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

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.

SUBMITTER: Gao M

PROVIDER: S-EPMC4679041 | biostudies-other | 2015 Dec

REPOSITORIES: biostudies-other

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Project description:In diabetes-associated chronic wounds, the normal response to hypoxia is impaired and many cellular processes involved in wound healing are hindered. Central to the hypoxia response is hypoxia-inducible factor-1α (HIF-1α), which activates multiple factors that enhance wound healing by promoting cellular motility and proliferation, new vessel formation, and re-epithelialization. Prolyl hydroxylase domain-containing protein 2 (PHD2) regulates HIF-1α activity by targeting it for degradation under normoxia. HIF-1α also upregulates microRNA miR-210, which in turn regulates proteins involved in cell cycle control, DNA repair, and mitochondrial respiration in ways that are antagonistic to wound repair. We have identified a highly potent short synthetic hairpin RNA (sshRNA) that inhibits expression of PHD2 and an antisense oligonucleotide (antimiR) that inhibits miR-210. Both oligonucleotides were chemically modified for improved biostability and to mitigate potential immunostimulatory effects. Using the sshRNA to silence PHD2 transcripts stabilizes HIF-1α and, in combination with the antimiR targeting miR-210, increases proliferation and migration of keratinocytes in vitro. To assess activity and delivery in an impaired wound healing model in diabetic mice, PHD2-targeting sshRNAs and miR-210 antimiRs both alone and in combination were formulated for local delivery to wounds using layer-by-layer (LbL) technology. LbL nanofabrication was applied to incorporate sshRNA into a thin polymer coating on a Tegaderm mesh. This coating gradually degrades under physiological conditions, releasing sshRNA and antimiR for sustained cellular uptake. Formulated treatments were applied directly to splinted full-thickness excisional wounds in db/db mice. Cellular uptake was confirmed using fluorescent sshRNA. Wounds treated with a single application of PHD2 sshRNA or antimiR-210 closed 4 days faster than untreated wounds, and wounds treated with both oligonucleotides closed on average 4.75 days faster. Markers for neovascularization and cell proliferation (CD31 and Ki67, respectively) were increased in the wound area following treatment, and vascular endothelial growth factor (VEGF) was increased in sshRNA-treated wounds. Our results suggest that silencing of PHD2 and miR-210 either together or separately by localized delivery of sshRNAs and antimiRs is a promising approach for the treatment of chronic wounds, with the potential for rapid clinical translation.

Project description:IntroductionPharmacologic approaches for promoting angiogenesis have been utilized to accelerate healing of chronic wounds in diabetic patients with varying degrees of success. We hypothesize that the distribution of proangiogenic drugs in the wound area critically impacts the rate of closure of diabetic wounds. To evaluate this hypothesis, we developed a mathematical model that predicts how spatial distribution of VEGF-A produced by delivery of a modified mRNA (AZD8601) accelerates diabetic wound healing.MethodsWe modified a previously published model of cutaneous wound healing based on coupled partial differential equations that describe the density of sprouting capillary tips, chemoattractant concentration, and density of blood vessels in a circular wound. Key model parameters identified by a sensitivity analysis were fit to data obtained from an in vivo wound healing study performed in the dorsum of diabetic mice, and a pharmacokinetic model was used to simulate mRNA and VEGF-A distribution following injections with AZD8601. Due to the limited availability of data regarding the spatial distribution of AZD8601 in the wound bed, we performed simulations with perturbations to the location of injections and diffusion coefficient of mRNA to understand the impact of these spatial parameters on wound healing.ResultsWhen simulating injections delivered at the wound border, the model predicted that injections delivered on day 0 were more effective in accelerating wound healing than injections delivered at later time points. When the location of the injection was varied throughout the wound space, the model predicted that healing could be accelerated by delivering injections a distance of 1-2 mm inside the wound bed when compared to injections delivered on the same day at the wound border. Perturbations to the diffusivity of mRNA predicted that restricting diffusion of mRNA delayed wound healing by creating an accumulation of VEGF-A at the wound border. Alternatively, a high mRNA diffusivity had no effect on wound healing compared to a simulation with vehicle injection due to the rapid loss of mRNA at the wound border to surrounding tissue.ConclusionsThese findings highlight the critical need to consider the location of drug delivery and diffusivity of the drug, parameters not typically explored in pre-clinical experiments, when designing and testing drugs for treating diabetic wounds.Supplementary informationThe online version contains supplementary material available at 10.1007/s12195-021-00678-9.

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

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