Abstract
Diabetic wounds have limited effective therapies to restore tissue repair and resolve excessive inflammation. This study aimed to identify mechanisms of diabetic wound healing defects and test a therapeutic intervention using diabetic mouse and pig models. Here, we show that Smad7 transgene expression in mouse epidermis promotes wound healing in diabetic mice. To restrict the therapeutic effects of Smad7 on wounds, we develop a Smad7-based biologic (Tat-PYC-Smad7) that penetrates cells of the wound. Topical Tat-PYC-Smad7 treatment to diabetic pig and mouse wounds accelerates healing. Tat-PYC-Smad7-treated wounds exhibit reduced TGFβ/NFκB signaling, faster re-epithelialization, and better extracellular matrix remodeling compared to vehicle controls. Tat-PYC-Smad7 also attenuates neutrophil extracellular trap (NET) formation, potentially acting through reductions in MPO enzymatic activity and MPO nuclear entry, consequently reducing chromatin decondensation and the release of NET components. Our study reveals that keratinocytes and neutrophils are the two major cell types targeted by Tat-PYC-Smad7 to promote diabetic wound healing, providing insight into mechanisms of diabetic wound healing defects targetable by Smad7-based therapy.
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Data availability
RNA-seq data generated in this study are available in the Gene Expression Omnibus (under accession code GSE274513) [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE274513]. Mass spectrometry data generated in this study are available in MassIVE under accession code (MSV000095545)[https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=d2212a53e36d40ad9b872f22d98814b4]. Source data are provided with this paper.
Code availability
A customized Python script for semi-automatic wound area segmentation has been deposited and is publicly accessible at https://zenodo.org/records/18341887. For RNAseq analysis, Quality control was conducted by FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and FastQ Screen (https://www.bioinformatics.babraham.ac.uk/projects/fastq_screen/). The RNA-seq reads were processed with BBDuk (BBMap – Bushnell B. – sourceforge.net/projects/bbmap/) and aligned to mouse genome GRCm38.p6 (release 96) with STAR [https://www.ensembl.org/Mus_musculus/Info/Index]57. The differential expression analysis was performed on TMM (trimmed-mean M values) normalized count data using a negative binomial generalized linear model and likelihood ratio test58,59 implemented in the edgeR package (version 4.0.11, https://doi.org/10.5281/zenodo.3748085)60. The Benjamini–Hochberg multiplicity correction was applied on p-values to calculate false discovery rates. GSEA was performed using pre-ranked GSEA61 and Enrichr62 implemented by the GSEApy package (version 1.1.2, https://doi.org/10.5281/zenodo.3748085), incorporating pathway collections from the Molecular Signatures Database (MSigDB) [https://www.gsea-msigdb.org/gsea/msigdb], including Hallmark, KEGG, and Gene Ontology biological process gene sets.
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Acknowledgements
This work was funded by National Institutes of Health grants DE024659, DE028718, and AR078669 to X.J.W. and C.D.Y, and R01GM148769 to S.R.C. Y.K. received funding through the Milstein Medical Asian American Partnership Foundation Awards in Dermatology and a Sequencing Plot Award from the Genomics and Microarray Core at the University of Colorado Denver. We thank Dr. David Orlicky for pathology support. We thank Sinclair Research Center and Sinclair BioResources for the establishment of diabetic pig models and treatment tests. We thank Ivan Lu for his assistance with HL60 cell culture, Yi Wang for her assistance with organizing the gross images of the wounds, Qian Chen with cell pellet embedding, and Dr. Ingrid Brust-Mascher from the Advanced Imaging Facility at the UC Davis School of Veterinary Medicine for her expert support with confocal imaging. We extend our appreciation to several entities for their valuable contributions: the University of Colorado Cancer Center Cell Technologies Shared Resource for their assistance with cell culture and use of the IncuCyte, the Mass Spectrometry Proteomics Shared Resource Facility at the University of Colorado for their mass spectrometry proteomic analyses, the Gates Center for Regenerative Medicine at the University of Colorado’s Anschutz Medical Campus for their histological support, and the RNA Biosciences Initiative at the University of Colorado for their support with RNA sequencing. Additionally, we acknowledge the Biostatistics and Bioinformatics Shared Resource at the University of Colorado Cancer Center for their assistance with processing RNAseq data. These shared resources are supported by the University of Colorado Cancer Center Support Grant (P30CA046934). The study used the UC Davis Comprehensive Cancer Center Molecular Pharmacology and Chemical Biology Shared Resource, supported by the National Cancer Institute of the National Institutes of Health under award number P30CA093373.
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Y.K., C.D.Y., and X.J.W. conceived and designed the study. Y.K. performed the experiments, analyzed the data, and wrote the manuscript. B.Z.L. performed bioinformatics analyses and created all elements in Fig. 1h and the pig illustration in Fig. 2a. F.L. conducted a subset of the K5.Smad7/db/db mouse wound experiments and analyzed the data. R.K.R. performed ELISA and ADA assays and analyzed the data. D.W. and S.W. purified the Tat-PYC-Smad7 protein. S.R.C. provided expertise in neutrophil and NETosis functional studies and contributed to data interpretation. S.T.H., S.S., and S.R.C. assisted with manuscript editing. C.D.Y. and X.J.W. supervised the project and contributed to manuscript writing and editing.
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Christian Young and Xiao-Jing Wang are inventors of a patent application filed to the US Patent and Trademark Office pertaining to the Tat-PYC-Smad7 drug product aspect(s) of this work (PCT/US2022/076242). Yao Ke and Xiao-Jing Wang are inventors of a patent application filed to the US Patent and Trademark Office pertaining to the Tat-PYC-Smad7 treatment to netosis-related indications (application # US63/792,252). The remaining authors declare no competing interests.
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Ke, Y., Li, BZ., Li, F. et al. Smad7-based biologic targeting epidermis and stroma promotes healing of diabetic wounds in mice and pigs. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70790-9
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DOI: https://doi.org/10.1038/s41467-026-70790-9
