Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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Primed Mesenchymal Stem Cells Prevent Endothelial Activation And Improve Allograft Perfusion Following Transplantation
Jessica B. Chang, BS, Marc A. Soares, MD, Jonathan P. Massie, BS, April Duckworth, MD, Nakul Rao, MD, Camille Kim, BS, Karan Mehta, BS, Amanda Hua, BS, Piul Rabbani, PhD, Pierre B. Saadeh, MD, Daniel J. Ceradini, MD.
NYU Department of Plastic Surgery, New York, NY, USA.

PURPOSE:
Endothelial activation following ischemia-reperfusion injury (IRI) in transplantation triggers the inflammatory cascade, compromising allograft perfusion. Additionally, IRI is a critical factor that contributes to the incidence and severity of both acute and chronic rejection. We have previously demonstrated that mesenchymal stem cells (MSCs) can be seeded into allografts ex vivo where they take up residence in the perivascular space. While conventional expansion of MSCs produces an innate immunomodulatory phenotype, conditions that enhance this phenotype may be utilized to attenuate endothelial failure following ischemic insult during transplantation. We hypothesized that expansion under hypoxic conditions or with inflammatory cytokines primes the immunosuppressive functions of MSCs and improves allograft perfusion subsequent to ex vivo delivery.
METHODS:
MSCs were expanded during exposure to hypoxia (5% O2) or inflammatory cytokines IFNγ and/or TNFα prior to assessment of the immunomodulator indoleamine 2,3-dioxygenase (IDO) expression. MSC-only, endothelial cell (EC)-only, and MSC-EC co-cultures were exposed to IRI and analyzed for changes in expression of pro-inflammatory (MCP-1), anti-inflammatory (IDO), and permeability (Cadherin 5) markers by quantitative real-time RT-PCR. Results were normalized to 18s rRNA expression and expressed as arbitrary units (a.u.). Leukocyte-endothelium adhesion and endothelial permeability assays were also performed to determine functional modulation of MSCs on endothelial activation. In an ex vivo allogeneic transplant model, donor Brown-Norway rat allografts were seeded with recipient Lewis rat MSCs prior to transplantation into Lewis rats. Postoperative perfusion was then assessed via clinical inspection and Doppler imaging.
RESULTS:
Hypoxic conditions significantly increased expression of IDO (2.81E+04 a.u.) by MSCs compared to normoxia (1.24E-03 a.u., p <0.01). Co-culture with ECs further increased IDO expression (8.59E+04 a.u., p <0.01). Exposure to inflammatory cytokines IFNγ and TNFα also markedly increased IDO expression (2.77E-02 and 5.38E-03 a.u. compared to 1.24E-03 a.u. untreated, or 4.33- and 22.3-fold change, respectively). Co-cultured MSC-ECs demonstrated a 49.8% reduction in endothelial MCP-1 (1.23 vs. 2.45 a.u.) and 230% increase in Cadherin 5 expression compared to ECs alone (p <0.05) following IR injury. Enhanced immunomodulation was functionally demonstrated in diminished leukocyte adhesion to ECs (49%, p <0.01) and mitigated IRI-related permeability through an endothelial barrier. MSCs perfused in the ex vivo period were identified in the perivascular space throughout the allograft and improved postoperative perfusion (normalized perfusion index of 83±12 compared to 42±8 in controls; p <0.01) consequent to increased eNOS expression (3.66 vs. 1.48 a.u. in controls, p <0.01) and vasodilation.
CONCLUSION:
Here we demonstrate that ex vivo MSC therapy can attenuate IRI in a composite tissue transplant model. MSCs primed with a hypoxic environment and/or exposure to inflammatory cytokines exhibit enriched immunoregulatory and vasoprotective functions that mitigate activation of the endothelial barrier, which significantly improves post-operative allograft perfusion. This strategy is clinically feasible and may reduce IRI in composite tissue allotransplantation.


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