Engineered Exosomes Containing Cathelicidin/LL-37 Exhibit Multiple Biological Functions.

Exosomes show great potential in diagnostic and therapeutic applications. Inspired by the human innate immune defense, herein, we report engineered exosomes derived from monocytic cells treated with immunomodulating compounds 1α,25-dihydroxyvitamin D3, and CYP24A1 inhibitor VID400 which are slowly released from electrospun nanofiber matrices. These engineered exosomes contain significantly more cathelicidin/LL-37 when compared with exosomes derived from either untreated cells or Cathelicidin Human Tagged ORF Clone transfected cells. In addition, such exosomes exhibit multiple biological functions evidenced by killing bacteria, facilitating human umbilical vein endothelial cell tube formation, and enhancing skin cell proliferation and migration. Taken together, the engineered exosomes developed in this study can be used as therapeutics alone or in combination with other biomaterials for effective infection management, wound healing, and tissue regeneration.

Selective Ablation of BCL11A in Epidermal Keratinocytes Alters Skin Homeostasis and Accelerates Excisional Wound Healing In Vivo.

Transcriptional regulator BCL11A plays a crucial role in coordinating a suite of developmental processes including skin morphogenesis, barrier functions and lipid metabolism. There is little or no reports so far documenting the role of BCL11A in postnatal adult skin homeostasis and in the physiological process of tissue repair and regeneration. The current study establishes for the first time the In Vivo role of epidermal BCL11A in maintaining adult epidermal homeostasis and as a negative regulator of cutaneous wound healing. Conditional ablation of Bcl11a in skin epidermal keratinocytes (Bcl11aep-/-mice) enhances the keratinocyte proliferation and differentiation program, suggesting its critical role in epidermal homeostasis of adult murine skin. Further, loss of keratinocytic BCL11A promotes rapid closure of excisional wounds both in a cell autonomous manner likely via accelerating wound re-epithelialization and in a non-cell autonomous manner by enhancing angiogenesis. The epidermis specific Bcl11a knockout mouse serves as a prototype to gain mechanistic understanding of various downstream pathways converging towards the manifestation of an accelerated healing phenotype upon its deletion.

Codelivery of 1α,25-Dihydroxyvitamin D3 and CYP24A1 Inhibitor VID400 by Nanofiber Dressings Promotes Endogenous Antimicrobial Peptide LL-37 Induction.

Surgical site infections represent a significant clinical problem. Herein, we report a nanofiber dressing for topical codelivery of immunomodulating compounds including 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and VID400, a CYP24A1 inhibitor in a sustained manner, for inducing the expression of the endogenous cathelicidin antimicrobial peptide (CAMP) gene encoding the hCAP18 protein, which is processed into the LL-37 peptide. Nanofiber wound dressings with coencapsulation of 1,25(OH)2D3 and VID400 were generated by electrospinning. Both 1,25(OH)2D3 and VID400 were coencapsulated into nanofibers with loading efficiencies higher than 90% and exhibited a prolonged release from nanofiber membranes longer than 28 days. Incubation with 1,25(OH)2D3/VID400-coencapsulated poly(ϵ-caprolactone) nanofiber membranes greatly induced the hCAP18/LL-37 gene expression in monocytes, neutrophils, and keratinocytes in vitro. Moreover, the administration of 1,25(OH)2D3/VID400-coencapsulated nanofiber membranes dramatically promoted the hCAP18/LL-37 expression in dermal wounds created in both human CAMP transgenic mice and human skin tissues. The 1,25(OH)2D3- and VID400-coencapsulated nanofiber dressings enhanced innate immunity via the more effective induction of antimicrobial peptide than the free drug alone or 1,25(OH)2D3-loaded nanofibers. Together, 1,25(OH)2D3/VID400-embedded nanofiber dressings presented in this study show potential in preventing surgical site infections.

Bioengineered Efficacy Models of Skin Disease: Advances in the Last 10 Years.

Models of skin diseases, such as psoriasis and scleroderma, must accurately recapitulate the complex microenvironment of human skin to provide an efficacious platform for investigation of skin diseases. Skin disease research has been shifting from less complex and less relevant 2D (two-dimensional) models to significantly more relevant 3D (three-dimensional) models. Three-dimensional modeling systems are better able to recapitulate the complex cell-cell and cell-matrix interactions that occur in vivo within skin. Three-dimensional human skin equivalents (HSEs) have emerged as an advantageous tool for the study of skin disease in vitro. These 3D HSEs can be highly complex, containing both epidermal and dermal compartments with integrated adnexal structures. The addition of adnexal structures to 3D HSEs has allowed researchers to gain more insight into the complex pathology of various hereditary and acquired skin diseases. One method of constructing 3D HSEs, 3D bioprinting, has emerged as a versatile and useful tool for generating highly complex HSEs. The development of commercially available 3D bioprinters has allowed researchers to create highly reproducible 3D HSEs with precise integration of multiple adnexal structures. While the field of bioengineered models for study of skin disease has made tremendous progress in the last decade, there are still significant efforts necessary to create truly biomimetic skin disease models. In future studies utilizing 3D HSEs, emphasis must be placed on integrating all adnexal structures relevant to the skin disease under investigation. Thorough investigation of the intricate pathology of skin diseases and the development of effective treatments requires use of highly efficacious models of skin diseases.

Thioredoxin Reductase 1 Modulates Pigmentation and Photobiology of Murine Melanocytes in vivo.

Pigment-producing melanocytes overcome frequent oxidative stress in their physiological role of protecting the skin against the deleterious effects of solar UV irradiation. This is accomplished by the activity of several endogenous antioxidant systems, including the thioredoxin antioxidant system, in which thioredoxin reductase 1 (TR1) plays an important part. To determine whether TR1 contributes to the redox regulation of melanocyte homeostasis, we have generated a selective melanocytic Txnrd1-knockout mouse model (Txnrd1mel‒/‒), which exhibits a depigmentation phenotype consisting of variable amelanotic ventral spotting and reduced pigmentation on the extremities (tail tip, ears, and paws). The antioxidant role of TR1 was further probed in the presence of acute neonatal UVB irradiation, which stimulates melanocyte activation and introduces a spike in oxidative stress in the skin microenvironment. Interestingly, we observed a significant reduction in overall melanocyte count and proliferation in the absence of TR1. Furthermore, melanocytes exhibited an elevated level of UV-induced DNA damage in the form of 8-oxo-2'-deoxyguanosine after acute UVB treatment. We also saw an engagement of compensatory antioxidant mechanisms through increased nuclear localization of transcription factor NRF2. Altogether, these data indicate that melanocytic TR1 positively regulates melanocyte homeostasis and pigmentation during development and protects against UVB-induced DNA damage and oxidative stress.

CTIP2 and lipid metabolism: regulation in skin development and associated diseases.

INTRODUCTION: COUP-TF INTERACTING PROTEIN 2 (CTIP2) is a crucial transcription factor exhibiting its control through coupled modulation of epigenetic modification and transcriptional regulation of key genes related to skin, immune, and nervous system development. Previous studies have validated the essential role of CTIP2 in skin development and maintenance, propagating its effects in epidermal permeability barrier (EPB) homeostasis, wound healing, inflammatory diseases, and epithelial cancers. Lipid metabolism dysregulation, on the other hand, has also established its independent emerging role over the years in normal skin development and various skin-associated ailments. This review focuses on the relatively unexplored connections between CTIP2-mediated control of lipid metabolism and alteration of EPB homeostasis, delayed wound healing, inflammatory diseases exacerbation, and cancer promotion and progression. AREAS COVERED: Here we have discussed the intricate interplay of various endogenous lipids and lipoproteins accompanying skin development and associated disease processes and the possible link to CTIP2-mediated regulation of lipid metabolism. EXPERT OPINION: Establishing the link between CTIP2 and lipid metabolism alterations in the context of skin morphogenesis and diverse types of skin diseases including cancer can help us identify novel targets for effective therapeutic intervention.

Healing of Full-Thickness Murine Skin Wounds Containing Nanofibers Using Splints for Efficient Reepithelialization and to Avoid Contracture.

Wound healing process is the outcome of a series of actions and combined with collaborative process involving concerted efforts of multiple cell types. The dynamic series of events constituting each of these overlapping rather than discrete stages of wound healing increases its complexity and the necessity to understand it. The contrasting mechanisms of wound healing employed by mouse (via wound contraction) and humans (via reepithelialization) puts forth the need of a model closely mimicking human wound-healing and hence comes the applicability of the mouse excisional wound splinting model. Use of silicone-based splints has demonstrated their effectiveness in aptly resembling the human reepithelialization mediated wound healing by preventing contraction during healing. The rising popularity of nanofiber-based treatments for wound healing through sustained release of factors/molecules promoting wound closure can be potentially implemented in association with this model to determine its efficacy in wound management in a more humanized way.

A mouse model for vitamin D-induced human cathelicidin antimicrobial peptide gene expression.

In humans and other primates, 1,25(OH)2vitamin D3 regulates the expression of the cathelicidin antimicrobial peptide (CAMP) gene via toll-like receptor (TLR) signaling that activates the vitamin D pathway. Mice and other mammals lack the vitamin D response element (VDRE) in their CAMP promoters. To elucidate the biological importance of this pathway, we generated transgenic mice that carry a genomic DNA fragment encompassing the entire human CAMP gene and crossed them with Camp knockout (KO) mice. We observed expression of the human transgene in various tissues and innate immune cells. However, in mouse CAMP transgenic macrophages, TLR activation in the presence of 25(OH)D3 did not induce expression of either CAMP or CYP27B1 as would normally occur in human macrophages, reinforcing important species differences in the actions of vitamin D. Transgenic mice did show increased resistance to colonization by Salmonella typhimurium in the gut. Furthermore, the human CAMP gene restored wound healing in the skin of Camp KO mice. Topical application of 1,25(OH)2vitamin D3 to the skin of CAMP transgenic mice induced CAMP expression and increased killing of Staphylococcus aureus in a wound infection model. Our model can help elucidate the biological importance of the vitamin D-cathelicidin pathway in both pathogenic and non-pathogenic states.

Transcriptional control and transcriptomic analysis of lipid metabolism in skin barrier formation and atopic dermatitis (AD).

Introduction: Atopic dermatitis (AD) is a multifactorial ailment associated with barrier breach and intense systemic inflammation. Several studies over the years have shown the complex interplay of a large number of factors in governing the progression and outcome of AD. In addition to the diverse types of AD resulting due to variation in the intrinsic mechanisms giving rise to AD such as single nucleotide polymorphisms (SNPs), epigenetic alterations or transcriptional changes, extrinsic factors such as age, ancestry, ethnicity, immunological background of the subject, the interactions of the subject with environmental stimuli and existing microbiome in the periphery surrounding the subject account for further heterogeneity in the clinical manifestations of the disease. Areas covered: Here we have selectively discussed transcriptional regulation of genes associated with skin lipid metabolism in the context of AD. Transcriptional control and transcriptomic changes are just one face of this multifaceted disease known to affect humans and a detailed study concerning those will enable us to develop targeted therapies to deal with the disease. Expert opinion: Large-scale integration of different omics approaches (genomics, epigenomics, transcriptomics, lipidomics, proteomics, metabolomics, effect of exposome) will help identify the potential candidate gene(s) associated with the development of various endotypes of AD.

Mitochondrial complex I inhibitor deguelin induces metabolic reprogramming and sensitizes vemurafenib-resistant BRAFV600E mutation bearing metastatic melanoma cells.

Treatment with vemurafenib, a potent and selective inhibitor of mitogen-activated protein kinase signaling downstream of the BRAFV600E oncogene, elicits dramatic clinical responses in patients with metastatic melanoma. Unfortunately, the clinical utility of this drug is limited by a high incidence of drug resistance. Thus, there is an unmet need for alternative therapeutic strategies to treat vemurafenib-resistant metastatic melanomas. We have conducted high-throughput screening of two bioactive compound libraries (Siga and Spectrum libraries) against a metastatic melanoma cell line (A2058) and identified two structurally analogous compounds, deguelin and rotenone, from a cell viability assay. Vemurafenib-resistant melanoma cell lines, A2058R and A375R (containing the BRAFV600E mutation), also showed reduced proliferation when treated with these two compounds. Deguelin, a mitochondrial complex I inhibitor, was noted to significantly inhibit oxygen consumption in cellular metabolism assays. Mechanistically, deguelin treatment rapidly activates AMPK signaling, which results in inhibition of mTORC1 signaling and differential phosphorylation of mTORC1's downstream effectors, 4E-BP1 and p70S6 kinase. Deguelin also significantly inhibited ERK activation and Ki67 expression without altering Akt activation in the same timeframe in the vemurafenib-resistant melanoma cells. These data posit that treatment with metabolic regulators, such as deguelin, can lead to energy starvation, thereby modulating the intracellular metabolic environment and reducing survival of drug-resistant melanomas harboring BRAF V600E mutations.

Epidermal Lipids: Key Mediators of Atopic Dermatitis Pathogenesis.

The skin barrier keeps the 'inside in' and the 'outside out', forming a protective blanket against external insults. Epidermal lipids, such as ceramides, fatty acids (FAs), triglycerides, and cholesterol, are integral components driving the formation and maintenance of the epidermal permeability barrier (EPB). A breach in this lipid barrier sets the platform for the subsequent onset and progression of atopic dermatitis (AD). Such lipids are also important in the normal functioning of organisms, both plants and animals, and in diseases, including cancer. Given the doubling of the number of cases of AD in recent years and the chronic nature of this disorder, here we shed light on the multifaceted role of diverse types of lipid in mediating AD pathogenesis.

Stearoyl CoA Desaturase Is Essential for Regulation of Endoplasmic Reticulum Homeostasis and Tumor Growth in Glioblastoma Cancer Stem Cells.

Inherent plasticity and various survival cues allow glioblastoma stem-like cells (GSCs) to survive and proliferate under intrinsic and extrinsic stress conditions. Here, we report that GSCs depend on the adaptive activation of ER stress and subsequent activation of lipogenesis and particularly stearoyl CoA desaturase (SCD1), which promotes ER homeostasis, cytoprotection, and tumor initiation. Pharmacological targeting of SCD1 is particularly toxic due to the accumulation of saturated fatty acids, which exacerbates ER stress, triggers apoptosis, impairs RAD51-mediated DNA repair, and achieves a remarkable therapeutic outcome with 25%-100% cure rate in xenograft mouse models. Mechanistically, divergent cell fates under varying levels of ER stress are primarily controlled by the ER sensor IRE1, which either promotes SCD1 transcriptional activation or converts to apoptotic signaling when SCD1 activity is impaired. Taken together, the dependence of GSCs on fatty acid desaturation presents an exploitable vulnerability to target glioblastoma.

1α,25-dihydroxyvitamin D3-eluting nanofibrous dressings induce endogenous antimicrobial peptide expression.

AIM: The aim of this study was to develop a nanofiber-based dressing capable of local sustained delivery of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and augmenting human CAMP induction. MATERIALS & METHODS: Nanofibrous wound dressings containing 1,25(OH)2D3 were successfully prepared by electrospinning, which were examined in vitro, in vivo and ex vivo. RESULTS: 1,25(OH)2D3 was successfully loaded into nanofibers with encapsulation efficiency larger than 90%. 1,25(OH)2D3 showed a sustained release from nanofibers over 4 weeks. Treatment of U937 and HaCaT cells with 1,25(OH)2D3-loaded poly(ϵ-caprolactone) nanofibers significantly induced hCAP18/LL37 expression in monocytes and keratinocytes, skin wounds of humanized transgenic mice and artificial wounds of human skin explants. CONCLUSION: 1,25(OH)2D3 containing nanofibrous dressings could enhance innate immunity by inducing antimicrobial peptide production.

Photoprotective Properties of Isothiocyanate and Nitrile Glucosinolate Derivatives From Meadowfoam (Limnanthes alba) Against UVB Irradiation in Human Skin Equivalent.

Exposure to ultraviolet B (UVB) irradiation of the skin leads to numerous dermatological concerns including skin cancer and accelerated aging. Natural product glucosinolate derivatives, like sulforaphane, have been shown to exhibit chemopreventive and photoprotective properties. In this study, we examined meadowfoam (Limnanthes alba) glucosinolate derivatives, 3-methoxybenzyl isothiocyanate (MBITC) and 3-methoxyphenyl acetonitrile (MPACN), for their activity in protecting against the consequences of UV exposure. To that end, we have exposed human primary epidermal keratinocytes (HPEKs) and 3D human skin reconstructed in vitro (EpiDermTM FT-400) to UVB insult and investigated whether MBITC and MPACN treatment ameliorated the harmful effects of UVB damage. Activity was determined by the compounds' efficacy in counteracting UVB-induced DNA damage, matrix-metalloproteinase (MMP) expression, and proliferation. We found that in monolayer cultures of HPEK, MBITC and MPACN did not protect against a UVB-induced loss in proliferation and MBITC itself inhibited cell proliferation. However, in human reconstructed skin-equivalents, MBITC and MPACN decrease epidermal cyclobutane pyrimidine dimers (CPDs) and significantly reduce total phosphorylated γH2A.X levels. Both MBITC and MPACN inhibit UVB-induced MMP-1 and MMP-3 expression indicating their role to prevent photoaging. Both compounds, and MPACN in particular, showed activity against UVB-induced proliferation as indicated by fewer epidermal PCNA+ cells and prevented UVB-induced hyperplasia as determined by a reduction in reconstructed skin epidermal thickness (ET). These data demonstrate that MBITC and MPACN exhibit promising anti-photocarcinogenic and anti-photoaging properties in the skin microenvironment and could be used for therapeutic interventions.

Ablation of epidermal RXRα in cooperation with activated CDK4 and oncogenic NRAS generates spontaneous and acute neonatal UVB induced malignant metastatic melanomas.

BACKGROUND: Understanding the underlying molecular mechanisms involved in the formation of cutaneous malignant melanoma is critical for improved diagnosis and treatment. Keratinocytic nuclear receptor Retinoid X Receptor α (RXRα) has a protective role against melanomagenesis and is involved in the regulation of keratinocyte and melanocyte homeostasis subsequent acute ultraviolet (UV) irradiation. METHODS: We generated a trigenic mouse model system (RXRα ep-/- | Tyr-NRAS Q61K | CDK4 R24C/R24C ) harboring an epidermal knockout of Retinoid X Receptor α (RXRα ep-/- ), combined with oncogenic NRAS Q61K (constitutively active RAS) and activated CDK4 R24C/R24C (constitutively active CDK4). Those mice were subjected to a single neonatal dose of UVB treatment and the role of RXR α was evaluated by characterizing the molecular and cellular changes that took place in the untreated and UVB treated trigenic RXRα ep-/- mice compared to the control mice with functional RXRα. RESULTS: Here we report that the trigenic mice develops spontaneous melanoma and exposure to a single neonatal UVB treatment reduces the tumor latency in those mice compared to control mice with functional RXRα. Melanomas from the trigenic RXRα ep-/- mice are substantial in size, show increased proliferation, exhibit increased expression of malignant melanoma markers and exhibit enhanced vascularization. Altered expression of several biomarkers including increased expression of activated AKT, p21 and cyclin D1 and reduced expression of pro-apoptotic marker BAX was observed in the tumor adjacent normal (TAN) skin of acute ultraviolet B treated trigenic RXRα ep-/- mice. Interestingly, we observed a significant increase in p21 and Cyclin D1 in the TAN skin of un-irradiated trigenic RXRα ep-/- mice, suggesting that those changes might be consequences of loss of functional RXRα in the melanoma microenvironment. Loss of RXRα in the epidermal keratinocytes in combination with oncogenic NRAS Q61K and CDK4 R24C/R24C mutations in trigenic mice led to significant melanoma invasion into the draining lymph nodes as compared to controls with functional RXRα. CONCLUSIONS: Our study demonstrates the protective role of keratinocytic RxRα in (1) suppressing spontaneous and acute UVB-induced melanoma, and (2) preventing progression of the melanoma to malignancy in the presence of driver mutations like activated CDK4 R24C/R24C and oncogenic NRAS Q61K .

Transcription Factor CTIP1/ BCL11A Regulates Epidermal Differentiation and Lipid Metabolism During Skin Development.

The epidermal permeability barrier (EPB) prevents organisms from dehydration and infection. The transcriptional regulation of EPB development is poorly understood. We demonstrate here that transcription factor COUP-TF-interacting protein 1 (CTIP1/BCL11A; hereafter CTIP1) is highly expressed in the developing murine epidermis. Germline deletion of Ctip1 (Ctip1 -/-) results in EPB defects accompanied by compromised epidermal differentiation, drastic reduction in profilaggrin processing, reduced lamellar bodies in granular layers and significantly altered lipid composition. Transcriptional profiling of Ctip1 -/- embryonic skin identified altered expression of genes encoding lipid-metabolism enzymes, skin barrier-associated transcription factors and junctional proteins. CTIP1 was observed to interact with genomic elements within the regulatory region of the gene encoding the differentiation-associated gene, Fos-related antigen2 (Fosl2) and lipid-metabolism-related gene, Fatty acid elongase 4 (Elvol4), and the expression of both was altered in Ctip1 -/- mice. CTIP1 appears to play a role in EPB establishment of via direct or indirect regulation of a subset of genes encoding proteins involved in epidermal differentiation and lipid metabolism. These results identify potential, CTIP1-regulated avenues for treatment of skin disorders involving EBP defects.

Lipidomic analysis of epidermal lipids: a tool to predict progression of inflammatory skin disease in humans.

INTRODUCTION: Lipidomics is the large-scale profiling and characterization of lipid species in a biological system using mass spectrometry. The skin barrier is mainly comprised of corneocytes and a lipid-enriched extracellular matrix. The major skin lipids are ceramides, cholesterol and free fatty acids (FFA). Lipid compositions are altered in inflammatory skin disorders with disrupted skin barrier such as atopic dermatitis (AD). AREAS COVERED: Here we discuss some of the recent applications of lipidomics in human skin biology and in inflammatory skin diseases such as AD, psoriasis and Netherton syndrome. We also review applications of lipidomics in human skin equivalent and in pre-clinical animal models of skin diseases to gain insight into the pathogenesis of the skin disease. Expert commentary: Skin lipidomics analysis could be a fast, reliable and noninvasive tool to characterize the skin lipid profile and to monitor the progression of inflammatory skin diseases such as AD.

Transcription Factor CTIP2 Maintains Hair Follicle Stem Cell Pool and Contributes to Altered Expression of LHX2 and NFATC1.

Transcription factor CTIP2 (chicken ovalbumin upstream promoter transcription factor-interacting protein 2), also known as BCL11B, is expressed in hair follicles (HFs) of embryonic and adult skin. Ctip2-null mice exhibit reduced HF density during embryonic development. In contrast, conditional inactivation of Ctip2 in the epidermis (Ctip2(ep-/-) mice) leads to a shorter telogen and a premature entry into anagen during the second phase of hair cycling without a detectable change in the number of HFs. Keratinocytes of the bulge stem cells (SCs) niche of Ctip2(ep-/-) mice proliferate more and undergo reduced apoptosis compared with the corresponding cells of wild-type mice. However, premature activation of follicular SCs in mice lacking CTIP2 leads to the exhaustion of this SC compartment in comparison with Ctip2(L2/L2) mice, which retained quiescent follicle SCs. CTIP2 modulates expression of genes encoding EGFR and NOTCH1 during formation of HFs and those encoding nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 1 and LIM homeobox 2 during normal hair cycling in adult skin. The expression of most of these genes is disrupted in mice lacking CTIP2, and these alterations may underlie the phenotype of Ctip2-null and Ctip2(ep-/-) mice. CTIP2 appears to serve as a transcriptional organizer that integrates input from multiple signaling cues during HF morphogenesis and hair cycling.