Topical GDF11 accelerates skin wound healing in both type 1 and 2 diabetic mouse models

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Highlights

  • GDF11 has similar effect on the healing of diabetic wounds as the other three growth factors (PDGF, bFGF and EGF).

  • GDF11 can promote the expression of CTGF by promoting YAP and p-Smad2/3 forming a nuclear translocation complex.

  • The truncated GDF11 synthesized ourselves has a similar effect to the purchased one in promoting diabetic wound healing.

  • The truncated GDF11 might be considered a potential new agent for the treatment of diabetic wounds.

Abstract

This study aimed to investigate the role of truncated growth differentiation factor 11 (GDF11), in which the recognition site of Furin from wild-type GDF11 was deleted to enhance the cellular stability, in skin wound healing in the setting of diabetes mellitus (DM) and the underlying mechanisms. Our study found that both truncated and natural GDF11s effectively accelerated wound healing processes in both T1DM and T2DM mice with a potency compatible to PDGF, bFGF, and EGF, but being much higher than GDF8. At the cellular level, GDF11 stimulated the proliferation and suppressed HG-induced apoptosis of HSFs. Further study revealed that GDF11 activated the YAP—Smad2/3—CTGF fibrotic signaling pathway by reversing HG-induced upregulation of phosphorylated form of YAP (p-YAP), increases p-Smad2/3 levels, and restoring HG-induced repression of CTGF expression by GDF11. Overall, the study shows that both natural and truncated GDF11s promote the healing process of skin wound in mice of both T1DM and T2DM partly via stimulating dermal fibrosis via the YAP—Smad2/3—CTGF pathway, suggesting it a potential agent for treating skin wound in diabetic population.

Introduction

Patients with diabetes mellitus (DM) are highly fragile to various environmental insults and skin damage is one of the most frequently occurred problems in DM subjects because of the non-healing property of skin wounds. Diabetic wound (DW) developing into diabetic foot ulcers (DFU) is the leading cause of hospital admission in diabetic patients, significantly adding to global economic, social, and clinical burdens worldwide [1]. Compared to euglycemic subjects, diabetic patients have more than a 100 times greater risk of suffering a lower extremity amputation due to non-healing DFU. However, effective treatment of diabetic wounds has been lacking [2].

As one of the relatively effective methods, growth factor substances have been shown to be effective in promoting wound healing. They can play a role in promoting angiogenesis and promoting fibroblast proliferation, thereby achieving wound healing. Including platelet-derived growth factor (PDGF), which is the only drug approved by the US Food and Drug Administration, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Growth differentiation factor 11 (GDF11), a member of the transforming growth factor β (TGF-β) superfamily, is a regulator of cell growth and differentiation in both embryonic and adult tissues [3]. It has emerged as a youth factor that produces anti-aging effects in both animals and humans although contradictory or contrary observations [4]. Our recent study demonstrates that topical application of GDF11 accelerates the healing process of diabetic skin wounds via accelerating vasculogenesis suggesting GDF11as a potential therapeutic drug for would healing in patients (unpublished observations). Nonetheless, the presence of a recognition site of Furin, a paired basic amino acid cleaving enzyme or a protease, at Arg312 of wild-type GDF11 could make this protein susceptible to enzyme digestion at the disulfide bond at between positions 313 and 372 thereby impairing its cellular stability and activity. Such an issue could hinder the translational potential of GDF11 for clinical applications. To tackle this problem, we carried out deletion mutation of GDF11 by removing N-terminal 14 amino acids (AA) from natural GDF11 to become a 95-AA peptide (Supplementary Fig. S1). Such a truncation aims to delete the Furin recognition site at Arg312 while retaining the intact disulfide bond, thereby enhancing the cellular stability of this protein [5]. Our previous experimental study has confirmed the greater efficacy of the truncated version of GDF11 (tGDF11) than natural GDF11 in activating Smad2/3, the typical downstream signalling mediator of GDF11 [5].

The present study was set out to investigate the effects of tGDF11 on diabetic skin would healing and compare the relative efficacies of tGDF11 with those of wild-type or natural GDF11, PDGF, bFGF and EGF in full-thickness skin wound healing. In addition, we also investigated fibrosis-promoting effect of GDF11 as an alternative mechanism for its wound healing-accelerating property. Our experiments were carried out on a mouse model of pharmacologically induced type 1 DM (T1DM) and genetically engineered type 2 diabetic db/db mice (T2DM).

Section snippets

Ethics statement

The experimental procedures involving the use of animals in this study were approved by the Animal Care and Use Committee of Harbin Medical University (HMUIRB-2008-06), and conformed to the Guidelines for the Care and Use of Laboratory Animals set forth by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996).

Type 1 DM mice model

Kunming male mice (29–31 g) were injected 1.8 mg/g streptozocin (STZ; Sigma, USA) after fasted and DM model was considered successfully established with blood

GDF11 accelerates diabetic wound healing in T1DM mice

To explore the role of GDF11 in skin would healing, we began with a mouse model of T1DM or pharmacological T1DM induced by injection of streptozocin (STZ). We then verified the successful establishment of T1DM by monitoring the changes of fasting blood glucose (FBG) levels (Supplementary Fig. S2). We subsequently confirmed the bioactivities of natural and truncated GDF11s used in our experiments in human skin fibroblasts (HSFs) by demonstrating the remarkable activation of Smad2/3 (

Discussion

The non-healing nature of diabetic wounds affects millions of people worldwide, and offers a substantial unmet clinical need. The aims of the present study was to examine the efficacy of GDF11 in controlling diabetic skin wound healing by comparing with the effects of three well-recognized wound healing-promoting growth factors (including PDGF, bFGF and EGF) and of another myokine growth factor myostatin (GDF8) and to shed light on the cellular and molecular mechanisms for its beneficial

Declaration of competing interest

All the authors declare no conflict of interest.

Acknowledgements

This work was supported in part by the National Key R&D Program of China (2017YFC1702003), the National Natural Science Foundation of China (81970320, 81570399, 81670238, and 81773735).

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