Inflammation, following injury, induces cellular plasticity as an inherent component of physiological tissue repair. The dominant fate of wound macrophages is unclear and debated. Here we show that two-thirds of all granulation tissue fibroblasts, otherwise known to be of mesenchymal origin, are derived from myeloid cells which are likely to be wound macrophages. Conversion of myeloid to fibroblast-like cells is impaired in diabetic wounds. In cross-talk between keratinocytes and myeloid cells, miR-21 packaged in extracellular vesicles (EV) is required for cell conversion. EV from wound fluid of healing chronic wound patients is rich in miR-21 and causes cell conversion more effectively compared to that by fluid from non-healing patients. Impaired conversion in diabetic wound tissue is rescued by targeted nanoparticle-based delivery of miR-21 to macrophages. This work introduces a paradigm wherein myeloid cells are recognized as a major source of fibroblast-like cells in the granulation tissue.

Human lungs single-cell RNA sequencing data from healthy donors (elderly and young; GEO accession no. GSE122960) were analyzed to isolate and specifically study gene expression in alveolar type II cells. Colocalization of angiotensin-converting enzyme 2 ( ACE2 ) and TMPRSS2 enables severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2) to enter the cells. Expression levels of these genes in the alveolar type II cells of elderly and young patients were comparable and, therefore, do not seem to be responsible for worse outcomes observed in coronavirus disease 2019 (COVID-19) affected elderly. In cells from the elderly, 263 genes were downregulated and 95 upregulated. Superoxide dismutase 3 ( SOD3 ) was identified as the top-ranked gene that was most downregulated in the elderly. Other redox-active genes that were also downregulated in cells from the elderly included activating transcription factor 4 ( ATF4 ) and metallothionein 2A ( M2TA ). ATF4 is an endoplasmic reticulum stress sensor that defends lungs via induction of heme oxygenase 1. The study of downstream factors known to be induced by ATF4 , according to Ingenuity Pathway Analysis™, identified 24 candidates. Twenty-one of these were significantly downregulated in the cells from the elderly. These downregulated candidates were subjected to enrichment using the Reactome Database identifying that in the elderly, the ability to respond to heme deficiency and the ATF4 -dependent ability to respond to endoplasmic reticulum stress is significantly compromised. SOD3 -based therapeutic strategies have provided beneficial results in treating lung disorders including fibrosis. The findings of this study propose the hypotheses that lung-specific delivery of SOD3 / ATF4 -related antioxidants will work in synergy with promising antiviral drugs such as remdesivir to further improve COVID-19 outcomes in the elderly.

Hyperglycemia (HG) induces genome-wide cytosine demethylation. Our previous work recognized miR-200b as a critical angiomiR, which must be transiently downregulated to initiate wound angiogenesis. Under HG, miR-200b downregulation is not responsive to injury. Here, we demonstrate that HG may drive vasculopathy by epigenetic modification of a miR promoter. In human microvascular endothelial cells (HMECs), HG also lowered DNA methyltransferases (DNMT-1 and DNMT-3A) and compromised endothelial function as manifested by diminished endothelial nitric oxide (eNOS), lowered LDL uptake, impaired Matrigel tube formation, lower NO production, and compromised VE-cadherin expression. Bisulfite-sequencing documented HG-induced miR-200b promoter hypomethylation in HMECs and diabetic wound-site endothelial cells. In HMECs, HG compromised endothelial function. Methyl donor S-adenosyl-L-methionine (SAM) corrected miR-200b promoter hypomethylaton and rescued endothelial function. In vivo, wound-site administration of SAM to diabetic mice improved wound perfusion by limiting the pathogenic rise of miR-200b. Quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomics and ingenuity pathway analysis identified HG-induced proteins and principal clusters in HMECs sensitive to the genetic inhibition of miR-200b. This work presents the first evidence of the miR-200b promoter methylation as a critical determinant of diabetic wound angiogenesis.

The Toll-Like receptor 4 (TLR4) plays an important role in immunity, tissue repair, and regeneration. The objective of the present work was to evaluate the association of TLR4 single nucleotide polymorphisms (SNPs) rs4986790, rs4986791, rs11536858 (merged into rs10759931), rs1927911, and rs1927914 with increased diabetic foot ulcer (DFU) risk in patients with type 2 diabetes mellitus (T2DM). PCR-RFLP was used for genotyping TLR4 SNPs in 125 T2DM patients with DFU and 130 controls. The haplotypes and linkage disequilibrium between the SNPs were determined using Haploview software. Multivariate linear regression (MLR) and artificial neural network (ANN) modeling was done to observe their predictability for the risk of DFU in T2DM patients. Risk genotypes of all SNPs except rs1927914 were significantly associated with DFU. Haplotype ACATC (Pvalue =9.3E-5) showed strong association with DFU risk. Two haplotypes ATATC (Pvalue = 0.0119) and ATGTT (Pvalue = 0.0087) were found to be protective against DFU. In conclusion TLR4 SNPs and their haplotypes may increase the risk of impairment of wound healing in T2DM patients. ANN model (83%) is found to be better than the MLR model (76%) and can be used as a tool for the DFU risk assessment in T2DM patients.

Epithelial to mesenchymal transition (EMT) and wound vascularization are two critical interrelated processes that enable cutaneous wound healing. Zinc finger E-box binding homeobox 1 (ZEB1), primarily studied in the context of tumor biology, is a potent EMT activator. ZEB1 is also known to contribute to endothelial cell survival as well as stimulate tumor angiogenesis. The role of ZEB1 in cutaneous wounds was assessed using Zeb1+/− mice, as Zeb1−/− mice are not viable. Quantitative stable isotope labeling by amino acids in cell culture (SILAC) proteomics was used to elucidate the effect of elevated ZEB1, as noted during hyperglycemia. Under different glycemic conditions, ZEB1 binding to E-cadherin promoter was investigated using chromatin immunoprecipitation. Cutaneous wounding resulted in loss of epithelial marker E-cadherin with concomitant gain of ZEB1. The dominant proteins downregulated after ZEB1 overexpression functionally represented adherens junction pathway. Zeb1+/− mice exhibited compromised wound closure complicated by defective EMT and poor wound angiogenesis. Under hyperglycemic conditions, ZEB1 lost its ability to bind E-cadherin promoter. Keratinocyte E-cadherin, thus upregulated, resisted EMT required for wound healing. Diabetic wound healing was improved in ZEB+/− as well as in db/db mice subjected to ZEB1 knockdown. This work recognizes ZEB1 as a key regulator of cutaneous wound healing that is of particular relevance to diabetic wound complication.