A mechanistic evaluation of the angiogenic properties of a dehydrated amnion chorion membrane in vitro and in vivo

Angiogenesis is essential for the successful repair of tissues; however, in many chronic conditions, angiogenesis is inhibited. Placental tissues have been shown to illicit an angiogenic response both in vitro and in vivo, and the angiogenic properties of these tissues likely contribute to observed clinical outcomes. Although there is some work describing the angiogenic effects of these tissues, comparatively little has been done to determine the possible mechanisms responsible for this effect. The purpose of this study was to conduct a thorough evaluation of a commercially available dehydrated amnion chorion membrane to better understand how these tissues may promote angiogenesis. The proteomic content of this tissue was evaluated using a high throughput proteomic microarray, and then the effects of these grafts were evaluated in vivo using subcutaneous gelfoam sponge implants containing conditioned media (CM) from the graft. Human microvascular endothelial cells were then used to determine how released factors effect migration, proliferation, gene expression, and protein production in vitro. Finally, to elucidate potential signaling‐pathways through which tissue‐derived factors act to induce pro‐angiogenetic phenotypes in endothelial cells in vitro, we performed a global analysis of both serine/threonine and tyrosine kinase activity. Kinomic and proteomic data were then combined to generate protein–protein interaction networks that enabled the identification of multiple growth factors and cytokines with both pro‐ and anti‐angiogenetic properties. In vivo, the addition of CM resulted in increased CD31 and αSMA staining and increases in pro‐angiogenic gene expression. In vitro, CM resulted in significant increases in endothelial proliferation, migration, and the expression of granulocyte‐macrophage colony‐stimulating factor, hepatocyte growth factor, and transforming growth factor beta‐3. Integrated kinomic analysis implicated ERK1/2 signaling as the primary pathway activated following culture of endothelial cells with dehydrated amnion/chorion membrane (dACM) CM. In conclusion, dACM grafts triggered pro‐angiogenic responses both in vitro and in vivo that are likely at least partially mediated by ERK1/2 signaling.