A multifactorial approach to targeting signalling pathways in diabetic foot ulcers

Diabetic foot ulcers (DFU) are one of the most debilitating complication of diabetes that adversely impacts the health, economics and quality of life of the afflicted individual. The primary pathogenic factor of DFU is hyperglycemia, and its negative effects on normal signaling pathways is still being investigated. As such, there is no specific therapy that could target the underlying dysregulations caused by hyperglycemia. So, it is important to delve into various pathways that are altered by hyperglycemia in diabetic foot in order to successfully establish novel treatment paradigms. Wound healing consists of various phases where different cellular processes such as cell proliferation, migration, angiogenesis and apoptosis coordinate to achieve a swift healing of the wound. In my thesis, I have investigated several signaling pathways that play key roles in wound healing and are profoundly disturbed by hyperglycemia in diabetes.

Notch signaling pathway is an important pathway where receptors and ligands from juxtaposed cells activate signal transduction. Upon activation, an intracellular domain of Notch (NICD) translocates to the nucleus and initiates the transcription of specific targets to control cell proliferation, cell migration, angiogenesis, differentiation and apoptosis. In paper-I, we show that Notch1 is activated in human and rodent skin and several processes central to wound healing are impaired in response to hyperglycemia in a Notch1 dependent manner. Mechanistically, we show that hyperglycemia activates a Dll4-Notch1 feedforward loop that impairs wound healing in diabetes. Inhibition of Notch signaling by chemical and genetic approaches improved wound healing in diabetic mice significantly. IGF-I is a growth hormone that is expressed in every cell of our body. The circulating IGF-I is however derived mainly from the liver. IGF-I promotes wound healing and its levels are decreased in diabetic wounds. However, the contribution of circulating IGF-I to wound healing is unknown. In Paper II, we generated a liver-specific IGF-I knockout mice and induced diabetes in these mice to study the effect of liver-derived IGF-I on wound healing. We found that the lack of liver-derived IGF-I did not affect healthy wound healing. Although diabetes delayed wound healing, there was no difference between knock-out mice and control mice. In addition, the processes contributing to wound healing were not altered by the liver-derived IGF-I deficiency. In summary, we found that a lack of liver-derived IGF-I did not affect wound healing. Future therapies using IGF-I can be designed to be delivered locally since systemic IGF-I therapy is known to carry risks of unfavorable side-effects.

In papers-III and IV, I have investigated the roles of miRNA-210 and miRNA-34a in diabetic wound healing respectively. miR-210 is induced by transcription factor HIF-1 in response to hypoxia. miR-210 mirrors HIF function in hypoxia by regulating important processes such as cell proliferation, migration, apoptosis, metabolism and angiogenesis. We found that miR-210 expression is reduced in diabetic wounds and locally reconstituting miR-210 using mimics improves diabetic wound healing significantly. miR-210 reconstitution led to a reduction in the oxygen consumption rate in the wounds that led to a decrease in ROS levels in the wound tissue. This metabolic reprogramming by miR-210 ultimately resulted in the improvement in different cellular processes central to wound healing. miR-34a plays important roles in cell cycle and DNA repair. Importantly, miR-34a has been shown to regulate Notch1 directly. Although there are contrasting reports on their function in hypoxia and diabetes, their role in diabetic wound healing has not been elucidated. In paper-IV, we show that miR-34a was reduced in DFUs and in the wounds of diabetic mice. We also found that a long exposure to hypoxia increased miR-34a expression exclusively in keratinocytes but exposing cells to high glucose decreased its expression in hypoxia. Reciprocally, Notch1 expression levels increased in keratinocytes under hypoxic and high glucose levels in a time-dependent manner. Finally, we found that diabetic wounds injected with miR-34a mimic showed significantly lower expression of Notch1, directly correlating with paper-I, indicating that reconstitution of miR-34a could be a potential therapeutic strategy for diabetic wounds.

List of scientific papers

I. Zheng X*, Narayanan S*, Sunkari VG*, Eliasson S, Botusan IR, Grünler J, Catrina AI, Radtke F, Xu C, Zhao A, Ekberg NR, Lendahl U, Catrina SB. Triggering of a Dll4-Notch1 loop impairs wound healing in diabetes. Proceedings of the National Academy of Sciences (PNAS). 2019. 116 (14): 6985-6994. *Equally contributed authors.
https://doi.org/10.1073/pnas.1900351116

II. Botusan IR*, Zheng X*, Narayanan S, Grünler J, Sunkari VG, Calissendorf FS, Ansurudeen I, Illies C, Svensson J, Jansson JO, Ohlsson C, Brismar K, Catrina SB. Deficiency of liver-derived insulin-like growth factor-I (IGF-I) does not interfere with the skin wound healing rate. PLoS One. 2018. 13 (3): e0193084 *Equally contributed authors.
https://doi.org/10.1371/journal.pone.0193084

III. Narayanan S, Eliasson S, Xu C, Grünler J, Zhao A, Zhu W, Landén NX, Ståhle M, Botusan IR, Ekberg NR, Zheng X*, Catrina SB*. HypoxamiR-210 accelerates wound healing in diabetes by improving mitochondrial energy metabolism. *Equally contributed authors. [Manuscript]

IV. Narayanan S*, Eliasson S*, Grünler J, Xu C, Li D, Landén NX, Ståhle M, Botusan IR, Ekberg NR, Zheng X#, Catrina SB#. Downregulated miR-34a contributes to the increased Notch1 signaling in diabetic wounds. *,#Equally contributed authors. [Manuscript]