Highlights d Epidermal Nrf2 initiates a regenerative response through Ccl2 regulation d Ccl2 release by basal stem/progenitor keratinocytes prompts macrophage trafficking d Ccl2 regulates EGF production in macrophages trafficked to the injury site d EGF from macrophages stimulates basal keratinocyte proliferation

Background Cutaneous wounds in patients with diabetes exhibit impaired healing due to physiological impediments and conventional care options are severely limited. Multipotent stromal cells (MSCs) have been touted as a powerful new therapy for diabetic tissue repair owing to their trophic activity and low immunogenicity. However, variations in sources and access are limiting factors for broader adaptation and study of MSC-based therapies. Amniotic fluid presents a relatively unexplored source of MSCs and one with wide availability. Here, we investigate the potential of amniotic fluid-derived multipotent stromal cells (AFMSCs) to restore molecular integrity to diabetic wounds, amend pathology and promote wound healing. Method We obtained third trimester amniotic fluid from term cesarean delivery and isolated and expanded MSCs in vitro. We then generated 10 mm wounds in Leprdb/db diabetic mouse skin, and splinted them open to allow for humanized wound modeling. Immediately after wounding, we applied AFMSCs topically to the sites of injuries on diabetic mice, while media application only, defined as vehicle, served as controls. Post-treatment, we compared healing time and molecular and cellular events of AFMSC-treated, vehicle-treated, untreated diabetic, and non-diabetic wounds. A priori statistical analyses measures determined significance of the data. Result Average time to wound closure was approximately 19 days in AFMSC-treated diabetic wounds. This was significantly lower than the vehicle-treated diabetic wounds, which required on average 27.5 days to heal (p < 0.01), and most similar to time of closure in wild type untreated wounds (an average of around 18 days). In addition, AFMSC treatment induced changes in the profiles of macrophage polarizing cytokines, resulting in a change in macrophage composition in the diabetic wound bed. We found no evidence of AFMSC engraftment or biotherapy induced immune response. Conclusion Treatment of diabetic wounds using amniotic fluid-derived MSCs encourages cutaneous tissue repair through affecting inflammatory cell behavior in the wound site. Since vehicle-treated diabetic wounds did not demonstrate accelerated healing, we determined that AFMSCs were therapeutic through their paracrine activities. Future studies should be aimed towards validating our observations through further examination of the paracrine potential of AFMSCs. In addition, investigations concerning safety and efficacy of this therapy in clinical trials should be pursued.

Background Chronic venous insufficiency (CVI) stems from venous hypertension, extravasation of blood, and iron-rich skin deposits. The latter is central to ulcer development through generating reactive oxygen species (ROS) that drive persistent local inflammation and the development of lipodermatosclerosis. The ability to study CVI cutaneous inflammation is fundamental to advancing therapies. To address this end, a novel protocol was adapted to investigate cutaneous wound healing in iron-induced inflammation. Methods: Mice were injected subcutaneously or intraperitoneally with iron-dextran, and excisional wounding was performed. Histologic and biomolecular analysis was performed. Results: Iron loading was associated with dense iron deposits similar to those in chronic venous stasis. Subcutaneous but not intraperitoneal loading resulted in dermal collagen expansion. Iron overload was associated with atypical antioxidant expression as compared to vehicle controls ( p < 0.0001) as well as delayed wound healing by 3-4 days. A potent activator of Nuclear factor erythroid 2-related factor 2 (Nrf2), a major transcriptional regulator of redox status, was applied to establish therapeutic efficacy. Nrf2 activation in the wound resulted in significant reduction of closure times across all experimental arms. Antioxidant expression following topical treatment was significantly increased for intraperitoneally iron-loaded mice ( p < 0.0001) but did not achieve significance for the subcutaneously-loaded animals. Conclusions: We have characterized a novel model of cutaneous iron-overload designed to advance our understanding of dysfunctional wound healing in CVI. Cutaneous changes of iron overload coincide with redox imbalance and delayed wound healing. By activating Nrf2, we demonstrate the regenerative potential of pro-antioxidant mediators in treating CVI related wound complications.