Triggering of a Dll4–Notch1 loop impairs wound healing in diabetes

The incidence of diabetes is increasing annually and will likely increase over 50% by 2030. Diabetes currently affects approximately 9.5% of the world population and diabetic foot ulceration (DFU), a chronic diabetes complication, represents a major medical, social and economic problem. The lifetime risk of a person with type 1 and type 2 diabetes developing a foot ulcer is around 15%. DFUs are difficult to heal and options for treatment are limited due to a poor understanding of the pathogenic mechanisms. Growth factors, cytokines, the extracellular matrix (ECM) and different cell types in the skin coordinate wound healing. Homeostasis and inflammation are followed by keratinocyte and fibroblast proliferation and migration then angiogenesis and ECM deposition. The last stage involves remodeling and reepithelization in the wound area.

A recent report by Zheng et al highlights a mechanism that can explain the impaired wound healing in diabetes which can lead to DFUs. The study identified the Notch1 signaling pathway as a crucial mediator of impaired wound healing in diabetes.

Using a specific antibody for activated Notch1, increased Notch1 ICD levels were detected in skin from patients with diabetes and in skin from db/db diabetic mice, mainly localised in the epidermis in both patients and mice.

Increased mRNA expression of Notch target genes, Hey1, Hey2, Hes1 and Hes5 was demonstrated in patient skin and in animal models of types 1 and 2 diabetes. Dll4, a Notch ligand was also increased in the skin of diabetic patients and animal models however Jagged1 expression was unchanged. Notch Signaling Target RT2 Profiler PCR Array analysis confirmed increased expression of many Notch target genes in the skin of diabetic mouse models.

Keratinocytes, the major cell type of the epidermis and fibroblasts and epithelial cells, the main cell types in the dermis, had increased expression of hey1 and Notch1 ICD and Hes1 protein levels after treatment with high glucose (hallmark of diabetes). No changes in Notch2 or 3 ICDs were detected and siRNA directed to Notch1 confirmed its involvement in hyperglycemic signaling.

Activation of the Notch1 pathway was dependent on γ-secretase-mediated cleavage of the Notch1 receptor, revealed by treatment with the γ-secretase inhibitor DAPT. Notch1 ICD levels in cells expressing a truncated version of Notch1, which can be cleaved by γ-secretase in a ligand free manner, was not affected by high glucose levels suggesting that glucose does not directly affect γ-secretase activity.

High glucose levels significantly inhibited migration of keratinocytes and fibroblasts as well as tube formation by the endothelial cells. Exposure of the cells to DAPT negated the inhibitory effects of hyperglycemia.

Local application of γ-secretase inhibitors DAPT and L-685,458 onto the full excision wounds created in db/db or STZ-induced diabetic mice improved the rate of delayed wound healing but interestingly did not affect the wound healing rate in non-diabetic controls.

Notch1 ICD expression in diabetic wounds was significantly increased compared to control wounds and was inhibited by local γ-secretase inhibitor treatment. ICDs for Notch2, 3, and 4 and Dll4 in diabetic wounds was also inhibited. Jagged1, 2, and Dll1 expression remained unchanged.

The improvement of the wound healing rate resulted from increased skin cell proliferation (as demonstrated by increased nuclear proliferating cell nuclear antigen (PCNA) staining) and angiogenesis (as shown by increased levels of angiogenic markers CD31, VEGRF-2, VEGRF-3). Treatment with γ-secretase inhibitors also increased granulation tissue in healing wounds.

High glucose levels increased the protein expression of Notch ligand Dll4 in the epidermis of both patients and mouse models. Treatment with siRNA directed to Notch1 blocked induction of Dll4 suggesting a positive Dll4-Notch1 feedback loop was induced by high glucose levels. This feedback loop was also observed in endothelial cells.

Genetically modified KRT14-Cre; Notch1fl/fl mice showed significantly decreased Notch1 signaling in the skin, as demonstrated by reduced Hes1 and DLL4 mRNA expression. Notch1 and Dll4 expression was induced by STZ in wild type mice and a more rapid wound healing rate observed in diabetic Notch1 knockouts compared to diabetic wild-type controls despite similar blood glucose levels. Ablation of Notch1 improved angiogenesis and proliferation in the granulation tissue in diabetic wounds.

Together the results demonstrated that glucose activates a DLL4-Notch1 positive feedback loop that contributes to delayed wound healing in diabetes. These observations can serve as the basis for the future development of new therapeutic approaches for DFUs in addition to improvement to current standard interventions e.g. better glycemic control in diabetic patients. In addition, authors speculated that specific inhibition of Notch1 in the skin utilizing Notch1 inhibitors that are already in the clinic for other indications could serve as potential treatments for diabetic wounds.

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Xiaowei Zheng et al (2019). “Triggering of a DLL4-Notch1 loop impairs wound healing in diabetes”. PNAS, DOI: 10.1073/pnas.1900351116

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