Acceleration of diabetic wound healing using a novel protease-anti-protease combination therapy - PubMed (original) (raw)

Acceleration of diabetic wound healing using a novel protease-anti-protease combination therapy

Ming Gao et al. Proc Natl Acad Sci U S A. 2015.

Abstract

Nonhealing chronic wounds are major complications of diabetes resulting in >70,000 annual lower-limb amputations in the United States alone. The reasons the diabetic wound is recalcitrant to healing are not fully understood, and there are limited therapeutic agents that could accelerate or facilitate its repair. We previously identified two active forms of matrix metalloproteinases (MMPs), MMP-8 and MMP-9, in the wounds of db/db mice. We argued that the former might play a role in the body's response to wound healing and that the latter is the pathological consequence of the disease with detrimental effects. Here we demonstrate that the use of compound ND-336, a novel highly selective inhibitor of gelatinases (MMP-2 and MMP-9) and MMP-14, accelerates diabetic wound healing by lowering inflammation and by enhancing angiogenesis and re-epithelialization of the wound, thereby reversing the pathological condition. The detrimental role of MMP-9 in the pathology of diabetic wounds was confirmed further by the study of diabetic MMP-9-knockout mice, which exhibited wounds more prone to healing. Furthermore, topical administration of active recombinant MMP-8 also accelerated diabetic wound healing as a consequence of complete re-epithelialization, diminished inflammation, and enhanced angiogenesis. The combined topical application of ND-336 (a small molecule) and the active recombinant MMP-8 (an enzyme) enhanced healing even more, in a strategy that holds considerable promise in healing of diabetic wounds.

Keywords: MMP-8; MMP-9; ND-336; diabetic wound healing; inhibition.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Scheme 1.

Scheme 1.

Structures of compounds 1 and 2.

Fig. S1.

Fig. S1.

Synthesis of ND-336.

Fig. 1.

Fig. 1.

Effect of MMP-9 inhibition, topical treatment with exogenously added MMP-8, and combined MMP-9 inhibition and exogenous MMP-8 on diabetic wound healing. A single 8-mm wound was made on the dorsal thorax of db/db mice. *P < 0.05, #P < 0.01, &P < 0.001 indicate statistically significant differences in wound closure between the indicated groups. Statistical significance was evaluated by the two-tailed Mann–Whitney u test. (A) Wound healing after treatment with ND-336 (0.1 mg per wound per day), ND-322 (0.1 mg per wound per day) as a positive control, or vehicle. Data are shown as mean ± SEM (n = 8 mice per group on days 7, 10, and 14). (B) Wound healing after exogenously added MMP-8 (1 µg per wound per day). Data are shown as mean ± SEM (n = 20, 9, and 9 mice on days 7, 10, and 14, respectively, for the vehicle group; n = 20, 10, and 10 mice on days 7, 10, and 14, respectively, for the MMP-8 group). (C) Wound healing after treatment with combined ND-336 (0.05 mg per wound per day) and MMP-8 (1 µg per wound per day). Data are shown as mean ± SEM; n = 13, 14, 12, and 12 for the groups treated with vehicle, ND-336, MMP-8, and ND-336 + MMP-8, respectively.

Fig. S2.

Fig. S2.

Effect of MMP-9 inhibition on diabetic wound healing. A single 8-mm wound was made on the dorsal thorax. Wounds were treated with ND-336 (0.1 mg per wound per day), ND-322 (0.1 mg per wound per day) as a positive control, or vehicle. H&E staining, TUNEL, and in situ zymography with DQ-gelatin and DQ-collagen were performed (n = 3 mice per group). (A) H&E staining on day 14. Re-epithelialization is indicated by the black line. Pictures were taken with a 10× lens. (Scale bars, 50 µm.) (B) TUNEL images of wounds on day 14. Arrows point to representative TUNEL+ (apoptotic) cells. Pictures were taken with a 10× lens. (Scale bars, 50 µm.) (C) In situ zymography with gelatinase fluorogenic substrate DQ-gelatin (Left, green), and merged with nuclear DNA staining by DAPI (Right, blue). Pictures were taken with a 40× lens. (Scale bars, 50 µm.) (D) In situ zymography with collagenase fluorogenic substrate DQ-collagen (Left, green) and merged with nuclear DNA staining by DAPI (Right, blue). Pictures were taken with a 40× lens. (Scale bars, 50 µm.)

Fig. S3.

Fig. S3.

Effect of MMP-9 gene ablation on diabetic wound healing. Diabetes was induced in wild-type and MMP-9–knockout mice by treatment with streptozotocin (150 mg/kg i.p.) and confirmed by a fasting blood glucose of >300 mg/dL. Excisional 8-mm wounds were inflicted 2 wk later. (A) Wound healing in wild-type and MMP-9–knockout streptozotocin-induced diabetic mice. Data are shown as mean ± SEM (n = 13 and 7 mice per group on days 7 and 14, respectively; total, 26 mice). #P < 0.01 indicates statistically significant differences in wound healing between the two indicated groups using the two-tailed Mann–Whitney u test. (B) Representative wound images (all to the same scale) on days 0, 7, and 14. (C) H&E staining for representative wounds in wild-type and MMP-9–knockout streptozotocin-induced diabetic mice (n = 3 mice per group per time point). Re-epithelialization is indicated by the black line. Pictures were taken with a 10× lens. (Scale bars, 50 µm.) (D) TUNEL images of representative wounds on day 7 (n = 3 mice per group per time point). Arrows point to representative TUNEL+ (apoptotic) cells. Pictures were taken with a 10× lens. (Scale bars, 50 µm.)

Fig. S4.

Fig. S4.

Topical treatment with exogenously added MMP-8 accelerates wound healing in db/db mice. A single 8-mm punch biopsy lesion on the dorsal thorax was administered to mice. Wounds were treated with MMP-8 (1 µg per wound per day; n = 20 mice) or vehicle (reaction buffer, n = 20 mice). H&E staining and in situ zymography with DQ-collagen were performed with on day 14 after wound infliction (n = 3 mice per group). (A) H&E staining for representative wounds treated with vehicle or MMP-8. Re-epithelialization is indicated by the black line. Pictures were taken with a 10× lens. (Scale bars, 50 µm.) (B) In situ zymography with collagenase fluorogenic substrate DQ-collagen (Left, green) and merged with nuclear DNA staining by DAPI (Right, blue). Pictures were taken with a 40× lens. (Scale bars, 50 µm.)

Fig. 2.

Fig. 2.

Effect of MMP-9 inhibition and exogenous MMP-8 on diabetic wound healing. Mice received a single 8-mm excisional wound on the dorsal thorax. Wounds were treated with vehicle, ND-336 (0.05 mg per wound per day), MMP-8 (1 µg per wound per day), or ND-336 (0.05 mg per wound per day) plus MMP-8 (1 µg per wound per day). H&E staining, TUNEL, and in situ zymography with DQ-gelatin and DQ-collagen were performed on day 14 (n = 3 mice per group). (A) H&E staining for representative wounds on day 14. Re-epithelialization is indicated by the black line. Pictures were taken with a 10× lens. (Scale bars, 50 µm.) (B) TUNEL images of representative wounds on day 14. Arrows point to representative TUNEL+ (apoptotic) cells. Pictures were taken with a 10× lens. (Scale bars, 50 µm.) (C) In situ zymography with gelatinase fluorogenic substrate DQ-gelatin (Left, green) and merged with nuclear DNA staining by DAPI (Right, blue). Pictures were taken with a 40× lens. (Scale bars, 50 µm.) (D) In situ zymography with collagenase fluorogenic substrate DQ-collagen (Left, green) and merged with nuclear DNA staining by DAPI (Right, blue). Pictures were taken with a 40× lens. (Scale bars, 50 µm.)

Fig. S5.

Fig. S5.

Dose–response of ND-336 in diabetic wound healing. (A) Wound healing in db/db mice treated with ND-336 at 0.05, 0.025, and 0.01 mg per wound per day. Data are shown as mean ± SEM (n = 7, 6, 7, and 7 mice, respectively, for vehicle, 0.05-mg, 0.025-mg, and 0.01-mg treatments; total, 27 mice). *P < 0.05 indicates statistically significant differences in wound healing by the two-tailed Mann–Whitney u test. (B) Representative wound images (all to the same scale) on days 0, 7, 10, and 14.

Fig. S6.

Fig. S6.

Dose–response of MMP-8 in diabetic wound healing. (A) Wound healing in db/db mice treated with MMP-8 at 1, 5, and 10 µg per wound per day. Data are shown as mean ± SEM (n = 20, 9, and 9 mice on days 7, 10, and 14, respectively, for the vehicle-treated group; n = 20, 10, and 10 mice on days 7, 10, and 14, respectively, for the group treated with 1 µg MMP-8 per wound per day; n = 10, 10, and 10 mice on days 7, 10, and 14, respectively, for the group treated with 5 µg MMP-8 per wound per day; n = 10, 10, and 9 mice on days 7, 10, and 14, respectively, for the group treated with 10 µg MMP-8 per wound per day). *P < 0.05 indicates statistically significant differences in wound healing using the two-tailed Mann–Whitney u test. (B) Representative wound images (all to the same scale) on days 0, 7, 10, and 14.

Fig. 3.

Fig. 3.

MMP-9 inhibition and/or exogenous MMP-8 result in decreased inflammation and increased angiogenesis. Data represent the mean ± SD (n = 3 mice per group per time point; total, 36 mice). *P < 0.05 and #P < 0.01 indicate statistically significant differences between the indicated groups. Statistical significance was evaluated by the Student’s t test using a two-tail distribution and unequal variance. (A) Concentrations of IL-6 as a function of time after wound infliction. (B) The AUC for IL-6 showed that IL-6 levels were reduced significantly upon treatment with ND-336, MMP-8, or combined ND-336 and MMP-8. (C) Concentrations of TGF-β1 as a function of time after wound infliction. (D) The AUC for TGF-β1 showed that TGF-β1 levels were reduced significantly upon treatment with ND-336, MMP-8, or combined ND-336 and MMP-8. (E) Concentrations of VEGF as a function of time after wound infliction. (F) AUC for VEGF showed that VEGF levels were increased significantly upon treatment with ND-336, MMP-8, or combined ND-336 and MMP-8.

Fig. S7.

Fig. S7.

MMP-9 inhibition, exogenous MMP-8 treatment, and combined MMP-9 inhibition and exogenous MMP-8 increase angiogenesis as measured by anti-CD31. Tissues were collected 14 d after wound infliction (n = 3 mice per group).

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References

    1. Centers for Disease Control and Prevenion 2014. National Diabetes Statistics Report: Estimates of diabetes and its burden in the United States. (Centers for Disease Control and Prevention, Atlanta, GA)
    1. Alexiadou K, Doupis J. Management of diabetic foot ulcers. Diabetes Ther. 2012;3(1):4. - PMC - PubMed
    1. Ziyadeh N, Fife D, Walker AM, Wilkinson GS, Seeger JD. A matched cohort study of the risk of cancer in users of becaplermin. Adv Skin Wound Care. 2011;24(1):31–39. - PubMed
    1. Wilgus TA. Growth Factor-Extracellular Matrix Interactions Regulate Wound Repair. Adv Wound Care (New Rochelle) 2012;1(6):249–254. - PMC - PubMed
    1. Nagase H, Visse R, Murphy G. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res. 2006;69(3):562–573. - PubMed

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