Abstract 900: HoxB5 Induces Both Sprouting And Intussusceptive (non-sprouting) Angiogenesis In An Angiopoietin-2 (Ang2) Dependent Manner
Objective: HoxB5 belongs to the group of homeobox transcription factors and has been shown to be an important regulator of vasculogenesis by direct interaction with a flk1 enhancer element. Previous studies indicate that overexpression of HoxB5 results in enhanced formation of endothelial progenitor cells. Here we hypothesize that HoxB5 also plays a role in angiogenesis.
Methods: Endothelial cell sprouting was investigated in a human umbilical vein endothelial cell (HUVEC) spheroid assay. In vivo angiogenesis and intussusceptive capillary growth were examined in the chorioallantoic membrane (CAM) assay in chicken embryos four days after transduction of membranes. Specimens were stained for endothelial cells by sambucus nigra agglutinin and for vascular smooth muscle cells by alpha-smooth muscle actin antibody. HoxB5 and Angiopoietin-2 (Ang2) were overexpressed using adenovirus constructs. Ang2 promoter regulation was investigated in bovine aortic endothelial cells (BAEC) utilizing a luciferase reporter gene assay.
Results: Overexpression of HoxB5 resulted in a vascular phenotype of the capillary plexus within the chorionic epithelial layer of the CAM. The regular dense capillary mesh structure was abolished and more mature blood vessels surrounded by vascular smooth muscle cells were observed instead. The number of vessel intersections decreased significantly in favor of enlarged intussusceptive pillars and a more arborized vessel architecture. The same blood vessel phenotype was observed when Ang2 was overexpressed in the CAM. This observation led us to investigate the association between HoxB5 and Ang2. In HUVEC spheroids, overexpression of Ang2 induced sprouting. This effect could also be achieved by overexpression of HoxB5 and was antagonized by the Ang2 antagonist soluble tie2, indicating that Ang2 may be a downstream mediator of HoxB5. Indeed, overexpression of HoxB5 in HUVECs resulted in increased expression of Ang2 at the RNA and protein level. Furthermore, HoxB5 transactivated the Ang2 promoter in BAEC. These data taken together suggest that HoxB5 induces non-sprouting (intussusceptive) and sprouting capillary growth as well as blood vessel maturation and that Ang2 is an important downstream mediator in this process.