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The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Diabetic Foot Consortium (DFC) was established in September 2018 by the NIDDK to build an organization to facilitate the highest quality of clinical research on diabetic foot ulcers (DFUs) that will answer clinically significant questions to improve DFU healing and prevent amputations. The initial focus of the DFC is to develop and validate biomarkers for DFUs that can be used in clinical care and research. The DFC consists of a data coordinating center (DCC) for operational oversight and statistical analysis, clinical sites for participant recruitment and evaluation, and biomarker analysis units (BAUs). The DFC is currently studying biomarkers to predict wound healing and recurrence and is collecting biosamples for future studies through a biorepository. The DFC plans to address the challenges of recruitment and eligibility criteria for DFU clinical trials by taking an approach of “No DFU Patient Goes Unstudied.” In this platform approach, clinical history, DFU outcome, wound imaging, and biologic measurements from a large number of patients will be captured and the in-depth longitudinal data set will be analyzed to develop a computational-based DFU risk factor profile to facilitate scientifically sound clinical trial design. The DFC will expand its platform to include studies of the role of social determinants of health, such as food insecurity, housing instability, limited health literacy, and poor social support. The DFC is starting partnerships with the broad group of stakeholders in the wound care community.

Diabetic foot ulcers and other chronic wounds with impaired healing can be treated with bioengineered skin or with growth factors. However, most patients do not benefit from these treatments. Here we report the development and preclinical therapeutic performance of a strain-programmed patch that rapidly and robustly adheres to diabetic wounds, and promotes wound closure and re-epithelialization. The patch consists of a dried adhesive layer of crosslinked polymer networks bound to a pre-stretched hydrophilic elastomer backing, and implements a hydration-based shape-memory mechanism to mechanically contract diabetic wounds in a programmable manner on the basis of analytical and finite-element modelling. In mouse and human skin, and in mini-pigs and humanized mice, the patch enhanced the healing of diabetic wounds by promoting faster re-epithelialization and angiogenesis, and the enrichment of fibroblast populations with a pro-regenerative phenotype. Strain-programmed patches might also be effective for the treatment of other forms of acute and chronic wounds.

Diabetic foot ulcers are a health crisis that affect millions of individuals worldwide. Current standard of care involves diligent wound care with adjunctive antibiotics and surgical debridement. However, despite this, the majority will still become infected and fail to heal. Recent efforts using bioengineered skin initially appeared promising, but randomized clinical trials have disappointed. Scientists have now begun to understand that the normal wound healing physiology does not apply to diabetic foot ulcers as they maintain a chronic state of inflammation and fail to progress in a linear pathway. Using transcriptomics, research over the past decade has started identifying master genes and protein pathways that are dysregulated in patients with diabetes. This review paper discusses those genes involved and how novel advancements are using this information to create new biologically based compounds to accelerate wound healing in patients with diabetic foot ulcers.

Mast cells (MCs) are granulated, immune cells of the myeloid lineage that are present in connective tissues. Apart from their classical role in allergies, MCs also mediate various inflammatory responses due to the nature of their secretory products. They are involved in important physiological and pathophysiological responses related to inflammation, chronic wounds, and autoimmune diseases. There are also indications that MCs are associated with diabetes and its complications. MCs and MC-derived mediators participate in all wound healing stages and are involved in the pathogenesis of non-healing, chronic diabetic foot ulcers (DFUs). More specifically, recent work has shown increased degranulation of skin MCs in human diabetes and diabetic mice, which is associated with impaired wound healing. Furthermore, MC stabilization, either systemic or local at the skin level, improves wound healing in diabetic mice. Understanding the precise role of MCs in wound progression and healing processes can be of critical importance as it can lead to the development of new targeted therapies for diabetic foot ulceration, one of the most devastating complications of diabetes.

Cutaneous wound healing is a highly coordinated process involving numerous molecular pathways that regulate the function of specific skin cell types. One of the key decisions in acute and/or chronic wound healing management is to choose the skin substitute that, based on its composition and/or properties, may act as permanent skin replacement or temporary wound cover. The current products however are limited in their action, especially in the context of large chronic wounds or extensive second-/third-degree burns, due to the risk of developing infection, the limited vascularization, the inability to integrate to host tissue, and the lack in the healed area of skin apparatus responsible for temperature control, pigmentation, immune regulation, and nerve supply. In addition, the high cost of skin substitutes precludes their use in small- and moderate-size burns because they are economically impractical and protract the time to definitive wound closure. Thus, the need to develop cost-effective substitutes of high quality is imperative. The purpose of this review is to discuss current available products, their limitations and to provide some perspectives on future research toward generation of cost-effective, high-quality substitutes.

Diabetic foot ulceration is a devastating diabetic complication with unmet needs. We explored the efficacy of calcium-crosslinked alginate dressings in topically delivering primary macrophages and their secretome to diabetic wounds. The alginate bandages had a microporous structure that enabled even cell loading with prolonged cell survival and egress following wound placement. In vitro experiments showed that we could successfully differentiate and polarize primary murine bone marrow derived monocytes into M0, M1, M2a and M2c defined states with distinct gene expression, surface protein and secretome profiles. The primary macrophages were delivered in the bandages, migrated within the wounds and were still present for as long as 16 days post-injury. In wounds of db/db mice, treatment with all macrophage subtypes and their secretome, when compared to control, accelerated wound healing. Bulk RNA sequencing analysis and multiplex protein quantification of wound lysates revealed that M2c macrophages conditioned media had the most impact in wound healing affecting processes like neurogenesis, while M1 conditioned media promoted keratinization and epidermal differentiation. Collectively, our results indicate that alginate dressings can serve as a delivery platform for topical treatment of diabetic wounds and that conditioned media from distinctly polarized macrophages is equally or more effective than their parental cells in advancing wound healing and could therefore be a promising and technically advantageous alternative to cell therapy.

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., Diabetic foot ulcers (DFUs) remain a complication of diabetes that are difficult to heal and lead to disability. Here the authors use single-cell RNA-sequencing and spatial transcriptomics to characterize the DFU cellular landscape and identify a population of fibroblasts that is associated with successful wound closure.

High-parameter tissue imaging enables detailed molecular analysis of single cells in their spatial environment. However, the comprehensive characterization and mapping of tissue states through multimodal imaging across different physiological and pathological conditions requires data integration across multiple imaging systems. Here, we introduce MIAAIM (Multi-omics Image Alignment and Analysis by Information Manifolds) a modular, reproducible computational framework for aligning data across bioimaging technologies, modeling continuities in tissue states, and translating multimodal measures across tissue types. We demonstrate MIAAIM’s workflows across diverse imaging platforms, including histological stains, imaging mass cytometry, and mass spectrometry imaging, to link cellular phenotypic states with molecular microenvironments in clinical biopsies from multiple tissue types with high cellular complexity. MIAAIM provides a robust foundation for the development of computational methods to integrate multimodal, high-parameter tissue imaging data and enable downstream computational and statistical interrogation of tissue states.

Diabetic foot ulceration is a major diabetic complication with unmet needs. We investigated the efficacy of epidermal stem cells and epidermal stem cells-derived exosomes (ESCs-Exo) in improving impaired diabetic wound healing and their mechanisms of action. In vitro experiments showed that ESCs-Exo enhanced the proliferation and migration of diabetic fibroblasts and macrophages and promoted alternative or M2 macrophage polarization. In wounds of db/db mice, treatment with both epidermal stem cells and ESCs-Exo, when compared with fibroblast exosomes and PBS control, accelerated wound healing by decreasing inflammation, augmenting wound cell proliferation, stimulating angiogenesis, and inducing M2 macrophage polarization. Multiplex protein quantification of wound lysates revealed TGFβ signaling influenced by ESCs-Exo. High-throughput sequencing of small RNAs contained in the ESCs-Exo showed higher proportions of microRNAs than those contained in fibroblast exosomes. In silico functional analysis showed that the ESCs-Exo microRNAs‒target genes were primarily involved in homeostatic processes and cell differentiation and highlighted regulatory control of phosphatidylinositol-3 kinase/protein kinase B and TGFβ signaling pathways. This was also validated in vitro. Collectively, our results indicate that epidermal stem cells and ESCs-Exo are equally effective in promoting impaired diabetic wound healing and that ESCs-Exo treatment may be a promising and technically advantageous alternative to stem cell therapies.

Neuropathy is a major cause of morbidity and mortality in individuals with diabetes, with no effective therapy to alter the inevitable progression of nerve damage. We hypothesized that mesenchymal stroma cell-like populations, that are characterized as immune modulators also have the potential of inducing angiogenesis and neurite outgrowth, might be useful in treating diabetic peripheral neuropathy (DPN). The aims of this study were to investigate the efficacy and safety of mesenchymal stem cell-like product (PDA-002) in treating DPN. A phase-2 randomized placebo-controlled trial was conducted in 26 patients with DPN. Treatment consisted of three rounds of intramuscular injections in one lower limb using one of the three randomized treatment arms PDA-002 (low-dose 3 × 106 cells), PDA-002 (high-dose 30 × 106 cells), or placebo. Three treatments per patient occurred on days 1, 29, and 57. Study endpoints included efficacy and safety of PDA-002 in treating DPN in both lower extremities following unilateral local injection. Outcome measures included intra-epidermal nerve fiber density (IENFD) up to 1 year from the day of treatment with 6-month as the primary outcome measurement. In this phase 2 study of DPN, PDA-002 was well tolerated in both doses. No significant changes were noted in IENFD in both the treated and untreated leg in the NIS-LL, NTSS-6, or UENS. Mesenchymal stem cells represent a novel mechanism for treating diabetic neuropathy and are well tolerated. Preliminary results highlight the need of further investigation of PDA-001 as a disease modifying agent for treatment of DPN.

To understand the diabetic wound healing microenvironment, we profiled 174,962 single cells from foot, forearm, and PBMCs using single-cell RNA sequencing (scRNASeq) approach. Our analysis shows enrichment of a unique population of fibroblasts overexpressing MMP1, MMP3, MMP11, HIF1A, CHI3L1, and TNFAIP6 genes and M1 macrophage polarization in the DFU patients with healing wounds. Further, scRNASeq of spatially separated samples from same patient and spatial transcriptomics (ST) revealed preferential localization of these healing associated fibroblasts toward deep wound/ulcer bed as compared to wound edge or non-wounded skin. ST also validated our findings of higher enrichment 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.

A major challenge in the management of patients suffering from diabetes is the risk of developing nonhealing foot ulcers. Most in vitro methods to screen drugs for wound healing therapies rely on conventional 2D cell cultures that do not closely mimic the complexity of the diabetic wound environment. In addition, while three-dimensional (3D) skin tissue models of human skin exist, they have not previously been adapted to incorporate patient-derived macrophages to model inflammation from these wounds. In this study, we present a 3D human skin equivalent (HSE) model incorporating blood-derived monocytes and primary fibroblasts isolated from patients with diabetic foot ulcers (DFUs). We demonstrate that the monocytes differentiate into macrophages when incorporated into HSEs and secrete a cytokine profile indicative of the proinflammatory M1 phenotype seen in DFUs. We also show how the interaction between fibroblasts and macrophages in the HSE can guide macrophage polarization. Our findings take us a step closer to creating a human, 3D skin-like tissue model that can be applied to evaluate the response of candidate compounds needed for potential new foot ulcer therapies in a more complex tissue environment that contributes to diabetic wounds. IMPACT STATEMENT: This study is the first to incorporate disease-specific, diabetic macrophages into a three-dimensional (3D) model of human skin. We show how to fabricate skin that incorporates macrophages with disease-specific fibroblasts to guide macrophage polarization. We also show that monocytes from diabetic patients can differentiate into macrophages directly in this skin disease model, and that they secrete a cytokine profile mimicking the proinflammatory M1 phenotype seen in diabetic foot ulcers. The data presented here indicate that this 3D skin disease model can be used to study macrophage-related inflammation in diabetes and as a drug testing tool to evaluate new treatments for the disease.

Current treatment options for foot ulcers, a serious and prevalent complication of diabetes, remain nonspecific. In this Perspective, we present recent advances in understanding the pathophysiology of diabetic wound healing and the emergence of previously unidentified targets. We discuss wound dressings tailored to the diabetic wound environment currently under development.

Nonhealing diabetic foot ulcers (DFUs) are characterized by low-grade chronic inflammation, both locally and systemically. We prospectively followed a group of patients who either healed or developed nonhealing chronic DFUs. Serum and forearm skin analysis, both at the protein expression and the transcriptomic level, indicated that increased expression of factors such as interferon-γ (IFN-γ), vascular endothelial growth factor, and soluble vascular cell adhesion molecule-1 were associated with DFU healing. Furthermore, foot skin single-cell RNA sequencing analysis showed multiple fibroblast cell clusters and increased inflammation in the dorsal skin of patients with diabetes mellitus (DM) and DFU specimens compared with control subjects. In addition, in myeloid cell DM and DFU upstream regulator analysis, we observed inhibition of interleukin-13 and IFN-γ and dysregulation of biological processes that included cell movement of monocytes, migration of dendritic cells, and chemotaxis of antigen-presenting cells pointing to an impaired migratory profile of immune cells in DM skin. The SLCO2A1 and CYP1A1 genes, which were upregulated at the forearm of nonhealers, were mainly expressed by the vascular endothelial cell cluster almost exclusively in DFU, indicating a potential important role in wound healing. These results from integrated protein and transcriptome analyses identified individual genes and pathways that can potentially be targeted for enhancing DFU healing.

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.

Rationale: Although both type 2 diabetes mellitus (T2DM) and obstructive sleep apnea (OSA) are independently recognized as risk factors for cardiovascular disease, little is known about their interaction.Objectives: We hypothesized that T2DM and OSA act synergistically to increase vascular risk, and that treatment of OSA would improve vascular reactivity in patients with T2DM plus OSA.Methods: Cross-sectional study of 141 adults with T2DM, OSA, T2DM plus OSA, and control subjects, followed by a 3-month, parallel-arm, randomized, placebo-controlled trial comparing active and sham continuous positive airway pressure (CPAP) in 53 adults with T2DM plus OSA. Endothelium-dependent macro- and microvascular reactivity (flow-mediated dilation [FMD] of the brachial artery and acetylcholine-induced dilation of forearm microvasculature, respectively) and cardiovascular magnetic resonance to assess left- and right-ventricular mass/volume.Results: Mean (±SD) FMD was 6.1 (±4.0)%, 7.3 (±3.6)%, 6.8 (±4.5)%, and 4.8 (±2.9)% in control subjects, T2DM only, OSA only, and T2DM plus OSA, respectively. We observed a significant T2DM × OSA interaction on FMD, such that the mean effect of OSA in those with T2DM was 3.1% (95% confidence interval [CI], 0.6 to 5.6) greater than the effect of OSA in those without T2DM. A total of 3 months of CPAP resulted in a mean absolute increase in FMD of 0.3% (95% CI, -1.9 to 2.5; primary endpoint), with a net improvement of 1.1% (95% CI, -1.4 to 3.6) among those with adherence of 4 h/night or greater. A significant T2DM × OSA interaction was found for both left ventricular (LV) and right ventricular end-diastolic volume, such that OSA was associated with a 22.4 ml (95% CI, 3.2 to 41.6) greater LV end-diastolic volume and 23.2 ml (95% CI, 2.6 to 43.8) greater right ventricular end-diastolic volume in those with T2DM compared with the impact of OSA in those without T2DM. We observed a net improvement in LV end-diastolic volume of 8.7 ml (95% CI, -7.0 to 24.4).Conclusions: The combination of T2DM plus OSA is associated with macrovascular endothelial dysfunction beyond that observed with either disease alone. CPAP for 3 months did not significantly improve macrovascular endothelial function in the intent-to-treat analysis; however, cardiovascular magnetic resonance results suggest that there may be a beneficial effect of CPAP on LV diastolic volume.Clinical trial registered with www.clinicaltrials.gov (NCT01629862).

Impaired wound healing in the diabetic foot is a major problem often leading to amputation. Mast cells have been shown to regulate wound healing in diabetes. We developed an indole-carboxamide type mast cell stabilizer, MCS-01, which proved to be an effective mast cell degranulation inhibitor in vitro and can be delivered topically for prolonged periods through controlled release by specifically designed alginate bandages. In diabetic mice, both pre- and post-wounding, topical MCS-01 application accelerated wound healing comparable to that achieved with systemic mast cell stabilization. Moreover, MCS-01 altered the macrophage phenotype, promoting classically activated polarization. Bulk transcriptome analysis from wounds treated with MCS-01 or placebo showed that MCS-01 significantly modulated the mRNA and microRNA profile of diabetic wounds, stimulated upregulation of pathways linked to acute inflammation and immune cell migration, and activated the NF-κB complex along with other master regulators of inflammation. Single-cell RNA sequencing analysis of 6,154 cells from wounded and unwounded mouse skin revealed that MCS-01 primarily altered the gene expression of mast cells, monocytes, and keratinocytes. Taken together, these findings offer insights into the process of diabetic wound healing and suggest topical mast cell stabilization as a potentially successful treatment for diabetic foot ulceration.

Lower extremity ulcerations represent a major complication in diabetes mellitus and involve multiple physiological factors that lead to impairment of wound healing. Neuropeptides are neuromodulators implicated in various processes including diabetic wound healing. Diabetes causes autonomic and small sensory nerve fibers neuropathy as well as inflammatory dysregulation, which manifest with decreased neuropeptide expression and a disproportion in pro- and anti- inflammatory cytokine response. Therefore to fully understand the contribution of autonomic nerve dysfunction in diabetic wound healing it is crucial to explore the implication of neuropeptides. Here, we will discuss recent studies elucidating the role of specific neuropeptides in wound healing.

Obstructive sleep apnea (OSA) affects a large proportion of adults, and is as an independent risk factor for cerebrovascular and cardiovascular disease. The repetitive airway obstruction that characterizes OSA results in intermittent hypoxia, intrathoracic pressure swings, and sleep fragmentation, which in turn lead to sympathetic activation, oxidative stress, inflammation, and endothelial dysfunction. This review outlines the associations between OSA and vascular diseases and describes basic mechanisms that may be responsible for this association, in both the micro- and macrocirculation. It also reports on interventional studies that aim to ameliorate OSA and thereby reduce vascular disease burden. © 2016 American Physiological Society. Compr Physiol 6:1519-1528, 2016.

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.

Purpose To assess differences in the phosphorus-31 (31P) metabolism and morphology in multiple muscle regions in the forefoot of diabetic patients and normal subjects. Materials and Methods Fifteen diabetic patients and 15 normal subjects were assessed for muscle atrophy by 1H magnetic resonance imaging (MRI) at 3T to grade the flexor hallucis, adductor hallucis, interosseous regions, and entire foot cross-section. Each region and the entire foot were also quantitatively evaluated for metabolic function using 31P imaging for spatial mapping of the inorganic phosphate (Pi) to phosphocreatine (PCr) ratio (Pi/PCr). The ratio of viable muscle area to the predefined region areas (31P/1H) was calculated. The variability of each method was assessed by its coefficient of variation (CV). Results Muscle atrophy was significantly more severe in diabetic compared to normal subjects in all regions (P < 0.01). The 31P/1H area ratio was significantly larger in the adductor hallucis than in the other two regions (P < 0.05). The Pi/PCr ratio was significantly different between the two groups in the flexor hallucis and interosseous regions (P < 0.05) but not adductor hallucis region. The CV for Pi/PCr ranged from 10.13 to 55.84, while it ranged from 73.40 to 263.90 for qualitative grading. Conclusion Changes in atrophy and metabolism appear to occur unequally between different regions of the forefoot in diabetes. The adductor hallucis region appears more capable of maintaining structural and metabolic integrity than the flexor hallucis or interosseous regions. The CV analysis suggests that the quantitative 31P methods have less variability than the qualitative grading. J. Magn. Reson. Imaging 2016;44:1132–1142.

Diabetic foot ulceration is a severe complication of diabetes that lacks effective treatment. Mast cells (MCs) contribute to wound healing, but their role in diabetes skin complications is poorly understood. Here we show that the number of degranulated MCs is increased in unwounded forearm and foot skin of patients with diabetes and in unwounded dorsal skin of diabetic mice (P < 0.05). Conversely, postwounding MC degranulation increases in nondiabetic mice, but not in diabetic mice. Pretreatment with the MC degranulation inhibitor disodium cromoglycate rescues diabetes-associated wound-healing impairment in mice and shifts macrophages to the regenerative M2 phenotype (P < 0.05). Nevertheless, nondiabetic and diabetic mice deficient in MCs have delayed wound healing compared with their wild-type (WT) controls, implying that some MC mediator is needed for proper healing. MCs are a major source of vascular endothelial growth factor (VEGF) in mouse skin, but the level of VEGF is reduced in diabetic mouse skin, and its release from human MCs is reduced in hyperglycemic conditions. Topical treatment with the MC trigger substance P does not affect wound healing in MC-deficient mice, but improves it in WT mice. In conclusion, the presence of nondegranulated MCs in unwounded skin is required for proper wound healing, and therapies inhibiting MC degranulation could improve wound healing in diabetes.

Neuropathies of the peripheral and autonomic nervous systems affect up to half of all people with diabetes mellitus, and are major risk factors for foot ulceration, amputation and cardiovascular dysfunction. Peripheral neuropathies manifest with either painful or painless symptoms, but many patients experience both. Once diagnosed appropriately, painful diabetic neuropathy management presents a unique challenge for physicians and should be considered as a syndrome, clinically distinct from diabetic peripheral neuropathy. The aetiology is multifactorial: metabolic changes in diabetes may directly affect neural tissue and neurodegenerative changes are precipitated by compromised nerve vascular supply. Metabolic changes include the elevated polyol pathway activity, the increased oxidative stress, the formation of advanced glycation and lipoxidation end products, and various pro-inflammatory changes. These mechanisms work in combination and interact in a mutually facilitatory fashion. This review focuses on the current therapies for the management of peripheral and cardiovascular autonomic neuropathy and of painful neuropathy as a distinct entity, based on the current knowledge of diabetic neuropathy. Moreover, the role of ACE inhibition has been explored in the treatment of Cardiovascular Autonomic Neuropathy.

Fifteen percent of diabetes mellitus (DM) patients are expected to develop a diabetic foot ulcer (DFU) within their lifetime. DFUs represent an ever increasing burden for the healthcare system. Chronic wounds, lack the linear progression from one classical phase of healing to the next and are mainly characterized by the persistence of the inflammatory phase. To define the transcriptome landscape of healthy uninjured skin, DM and DFU skin, we performed transcriptome profiling on skin specimens from foot surgeries obtained from 4 controls, 4 DM patients without DFU and on samples from 2 DM patients after DFU debridement . We generated the single cell transcriptome profiles of 3,766 and 3,469 cells from non-DM and DM patients and of 660 cells from patients with active DFUs. Further, bioinformatics analysis identified clusters of cells with similar transcriptome states. The most abundant cell type was fibroblast. Other clusters included adipocytes and endothelial cells. T-cells, monocytes, and macrophages were the main immune cell types identified. As macrophages are critical for healing of DFUs, we estimated the abundance of total macrophages and their polarization. Approximately 10% of cells were macrophages in each group while DFU group had significantly more M1 polarized as compared to M2 (M1/M2=2.4 times). Supervised analysis identified a set of 66 and 1genes that are significantly differentially expressed in macrophages of DFU and DM patients respectively as compared to controls. A master regulator (MR) analysis in DFU patients identified activation of inflammatory and angiogenesis related MRs (e.g., STAT3, IL15). while in DM patients it identified activation of distinct inflammatory MRs (e.g., SMAD3, CD44). In summary, we present for the first time sc-RNAseq data on lower extremity skin samples that could help delineate the molecular mechanisms of dysregulated wound healing and identify novel therapeutic targets. Disclosure G. Theocharidis: None. S.S. Bhasin: None. K. Kounas: None. M.K. Bhasin: None. A. Veves: Advisory Panel; Self; HyperMed Imaging, Inc., AOBiome.

Diabetes, especially type 2, is characterized by systemic inflammation. At the skin level, there is increased infiltration by inflammatory cells and polarization of the macrophages toward the M1 inflammatory type. In addition, there is increased expression of MMP-9 and protein tyrosine phosphatase-1B (PTP1B). Other dermatologic conditions include acanthosis nigricans, characterized by a hyperpigmented, velvety, cutaneous thickening that appears predominantly in the neck, axilla, and groin areas; necrobiosis lipoidica (NL), a chronic, necrotizing, granulomatous skin disease; granuloma annulare; diabetic bullae; and diabetic dermopathy. As these conditions can be present in the lower extremity, they should be sought and easily recognized by the health care providers who manage the diabetic lower extremity.

Diabetic peripheral neuropathy and vascular disease, along with trauma, have long been recognized as major risk factors for the development of diabetic foot ulcerations (DFUs). More recently, chronic inflammation, abnormal extracellular matrix (ECM) remodeling, and reduced wound neovascularization, as a result of dysregulated cell function with imbalanced secretion of cytokines, matrix metalloproteinases, and growth factors, have been implicated in DFU failure to heal. Therefore, researchers are now focusing their efforts on further understanding the cellular and molecular mechanisms of diabetes-associated impaired wound healing, in an attempt to identify new targets and novel potential therapeutic approaches for DFUs, which remain a serious unmet clinical need. A growing body of evidence suggests an important role of neuropeptides in skin repair, particularly in diabetes, where neuropeptide levels are diminished. On the other hand, there is emerging interest in dissecting the mechanisms of dysregulated inflammation, namely the changes in immune cells, such as macrophages and mast cells (MCs), in diabetic wound healing. Studies using in vitro and in vivo models of diabetic wound healing have considerably improved our understanding of the healing process. However, the currently available models have major caveats and are not ideal to study chronic, complicated, and multifactorial wounds, such as DFUs. In this chapter we summarize the involvement of neuropeptides and mast cells in diabetic wound healing, highlighting the most recent findings. We also discuss the benefits and limitations of the current wound healing models, emphasizing the need for confirmation and/or validation in multiple models and/or tissue specimens from human subjects.

Normal vascular function is characterized by a continuous interaction between the endothelium and vascular smooth muscle that maintains optimal vascular tone and organ perfusion via balanced vasodilatory and vasoconstrictive mechanisms. In an environment of chronic hyperglycemia, such as that occurring in diabetes mellitus, normal vascular function is disrupted most likely due to elevations in reactive oxygen species and the compounding effects of vascular inflammation. Such vascular dysfunctions represent key mechanisms for atherosclerotic changes and primary events in the pathogenesis of cardiovascular disease, which remains the single leading cause of mortality for those with type 1 or type 2 diabetes. Exercise has been described as a cornerstone in the management and treatment of diabetes due to its prominent role in improving both metabolic and cardiovascular health. Evidence suggests that habitual physical activity disrupts chronic hyperglycemic by improving glycemic control via enhanced GLUT protein activation and increased insulin sensitivity. Furthermore, exercise-mediated shear stress may enhance vasodilatory capacity by improving expression of endothelial nitric oxide synthase proteins and enhancing nitric oxide bioavailability. Although some specific precautions must be adhered to when prescribing exercise to diabetic populations, the benefits significantly outweigh the consequences of continuing a sedentary lifestyle. This chapter will present a non-exhaustive review focusing on changes in vascular function and inflammatory status from healthy to diabetic states, techniques that are often implemented to quantify such changes in vascular function, and the role of exercise in improving underlying mechanisms critical to maintaining normal vascular function and disrupting the pathogenesis of cardiovascular disease.

Diabetic foot ulcers (DFUs) are one of the most common and serious complications of diabetes mellitus, as wound healing is impaired in the diabetic foot. Wound healing is a dynamic and complex biological process that can be divided into four partly overlapping phases: hemostasis, inflammation, proliferative and remodeling. These phases involve a large number of cell types, extracellular components, growth factors and cytokines. Diabetes mellitus causes impaired wound healing by affecting one or more biological mechanisms of these processes. Most often, it is triggered by hyperglycemia, chronic inflammation, micro- and macro-circulatory dysfunction, hypoxia, autonomic and sensory neuropathy, and impaired neuropeptide signaling. Research focused on thoroughly understanding these mechanisms would allow for specifically targeted treatment of diabetic foot ulcers. The main principles for DFU treatment are wound debridement, pressure off-loading, revascularization and infection management. New treatment options such as bioengineered skin substitutes, extracellular matrix proteins, growth factors, and negative pressure wound therapy, have emerged as adjunctive therapies for ulcers. Future treatment strategies include stem cell-based therapies, delivery of gene encoding growth factors, application of angiotensin receptors analogs and neuropeptides like substance P, as well as inhibition of inflammatory cytokines. This review provides an outlook of the pathophysiology in diabetic wound healing and summarizes the established and adjunctive treatment strategies, as well as the future therapeutic options for the treatment of DFUs.

NIDDK, JDRF, and the Diabetic Neuropathy Study Group of EASD sponsored a meeting to explore the current status of animal models of diabetic peripheral neuropathy. The goal of the workshop was to develop a set of consensus criteria for the phenotyping of rodent models of diabetic neuropathy. The discussion was divided into five areas: (1) status of commonly used rodent models of diabetes, (2) nerve structure, (3) electrophysiological assessments of nerve function, (4) behavioral assessments of nerve function, and (5) the role of biomarkers in disease phenotyping. Participants discussed the current understanding of each area, gold standards (if applicable) for assessments of function, improvements of existing techniques, and utility of known and exploratory biomarkers. The research opportunities in each area were outlined, providing a possible roadmap for future studies. The meeting concluded with a discussion on the merits and limitations of a unified approach to phenotyping rodent models of diabetic neuropathy and a consensus formed on the definition of the minimum criteria required for establishing the presence of the disease. A neuropathy phenotype in rodents was defined as the presence of statistically different values between diabetic and control animals in 2 of 3 assessments (nocifensive behavior, nerve conduction velocities, or nerve structure). The participants propose that this framework would allow different research groups to compare and share data, with an emphasis on data targeted toward the therapeutic efficacy of drug interventions.

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxemia, recurrent arousals, and intra-thoracic pressure swings, all of which can contribute to sympathetic activation and/or cardiovascular risk. The intermittent hypoxemia can lead to inflammation and oxidative stress, while pressure swings can place shear stress on the myocardium and intrathoracic vessel walls.1 Taken together, these pathways can result in endothelial dysfunction, a recognized early marker of cardiovascular disease.2 At the macrovascular level, cross-sectional studies have reported impaired brachial artery flow-mediated dilation (FMD), increased carotid intima-media thickness, and arterial stiffness quantified by the augmentation index (AIx) in OSA subjects compared with controls.3-8 This association is strengthened by reports of a dose-response relationship between these outcomes and OSA severity and duration.3,8,10 Microvascular reactivity has been less studied in OSA; an association between OSA and reduced capillary density has been demonstrated,11 and two small studies have reported differences between OSA subjects and controls in microcirculatory flow following administration of the endothelium-dependent vasodilator acetylcholine (ACh) delivered either non-invasively12 or via intra-arterial infusion.13 Two studies conducted in our laboratories, however, have not replicated these findings in the microvasculature.4,14 In addition to this observational evidence, a number of randomized controlled trials have found improvements in FMD and the AIx with continuous positive airway pressure (CPAP),15-18 while CPAP-withdrawal studies have demonstrated a deterioration.15,19,20 Again, the effects of CPAP on endothelium-dependent microvascular reactivity have not been widely studied. Only one study has reported a significant improvement with CPAP in microvascular reactivity measured non-invasively;12 this trial was of a short duration (two months) and the average BMI was

Aberrant activation of mast cells contributes to the development of numerous diseases including cancer, autoimmune disorders, as well as diabetes and its complications. The influx of extracellular calcium via the highly calcium selective calcium-release activated calcium (CRAC) channel controls mast cell functions. Intracellular calcium homeostasis in mast cells can be maintained via the modulation of the CRAC channel, representing a critical point for therapeutic interventions. We describe the structure-activity relationship study (SAR) of indazole-3-carboxamides as potent CRAC channel blockers and their ability to stabilize mast cells. Our SAR results show that the unique regiochemistry of the amide linker is critical for the inhibition of calcium influx, the release of the pro-inflammatory mediators β-hexosaminidase and tumor necrosis factor α by activated mast cells. Thus, the indazole-3-carboxamide 12d actively inhibits calcium influx and stabilizes mast cells with sub-μM IC 50 . In contrast, its reverse amide isomer 9c is inactive in the calcium influx assay even at 100 μM concentration. This requirement of the specific 3-carboxamide regiochemistry in indazoles is unprecedented in known CRAC channel blockers. The new structural scaffolds described in this report expand the structural diversity of the CRAC channel blockers and may lead to the discovery of novel immune modulators for the treatment of human diseases.

Diabetic foot ulceration (DFU) is one of the most costly and debilitating complications of diabetes and is the leading cause of non-traumatic amputations, affecting 15% of the diabetic population. Impaired wound healing in diabetic patients without large-vessel disease has been attributed to microvascular dysfunction, neuropathy, and abnormal cellular and inflammatory responses. These abnormalities have been examined mainly in animal models although a few studies have been undertaken in diabetic patients. This review provides an overview of the inflammatory and vascular abnormali- ties in DFU and emphasises the role of angiogenic growth factors, endothelial progenitor cells (EPCs), and neuropeptides as mediators of wound healing and potential therapeutic agents for these chronic, non-healing ulcers.

SUMMARY Diabetic foot ulcers result from multiple risk factors including peripheral neuropathy, arterial insufficiency and foot deformities. Recent investigation has also revealed a chronic wound environment with diminished expression of growth factors and cytokines integral to the wound healing process. Current accepted standard of care for the treatment of diabetic foot ulcerations focuses on periodic debridement of the wound, appropriate topical wound therapy, pressure off-loading and treatment of infection. Owing to increased cost and equivocal effectiveness, topical growth factors, bioengineered living skin equivalents, hyperbaric oxygen therapy and negative pressure wound therapy are proposed as adjuncts to standard of care and may be added to the treatment regimen when healing of the wound has stalled. Other future therapies currently under investigation include stem cell therapy, platelet-rich plasma, extracorporeal shock-wave therapy and laser treatment. These modalities continue to be developed ...

We examined the influences of obesity and diabetes on endothelium-dependent and -independent vasodilation, inflammatory cytokines, and growth factors. We included 258 subjects, age 21–80 years in four groups matched for age and gender: 40 healthy nonobese (BMI 30) nondiabetic subjects, and 105 obese (BMI >30) diabetic patients. The flow-mediated dilation (FMD, endothelium-dependent) and nitroglycerin-induced dilation (NID, endothelium-independent) in the brachial artery, the vascular reactivity at the forearm skin and serum growth factors and inflammatory cytokines were measured. FMD was reduced in the nonobese diabetic patients, obese nondiabetic controls, and obese diabetic patients (P < 0.0001). NID was different among all four groups, being highest in the obese nondiabetic subjects and lowest in the obese diabetic patients (P < 0.0001). The resting skin forearm blood flow was reduced in the obese nondiabetic subjects (P < 0.01). Vascular endothelial growth factor (VEGF) was higher in the obese nondiabetic subjects (P < 0.05), tumor necrosis factor–α was higher in the obese diabetic patients (P < 0.0001) and C-reactive protein was higher in both the obese nondiabetic and diabetic subjects (P < 0.0001). Soluble intercellular adhesion molecule-1 was elevated in the two diabetic groups and the obese nondiabetic subjects (P < 0.05). We conclude that diabetes and obesity affect equally the endothelial cell function but the smooth muscle cell function is affected only by diabetes. In addition, the above findings may be related to differences that were observed in the growth factors and inflammatory cytokines.

OBJECTIVE To determine the relationships among large, small, and autonomic fiber neurophysiological measures in a cross-sectional study of patients with diabetes. RESEARCH DESIGN AND METHODS We assessed 130 individuals: 25 healthy subjects and 105 subjects with diabetes. Subjects were classified by the presence or absence of neuropathy by physical examination. All subjects underwent autonomic testing, nerve conduction studies, quantitative sensory testing, and nerve-axon reflex vasodilation in addition to quantifiable neurological examination and symptom scores. Correlation and cluster analysis were used to determine relationships between and among different neurophysiological testing parameters. RESULTS Results of neurophysiological tests were abnormal in patients with clinical evidence of diabetic neuropathy compared with results in healthy control subjects and in those without neuropathy (P < 0.01, all tests). The correlations among individual tests varied widely, both within (r range 0.9, NS to 0.5, NS to CONCLUSIONS The modest correlation coefficients seen between the different testing modalities suggest that these techniques measure different neurophysiological parameters and are therefore not interchangeable. However, the data suggest that only a small number of neurophysiological tests are actually required to clinically differentiate individuals with neuropathy from those without. The natural clustering of both patients and healthy control subjects suggests that variations in the population will need to be considered in future studies of diabetic neuropathy.

Peripheral arterial disease is characterized by a gradual reduction in blood to the extremities secondary to atherosclerosis. In diabetes, the pattern of atherosclerotic occlusion typically shows a propensity toward the infrapopliteal vessels. Additionally, impairment of the microcirculation manifests in diminished vasoreactivity and a functional ischemia that is not always correctable with surgery. However, when a nonhealing wound is complicated by peripheral arterial disease, revascularization is paramount to wound healing. Revascularization can be accomplished through traditional bypass surgery or newer endovascular interventions, such as angioplasty and stenting. These less invasive techniques of revascularization offer the advantages of quicker recovery and lower morbidity but durability may be compromised. Ultimately, the choice of revascularization procedure should be based on the clinical characteristics of the atherosclerotic lesion along with the individual patient history.

OBJECTIVE To determine how thermoregulation of the feet is affected by diabetes and diabetic polyneuropathy in both wakefulness and sleep. RESEARCH DESIGN AND METHODS Normal subjects, diabetic subjects without neuropathy, diabetic subjects with small-fiber diabetic polyneuropathy, and those with advanced diabetic polyneuropathy were categorized based on neurological examination, nerve conduction studies, and quantitative sensory testing. Subjects underwent foot temperature monitoring using an iButton device attached to the foot and a second iButton for recording of ambient temperature. Socks and footwear were standardized, and subjects maintained an activity diary. Data were collected over a 32-h period and analyzed. RESULTS A total of 39 normal subjects, 28 patients with diabetes but without diabetic polyneuropathy, 14 patients with isolated small-fiber diabetic polyneuropathy, and 27 patients with more advanced diabetic polyneuropathy participated. No consistent differences in foot temperature regulation between the four groups were identified during wakefulness. During sleep, however, multiple metrics revealed significant abnormalities in the diabetic patients. These included reduced mean foot temperature (P < 0.001), reduced maximal temperature (P < 0.001), increased rate of cooling (P < 0.001), as well as increased frequency of variation (P = 0.005), supporting that patients with diabetic polyneuropathy and even those with only diabetes but no diabetic polyneuropathy have impaired nocturnal thermoregulation. CONCLUSIONS Nocturnal foot thermoregulation is impaired in patients with diabetes and diabetic polyneuropathy. Because neurons are highly temperature sensitive and because foot warming is part of the normal biology of sleep onset and maintenance, these findings suggest new potentially treatable mechanisms of diabetes-associated nocturnal pain and sleep disturbance.

The role of dipeptidyl peptidase-IV (DPP4) as both a regulatory enzyme and a signalling factor has been evaluated and described in many studies. DPP4 inhibition results in increased blood concentration of the incretin hormones glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP). This causes an increase in glucose-dependent stimulation of insulin secretion, resulting in a lowering of blood glucose levels. Recent studies have shown that DPP4 inhibitors can induce a significant reduction in glycosylated haemoglobin (HbA(1c)) levels, either as monotherapy or as a combination with other antidiabetic agents. Research has also demonstrated that DPP4 inhibitors portray a very low risk of hypoglycaemia development. This review article focuses on the two leading agents of this category (sitagliptin and vildagliptin), providing an overview of their function along with the latest data regarding their clinical efficacy as antidiabetic agents.

1. Chaired this panel,2. University of Pittsburgh/UPMC, Pittsburgh, Pennsylvania,3. Georgetown University Hospital, Washington, DC,4. Columbia University College of Physicians and Surgeons, Department of Surgery, New York, New York,5. Colaizzi Pedorthic Center, Pittsburgh, Pennsylvania,6. HCA Richmond Retreat Hospital, Richmond, Virginia,7. University of Michigan Hospital, Ann Arbor, Michigan,8. University of Texas Health Science Center, San Antonio, Texas,9. Covance, Princeton, New Jersey,10. St. Joseph’s Hospital, Belleville, Illinois,11. University of South Florida, Tampa, Florida,12. Penn North Centers for Advanced Wound Care, Warren, Pennsylvania,13. Cabrini Medical Center, New York, New York,14. Beth Israel Deaconess Medical Center, Boston, Massachusetts, and15. Barnes-Jewish Hospital at Washington University Medical Center, St. Louis, Missouri