Search Results
Influence of redox stress on crosstalk between fibroblasts and keratinocytes
2021, Bhartiya, Pradeep, Masur, Kai, Shome, Debarati, Kaushik, Neha, Nguyen, Linh N., Kaushik, Nagendra Kumar, Choi, Eun Ha
Although the skin is constantly subjected to endogenous and exogenous stress, it maintains a homeostatic state through wound repair and regeneration pathways. Treatment for skin diseases and injury requires a significant understanding of the various mechanisms and interactions that occur within skin cells. Keratinocytes and fibroblasts interact with each other and act as key players in the repair process. Although fibroblasts and keratinocytes are widely studied in wound healing and skin remodeling under different conditions, the influence of redox stress on keratinocyte-fibroblast crosstalk has not been thoroughly investigated. In this study, we used cold atmospheric plasma (CAP) to generate and deliver oxidative stress to keratinocytes and fibroblasts and to assess its impact on their interactions. To this end, we used a well-established in vitro 3D co-culture model imitating a realistic scenario. Our study shows that low CAP exposure is biocompatible and does not affect the viability or energetics of fibroblasts and keratinocytes. Exposure to low doses of CAP enhanced the proliferation rate of cells and stimulated the expression of key genes (KGF, MMP2, GMCSF, IL-6, and IL-8) in fibroblasts, indicating the activation and initiation of the skin repair process. Additionally, enhanced migration was observed under co-culture conditions under the given redox stress conditions, and expression of the upstream regulator and the effectors of the Hippo pathway (YAP and CYR61, respectively), which are associated with enhanced migration, were elevated. Overall, this study reinforces the application of CAP and redox stress in skin repair physiology.
Neutrophil extracellular trap formation is elicited in response to cold physical plasma
2016, Bekeschus, Sander, Winterbourn, VChristine C., Kolata, Julia, Masur, Kai, Hasse, Sybille, Bröker, Barbara M., Parker, Heather A.
Cold physical plasma is an ionized gas with a multitude of components, including hydrogen peroxide and other reactive oxygen and nitrogen species. Recent studies suggest that exposure of wounds to cold plasma may accelerate healing. Upon wounding, neutrophils are the first line of defense against invading microorganisms but have also been identified to play a role in delayed healing. In this study, we examined how plasma treatment affects the functions of peripheral blood neutrophils. Plasma treatment induced oxidative stress, as assessed by the oxidation of intracellular fluorescent redox probes; reduced metabolic activity; but did not induce early apoptosis. Neutrophil oxidative burst was only modestly affected after plasma treatment, and the killing of Pseudomonas aeruginosa and Staphylococcus aureus was not significantly affected. Intriguingly, we found that plasma induced profound extracellular trap formation. This was inhibited by the presence of catalase during plasma treatment but was not replicated by adding an equivalent concentration of hydrogen peroxide. Plasma-induced neutrophil extracellular trap formation was not dependent on the activity of myeloperoxidase or NADPH oxidase 2 but seemed to involve short-lived molecules. The amount of DNA release and the time course after plasma treatment were similar to that with the common neutrophil extracellular trap inducer PMA. After neutrophil extracellular traps had formed, concentrations of IL-8 were also significantly increased in supernatants of plasma-treated neutrophils. Both neutrophil extracellular traps and IL-8 release may aid antimicrobial activity and spur inflammation at the wound site. Whether this aids or exacerbates wound healing needs to be tested.
Effect of Cold Atmospheric Plasma Therapy vs Standard Therapy Placebo on Wound Healing in Patients With Diabetic Foot Ulcers: A Randomized Clinical Trial
2020, Stratmann, Bernd, Costea, Tania-Cristina, Nolte, Catharina, Hiller, Jonas, Schmidt, Jörn, Reindel, Jörg, Masur, Kai, Motz, Wolfgang, Timm, Jürgen, Kerner, Wolfgang, Tschoepe, Diethelm
Importance: Diabetic foot ulcers are a common complication of diabetes and require specialized treatment. Cold atmospheric plasma (CAP) has been associated with benefits in wound infection and healing in previous smaller series of case reports. Yet the effect of CAP compared with standard care therapy in wound healing in diabetic foot ulcers remains to be studied. Objective: To determine whether the application of CAP accelerates wound healing in diabetic foot ulcers compared with standard care therapy. Design, Setting, and Participants: A prospective, randomized, placebo-controlled, patient-blinded clinical trial was conducted at 2 clinics with recruitment from August 17, 2016, to April 20, 2019. Patients were scheduled to remain in follow-up until April 30, 2024. Patients with diabetes and diabetic foot ulcers described using the combined Wagner-Armstrong classification of 1B or 2B (superficial or infected diabetic foot ulcers extending to tendon) were eligible. A patient could participate with 1 or more wounds in both groups in both intervention and control groups. Wounds were randomized separately, allowing a participant to be treated several times within the study following a 2 × 2 × 2 randomization strata considering sex, smoking status, and age (≤68 years and >68 years). Interventions: Standard care treatment with 8 applications of either CAP generated from argon gas in an atmospheric pressure plasma jet or 8 applications of placebo treatment in a patient-blinded manner. Main Outcomes and Measures: Primary end points were reduction in wound size, clinical infection, and microbial load compared with treatment start. Secondary end points were time to relevant wound reduction (>10%), reduction of infection, parameters of patient’s well-being, and treatment-associated adverse events. Results: Of 65 diabetic foot ulcer wounds from 45 patients assessed for study, 33 wounds from 29 patients were randomized to CAP and 32 wounds from 28 to placebo, with 62 wounds from 43 patients (31 wounds per group) included for final evaluation (mean [SD] age, 68.5 [9.1] years for full sample). Four patients with 5 wounds of 31 (16.1%) wounds in the CAP group and 3 patients with 4 wounds of 31 (13%) wounds in the placebo group were active smokers. CAP therapy yielded a significant increase in wound healing, both in total mean (SD) area reduction (CAP vs placebo relative units, −26.31 [11.72]; P = .03) and mean (SD) time to relevant wound area reduction (CAP vs placebo relative units, 10% from baseline, 1.60 [0.58]; P = .009). Reduction of infection and microbial load was not significantly different between CAP and placebo. No therapy-related adverse events occurred during therapy; patient’s perceptions during therapy were comparable. Conclusions and Relevance: In this randomized clinical trial, CAP therapy resulted in beneficial effects in chronic wound treatment in terms of wound surface reduction and time to wound closure independent from background infection.
Non-thermal plasma treatment induces MAPK signaling in human monocytes
2014, Bundscherer, Lena, Nagel, Stefanie, Hasse, Sybille, Tresp, Helena, Wende, Kristian, Walther, Reinhard, Reuter, Stephan, Weltmann, Klaus-Dieter, Masur, Kai, Lindequist, Ulrike
The application of non-thermal atmospheric pressure plasma raises a hope for the new wound healing strategies. Next to its antibacterial effect it is known to stimulate skin cells. However, monocytes are also needed for the complex process of a wound healing. This study investigates the impact of plasma on the intracellular signaling events in the primary human monocytes. The proliferative MEK-ERK (MAPK/ERK kinase-extracellular signal-regulated kinase) pathway was activated by short plasma treatment times. In contrast, an induction of the apoptotic JNK (c-Jun N-terminal kinase) cascade as well as activation of caspase 3 were observed after long plasma exposure. These findings indicate that monocytes can be differentially stimulated by plasma treatment and may contribute to the proper wound recovery.
Detecting Bacteria on Wounds with Hyperspectral Imaging in Fluorescence Mode
2020, Hornberger, Christoph., Herrmann, Bert. H., Daeschlein, Georg, Podewils, Sebastian von, Sicher, Claudia, Kuhn, Jana, Masur, Kai, Meister, Mareike, Wahl, Philip
Chronic non-healing wounds represent an increasing problem. In order to enable physicians and nurses to make evidence based decisions on wound treatment, the professional societies call for supporting tools to be offered to physicians. Oxygen supply, bacteria colonization and other parameters influence the healing process. So far, these parameters cannot be monitored in an objective and routinely manner. Existing methods like the microbiological analysis of wound swabs, mean a great deal of effort and partly a long delay. In this paper 42 fluorescence images from 42 patients with diabetic foot ulcer, recorded with a hyperspectral imaging system (TIVITA®), converted for fluorescence imaging, were analysed. Beside the fluorescence images, information about the bacterial colonization is available from microbiological analysis of wound swabs. After preprocessing, principal component analysis, PCA, is used for data analysis with a 405 nm excitation wavelength, the emission wavelength range 510 - 745 nm is used for analysis. After dividing the data into a training and a test dataset it could be shown, that bacteria are detectable in the wound area. A quantification in bacterial colonization counts (BCC) was not in the focus of the research in this study stage.
Non-thermal plasma activates human keratinocytes by stimulation of antioxidant and phase II pathways
2015, Schmidt, Anke, Dietrich, Stephan, Steuer, Anna, Weltmann, Klaus-Dieter, von Woedtke, Thomas, Masur, Kai, Wende, Kristian
Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application.
Uv absorption spectroscopy for the diffusion of plasma-generated reactive species through a skin model
2021, Ki, Se Hoon, Masur, Kai, Baik, Ku Youn, Choi, Eun Ha
Skin applications of non-thermal atmospheric pressure plasma (NTAPP) have been at-tracting attention from medical and cosmetic aspects. The reactive species generated from plasma sources have been known to play important roles in the skin. For proper applications, it is essential to know how they diffuse into the skin. In this study, the penetration of active species from NTAPP through a skin model was analyzed by UV absorption spectroscopy. The diffusions of hydrogen peroxide, nitrite, and nitrate were quantified through curve fitting. We utilized an agarose gel to mimic epidermis and dermis layers, and we used a lipid film or a pig skin sample to mimic the stratum corneum (SC). The diffusion characteristics of reactive species through this skin model and the limitations of this method were discussed.
Redox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma
2015, Bekeschus, Sander, Schmidt, Anke, Bethge, Lydia, Masur, Kai, von Woedtke, Thomas, Hasse, Sybille, Wende, Kristian
In plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α), differentiation (retinoic acid signaling and interferon inducible factors), and cell growth (Yin Yang 1). Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1) and of the neutrophil attractant chemokine interleukin-8 (IL-8). Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.
Periodic Exposure of Plasma-Activated Medium Alters Fibroblast Cellular Homoeostasis
2022, Bhartiya, Pradeep, Kaushik, Neha, Nguyen, Linh N., Bekeschus, Sander, Masur, Kai, Weltmann, Klaus-Dieter, Kaushik, Nagendra Kumar, Choi, Eun Ha
Excess amounts of redox stress and failure to regulate homeostatic levels of reactive species are associated with several skin pathophysiologic conditions. Nonmalignant cells are assumed to cope better with higher reactive oxygen and nitrogen species (RONS) levels. However, the effect of periodic stress on this balance has not been investigated in fibroblasts in the field of plasma medicine. In this study, we aimed to investigate intrinsic changes with respect to cellular proliferation, cell cycle, and ability to neutralize the redox stress inside fibroblast cells following periodic redox stress in vitro. Soft jet plasma with air as feeding gas was used to generate plasma-activated medium (PAM) for inducing redox stress conditions. We assessed cellular viability, energetics, and cell cycle machinery under oxidative stress conditions at weeks 3, 6, 9, and 12. Fibroblasts retained their usual physiological properties until 6 weeks. Fibroblasts failed to overcome the redox stress induced by periodic PAM exposure after 6 weeks, indicating its threshold potential. Periodic stress above the threshold level led to alterations in fibroblast cellular processes. These include consistent increases in apoptosis, while RONS accumulation and cell cycle arrest were observed at the final stages. Currently, the use of NTP in clinical settings is limited due to a lack of knowledge about fibroblasts’ behavior in wound healing, scar formation, and other fibrotic disorders. Understanding fibroblasts’ physiology could help to utilize nonthermal plasma in redox-related skin diseases. Furthermore, these results provide new information about the threshold capacity of fibroblasts and an insight into the adaptation mechanism against periodic oxidative stress conditions in fibroblasts.
A New CYP2E1 Inhibitor, 12-Imidazolyl-1-dodecanol, Represents a Potential Treatment for Hepatocellular Carcinoma
2021, Diesinger, Torsten, Lautwein, Alfred, Bergler, Sebastian, Buckert, Dominik, Renz, Christian, Dvorsky, Radovan, Buko, Vyacheslav, Kirko, Siarhei, Schneider, Edith, Kuchenbauer, Florian, Kumar, Mukesh, Günes, Cagatay, Genze, Felicitas, Büchele, Berthold, Simmet, Thomas, Haslbeck, Martin, Masur, Kai, Barth, Thomas, Müller-Enoch, Dieter, Wirth, Thomas, Haehner, Thomas, Granito, Alessandro
Cytochrome P450 2E1 (CYP2E1) is a key target protein in the development of alcoholic and nonalcoholic fatty liver disease (FLD). The pathophysiological correlate is the massive production of reactive oxygen species. The role of CYP2E1 in the development of hepatocellular carcinoma (HCC), the final complication of FLD, remains controversial. Specifically, CYP2E1 has not yet been defined as a molecular target for HCC therapy. In addition, a CYP2E1-specific drug has not been developed. We have already shown that our newly developed CYP2E1 inhibitor 12-imidazolyl-1-dodecanol (I-ol) was therapeutically effective against alcoholic and nonalcoholic steatohepatitis. In this study, we investigated the effect of I-ol on HCC tumorigenesis and whether I-ol could serve as a possible treatment option for terminal-stage FLD. I-ol exerted a very highly significant antitumour effect against hepatocellular HepG2 cells. Cell viability was reduced in a dose-dependent manner, with only the highest doses causing a cytotoxic effect associated with caspase 3/7 activation. Comparable results were obtained for the model colorectal adenocarcinoma cell line, DLD-1, whose tumorigenesis is also associated with CYP2E1. Transcriptome analyses showed a clear effect of I-ol on apoptosis and cell-cycle regulation, with the increased expression of p27Kip1 being particularly noticeable. These observations were confirmed at the protein level for HepG2 and DLD-1 cells grafted on a chorioallantoic membrane. Cell-cycle analysis showed a complete loss of proliferating cells with a simultaneous increase in S-phase arrest beginning at a threshold dose of 30 μM. I-ol also reduced xenograft tumour growth in nude mice. This antitumour effect was not associated with tumour cachexia. I-ol was not toxic to healthy tissues or organs. This study demonstrates for the first time the therapeutic effect of the specific CYP2E1 inhibitor I-ol on the tumorigenesis of HCC. Our findings imply that I-ol can potentially be applied therapeutically on patients at the final stage of FLD. © 2021 Torsten Diesinger et al.