; fe, femur; ti, tibia; fi, fibula; pa, patella; co, condyle; pf, patellar face.