Abstract 594: E-selectin-Overexpressing Mesenchymal Stem Cell-based Therapy Augments Cutaneous Wound Healing In Ischemic Limbs

Abstract

Objective: Tissue loss considerably worsens the risk of extremity amputation in patients with chronic limb-threatening ischemia (CLTI), and unmodified mesenchymal stem cell (MSC) therapies have failed to demonstrate more than minor clinical benefits for inducing therapeutic angiogenesis and wound healing. Here we describe the effects of MSCs engineered to overexpress E-selectin, a cell-adhesion molecule capable of stimulating neovascularization, using a translational murine model recapitulating hindlimb ischemia with cutaneous tissue loss. Methods: MSCs were harvested from bone marrow of 8-10-week-old FVB/Rosa26Sor mTmG donor mice and verified as CD44 + /CD73+/CD105 + /CD29 + /Sca-1 + by flow cytometry. MSCs underwent viral transduction with E-selectin-GFP/AAV or GFP/AAV. Femoral artery ligation was performed in 12-14-week-old recipient FVB mice followed by a 4 mm cutaneous wound in the ipsilateral limb and local injection of PBS or 1x10^6 donor GFP + /MSCs or E-selectin + /MSCs (E-sel + /MSC). Wounds were monitored daily for 7 days and then harvested. Results: Unmodified MSCs do not express E-selectin, and E-sel + /MSCs retained MSC phenotype. Mice receiving E-sel + /MSC treatment (n=10) demonstrated accelerated wound closure compared to both GFP + /MSCs (n=10) and PBS (n=10) treated mice at each post-operative day (POD) with the highest degree of difference observed at POD 5 (94 ± 3% vs. 79 ± 10% GFP vs. 72 ± 9% PBS, p<0.001) and 7 (98 ± 2% vs. 86 ± 8% GFP vs. 87 ± 6% PBS; p<0.010). Collagen deposition was greater in wounds treated with E-sel + /MSCs (21 ± 4% vs. 5 ± 1% GFP vs. 9 ± 1% PBS). DiI perfusion at POD 10 demonstrated greater vessel density in wounds treated with E-sel + /MSCs (76 ± 15% vs. 29 ± 7% GFP vs. 9 ± 5% PBS relative vascular density). E-sel + /MSCs demonstrated improved viability with more mTmG + -E-sel + /MSCs (13 ± 3 cells/high powered field (HPF) vs. GFP + /MSCs 3 ± 2 cells/HPF vs. PBS 0 ± 0 cells/HPF, p<0.010) at POD 7 on immunofluorescent microscopy. Conclusion: Cell therapy using E-selectin-overexpressing MSCs overcomes impairments in wound healing of ischemic limbs. These data exhibit the potential role for E-selectin-modified MSCs as a novel cell therapy in future clinical applications for delayed and non-healing wounds associated with CLTI.