MnSOD is implicated in accelerated wound healing upon Negative Pressure Wound Therapy (NPWT): A case in point for MnSOD mimetics as adjuvants for wound management

Abstract

Negative Pressure Wound Therapy (NPWT), a widely used modality in the management of surgical and trauma wounds, offers clear benefits over conventional wound healing strategies. Despite the wide-ranging effects ascribed to NPWT, the precise molecular mechanisms underlying the accelerated healing supported by NPWT remains poorly understood. Notably, cellular redox status-a product of the balance between cellular reactive oxygen species (ROS) production and anti-oxidant defense systems-plays an important role in wound healing and dysregulation of redox homeostasis has a profound effect on wound healing. Here we investigated potential links between the use of NPWT and the regulation of antioxidant mechanisms. Using patient samples and a rodent model of acute injury, we observed a significant accumulation of MnSOD protein as well as higher enzymatic activity in tissues upon NPWT. As a proof of concept and to outline the important role of SOD activity in wound healing, we replaced NPWT by the topical application of a MnSOD mimetic, Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP⁵⁺, MnE, BMX-010, AEOl10113) in the rodent model. We observed that MnE is a potent wound healing enhancer as it appears to facilitate the formation of new tissue within the wound bed and consequently advances wound closure by two days, compared to the non-treated animals. Taken together, these results show for the first time a link between NPWT and regulation of antioxidant mechanism through the maintenance of MnSOD activity. Additionally this discovery outlined the potential role of MnSOD mimetics as topical agents enhancing wound healing.

Keywords: Manganese superoxide dismutase; MnSOD mimetics; MnTE-2-PyP(5+); Negative pressure wound therapy; Skin wound healing.

Fig. 1

Negative pressure increases total antioxidant capacity and MnSOD protein level in human tissues from wounds treated by NPWT. A) Pictures of traumatic wound pre- and post-debridement and after 5 days of NPWT treatment. Deep red granulation tissue formation can be observed following negative pressure application which is one of the main characteristic of this technique. B) Measure of total antioxidant capacity (in Trolox equivalent/µg of proteins) in patient samples at day 0 and after 2 and 3 days of NPWT treatment. Statistical analysis performed comparing day 2 and 3 to day 0 using one-way ANOVA followed by post hoc Dunnett multiple comparison test; * p < 0.05. C) Western blot analysis of patient biopsies at day 0 and after 2 and 3 days of NPWT treatment. Evaluation of the effect of NPWT on NRF2, Catalase, Angiopoietin 2, VEGF (dimer), MnSOD and Cu/ZnSOD with actin used as a loading control. Red lanes outline the main changes observed in patients for NRF2 and MnSOD. Numbers represent individual patients.

Fig. 2

MnSOD (SOD2) activity is increased in human tissues from wounds following 2 days of NPWT application. A) Total SOD activity (in mU/µg of proteins) in patient samples at day 0 and after 2 of negative pressure treatment. *p < 0.05. B) Validation of the inhibition of Cu/ZnSOD (SOD1) by 3 mM KCN to discriminate between both SOD1 and SOD2 activities. C) Discrimination of both SOD1 and SOD2 activities in patient samples at day 0 and after 2 of negative pressure treatment. Total SOD activity as the sum of SOD1 and SOD2 in human biopsies (upper bar graph). SOD1 activity (bottom left bar graph) and SOD2 activity as measured in patient samples at day 0 and after 2 of negative pressure treatment. *p < 0.05.

Fig. 3

Negative pressure maintains total antioxidant capacity and higher MnSOD protein level in rats treated by NPWT compared to the control rats. A) Pictures of surgical wound at day 0 and after 2 days of NPWT treatment. Characteristic granulation tissue formation can be observed following negative pressure application as indicated by the arrow. B) Measure of total antioxidant capacity (in Trolox equivalent/µg of proteins) in rat samples at day 0 and after 2 and 4 days in control and NPWT groups. Statistical analysis performed comparing day 2 and 4 to day 0 using one-way ANOVA followed by post hoc Dunnett multiple comparison test; * p < 0.05. C) Western blot analysis of rat samples from control and NPWT-treated groups at day 0, day 2 and day 4. Evaluation of the effect of NPWT on NRF2, HIF1α, Angiopoietin 2, VEGF (dimer) and MnSOD with actin used as a loading control.

Fig. 4

Negative pressure treatment maintains a higher MnSOD activity level in the NPWT-treated group compared to the control group in the rat model of acute wound. A) Total SOD activity (in mU/µg of proteins) in rat samples at day 0 and after 2 and 4 days in control and NPWT groups. Statistical analysis performed comparing day 2 and 4 to day 0 using one-way ANOVA followed by post hoc Dunnett multiple comparison test; * p < 0.05. B) Measure of SOD1 (upper bar graph) and SOD2 (lower bar graph) activities in rat samples at day 0 and after 2 and 4 days in control and NPWT groups. Statistical analysis performed comparing day 2 and 4 to day 0 using one-way ANOVA followed by post hoc Dunnett multiple comparison test; * p < 0.05, * * p < 0.01.

Fig. 5

Replacement of NPWT by the topical application of a MnSOD mimetic (MnE). A) Picture of the rodent model of acute wound treated with MnE showing the wound creation step, the topical application of the SOD mimetic and the final dressing. B) Pictures of the wounds of 1 representative rat from both control and MnE-treated rats. Pictures were taken every 2 days during dressing change and MnE application. The purple area represents the open wound area as explained in material and methods section and shown in Fig. S1.

Fig. 6

MnE treatment promotes wound closure. A) Measure of the total wound area over time for both control (PBS) and MnE-treated groups (in cm²). B) Measure of the percentage of wound contraction over time for control and MnE-treated animals. C) Measure of the area corresponding to newly formed tissues filling the wound during the wound healing process for control and MnE-treated rats (in cm²). D) Measure of the open wound area in control and MnE-treated rats (in cm²). Statistical analysis performed comparing control and MnE-treated rats at day n using one-way ANOVA followed by post hoc Sidak multiple comparison test; * p < 0.05. Each graph is accompanied by the actual values measured or calculated as explained in the material and methods, SEM and n number of animals (missing n values in percentage of contraction correspond to a “negative contraction” calculated which would not be possible).

Fig. 7

Topical application of MnE accelerates wound healing compared to untreated animals. A) Measure of the percentage of epithelialization for both control (PBS) and MnE-treated groups. Statistical analysis performed comparing control and MnE-treated rats at day n using one-way ANOVA followed by post hoc Sidak multiple comparison test; * **p < 0.005. B) Estimation of ET25, ET50 and ET80 in control and MnE-treated animals: effective times observed to achieve 25%, 50% and 80% of epithelialization of the wounds respectively. * p < 0.05. C) Measure of the percentage of wound healing for both control (PBS) and MnE-treated groups. Statistical analysis performed comparing control and MnE-treated rats at day n using one-way ANOVA followed by post hoc Sidak multiple comparison test; * ** * p < 0.005. D) Estimation of ET25, ET50 and ET80 in control and MnE-treated animals: effective times observed to achieve 25%, 50% and 80% of wound healing respectively. * p < 0.05, * *p < 0.01. Graph A and C are accompanied by the actual values for the percentage of epithelialization and wound healing respectively, calculated as explained in the material and methods, with SEM and n number of animals as additional information (remark: the missing value for the second day of MnE treatment was identified as an outlier using the ROUT method and was consequently removed for the accuracy of the analysis).