Background
Endothelial failure following ischemia-reperfusion injury (IRI) in transplantation triggers the inflammatory cascade, compromising allograft perfusion and contributing to acute rejection. Culture conditions that activate the innate immunomodulatory phenotype of mesenchymal stem cells (MSCs) may attenuate IRI-mediated endothelial failure. We hypothesized that expansion in hypoxia or with inflammatory cytokines primes immunosuppressive functions of MSCs and improves allograft perfusion after ex-vivo delivery.
Methods
MSCs exposed to hypoxia (5% O2) or inflammatory cytokines (IFNγ/TNFα) were assessed for immunomodulator indoleamine 2,3-dioxygenase (IDO) and COX-2 expression. MSC-endothelial cell (EC) co-cultures were exposed to IRI and analyzed for changes in expression of pro-inflammatory (MCP-1), anti-inflammatory (IDO), and permeability (VE-cadherin) markers by qRT-PCR. Leukocyte-endothelium adhesion and endothelial permeability assays were performed to determine MSC modulation of endothelial activation. In an ex-vivo allogeneic transplant model, Brown-Norway allografts were seeded with Lewis MSCs, then transplanted into Lewis rats. Postoperative perfusion was assessed via clinical inspection and Doppler imaging.
Results
Hypoxic conditions increased IDO (2.81E + 04 a.u.) in 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/TNFα resulted in 4.33- and 22.3-fold changes in IDO expression, respectively (P < 0.01). Hypoxia decreased COX-2 expression by 2.9-fold, and IFNγ/TNFα incubation reduced it by half (P < 0.01 for all). Co-cultured MSC-ECs demonstrated a 49.8% reduction in endothelial MCP-1 and 230% increase in VE-cadherin expression compared to ECs alone (P < 0.05) following IR injury. Enhanced immunomodulation was functionally demonstrated by diminished leukocyte-EC adhesion (49%, P < 0.01) and endothelial permeability (60.8%). Ex-vivo perfused MSCs improved postoperative perfusion (normalized perfusion index of 83 ± 12 compared to 42 ± 8 in controls; P < 0.01) following increased eNOS expression (3.66 vs. 1.48 a.u. in controls, P < 0.01) and vasodilation.
Conclusion
Ex-vivo MSC therapy attenuates IRI in a composite-tissue transplant model. MSCs primed with hypoxia and/or inflammatory cytokines exhibit enriched immunoregulatory and vasoprotectivefunctions that mitigate endothelial activation, which improves post-operative allograft perfusion. This strategy is rapidly translatable and may reduce chronic immunotherapy requirements.