Adrenergic receptor modulation of MSC exosome cargo to improve wound healing

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Chronic wounds, defined as those that do not progress through the normal healing process and remain unhealed after one month of standard care, pose tremendous health problems- clinically and economically. There is growing interest in isolating ttherapeutic agents directly from mesenchymal stem cells: particularly the exosome enriched fractions that are now understood to carry the active immunomodulatory and regenerative factors. Their cargo of miRNAs, Wnt ligands, growth factors, cytokines and signaling lipids provide the mechanism for paracrine stimulation of wound resident cells. However, cargo contents of MEX are dependent on culture conditions. MSCs express a full complement of AR, including the β2-AR. Our prior work has demonstrated that β2-AR agonists increase inflammatory cytokine secretion MSCs and conversely, treating MSC with a β2-AR antagonist, timolol, substantially enhances their anti-inflammatory and wound reparative properties. Here we hypothesize that activation of MSC β2-AR by stress catecholamine agonists alters their exosomal cargo contents, to be more pro-inflammatory, and conversely, β2-AR antagonists block agonist- induced changes, and revert exosomal cargo to pro-reparative, anti-inflammatory phenotype. Our long-term goal is to determine whether priming MSC with a β2-AR antagonist can generate MSC exosomes (MEX) that are a viable therapeutic candidate for improving wound healing. Our major objective is to evaluate the effects of β2-AR activation and antagonism on MEX cargo contents and function in improving healing. In Specific Aim 1a: we will valuate the effects of activation of the β2-AR on MEX cargo. Determine if stress catecholamine ligands modify the cargo to become more pro-inflammatory. Proteomic, and transcriptomic analyses of MEX contents will be performed. Their pro-inflammatory potential will be evaluated by cytokine release and in vitro T cell proliferation. In Aim 1b we will evaluate the effects of blockade of the β2-AR on MEX cargo. The endogenous generation of catecholamines by MSC will be determined, and the effect of blockade of their activation of MSC on exosome cargo will be analyzed as in Aim 1a. In Specific Aim 2: we will determine whether β2-AR agonists and antagonists alternatively result in MEX with diminished or enhanced wound healing properties, respectively, in an in vivo impaired healing wound model (db/db mouse). This work will provide foundational information for the future development of MEX as a wound therapeutic.