Diabetic foot ulcers (DFUs) are a serious complication of diabetes. Previous exposure to hyperglycemic conditions accelerates a decline in cellular function through metabolic memory despite normalization of glycemic control. Persistent, hyperglycemia-induced epigenetic patterns are considered a central mechanism that activates metabolic memory; however, this has not been investigated in patient-derived fibroblasts from DFUs. We generated a cohort of patient-derived lines from DFU fibroblasts (DFUF), and site- and age-matched diabetic foot fibroblasts (DFF) and non-diabetic foot fibroblasts (NFF) to investigate global and genome-wide DNA methylation patterns using liquid chromatography/mass spectrometry and the Illumina Infinium HumanMethylation450K array. DFFs and DFUFs demonstrated significantly lower global DNA methylation compared to NFFs (p = 0.03). Hierarchical clustering of differentially methylated probes (DMPs, p = 0.05) showed that DFFs and DFUFs cluster together and separately from NFFs. Twenty-five percent of the same probes were identified as DMPs when individually comparing DFF and DFUF to NFF. Functional annotation identified enrichment of DMPs associated with genes critical to wound repair, including angiogenesis (p = 0.07) and extracellular matrix assembly (p = 0.035). Identification of sustained DNA methylation patterns in patient-derived fibroblasts after prolonged passage in normoglycemic conditions demonstrates persistent metabolic memory. These findings suggest that epigenetic-related metabolic memory may also underlie differences in wound healing phenotypes and can potentially identify therapeutic targets.
Keywords: ANOVA, Analysis of Variance; BMP, Bone Morphogenic Protein; COL4A1, Collagen 4A1; DAVID, Database for Annotation, Visualization, and Integrative Discovery; DCCT, Diabetes Control and Complications Trial; DFF, Diabetic Foot Fibroblast; DFU, Diabetic Foot Ulcer; DFUF, Diabetic Foot Ulcer Fibroblast; DHS, DNase Hypersensitive Site; DMP, Differentially Methylated Probe; DNA methylation; ECM, Extracellular Matrix; EDIC, Epidemiology of Diabetes Interventions and Complications; ENCODE, Encyclopedia of DNA Elements; FGF1, Fibroblast Growth Factor 1; HbA1c, Hemoglobin A1c; NFF, Non-diabetic Foot Fibroblast; NHLF, Normal Human Lung Fibroblast; PLAU, Plasminogen Activator Urokinase; SNP, Single Nucleotide Polymorphism; TFBS, Transcription Factor Binding Site; TGFb, Transforming Growth Factor b; TNFa, Tumor Necrosis Factor a; TSS, Transcription Start Site; UTR, Untranslated Region.; dNTPs, deoxynucleotide; diabetes; diabetic foot ulcer; epigenetics; fibroblast; metabolic memory; wound healing.