Non-irradiated Skin Research Articles

Long Noncoding VIM-AS1: Biomarker of Breast Fibrosis Susceptibility After Radiation Therapy and Promoter of Transforming Growth Factor Beta1–Driven Fibrosis

Fibrosis is a common late complication of radiation therapy. Molecular dysregulations leading to fibrosis have been characterized for the coding part of the genome, notably those involving the TGFB1 gene network. However, because a large part of the human genome encodes RNA transcripts that are not translated into proteins, exploring the involvement of the noncoding part of the genome in fibrosis susceptibility and development was the aim of this work. Breast cancer patients having or not having developed severe breast fibrosis after radiation therapy were retrospectively selected from the COPERNIC collection. Exome sequencing and RNA-seq transcriptomic profiling were performed on 19 primary dermal fibroblast strains isolated from the patients' nonirradiated skin. Functional experiments were based on fibrogenic induction by transforming growth factor-Beta1 (TGFB1) and gene knockdown in healthy donor fibroblasts. Coding and noncoding transcriptomes discriminated fibrosis from nonfibrosis conditions, and a signature of breast fibrosis susceptibility comprising 15 long noncoding RNAs (lncRNAs) was identified. A hazard ratio validation showed that the lncRNA vimentin antisense long noncoding RNA 1 (VIM-AS1) was the best biomarker associated with fibrosis risk. This lncRNA has not been previously associated with any fibrotic disorder, but we found it upregulated in data sets from cardiac fibrosis and scleroderma, suggesting a general role in tissue fibrosis. Functional experiments demonstrated a profibrotic action of VIM-AS1 because its knockdown reduced myofibroblast activation, collagen matrix production, and dermal organoid contraction. RNA-seq data analysis after VIM-AS1 silencing also pointed out the regulation of replication, cell cycle, and DNA repair. Mechanistically, because VIM-AS1 was found coregulated with the vimentin gene, these data support a profibrotic function of the TGFB1/VIM-AS1/vimentin axis, targeting the dynamics of fibroblast-myofibroblast transition. Noncoding RNA analysis can provide specific biomarkers relevant to the prediction of normal tissue responses after radiation therapy, which opens perspectives of next-generation approaches for treatment, in the frame of the recent developments of RNA-based technologies.

Single-cell transcriptional analysis of irradiated skin reveals changes in fibroblast subpopulations and variability in caveolin expression

BackgroundRadiation-induced fibrosis (RIF) is an important late complication of radiation therapy, and the resulting damaging effects of RIF can significantly impact reconstructive outcomes. There is currently a paucity of effective treatment options available, likely due to the continuing knowledge gap surrounding the cellular mechanisms involved. In this study, detailed analyses of irradiated and non-irradiated human skin samples were performed incorporating histological and single-cell transcriptional analysis to identify novel features guiding development of skin fibrosis following radiation injury.MethodsPaired irradiated and contralateral non-irradiated skin samples were obtained from six female patients undergoing post-oncologic breast reconstruction. Skin samples underwent histological evaluation, immunohistochemistry, and biomechanical testing. Single-cell RNA sequencing was performed using the 10X single cell platform. Cells were separated into clusters using Seurat in R. The SingleR classifier was applied to ascribe cell type identities to each cluster. Differentially expressed genes characteristic to each cluster were then determined using non-parametric testing.ResultsComparing irradiated and non-irradiated skin, epidermal atrophy, dermal thickening, and evidence of thick, disorganized collagen deposition within the extracellular matrix of irradiated skin were readily appreciated on histology. These histologic features were associated with stiffness that was higher in irradiated skin. Single-cell RNA sequencing revealed six predominant cell types. Focusing on fibroblasts/stromal lineage cells, five distinct transcriptional clusters (Clusters 0–4) were identified. Interestingly, while all clusters were noted to express Cav1, Cluster 2 was the only one to also express Cav2. Immunohistochemistry demonstrated increased expression of Cav2 in irradiated skin, whereas Cav1 was more readily identified in non-irradiated skin, suggesting Cav1 and Cav2 may act antagonistically to modulate fibrotic cellular responses.ConclusionIn response to radiation therapy, specific changes to fibroblast subpopulations and enhanced Cav2 expression may contribute to fibrosis. Altogether, this study introduces a novel pathway of caveolin involvement which may contribute to fibrotic development following radiation injury.

Non-pigmented laser hair removal mediated via sepia melanin nanoparticles: in vivo study on albino mice

Objectives Melanin is considered the main chromophore for laser hair removal. Due to a lack of laser-absorbing chromophores, removing non-pigmented hair with laser is quite problematic with unsatisfactory outcomes. This problem could be solved by delivering more melanin to the area around the hair follicle and enhancing that area as a target for light absorption. The insolubility of Sepia melanin as an exogenous dye, in most solvents, limits its bioavailability and thus its clinical use. Methods In our study, to overcome the solubility problems and increase the bioavailability of melanin for biomedical and cosmetic applications, natural sepia melanin was loaded in different nano-delivery systems (spanlastics and transfersomes) to be delivered to the hair follicles. The different formulations of melanin were prepared and characterized. In vivo skin deposition and histopathological studies were conducted on albino mice. Results Transmission electron microscopy (TEM) showed the spherical shape of the prepared vesicles with an average particle size of 252 and 262 nm and zeta potential of −22.5 and −35 mV for melanin spanlastics and melanin transfersomes, respectively. Histopathological examination of hair follicles and pilosebaceous glands for the irradiated and non-irradiated albino mice skin was studied post the application of the prepared formulations topically and subcutaneously. Qualitative statistical analysis was conducted and melanin transfersomes and melanin spanlastics showed significant damage to pilosebaceous glands and hair follicles with a p-value of 0.031 and 0.009 respectively. Conclusion Melanin nanovesicles as transfersomes and spanlastics could be considered a promising approach for the removal of non-pigmented hair.

Ferroptosis Inhibition with Deferoxamine Alleviates Radiation-Induced Fibrosis.

Background Radiation-induced fibrosis (RIF) is a debilitating sequelae of radiation therapy that has been shown to improve with topical treatment with the iron chelator deferoxamine (DFO). We investigated whether DFO exerts this effect through attenuation of ferroptosis, a recently described iron-dependent pathway of cell death. Methods Adult C57BL/6J mice were treated with topical DFO or ferrostastin-1 (Fer-1) and irradiated with 30 Grays of ionizing radiation to the dorsal skin to promote development of chronic RIF. Immunofluorescent staining with 4-hydroxynonenal (4-HNE) antibody was carried out directly following irradiation to assess ferroptosis activity. Perfusion testing with laser Doppler was performed throughout the healing interval. Eight weeks following radiation, dorsal skin was harvested and analyzed histologically and biomechanically. Results Immunohistochemical staining demonstrated lower presence of 4-HNE in non-irradiated skin, DFO-treated skin, and Fer-1-treated skin compared to irradiated, untreated skin. DFO resulted in histological measurements (dermal thickness and collagen content) that resembled normal skin, while Fer-1 treatment yielded less significant improvements. These results were mirrored by analysis of extracellular matrix ultrastructure and biomechanical testing, which recapitulated the ability of topical DFO treatment to alleviate RIF across these parameters while Fer-1 resulted in less notable improvement. Finally, perfusion levels in DFO treated irradiated skin were similar to measurements in normal skin, while Fer-1 treatment did not impact this feature. Conclusions Ferroptosis contributes to the development of RIF and attenuation of this process leads to reduced skin injury. DFO further improves RIF through additional enhancement of perfusion not seen with Fer-1.

Evaluating hyperpigmentation: is invasion of the skin necessary?

BackgroundThe combination of long wavelength ultraviolet A1 radiation (LWUVA1) and visible light (VL) has been shown to produce photodamage and the majority of organic sunscreens lack protection against this spectrum. Currently, established testing protocols for VL photoprotection are lacking.ObjectiveTo compare pigmentation assessment methods, including Investigator’s Global Assessment (IGA) scoring, Diffuse Reflectance Spectroscopy (DRS), and immunohistochemistry, to assess the utility of each in evaluating VL + LWUVA1 induced pigmentation.MethodsAnonymized IGA, DRS, and immunohistochemistry data for 37 subjects (Fitzpatrick skin phototypes IV-VI) was retrospectively analyzed for pigment evaluation. Pigmentation was induced on the subjects’ backs with VL + LWUVA1 (0%–0.5% UVA1) irradiation at a dose of 480 J/cm2. Comparisons were made for all three assessment techniques between non-irradiated skin and VL + LWUVA1 irradiated skin, as well as between pure VL (0% UVA1) and VL + LWUVA1 (0.5% UVA1) irradiated sites. All comparisons were made for data collected approximately 24 h after irradiation to evaluate persistent pigment darkening (PPD).ResultsAmong all 37 subjects, both IGA scores and DRS detected a statistically significant difference in PPD between irradiated and non-irradiated sites, as well as between VL + LWUVA1 and pure VL irradiated sites. However, MART-1/Melan-A did not indicate a statistically significant difference in PPD between irradiated and non-irradiated sites or between VL + LWUVA1 and pure VL irradiated sites.ConclusionSubjective and objective noninvasive assessments were more sensitive in detecting VL + LWUVA1 induced pigmentation and should be preferred over invasive methods. Researchers are advised to initiate assessments with IGA and subsequently incorporate DRS for more objective and comprehensive insights in pigment evaluation.

The Effects of Multiple Power Densities of Carbon Dioxide Laser on Photothermal Damage in Rat Skin Tissue

A CO2 laser produces infrared photons that are largely absorbed by the skin and cause morphological alterations. Twenty-four (Wistar) rats weighing 290-380 g and ranging in age from 8 months to a year were chosen at random and divided into sixteen rats for histological examination and eight rats for tensile testing to determine the extent of injury caused by photothermal damage induced by multiple doses of a CO2 laser. Anesthesia was achieved with intramuscular doses of 10 mg/kg ketamine and 60 mg/kg Xylazine. Two equal 0.5 cm surgical incisions of rat dorsal skin were performed on the left and right sides. One was utilized as a control while the other was subjected to a 10600 nm CO2 laser at various power levels (12.5, 14.1, 15.6, and 17.2) W/cm2. According to the histological analysis, the non-irradiated skin appeared to be flawless, and normal skin layers were observed. The amount of radiation in the irradiated skin samples was closely related to tissue damage. Higher dosages of irradiation resulted in the most severe cellular mutilation. Tissue injury manifested as epidermal obliteration, coagulation, homogeneous hyalinization, and hair loss. The effects of CO2 laser interaction with the skin were explored in-depth in this study. Exposure to the CO2 laser resulted in severe burns and coagulation.

Radiation-Induced Localized Bullous Pemphigoid.

Radiation-Induced Localized Bullous Pemphigoid.

Melatonin immunoreactivity of epidermal skin is higher in the evening than morning but does not account for erythema sensitivity

ABSTRACT The skin is a site of melatonin synthesis, and melatonin has a role in protecting against ultraviolet radiation-induced damage. Ultraviolet B (UVB) induced erythema seems to vary between morning and evening. We investigated whether epidermal melatonin immunoreactivities in the morning differed from those in the evening, and whether UVB-induced erythema was associated with these melatonin immunoreactivities in healthy volunteers. Erythema sensitivity of the skin was determined in the morning and in the evening by scoring the Minimal Erythema Dose and quantifying the erythema index (EI). We took biopsies from the non-UVB-exposed skin of healthy volunteers (n = 39) in the morning and in the evening to study melatonin immunoreactivity with immunohistochemistry (IHC). In the IHC staining, there was more melatonin immunoreactivity in the evening than in the morning (p < .001). Erythema was more pronounced in the evening than in the morning irradiated skin (p < .001). The graded amount of melatonin immunoreactivity in the samples was not associated with the EI. We discovered melatonin immunoreactivity of the non-irradiated skin to vary diurnally. However, endogenous skin melatonin does not seem to be the reason why NB-UVB induces more erythema in the evening than in the morning.

The Effect of Irradiation on the Skin upon Breast Cancer Radiotherapy Studied by FTIR Spectroscopy

Breast cancer affects the female population worldwide. Radiotherapy (RT) is part of the therapeutic modality in the management of breast cancer, after radical mastectomy or conserving surgery. The FTIR spectroscopic “marker bands” will lead us to approach the mechanism of skin damage due to the interaction of ionizing radiation and skin, on a molecular level at the very early stages. FT-IR spectroscopy, breast digital pictures, and ImageJ software were used in the study. Healthy breast skin was irradiated &lt;em&gt;ex-vivo&lt;/em&gt; with a 4 Gy dose of a γ-&lt;sup&gt;60&lt;/sup&gt;Co course Gammachamber 4000A. The FT-IR spectra showed that the low-dose irradiation induces skin dehydration, collagen secondary structure changes and advanced glycation end products (AGEs) as a result of free radicals as mediated products. The infrared “marker bands” at about 1743, 1160, and 870 cm&lt;sup&gt;-1&lt;/sup&gt; are characteristic, indicating the development of inflammation, glycations, and peroxidations respectively, due to ionizing radiation-induced oxidative stress. ImageJ analysis provided the sharp surface of the skin after RT irradiation in contrast to the smooth surface of the non-irradiated healthy skin. The most important damages, induced by radiotherapy, were connective tissue lesions, glycosylation, and phosphorylation processes in the skin. The reactive oxygen species (ROS) free radicals prefer to abstract H atoms from lipids, sugar rings of glycoproteins, and base ribose of DNA. The produced intermediate free radicals, as a result of ROS reactions, led to the formation of AGEs and peroxides.

592 Epigenetic memory of radiation injury in skin fibroblasts of cancer patients

Radiotherapy (RT) is a common cancer treatment but often results in an unintended injury to overlying skin and contributes to impaired wound healing. Skin fibroblasts (FBs) play critical roles in wound repair. We investigated the long-term effects of RT on FB functions to understand better the pathological mechanisms of adverse skin reactions to RT. We collected tissue biopsies from non-irradiated (RT-) and irradiated skin (RT+) of breast cancer patients who received RT 1-12 years ago. We found that ex vivo wound healing and FB motility were significantly reduced in the RT+ skin compared to the paired RT- skin.

Biological Cover Mitigates Disruption of the Dermal Structure in Mechanically Expanded Skin in a Porcine Model.

Tissue expansion is an integral procedure of the vast majority of breast reconstruction and has a significant impact on the final clinical outcomes. Therefore, technological advances leading to a fewer number of unfavorable outcomes and a decrease in complication rates are imperative. In this study, using a porcine model, we investigated an effect of acellular dermal matrix (ADM) used as a tissue expander cover on the dermal changes induced by mechanical forces during tissue expansion. After 14 days of expansion, skin samples were collected from one animal, while the second animal underwent radiation, and tissue was collected 8 weeks later. Tissue expanded without the use of ADM and unexpanded skin served as the controls. Collected skin biopsies were used for histological and immunohistochemical evaluation, and for gene expression analysis. We revealed that the biological cover incorporation into host tissue is facilitated by macrophages without inducing a broad inflammatory response. The utilization of ADM mitigated disruption in the dermal structure, excessive collagen deposition, and capsule formation in non-irradiated expanded skin. The protective effect was not fully maintained in irradiated skin. These results demonstrate that tissue expansion might be improved by using the tissue expander cover.

Effect of hyperbaric oxygen treatment on skin elasticity in irradiated patients.

Hyperbaric oxygen treatment (HBOT) is often used in an attempt to reverse/treat late radiation-induced tissue fibrosis (LRITF). This study aimed to quantify the effects on skin elasticity. Skin retraction time was used as a marker of skin elasticity in 13 irradiated breast cancer patients. The measurements were carried out on the affected side as well as the unaffected/healthy side at a mirrored location. Readings were taken at the start and end of HBOT (mean 43 sessions, 80 min at 243 kPa). Patient age ranged from 39-70 years. All patients underwent surgical lumpectomy and radiotherapy prior to undergoing HBOT. The mean time between radiotherapy and HBOT was 70 months. Seven of the 13 patients underwent chemotherapy. Mean irradiated skin retraction time improved from 417 (SD 158) pre-HBOT to 171 (24) msec post-HBOT (P < 0.001). Mean pre-HBOT retraction time in the non-irradiated skin was 143 (20) msec and did not change. This promising pilot study that suggests that HBOT may improve skin elasticity in patients with LRITF.

Cultured Human Foreskin as a Model System for Evaluating Ionizing Radiation-Induced Skin Injury.

Purpose: Precise molecular and cellular mechanisms of radiation-induced dermatitis are incompletely understood. Histone variant H2A.J is associated with cellular senescence and modulates senescence-associated secretory phenotype (SASP) after DNA-damaging insults, such as ionizing radiation (IR). Using ex vivo irradiated cultured foreskin, H2A.J was analyzed as a biomarker of radiation-induced senescence, potentially initiating the inflammatory cascade of radiation-induced skin injury. Methods: Human foreskin explants were collected from young donors, irradiated ex vivo with 10 Gy, and cultured in air-liquid interphase for up to 72 h. At different time-points after ex vivo IR exposure, the foreskin epidermis was analyzed for proliferation and senescence markers by immunofluorescence and immunohistochemical staining of sectioned tissue. Secretion of cytokines was measured in supernatants by ELISA. Using our mouse model with fractionated in vivo irradiation, H2A.J expression was analyzed in epidermal stem/progenitor cell populations localized in different regions of murine hair follicles (HF). Results: Non-vascularized foreskin explants preserved their tissue homeostasis up to 72 h (even after IR exposure), but already non-irradiated foreskin epithelium expressed high levels of H2A.J in all epidermal layers and secreted high amounts of cytokines. Unexpectedly, no further increase in H2A.J expression and no obvious upregulation of cytokine secretion was observed in the foreskin epidermis after ex vivo IR. Undifferentiated keratinocytes in murine HF regions, by contrast, revealed low H2A.J expression in non-irradiated skin and significant radiation-induced H2A.J upregulations at different time-points after IR exposure. Based on its staining characteristics, we presume that H2A.J may have previously underestimated the importance of the epigenetic regulation of keratinocyte maturation. Conclusions: Cultured foreskin characterized by highly keratinized epithelium and specific immunological features is not an appropriate model for studying H2A.J-associated tissue reactions during radiation-induced dermatitis.

Fenofibrate Attenuates Radiation-Induced Oxidative Damage to the Skin through Fatty Acid Binding Protein 4 (FABP4).

Radiation facilities and radioactive materials have been widely used in military, industry, medicine, science and nuclear facilities, which has significantly increased the potential of large-scale, uncontrolled exposure to radiation. The skin is one of the radiosensitive organ systems and radiation-induced skin injury remains a serious concern after ionizing radiation exposure. Our previous report indicates the involvement of the peroxisome proliferator-activated receptor pathway in the response of skin tissues to ionizing radiation. PPARα is a member of the PPAR nuclear hormone receptor superfamily, which can be activated by fibrate ligands. However, the protection of fenofibrate against ionizing radiation in skin keratinocytes and fibroblasts has not been described. The PPARα mRNA levels in irradiated and nonirradiated skin tissues of rats were determined by real-time assay. The expression of PPARα, and FABP4 were evaluated by western blot and IHC assay. The cell proliferation was detected by colony formation. The γH2AX foci and ROS levels in irradiated WS1 cells with FABP4 overexpression than in control cells were performed by Immunofluorescence assay. We found that PPARα expression was lower in the irradiated skin tissues of mouse, rat, monkey, and human patients than in their nonirradiated counterparts. PPARα fenofibrate significantly decreased radiation-induced ROS and apoptosis in a dose-dependent manner in human keratinocyte HaCaT and skin fibroblast WS1 cells. Moreover, fenofibrate significantly decreased radiation-induced ROS and malondialdehyde (MDA) levels in electron beam irradiated skin tissues of rats. Mechanistically, the proximal promoter of fatty acid binding protein 4 (FABP4) harbored three binding sites of PPARα and fenofibrate stimulated the transcription of FABP4 in skin cells. FABP4 overexpression decreased radiation-induced ROS and γH2AX foci. FABP4 inhibitor BMS309403 abrogated the ROS-eliminating activity as well as the lipid-accumulating role of fenofibrate, indicating that FABP4 mediates the radioprotective role of fenofibrate. In addition, FABP4 overexpression significantly decreased radiation-induced oxidative damage in vivo. These results confirm that fenofibrate attenuated radiation-induced oxidative damage to the skin by stimulating FABP4.

112. Deferoxamine Transdermal Application Decreases Fibrosis and Improves Elasticity in Irradiated Porcine Skin

Purpose: Radiation therapy is notorious for its effectiveness in oncologic treatment but its devastating side effects to the surrounding soft tissue. We have previously demonstrated the efficacy of a transdermal drug delivery system (TDDS) to deliver deferoxamine (DFO) cutaneously to irradiated porcine skin safely without any systemic effects. With this study, we aim to demonstrate the beneficial effects of deferoxamine on irradiated porcine skin. Methods: Six red Duroc pigs (three months old) were irradiated with a single fraction of 30 Gy bilaterally. The pigs were allowed to recover for 12 weeks to allow chronic fibrosis to develop. The pigs were then divided into 4 different treatment conditions: normal, non-irradiated skin, irradiated skin with no patch (IR control), vehicle patch, and 1.0mg DFO TDDS patch. The pigs were treated with daily patch changes for 4 weeks and harvested immediately following the 4 weeks of treatment of after waiting another 4 weeks post-treatment. Histological analysis and suction cutometer analysis were performed. P<0.05 was considered significant on statistical analysis. Results: Dermal thickness was noted to be significantly lower in the DFO cohort than the IR control or vehicle cohort at both 4 and 8 weeks post-treatment. Collagen ultrastructure was significantly different between the DFO treatment groups, more closely resembling the normal skin groups, compared to the no patch or vehicle patch groups. Collagen density was also significantly lower in the DFO cohort compared the vehicle or IR control groups. CD31+ expression was significantly higher in the DFO group compared to all other conditions except the normal skin. DFO also had significantly more elasticity than the IR control or vehicle group. Conclusion: DFO effectively treats radiation-induced fibrosis in a porcine model, allowing the skin to more closely resemble normal skin than irradiated skin.

Narrow-band ultraviolet B therapy attenuates cutaneous T-cell responses in hapten-induced, experimental contact dermatitis in beagles.

In human medicine, narrow-band ultraviolet B (NB-UVB) phototherapy has been used to treat various T-cell-mediated skin diseases. However, the effect of NB-UVB on inflamed canine skin remains uncertain. To investigate the effect of NB-UVB phototherapy on the skin of dogs with hapten-induced contact dermatitis. Seven healthy beagles without skin problems. Dogs were irradiated with varying doses of NB-UVB to determine the minimal erythema dose (MED). After determining the MEDs of six dogs (excluding one of the seven whose skin did not show a visible reaction), we investigated the effect of NB-UVB on their inflamed skin by topically applying 2,4-dinitrochlorobenzene (DNCB), which causes type 1 helper T cell (Th1)- and cytotoxic T-cell (Tc)1-induced skin inflammation. We then irradiated the skin with NB-UVB. We analysed the treated skin samples via histopathological and immunohistochemical methods, and TdT-mediated dUTP nick-end labelling (TUNEL) to demonstrate apoptotic cells. We also analysed the cytokine gene transcription via real-time quantitative reverse transcription PCR. The NB-UVB MEDs caused mild inflammatory changes yet no severe epidermal exfoliations in the irradiated skin. In DNCB-treated skin irradiated by the NB-UVB MEDs, TUNEL-positive dermal apoptotic cells were increased significantly compared with those of DNCB-treated, nonirradiated skin. INF-γ and TNF-α transcription levels in DNCB-treated, irradiated skin were significantly lower than those in the DNCB-treated, nonirradiated skin. Phototherapy using NB-UVB MEDs attenuated cutaneous Th1 and Tc1 cytokine responses with minimal skin damage in a canine model of hapten-induced contact dermatitis.

Comparative Cytokine Profiling of Wound Tissue Homogenate From Irradiated and Non-irradiated Skin

Comparative Cytokine Profiling of Wound Tissue Homogenate From Irradiated and Non-irradiated Skin

349 Long non-coding RNAs and skin fibrosis consequent to radiotherapy

In this study, we explore the role of long non-coding RNAs (lncRNAs) as biomarkers of radiation- induced skin fibrosis. Skin fibrosis, a common late complication of radiotherapy, is characterized by myofibroblast proliferation and pathological deposition of extracellular matrix, with TGFB1 pathway as a known master switch. However, epigenetic mechanisms of fibrosis development are still poorly described. Transcriptome sequencing (NGS RNA-seq) was performed for untransformed fibroblast strains isolated from non-irradiated skin samples of 85 patients presenting with severe complications of radiotherapy. Clustering analysis of gene expression data was used to define sub-populations of patients and differentially expressed genes between sub-populations were identified using EdgeR. Functional enrichment analysis was performed via Gene Ontology and Reactome. Co-expressed lncRNAs and fibrosis- and matrix modeling-related transcripts were identified by WGCNA R package. Two sub-populations of patients were found by clustering analysis of gene expression data: cluster 1 (45 patients) and cluster 2 (40 patients), which differed by 2 163 messenger RNAs (mRNAs) and 291 lncRNAs. Cluster 1 was characterized by the enrichment of a set of 272 mRNAs relative to extracellular matrix remodeling and fibrosis, when compared to cluster 2. A weighted co-expression network analysis identified 12 lncRNAs co-expressed in cluster 1 with 137 major fibrosis-related genes, including TGFB1, metalloproteinases and collagens. These candidate lncRNAs are currently investigated for their response to TGFB1 and radiation exposure, their involvement in myofibroblast activation, and their functional role in fibrosis gene regulation. From the exploration of this set of new molecular regulators, we anticipate the description of original skin fibrosis susceptibility biomarkers and effectors, and the discovery of potential anti-fibrosis therapeutic targets.

Mechanical sensitization, increased axonal excitability, and spontaneous activity in C-nociceptors after ultraviolet B irradiation in pig skin.

Ultraviolet B (UVB) irradiation induces hyperalgesia in human and animal pain models. We investigated mechanical sensitization, increase in axonal excitability, and spontaneous activity in different C-nociceptor classes after UVB in pig skin. We focused on units with receptive fields covering both irradiated and nonirradiated skin allowing intraindividual comparisons. Thirty-five pigs were irradiated in a chessboard pattern, and extracellular single-fibre recordings were obtained 10 to 28 hours later (152 fibers). Units from the contralateral hind limb served as a control (n = 112). Irradiated and nonirradiated parts of the same innervation territory were compared in 36 neurons; low threshold C-touch fibers (n = 10) and sympathetic efferents (n = 2) were unchanged, but lower mechanical thresholds and higher discharge frequency at threshold were found in mechanosensitive nociceptors (n = 12). Half of them could be activated with nonnoxious brush stimuli in the sunburn. Four of 12 mechanoinsensitive nociceptors were found sensitized to mechanical stimulation in the irradiated part of the receptive field. Activity-dependent slowing of conduction was reduced in the irradiated and in the nonirradiated skin as compared with the control leg, whereas increased ability to follow high stimulation frequencies was restricted to the sunburn (108.5 ± 37 Hz UVB vs 6.3 ± 1 Hz control). Spontaneous activity was more frequent in the sunburn (72/152 vs 31/112). Mechanical sensitization of primary nociceptors and higher maximum after frequency are suggested to contribute to primary hyperalgesia, whereas the spontaneous activity of silent nociceptors might offer a mechanistic link contributing to ongoing pain and facilitated induction of spinal sensitization.

NFATC2 Modulates Radiation Sensitivity in Dermal Fibroblasts From Patients With Severe Side Effects of Radiotherapy.

Although it is well established that 5 to 15% of radiotherapy patients exhibit severe side-effects in non-cancerous tissues, the molecular mechanisms involved are still poorly known, and the links between cellular and tissue radiosensitivity are still debated. We here studied fibroblasts from non-irradiated skin of patients with severe sequelae of radiotherapy, to determine whether specific basal cell activities might be involved in susceptibility to side-effects in normal tissues. Compared to control cells, patient fibroblasts exhibited higher radiosensitivity together with defects in DNA repair. Transcriptome profiling of dermal fibroblasts from 16 radiotherapy patients with severe side-effects and 8 healthy individuals identified 540 genes specifically deregulated in the patients. Nuclear factor of activated T cells 2 (NFATC2) was the most differentially expressed gene, poorly expressed at both transcript and protein level, whereas the NFATC2 gene region was hypermethylated. Furthermore, NFATC2 expression correlated with cell survival after irradiation. Finally, silencing NFATC2 in normal cells by RNA interference led to increased cellular radiosensitivity and defects in DNA repair. This study demonstrates that patients with clinical hypersensitivity also exhibit intrinsic cellular radiosensitivity in their normal skin cells. It further reveals a new role for NFATC2 as a potential regulator of cellular sensitivity to ionizing radiation.