An integrated method for IgG N-glycans enrichment and analysis: Understanding the role of IgG glycosylation in diabetic foot ulcer.

Diabetic foot ulcer (DFU) is the most common and serious complication of diabetes, and its incidence, disability, and mortality rates are increasing worldwide. The pathogenesis of DFU is associated with dysregulated inflammation mediated by abnormal immunoglobulin G (IgG) glycosylation. In this study, we developed a comprehensive method for IgG N-linked glycosylation in the serum of DFU patients. Through analysis, we identified 31 IgG1 glycans, 32 IgG2 glycans, and 30 IgG4 glycans in the DFU serum. Furthermore, 13 IgG1 glycans, 12 IgG2 glycans, and 5 IgG4 glycans in the DFU groups were found to be significantly different from those of the control groups (p < 0.05). Of these, compared with the control group, one glycan was unique to DFU patients, and seven glycans were not detected in the DFU group. In terms of glycan characteristics, we observed a substantial decrease in galactosylation, sialylation and bisecting GlcNAcylation, and a significant increase in agalactosylation. Abnormal IgG N-glycosylation modifications were significantly associated with the chronic inflammation that is characteristic of DFU. Further, this is the first comprehensive analysis of subclass-specific IgG N-glycosylation in DFU patients, which not only fills the gap of DFU in terms of the pathological mechanisms related to IgG glycosylation but also may provide valuable clues for the immunotherapeutic pathway of DFU.

Clinical Study on the Efficacy of Silver Ion Dressing Combined with Prontosan Gel Dressing in the Treatment of Diabetic Foot Ulcers and the Effect on Serum Inflammatory Factors.

Diabetic foot ulcers (DFUs) have a high disability rate and have a great impact on patients and society, and the search for effective and economical treatment options is a major clinical concern. In this study, 112 patients with DFU admitted to two hospitals from October 2018 to November 2020 were randomly divided into 56 cases each in the single group treated with Prontosan gel dressing and the joint group treated on silver ion dressing combined with Prontosan gel dressing. Both groups of patients were evaluated for efficacy after 30 days of treatment. The number of days for debridement, granulation tissue growth time, epithelial tissue formation time, and wound healing time were observed and recorded in both groups. The trauma area, visual analogue score (VAS), and levels of inflammatory factors such as vascular endothelial adhesion molecule-1 (VCAM-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP) were recorded before and after treatment in both groups. The occurrence of adverse reactions such as edema, fever, infection, and rash during treatment was recorded in both groups for safety assessment. Comparison of the abovementioned data showed that the clinical efficacy of the joint group was significantly higher than that of the single group. The number of days to clear wounds, granulation tissue growth time, epithelial tissue formation time, and wound healing time were significantly lower in the joint group than in the single group. The trauma area, VAS score, VCAM-1, IL-6, TNF-α, and CRP levels decreased in both groups after treatment compared with the pretreatment levels, with the joint group being lower than the single group. The results also showed that the difference in the overall incidence of adverse reactions between the two groups was not statistically significant, and the incidence was low and transient. In addition to the usual treatment regimen of blood glucose control and improvement of microcirculation for patients with DFU, combined treatment with silver ionomer dressings and Prontosan gel dressings can promote ulcer healing and improve foot wound regression. It has a stronger antibacterial effect and can more effectively reduce the inflammatory response of the ulcerated surface with fewer adverse effects, making it an effective and safe method for the treatment of DFU, and has implications for promotion.

miR-19a/b and miR-20a Promote Wound Healing by Regulating the Inflammatory Response of Keratinocytes.

Persistent and impaired inflammation impedes tissue healing and is a characteristic of chronic wounds. A better understanding of the mechanisms controlling wound inflammation is needed. In this study, we show that in human wound-edge keratinocytes, the expressions of microRNA (miR)-17, miR-18a, miR-19a, miR-19b, and miR-20a, which all belong to the miR-17∼92 cluster, are upregulated during wound repair. However, their levels are lower in chronic ulcers than in acute wounds at the proliferative phase. Conditional knockout of miR-17∼92 in keratinocytes as well as injection of miR-19a/b and miR-20a antisense inhibitors into wound edges enhanced inflammation and delayed wound closure in mice. In contrast, conditional overexpression of the miR-17∼92 cluster or miR-19b alone in mice keratinocytes accelerated wound closure in vivo. Mechanistically, miR-19a/b and miR-20a decreased TLR3-mediated NF-κB activation by targeting SHCBP1 and SEMA7A, respectively, reducing the production of inflammatory chemokines and cytokines by keratinocytes. Thus, miR-19a/b and miR-20a being crucial regulators of wound inflammation, the lack thereof may contribute to sustained inflammation and impaired healing in chronic wounds. In line with this, we show that a combinatory treatment with miR-19b and miR-20a improved wound healing in a mouse model of type 2 diabetes.

Circular RNA hsa_circ_0084443 Is Upregulated in Diabetic Foot Ulcer and Modulates Keratinocyte Migration and Proliferation.

Objective: Insufficient knowledge about the molecular pathology of diabetic foot ulcer (DFU) impedes the development of effective wound treatment. Circular RNAs (circRNAs) are a novel class of RNA recently discovered to be widely expressed and have important biological functions; however, their role in skin wound healing remains largely unexplored. In this study, we investigated the role of circRNAs in DFU. Approach: CircRNA expression was profiled in normal wounds (NWs) and DFUs by microarray analysis, and hsa_circ_0084443 was identified as differentially expressed. The circularity and subcellular localization of hsa_circ_0084443 were characterized by northern blotting, real-time PCR, and fluorescence in situ hybridization. Cell migration, cell growth, and the transcriptome of human primary keratinocytes were analyzed after overexpression or RNA interference of hsa_circ_0084443. Results: hsa_circ_0084443 is downregulated in NWs compared with intact skin, and its level is higher in DFUs than NWs. We confirmed its circularity and presence in the cytoplasm of human epidermal keratinocytes. We showed that hsa_circ_0084443 reduced motility while enhancing the growth of keratinocytes. Furthermore, we identified a gene network with the potential to mediate the biological effect of hsa_circ_0084443. Innovation: CircRNAs have a functional role and a potential clinical significance in skin wound healing. Conclusions: We identified hsa_circ_0084443, a circRNA downregulated during NW healing, as a negative regulator of keratinocyte migration. Higher levels of hsa_circ_0084443 were detected in DFU samples, suggesting that it plays a role in pathology. These findings pave the way to understanding the functional role of circRNAs in human skin wound healing.

A Randomized, Double-Blind, Placebo-Controlled Phase II Study of Hepatocyte Growth Factor in the Treatment of Critical Limb Ischemia.

NL003 is a plasmid engineered to simultaneously express two isoforms of hepatocyte growth factor. This phase II study was performed to assess the clinical safety and efficacy of intramuscular injection of NL003 in critical limb ischemia (CLI) patients for 6 months. Two hundred patients (Rutherford scale 4-5) were randomly assigned: placebo (n = 50), low-dose NL003 (n = 50), middle-dose NL003 (n = 50), or high-dose NL003 (n = 50). The drug was administered in the affected limb of 197 patients on days 0, 14, and 28. No significant differences in the incidence of adverse events (AEs) or serious AEs were found among the groups. At 6 months, pain severity was significantly reduced in all NL003 groups, but not in the placebo group (p < 0.05). The proportion of patients with complete ulcer healing in the high-dose group was significantly higher than that of the placebo group (p = 0.0095). There were no statistically significant differences in transcutaneous oxygen pressure (TcPO2), ankle-brachial index (ABI), or toe-brachial index (TBI) value among the four groups throughout the study period. These results provide the first effective evidence of significant improvements in total healing of ulcers in treated legs, complete pain relief without analgesics, and safety for NL003 in patients with Rutherford stage 4-5.

Effect of miR-181a-3p on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting BMP10.

Objective: To explore the regulation relationship between miR-181a-3p and BMP10, and their mechanism of osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs).Methods: After osteogenic induction of MSCs, the ALP activity was detected by ELISA. The expression of miRNA-181a-3p and BMP10 was detected by RT-qPCR, and the protein levels of BMP10 and osteogenic differentiation marker proteins ALK and RUNX2 were detected by Western blot. The TargetScan online website was used to predict the putative target of miR-181a-3p, and dual luciferase reporter assay was performed to validate the targeting relationship between miR-181a-3p and BMP10.Results: In osteogenic differentiation of MSCs, ALP activity, the level of ALK and RUNX2 was evidently increased (p < .05), and the expression of miR-181a-3p was significantly downregulated (p < .05). Moreover, overexpression of miR-181a-3p obviously decreased the expression of BMP10 (p < .05), miR-181a-3p knockdown increased the expression of BMP10 prominently (p < .05). The transfection of miR-181a-3p mimics resulted in significantly downregulation of ALP activity and RUNX2 protein expression in MSCs (p < .05). In addition, overexpression of BMP10 could reverse the inhibitory effect of miR-181a-3p on osteogenic differentiation (p < .05).Conclusions: In conclusion, we found that miR-181a-3p inhibited osteogenic differentiation of MCSs by targeting BMP10.

Human skin long noncoding RNA WAKMAR1 regulates wound healing by enhancing keratinocyte migration.

An increasing number of studies reveal the importance of long noncoding RNAs (lncRNAs) in gene expression control underlying many physiological and pathological processes. However, their role in skin wound healing remains poorly understood. Our study focused on a skin-specific lncRNA, LOC105372576, whose expression was increased during physiological wound healing. In human nonhealing wounds, however, its level was significantly lower compared with normal wounds under reepithelialization. We characterized LOC105372576 as a nuclear-localized, RNAPII-transcribed, and polyadenylated lncRNA. In keratinocytes, its expression was induced by TGF-ß signaling. Knockdown of LOC105372576 and activation of its endogenous transcription, respectively, reduced and increased the motility of keratinocytes and reepithelialization of human ex vivo skin wounds. Therefore, LOC105372576 was termed "wound and keratinocyte migration-associated lncRNA 1" (WAKMAR1). Further study revealed that WAKMAR1 regulated a network of protein-coding genes important for cell migration, most of which were under the control of transcription factor E2F1. Mechanistically, WAKMAR1 enhanced E2F1 expression by interfering with E2F1 promoter methylation through the sequestration of DNA methyltransferases. Collectively, we have identified a lncRNA important for keratinocyte migration, whose deficiency may be involved in the pathogenesis of chronic wounds.

WAKMAR2, a Long Noncoding RNA Downregulated in Human Chronic Wounds, Modulates Keratinocyte Motility and Production of Inflammatory Chemokines.

Chronic wounds represent a major and growing health and economic burden worldwide. A better understanding of molecular mechanisms of normal as well as impaired wound healing is needed to develop effective treatment. Herein we studied the potential role of long noncoding RNA LOC100130476 in skin wound repair. LOC100130476 is an RNA polymerase II-encoded polyadenylated transcript present in both cytoplasm and nucleus. We found that its expression was lower in wound-edge keratinocytes of human chronic wounds compared to normal wounds of healthy donors and intact skin. In cultured keratinocytes, LOC100130476 expression was induced by TGF-ß signaling. By reducing LOC100130476 expression with antisense oligos or activating its transcription with CRISPR/Cas9 Synergistic Activation Mediator system, we showed that LOC100130476 restricted the production of inflammatory chemokines by keratinocytes, while enhancing cell migration. In line with this, knockdown of LOC100130476 impaired re-epithelization of human ex vivo wounds. Based on these results, we named LOC100130476 wound and keratinocyte migration-associated long noncoding RNA 2 (WAKMAR2). Moreover, we identified a molecular network that may mediate the biological function of WAKMAR2 in keratinocytes using microarray. In summary, our data suggest that WAKMAR2 is an important regulator of skin wound healing and its deficiency may contribute to the pathogenesis of chronic wounds.

MicroRNA-132 with Therapeutic Potential in Chronic Wounds.

Chronic wounds represent a major and rising health and economic burden worldwide. There is a continued search toward more effective wound therapy. We found significantly reduced microRNA-132 (miR-132) expression in human diabetic ulcers compared with normal skin wounds and also in skin wounds of leptin receptor-deficient (db/db) diabetic mice compared with wild-type mice. Local replenishment of miR-132 in the wounds of db/db mice accelerated wound closure effectively, which was accompanied by increased proliferation of wound edge keratinocytes and reduced inflammation. The pro-healing effect of miR-132 was further supported by global transcriptome analysis, which showed that several inflammation-related signaling pathways (e.g., NF-κB, NOD-like receptor, toll-like receptor, and tumor necrosis factor signaling pathways) were the top ones regulated by miR-132 in vivo. Moreover, we topically applied liposome-formulated miR-132 mimics mixed with pluronic F-127 gel on human ex vivo skin wounds, which promoted re-epithelialization. Together, our study showed the therapeutic potential of miR-132 in chronic wounds, which warrants further evaluation in controlled clinical trials.