Ex Vivo Study of the Angiogenic Effect of the Extracellular Matrix Protein Amelogenin

Paper in proceeding, 2008

Introduction: Angiogenesis is crucial for wound healing but is often impaired in chronic wounds. The process is dependent on the interaction of endothelial cells and the extracellular matrix (ECM), which is mediated by cell membrane integrins. Amelogenin is an extracellular matrix protein that has been reported to promote formation of granulation tissue and repair of chronic venous leg ulcers and elevate the pro-angiogenic vascular endothelial growth factor in dermal fibroblasts.1-3 This study investigated the effect of amelogenin on angiogenesis in an ex vivo sprouting assay and related the findings to the cell surface integrin expression. Methods: Chick aortic arch assay: Transverse sections of the aortic arch of 13-day-old chick embryos were attached and sealed with Matrigel to the bottom of a 48-well plate. Amelogenin was added (0.01 mg/ml, 0.1 mg/ml and 1 mg/ml) in serum-free endothelial basal growth medium. Porcine serum albumin was used as control for unspecific protein effects. The plates were incubated at 37°C and sprouting was assessed at 24 h and 48 h by microscopy and scored from 0 to 6 (arbitrary units) by a blinded observer. Integrin assay: Human dermal microvascular endothelial cells (Promocell) were seeded in complete cell growth medium alone or supplemented with 0.1 mg/ml, 1 mg/ml amelogenin or 20 µg/ml fibronectin. After 24 h incubation in 37°C, cells were gently harvested with the non-enzymatic buffer (EDTA/PBS). Upregulated integrins/subunits were detected by an Integrin-Mediated Cell Adhesion Array (Chemicon), where cells expressing specific integrins (α1, α2, α3, α4, α5, αv, β1, β2, β3, β4, β6, αvβ3, αvβ5 and α5b1) are captured by surface immobilized antibodies. Results and Discussion: Amelogenin at 0.1 mg/ml significantly (p = 0.001) increased micro-vessel outgrowth by 76 % from the explants compared with control explants after 48 h of incubation. No significant sprouting was observed with the non-specific protein control porcine serum albumin or medium only. The preliminary data from the integrin assay show that amelogenin at 0.1 mg/ml also displays a broad up-regulation of several integrins/subunits. This result is comparable to the positive control, fibronectin, an ECM protein involved in all phases of tissue repair. Taken together, the present observations suggest that the angiogenic effects might be explained by the cell binding properties of amelogenin. Conclusions: Amelogenin stimulated micro-vessel outgrowth in the chick aortic arch assay possibly through up-regulation of several integrins and subunits important for cell interaction with the ECM. The pro-angiogenic property may contribute to the beneficial effects reported after treatment of chronic ulcers with the novel ECM therapy containing amelogenin. Acknowledgements: The studies were supported by the Swedish Research Council (grant K2006-73X-09495-16-3), Mölnlycke Health Care AB, the VINNOVA VinnVäxt Program Biomedical Development in Western Sweden, and the Danish Medical Research Council (22-02-0287). References: 1. Mirastschijski U. et al. (2004) Wound Repair Regen. 12:100-108. 2. Vowden P. et al. (2006) Wound Repair Regen. 14:240-248. 3. Ågren M. S. et al. (2007) Wound Repair Regen. 15:A139.