Abstract
Cellular therapy with mesenchymal stem cells (MSC) is emerging as a promising approach in wound healing. Understanding physiologic cues that modulate MSC migration and recruitment to wound sites is critical to maximizing their clinical use. Stromal cell-derived factor 1 (SDF-1), has been shown to promote MSC chemotactic migration and thus could potentially enhance wound healing. On the other hand, stress-induced epinephrine, whose systemic level can be up to 10-fold elevated in stressed patients, and is generated within the wound, has been shown to impair wound healing. The purpose of this study was to evaluate how stress-induced epinephrine impacts on MSC migratory response to SDF-1. MSC were treated with different concentrations of epinephrine, timolol (β1/2-adrenergic receptor blocker) and/or SDF-1. Individual cells’ images were captured using Volocity software package and manually tracked in OpenLab software and migration rates were analyzed. Immunoblotting and Immunocytochemistry were performed using standard protocols. MSC were seeded and incubated for 36, 60, and 84 hours and stained with Calcein acetoxymethylester (AM) fluorescent dye to label viable cells and incubated at 37oC for one hour to evaluate proliferation rates. It was our findings that SDF-1 and epinephrine synergistically enhanced MSC proliferation. SDF-1 significantly increased the migratory speed of MSC in a dose-dependent manner. Immunolocalization of actin and vinculin showed cytoskeleton reorganizations and increase in focal adhesions in MSC treated with SDF-1. We also found that MSC migratory speed was significantly reduced by treatment with epinephrine alone. This response was reversed when the β1/2AR antagonist timolol was co-incubated with the cells, demonstrating the βAR specificity of the response. Immunoblotting demonstrated a significant decrease in phosphorylation of the ERK signaling pathway in epinephrine-treated MSC, correlating to the decreased migration. Surprisingly, co-incubation of MSC with both SDF-1 and epinephrine resulted in an increase the migratory speed relative to incubation with SDF-1 alone. This finding is important because it suggests that stress-induced high catecholamine environment at the wound site does not alter the positive effects of SDF-1 on MSC migration. Overall, our study sheds new light on the potential interactions between the two signaling pathways in MSC and provides initial evidence as to how they may be manipulated to improve healing.