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.

Downregulation of Caveolae-Associated Proteins in Psoriasis: A Case Series Study.

We have previously identified that a structural membrane protein Caveolin-1 (Cav1) is involved in the regulation of aberrant keratinocyte proliferation and differentiation. The aim of this study was to elucidate the role of Cav1, Caveolin-2 (Cav2), and Cavin-1 in the pathogenesis of psoriasis vulgaris and between psoriasis subtypes. We utilized human biopsies from validated cases of psoriasis vulgaris (n = 21) at the University of Miami Hospital and compared the expression of Cav1, Cav2, and Cavin-1 by immunohistochemistry staining with that in normal healthy age-/sex-/location-matched skin (n = 15) and chronic spongiotic dermatitis skin samples (as control inflammatory skin condition) and quantified using QuPath. Distinct subtypes of psoriasis included guttate, inverse, nail, plaque, palmoplantar, and pustular. All biopsy samples exhibited a trend toward downregulation of Cav1, with nail, plaque, and palmoplantar psoriasis exhibiting the most pronounced effects. Only nail and pustular psoriasis samples exhibited significant downregulation of Cav2 and Cavin-1, suggesting Cav1 to be the main caveolar contributor to the pathogenesis of psoriasis. Together, these data support caveolae as pathophysiological targets in nail and pustular psoriasis, whereas Cav1 seems to be a general biomarker of multiple subtypes of psoriasis.

Plantar Skin Exhibits Altered Physiology, Constitutive Activation of Wound-Associated Phenotypes, and Inherently Delayed Healing.

Wound research has typically been performed without regard for where the wounds are located on the body, despite well-known heterogeneities in physical and biological properties between different skin areas. The skin covering the palms and soles is highly specialized, and plantar ulcers are one of the most challenging and costly wound types to manage. Using primarily the porcine model, we show that plantar skin is molecularly and functionally more distinct from nonplantar skin than previously recognized, with unique gene and protein expression profiles, broad alterations in cellular functions, constitutive activation of many wound-associated phenotypes, and inherently delayed healing. This unusual physiology is likely to play a significant but underappreciated role in the pathogenesis of plantar ulcers as well as the last 25+ years of futility in therapy development efforts. By revealing this critical yet unrecognized pitfall, we hope to contribute to the development of more effective therapies for these devastating nonhealing wounds.

Upregulation of Caveolae-Associated Proteins in Lesional Samples of Hidradenitis Suppurativa: A Case Series Study.

Hidradenitis suppurativa (HS) is a chronic, inflammatory skin condition. HS disease management has proven difficult owing to an insufficient understanding of the immunological processes that drive its pathogenesis. We have demonstrated that misregulation of caveolae perturbs inflammatory responses, inhibits cutaneous wound healing, and contributes to immune privilege collapse in other hair follicle-related diseases. However, nothing is known about its role or the role of structural components of caveolae (caveolin [Cav1] 1, Cav2, and Cavin-1) in the pathophysiology of HS. We aimed to identify whether Cav1, Cav2, and Cavin-1 may serve as immunohistochemical markers of HS. Lesional and perilesional HS skin samples from patients (n = 7, mean age = 35.7 years, range = 20-57 years) with active HS and normal skin from control participants (n = 4, mean age = 36.7 years, range = 23-49 years) were used to assess Cav1, Cav2, and Cavin-1 expression and localization by immunofluorescence staining. HS samples demonstrated increased levels of Cav1 compared with normal skin, whereas Cav1, Cav2, and Cavin-1 were all elevated in hair follicles of lesional versus perilesional HS samples, suggesting a potentially novel therapeutic target and highlighting caveolae as potential biomarkers of HS.

The Application of GHRH Antagonist as a Treatment for Resistant APL.

GHRH is a hypothalamic peptide shown to stimulate the proliferation of malignant cells in humans. We have previously shown that the use of GHRH antagonist MIA-602 successfully suppressed the growth of many human cancer cell lines, spanning more than 20 types of cancers. In this study, we demonstrate the presence of GHRH-R in the NB4, NB4-RAA, and K-562 model cell lines. Furthermore, we demonstrate the inhibited proliferation of all three cell lines in vitro after incubation with MIA-602. The treatment of xenografts of human APL cell lines with MIA-602 led to a significant reduction in tumor growth. Additionally, combination therapy with both doxorubicin (DOX) and MIA-602 showed a marked synergistic effect in reducing the proliferation of the K-562 AML cell line. These findings suggest that MIA-602 could be utilized to address resistance to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) therapies, as well as in augmenting anthracycline-based regimens.

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.

Frontiers in Lichen Planopilaris and Frontal Fibrosing Alopecia Research: Pathobiology Progress and Translational Horizons.

Lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA) are primary, lymphocytic cicatricial hair loss disorders. These model epithelial stem cell (SC) diseases are thought to result from a CD8+ T-cell‒dominated immune attack on the hair follicle (HF) SC niche (bulge) after the latter has lost its immune privilege (IP) for as yet unknown reasons. This induces both apoptosis and pathological epithelial‒mesenchymal transition in epithelial SCs, thus depletes the bulge, causes fibrosis, and ultimately abrogates the HFs' capacity to regenerate. In this paper, we synthesize recent progress in LPP and FFA pathobiology research, integrate our limited current understanding of the roles that genetic, hormonal, environmental, and other factors may play, and define major open questions. We propose that LPP and FFA share a common initial pathobiology, which then bifurcates into two distinct clinical phenotypes, with macrophages possibly playing a key role in phenotype determination. As particularly promising translational research avenues toward direly needed progress in the management of these disfiguring, deeply distressful cicatricial alopecia variants, we advocate to focus on the development of bulge IP and epithelial SC protectants such as, for example, topically effective, HF‒penetrating and immunoinhibitory preparations that contain tacrolimus, peroxisome proliferator-activated receptor-γ, and/or CB1 agonists.

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.

Diabetic Wound-Healing Science.

Diabetes mellitus is an increasingly prevalent chronic metabolic disease characterized by prolonged hyperglycemia that leads to long-term health consequences. It is estimated that impaired healing of diabetic wounds affects approximately 25% of all patients with diabetes mellitus, often resulting in lower limb amputation, with subsequent high economic and psychosocial costs. The hyperglycemic environment promotes the formation of biofilms and makes diabetic wounds difficult to treat. In this review, we present updates regarding recent advances in our understanding of the pathophysiology of diabetic wounds focusing on impaired angiogenesis, neuropathy, sub-optimal chronic inflammatory response, barrier disruption, and subsequent polymicrobial infection, followed by current and future treatment strategies designed to tackle the various pathologies associated with diabetic wounds. Given the alarming increase in the prevalence of diabetes, and subsequently diabetic wounds, it is imperative that future treatment strategies target multiple causes of impaired healing in diabetic wounds.

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.

A Cell Membrane-Level Approach to Cicatricial Alopecia Management: Is Caveolin-1 a Viable Therapeutic Target in Frontal Fibrosing Alopecia?

Irreversible destruction of the hair follicle (HF) in primary cicatricial alopecia and its most common variant, frontal fibrosing alopecia (FFA), results from apoptosis and pathological epithelial-mesenchymal transition (EMT) of epithelial HF stem cells (eHFSCs), in conjunction with the collapse of bulge immune privilege (IP) and interferon-gamma-mediated chronic inflammation. The scaffolding protein caveolin-1 (Cav1) is a key component of specialized cell membrane microdomains (caveolae) that regulates multiple signaling events, and even though Cav1 is most prominently expressed in the bulge area of human scalp HFs, it has not been investigated in any cicatricial alopecia context. Interestingly, in mice, Cav1 is involved in the regulation of (1) key HF IP guardians (TGF-ß and α-MSH signaling), (2) IP collapse inducers/markers (IFNγ, substance P and MICA), and (3) EMT. Therefore, we hypothesize that Cav1 may be an unrecognized, important player in the pathobiology of cicatricial alopecias, and particularly, in FFA, which is currently considered as the most common type of primary lymphocytic scarring alopecia in the world. We envision that localized therapeutic inhibition of Cav1 in management of FFA (by cholesterol depleting agents, i.e., cyclodextrins/statins), could inhibit and potentially reverse bulge IP collapse and pathological EMT. Moreover, manipulation of HF Cav1 expression/localization would not only be relevant for management of cicatricial alopecia, but FFA could also serve as a model disease for elucidating the role of Cav1 in other stem cell- and/or IP collapse-related pathologies.

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.

Wound Healing Assay for Melanoma Cell Migration.

Cell migration is a critical process involved in morphogenesis, inflammation, and cancer metastasis. Wound healing assay is a simple, non-expensive, and highly reproducible method to study cancer cell migration in vitro. It is based on the observation that cells growing in a monolayer migrate to re-establish cell contacts after the development of an artificial wound. The assay involves creation of a wound in a monolayer, image acquisition during wound closure, and comparison of migrated area at initial and final time points.

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.

The importance of caveolins and caveolae to dermatology: Lessons from the caves and beyond.

Caveolae are flask-shaped invaginations of the cell membrane rich in cholesterol and sphingomyelin, with caveolin proteins acting as their primary structural components that allow compartmentalization and orchestration of various signalling molecules. In this review, we discuss how pleiotropic functions of caveolin-1 (Cav1) and its intricate roles in numerous cellular functions including lipid trafficking, signalling, cell migration and proliferation, as well as cellular senescence, infection and inflammation, are integral for normal development and functioning of skin and its appendages. We then examine how disruption of the homeostatic levels of Cav1 can lead to development of various cutaneous pathophysiologies including skin cancers, cutaneous fibroses, psoriasis, alopecia, age-related changes in skin and aberrant wound healing and propose how levels of Cav1 may have theragnostic value in skin physiology/pathophysiology.

Multimodal, in Situ Imaging of Ex Vivo Human Skin Reveals Decrease of Cholesterol Sulfate in the Neoepithelium during Acute Wound Healing.

Skin repair is a significant aspect of human health. While the makeup of healthy stratum corneum and epidermis is generally understood, the mobilization of molecular components during skin repair remains largely unknown. In the present work, we utilize multimodal, in situ, mass spectrometry, and immunofluorescence imaging for the characterization of newly formed epidermis, following an initial acute wound for the first 96 h of epithelization. In particular, TOF-SIMS and confirmatory MALDI FT-ICR MS (/MS) analysis permitted the mapping of several lipid classes, including phospholipids, neutral lipids, cholesterol, ceramides, and free fatty acids. Endogenous lipid species were localized in discrete epidermal skin layers, including the stratum corneum (SC), stratum granulosum (SG), stratum basale (SB), and dermis. Experiments revealed that healthy re-epithelializing skin is characterized by diminished cholesterol sulfate signal along the stratum corneum toward the migrating epithelial tongue. The spatial distribution and relative abundances of cholesterol sulfate are reported and correlated with the healing time. The multimodal imaging approach enabled in situ high-confidence chemical mapping based on accurate mass and fragmentation pattern of molecular components. The use of postanalysis immunofluorescence imaging from the same tissue confirmed the localization of endogenous lipid species and Filaggrin and Cav-1 proteins at high spatial resolution (approximately a few microns).

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.

Pharmacological and Genetic Inhibition of Caveolin-1 Promotes Epithelialization and Wound Closure.

Chronic wounds-including diabetic foot ulcers, venous leg ulcers, and pressure ulcers-represent a major health problem that demands an urgent solution and new therapies. Despite major burden to patients, health care professionals, and health care systems worldwide, there are no efficacious therapies approved for treatment of chronic wounds. One of the major obstacles in achieving wound closure in patients is the lack of epithelial migration. Here, we used multiple pre-clinical wound models to show that Caveolin-1 (Cav1) impedes healing and that targeting Cav1 accelerates wound closure. We found that Cav1 expression is significantly upregulated in wound edge biopsies of patients with non-healing wounds, confirming its healing-inhibitory role. Conversely, Cav1 was absent from the migrating epithelium and is downregulated in acutely healing wounds. Specifically, Cav1 interacted with membranous glucocorticoid receptor (mbGR) and epidermal growth factor receptor (EGFR) in a glucocorticoid-dependent manner to inhibit cutaneous healing. However, pharmacological disruption of caveolae by MßCD or CRISPR/Cas9-mediated Cav1 knockdown resulted in disruption of Cav1-mbGR and Cav1-EGFR complexes and promoted epithelialization and wound healing. Our data reveal a novel mechanism of inhibition of epithelialization and wound closure, providing a rationale for pharmacological targeting of Cav1 as potential therapy for patients with non-healing chronic wounds.

Single cell analyses reveal specific distribution of anti-bacterial molecule Perforin-2 in human skin and its modulation by wounding and Staphylococcus aureus infection.

Perforin-2 (P-2) is a recently described antimicrobial protein with unique properties to kill intracellular bacteria. We investigated P-2 expression pattern and cellular distribution in human skin and its importance in restoration of barrier function during wound healing process and infection with the common wound pathogen Staphylococcus aureus. We describe a novel approach for the measurement of P-2 mRNA within individual skin cells using an amplified fluorescence in situ hybridization (FISH) technique. The unique aspect of this approach is simultaneous detection of P-2 mRNA in combination with immune-phenotyping for cell surface proteins using fluorochrome-conjugated antibodies. We detected P-2 transcript in both hematopoietic (CD45+ ) and non-hematopoietic (CD45- ) cutaneous cell populations, confirming the P-2 expression in both professional and non-professional phagocytes. Furthermore, we found an induction of P-2 during wound healing. P-2 overexpression resulted in a reduction of intracellular S. aureus, while infection of human wounds by this pathogen resulted in P-2 suppression, revealing a novel mechanism by which S. aureus may escape cutaneous immunity to cause persistent wound infections.

A Modeling Conundrum: Murine Models for Cutaneous Wound Healing.

The complexity of the cutaneous wound healing process and its impairment in disease states, combined with the urgent clinical need for new therapies demand well-defined preclinical models that facilitate translation of research findings to clinical use. Many murine wound models are well suited for studying mechanisms of various aspects of wound healing, but they have shown limited utility for translating research findings to human conditions, thereby impeding therapeutic developments. Ansell et al. underscore the need for standardization of preclinical murine models by showing the impact of duration of diabetes, animal sex, and assessment methods on healing outcomes in the streptozotocin-induced diabetes mellitus rat model. Recognizing and understanding the limitations and advantages of preclinical murine wound models will facilitate more effective translation of experimental results to improved treatment of human chronic wounds.