The HIPPO Transducer YAP and Its Targets CTGF and Cyr61 Drive a Paracrine Signalling in Cold Atmospheric Plasma-Mediated Wound Healing

Shome, Debarati; von Woedtke, Thomas; Riedel, Katharina; Masur, Kai

doi:https://doi.org/10.1155/2020/4910280

Oxidative Medicine and Cellular Longevity

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Abstract Introduction Materials and Methods Results Discussion Conclusions Abbreviations Data Availability Conflicts of Interest Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2020 | Article ID 4910280 | https://doi.org/10.1155/2020/4910280

The HIPPO Transducer YAP and Its Targets CTGF and Cyr61 Drive a Paracrine Signalling in Cold Atmospheric Plasma-Mediated Wound Healing

Debarati Shome,¹Thomas von Woedtke,¹Katharina Riedel,²and Kai Masur¹

Academic Editor: Reiko Matsui

Received09 Sept 2019

Revised27 Jan 2020

Accepted29 Jan 2020

Published13 Feb 2020

Abstract

Reactive species play a pivotal role in orchestrating wound healing responses. They act as secondary messengers and drive redox-signalling pathways that are involved in the homeostatic, inflammatory, proliferative, and remodelling phases of wound healing. The application of Cold Atmospheric Plasma (CAP) to the wound site produces a profusion of short- and long-lived reactive species that have been demonstrated to be effective in promoting wound healing; however, knowledge of the mechanisms underlying CAP-mediated wound healing remains scarce. To address this, an in vitro coculture model was used to study the effects of CAP on wound healing and on paracrine crosstalk between dermal keratinocytes and fibroblasts. Using this coculture model, we observed a stimulatory effect on the migration ability of HaCaT cells that were cocultured with dermal fibroblasts. Additionally, CAP treatment resulted in an upregulation of the HIPPO transcription factor YAP in HaCaTs and fibroblasts. Downstream effectors of the HIPPO signalling pathway (CTGF and Cyr61) were also upregulated in dermal fibroblasts, and the administration of antioxidants could inhibit CAP-mediated wound healing and abrogate the gene expression of the HIPPO downstream effectors. Interestingly, we observed that HaCaT cells exhibited an improved cell migration rate when incubated with CAP-treated fibroblast-conditioned media compared to that observed after incubation with untreated media. An induction of CTGF and Cyr61 secretion was also observed upon CAP treatment in the fibroblast-conditioned media. Finally, exposure to recombinant CTGF and Cyr61 could also significantly improve HaCaT cell migration. In summary, our results validated that CAP activates a regenerative signalling pathway at the onset of wound healing. Additionally, CAP also stimulated a reciprocal communication between dermal fibroblasts and keratinocytes, resulting in improved keratinocyte wound healing in coculture.

1. Introduction

Cold Atmospheric Plasma (CAP) is an emerging tool of biomedical and clinical importance that controls cellular processes such as wound healing, immunomodulation, cell death, and cancer. CAP is a partially ionized molecular or noble gas or gas mixture that generates a plethora of reactive oxygen and nitrogen species (ROS/RNS), radiation (ultraviolet (UV), visible, and infrared), and small chemical entities such as neutral molecules, ions, electrons, and exited atoms under atmospheric pressure and ambient temperature [1]. The short- and long-lived ROS and RNS produced by CAP are involved in redox signalling in vitro and in vivo. Wound healing involves a coordinated infiltration of dermal cells that is followed by infiltration of immune cells, extracellular matrix deposition, and reepithelization over distinct yet interdependent phases that are regulated by a large number of transcription factors or transcriptional events [2–5]. ROS are critical secondary messengers that orchestrate wound healing processes by regulating the recruitment of immune cells, angiogenesis, and optimal perfusion of blood at the wound site [6]. Recent reports also indicate that CAP supports wound healing by influencing cell viability [7, 8], proliferation [9], migration [10], inflammation [11], dermal regeneration, and reepithelization [12]. Currently, only a small number of studies have examined the cellular and molecular effects of CAP-mediated wound healing [1, 13, 14] and the mechanisms underlying CAP-mediated wound healing in the context of regenerative pathways require further investigation.

The HIPPO signalling pathway was initially identified in Drosophila melanogaster, and this pathway regulates cellular proliferation, organ size, and survival and is highly conserved in mammals [15–17]. The core HIPPO pathway in vertebrates consists of a kinase cascade where one of the major transcriptional coactivators is Yes-associated protein (YAP) [18, 19]. Nonphosphorylated YAP translocates to the nucleus and induces target gene transcription by interacting with the transcription factors TEA domain family member proteins (TEAD 1-4) [20]. In particular, YAP-mediated target gene transcription depends upon its cellular localization (cytoplasmic or nuclear) [21–24].

The HIPPO signalling pathway is of paramount importance in the oxidative stress response and wound healing. YAP is involved in redox homeostasis regulation, and its deletion can lead to redox sensitivity [22]. Certain YAP-targeting genes encode secretory proteins and may possess regenerative potential [25]. The downstream target genes of YAP that are of primary interest in this study are Connective Tissue Growth Factor (CTGF) and Cysteine-rich angiogenic protein 61 (Cyr61), members of CCN family proteins. According to literature, YAP moves to the nucleus and interacts with TEAD to promote the transcription of these matricellular proteins that are involved in cell adhesion and migration [26, 27]. These extracellular matrix-associated signalling proteins are structurally related heparin-binding proteins and are synthesized during cutaneous wound healing in dermal fibroblasts [28]. Due to its ability to modulate the activities of several growth factors and cytokines, CTGF possesses the potential to regulate diverse biological processes including cell adhesion, migration [29], proliferation [30], angiogenesis [31], and early wound healing and repair [32]. A physiological role of Cyr61 in regulating inflammation, adhesion, angiogenesis, proliferation, matrix remodelling, and cell-ECM interaction during wound repair has also been identified [26, 28, 33, 34]. Oxidative stress plays a role in the alteration of CTGF expression in fibroblasts [35]. Qin et al. found that Cyr61 expression is induced by oxidative stress and that a positive feedback loop continues to generate ROS and upregulate Cyr61 in fibroblasts [36]. Taken together, these reports strongly suggest the presence of a redox-induced HIPPO signalling axis in wound healing that is complex and poorly understood.

In this study, for the first time, we demonstrate the activation of the YAP-CTGF-Cyr61 axis in response to CAP treatment in a well-characterized human epithelial cell line (HaCaT) and in a human nonneonatal skin fibroblast cell line (GM00637). CAP-mediated acceleration of wound healing indicates the presence of a paracrine signalling pathway that is driven by the extracellular matrix-associated signalling proteins CTGF and Cyr61 in a coculture model. The activation of organ survival and regenerative pathways further establishes the usefulness of CAP in redox-mediated wound management and repair.

2. Materials and Methods

2.1. Cell Culture

Human skin keratinocytes (HaCaT) and the immortalized skin fibroblast cell line GM00637 (GM Fbs) were cultured in Roswell Park Memorial Institute 1640 cell culture medium (RPMI 1640, Invitrogen) supplemented with 10% fetal calf serum (FCS, Invitrogen), 0.1 mg/ml penicillin/streptomycin, and 2 mM L-glutamine (PAN Biotech, Germany). For 2D scratch assays, the indicated number of cells (either in mono- or coculture), as per experimental requirements, was incubated in 96-well image lock culture plates (Essen BioScience) in a starved cell culture medium (supplemented with 1% FCS) 16-18 hr prior to their experimental use.

2.1.1. Coculture

Direct coculture of HaCaTs and GM Fbs was used for cell migration assays and metabolic assays in 96-well image lock culture plates (Essen BioScience) or normal 96-well plates (Sarstedt), respectively. For coculture experiments, GM Fbs were seeded first into a 96-well plate, and once they adhered to the plate, the indicated number of HaCaTs was seeded on top of the fibroblasts in a manner that was dependent upon experimental type. The exact cell numbers are described in the respective method sections. The cell numbers (in mono- or coculture) and the experimental type are also summarized in Figure S1.

2.2. Harvest of GM-Fb-Conditioned Medium (GM-Fb-CM)

To harvest the conditioned medium (CM), GM Fbs (/well) were seeded onto a 24-well plate in complete RPMI 1640 (supplemented with 10% FCS) until the cells were approximately 80% confluent. The medium was then replaced with 1% FCS medium for 16-18 hr. Next, fibroblasts were incubated with untreated or CAP-treated medium for another 18 hr. Following the incubation, conditioned medium was collected. The CM was centrifuged at 2000 rpm for 5 min, filtered through a 0.22 μm syringe filter, and kept at 4°C for immediate use. For longer storage, the medium was stored at −80°C.

2.3. CAP Treatment

CAP treatment was performed using an atmospheric pressure argon plasma jet kINPen MED (neoplas tools, Greifswald, Germany). The electrical safety of the kINPen was certified and complies with EU standards [37]. This is the first jet worldwide to be accredited as a medical device class IIa according to European Council Directive 93/42/EEC for use in wound treatment, and this device lacks mutagenic and genotoxic effects [37–39]. The jet was operated at a voltage of 2-6 kVpp and a frequency of 1 MHz. The gas flow rate was set at 5 SLM (standard litres per minute) to treat 5 ml of RPMI 1640 supplemented with 1% FCS in a 60 mm dish for 10, 30, and 60 s. CAP-treated media were immediately used to treat the cells for further experiments. Cells incubated with untreated RPMI medium served as negative controls for all experiments.

2.4. Metabolic Activity Assay

To determine metabolic activity, cells (in monoculture) were seeded into a 96-well plate (Sarstedt) 24 hours prior to the start of the experiment. For coculture, GM Fbs and HaCaTs were seeded onto the plate. After incubation with CAP-treated media for 3 and 24 hr, the wells were loaded with 100 μM of resazurin (Alfa Aesar) that is transformed to fluorescent resorufin by metabolically active cells. The plate was incubated for 2 h at 37°C, and fluorescence was measured using a Tecan plate reader (Infinite M200 Pro) at 535 nm and 590 nm. The fluorescence values were normalized to those of the untreated controls.

2.5. Cell Migration Assay

The cell migratory behaviour of HaCaTs and GM Fbs was assayed using a cell migration assay that incorporated an Essen BioScience wound maker. For this assay, separate monocultures of HaCaT and GM Fbs and coculture of HaCaT and GM Fbs were used. For the monocultures, either HaCaTs/well or GM Fbs/well were seeded into Essen BioScience 96-well image lock plates. For the coculture, GM Fbs/well were seeded into image lock plates and incubated until the fibroblasts attached to the plates, and then HaCaTs/well were seeded onto the top of the fibroblasts at a ratio of 1 : 2 fibroblasts: keratinocytes within the coculture. Several other ratios (1 : 3, 1 : 5) of HaCaTs and GM Fbs were also tested for the cell migration assays (data not shown), but in further experiments, a 1 : 2 ratio of HaCaT and GM Fbs was used. After complete adherence of HaCaTs and GM-Fbs in mono- and cocultures, the cells were starved using 1% FCS for 16-18 hr prior to the experiment. The Essen BioScience wound maker was used to create scratches, and the cells were then incubated with either 10 or 30 s plasma-treated medium or untreated medium as indicated.

For other experiments, recombinant CTGF (10-50 ng/ml) and Cyr61 (100-1000 ng/ml) (PeproTech GmBH) and GM-Fb-CMs were used on HaCaT monocultures seeded at a density of cells/well.

The cell migration rate was monitored using an IncuCyte S3 live cell analysis system for up to 24 hr. The relative wound density was measured with the in-built software of the IncuCyte S3 2018A. The relative wound density (%) is the ratio of the occupied area of the initially scratched area with respect to the total scratched area. The graphs are plotted as a function of relative wound density (%) in -axis in comparison to the time at the -axis.

2.5.1. mRNA Expression Analysis

Quantitative real-time PCR was performed using a 96-well LightCycler 480 qPCR system according to the manufacturer’s protocol (Roche Diagnostics, Germany). Briefly, HaCaTs or GM Fbs were seeded into a 6-well plate, and once the monocultures reached confluence, the cells were starved for 16-18 hours, scratches were made using a 10 μl pipette tip, and the cells were subsequently incubated with CAP-treated medium or untreated control medium. Total RNA was isolated with an RNA mini kit (Bio & Sell, Germany) after 3, 6, 18, and 24 hr. 1 μg of RNA was transcribed to cDNA using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and qPCR was performed in triplicate with SYBR Green I Master mix (Roche Diagnostics, Germany) specific for the genes YAP, CTGF, and Cyr61. Primer sequences are provided in Fig S2. The housekeeping gene β-actin was used as the internal control for normalization. Gene expression analysis was performed using the ΔΔCT method [40]. The final fold change in gene expression upon plasma treatment was determined according to the ratio of expression in the respective sample corresponding to the control.

2.6. Immunoblotting

HaCaTs and GM Fbs were seeded at a density of cells in a 6-well plate. After the cells reached confluency, they were serum starved for 16-18 hours, scratches were created using a 10 μl pipette tip, and the cells were subsequently incubated with CAP-treated medium or untreated medium. Cells were harvested into ice-cold PBS after 3, 6, and 24 hr. Next, the samples were lysed in RIPA buffer that was supplemented with protease and phosphatase inhibitors (Roche) for 20 min on ice. Lysates were then centrifuged at 15000 g for 15 min at 4°C, and total extracted protein was quantified using the DC protein assay (Bio-Rad). Total protein (25 μg) was resolved by 10% SDS-PAGE (Bio-Rad) and then transferred and blotted on PVDF membranes. The membranes were probed with antitotal YAP (Santa Cruz Biotechnology), phospho-YAP (CST), CTGF (Novex Biotechnology), Cyr61 (CST), and β-actin (CST) primary antibodies, and this was followed by incubation with secondary horse radish peroxide- (HRP-) coupled antibodies (CST) and subsequent detection of the bands using chemiluminescence (Applied Biosystems). Band intensities were quantified with Image Studio Lite (version 5.2), normalized to housekeeping β-actin, and expressed as a fold change compared to corresponding untreated control.

2.7. N-Acetyl Cysteine (NAC) Treatment

A 100 mM stock solution was prepared by dissolving NAC powder (Sigma-Aldrich) into 1x sterile PBS. This solution was sterilized using a 0.2 μM sterile membrane filter, and aliquots of the stock solution were stored at −20°C. HaCaTs and GM Fbs were incubated with 2.5 mM NAC in the presence or absence of CAP-treated media for the designated times and then harvested for further experiments.

2.8. Enzyme-Linked Immunosorbent Assay (ELISA)

GM-Fb-CM was collected 18 hours after incubation with 10 and 30 s of CAP-treated or untreated medium and analysed using enzyme-linked immunosorbent assay (ELISA). The concentrations of secreted CTGF (PeproTech GmBH, catalogue no: 900-K317) and Cyr61 (R&D systems, catalogue no: DCYR10) were quantitatively determined according to the manufacturer’s protocol. ELISA values were measured in duplicate from two different biological experiments for each sample to minimize the intra- and interassay variability.

2.9. Statistical Analysis

Graphics and statistics were performed using prism 8.02 (GraphPad PRISM software, San Diego, USA). Mean and standard deviation were calculated and analysed according to unpaired -test with Welch’s correction and ordinary one-way or 2-way analysis of variance (ANOVA). A value < 0.05 was considered statistically significant (, , , and ).

3. Results

3.1. Influence of CAP on Metabolic Activity in Mono- and Coculture

There are compelling evidences within the literature indicating that among all the short- and long-lived ROS produced by CAP, H₂O₂ is one of the most stable oxidants produced by kINPen MED [41]. Based on this, we quantified the H₂O₂ concentration (in μM) and found that there is a linear increase in H₂O₂ concentration according to treatment time (Fig S3). CAP did not significantly alter metabolic activity after 10 and 30 s of treatment; however, a decrease was observed in response to a 60 s of treatment after 3 hr of incubation in all cell types (Figure 1(a)). Further significant reduction was not observed in the metabolic activity of HaCaTs and cocultured cells after 24 hr, but a 30 and 60 s treatment resulted in a significant reduction in metabolic activity in GM Fbs after 24 hr (Figure 1(b)). Based on this, we chose 10 and 30 s treatments for all subsequent experiments.

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3.2. CAP Accelerates Cell Migration in Coculture

Migration and proliferation of keratinocytes and fibroblasts are key biological processes that occur during various steps of wound healing, including wound repair and reepithelization. To exclusively determine cell migration, all cell types were starved prior to performing the scratch assay. Based on the results, CAP did not significantly alter relative wound density in either of the cell lines after 10 and 30 s of treatments (Figures 2(a) and 2(b), first and second column); however, CAP significantly accelerated relative wound density in coculture after 12 hours (Figures 2(a) and 2(b), third column). Wound closure images of both the monocultures and the coculture are provided in the supplementary figures (S3, S4, and S5). We also compared the relative wound density between HaCaTs alone and those cocultured with fibroblasts, and we observed that after CAP treatment, relative wound density was significantly increased in coculture compared to that observed in HaCaT monoculture (Figure 2(c)). These results indicated that the CAP-mediated effect on wound closure may differ between mono- and coculture. However, as coculture is more closely related to skin models due to the inclusion of both keratinocytes and fibroblasts, we assessed the signalling pathways that were active in each cell type to determine the stimulatory effect of CAP on coculture migration.

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Figure 2

CAP accelerates cell migration in coculture: (a) depicts the representative time course of wound closure in untreated or CAP-treated HaCaTs, GM Fbs, and coculture, respectively, (upper row) up to 24 h. (b) shows the summation of relative wound density of untreated and CAP-treated HaCaTs, GM Fbs, and coculture, respectively, (middle row) at specific time points (6, 12, 18, and 24 h). (c) details the comparison of relative wound density between untreated and CAP-treated HaCaTs and coculture at 6, 12, 18, and 24 hr (lower row). Data are presented as of three independent experiments performed in triplicate. Statistical comparisons were performed using 2-way ANOVA.

3.3. CAP Accelerates Migration through the Activation of the YAP-CTGF-Cyr61 Axis

Quantitative real-time PCR revealed that CAP significantly stimulated YAP mRNA expression at 6 hr in GM Fbs (Figure 3(b)). CAP also tends to increase YAP at late time points in GM Fbs (18 hr) and HaCaTs (at 24 hr) (Figures 3(a) and 3(b)), and CAP minimally induced CTGF and Cyr61 in HaCaTs at 3 hr (Figure 3(a)). However, CAP increased CTGF and Cyr61 gene expression to more than threefold in GM Fbs, and this peaked 18 hr after treatment and then declined to near baseline by 24 hr (Figure 3(b)).

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Figure 3

mRNA expression of HIPPO signalling effectors after CAP treatment: mRNA expression of YAP, CTGF, and Cyr61 in HaCaTs (a) and GM Fbs (b) measured 3, 6, 18, and 24 hr after CAP treatment by qPCR and normalized to relative gene expression (ΔΔCT values on a log2 scale). The -axis represents CAP treatment time and incubation time after plasma treatment. Data are represented as of either three (a) or four (b) independent experiments. Statistical analysis was performed using unpaired -test with Welch’s correction for multiple comparisons, along with normalization to the untreated control.

Under serum-starved conditions, the ratio of phosphorylated YAP compared to total YAP (pYAP/YAP) protein was not significantly altered in GM Fbs (Figure 4(a)); however, there was a tendency for increased phosphorylation in HaCaT cells (Figure 4(b)). We also observed an increase in total cellular CTGF (3 hr) and Cyr61 protein (3 hr and 6 hr) levels after CAP treatment in GM Fbs (Figures 4(c) and 4(d)); however, unlike in GM Fbs, we could only detect a minor increase of CTGF in HaCaT cells upon CAP treatment (Figure 4(e)) and Cyr61 protein expression was completely undetectable (data not shown).

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Figure 4

Effect of CAP on the HIPPO signalling pathway: the relative phosphorylation level of YAP is shown in GM Fbs (a) and HaCaT cells (b). CTGF and Cyr61 protein expression was enhanced after 10 and 30 s of CAP treatment in GM Fbs (c, d). CTGF expression slightly increased after 10 and 30 s of CAP treatments in HaCaT cells after approximately 3 hours (e). Representative blots are shown. The -axis represents CAP treatment time and incubation time after plasma treatment. Data are represented as of at least three independent experiments. Statistical analysis was performed using unpaired -test with Welch’s correction for multiple comparisons, along with normalization to the untreated control.

3.4. Effect of NAC on Metabolic Activity and Cell Migration

NAC is a well-known inhibitor of reactive species, and to determine the effect of CAP-produced ROS on cell signalling, cells were incubated with 2.5 mM NAC alone or in combination with differential CAP-treated media for 24 hr. After 24 hr, NAC alone did not significantly alter the metabolic activity of the cells. In HaCaTs, NAC significantly counteracted the 30 and 60 s CAP treatment effects. In GM Fbs, NAC counteracted 10, 30, and 60 s CAP treatments and increased the metabolic activity. In coculture, NAC significantly counteracted the effects of 30 s of CAP treatment and increased the metabolic activity of these cells (Figure 5(a)). Although metabolic activity was restored, we still sought to determine if CAP-induced wound healing signalling is affected after NAC administration. Therefore, cell migration assays were performed using HaCaT monoculture and coculture. The cells were incubated with CAP-treated media with or without 2.5 mM NAC. Interestingly, we observed that NAC alone did not improve HaCaT cell migration. CAP treatment actually resulted in a modest acceleration in HaCaT cell migration that was significantly reduced after NAC administration (Figure 5(b)). Similar results were obtained in coculture experiments, where NAC reduced CAP-mediated cell migration to ~40-50%. (Figure 5(c)).

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Figure 5

Effect of NAC on CAP-mediated wound healing and metabolic activity: metabolic activity of HaCaTs, GM Fbs, and coculture after 24 hr incubation with 2.5 mM NAC alone or in combination with CAP-treated media (a). Relative wound density of HaCaTs after treatment with NAC alone or in combination with CAP-treated media at 6, 12, 18, and 24 hr (b). Relative wound density of coculture after treatment with NAC alone or in combination with CAP-treated media at 6, 12, 18, and 24 hr (c). Data are presented as from at least three independent experiments performed in triplicate (a–c). Statistical analysis was performed using either unpaired -test with Welch’s correction (a) or 2-way ANOVA (b, c).

3.5. Effect of NAC on YAP-CTGF-Cyr61 Axis

We previously observed a CAP-mediated activation of the YAP-CTGF-Cyr61 signalling cascade. To confirm that the upregulation of this signalling is mediated by CAP-induced reactive species, total RNA was isolated from HaCaTs and GM Fbs after incubation with NAC alone or with CAP-treated media. An 18-hour incubation time in the presence of CAP-treated media was chosen for gene expression analysis. NAC in combination with 10 and 30 s CAP-treated media significantly reduced the expression of CTGF (~2.4 fold) and Cyr61 (~1.9 fold) in GM Fbs. YAP expression was also reduced; however, NAC alone did not significantly impact these signalling molecules (Figure 6(b)). Similar results were obtained in HaCaT cells that revealed that NAC significantly reduced the CAP-mediated Cyr61 increase; however, YAP expression remained unaltered and CTGF expression was slightly but not significantly reduced (Figure 6(a)).

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3.6. CAP Promotes Migration in Coculture through Secreted CTGF and Cyr61

CAP-induced migration was higher in HaCaTs cocultured with GM Fbs than in monoculture alone (see Figure 2), indicating that CAP regulates the interaction between these cells in coculture. We assumed that the major regulatory factors are CTGF and Cyr61 in coculture based on these data. We performed a cell migration assay to determine the migratory behaviour of HaCaTs grown in media conditioned from untreated or CAP-treated GM Fbs. Conditioned medium harvested from CAP-treated GM Fbs at 18 hr significantly accelerated HaCaT cell migration compared to conditioned medium from untreated GM Fbs and normal medium (Figure 7(a)). We also analysed the GM-Fb-CM by ELISA, and we observed an increase in secreted CTGF and Cyr61 after CAP treatment (Figure 7(b)).

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Figure 7

Effect of CAP on paracrine signalling between keratinocytes and fibroblasts: HaCaT cells were incubated with conditioned media harvested from untreated or 10 and 30 s CAP-treated GM Fbs. Relative wound density of HaCaT cells was monitored after 6, 12, 18, and 24 hr (a). Secreted CTGF and Cyr61 (pg/ml) was analysed by ELISA (b). Relative wound density in HaCaT cells was monitored after treatment with 10 and 50 ng/ml CTGF and 100 ng/ml and 1 μg/ml Cyr61 (c, d). Data are presented as from three (a, c, d) or two (b) independent experiments, and statistical analysis was performed using 1- (b) or 2-way ANOVA (a, c, d).

Recombinant CTGF and Cyr61 were also used to assess cell migration. HaCaT monoculture was treated with 10 and 50 ng/ml recombinant CTGF and 100 ng/ml and 1 μg/ml of Cyr61. Treatment with 10 ng/ml CTGF and treatment with 50 ng/ml significantly accelerated cell migration (Figure 7(c)). Treatment with either 100 ng/ml or 1 μg/ml Cyr61 also accelerated cell migration at early time point (Figure 7(d)).

4. Discussion

During the past few years, plasma medicine has emerged as an important field in biomedical science. Cold atmospheric plasma generates a mixture of reactive oxygen and nitrogen species, including singlet oxygen, atomic oxygen, hydroxyl radicals, and hydrogen peroxide, as major (secondary) contributors. Further, especially long-living species can be generated upon contact with liquids [42, 43]. Various studies have focused on the anti-inflammatory, antimicrobial, antineoplastic, and wound healing promoting properties of CAP [13, 14, 44, 45]. In recent years, CAP has been used for the management of acute and chronic wounds in clinics; however, the mechanistic insight of CAP-mediated wound healing responses is still not investigated enough. Here, not only did we demonstrate that CAP enhances wound healing in vitro, but we also provided evidence regarding the CAP-mediated induction of differentially regulated genes and the paracrine effectors that promote wound healing via the HIPPO pathway.

Immortalized dermal keratinocytes and dermal fibroblasts were used for our present study, as these cells are primarily affected during skin/wound treatment. We also chose the in vitro keratinocyte-fibroblast coculture model for a portion of our studies, as this model provides the ability to examine the influence of fibroblasts on cocultured keratinocytes while omitting additional influences that are present in intact skin. For our experiments, we used the comparison of monocultured fibroblasts and keratinocytes on the one hand side with the coculture of both cell types, in order to identify possible paracrine interactions of those cells after treatment with CAP. These data are intended to support further research in chronic wound healing—mostly to compare data from diabetic patients. Therefore, it is important to understand how fibroblasts and keratinocytes influence each other to support wound healing after cold plasma treatment.

H₂O₂ is an important second messenger that is generated by CAP which triggers the release of growth factors during the wound healing process and has been shown to increase keratinocyte viability and migration in wound models [46]. Previous studies have revealed an increase in H₂O₂ in response to increasing CAP treatments [47]. Intracellular ROS levels also increase in keratinocytes after plasma treatment [48, 49]. High amounts of ROS/RNS species can affect human cells and lead to cell death and reduction in cell viability and proliferation [50]. Indeed, we observed that a 60 s CAP treatment significantly reduced cell viability in HaCaTs, GM Fbs, and cocultures within 3 hr. Cell viability in GM Fbs significantly decreased by greater than 50% after 24 hr, and based on this, we excluded the 60 s treatment from our subsequent experiments. However, short- and long-lived radicals also act as secondary messengers and contribute to wound healing processes [51]. Therefore, the aim of this study was to identify the stimulatory effects of CAP-generated reactive species on wound healing.

The epidermal layer functions as a barrier to the outside of the skin, and upon damage, newly divided keratinocytes migrate across the dermis [52]. Here, we optimized an in vitro coculture model that incorporated keratinocytes and fibroblasts. Unlike in single cell studies, this model for migration studies using coculture provides the opportunity to monitor the influence of each cell type on the other after CAP treatment. We observed that cells in coculture closely mimic real skin, and this system also displayed better CAP stimulation effects compared to those observed in the monocultures, indicating a crosstalk between the plasma-treated cell types. Many growth factors and cytokines are released from fibroblasts after injury, and this could provide an explanation for the acceleration of coculture wound closure [53–55].

To more thoroughly assess the paracrine signalling axis, we examined the signalling pathways involved in tissue homeostasis and regeneration. Emerging evidence suggests that the HIPPO pathway is regulated by a variety of signals, including mechanical stress, DNA damage, cellular stress, and oxidative stress [56]. Hence, we were interested in the HIPPO signalling pathway, as it plays an important role in cell growth, proliferation, and tissue homeostasis and is under redox control [25, 57]. YAP, a multifunctional adapter protein that possesses one coactivator domain, is a nuclear executer of the HIPPO signalling pathway. Based on our results, there was an increase in the mRNA expression of YAP in GM Fbs and HaCaT cells. We also detected a major upregulation of the YAP target genes CTGF and Cyr61 that was present primarily in GM Fbs and minimally in HaCaT cells. In accordance with this gene upregulation, CTGF and Cyr61 protein expression levels were also increased in GM Fbs. Upon phosphorylation of YAP, the HIPPO signalling cascade is blocked and phosphorylated YAP is retained in the cytoplasm. As YAP phosphorylation is increased after CAP treatment in HaCaT cells, this could provide a possible explanation for the minimal increase in CTGF protein and lack of Cyr61 detection in HaCaT cells; however, a significant decrease in YAP phosphorylation was not observed in GM Fbs. Therefore, it is likely that the interaction of cytoplasmic phosphorylated YAP with other signalling pathways (like Wnt, Notch, and TGF-β) can also contribute to the increased translation of CTGF and Cyr61 in GM Fbs [58–62]. Further studies are required to examine other upstream regulators that can induce CTGF and Cyr61 expressions.

The effect of NAC on restoring metabolic activity upon reduction of oxidative stress has been studied previously in other cell types [63]. In accordance with those results, in our immortalized skin cells, we also found that NAC restored and enhanced metabolic activity after reduction of CAP-mediated reactive species. This can be directly attributed to the ability of NAC to scavenge excess ROS within the media. CAP treatment can also reduce intracellular GSH levels in HaCaT cells as described previously by Dezest et al. [64]. As a cysteine-donating compound, NAC may act as a precursor to replenish the depleted cellular GSH upon CAP treatment, which in turn could induce the metabolic activity. Previous studies have also demonstrated the inhibitory effect of NAC on epithelial cell migration [65, 66]. Similar to these studies, we found that CAP-induced cell migration decreased by approximately 50% in response to 2.5 mM NAC treatment in HaCaT alone and in HaCaT cocultured with GM Fbs. Treatment with NAC reduced the mRNA expression of CTGF and Cyr61 in GM Fbs and Cyr61 in HaCaTs, and this confirms the effect of CAP in promoting the upregulation of this signalling pathway at the onset of wound healing. These findings are consistent with those of previous studies which observed that antioxidant treatment caused downregulation of CTGF and Cyr61 expressions in GO orbital fibroblasts and dermal primary fibroblasts, respectively [35, 36]. Therefore, we hypothesized that CAP could stimulate wound healing by triggering crosstalk between fibroblasts and keratinocytes that is mediated by HIPPO signalling.

Increasing numbers of studies are now focusing on the paracrine signalling and interaction between keratinocytes and fibroblasts in the context of wound repair [52, 67]. To test our hypothesis that CAP-induced stimulatory effects in coculture are a result of paracrine crosstalk, we monitored the cell migration of HaCaTs that were incubated with conditioned media harvested from CAP-treated GM Fbs (GM-Fb-CM) collected after 18 hr of CAP treatment. GM-Fb-CM was capable of accelerating HaCaT cell migration to a greater degree compared to that of CM harvested from untreated Fbs and no CM at all. This phenomenon supports our hypothesis that CAP induces paracrine signalling between these two cell types by releasing paracrine effectors and hence promoting keratinocyte migration. As expected, we could also detect an increase in secreted CTGF and Cyr61 expression in GM-Fb-CM treated with plasma. Additionally, HaCaT monocultures treated with recombinant CTGF and Cyr61 also exhibited improved cell migration. In particular, these data indicate that CTGF and Cyr61 secreted from CAP-treated fibroblasts act as paracrine effectors in wound healing. These results further confirm our hypothesis that CAP promotes a beneficial interaction between keratinocytes and fibroblasts that ultimately contributes to wound healing.

Here, for the first time, we demonstrated that CAP can stimulate a regenerative signalling pathway in dermal cells. We observed that the cold plasma-mediated effect on skin repair is mainly due to the activation of fibroblasts that when stimulated by CAP causes a paracrine stimulation of keratinocytes by releasing extracellular proteins as indicated by our coculture experiments. Together, these results confirm the role of the HIPPO pathway in plasma-mediated wound healing that is dominantly triggered by long-living ROS that are generated by cold plasma.

An overview of the impact of CAP treatment on the cellular redox signalling based on the present data is provided in Figure 8.

Figure 8

Schematic of keratinocyte activation by CAP modulated fibroblasts: the primary event in this scheme is the generation of reactive species in response to CAP treatment. These reactive species function as secondary messengers and stimulate the production of HIPPO signalling effectors such as CTGF in HaCaT and CTGF and Cyr61 by dermal fibroblasts in the vicinity. These extracellular matrix proteins that are released from fibroblasts in turn activate keratinocytes, accelerate migration, and promote wound healing.

5. Conclusions

CAP treatment results into complex crosstalk between redox signalling pathways in mammalian cells via the impact of short- and long-lived redox species. kINPen Med has already received approval for clinical applications in wound healing [12, 68]. Here, we revealed a mechanistic insight into wound healing where CAP induced a YAP-CTGF-Cyr61 signalling axis in dermal fibroblasts that was inhibited by antioxidant treatment. Although dermal keratinocytes could minimally produce HIPPO downstream effector CTGF, it is the secretion of CTGF and Cyr61 from fibroblasts that influences the activation of keratinocytes through paracrine signalling and improves cell migration upon wound onset.

Abbreviations

CAP:	Cold atmospheric plasma
CM:	Conditioned medium
CTGF:	Connective Tissue Growth Factor
Cyr61:	Cysteine-rich angiogenic protein 61
YAP:	Yes-associated protein 1
TEAD:	TEA domain family member
GM Fbs:	GM00637 fibroblasts
NAC:	N-Acetyl L cysteine
TGF-β:	Transforming growth factor β
RNS:	Reactive nitrogen species
ROS:	Reactive oxygen species.

Data Availability

The majority of the data can be found in the manuscript, and further data used to support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest

The authors declare no conflict of interest regarding the publication of this paper.

Acknowledgments

The authors acknowledge the technical assistance of Liane Kantz. This work was partly supported by TBI-V-1-234-VBW-081 from the Federal Government of Mecklenburg, Western Pomerania.

Supplementary Materials

Figure S1: summary of cell count in monoculture and coculture. Figure S2: forward and reverse primer sequences used in real-time PCR for YAP, Cyr61, and CTGF. Figure S3: Amplex Red assay was performed to quantitatively measure the production of H₂O₂ in response to CAP treatment. Figure S4: wound healing images at times 0, 12, and 24 hr in untreated and CAP-treated coculture. Figure S5: wound healing images at times 0, 12, and 24 hr in untreated and CAP-treated HaCaT cells. Figure S6: wound healing images at times 0, 12, and 24 hr in untreated and CAP-treated GM Fbs. (Supplementary Materials)

References

T. von Woedtke, A. Schmidt, S. Bekeschus, K. Wende, and K.-D. Weltmann, “Plasma medicine: a field of applied redox biology,” In Vivo, vol. 33, no. 4, pp. 1011–1026, 2019.
View at: Publisher Site | Google Scholar
L. E. Reynolds, F. J. Conti, M. Lucas et al., “Accelerated re-epithelialization in β3-integrin-deficient- mice is associated with enhanced TGF-β1 signaling,” Nature Medicine, vol. 11, no. 2, pp. 167–174, 2005.
View at: Publisher Site | Google Scholar
A. Schmidt, S. Dietrich, A. Steuer et al., “Non-thermal plasma activates human keratinocytes by stimulation of antioxidant and phase II pathways,” Journal of Biological Chemistry, vol. 290, no. 11, pp. 6731–6750, 2015.
View at: Publisher Site | Google Scholar
R. Melchionna, G. Bellavia, M. Romani et al., “C/EBPγ regulates wound repair and EGF receptor signaling,” Journal of Investigative Dermatology, vol. 132, no. 7, pp. 1908–1917, 2012.
View at: Publisher Site | Google Scholar
H. R. Rezvani, N. Ali, M. Serrano-Sanchez et al., “Loss of epidermal hypoxia-inducible factor-1α accelerates epidermal aging and affects re-epithelialization in human and mouse,” Journal of Cell Science, vol. 124, no. 24, pp. 4172–4183, 2011.
View at: Publisher Site | Google Scholar
C. Dunnill, T. Patton, J. Brennan et al., “Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process,” International Wound Journal, vol. 14, no. 1, pp. 89–96, 2017.
View at: Publisher Site | Google Scholar
L. Bundscherer, K. Wende, K. Ottmüller et al., “Impact of non-thermal plasma treatment on MAPK signaling pathways of human immune cell lines,” Immunobiology, vol. 218, no. 10, pp. 1248–1255, 2013.
View at: Publisher Site | Google Scholar
K. Wende, S. Straßenburg, B. Haertel et al., “Atmospheric pressure plasma jet treatment evokes transient oxidative stress in HaCaT keratinocytes and influences cell physiology,” Cell Biology International, vol. 38, no. 4, pp. 412–425, 2014.
View at: Publisher Site | Google Scholar
S. Bekeschus, A. Schmidt, L. Bethge et al., “Redox stimulation of human THP-1 monocytes in response to cold physical plasma,” Oxidative Medicine and Cellular Longevity, vol. 2016, Article ID 5910695, 11 pages, 2016.
View at: Publisher Site | Google Scholar
S. Arndt, M. Landthaler, J. L. Zimmermann et al., “Effects of cold atmospheric plasma (CAP) on ß-defensins, inflammatory cytokines, and apoptosis-related molecules in keratinocytes in vitro and in vivo,” PLoS One, vol. 10, no. 3, article e0120041, 2015.
View at: Publisher Site | Google Scholar
A. Kramer, S. Bekeschus, R. Matthes et al., “Cold physical plasmas in the field of hygiene—relevance, significance, and future applications,” Plasma Processes and Polymers, vol. 12, no. 12, pp. 1410–1422, 2015.
View at: Publisher Site | Google Scholar
A. Schmidt, S. Bekeschus, K. Wende, B. Vollmar, and T. von Woedtke, “A cold plasma jet accelerates wound healing in a murine model of full-thickness skin wounds,” Experimental Dermatology, vol. 26, no. 2, pp. 156–162, 2017.
View at: Publisher Site | Google Scholar
S. Arndt, A. Schmidt, S. Karrer, and T. von Woedtke, “Comparing two different plasma devices kINPen and Adtec SteriPlas regarding their molecular and cellular effects on wound healing,” Clinical Plasma Medicine, vol. 9, pp. 24–33, 2018.
View at: Publisher Site | Google Scholar
A. Schmidt, T. von Woedtke, B. Vollmar, S. Hasse, and S. Bekeschus, “Nrf2 signaling and inflammation are key events in physical plasma-spurred wound healing,” Theranostics, vol. 9, no. 4, pp. 1066–1084, 2019.
View at: Publisher Site | Google Scholar
G. Halder and R. L. Johnson, “Hippo signaling: growth control and beyond,” Development, vol. 138, no. 1, pp. 9–22, 2011.
View at: Publisher Site | Google Scholar
D. Pan, “The hippo signaling pathway in development and cancer,” Developmental Cell, vol. 19, no. 4, pp. 491–505, 2010.
View at: Publisher Site | Google Scholar
F. X. Yu and K. L. Guan, “The Hippo pathway: regulators and regulations,” Genes & Development, vol. 27, no. 4, pp. 355–371, 2013.
View at: Publisher Site | Google Scholar
W. Hong and K. L. Guan, “The YAP and TAZ transcription co-activators: key downstream effectors of the mammalian Hippo pathway,” Seminars in Cell & Developmental Biology, vol. 23, no. 7, pp. 785–793, 2012.
View at: Publisher Site | Google Scholar
M. J. Lee, M. R. Byun, M. Furutani-Seiki, J. H. Hong, and H. S. Jung, “YAP and TAZ regulate skin wound healing,” Journal of Investigative Dermatology, vol. 134, no. 2, pp. 518–525, 2014.
View at: Publisher Site | Google Scholar
X. Luo, Y. Liu, W. Feng et al., “NUP37, a positive regulator of YAP/TEAD signaling, promotes the progression of hepatocellular carcinoma,” Oncotarget, vol. 8, no. 58, pp. 98004–98013, 2017.
View at: Publisher Site | Google Scholar
E. R. Barry and F. D. Camargo, “The Hippo superhighway: signaling crossroads converging on the Hippo/Yap pathway in stem cells and development,” Current Opinion in Cell Biology, vol. 25, no. 2, pp. 247–253, 2013.
View at: Publisher Site | Google Scholar
D. P. Del Re, Y. Yang, N. Nakano et al., “Yes-associated protein isoform 1 (Yap1) promotes cardiomyocyte survival and growth to protect against myocardial ischemic injury,” Journal of Biological Chemistry, vol. 288, no. 6, pp. 3977–3988, 2013.
View at: Publisher Site | Google Scholar
B. Zhao, L. Li, Q. Lu et al., “Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein,” Genes & Development, vol. 25, no. 1, pp. 51–63, 2011.
View at: Publisher Site | Google Scholar
B. Zhao, X. Wei, W. Li et al., “Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control,” Genes & Development, vol. 21, no. 21, pp. 2747–2761, 2007.
View at: Publisher Site | Google Scholar
Y. Wang, A. Yu, and F. X. Yu, “The Hippo pathway in tissue homeostasis and regeneration,” Protein & Cell, vol. 8, no. 5, pp. 349–359, 2017.
View at: Publisher Site | Google Scholar
C. C. Chen, F. E. Mo, and L. F. Lau, “The angiogenic factor Cyr61 activates a genetic program for wound healing in human skin fibroblasts,” Journal of Biological Chemistry, vol. 276, no. 50, pp. 47329–47337, 2001.
View at: Publisher Site | Google Scholar
B. Zhao, X. Ye, J. Yu et al., “TEAD mediates YAP-dependent gene induction and growth control,” Genes & Development, vol. 22, no. 14, pp. 1962–1971, 2008.
View at: Publisher Site | Google Scholar
C.-C. Chen and L. F. Lau, “Functions and mechanisms of action of CCN matricellular proteins,” The International Journal of Biochemistry & Cell Biology, vol. 41, no. 4, pp. 771–783, 2009.
View at: Publisher Site | Google Scholar
W. H. Fan, M. Pech, and M. J. Karnovsky, “Connective tissue growth factor (CTGF) stimulates vascular smooth muscle cell growth and migration in vitro,” European Journal of Cell Biology, vol. 79, no. 12, pp. 915–923, 2000.
View at: Publisher Site | Google Scholar
Z. Yang, Z. Sun, H. Liu et al., “Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis,” Molecular Medicine Reports, vol. 12, no. 1, pp. 1091–1097, 2015.
View at: Publisher Site | Google Scholar
D. R. Brigstock, “Regulation of angiogenesis and endothelial cell function by connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61),” Angiogenesis, vol. 5, no. 3, pp. 153–165, 2002.
View at: Publisher Site | Google Scholar
M. P. Alfaro, D. L. Deskins, M. Wallus et al., “A physiological role for connective tissue growth factor in early wound healing,” Laboratory Investigation, vol. 93, no. 1, pp. 81–95, 2013.
View at: Publisher Site | Google Scholar
J. I. Jun and L. F. Lau, “The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing,” Nature Cell Biology, vol. 12, no. 7, pp. 676–685, 2010.
View at: Publisher Site | Google Scholar
J. M. Schober, L. F. Lau, T. P. Ugarova, and S. C.-T. Lam, “Identification of a novel integrin α_Mβ₂ binding site in CCN1 (CYR61), a matricellular protein expressed in healing wounds and atherosclerotic lesions,” Journal of Biological Chemistry, vol. 278, no. 28, pp. 25808–25815, 2003.
View at: Publisher Site | Google Scholar
C. C. Tsai, S. B. Wu, H. C. Kau, and Y. H. Wei, “Essential role of connective tissue growth factor (CTGF) in transforming growth factor-β1 (TGF-β1)-induced myofibroblast transdifferentiation from Graves' orbital fibroblasts,” Scientific Reports, vol. 8, no. 1, p. 7276, 2018.
View at: Publisher Site | Google Scholar
Z. Qin, P. Robichaud, T. He, G. J. Fisher, J. J. Voorhees, and T. Quan, “Oxidant exposure induces cysteine-rich protein 61 (CCN1) via c-Jun/AP-1 to reduce collagen expression in human dermal fibroblasts,” PLoS One, vol. 9, no. 12, article e115402, 2014.
View at: Publisher Site | Google Scholar
S. Bekeschus, A. Schmidt, K. D. Weltmann, and T. von Woedtke, “The plasma jet kINPen – A powerful tool for wound healing,” Clinical Plasma Medicine, vol. 4, no. 1, pp. 19–28, 2016.
View at: Publisher Site | Google Scholar
A. Schmidt, T. Woedtke, J. Stenzel et al., “One year follow-up risk assessment in SKH-1 mice and wounds treated with an argon plasma jet,” International Journal of Molecular Sciences, vol. 18, no. 4, p. 868, 2017.
View at: Publisher Site | Google Scholar
K. Wende, S. Bekeschus, A. Schmidt et al., “Risk assessment of a cold argon plasma jet in respect to its mutagenicity,” Mutation Research/Genetic Toxicology and Environmental Mutagenesis, vol. 798-799, pp. 48–54, 2016.
View at: Publisher Site | Google Scholar
K. J. Livak and T. D. Schmittgen, “Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔC_T method,” Methods, vol. 25, no. 4, pp. 402–408, 2001.
View at: Publisher Site | Google Scholar
K. Wende, P. Williams, J. Dalluge et al., “Identification of the biologically active liquid chemistry induced by a nonthermal atmospheric pressure plasma jet,” Biointerphases, vol. 10, no. 2, article 029518, 2015.
View at: Publisher Site | Google Scholar
P. J. Bruggeman, M. J. Kushner, B. R. Locke et al., “Plasma–liquid interactions: a review and roadmap,” Plasma Sources Science and Technology, vol. 25, no. 5, article 053002, 2016.
View at: Publisher Site | Google Scholar
K. Wende, T. von Woedtke, K. D. Weltmann, and S. Bekeschus, “Chemistry and biochemistry of cold physical plasma derived reactive species in liquids,” Biological Chemistry, vol. 400, no. 1, pp. 19–38, 2018.
View at: Publisher Site | Google Scholar
S. Arndt, E. Wacker, Y. F. Li et al., “Cold atmospheric plasma, a new strategy to induce senescence in melanoma cells,” Experimental Dermatology, vol. 22, no. 4, pp. 284–289, 2013.
View at: Publisher Site | Google Scholar
A. M. Hirst, F. M. Frame, M. Arya, N. J. Maitland, and D. O'Connell, “Low temperature plasmas as emerging cancer therapeutics: the state of play and thoughts for the future,” Tumour Biology, vol. 37, no. 6, pp. 7021–7031, 2016.
View at: Publisher Site | Google Scholar
A. E. Loo, Y. T. Wong, R. Ho et al., “Effects of hydrogen peroxide on wound healing in mice in relation to oxidative damage,” PLoS One, vol. 7, no. 11, article e49215, 2012.
View at: Publisher Site | Google Scholar
R. K. Gandhirajan, K. Rödder, Y. Bodnar et al., “Cytochrome C oxidase inhibition and cold plasma-derived oxidants synergize in melanoma cell death induction,” Scientific Reports, vol. 8, no. 1, article 12734, 2018.
View at: Publisher Site | Google Scholar
A. Schmidt, S. Bekeschus, K. Jarick, S. Hasse, T. von Woedtke, and K. Wende, “Cold physical plasma modulates p53 and mitogen-activated protein kinase signaling in keratinocytes,” Oxidative Medicine and Cellular Longevity, vol. 2019, Article ID 7017363, 16 pages, 2019.
View at: Publisher Site | Google Scholar
A. Schmidt, T. von Woedtke, and S. Bekeschus, “Periodic exposure of keratinocytes to cold physical plasma: an in vitro model for redox-related diseases of the skin,” Oxidative Medicine and Cellular Longevity, vol. 2016, Article ID 9816072, 17 pages, 2016.
View at: Publisher Site | Google Scholar
A. K. Kaushik, S. K. Vareed, S. Basu et al., “Metabolomic profiling identifies biochemical pathways associated with castration-resistant prostate cancer,” Journal of Proteome Research, vol. 13, no. 2, pp. 1088–1100, 2014.
View at: Publisher Site | Google Scholar
A. Soneja, M. Drews, and T. Malinski, “Role of nitric oxide, nitroxidative and oxidative stress in wound healing,” Pharmacological Reports, vol. 57, pp. 108–119, 2005.
View at: Google Scholar
G. Seo, C. Hyun, D. Koh et al., “A novel synthetic material, BMM, accelerates wound repair by stimulating re-epithelialization and fibroblast activation,” International Journal of Molecular Sciences, vol. 19, no. 4, p. 1164, 2018.
View at: Publisher Site | Google Scholar
N. Maas-Szabowski, H. J. Stark, and N. E. Fusenig, “Keratinocyte growth regulation in defined organotypic cultures through IL-1-induced keratinocyte growth factor expression in resting fibroblasts,” Journal of Investigative Dermatology, vol. 114, no. 6, pp. 1075–1084, 2000.
View at: Publisher Site | Google Scholar
N. Maas-Szabowski, A. Szabowski, H. J. Stark et al., “Organotypic cocultures with genetically modified mouse fibroblasts as a tool to dissect molecular mechanisms regulating keratinocyte growth and differentiation,” Journal of Investigative Dermatology, vol. 116, no. 5, pp. 816–820, 2001.
View at: Publisher Site | Google Scholar
Z. Wang, Y. Wang, F. Farhangfar, M. Zimmer, and Y. Zhang, “Enhanced keratinocyte proliferation and migration in co-culture with fibroblasts,” PLoS One, vol. 7, no. 7, article e40951, 2012.
View at: Publisher Site | Google Scholar
B. Mao, Y. Gao, Y. Bai, and Z. Yuan, “Hippo signaling in stress response and homeostasis maintenance,” Acta Biochimica et Biophysica Sinica, vol. 47, no. 1, pp. 2–9, 2015.
View at: Publisher Site | Google Scholar
D. Shao, P. Zhai, D. P. del Re et al., “A functional interaction between Hippo-YAP signalling and FoxO1 mediates the oxidative stress response,” Nature Communications, vol. 5, no. 1, p. 3315, 2014.
View at: Publisher Site | Google Scholar
O. Ferrigno, F. Lallemand, F. Verrecchia et al., “Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-β/Smad signaling,” Oncogene, vol. 21, no. 32, pp. 4879–4884, 2002.
View at: Publisher Site | Google Scholar
W. M. Konsavage and G. S. Yochum, “Intersection of Hippo/YAP and Wnt/β-catenin signaling pathways,” Acta Biochimica et Biophysica Sinica, vol. 45, no. 2, pp. 71–79, 2013.
View at: Publisher Site | Google Scholar
J. Tao, D. F. Calvisi, S. Ranganathan et al., “Activation of β-catenin and Yap1 in human hepatoblastoma and induction of hepatocarcinogenesis in mice,” Gastroenterology, vol. 147, no. 3, pp. 690–701, 2014.
View at: Publisher Site | Google Scholar
D. F. Tschaharganeh, X. Chen, P. Latzko et al., “Yes-associated protein up-regulates jagged-1 and activates the NOTCH pathway in human hepatocellular carcinoma,” Gastroenterology, vol. 144, no. 7, pp. 1530–1542.e12, 2013.
View at: Publisher Site | Google Scholar
X. Varelas, P. Samavarchi-Tehrani, M. Narimatsu et al., “The Crumbs complex couples cell density sensing to Hippo-dependent control of the TGF-β-SMAD pathway,” Developmental Cell, vol. 19, no. 6, pp. 831–844, 2010.
View at: Publisher Site | Google Scholar
Y. Jiao, S. Ma, Y. Wang et al., “N-Acetyl cysteine depletes reactive oxygen species and prevents dental monomer-induced intrinsic mitochondrial apoptosis in vitro in human dental pulp cells,” PLoS One, vol. 11, no. 1, article e0147858, 2016.
View at: Publisher Site | Google Scholar
M. Dezest, L. Chavatte, M. Bourdens et al., “Mechanistic insights into the impact of cold atmospheric pressure plasma on human epithelial cell lines,” Scientific Reports, vol. 7, no. 1, article 41163, 2017.
View at: Publisher Site | Google Scholar
Y. Huo, W. Y. Qiu, Q. Pan, Y. F. Yao, K. Xing, and M. F. Lou, “Reactive oxygen species (ROS) are essential mediators in epidermal growth factor (EGF)-stimulated corneal epithelial cell proliferation, adhesion, migration, and wound healing,” Experimental Eye Research, vol. 89, no. 6, pp. 876–886, 2009.
View at: Publisher Site | Google Scholar
T. Ramaesh, K. Ramaesh, S. C. Riley, J. D. West, and B. Dhillon, “Effects of N-acetylcysteine on matrix metalloproteinase-9 secretion and cell migration of human corneal epithelial cells,” Eye, vol. 26, no. 8, pp. 1138–1144, 2012.
View at: Publisher Site | Google Scholar
G. Y. Seo, Y. Lim, D. Koh et al., “TMF and glycitin act synergistically on keratinocytes and fibroblasts to promote wound healing and anti-scarring activity,” Experimental & Molecular Medicine, vol. 49, no. 3, article e302, 2017.
View at: Publisher Site | Google Scholar
K.-D. Weltmann and T. von Woedtke, “Plasma medicine—current state of research and medical application,” Plasma Physics and Controlled Fusion, vol. 59, no. 1, article 014031, 2017.
View at: Publisher Site | Google Scholar

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