Dehydrated Amnion Chorion Membranes Induce Broad-scale Changes In Kinase Activity Of Endothelial Cells
Miranda Burnette, John McQuilling, MaryRose Kammer, Kelly Kimmerling, Katie Mowry.
Organogenesis Inc, Birmingham, AL, USA.
Angiogenesis is critical for successful wound healing; low oxygen levels and decreased perfusion in chronic wounds increase risk of impaired healing and infection. While a variety of approaches have been used to promote healing through acute oxygenation of wound beds with hyperbaric oxygen or negative pressure therapy, promotion of endogenous vasculature by releasing proangiogenic growth factors to the wound environment remains a promising alternative. Placental membranes have been shown to promote wound repair responses in vitro through a variety of mechanisms including promotion of angiogenesis. In the current study, we have evaluated pro-angiogenic responses in endothelial cells. To identify the signaling-pathways through which dACM-derived factors induce these responses, we performed a global analysis of both serine/threonine and tyrosine kinase activity using a high-throughput commercial platform (PamGene). Human microvascular endothelial cells (HMVECs) were treated for 30 minutes with either assay media or dACM conditioned media (CM). CM was obtained by incubating dACM grafts in basal media for 3-5 days at 4°C at a ratio of 1 cm2 dACM to 1 mL media. Cell lysate was run on the “kinomics” platform to directly detect phosphorylation of ~290 peptide probes by active kinases in each sample. dACM-derived factors were found to drive gross changes in kinase activity, with ERK1, ERK2, TEX14, and PDK1 most significantly upregulated in dACM-treated cells. Pathways functionally enriched in kinases activated by dACM include MAPK signaling, FC-epsilon receptor signaling, and toll-like receptor (TLR) 5 signaling pathway. The activation of VEGFR-2 by pro-angiogenic VEGF-A has been shown to preferentially signal through the PLCj-PKC-MAPK pathway. While generally associated with pathogen recognition and innate immunity, TLR4 signaling has been implicated in angiogenesis through an inflammatory pathway. This work provides an unbiased identification of pathways important for induction of angiogenesis by dACM in vitro.
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