Multimodal Identification of Molecular Factors Linked to Severe Diabetic Foot Ulcers Using Artificial Intelligence.

Diabetic foot ulcers (DFUs) are a severe complication of diabetes mellitus (DM), which often lead to hospitalization and non-traumatic amputations in the United States. Diabetes prevalence estimates in South Texas exceed the national estimate and the number of diagnosed cases is higher among Hispanic adults compared to their non-Hispanic white counterparts. San Antonio, a predominantly Hispanic city, reports significantly higher annual rates of diabetic amputations compared to Texas. The late identification of severe foot ulcers minimizes the likelihood of reducing amputation risk. The aim of this study was to identify molecular factors related to the severity of DFUs by leveraging a multimodal approach. We first utilized electronic health records (EHRs) from two large demographic groups, encompassing thousands of patients, to identify blood tests such as cholesterol, blood sugar, and specific protein tests that are significantly associated with severe DFUs. Next, we translated the protein components from these blood tests into their ribonucleic acid (RNA) counterparts and analyzed them using public bulk and single-cell RNA sequencing datasets. Using these data, we applied a machine learning pipeline to uncover cell-type-specific and molecular factors associated with varying degrees of DFU severity. Our results showed that several blood test results, such as the Albumin/Creatinine Ratio (ACR) and cholesterol and coagulation tissue factor levels, correlated with DFU severity across key demographic groups. These tests exhibited varying degrees of significance based on demographic differences. Using bulk RNA-Sequenced (RNA-Seq) data, we found that apolipoprotein E (APOE) protein, a component of lipoproteins that are responsible for cholesterol transport and metabolism, is linked to DFU severity. Furthermore, the single-cell RNA-Seq (scRNA-seq) analysis revealed a cluster of cells identified as keratinocytes that showed overexpression of APOE in severe DFU cases. Overall, this study demonstrates how integrating extensive EHRs data with single-cell transcriptomics can refine the search for molecular markers and identify cell-type-specific and molecular factors associated with DFU severity while considering key demographic differences.

Enhancing diabetic foot ulcer healing: Impact of the regulation of the FUS and ILF2 RNA­binding proteins through negative pressure wound therapy.

Diabetic foot ulcer (DFU) is a destructive complication of diabetes. Negative pressure wound therapy (NPWT) promotes DFU wound healing through an undetermined mechanism. In the present study, RNA sequencing was performed on wound granulation tissue from 3 patients with DFU before and after 1 week of NPWT. The fused in sarcoma (FUS) and interleukin enhancer binding factor 2 (ILF2) encoding RNA­binding proteins (RBPs) were screened from the sequencing data, and wound tissue samples from 24 patients with DFU were validated and analyzed before and after receiving NPWT by reverse transcription­quantitative PCR, western blotting and immunohistochemistry. In addition, in vitro and in vivo experiments were conducted to determine the effect of the expression of FUS and ILF2 on the function of human epidermal keratinocyte cells (HaCaT cells) and the healing of diabetic skin wounds. The results indicated that NPWT induced the upregulation of 101 genes and the downregulation of 98 genes in DFU wound granulation tissue. After NPWT, the expression of FUS and ILF2 was significantly upregulated (P<0.05). Pearson's correlation coefficient showed that the changes in FUS and ILF2 before and after NPWT were negatively correlated with changes in white blood cells, the neutrophil percentage, C­reactive protein, tumor necrosis factor­α, reactive oxygen species, lipid peroxides, matrix metalloproteinase (MMP) 2 and MMP9 (P<0.05), but positively correlated with the anti­inflammatory factor, IL­4 (P<0.01). There was also a positive correlation (P<0.05) with the 4­week ulcer healing rate. Additionally, the knockdown of FUS and ILF2 expression inhibited the proliferation and migration of HaCaT cells, while increasing cell apoptosis. In vivo, the knockdown of FUS and ILF2 significantly reduced the rate of skin wound healing in diabetic mice. The results of the present study therefore provide new insights into the mechanism by which NPWT promotes DFU wound healing. In conclusion, the RBPs, FUS and ILF2, promoted DFU wound healing by regulating the function of keratinocytes and reducing the inflammatory response and oxidative stress.

Epigenetic regulation of Nrf2-Mediated angiogenesis in diabetic foot ulcer progression: Role of histone deacetylases.

Nuclear factor E2-related factor 2 (Nrf2), a redox-sensitive transcription factor, regulates proangiogenic mediators, and antioxidant and detoxification enzymes. However, hitherto its regulation in the progression of DFU was poorly examined. The regulation of Nrf2 has been reported to be affected by various factors, including histone deacetylase (HDACs) and DNA methylation. The present study aimed to profile all classes of HDACs and correlate them with Nrf2 and angiogenic markers in the tissue biopsies of different grades of DFU patients (n = 20 in each grade). The gene expression profile of Nrf2 and its downstream targets, angiogenic markers, and all classes of HDACs were assessed using qPCR. Spearman's correlation was performed to analyze the correlation of HDACs with Nrf2 and its downstream targets along with angiogenic markers. We observed a progressive decrease in the gene expression of Nrf2 and angiogenic markers such as VEGF, HIF-1α, and SDF-1α and also an increase in the TSP-2 expression in different grades of DFU. In parallel, a significant downregulation of HDAC2/8 and SIRT1/2/4 has been observed in various grades of DFU subjects. On the other hand, HDAC1/3/4/11 and SIRT3/5/6/7 showed upregulation in different grades of DFU and the maximum increase was observed in Grade 3 patients. A significant negative correlation between Nrf2 and HDAC4, angiogenic markers, and HDAC4 suggested the pivotal role of the HDAC4-regulated Nrf2-mediated angiogenesis among DFU subjects. We have generated a first line of evidence on the epigenetic regulation of Nrf2 and its correlation with angiogenesis in the progression of diabetic foot ulcers.

Identification of CGNL1 as a diagnostic marker in fibroblasts of diabetic foot ulcers: Insights from single cell RNA sequencing and bulk sequencing data.

OBJECTIVES: This study aimed to explore the unique transcriptional feature of fibroblasts subtypes and the role of ferroptosis in diabetic foot ulcers (DFUs). METHODS: The GEO (Gene Expression Omnibus) was searched to obtain the DFUs single-cell and transcriptional datasets. After identifying cell types by classic marker genes, the integrated single-cell dataset was used to run trajectory inference, RNA velocity, and ligand-receptor interaction analysis. Next, bulk RNA-seq datasets of DFUs were analyzed to the key ferroptosis genes. RESULTS: Here, we profile 83529 single transcriptomes from the foot samples utilizing single-cell sequencing (scRNA-seq) data of DFU from GEO database and identified 12 cell types, with fibroblasts exhibiting elevated levels of ferroptosis activity and substantial cellular heterogeneity. Our results defined six main fibroblast subsets that showed mesenchymal, secretory-reticular, secretory-papillary, pro-inflammatory, myogenesis, and healing-enriched functional annotations. Trajectory inference and cell-cell communication analysis revealed two major cell fates with subpopulations of fibroblasts and altered ligand-receptor interactions. Bulk RNA sequencing data identified CGNL1 as a distinctive diagnostic signature in fibroblasts. Notably, CGNL1 positively correlated with pro-inflammatory fibroblasts. CONCLUSIONS: Overall, our analysis delineated the heterogeneity present in cell populations of DFUs, showing distinct fibroblast subtypes characterized by their own unique transcriptional features and enrichment functions. Our study will help us better understand DFUs pathogenesis and identifies CGNL1 as a potential target for DFUs therapies.

Association of vitamin D status and vitamin D receptor polymorphism in diabetic foot ulcer patients: A prospective observational study in a South-Indian tertiary healthcare facility.

Objective of the study was to find the association of vitamin D receptor (VDR) polymorphisms (Fokl, Taql and Apal) with vitamin D levels in diabetic foot ulcer (DFU) patients in South India. In this case-control study, plasma vitamin D levels and VDR genotype frequencies of 70 cases (DFU patients) were compared with 70 diabetic (diabetes mellitus [DM] [non-DFU]) patients and 70 apparently healthy controls (HC) from South India. Plasma vitamin D levels were measured using the ELISA technique, and genotyping of VDR polymorphisms was carried out using real-time polymerase chain reaction. Logistic regression was used to find the association between DFU versus HC and DFU versus DM traits. Association analysis was performed based on additive, dominant and recessive models with age and gender as covariates. A 45.7% of DFU patients have sufficient vitamin D levels than 48.6% and 40% of DM patients and HC, respectively. Linkage disequilibrium analysis for DFU versus HC and DFU versus DM traits shows that single nucleotide polymorphisms (SNPs) Taq1 (rs731236) and Apal (rs7975232) are in strong linkage disequilibrium in DFU patients. The alleles and genotype frequencies were similar in all three groups. Although the additive model does not show statistical significance, age and sex correlate with the three SNPs (Fokl, Taql and Apal). No association was found between VDR gene polymorphisms and vitamin D levels in DFU patients in Southern India. On the other hand, age and sex correlate with the three SNPs.

Angio-microRNAs in diabetic foot ulcer-: Mechanistic insights and clinical perspectives.

Diabetic foot ulcers, as one of the chronic wounds, are a serious challenge in the global healthcare system which have shown notable growth in recent years. DFU is associated with impairment in various stages of wound healing, including angiogenesis. Aberrant expression of microRNAs (miRNAs) involved in the disruption of the balance between angiogenic and anti-angiogenic factors, plays a crucial role in angiogenesis dysfunction. Alteration in the expression of angiomiRNAs (angiomiRs) have the potential to function as biomarkers in chronic wounds. Additionally, considering the rising importance of therapeutic RNAs, there is potential for utilizing angiomiRs in wound healing to induce angiogenesis. This review aims to explore angiogenesis in chronic wounds and investigate the mechanisms mediated by pro- and anti-angiomiRs in the context of diabetic foot ulcers.

A single dose of VEGF-A circular RNA sustains in situ long-term expression of protein to accelerate diabetic wound healing.

Diabetic foot ulcer (DFU), which is characterised by damage to minute blood vessels or capillaries around wounds, is one of the most serious and dreaded complications of diabetes. It is challenging to repair chronic non-healing DFU wounds. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and promotes wound healing in DFU. However, it is difficult to sustainably deliver VEGF to the wound site owing to its poor stability and easy degradation. To overcome this challenge, lipid nanoparticles (LNP) encapsulating circular RNA (circRNA) encoding VEGF-A have been developed to continuously generate and release VEGF-A and accelerate diabetic wound healing. First, VEGF-A circRNA was synthesized using group I intron autocatalysis strategy and confirmed by enzyme digestion, polymerase chain reaction, and sequencing assay. VEGF-A circRNA was encapsulated in ionizable lipid U-105-derived LNP (U-LNP) using microfluidic technology to fabricate U-LNP/VEGF-A circRNA. For comparison, a commercially ionizable lipid ALC-0315-derived LNP (A-LNP) encapsulating circRNA (A-LNP/circRNA) was used. Dynamic light scattering and transmission electron microscopy characterization indicated that U-LNP/circRNA had spherical structure with an average diameter of 108.5 nm, a polydispersity index of 0.22, and a zeta potential of -3.31 mV. The messenger RNA (mRNA) encapsulation efficiency (EE%) of U-LNP was 87.12%. In vitro transfection data confirmed better stability and long-term VEGF-A expression of circRNA compared with linear mRNA. Assessment of cytotoxicity and innate immunity further revealed that U-LNP/circRNA was biocompatible and induced a weak congenital immune response. Cell scratch and angiogenesis tests demonstrated the bioactivity of U-LNP/VEGF-A circRNA owing to its VEGF-A expression. In situ bioluminescence imaging of firefly luciferase (F-Luc) probe and ELISA demonstrated that circRNA had long-term and strong expression of VEGF-A in the first week, and a gradual decrease in the next week at the wound site and surrounding areas. Finally, a diabetic mouse model was used to validate the healing effect of U-LNP/VEGF-A circRNA formulation. The results showed that a single dose of U-LNP/VEGF-A circRNA administered by dripping resulted in almost complete wound recovery on day 12, which was significantly superior to that of U-LNP/VEGF-A linear mRNA, and it also outperformed recombinant human vascular endothelial growth factor (rhVEGF) injection and A-LNP/circRNA dripping. Histological analysis confirmed the healing efficiency and low toxicity of U-LNP/VEGF-A circRNA formulation. Together, VEGF-A circRNA delivered by U-105-derived LNP showed good performance in wound healing, which was ascribed to the long-term expression and continuous release of VEGF-A, and has potential applications for the treatment of diabetic foot ulcer wounds.

miR-200b-3p accelerates diabetic wound healing through anti-inflammatory and pro-angiogenic effects.

The poor healing characteristics of diabetic foot ulcers are partially attributed to diabetes-induced pro-inflammatory wounds. Our previous study reported that both miR-146a-5p and miR-200b-3p decrease endothelial inflammation in human aortic endothelial cells and db/db diabetic mice. Although miR-146a-5p has been reported to improve diabetic wound healing, the role of miR-200b-3p is not clear. This study compared the roles of these miRNAs in diabetic wound healing. Two 8-mm full-thickness wounds were created in 12-week-old male db/db mice on the left and right back. After surgery, 100 ng miR-146a-5p, miR-200b-3p, or miR-negative control (NC) was injected in each wound. Full-thickness skin samples were harvested from mice at the 14th day for real-time polymerase chain reaction and immunohistochemistry analyses. At the 14th day, the miR-200b-3p group showed better wound healing and greater granulation tissue thickness than the miR-146a-5p group. The miR-200b-3p group showed a significant decrease of IL-6 and IL-1ß gene expression and a significant increase of Col3α1 gene expression compared to those in the miR-NC group. The miR-200b-3p group had the lowest gene expression of TGF-ß1, followed by the miR-146a-5p and miR-NC groups. Our findings suggest that the miR-200b-3p group had better healing characteristics than the other two groups. Immunohistochemical staining revealed that CD68 immunoreactivity was significantly decreased in both the miR-146a-5p and miR-200b-3p groups compared with that in the miR-NC group. In addition, CD31 immunoreactivity was significantly higher in the miR-200b-3p group than in the miR-146a-5p group. In conclusion, these results suggest that miR-200b-3p is more effective than miR-146a-5p in promoting diabetic wound healing through its anti-inflammatory and pro-angiogenic effects.

Therapeutic potential of microRNA-engineered exosomes in diabetic wound healing: a meta-analysis.

Diabetic wounds, a prevalent diabetes complication, pose significant challenges in treatment. MicroRNA-engineered exosomes (miR-exo) are a promising new treatment for diabetic wounds; however, their mechanism remains to be completely understood. Therefore, we aimed to conduct a meta-analysis to evaluate the efficacy of miR-exo treatment in the management of diabetic wounds. To achieve this aim, academic databases, including PubMed, Embase, Web of Science, and the Cochrane Library, were searched for papers published before July 4, 2023. Outcome indicators (e.g., rate of wound healing, neovascular count, rate of re-epithelialization, deposition of collagen, breadth of scar, and inflammatory factors) were assessed. Six studies (total of 72 animals) met inclusion criteria and were analyzed. The amalgamated data revealed that miR-exo treatment exhibited superior results compared to those of control therapy. miR-exo treatment significantly enhanced the rate of wound healing, increased the number of neovascular formations, accelerated the rate of re-epithelialization, increased collagen deposition, reduced scar width, while significantly downregulating the expression of inflammatory factors. Our findings indicate that miR-exo treatment augments overall diabetic wound healing, especially when administered in conjunction with innovative dressings. To ascertain the optimal parameters for miR-exo treatment in managing diabetic wounds, future studies must encompass rigorous, large-scale, double-blinded clinical trials while incorporating long-term follow-up assessments for enhanced reliability and accuracy.

Inhibition of CYP1A1 expression enhances diabetic wound healing by modulating inflammation and oxidative stress in a rat model.

OBJECTIVE: Wound therapies utilizing gene delivery to the skin offer considerable promise owing to their localized treatment benefits and straightforward application. This study investigated the impact of skin electroporation of CYP1A1 shRNA lentiviral particles on diabetic wound healing in a streptozotocin (STZ)-induced rat model. METHODS: Male Sprague Dawley (SD) rats were made diabetic by injecting STZ and subsequently creating foot skin wounds. The rats were randomly divided into four groups: normal, diabetic foot ulcers (DFU), DFU + control shRNA (electroporation of control shRNA lentiviral particles), and DFU + CYP1A1 shRNA (electroporation of CYP1A1 shRNA lentiviral particles). Wound healing progress was monitored at multiple time points (0, 1, 3, 5, 7, 10, 14 days). On day 14, wound tissue specimens were collected for histological examination. Wound samples collected at days 7 and 14 were used for gene expression analysis via qRT-PCR, assessment of CYP1A1 protein levels using western blotting, and evaluation of oxidative stress markers. RESULTS: Treatment with CYP1A1 shRNA significantly enhanced diabetic wound healing rates compared to untreated controls over the observation period. Histological analysis revealed improved wound characteristics in the CYP1A1 shRNA-treated group, including enhanced epithelial regeneration, reduced inflammation, and increased collagen deposition, indicative of improved tissue repair. Furthermore, suppression of CYP1A1 corresponded with decreased expression levels of pro-inflammatory cytokines (interleukin-1ß, tumor necrosis factor-α, and interleukin-6) and diminished oxidative stress markers (malondialdehyde, superoxide dismutase) within wound tissues. CONCLUSION: Targeted suppression of CYP1A1 represents a promising therapeutic strategy to enhance diabetic wound healing by modulating inflammation and oxidative stress.

Key extracellular proteins and TF-miRNA co-regulatory network in diabetic foot ulcer: Bioinformatics and experimental insights.

BACKGROUND: Diabetic foot ulcers (DFUs), a serious complication of diabetes, are associated with abnormal extracellular protein (EP) metabolism. The identification of key EPs and their regulatory networks is crucial for the understanding of DFU formation and development of effective treatments. In this study, a large-scale bioinformatics analysis was conducted to identify potential therapeutic targets and experimental validation was performed to ensure the reliability and biological relevance of the findings. METHODS: Due to the comprehensive profiling of DFU samples provided by the GSE80178 dataset, we initially selected it to derive differentially expressed genes (DEGs) associated with DFU. Subsequently, utilizing the UniProt database and annotated EP list from the Human Protein Atlas annotation database, we screened for extracellular protein-related differentially expressed genes (EP-DEGs) due to their crucial role in the pathogenesis and healing of DFU. We examined EP-DEG pathway enrichment and protein-protein interaction networks, analyzed paired full-thickness skin tissue samples from 24 patients with DFUs and healthy controls, and performed polymerase chain reaction (PCR) experiments to validate candidate genes. Ultimately, we constructed a transcription factor (TF)-microRNA (miRNA)-hub gene co-regulatory network to explore upstream and downstream regulatory connections based on validated DEGs. RESULTS: Four crucial candidate genes (FMOD, LUM, VCAN, and S100A12) were identified and verified via PCR analysis. The TF-miRNA-hub EP-DEG regulatory network contained the pivotal TFs TRIM28 and STAT3 and the miRNAs hsa-mir-20a-5p, hsa-miR-21, and hsa-miR-203. CONCLUSION: The findings of this study advance our understanding of the pathology of DFU by defining key roles of specific EPs and elucidating a comprehensive regulatory network. These insights pave the way for novel approaches to improve DFU treatment outcomes.

Combined analysis of single-cell sequencing and bulk transcriptome sequencing reveals new mechanisms for non-healing diabetic foot ulcers.

Diabetic foot ulcers (DFUs) pose a significant challenge in diabetes care. Yet, a comprehensive understanding of the underlying biological disparities between healing and non-healing DFUs remains elusive. We conducted bioinformatics analysis of publicly available transcriptome sequencing data in an attempt to elucidate these differences. Our analysis encompassed differential analysis to unveil shifts in cell composition and gene expression profiles between non-healing and healing DFUs. Cell communication alterations were explored employing the Cellchat R package. Pseudotime analysis and cytoTRACE allowed us to dissect the heterogeneity within fibroblast subpopulations. Our findings unveiled disruptions in various cell types, localized low-grade inflammation, compromised systemic antigen processing and presentation, and extensive extracellular matrix signaling disarray in non-healing DFU patients. Some of these anomalies partially reverted in healing DFUs, particularly within the abnormal ECM-receptor signaling pathway. Furthermore, we distinguished distinct fibroblast subpopulations in non-healing and healing DFUs, each with unique biological functions. Healing-associated fibroblasts exhibited heightened extracellular matrix (ECM) remodeling and a robust wound healing response, while non-healing-associated fibroblasts showed signs of cellular senescence and complement activation, among other characteristics. This analysis offers profound insights into the wound healing microenvironment, identifies pivotal cell types for DFU healing promotion, and reveals potential therapeutic targets for DFU management.

Analysis of Circulating miRNA Expression Profiles in Type 2 Diabetes Patients with Diabetic Foot Complications.

Type 2 diabetes mellitus (T2DM) is associated with various complications, including diabetic foot, which can lead to significant morbidity and mortality. Non-healing foot ulcers in diabetic patients are a major risk factor for infections and amputations. Despite conventional treatments, which have limited efficacy, there is a need for more effective therapies. MicroRNAs (miRs) are small non-coding RNAs that play a role in gene expression and have been implicated in diabetic wound healing. miR expression was analyzed through RT-qPCR in 41 diabetic foot Mexican patients and 50 controls. Diabetic foot patients showed significant increases in plasma levels of miR-17-5p (p = 0.001), miR-191-5p (p = 0.001), let-7e-5p (p = 0.001), and miR-33a-5p (p = 0.005) when compared to controls. Elevated levels of miR-17, miR-191, and miR-121 correlated with higher glucose levels in patients with diabetic foot ulcers (r = 0.30, p = 0.004; r = 0.25, p = 0.01; and r = 0.21, p = 0.05, respectively). Levels of miR-17 showed the highest diagnostic potential (AUC 0.903, p = 0.0001). These findings underscore the possible role of these miRs in developing diabetes complications. Our study suggests that high miR-17, miR-191, and miR-121 expression is strongly associated with higher glucose levels and the development of diabetic foot ulcers.

Comprehensive transcriptomic analysis of immune-related genes in diabetic foot ulcers: New insights into mechanisms and therapeutic targets.

BACKGROUND: Diabetic foot ulcers (DFU), affecting a quarter of diabetic patients and leading to high rates of amputation and mortality, pose significant health and economic burdens. Wound healing in DFU is often compromised by chronic inflammation, underscoring the critical role of immune cells. However, the systematic investigation of immune-related genes (IRGs) in DFU pathogenesis remains elusive. To address this gap, our study aims to explore the association between IRGs and DFU. METHODS: To explore biological changes in immune related gene expression in DFU, RNA-seq was performed on wound biopsies derived from 10 DFU patients and 11 healthy controls. Differentially expressed genes (DEGs) between DFU and normal samples were obtained by DEseq2. By intersecting the IRG list from the ImmPort database, the immune-related differentially expressed genes were identified. Function enrichment analysis and protein-protein interaction (PPI) analysis were applied by clusterProfiler and STRING database, and the hub genes of the PPI network were calculated by the cytoHubba plug-ins in Cytoscape. CIBERSORT algorithms was applied to analyze immune infiltration in DFU. And the correlation between immune cells infiltration and hub genes was explored by correlation analysis. Finally, to validate our findings, the transcriptional change of hub genes in DFU was confirmed using external scRNA-seq dataset and RT-qPCR. RESULTS: RNA-seq analysis detected 8,800 DEGs in DFUs, with 2,351 upregulated and 6,449 downregulated.526 differential IRGs were obtained from intersection of DEGs and IRGs. 526 differential IRGs were obtained from intersection of DEGs and IRGs. Enrichment function analysis of DEGs showed that they played a significant role in immune response. The PPI network was constructed, and the most significant module containing 4 hub genes was identified. CIBERSORT analysis showing that there was a significant difference between DFU and normal controls in the infiltration of immune cells. Compared with normal tissue, DFU tissue contained a higher proportion of resting NK cell, M0 macrophages, and activated mast cell, while resting dendritic cell, activated mast cell, and activated NK cell contributed to a relatively lower portion. Additionally, the analysis for M1/M2 polarization of macrophage cells shown that DFU tissue contained a higher M1/M2 ratio than normal group. Finally, the expression levels of 4 hub genes were confirmed by external scRNA-seq dataset and RT-qPCR. CONCLUSIONS: The immune related hub genes and the difference in immune infiltration between DFU tissue and normal controls might provide new insight for understanding DFU healing.

Exosomal miRNA-26b-5p from PRP suppresses NETs by targeting MMP-8 to promote diabetic wound healing.

The utilization of platelet-rich plasma (PRP) has exhibited potential as a therapeutic approach for the management of diabetic foot ulcers (DFUs). However, it is currently not well understood how the diabetic environment may influence PRP-derived exosomes (PRP-Exos) and their potential impact on neutrophil extracellular traps (NETs). This study aims to investigate the effects of the diabetic environment on PRP-Exos, their communication with neutrophils, and the subsequent influence on NETs and wound healing. Through bulk-seq and Western blotting, we confirmed the increased expression of MMP-8 in DFUs. Additionally, we discovered that miRNA-26b-5p plays a significant role in the communication between DFUs and PRP-Exos. In our experiments, we found that PRP-Exos miR-26b-5p effectively improved diabetic wound healing by inhibiting NETs. Further tests validated the inhibitory effect of miR-26b-5p on NETs by targeting MMP-8. Both in vitro and in vivo experiments showed that miRNA-26b-5p from PRP-Exos promoted wound healing by reducing neutrophil infiltration through its targeting of MMP-8. This study establishes the importance of miR-26b-5p in the communication between DFUs and PRP-Exos, disrupting NETs formation in diabetic wounds by targeting MMP-8. These findings provide valuable insights for developing novel therapeutic strategies to enhance wound healing in individuals suffering from DFUs.

Development of novel lysosome-related signatures and their potential target drugs based on bulk RNA-seq and scRNA-seq for diabetic foot ulcers.

BACKGROUND: Diabetic foot ulcers (DFU) is the most serious complication of diabetes mellitus, which has become a global health problem due to its high morbidity and disability rates and the poor efficacy of conventional treatments. Thus, it is urgent to identify novel molecular targets to improve the prognosis and reduce disability rate in DFU patients. RESULTS: In the present study, bulk RNA-seq and scRNA-seq associated with DFU were downloaded from the GEO database. We identified 1393 DFU-related DEGs by differential analysis and WGCNA analysis together, and GO/KEGG analysis showed that these genes were associated with lysosomal and immune/inflammatory responses. Immediately thereafter, we identified CLU, RABGEF1 and ENPEP as DLGs for DFU using three machine learning algorithms (Randomforest, SVM-RFE and LASSO) and validated their diagnostic performance in a validation cohort independent of this study. Subsequently, we constructed a novel artificial neural network model for molecular diagnosis of DFU based on DLGs, and the diagnostic performance in the training and validation cohorts was sound. In single-cell sequencing, the heterogeneous expression of DLGs also provided favorable evidence for them to be potential diagnostic targets. In addition, the results of immune infiltration analysis showed that the abundance of mainstream immune cells, including B/T cells, was down-regulated in DFUs and significantly correlated with the expression of DLGs. Finally, we found latamoxef, parthenolide, meclofenoxate, and lomustine to be promising anti-DFU drugs by targeting DLGs. CONCLUSIONS: CLU, RABGEF1 and ENPEP can be used as novel lysosomal molecular signatures of DFU, and by targeting them, latamoxef, parthenolide, meclofenoxate and lomustine were identified as promising anti-DFU drugs. The present study provides new perspectives for the diagnosis and treatment of DFU and for improving the prognosis of DFU patients.

A site-specific phosphorylation in FSTL1 determines its promigratory role in wound healing.

Follistatin like-1 (FSTL-1) is a secreted glycoprotein of mesenchymal in origin. In human skin, FSTL1 is upregulated in the epidermal keratinocytes upon acute injury and is required for the migration of keratinocytes. Failure to upregulate FSTL1 leads to the lack of keratinocyte migration and the non-healing nature of diabetic foot ulcer (DFU). FSTL1 undergoes extensive post-translational modification (PTM) at specific residues. Glycosylation at N144, N175 and N180, are the only experimentally demonstrated PTM in FSTL1, wherein, N180 and N144 glycosylations have been found to be critical for its function in cardiac tissue regeneration and pre-adipocyte differentiation, respectively. However, it is not known if PTMs other than glycosylation occurs in FSTL1 and how it impacts its pro-migratory function. Using in-silico analysis of mass spectrometric datasets, we found a novel PTM, namely, Serine 165 (S165) phosphorylation in FSTL1. To address the role of S165 phosphorylation in its pro-migratory function, a phosphorylation defective mutant of FSTL1 (S165A) was constructed by converting serine 165 to alanine and over expressed in 293T cells. S165A mutation did not affect the secretion of FSTL1 in vitro. However, S165A abolished the pro-migratory effect of FSTL1 in cultured keratinocytes likely via its inability to facilitate ERK signaling pathway. Interestingly, bacterially expressed recombinant FSTL1, trans-dominantly inhibited wound closure in keratinocytes highlighting the prime role of FSTL1 phosphorylation for its pro-migratory function. Further, under high glucose conditions, which inhibited scratchwound migration of keratinocytes, we noticed a significant decrease in S165 phosphorylation. Taken together, our results reveal a hitherto unreported role of FSTL1 phosphorylation PTM with profound implications in wound healing.

PROS1 is a crucial gene in the macrophage efferocytosis of diabetic foot ulcers: a concerted analytical approach through the prisms of computer analysis.

BACKGROUND: Diabetic foot ulcers (DFUs) pose a serious long-term threat because of elevated mortality and disability risks. Research on its biomarkers is still, however, very limited. In this paper, we have effectively identified biomarkers linked with macrophage excretion in diabetic foot ulcers through the application of bioinformatics and machine learning methodologies. These findings were subsequently validated using external datasets and animal experiments. Such discoveries are anticipated to offer novel insights and approaches for the early diagnosis and treatment of DFU. METHODS: In this work, we used the Gene Expression Omnibus (GEO) database's datasets GSE68183 and GSE80178 as the training dataset to build a gene model using machine learning methods. After that, we used the training and validation sets to validate the model (GSE134431). On the model genes, we performed enrichment analysis using both gene set variant analysis (GSVA) and gene set enrichment analysis (GSEA). Additionally, the model genes were subjected to immunological association and immune function analyses. RESULTS: In this study, PROS1 was identified as a potential key target associated with macrophage efflux in DFU by machine learning and bioinformatics approaches. Subsequently, the key biomarker status of PROS1 in DFU was also confirmed by external datasets. In addition, PROS1 also plays a key role in macrophage exudation in DFU. This gene may be associated with macrophage M1, CD4 memory T cells, naïve B cells, and macrophage M2, and affects IL-17, Rap1, hedgehog, and JAK-STAT signaling pathways. CONCLUSIONS: PROS1 was identified and validated as a biomarker for DFU. This finding has the potential to provide a target for macrophage clearance of DFU.

Identification and clinical validation of the role of anoikis-related genes in diabetic foot.

This study aims to investigate the role of anoikis-related genes in diabetic foot (DF) by utilizing bioinformatics analysis to identify key genes associated with anoikis in DF. We selected the GEO datasets GSE7014, GSE80178 and GSE68183 for the extraction and analysis of differentially expressed anoikis-related genes (DE-ARGs). GO analysis and KEGG analysis indicated that DE-ARGs in DF were primarily enriched in apoptosis, positive regulation of MAPK cascade, anoikis, focal adhesion and the PI3K-Akt signalling pathway. Based on the LASSO and SVM-RFE algorithms, we identified six characteristic genes. ROC curve analysis revealed that these six characteristic genes had an area under the curve (AUC) greater than 0.7, indicating good diagnostic efficacy. Expression analysis in the validation set revealed downregulation of CALR in DF, consistent with the training set results. GSEA results demonstrated that CALR was mainly enriched in blood vessel morphogenesis, endothelial cell migration, ECM-receptor interaction and focal adhesion. The HPA database revealed that CALR was moderately enriched in endothelial cells, and CALR was found to interact with 63 protein-coding genes. Functional analysis with DAVID suggested that CALR and associated genes were enriched in the phagosome component. CALR shows promise as a potential marker for the development and treatment of DF.

HMOX1 as a therapeutic target associated with diabetic foot ulcers based on single-cell analysis and machine learning.

Diabetic foot ulcers (DFUs) are a serious chronic complication of diabetes mellitus and a leading cause of disability and death in diabetic patients. However, current treatments remain unsatisfactory. Although macrophages are associated with DFU, their exact role in this disease remains uncertain. This study sought to detect macrophage-related genes in DFU and identify possible therapeutic targets. Single-cell datasets (GSE223964) and RNA-seq datasets (GSM68183, GSE80178, GSE134431 and GSE147890) associated with DFU were retrieved from the gene expression omnibus (GEO) database for this study. Analysis of the provided single-cell data revealed the distribution of macrophage subpopulations in the DFU. Four independent RNA-seq datasets were merged into a single DFU cohort and further analysed using bioinformatics. This included differential expression (DEG) analysis, multiple machine learning algorithms to identify biomarkers and enrichment analysis. Finally, key results were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western bolt. Finally, the findings were validated using RT-qPCR and western blot. We obtained 802 macrophage-related genes in single-cell analysis. Differential expression analysis yielded 743 DEGs. Thirty-seven macrophage-associated DEGs were identified by cross-analysis of marker genes with macrophage-associated DEGs. Thirty-seven intersections were screened and cross-analysed using four machine learning algorithms. Finally, HMOX1 was identified as a potentially valuable biomarker. HMOX1 was significantly associated with biological pathways such as the insulin signalling pathway. The results showed that HMOX1 was significantly overexpressed in DFU samples. In conclusion, the analytical results of this study identified HMOX1 as a potentially valuable biomarker associated with macrophages in DFU. The results of our analysis improve our understanding of the mechanism of macrophage action in this disease and may be useful in developing targeted therapies for DFU.