Primary Human Dermal Fibroblasts Research Articles

Three-dimensional co-culture model employing silica nonwoven fabrics to enhance cell-to-cell communication of paracrine signaling between hepatocytes and fibroblasts.

The realization that soluble factors secreted by heterotypic cells play an importanta role in paracrine signaling, which facilitates intercellular communication, enabled the development of physiologically relevant co-culture models for drug screening and the engineering of tissues, such as hepatic tissues. The most crucial issues confronting the use of conventional membrane inserts in segregated co-culture models that are used to study paracrine signaling between heterotypic cells have been identified as long-term viability and retention of cell-specific functions, especially when isolated primary cells are used. Herein, we present an in vitro segregated co-culture model consisting of a well plate incubated with rat primary hepatocytes and normal human dermal fibroblasts which were segregated using a membrane insert with silica nonwoven fabric (SNF) on it. SNF, which mimics a physiological environment much more effectively than a two-dimensional (2D) one, promotes cell differentiation and resultant paracrine signaling in a manner that is not possible in a conventional 2D culture, owing to high mechanical strength generated by its inorganic materials and interconnected network structure. In segregated co-cultures, SNF clearly enhanced the functions of hepatocytes and fibroblasts, thereby showing its potential as a measure of paracrine signaling. These results may advance the understanding of the role played by paracrine signaling in cell-to-cell communication and provide novel insights into the applications of drug metabolism, tissue repair, and regeneration.

Evaluating the role of protective creams on the cutaneous penetration of Ni nanoparticles

There is an increase of application of Nickel in the form of nanoparticles (NiNPs) in several fields including modern metallurgy, bioengineering, and medicine. Such growth of the areas of application is actually accompanied with an increase of exposure to Nickel, thus an intensification of the negative effects, the most frequent being the allergic contact dermatitis. Indeed, due to their smaller size, and therefore their higher surface area, NiNPs can release more Ni ions compared to bulk material, that can penetrate and permeate through the skin. To reduce the Ni cutaneous penetration, barrier creams (BC) are applied on the skin surface. There is little information, however, on the efficiency of such commercial protective creams on decreasing Ni cutaneous penetration. For this reason, the objective of the current study was to investigate the protective role of one commercially available formulation for Ni (Nik-L-Block™ containing a chelating agent) and one moisturizing cream (Ceramol 311 basic cream without chelating agent), following exposure to NiNPs, using in vitro Franz cells, as well as the cytotoxicity of NiNPs in primary human dermal fibroblasts was studied. Our results demonstrated that although both tested formulations can decrease Ni accumulation in the skin (4.13 ± 1.74 μg/cm2 for Nik-L-Block™ and 7.14 ± 1.46 μg/cm2 for Ceramol 311 basic cream); there are significant differences between the two creams (p = 0.004). Based on the experimental evidence, we therefore conclude that the composition of such formulations has an imperative role for dermal uptake of Ni. Finally, NiNPs showed no cytotoxic effect on cultured human dermal fibroblasts after 24 and 72 h.

The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity.

Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We next show that salivary sfRNA is protected in detergent-sensitive compartments, likely extracellular vesicles. In support of this hypothesis, we visualized viral RNAs in vesicles in mosquito saliva and noted a marked enrichment of signal from 3'UTR sequences, which is consistent with the presence of sfRNA. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in a human hepatoma cell line and human primary dermal fibroblasts. Transfection of 3'UTR RNA prior to DENV2 infection inhibited type I and III interferon induction and signaling, and enhanced viral replication. Therefore, we posit that sfRNA present in salivary extracellular vesicles is delivered to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.

Rosmarinic acid alleviates ultraviolet-mediated skin aging via attenuation of mitochondrial and ER stress responses.

Chronic exposure to Ultraviolet B radiation (UV-B) evokes a myriad of toxic signalling events in the irradiated skin. One of such response is ER stress, which is known to exacerbate photodamage responses. Also, recent literature has highlighted the adverse impact of environmental toxicants on mitochondrial dynamics and mitophagy. Impaired mitochondrial dynamics escalates oxidative damage and causes apoptosis. There have been evidences that support crosstalk between ER stress and mitochondrial dysfunction. However, mechanistic clarification is still needed to verify the interactions between UPR responses and mitochondrial dynamics impairment in UV-B-induced photodamage models. Lastly, plant-based natural agents have garnered attention as therapeutic agents against skin photodamage. Thus, gaining mechanistic insights of plant-based natural agents is required for their application and feasibility in clinical settings. With this aim in view, this study was performed in primary human dermal fibroblasts (HDFs) and Balb/C mice. Different parameters regarding mitochondrial dynamics, ER stress, intracellular damage and histological damage were analyzed using western blot, rt-PCR and microscopy. We demonstrated that UV-B exposure leads to induction of UPR responses, upregulation of Drp-1 and inhibition of mitophagy. Further, 4-PBA treatment leads to reversal of these noxious stimuli in irradiated HDF cells, thereby, indicating an upstream role of UPR induction in mitophagy inhibition. Also, we explored the therapeutic effect of Rosmarinic acid (RA) against ER stress and impaired mitophagy in photodamage models. RA prevents intracellular damage via alleviation of ER stress and mitophagic responses in HDFs and irradiated Balb/C mice skin. The current study summarizes the mechanistic insights into UVB-mediated intracellular damage and role of natural plant-based agent (RA) in ameliorating these toxic responses.

Potential Treatment of Dermatophyte Trichophyton rubrum in Rat Model Using Topical Green Biosynthesized Silver Nanoparticles with Achillea santolina Extract

Trichophyton rubrum is the most common dermatophyte, and can cause cutaneous infections in humans and animals (dermatophytosis). In this study, we investigated the anti-dermatophytic potential of green synthesized silver nanoparticles using Achillea santolina extract (AS-AgNPs) in an in vitro and in vivo rat model of dermal T. rubrum dermatophytosis (TRD). The green synthesis of AS-AgNPs was performed using A. santolina extract and characterized by UV-VIS spectroscopy, zeta potential, imaging (transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Energy dispersive X-ray analysis (EDX). The antifungal activity of AS-AgNPs was determined by the broth microdilution method, conidial germination, and hyphal growth inhibition. TEM and SEM were used to study the mode of the antifungal action of AS-AgNPs. AS-AgNPs inhibited the growth of T. rubrum with an MIC of 128 μg/mL, and suppressed the conidial germination and hyphal growth by 55.3% 84.6%, respectively. AS-AgNPs caused modified mycelial structures, increased cell membrane permeability, and cell wall damage. AS-AgNPs significantly increase the permeability of the fungal membrane, as revealed by reducing ergosterol biosynthesis. An increase in the intracellular ROS and the induction of apoptosis were also observed during AS-AgNP treatment. In addition, AS-AgNPs reduced the cell wall integrity, as shown by the reduction in the β-(1,3)-d-glucan synthase and chitin synthase activities. AS-AgNPs showed very low toxicity on primary human dermal fibroblasts (HDF) at the MIC. The topical treatment of the infected skin in the TRD rat model with AS-AgNPs showed a significant reduction in the fugal burden after 7 days and a complete clearance of fungal conidia, with a high recovery of epidermal and dermal structures after 14 days, compared to control rats. Interestingly, AS-AgNPs significantly attenuated the infiltrated inflammatory cells, in association with reducing the tissue proinflammatory cytokines including TNF-α, IL-1, IL-6, MOP and IL-17. In conclusion, our data prove AS-AgNPs to be a novel green topical therapy for dermatophytosis caused by T. rubrum.

In vitro assessment of Neuronal PAS domain 2 mitigating compounds for scarless wound healing.

The core circadian gene Neuronal PAS domain 2 (NPAS2) is expressed in dermal fibroblasts and has been shown to play a critical role in regulating collagen synthesis during wound healing. We have performed high throughput drug screening to identify genes responsible for downregulation of Npas2 while maintaining cell viability. From this, five FDA-approved hit compounds were shown to suppress Npas2 expression in fibroblasts. In this study, we hypothesize that the therapeutic suppression of Npas2 by hit compounds will have two effects: (1) attenuated excessive collagen deposition and (2) accelerated dermal wound healing without hypertrophic scarring. To test the effects of each hit compound (named Dwn1, 2, 3, 4, and 5), primary adult human dermal fibroblasts (HDFa) were treated with either 0, 0.1, 1, or 10 μM of a single hit compound. HDFa behaviors were assessed by picrosirius red staining and quantitative RT-PCR for in vitro collagen synthesis, cell viability assay, in vitro fibroblast-to-myofibroblast differentiation test, and cell migration assays. Dwn1 and Dwn2 were found to significantly affect collagen synthesis and cell migration without any cytotoxicity. Dwn3, Dwn4, and Dwn5 did not affect collagen synthesis and were thereby eliminated from further consideration for their role in mitigation of gene expression or myofibroblast differentiation. Dwn1 also attenuated myofibroblast differentiation on HDFa. Dwn1 and Dwn2 may serve as possible therapeutic agents for future studies related to skin wound healing.

Cytotoxic, cytostatic, and keratolytic activity of anti-dandruff shampoo formulations

Background: Dandruff is characterized by increased sebum production, yeast colonization, and hyper-keratinization leading to an itchy-flaky scalp. Ancillary non-microbial reasons for flaky scalp include ultraviolet rays and excessive heat from the sun. Thus, apart from traditional anti-fungal activity, anti-dandruff formulations should also possess the keratolytic activity and should be safe to use. Methods: Selsun, a shampoo formulation with 2.5% selenium sulfide (SS2.5), was tested in-vitro for its cytotoxic and cytostatic activities via cell viability of primary human dermal fibroblasts (HDFs). Its keratolytic activity was determined by assessing anti-proliferative activity on immortalized human keratinocyte (HaCaT) cell line. The shampoo was compared with 3 other shampoo formulations, one containing 2% ketoconazole (K2), second containing 2% ketoconazole with 1% zinc pyrithione (K2ZP1), and third containing 1% zinc pyrithione (ZP1). Results: Of the tested samples, SS2.5 and K2ZP1 showed cytostatic and keratolytic activity where the IC50 values on normal HDFs i.e., 0.521 mg/ml and 0.220 mg/ml, respectively, were higher than those on HaCaT cells (0.162 mg/ml and 0.198 mg/ml, respectively). SS2.5 displayed the highest keratolytic activity at IC50 of 0.162 mg/ml, followed by K2ZP1 (0.198 mg/ml) and K2 (0.267 mg/ml). On normal HDFs, SS2.5 showed the highest IC50 value (0.521 mg/ml), followed by K2 at 0.264 mg/mL, K2ZP1 at 0.220 mg/ml, and ZP1 at 0.139 mg/ml. Conclusions: SS2.5 was found to possess the highest cytostatic and keratolytic activities among all the shampoo formulations tested, thereby confirming its safety profile.

Lipase-Responsive Amphotericin B Loaded PCL Nanoparticles for Antifungal Therapies

Amphotericin B is an antifungal drug used for the treatment of invasive fungal infections. However, its clinical use is limited due to its serious side effects, such as renal and cardiovascular toxicity. Furthermore, amphotericin B is administered in high doses due to its poor water solubility. Hence, it is necessary to develop an on-demand release strategy for the delivery of amphotericin B to reduce cytotoxicity. The present report describes a novel encapsulation of amphotericin B into lipase-sensitive polycaprolactone to form a nanocomposite. Nanocomposites were produced by the oil-in-water method and their physicochemical properties such as size, hydrodynamic diameter, drug loading, and zeta potential were determined. The in vitro release of amphotericin B was characterized in the presence and absence of lipase. The antifungal activity of the nanocomposites was verified against lipase-secreting Candida albicans, and cytotoxicity was tested against primary human dermal fibroblasts. In the absence of lipase, the release of amphotericin B from the nanocomposites was minimal. However, in the presence of lipase, an enzyme that is abundant at infection sites, a fungicidal concentration of amphotericin B was released from the nanocomposites. The antifungal activity of the nanocomposites showed an enhanced effect against the lipase-secreting fungus, Candida albicans, in comparison to the free drug at the same concentration. Furthermore, nanoencapsulation significantly reduced amphotericin B-related cytotoxicity compared to the free drug. The synthesized nanocomposites can serve as a potent carrier for the responsive delivery of amphotericin B in antifungal applications.

Chitosan-collagen-fibrinogen uncrosslinked scaffolds possessing skin regeneration and vascularization potential.

Clinical success of regenerative medicine for treating deep-tissue skin injuries depends on the availability of skin grafts. Though bioengineered constructs are tested clinically, lack of neovascularization provide only superficial healing. Thus constructs, which promotes wound healing and supports vascularization has gained priority in tissue engineering. In this study, chitosan-collagen-fibrinogen (CCF) scaffold was fabricated using freeze-drying method without using any chemical crosslinkers. CCF scaffolds proved cytocompatibility and faster healing in in vitro scratch assay of primary human adult dermal fibroblasts cells with progressively increasing vascular endothelial growth factor-A and reducing vascular endothelial growth factor receptor 1 expressions. Skin regeneration evaluated on in vivo full thickness wound model confirmed faster remodeling with angiogenic signatures in CCF scaffold-implanted mice. Histopathological observations corroborated with stereo-zoom and SS-optical coherence tomography images of wound sites to prove the maturation of healing-bed, after 12 days of CCF implantation. Therefore, it is concluded that CCF scaffolds are promising for skin tissue regeneration and demonstrates pro-angiogenic potential.

Engineered Vasculogenic Extracellular Vesicles Drive Nonviral Direct Conversions of Human Dermal Fibroblasts into Induced Endothelial Cells and Improve Wound Closure.

Vasculogenic cell therapies have emerged as a powerful tool to increase vascularization and promote tissue repair/regeneration. Current approaches to cell therapies, however, rely mostly on progenitor cells, which pose significant risks (e.g., uncontrolled differentiation, tumorigenesis, and genetic/epigenetic abnormalities). Moreover, reprogramming methodologies used to generate induced endothelial cells (iECs) from induced pluripotent stem cells rely heavily on viral vectors, which pose additional translational limitations. This work describes the development of engineered human extracellular vesicles (EVs) capable of driving reprogramming-based vasculogenic therapies without the need for progenitor cells and/or viral vectors. The EVs were derived from primary human dermal fibroblasts (HDFs), and were engineered to pack transcription factor genes/transcripts of ETV2, FLI1, and FOXC2 (EFF). Our results indicate that in addition of EFF, the engineered EVs were also loaded with transcripts of angiogenic factors (e.g., VEGF-A, VEGF-KDR, FGF2). In vitro and in vivo studies indicate that such EVs effectively transfected HDFs and drove direct conversions towards iECs within 7-14 days. Finally, wound healing studies in mice indicate that engineered EVs lead to improved wound closure and vascularity. Altogether, our results show the potential of engineered human vasculogenic EVs to drive direct reprogramming processes of somatic cells towards iECs, and facilitate tissue repair/regeneration.

Benefits of Circulating Human Metabolites from Fish Cartilage Hydrolysate on Primary Human Dermal Fibroblasts, an Ex Vivo Clinical Investigation for Skin Health Applications

Due to its significant exposure to stressful environmental factors, the skin undergoes a high remodeling rate over time, which alters not only its appearance but also its functionality. This alteration of the skin, namely photoaging, is characterized by dryness and a loss of elasticity that mainly originates from the dysregulation of dermal fibroblast activities. In order to overcome such tissue outcome, cosmetic products have evolved toward nutricosmetics, thus promoting beauty from within. Among bio-actives of interest, bio-peptides deriving from plant or animal sources may exert various biological activities beyond their nutritional value. However, studies remain mostly descriptive and the mode of action at the cellular level in clinic remains a concern. In a recent clinical trial, it was showed that supplementation with a fish cartilage hydrolysate (FCH) improved signs of chronological and photoaging-induced skin changes in healthy women. Here, using an original ex vivo clinical approach adapted to nutricosmetic purpose, we further demonstrated that this fish cartilage hydrolysate was absorbed and that the circulating metabolites produced in humans following FCH intake stimulate human dermal fibroblast growth, promote specific hyaluronan production, up-regulate elastin synthesis and inhibit MMP-1 and 3 expression along with the enhancement of TGF-β release. Altogether, these data provide clues on the mechanisms likely contributing to the beneficial impact of FCH on human skin functionality by supporting hydration, elasticity and limiting the expression of catabolic factors involved in photoaging onset.

Glutaminase inhibitors rejuvenate human skin via clearance of senescent cells: a study using a mouse/human chimeric model.

Skin aging caused by various endogenous and exogenous factors results in structural and functional changes to skin components. However, the role of senescent cells in skin aging has not been clarified. To elucidate the function of senescent cells in skin aging, we evaluated the effects of the glutaminase inhibitor BPTES (bis-2-(5-phenylacetamido-1, 3, 4-thiadiazol-2-yl)ethyl sulfide) on human senescent dermal fibroblasts and aged human skin. Here, primary human dermal fibroblasts (HDFs) were induced to senescence by long-term passaging, ionizing radiation, and treatment with doxorubicin, an anticancer drug. Cell viability of HDFs was assessed after BPTES treatment. A mouse/human chimeric model was created by subcutaneously transplanting whole skin grafts from aged humans into nude mice. The model was treated intraperitoneally with BPTES or vehicle for 30 days. Skin samples were collected and subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, and histological analysis. BPTES selectively eliminated senescent dermal fibroblasts regardless of the method used to induce senescence; aged human skin grafts treated with BPTES exhibited increased collagen density, increased cell proliferation in the dermis, and decreased aging-related secretory phenotypes, such as matrix metalloprotease and interleukin. These effects were maintained in the grafts 1 month after termination of the treatment. In conclusion, selective removal of senescent dermal fibroblasts can improve the skin aging phenotype, indicating that BPTES may be an effective novel therapeutic agent for skin aging.

Nicotinamide and calcipotriol counteract UVB-induced photoaging on primary human dermal fibroblasts

BackgroundPhotoaging is mainly caused by ultraviolet radiations inasmuch they can damage the DNA, trigger ROS production, and activate p53/p21 pathway, which cause cell cycle arrest and senescence. The accumulation of senescent cells within the dermis contributes to tissue deregulation and skin carcinogenesis. However, the use of photoprotector molecules could reduce UV-induced damages and prevent photoaging. Therefore, the aim of this study is to evaluate whether the active forms of vitamin B3 (nicotinamide) and the analog of vitamin D3 (calcipotriol) might protect primary human dermal fibroblasts (HDFs) from UVB-induced photoaging. MethodsHDFs were isolated from a healthy adult donor and stimulated with nicotinamide (25 μM) and calcipotriol (100 nM) for 24h before UVB exposure, and then, cultured for further 24h on vitamin-supplemented media. Then, cell viability, ROS production, DNA damages, senescence markers, protein and gene expression were evaluated. ResultsHDFs treated with nicotinamide and calcipotriol showed better proliferation properties and lower DNA damages due to a reduced UVB-induced ROS production. Consequently, p53/p21 pathway was less active which enhanced cell cycle progression and reduced senescence and cell death. ConclusionsOverall, our results suggest that nicotinamide and calcipotriol can counteract UVB-induced effects responsible for the onset of skin photoaging.

610 Identification of the selective glucocorticoid receptor agonist Mapracorat as potential new drug for chronic wound therapy

We have shown that the effects of wound exudates on fibroblast cultures reflect their clinical healing phenotype and can thus serve as a functional biomarker. We used this assay for screening to identify compounds or compound classes with so far unknown potential for chronic wound therapy. The selective glucocorticoid receptor agonist (SEGRA) Mapracorat was found to reverse the inhibitory effect of several exudates. The compound was profiled in additional exudate-based cellular assays to investigate its suitability for wound therapy. Primary human dermal fibroblasts were grown in 384-well plates in 2D and 3D cultures. Mapracorat was added to the cells in the presence of wound exudates from different chronic wound patients. Incubation times were 72 hours for 2D cultures and 4 – 8 days for 3D cultures. Supernatants were then harvested for the determination of IL-1beta, RNA was extracted for RT-PCR, or cells were fixed and stained for cellular protein. Mapracorat rescued inhibition of wound exudate-induced fibroblast proliferation in 80% of 200 exudates tested. For 95/200 exudates from chronic wounds of different etiologies, cell proliferation was increased by Mapracorat by >150% compared to exudate alone, and complete recovery was demonstrated in 25% of these samples. This rescue effect was achieved at low nanomolar concentrations. The compound dose-dependently inhibited IL-1beta production and enhanced matrix formation as well as collagen 1 and collagen 3 mRNA expression, indicating transition from a chronic/non-healing to a healing phenotype ex vivo. In functional ex vivo assays for chronic wounds, Mapracorat reversed the detrimental effects of wound exudates on fibroblast proliferation and matrix formation, and reduced proinflammatory cytokine secretion. Mapracorat showed good or excellent activity with almost 50% of exudates. To select the patients best suited for therapy with this compound, theranostic pretesting is recommended.

616 Investigating if hydroxypyridone anti-fungals can target already established myofibroblasts in an in vitro model of hypertrophic scarring

Hypertrophic scars are a common complication of burn injury, yet there are no medications to prevent their formation. Myofibroblasts, a key cell involved in scar formation, are resistant to apoptosis. We have previously shown that hydroxypyridone anti-fungals can inhibit myofibroblast transformation in burn scar-derived fibroblasts. This study aimed to investigate if these drugs can target established myofibroblasts. Primary human dermal fibroblasts, derived from burn patient scars, were exposed to transforming growth factor beta-1 (TGF-β1) for 72 h to induce myofibroblast transformation.

286 Oxidative Stress Modulation by Carnosine in Scaffold Free Human Dermis Spheroids Model: A Proteomic Study

Carnosine is an endogenous dipeptide with antioxidant and scavenger properties, shown to prevent aging and photoaging process by in vivo and in vitro models on different human tissues. Here an advanced quantitative proteomic approach (high-resolution mass spectrometry and network analyses) was applied to a 3D scaffold-free dermal spheroids model to investigate the biological responses induced by carnosine in human dermis. Primary human dermal fibroblasts from a 50-years-old donor were seeded in hanging drops to develop dermis spheroids.

602 Development of an assay platform for personalized treatment of chronic wounds

Wound exudates are considered liquid biopsies of wounds and contain the pathogenic drivers of chronicity. The effects of different types of exudates on healthy primary human dermal fibroblasts (HDF) were investigated, demonstrating growth- and matrix-inhibitory as well as pro-inflammatory effects of chronic wound exudates. To identify potential drugs which could reverse these adverse effects, chemical compounds and growth factors were screened in a proliferation assay. Exudates from healing or chronic venous-, arterial-, mixed-, pressure- and diabetic ulcers, as well as surgical wounds were harvested by negative pressure therapy or using nylon-flocked swabs.

Trigonelline, a plant derived alkaloid prevents ultraviolet-B-induced oxidative DNA damage in primary human dermal fibroblasts and BALB/c mice via modulation of phosphoinositide 3-kinase-Akt-Nrf2 signalling axis

BackgroundDNA is the main target for UV-B-irradiation-induced skin photodamage and accounts for 90 % of all the non-melanoma skin cancers. PurposeIn this study, we explored the mechanistic basis of photoprotective effect of Trigonelline, a naturally occurring alkaloid from the Trigonella foenum-graecum, against UV-B-induced oxidative DNA Damage Response using Primary Human Dermal Fibroblasts (HDFs) and BALB/C mice as models of skin photodamage. MethodsPrimary HDFs were subjected to UV-B exposure (10 mJ/cm2) with or without TG for 24 h. Effect of UV-B exposure and TG treatment was evaluated by analyzing the cell survival, cellular morphology, oxidative stress & DNA damage response markers by performing biochemical studies, florescent microscopy & protein expression studies. In in-vivo study, TG pre-treated BALB/c mice were -irradiated with 180 mJ/cm2 of UV-B dose thrice a week on alternative days for four months, followed by topical application of different concentrations of TG. The photodamage caused by UV-B exposure and its ameleoriation by topical treatment of TG was studied by physical and morphological appearance and analyzing the oxidative stress & DNA damage response markers from skin. ResultsWe found that TG significantly alleviates UV-B–induced cell death effects in HDFs. TG protects HDF cells and BALB/c mice from UV-B-induced DNA damage by regulating the expression profile of key protein markers of DNA damage which include P53, ATM, ATR, ϒH2AX, Chk1 and Chk2. We found that TG offers geno-protection to UV-B–irradiated HDFs by alleviating CPD induction, reducing the number of TUNEL positive cells and by decreasing the expression levels of DNA damage marker protein ϒH2AX in immunocytochemistry. Further, we found that TG prevents the UVB induced oxidative stress by activating the PI3K-AKT-Nrf2 signalling pathway. On employing PI3K inhibitor, LY294002, we found the expression of ϒH2AX and p-P53 is significantly increased compared to UV-B treated only, indicating that TG mediates the geno-protection against UV-B irradiation via PI3K-AKT-Nrf2 signalling pathway. ConclusionCurrent study presents for the first time the photo-protective role of TG against UV-B–induced oxidative DNA damage and provides its mechanistic insights also and provide strong evidence for TG to be carried forward as a potential remedial and cosmeceutical agent against UV-B–induced skin photodamage disorders.

Phenotypic screening identifies hydroxypyridone anti-fungals as novel medicines for the prevention of hypertrophic scars

Although hypertrophic scarring affects ∼91% of burn patients annually, there is no drug to prevent this common complication. Hypertrophic scars are a result of dysregulated wound healing, characterised by persistent myofibroblast transformation and the excessive accumulation of extracellular matrix (ECM). Due to the multi-mechanistic nature of the scarring process, target-based approaches for identifying novel drugs have failed.Primary human dermal fibroblasts, derived from burn scar tissue, were exposed to transforming growth factor-beta 1 (TGF-β1) to induce myofibroblast transformation. A phenotypic screening assay, measuring alpha-smooth muscle actin (α-SMA) expression, was developed to screen 1,954 approved drugs. Drugs that elicited >80% inhibition of α-SMA expression, and >80% cell viability were progressed as candidate drugs. Anti-myofibroblast activity of the candidates was confirmed before investigating their effects on extracellular matrix (ECM) production and keratinocyte epithelial-mesenchymal transition (EMT).TGF-β1 induced myofibroblast transformation in primary human dermal fibroblasts (Emax = >3 ng/mL). The assay was optimised and validated (Z’ = 0.59), before screening 1,954 approved drugs. 90 drugs were identified as hits and hydroxypyridone anti-fungals selected for further testing. Concentration-response curves for these drugs confirmed their concentration-dependent anti-myofibroblast activity (IC50 = 1.4 – 16.7 μM). Hydroxypyridone anti-fungals were also found to successfully reduce ECM production and keratinocyte EMT.This is the first study to screen approved drugs in primary human dermal fibroblasts. Hydroxypyridone anti-fungals were found to prevent myofibroblast transformation, ECM production and keratinocyte EMT suggesting they could be repurposed to prevent hypertrophic scarring.

In-vitro cell culture model to determine toxic effects of soil Arsenic due to direct dermal exposure

Arsenic (As) is one of the most toxic environmental pollutants, classified as a Class I carcinogen. Anthropogenic activities have led to an increase in As contamination of soils. Using animal models to study the health impacts of As is time and cost-prohibitive, hence attempts have been made to develop in-vitro cell culture models. However, most studies so far have not represented realistic environmental exposure conditions. We exposed Human Immortalized Keratinocyte (HaCaT) and Primary Human Dermal Fibroblasts (HDFa) cells to the water-soluble fraction of arsenic extracted from As spiked Immokalee soil to study the effect of soil As on skin cells. The impact of As was determined through cell viability, cell migration, and quantitative assessment of the expression of cadherins, CD44, and Zeb1 using Western Blotting and In-cell Western. Our data indicated that HaCaT cells were more susceptible to As-induced cellular transformation than HDFa cells. At concentrations above 225 mg/kg of soil As, cellular responses were impacted, and changes in the expression of proteins were observed in HaCaT cells. The work presented here is a methodical approach to studying the impacts of toxic elements such as As through innovative cellular models using realistic environmental conditions, that can be readily adapted for different exposure pathways. • Human skin cells exposed to the water-soluble soil As showed decreased viability. • Uptake of soil As by keratinocytes was higher than that of fibroblasts. • Soil As impacted cell-to-cell adhesion and migration in skin cells. • Soil As induced epithelial to mesenchymal transformation in skin cells. • A human cell culture model to test soil As toxicity was developed.