An Optimized and Advanced Algorithm for the Quantification of Immunohistochemical Biomarkers in Keratinocytes.

Advancements in pathology have given rise to software applications intended to minimize human error and improve efficacy of image analysis. Still, the subjectivity of image quantification performed manually and the limitations of the most ubiquitous tissue stain analysis software requiring parameters tuned by the observer, reveal the need for a highly accurate, automated nuclear quantification software specific to immunohistochemistry, with improved precision and efficiency compared with the methods currently in use. We present a method for the quantification of immunohistochemical biomarkers in keratinocyte nuclei proposed to overcome these limitations, contributing sensitive shape-focused segmentation, accurate nuclear detection, and automated device-independent color assessment, without observer-dependent analysis parameters.

Calreticulin: a multifunctional protein with potential therapeutic applications for chronic wounds.

Calreticulin is recognized as a multifunctional protein that serves an essential role in diverse biological processes that include wound healing, modification and folding of proteins, regulation of the secretory pathway, cell motility, cellular metabolism, protein synthesis, regulation of gene expression, cell cycle regulation and apoptosis. Although the role of calreticulin as an endoplasmic reticulum-chaperone protein has been well described, several studies have demonstrated calreticulin to be a highly versatile protein with an essential role during wound healing. These features make it an ideal molecule for treating a complex, multifactorial diseases that require fine tuning, such as chronic wounds. Indeed, topical application of recombinant calreticulin to wounds in multiple models of wound healing has demonstrated remarkable pro-healing effects. Among them include enhanced keratinocyte and fibroblast migration and proliferation, induction of extracellular matrix proteins, recruitment of macrophages along with increased granulation tissue formation, all of which are important functions in promoting wound healing that are deregulated in chronic wounds. Given the high degree of diverse functions and pro-healing effects, application of exogenous calreticulin warrants further investigation as a potential novel therapeutic option for chronic wound patients. Here, we review and highlight the significant effects of topical application of calreticulin on enhancing wound healing and its potential as a novel therapeutic option to shift chronic wounds into healing, acute-like wounds.

Cutaneous changes in diabetic patients: Primed for aberrant healing?

Cutaneous manifestations affect most patients with diabetes mellitus, clinically presenting with numerous dermatologic diseases from xerosis to diabetic foot ulcers (DFUs). Skin conditions not only impose a significantly impaired quality of life on individuals with diabetes but also predispose patients to further complications. Knowledge of cutaneous biology and the wound healing process under diabetic conditions is largely limited to animal models, and studies focusing on biology of the human condition of DFUs remain limited. In this review, we discuss the critical molecular, cellular, and structural changes to the skin in the hyperglycaemic and insulin-resistant environment of diabetes with a focus specifically on human-derived data. Elucidating the breadth of the cutaneous manifestations coupled with effective diabetes management is important for improving patient quality of life and averting future complications including wound healing disorders.

Molecular Pathophysiology of Chronic Wounds: Current State and Future Directions.

Venous leg ulcers, diabetic foot ulcers, and pressure ulcers are complex chronic wounds with multifactorial etiologies that are associated with high patient morbidity and mortality. Despite considerable progress in deciphering the pathologies of chronic wounds using "omics" approaches, considerable gaps in knowledge remain, and current therapies are often not efficacious. We provide a comprehensive overview of current understanding of the molecular mechanisms that impair healing and current knowledge on cell-specific dysregulation including keratinocytes, fibroblasts, immune cells, endothelial cells and their contributions to impaired reepithelialization, inflammation, angiogenesis, and tissue remodeling that characterize chronic wounds. We also provide a rationale for further elucidation of ulcer-specific pathologic processes that can be therapeutically targeted to shift chronic nonhealing to acute healing wounds.

FOXM1 network in association with TREM1 suppression regulates NET formation in diabetic foot ulcers.

Diabetic foot ulcers (DFU) are a serious complication of diabetes mellitus and associated with reduced quality of life and high mortality rate. DFUs are characterized by a deregulated immune response with decreased neutrophils due to loss of the transcription factor, FOXM1. Diabetes primes neutrophils to form neutrophil extracellular traps (NETs), contributing to tissue damage and impaired healing. However, the role of FOXM1 in priming diabetic neutrophils to undergo NET formation remains unknown. Here, we found that FOXM1 regulates reactive oxygen species (ROS) levels in neutrophils and inhibition of FOXM1 results in increased ROS leading to NET formation. Next generation sequencing revealed that TREM1 promoted the recruitment of FOXM1+ neutrophils and reversed effects of diabetes and promoted wound healing in vivo. Moreover, we found that TREM1 expression correlated with clinical healing outcomes of DFUs, indicating TREM1 may serve as a useful biomarker or a potential therapeutic target. Our findings highlight the clinical relevance of TREM1, and indicates FOXM1 pathway as a novel regulator of NET formation during diabetic wound healing, revealing new therapeutic strategies to promote healing in DFUs.

Dichotomous role of miR193b-3p in diabetic foot ulcers maintains inhibition of healing and suppression of tumor formation.

Despite the hyperproliferative environment marked by activation of ß-catenin and overexpression of c-myc, the epidermis surrounding chronic diabetic foot ulcers (DFUs) is clinically hypertrophic and nonmigratory yet does not undergo malignant transformation. We identified miR193b-3p as a master regulator that contributes to this unique cellular phenotype. We determined that induction of tumor suppressor miR193b-3p is a unique feature of DFUs that is not found in venous leg ulcers, acute wounds, or cutaneous squamous cell carcinoma (SCC). Genomic analyses of DFUs identified suppression of the miR193b-3p target gene network that orchestrates cell motility. Inhibition of migration and wound closure was further confirmed by overexpression of miR193b-3p in human organotypic and murine in vivo wound models, whereas miR193b-3p knockdown accelerated wound reepithelialization in human ex vivo and diabetic murine wounds in vivo. The dominant negative effect of miR193b-3p on keratinocyte migration was maintained in the presence of promigratory miR31-5p and miR15b-5p, which were also overexpressed in DFUs. miR193b-3p mediated antimigratory activity by disrupting stress fiber formation and by decreasing activity of GTPase RhoA. Conversely, miR193b-3p targets that typically participate in malignant transformation were found to be differentially regulated between DFUs and SCC, including the proto-oncogenes KRAS (Kirsten rat sarcoma viral proto-oncogene) and KIT (KIT proto-oncogene). Although miR193b-3p acts as a tumor suppressor contributing to low tumor incidence in DFUs, it also acts as a master inhibitor of cellular migration and epithelialization in DFUs. Thus, miR193b-3p may represent a target for wound healing induction, cancer therapeutics, and diagnostics.

Intracellular Staphylococcus aureus triggers pyroptosis and contributes to inhibition of healing due to perforin-2 suppression.

Impaired wound healing associated with recurrent Staphylococcus aureus infection and unresolved inflammation are hallmarks of nonhealing diabetic foot ulcers (DFUs). Perforin-2, an innate immunity molecule against intracellular bacteria, limits cutaneous infection and dissemination of S. aureus in mice. Here, we report the intracellular accumulation of S. aureus in the epidermis of DFUs with no clinical signs of infection due to marked suppression of perforin-2. S. aureus residing within the epidermis of DFUs triggers AIM2 inflammasome activation and pyroptosis. These findings were corroborated in mice lacking perforin-2. The effects of pyroptosis on DFU clinical outcomes were further elucidated in a 4-week longitudinal clinical study in patients with DFUs receiving standard care. Increased AIM2 inflammasome and ASC-pyroptosome coupled with induction of IL-1ß were found in nonhealing DFUs compared with healing DFUs. Our findings revealed that perforin-2 suppression, intracellular S. aureus accumulation, and associated induction of pyroptosis contribute to healing inhibition and prolonged inflammation in patients with DFUs.

Cellular reprogramming of diabetic foot ulcer fibroblasts triggers pro-healing miRNA-mediated epigenetic signature.

Diabetic foot ulcers (DFUs), a prevalent complication of diabetes, constitute a major medical challenge with a critical need for development of cell-based therapies. We previously generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts derived from the DFU patients, location-matched skin of diabetic patients and normal healthy donors and re-differentiated them into fibroblasts. To assess the epigenetic microRNA (miR) regulated changes triggered by cellular reprogramming, we performed miRs expression profiling. We found let-7c, miR-26b-5p, -29c-3p, -148a-3p, -196a-5p, -199b-5p and -374a-5p suppressed in iPSC-derived fibroblasts in vitro and in 3D dermis-like self-assembly tissue, whereas their corresponding targets involved in cellular migration were upregulated. Moreover, targets involved in organization of extracellular matrix were induced after fibroblast reprogramming. PLAT gene, the crucial fibrinolysis factor, was upregulated in iPSC-derived fibroblasts and was confirmed as a direct target of miR-196a-5p. miR-197-3p and miR-331-3p were found upregulated specifically in iPSC-derived diabetic fibroblasts, while their targets CAV1 and CDKN3 were suppressed. CAV1, an important negative regulator of wound healing, was confirmed as a direct miR-197-3p target. Together, our findings demonstrate that iPSC reprogramming is an effective approach for erasing the diabetic non-healing miR-mediated epigenetic signature and promoting a pro-healing cellular phenotype.

Glucocorticoid-mediated induction of caveolin-1 disrupts cytoskeletal organization, inhibits cell migration and re-epithelialization of non-healing wounds.

Although impaired keratinocyte migration is a recognized hallmark of chronic wounds, the molecular mechanisms underpinning impaired cell movement are poorly understood. Here, we demonstrate that both diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs) exhibit global deregulation of cytoskeletal organization in genomic comparison to normal skin and acute wounds. Interestingly, we found that DFUs and VLUs exhibited downregulation of ArhGAP35, which serves both as an inactivator of RhoA and as a glucocorticoid repressor. Since chronic wounds exhibit elevated levels of cortisol and caveolin-1 (Cav1), we posited that observed elevation of Cav1 expression may contribute to impaired actin-cytoskeletal signaling, manifesting in aberrant keratinocyte migration. We showed that Cav1 indeed antagonizes ArhGAP35, resulting in increased activation of RhoA and diminished activation of Cdc42, which can be rescued by Cav1 disruption. Furthermore, we demonstrate that both inducible keratinocyte specific Cav1 knockout mice, and MßCD treated diabetic mice, exhibit accelerated wound closure. Taken together, our findings provide a previously unreported mechanism by which Cav1-mediated cytoskeletal organization prevents wound closure in patients with chronic wounds.

Epigenetic regulation of cellular functions in wound healing.

Stringent spatiotemporal regulation of the wound healing process involving multiple cell types is associated with epigenetic mechanisms of gene regulation, such as DNA methylation, histone modification and chromatin remodelling, as well as non-coding RNAs. Here, we discuss the epigenetic changes that occur during wound healing and the rapidly expanding understanding of how these mechanisms affect healing resolution in both acute and chronic wound milieu. We provide a focussed overview of current research into epigenetic regulators that contribute to wound healing by specific cell type. We highlight the role of epigenetic regulators in the molecular pathophysiology of chronic wound conditions. The understanding of how epigenetic regulators can affect cellular functions during normal and impaired wound healing could lead to novel therapeutic approaches, and we outline questions that can provide guidance for future research on epigenetic-based interventions to promote healing. Dissecting the dynamic interplay between cellular subtypes involved in wound healing and epigenetic parameters during barrier repair will deepen our understanding of how to improve healing outcomes in patients affected by chronic non-healing wounds.

Telomere Dynamics and Telomerase in the Biology of Hair Follicles and their Stem Cells as a Model for Aging Research.

In this review, we propose that telomere length dynamics play an important but underinvestigated role in the biology of the hair follicle (HF), a prototypic, cyclically remodeled miniorgan that shows an intriguing aging pattern in humans. Whereas the HF pigmentary unit ages quickly, its epithelial stem cell (ESC) component and regenerative capacity are surprisingly aging resistant. Telomerase-deficient mice with short telomeres display an aging phenotype of hair graying and hair loss that is attributed to impaired HF ESC mobilization. Yet, it remains unclear whether the function of telomerase and telomeres in murine HF biology translate to the human system. Therefore, we propose new directions for future telomere research of the human HF. Such research may guide the development of novel treatments for selected disorders of human hair growth or pigmentation (e.g., chemotherapy-induced alopecia, telogen effluvium, androgenetic alopecia, cicatricial alopecia, graying). It might also increase the understanding of the global role of telomeres in aging-related human disease.

Genomics of Human Fibrotic Diseases: Disordered Wound Healing Response.

Fibrotic disease, which is implicated in almost half of all deaths worldwide, is the result of an uncontrolled wound healing response to injury in which tissue is replaced by deposition of excess extracellular matrix, leading to fibrosis and loss of organ function. A plethora of genome-wide association studies, microarrays, exome sequencing studies, DNA methylation arrays, next-generation sequencing, and profiling of noncoding RNAs have been performed in patient-derived fibrotic tissue, with the shared goal of utilizing genomics to identify the transcriptional networks and biological pathways underlying the development of fibrotic diseases. In this review, we discuss fibrosing disorders of the skin, liver, kidney, lung, and heart, systematically (1) characterizing the initial acute injury that drives unresolved inflammation, (2) identifying genomic studies that have defined the pathologic gene changes leading to excess matrix deposition and fibrogenesis, and (3) summarizing therapies targeting pro-fibrotic genes and networks identified in the genomic studies. Ultimately, successful bench-to-bedside translation of observations from genomic studies will result in the development of novel anti-fibrotic therapeutics that improve functional quality of life for patients and decrease mortality from fibrotic diseases.

Deregulated immune cell recruitment orchestrated by FOXM1 impairs human diabetic wound healing.

Diabetic foot ulcers (DFUs) are a life-threatening disease that often result in lower limb amputations and a shortened lifespan. However, molecular mechanisms contributing to the pathogenesis of DFUs remain poorly understood. We use next-generation sequencing to generate a human dataset of pathogenic DFUs to compare to transcriptional profiles of human skin and oral acute wounds, oral as a model of "ideal" adult tissue repair due to accelerated closure without scarring. Here we identify major transcriptional networks deregulated in DFUs that result in decreased neutrophils and macrophages recruitment and overall poorly controlled inflammatory response. Transcription factors FOXM1 and STAT3, which function to activate and promote survival of immune cells, are inhibited in DFUs. Moreover, inhibition of FOXM1 in diabetic mouse models (STZ-induced and db/db) results in delayed wound healing and decreased neutrophil and macrophage recruitment in diabetic wounds in vivo. Our data underscore the role of a perturbed, ineffective inflammatory response as a major contributor to the pathogenesis of DFUs, which is facilitated by FOXM1-mediated deregulation of recruitment of neutrophils and macrophages, revealing a potential therapeutic strategy.

A bioengineered living cell construct activates metallothionein/zinc/MMP8 and inhibits TGFß to stimulate remodeling of fibrotic venous leg ulcers.

Venous leg ulcers (VLU) represent a major clinical unmet need, impairing quality of life for millions worldwide. The bioengineered bilayered living cell construct (BLCC) is the only FDA-approved therapy demonstrating efficacy in healing chronic VLU, yet its in vivo mechanisms of action are not well understood. Previously, we reported a BLCC-mediated acute wounding response at the ulcer edge; in this study we elucidated the BLCC-specific effects on the epidermis-free ulcer bed. We conducted a randomized controlled clinical trial (ClinicalTrials.gov NCT01327937) enrolling 30 subjects with nonhealing VLUs, and performed genotyping, genomic profiling, and functional analysis on wound bed biopsies obtained at baseline and 1 week after treatment with BLCC plus compression or compression therapy (control). The VLU bed transcriptome featured processes of chronic inflammation and was strikingly enriched for fibrotic/fibrogenic pathways and gene networks. BLCC application decreased expression of profibrotic TGFß1 gene targets and increased levels of TGFß inhibitor decorin. Surprisingly, BLCC upregulated metallothioneins and fibroblast-derived MMP8 collagenase, and promoted endogenous release of MMP-activating zinc to stimulate antifibrotic remodeling, a novel mechanism of cutaneous wound healing. By activating a remodeling program in the quiescent VLU bed, BLCC application shifts nonhealing to healing phenotype. As VLU bed fibrosis correlates with poor clinical healing, findings from this study identify the chronic VLU as a fibrotic skin disease and are first to support the development and application of antifibrotic therapies as a successful treatment approach.

Mevastatin promotes healing by targeting caveolin-1 to restore EGFR signaling.

Diabetic foot ulcers (DFUs) are a life-threatening disease that often results in lower limb amputations and a shortened life span. Current treatment options are limited and often not efficacious, raising the need for new therapies. To investigate the therapeutic potential of topical statins to restore healing in patients with DFUs, we performed next-generation sequencing on mevastatin-treated primary human keratinocytes. We found that mevastatin activated and modulated the EGF signaling to trigger an antiproliferative and promigratory phenotype, suggesting that statins may shift DFUs from a hyperproliferative phenotype to a promigratory phenotype in order to stimulate healing. Furthermore, mevastatin induced a migratory phenotype in primary human keratinocytes through EGF-mediated activation of Rac1, resulting in actin cytoskeletal reorganization and lamellipodia formation. Interestingly, the EGF receptor is downregulated in tissue biopsies from patients with DFUs. Mevastatin restored EGF signaling in DFUs through disruption of caveolae to promote keratinocyte migration, which was confirmed by caveolin-1 (Cav1) overexpression studies. We conclude that topical statins may have considerable therapeutic potential as a treatment option for patients with DFUs and offer an effective treatment for chronic wounds that can be rapidly translated to clinical use.

Staphylococcus aureus Triggers Induction of miR-15B-5P to Diminish DNA Repair and Deregulate Inflammatory Response in Diabetic Foot Ulcers.

Diabetic foot ulcers (DFUs) are a debilitating complication of diabetes in which bacterial presence, including the frequent colonizer Staphylococcus aureus, contributes to inhibition of healing. MicroRNAs (miRs) play a role in healing and host response to bacterial pathogens. However, the mechanisms by which miR response to cutaneous S. aureus contributes to DFU pathophysiology are unknown. Here, we show that S. aureus inhibits wound closure and induces miR-15b-5p in acute human and porcine wound models and in chronic DFUs. Transcriptome analyses of DFU tissue showed induction of miR-15b-5p to be critical, regulating many cellular processes, including DNA repair and inflammatory response, by suppressing downstream targets IKBKB, WEE1, FGF2, RAD50, MSH2, and KIT. Using a human wound model, we confirmed that S. aureus-triggered miR-15b-5p induction results in suppression of the inflammatory- and DNA repair-related genes IKBKB and WEE1. Inhibition of DNA repair and accumulation of DNA breaks was functionally confirmed by the presence of the pH2AX within colonized DFUs. We conclude that S. aureus induces miR-15b-5p, subsequently repressing DNA repair and inflammatory response, showing a mechanism of inhibition of healing in DFUs previously unreported, to our knowledge. This underscores a previously unknown role of DNA damage repair in the pathophysiology of DFUs colonized with S. aureus.

A bioengineered living cell construct activates an acute wound healing response in venous leg ulcers.

Chronic nonhealing venous leg ulcers (VLUs) are widespread and debilitating, with high morbidity and associated costs; about $15 billion is spent annually on the care of VLUs in the United States. Despite this, there is a paucity of treatments for VLUs because of the lack of pathophysiologic insight into ulcer development as well as the lack of knowledge regarding biologic actions of existing VLU-targeted therapies. The bioengineered bilayered living cellular construct (BLCC) skin substitute is a U.S. Food and Drug Administration-approved biologic treatment for healing VLUs. To elucidate the mechanisms through which the BLCC promotes healing of chronic VLUs, we conducted a clinical trial (NCT01327937) in which patients with nonhealing VLUs were treated with either standard of care (compression therapy) or the BLCC together with standard of care. Tissue was collected from the VLU edge before and 1 week after treatment, and the samples underwent comprehensive microarray mRNA and protein analyses. Ulcers treated with the BLCC skin substitute displayed three distinct transcriptomic patterns, suggesting that BLCC induced a shift from a nonhealing to a healing tissue response, involving modulation of inflammatory and growth factor signaling, keratinocyte activation, and attenuation of Wnt/ß-catenin signaling. In these ways, BLCC application orchestrated a shift from the chronic nonhealing ulcer microenvironment to a distinctive healing milieu resembling that of an acute, healing wound. Our findings provide in vivo evidence in VLU patients of pathways that can be targeted in the design of new therapies to promote healing of chronic VLUs.

Integrative analysis of miRNA and mRNA paired expression profiling of primary fibroblast derived from diabetic foot ulcers reveals multiple impaired cellular functions.

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.