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.

Molecular mechanisms regulating wound repair: Evidence for paracrine signaling from corneal epithelial cells to fibroblasts and immune cells following transient epithelial cell treatment with Mitomycin C.

In this paper, we use RNAseq to identify senescence and phagocytosis as key factors to understanding how mitomyin C (MMC) stimulates regenerative wound repair. We use conditioned media (CM) from untreated (CMC) and MMC treated (CMM) human and mouse corneal epithelial cells to show that corneal epithelial cells indirectly exposed to MMC secrete elevated levels of immunomodulatory proteins including IL-1α and TGFß1 compared to cells exposed to CMC. These factors increase epithelial and macrophage phagocytosis and promote ECM turnover. IL-1α supplementation can increase phagocytosis in control epithelial cells and attenuate TGFß1 induced αSMA expression by corneal fibroblasts. Yet, we show that epithelial cell CM contains factors besides IL-1α that regulate phagocytosis and αSMA expression by fibroblasts. Exposure to CMM also impacts the activation of bone marrow derived dendritic cells and their ability to present antigen. These in vitro studies show how a brief exposure to MMC induces corneal epithelial cells to release proteins and other factors that function in a paracrine way to enhance debris removal and enlist resident epithelial and immune cells as well as stromal fibroblasts to support regenerative and not fibrotic wound healing.

Intrinsic Networks Regulating Tissue Repair: Comparative Studies of Oral and Skin Wound Healing.

Wound repair is a systematic biological program characterized by four overlapping phases: hemostasis, inflammation, proliferation, and remodeling. Notwithstanding differences between species and distinct anatomical sites, the fundamental phases in the wound healing process are conserved among mammalian species. Oral wound healing is defined as an ideal wound healing model because it resolves rapidly and without scar formation. Understanding the regulation and contribution of the different molecular events that drive rapid wound healing in oral mucosa compared with those of the skin will help us define how these lesions heal more efficiently and may provide new therapeutic strategies that can be translated to the clinical settings for patients with chronic nonhealing wounds. Although all epithelial tissues have remarkable ability for tissue repair, the efficiency of such repair differs between epithelia (oral mucosa vs. cutaneous). This prompts the long-standing, fundamental biological and clinically relevant questions as to why and how does the oral mucosa achieve its enhanced wound healing capacity. In this review, we focus on (1) distinct innate wound healing capabilities of the oral mucosa, (2) lessons learned from comparative transcriptomic studies of oral mucosa versus skin, and (3) translation of findings to therapeutics for enhanced wound healing.

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.

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.

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.

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.

Topical mevastatin promotes wound healing by inhibiting the transcription factor c-Myc via the glucocorticoid receptor and the long non-coding RNA Gas5.

Diabetic foot ulcers (DFUs), a life-threatening complication of diabetes mellitus, have limited treatment options, often resulting in amputations. HMG-CoA reductase inhibitors such as statins are cholesterol-reducing agents that may provide a new therapeutic option. Statins target the cholesterol pathway and block the synthesis of the wound-healing inhibitors farnesyl pyrophosphate (FPP) and cortisol, ligands for the glucocorticoid receptor (GR). Here we demonstrate that the naturally occurring statin mevastatin reverses FPP's effects and promotes healing by using in vitro wound healing assays, human ex vivo and porcine in vivo wound models, and DFU tissue. Moreover, we measured cortisol levels by ELISA and found that mevastatin inhibited cortisol synthesis in keratinocytes and biopsies from patients with DFU. Of note, topical mevastatin stimulated epithelialization and angiogenesis in vivo Mevastatin also reversed FPP-mediated induction of the GR target, the transcription factor c-Myc (a biomarker of non-healing wounds), in porcine and human wound models. Importantly, mevastatin reversed c-Myc overexpression in DFUs. It induced expression of the long noncoding RNA Gas5 that blocks c-Myc expression, which was confirmed by overexpression studies. We conclude that topical mevastatin accelerates wound closure by promoting epithelialization via multiple mechanisms: modulation of GR ligands and induction of the long noncoding RNA Gas5, leading to c-Myc inhibition. In light of these findings, we propose that repurposing statin drugs for topical treatment of DFUs may offer another option for managing this serious condition.

Skin Metabolite, Farnesyl Pyrophosphate, Regulates Epidermal Response to Inflammation, Oxidative Stress, and Migration.

Skin produces cholesterol and a wide array of sterols and non-sterol mevalonate metabolites, including isoprenoid derivative farnesyl pyrophosphate (FPP). To characterize FPP action in epidermis, we generated transcriptional profiles of primary human keratinocytes treated with zaragozic acid (ZGA), a squalene synthase inhibitor that blocks conversion of FPP to squalene resulting in endogenous accumulation of FPP. The elevated levels of intracellular FPP resulted in regulation of epidermal differentiation and adherens junction signaling, insulin growth factor (IGF) signaling, oxidative stress response and interferon (IFN) signaling. Immunosuppressive properties of FPP were evidenced by STAT-1 downregulation and prominent suppression of its nuclear translocation by IFNγ. Furthermore, FPP profoundly downregulated genes involved in epidermal differentiation of keratinocytes in vitro and in human skin ex vivo. Elevated levels of FPP resulted in induction of cytoprotective transcriptional factor Nrf2 and its target genes. We have previously shown that FPP functions as ligand for the glucocorticoid receptor (GR), one of the major regulator of epidermal homeostasis. Comparative microarray analyses show significant but not complete overlap between FPP and glucocorticoid regulated genes, suggesting that FPP may have wider transcriptional impact. This was further supported by co-transfection and chromatin immunoprecipitation experiments where we show that upon binding to GR, FPP recruits ß-catenin and, unlike glucocorticoids, recruits co-repressor GRIP1 to suppress keratin 6 gene. These findings have many clinical implications related to epidermal lipid metabolism, response to glucocorticoid therapy as well as pleiotropic effects of cholesterol lowering therapeutics, statins. J. Cell. Physiol. 231: 2452-2463, 2016. © 2016 Wiley Periodicals, Inc.