Single cell transcriptomic landscape of diabetic foot ulcers.
ABSTRACT: Diabetic foot ulceration (DFU) is a devastating complication of diabetes whose pathogenesis remains incompletely understood. Here, we profile 174,962 single cells from the foot, forearm, and peripheral blood mononuclear cells using single-cell RNA sequencing. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 and increased M1 macrophage polarization in the DFU patients with healing wounds. Further, analysis of spatially separated samples from the same patient and spatial transcriptomics reveal preferential localization of these healing associated fibroblasts toward the wound bed as compared to the wound edge or unwounded skin. Spatial transcriptomics also validates our findings of higher abundance of M1 macrophages in healers and M2 macrophages in non-healers. Our analysis provides deep insights into the wound healing microenvironment, identifying cell types that could be critical in promoting DFU healing, and may inform novel therapeutic approaches for DFU treatment.
Project description:Current chronic wound treatments often fail to promote healing of diabetic foot ulcers (DFU), leading to amputation and increased patient morbidity. A critical mediator of proper wound healing is the production, assembly, and remodeling of the extracellular matrix (ECM) by fibroblasts. However, little is known about how these processes are altered in fibroblasts within the DFU microenvironment. Thus, we investigated the capacity of multiple, primary DFU-derived fibroblast strains to express, produce, and assemble ECM proteins compared to diabetic patient-derived fibroblasts and healthy donor-derived fibroblasts. Gene expression microarray analysis showed differential expression of ECM and ECM-regulatory genes by DFU-derived fibroblasts which translated to functional differences in a 3D in vitro ECM tissue model. DFU-derived fibroblasts produced thin, fibronectin-rich matrices, and responded abnormally when challenged with transforming growth factor-beta, a key regulator of matrix production during healing. These results provide novel evidence that DFU-derived fibroblasts contribute to the defective matrices of DFUs and chronic wound pathogenesis.
Project description:Diabetic foot ulcers (DFU) are a major, debilitating complication of diabetes mellitus. Unfortunately, many DFUs are refractory to existing treatments and frequently lead to amputation. The development of more effective therapies has been hampered by the lack of predictive in vitro methods to investigate the mechanisms underlying impaired healing. To address this need for realistic wound-healing models, we established patient-derived fibroblasts from DFUs and site-matched controls and used them to construct three-dimensional (3D) models of chronic wound healing. Incorporation of DFU-derived fibroblasts into these models accurately recapitulated the following key aspects of chronic ulcers: reduced stimulation of angiogenesis, increased keratinocyte proliferation, decreased re-epithelialization, and impaired extracellular matrix deposition. In addition to reflecting clinical attributes of DFUs, the wound-healing potential of DFU fibroblasts demonstrated in this suite of models correlated with in vivo wound closure in mice. Thus, the reported panel of 3D DFU models provides a more biologically relevant platform for elucidating the cell-cell and cell-matrix-related mechanisms responsible for chronic wound pathogenesis and may improve translation of in vitro findings into efficacious clinical applications.
Project description:Diabetic foot ulcers (DFUs) are a major complication of diabetes, and there is a critical need to develop novel cell- and tissue-based therapies to treat these chronic wounds. Induced pluripotent stem cells (iPSCs) offer a replenishing source of allogeneic and autologous cell types that may be beneficial to improve DFU wound-healing outcomes. However, the biologic potential of iPSC-derived cells to treat DFUs has not, to our knowledge, been investigated. Toward that goal, we have performed detailed characterization of iPSC-derived fibroblasts from both diabetic and nondiabetic patients. Significantly, gene array and functional analyses reveal that iPSC-derived fibroblasts from both patients with and those without diabetes are more similar to each other than were the primary cells from which they were derived. iPSC-derived fibroblasts showed improved migratory properties in 2-dimensional culture. iPSC-derived fibroblasts from DFUs displayed a unique biochemical composition and morphology when grown as 3-dimensional (3D), self-assembled extracellular matrix tissues, which were distinct from tissues fabricated using the parental DFU fibroblasts from which they were reprogrammed. In vivo transplantation of 3D tissues with iPSC-derived fibroblasts showed they persisted in the wound and facilitated diabetic wound closure compared with primary DFU fibroblasts. Taken together, our findings support the potential application of these iPSC-derived fibroblasts and 3D tissues to improve wound healing.-Kashpur, O., Smith, A., Gerami-Naini, B., Maione, A. G., Calabrese, R., Tellechea, A., Theocharidis, G., Liang, L., Pastar, I., Tomic-Canic, M., Mooney, D., Veves, A., Garlick, J. A. Differentiation of diabetic foot ulcer-derived induced pluripotent stem cells reveals distinct cellular and tissue phenotypes.
Project description:<h4>Background</h4>The accumulation of M1-polarized macrophages and excessive inflammation are important in the pathogenesis of diabetic foot ulcer (DFU). However, the underlying mechanism of DFU pathogenesis and the crucial regulators of DFU are less well known. Our previous study reported that kallikrein-binding protein (KBP), an angiogenesis inhibitor, was significantly upregulated in diabetic patients compared to its levels in controls. The effects of KBP on monocyte chemotaxis and macrophage M1 polarization were elucidated in this study.<h4>Methods</h4>Plasma KBP levels and monocyte counts were assessed by ELISA and flow cytometry. Wound closure rates in different groups were monitored daily. The phenotype and recruitment of macrophages were measured by real-time PCR, western blot and immunofluorescence assays. The expression of Notch and NF-κB signalling pathway members was determined by the methods mentioned above. ChIP and dual-luciferase reporter gene assays were employed to explore the binding and transcriptional regulation of Hes1 and iNOS.<h4>Results</h4>We found that plasma KBP levels and circulating monocytes were elevated in diabetic patients compared to those in nondiabetic controls, and both were higher in diabetic patients with DFU than in diabetic patients without DFU. KBP delayed wound healing in normal mice; correspondingly, KBP-neutralizing antibody ameliorated delayed wound healing in diabetic mice. Circulating monocytes and macrophage infiltration in the wound were upregulated in KBP-TG mice compared to those in control mice. KBP promoted the recruitment and M1 polarization of macrophages. Mechanistically, KBP upregulated iNOS by activating the Notch1/RBP-Jκ/Hes1 signalling pathway. Hes1 downregulated CYLD, a negative regulator of NF-κB signalling, and then activated the IKK/IκBα/NF-κB signalling pathway.<h4>Conclusions</h4>Our findings demonstrate that KBP is the key regulator of excessive inflammation in DFUs and provide a novel target for DFU therapy.
Project description:A variety of novel drugs and advanced therapeutic strategies have been developed for diabetic foot ulcers (DFUs); however, the clinical outcomes are unsatisfactory and the underlying mechanisms of DFU remain elusive. MicroRNAs (miRNA) regulate the pathological processes of many diseases. Fibroblasts are involved in each stage of wound healing, and the functions of fibroblasts may be regulated by miRNAs. In the present study, we found that the levels of miRNA-21-3p (miR-21-3p) were decreased in patients with diabetes as compared with those in the healthy control. Similarly, the level of miRNA-21-3p was decreased in fibroblasts that were stimulated with D-glucose as compared with that in the control fibroblasts. Furthermore, enhanced function was found in fibroblasts followed by the miR-21-3p agonist treatment, and a rapid wound healing process was achieved in the miR-21-3p agonist-treated mice. MiR-21-3p directly targeted protein sprout homolog 1 (SPRY1), and the miR-21-3p-regulated reduction in SPRY1 enhanced the function of fibroblasts and accelerated wound healing in vivo. These findings suggest that miR-21-3p may treat DFU by reducing SPRY1.
Project description:Diabetic foot ulcers (DFUs) are one of the major complications of diabetes. Its molecular pathology remains poorly understood, impeding the development of effective treatments. Although it has been established that multiple cell types, including fibroblasts, keratinocytes, macrophages, and endothelial cells, all contribute to inhibition of healing, less is known regarding contributions of individual cell type. Thus, we generated primary fibroblasts from nonhealing DFUs and evaluated their cellular and molecular properties in comparison to nondiabetic foot fibroblasts (NFFs). Specifically, we analyzed both micro-RNA and mRNA expression profiles of primary DFU fibroblasts. Paired genomic analyses identified a total of 331 reciprocal miRNA-mRNA pairs including 21 miRNAs (FC?>?2.0) along with 239 predicted target genes (FC?>?1.5) that are significantly and differentially expressed. Of these, we focused on three miRNAs (miR-21-5p, miR-34a-5p, miR-145-5p) that were induced in DFU fibroblasts as most differentially regulated. The involvement of these microRNAs in wound healing was investigated by testing the expression of their downstream targets as well as by quantifying cellular behaviors in prospectively collected and generated cell lines from 15 patients (seven DFUF and eight NFF samples). We found large number of downstream targets of miR-21-5p, miR-34a-5p, miR-145-5p to be coordinately regulated in mRNA profiles, which was confirmed by quantitative real-time PCR. Pathway analysis on paired miRNA-mRNA profiles predicted inhibition of cell movement and cell proliferation, as well as activation of cell differentiation and senescence in DFU fibroblasts, which was confirmed by cellular assays. We concluded that induction of miR-21-5p, miR-34a-5p, miR-145-5p in DFU dermal fibroblasts plays an important role in impairing multiple cellular functions, thus contributing to overall inhibition of healing in DFUs.
Project description:It is unclear why many with diabetes develop foot ulcers (DFU) and why some do not heal. It could be associated with genetic variation. We have previously shown that NOS1AP variation is associated with lower extremity amputation in those with diabetes and that circulating stem progenitor cell concentration (SPC) is associated with impaired foot ulcer healing in those with diabetes. The goal of this study was to determine if NOS1AP variation is associated with impaired wound healing and with SPC mobilization in those with DFU. In longitudinal cohort study we demonstrate that NOS1AP variants rs16849113 and rs19649113 are associated with impaired wound healing and with SPC mobilization in those with DFU. We believe that further study of NOS1AP is merited and that it NOS1AP might be associated with a functional impairment.
Project description:Diabetic foot ulcers (DFUs) lead to nearly 100,000 lower limb amputations annually in the United States. DFUs are colonized by complex microbial communities, and infection is one of the most common reasons for diabetes-related hospitalizations and amputations. In this study, we examined how DFU microbiomes respond to initial sharp debridement and offloading and how the initial composition associates with 4 week healing outcomes. We employed 16S rRNA next generation sequencing to perform microbial profiling on 50 samples collected from 10 patients with vascularized neuropathic DFUs. Debrided wound samples were obtained at initial visit and after one week from two DFU locations, wound bed and wound edge. Samples of the foot skin outside of the wounds were also collected for comparison. We showed that DFU wound beds are colonized by a greater number of distinct bacterial phylotypes compared to the wound edge or skin outside the wound. However, no significant microbiome diversity changes occurred at the wound sites after one week of standard care. Finally, increased initial abundance of Gram-positive anaerobic cocci (GPAC), especially Peptoniphilus (p < 0.05; n = 5 subjects), was associated with impaired healing; thus, GPAC's abundance could be a predictor of the wound-healing outcome.
Project description:The purpose of this clinical study was to assess, in a limited patient population, the potential for a novel advanced wound care treatment based on low-frequency (20 kHz) low-intensity (spatial peak temporal peak intensity <100 mW/cm2; i.e., pressure amplitude of 55 kPa) ultrasound (LFLI-US), to affect wound closure rate in human diabetic foot ulcers (DFUs) and to effect changes in the relative expression of pro-inflammatory and anti-inflammatory genes. The ratio of expression of these genes, termed the M1/M2 score because it was inspired by the transition of macrophages from pro-inflammatory (M1) to anti-inflammatory (M2) phenotypes as wound healing progresses, was previously presented as a potential healing indicator for DFUs treated with the standard of care. We previously found that non-cavitational, non-thermal LFLI-US delivered with a pulse repetition frequency of 25 Hz was effective at improving wound healing in a pilot study of 20 patients with chronic venous ulcers. In this study, we assessed the potential for weekly LFLI-US exposures to affect wound healing in patients with diabetic ulcers, and we analyzed temporal changes in the M1/M2 score in debrided diabetic wound tissue. Although this was a limited patient population of only 8 patients, wounds treated with LFLI-US exhibited a significantly faster reduction in wound size compared with sham-treated patients (p < 0.001). In addition, the value of the M1/M2 score decreased for all healing diabetic ulcers and increased for all non-healing diabetic ulcers, suggesting that the M1/M2 score could be useful as an indicator of treatment efficacy for advanced DFU treatments. Such an indicator would facilitate clinical decision making, ensuring optimal wound management and thus contributing to reduction of health care expenses. Moreover, the results presented may contribute to an understanding of the mechanisms underlying ultrasonically assisted chronic wound healing. Knowledge of these mechanisms could lead to personalized or patient-tailored treatment.
Project description:Diabetic foot ulcers (DFUs) are a serious complication of diabetes that results in significant morbidity and mortality. Mortality rates associated with the development of a DFU are estimated to be 5% in the first 12 months, and 5-year morality rates have been estimated at 42%. The standard practices in DFU management include surgical debridement, dressings to facilitate a moist wound environment and exudate control, wound off-loading, vascular assessment, and infection and glycemic control. These practices are best coordinated by a multidisciplinary diabetic foot wound clinic. Even with this comprehensive approach, there is still room for improvement in DFU outcomes. Several adjuvant therapies have been studied to reduce DFU healing times and amputation rates. We reviewed the rationale and guidelines for current standard of care practices and reviewed the evidence for the efficacy of adjuvant agents. The adjuvant therapies reviewed include the following categories: nonsurgical debridement agents, dressings and topical agents, oxygen therapies, negative pressure wound therapy, acellular bioproducts, human growth factors, energy-based therapies, and systemic therapies. Many of these agents have been found to be beneficial in improving wound healing rates, although a large proportion of the data are small, randomized controlled trials with high risks of bias.