Innovations

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Single-cell RNA-sequencing (scRNASeq) analysis provides deep insights into cell functions and disease pathophysiology by allowing the profiling of the transcriptomic signatures of individual cells in heterogeneous tissues. A recently completed study in our unit primarily focused on differences between DFU patients who heal their ulcers (Healers) and those who fail to heal them (non-Healers) and investigated molecular changes via scRNASeq analysis of surgically removed DFUs. Our analysis showed enrichment of a unique population of fibroblasts, named Healing Enhancing (HE)-fibroblasts, overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 genes in the Healers. Pathway analysis demonstrated the activation of multiple immune pathways including IL6, HIF1A and ILK signaling in the HE-fibroblasts. By leveraging the above scRNASeq data, we performed an in silico screeningof thousands of perturbagens, which indicated that metrifonate, an organophosphate cholinesterase inhibitor that can be used to treat schistosomiasis and has been tested in the past for the treatment of Alzheimer's disease, could be a suitable repurposed drug for DFU management. Subsequent experiments with diabetic db/db mice revealed that alginate gels releasing metrifonate at certain levels markedly enhanced wound healing and these results were confirmed in wounds of human skin that was transplanted onto diabetic nude mice and early pig experiments. Based on these findings, we are developing novel treatments for DFU that will be in the form of a wound bandage and will employ new biomaterials that can release appropriate doses of metrifonate in the wound area.

  The Orai calcium channel, a plasma membrane bound calcium-release activated channel is important for skin homeostasis by regulating the proliferation, migration, and differentiation of keratinocytes. Orai-mediated calcium influx controls the activation of immune cells that our previous studies have shown to be implicated in improving of diabetic wound healing, including mast cells and T cell. In particular, our previous experiments in diabetic mice showed that alginate gels releasing a prototype Orai inhibitor markedly enhanced wound healing and upregulated pathways linked to acute inflammation and immune cell migration. Further evidence of the possible beneficial effects of Orai inhibitors is provided by the fact that they activate the same interleukins inflammatory factors that were previously mentioned as improving diabetic wound healing. In addition, as reported recently, our second-generation Orai inhibitors are characterized by increased potency and selectivity, minimal cytotoxicity, and improved drug-like properties. Finally, an addition strength is that the Orai inhibitors will be delivered topically using specifically fabricated alginate hydrogels, which will avoid possible side effects that could be related to systemic exposure of these small molecules. We are developing topical treatment with a new Orai inhibitor that improves healing by modulation of skin homeostasis and singling pathways important to DFU healing.