ORIGINAL ARTICLE Detection of Hypoxia Inducible Factors and Angiogenic Growth Factors during Foetal Endochondral and Intramembranous Ossification W. De Spiegelaere 1* , P. Cornillie 1 , C. Casteleyn 1 , C. Burvenich 2 and W. Van den Broeck 1 Addresses of authors: 1 Faculty of Veterinary Medicine, Department of Morphology, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium; 2 Faculty of Veterinary Medicine, Department of Comparative Physiology and Biometrics, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Introduction Ossification of the foetal vertebrate skeleton is character- ized by the replacement of pre-existing connective tissue by a calcified bone matrix. This matrix is formed through the extracellular mineralization of osteoid that was depos- ited by osteoblasts. Two different mechanisms of ossifica- tion can be distinguished, depending on the nature of the primary connective tissue. When the pre-existing connec- tive tissue consists of condensed mesenchymal cells, the process is called intramembranous ossification. It takes place in a range of developing bones of the skull, including the calvarial bones, the palatine, maxillary, incisival and mandibular bones. The second mechanism is called endo- chondral ossification. During this process, a preformed cartilaginous mould serves as the primary connective tis- sue. Endochondral ossification is the most common and best studied mechanism. It gives rise to the bones of the axial and appendicular skeleton (Chung et al., 2004). Angiogenesis plays an important role during ossification because the presence of a well organized vascular plexus is crucial for the supply of oxygen and nutrients to the newly forming tissue (Dai and Rabie, 2007). However, angiogen- esis has almost exclusively been studied during the process of endochondral ossification (Dai and Rabie, 2007), whereas it is poorly investigated during intramembranous ossification. Hypoxia inducible factors (HIFs) are important regula- tors of angiogenesis (Hanze et al., 2007). Under hypoxic conditions, these factors regulate the expression of a *Correspondence: Tel.: (+32) 92647713; fax: (+32) 92647790; e-mail: Ward.DeSpiegelaere@UGent.be With 6 figures and 1 table Received March 2010; accepted for publication April 2010 doi: 10.1111/j.1439-0264.2010.01005.x Summary During skeletogenesis, the development of a new vascular network, i.e. angio- genesis, is triggered by hypoxia through the activation of the hypoxia inducible factors (HIFs) HIF-1a and HIF-2a. HIFs regulate the expression of several genes, including those coding for angiogenic growth factors such as VEGFA, angiopoietin-1 (ANGPT1) and angiopoietin-2 (ANGPT2). The expression of HIFs and angiogenic growth factors is well documented in endochondral ossifi- cation, but few data exist on their expression during intramembranous ossifica- tion. In this study, the localization of these factors was examined using immunohistochemistry and RT-PCR in bones of porcine foetuses. Immuno- staining for HIF-1a and HIF-2a was observed during endochondral ossifica- tion, whereas only HIF-2a was present at sites of intramembranous ossification. Furthermore, immunostaining for ANGPT2 was present at sites of endochondral and intramembranous ossification. In addition, gene transcripts for ANGPT1, ANGPT2 and VEGFA were detected with RT-PCR in laser cap- ture microdissected isolates from both types of ossification. These results indi- cate that angiogenesis plays an important role during endochondral and intramembranous ossification. However, the different expression pattern of the HIF-a subunits suggests that alternative regulatory pathways trigger angiogene- sis in these distinct types of ossification. Anatomia Histologia Embryologia 376 ª 2010 Blackwell Verlag GmbH • Anat. Histol. Embryol. 39 (2010) 376–384