Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival

Abstract

Breakdown or absence of vascular oxygen delivery is a hallmark of many common human diseases, including cancer, myocardial infarction, and stroke. The chief mediator of hypoxic response in mammalian tissues is the transcription factor hypoxia-inducible factor 1 (HIF-1), and its oxygen-sensitive component HIF-1alpha. A key question surrounding HIF-1alpha and the hypoxic response is the role of this transcription factor in cells removed from a functional vascular bed; in this regard there is evidence indicating that it can act as either a survival factor or induce growth arrest and apoptosis. To study more closely how HIF-1alpha functions in hypoxia in vivo, we used tissue-specific targeting to delete HIF-1alpha in an avascular tissue: the cartilaginous growth plate of developing bone. We show here the first evidence that the developmental growth plate in mammals is hypoxic, and that this hypoxia occurs in its interior rather than at its periphery. As a result of this developmental hypoxia, cells that lack HIF-1alpha in the interior of the growth plate die. This is coupled to decreased expression of the CDK inhibitor p57, and increased levels of BrdU incorporation in HIF-1alpha null growth plates, indicating defects in HIF-1alpha-regulated growth arrest occurs in these animals. Furthermore, we find that VEGF expression in the growth plate is regulated through both HIF-1alpha-dependent and -independent mechanisms. In particular, we provide evidence that VEGF expression is up-regulated in a HIF-1alpha-independent manner in chondrocytes surrounding areas of cell death, and this in turn induces ectopic angiogenesis. Altogether, our findings have important implications for the role of hypoxic response and HIF-1alpha in development, and in cell survival in tissues challenged by interruption of vascular flow; they also illustrate the complexities of HIF-1alpha response in vivo, and they provide new insights into mechanisms of growth plate development.

Figure 1

Detection of hypoxia and HIF-1α protein in wild-type growth plate. (a,b) Brightfield and immunofluorescence detection of EF-5 in proximal epiphysis of wild-type E15.5 tibia. (c) Overlay of a and b. (d) Immunofluorescence detection of EF-5 in E15.5 tibia and surrounding muscular tissue. (e) HIF-1α protein detection in the proximal epiphysis of wild-type E15.5 fetal tibia by immunoperoxidase analysis; the arrows indicate positively stained nuclei; for purposes of orientation, the border of the growth plate is drawn.

Figure 2

Generation of mutant animals lacking HIF-1α in growth plate chondrocytes. (a) Schematic representation of the colIICre transgene. (b,c) Whole-mount β-galactosidase staining of E15.5 embryo. (d) in situ hybridization analysis with antisense Cre riboprobe on histological sections of wild-type hindlimb from E15.5 embryo. (e) Picture of newborn wild-type ( left) and null mouse littermates (right). (f–h) Whole skeleton Alizarin Red S staining; newborn wild-type (left) and mutant (right) forelimbs (left) and hindlimbs (right) are shown in f; newborn wild-type rib cage is shown in g; newborn null rib cage is shown in h.

Figure 3

Histological analysis of long bone growth plates and trachea in wild-type and null animals. (a,b) H&E staining of wild-type and mutant newborn tibias; portions of the distal epiphysis of the femur and of fibula and calcaneus are also shown. (c,d) H&E staining of distal epiphysis of newborn wild-type and null tibias (higher power of a and b, respectively). (e–i) H&E staining of wild-type and null tracheas. g and i are higher power of f; h is higher power of e.

Figure 4

Analysis of rib cage and intercostal blood vessels in wild-type and null animals. (a,b) H&E staining of wild-type and mutant newborn sterna and ribs at their chondrosternal junction. (c,d) H&E staining of newborn wild-type and mutant ribs (higher power of a and b, respectively). (e,f) Brightfield pictures of wild-type and mutant rib cages adjacent to the chondrosternal junction; the intercostal blood vessels that surround the ribs are clearly evident in both wild-type and mutant specimens. (g,h) Immunostaining for von Willebrand factor in the perichondrium surrounding wild-type and mutant ribs in a region adjacent to the chondrosternal junction; the arrows indicate positively stained capillaries.

Figure 5

Evidence of massive cell death and decreased expression of p57 mRNA in mutant growth plate chondrocytes. (a) in situ hybridization analysis with collagen type II cRNA on histological sections from newborn wild-type and mutant tibias and rib cages, respectively; the darkfields of the proximal epiphyses and of the rib cages for both wild-type and mutant specimens are shown. (b) In situ hybridization analysis with collagen type X cRNA on histological sections from E15.5 wild-type and null tibias and newborn rib cages, respectively; the darkfields of the proximal epiphyses and of the rib cages for both wild-type and mutant specimens are shown. (c) In situ hybridization analysis with p57 cRNA on histological sections from E18.5 wild-type and mutant tibias; the darkfields for both wild-type and mutant specimens are shown. (d) Fluorescence detection (FITC) of TUNEL positive cells in the proximal epiphyses of wild-type and mutant newborn tibias; in comparison to the wild-type specimen, numerous TUNEL-positive cells are clearly evident in the mutant.

Figure 6

Evidence for loss of PGK expression and increased proliferation rate in mutant growth plate chondrocytes. (a,b) In situ hybridization analysis with PGK cRNA on histological sections from E15.5 wild-type and mutant tibias and fibulas; the darkfields for both wild-type and mutant specimens are shown. (c,d) Detection of BrdU positive cells on histological sections from E15.5 wild-type and mutant tibias, respectively. Sections were counterstained with methyl green. (e) Quantification of BrdU positive cells outside of regions of necrosis, defined as cells more than one cell layer from clearly necrotic regions; percent positive is as a function of total cells counted in the selected region. A significant increase in incorporation in nullizygous chondrocytes is seen. Error bars represent one standard error; counts were done on 10 growth plate regions from the long bones of five independent embryos for each genotype. (Open bar) Wild type (WT); (red bar) Null.

Figure 7

Evidence for HIF-1α-dependent and -independent regulation of VEGF expression, increase of EF-5 binding, and ectopic angiogenesis in the mutant growth plate. (a,b) In situ hybridization analysis with VEGF cRNA on histological sections E15.5 from wild-type and mutant tibias and fibulas; the darkfields for both wild-type and mutant specimens are shown (exposure to photoemulsion = 14 d). (c,d) In situ hybridization analysis with VEGF cRNA on histological sections from E15.5 mutant femur; brightfield and darkfield are shown, respectively (exposure to photoemulsion = 24 d) . (e,f) Immunofluorescence detection of EF-5 in proximal epiphysis of E18.5 wild-type and mutant tibia, respectively. (g–j) H&E staining of histological sections of proximal epiphyses of mutant newborn tibias; square and circles are drawn around areas of ectopic angiogenesis; h is a higher magnification image of g.