Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

MMP-9/Gelatinase B Is a Key Regulator of Growth Plate Angiogenesis and Apoptosis of Hypertrophic Chondrocytes

Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules

Angiogenesis and bone repair.

According to current dogma, chondrocytes and osteoblasts are considered independent lineages derived from a common osteochondroprogenitor. In endochondral bone formation, chondrocytes undergo a series of differentiation steps to form the growth plate, and it generally is accepted that death is the ultimate fate of terminally differentiated hypertrophic chondrocytes (HCs). Osteoblasts, accompanying vascular invasion, lay down endochondral bone to replace cartilage. However, whether an HC can become an osteoblast and contribute to the full osteogenic lineage has been the subject of a century-long debate. Here we use a cell-specific tamoxifen-inducible genetic recombination approach to track the fate of murine HCs and show that they can survive the cartilage-to-bone transition and become osteogenic cells in fetal and postnatal endochondral bones and persist into adulthood. This discovery of a chondrocyte-to-osteoblast lineage continuum revises concepts of the ontogeny of osteoblasts, with implications for the control of bone homeostasis and the interpretation of the underlying pathological bases of bone disorders.

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Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation

During endochondral bone formation, avascular cartilage differentiates to hypertrophic cartilage that then undergoes erosion and vascularization leading to bone deposition. Resting cartilage produces inhibitors of angiogenesis, shifting to production of angiogenic stimulators in hypertrophic cartilage. A major protein synthesized by hypertrophic cartilage both in vivo and in vitro is transferrin. Here we show that transferrin is a major angiogenic molecule released by hypertrophic cartilage. Endothelial cell migration and invasion is stimulated by transferrins from a number of different sources, including hypertrophic cartilage. Checkerboard analysis demonstrates that transferrin is a chemotactic and chemokinetic molecule. Chondrocyte-conditioned media show similar properties. Polyclonal anti-transferrin antibodies completely block endothelial cell migration and invasion induced by purified transferrin and inhibit the activity produced by hypertrophic chondrocytes by 50-70% as compared with controls. Function-blocking mAbs directed against the transferrin receptor similarly reduce the endothelial migratory response. Chondrocytes differentiating in the presence of serum produce transferrin, whereas those that differentiate in the absence of serum do not. Conditioned media from differentiated chondrocytes not producing transferrin have only 30% of the endothelial cell migratory activity of parallel cultures that synthesize transferrin. The angiogenic activity of transferrins was confirmed by in vivo assays on chicken egg chorioallantoic membrane, showing promotion of neovascularization by transferrins purified from different sources including conditioned culture medium. Based on the above results, we suggest that transferrin is a major angiogenic molecule produced by hypertrophic chondrocytes during endochondral bone formation.

/pdf/transferrin-promotes-endothelial-cell-migration-and-invasion-2gfzt0gben.pdf

Transferrin promotes endothelial cell migration and invasion: implication in cartilage neovascularization.

Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation. J Cell Sci 113(Pt 1):59-69

The effects of parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) on late events in chondrocyte differentiation were investigated by a dual in vitro model where conditions of suspension versus adhesion culturing are permissive either for apoptosis or for the further differentiation of hypertrophic chondrocytes to osteoblast- like cells. Chick embryo hypertrophic chondrocytes maintained in suspension synthesized type II and type X collagen and organized their extracellular matrix, forming a tissue highly reminiscent of true cartilage, which eventually mineralized. The formation of mineralized cartilage was associated with the expression of alkaline phosphatase (ALP), arrest of cell growth, and apoptosis, as observed in growth plates in vivo. In this system, PTH/PTHrP was found to repress type X collagen synthesis, ALP expression, and cartilage matrix mineralization. Cell proliferation was resumed, whereas apoptosis was blocked. Hypertrophic chondrocytes cultured in adherent conditions in the presence of retinoic acid underwent further differentiation to osteoblast-like cells (i.e., they resumed cell proliferation, switched to type I collagen synthesis, and produced a mineralizing bone-like matrix). In this system, PTH addition to culture completely inhibited the expression of ALP and matrix mineralization, whereas cell proliferation and expression of type I collagen were not affected. These data indicate that PTH/PTHrP inhibit both the mineralization of a cartilage-like matrix and apoptosis (mimicked in the suspension culture) and the production of a mineralizing bone-like matrix, characterizing further differentiation of hypertrophic chondrocytes to osteoblasts like cells (mimicked in adhesion culture). Treatment of chondrocyte cultures with PTH/PTHrP reverts cultured cells in states of differentiation earlier than hypertrophic chondrocytes (suspension), or earlier than mineralizing osteoblast-like cells (adhesion). However, withdrawal of hormonal stimulation redirects cells toward their distinct, microenvironment-dependent, terminal differentiation and fate.

Parathyroid hormone [PTH(1-34)] and parathyroid hormone-related protein [PTHrP(1-34)] promote reversion of hypertrophic chondrocytes to a prehypertrophic proliferating phenotype and prevent terminal differentiation of osteoblast-like cells.

Silvia Cermelli

Papers

Transferrin promotes endothelial cell migration and invasion: implication in cartilage neovascularization.

Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation. J Cell Sci 113(Pt 1):59-69

Parathyroid hormone [PTH(1-34)] and parathyroid hormone-related protein [PTHrP(1-34)] promote reversion of hypertrophic chondrocytes to a prehypertrophic proliferating phenotype and prevent terminal differentiation of osteoblast-like cells.

Expression of NRL/NGAL (neu-related lipocalin/neutrophil gelatinase-associated lipocalin) during mammalian embryonic development and in inflammation

The developmentally regulated avian Ch21 lipocalin is an extracellular fatty acid-binding protein.