Cold Atmospheric Plasma (CAP) Changes Gene Expression of Key Molecules of the Wound Healing Machinery and Improves Wound Healing In Vitro and In Vivo

Cold atmospheric plasma (CAP) has the potential to interact with tissue or cells leading to fast, painless and efficient disinfection and furthermore has positive effects on wound healing and tissue regeneration. For clinical implementation it is necessary to examine how CAP improves wound healing and which molecular changes occur after the CAP treatment. In the present study we used the second generation MicroPlaSter ß® in analogy to the current clinical standard (2 min treatment time) in order to determine molecular changes induced by CAP using in vitro cell culture studies with human fibroblasts and an in vivo mouse skin wound healing model. Our in vitro analysis revealed that the CAP treatment induces the expression of important key genes crucial for the wound healing response like IL-6, IL-8, MCP-1, TGF-ß1, TGF-ß2, and promotes the production of collagen type I and alpha-SMA. Scratch wound healing assays showed improved cell migration, whereas cell proliferation analyzed by XTT method, and the apoptotic machinery analyzed by protein array technology, was not altered by CAP in dermal fibroblasts. An in vivo wound healing model confirmed that the CAP treatment affects above mentioned genes involved in wound healing, tissue injury and repair. Additionally, we observed that the CAP treatment improves wound healing in mice, no relevant side effects were detected. We suggest that improved wound healing might be due to the activation of a specified panel of cytokines and growth factors by CAP. In summary, our in vitro human and in vivo animal data suggest that the 2 min treatment with the MicroPlaSter ß® is an effective technique for activating wound healing relevant molecules in dermal fibroblasts leading to improved wound healing, whereas the mechanisms which contribute to these observed effects have to be further investigated.     

等离子体医学及其在肿瘤治疗中的应用

大气压冷等离子体是近年来学术界兴起的新研究领域,由于其在大气压下产生,气体温度低、粒子活性高,在众多领域尤其是生物医学方面的应用引起了人们广泛的关注.等离子体医学是一个革新的、新兴的交叉学科研究领域,结合了等离子体物理学、化学、生命科学和临床医学等.本文首先介绍了大气压冷等离子体的产生及其粒子成分,与液体和生物组织的相互作用,并介绍了大气压冷等离子体在生物医学领域的一些主要应用,如杀菌消毒、凝血、牙科应用、伤口愈合及皮肤病治疗等方面.同时,重点介绍大气压冷等离子体在肿瘤治疗方面的研究进展.大气压冷等离子体可有效诱导肿瘤细胞死亡、抑制增殖及迁徙、诱导肿瘤细胞分化并抑制干细胞潜能,同时能提高化疗药物敏感性,在肿瘤治疗领域具有很好的应用前景.     

Programmed cell death is required for palate shelf fusion and is regulated by retinoic acid

Efficient wound healing including clotting and subsequent reepithelization is essential for animals ranging from insects to mammals to recover from epithelial injury. It is likely that genes involved in wound healing are conserved through the phylogeny and therefore, Drosophila may be an useful in vivo model system to identify genes necessary during this process. Furthermore, epithelial movement during specific developmental processes, such as dorsal closure, ressembles of those seen in mammalian wound healing. As puckered (puc) gene is a target of the JUN N-terminal kinase signaling pathway during dorsal closure, we investigated puc gene expression during wound healing in Drosophila. We showed that puc gene expression is induced at the edge of the wound in epithelial cells and Jun kinase is phosphorylated in wounded epidermal tissues, suggesting that the JUN N-terminal kinase signaling pathway is activated by a signal produced by an epidermal wound. In the absence of the Drosophila c-Fos homologue, puc gene expression is no longer induced. Finally, impaired epithelial repair in JUN N-terminal kinase deficient flies demonstrates that the JUN N-terminal kinase signaling is required to initiate the cell shape change at the onset of the epithelial wound healing. We conclude that the embryonic JUN N-terminal kinase gene cassette is induced at the edge of the wound. In addition, Drosophila appears as a good in vivo model to study morphogenetic processes requiring epithelial regeneration such as wound healing in vertebrates.     

Over-expression of ΔNp63α facilitates rat corneal wound healing in vivo

To investigate the roles of ΔNp63α during corneal wound healing and the genes regulated by ΔNp63α in limbal epithelial cells. Adenovirus or shRNA targeting ΔNp63α were pre-injected into the anterior chamber of rat eyeballs and the central corneal epithelium was then wounded with NaOH. The effects of ΔNp63α expression during wound healing were observed by propidium iodide staining. In addition, limbal epithelial cells were cultured and ectopically expressed ΔNp63α by transfecting Ad-ΔNp63α. Total RNA was extracted from transfected epithelial cells and subjected to a gene expression microarray assay. The results showed that over-expression of ΔNp63α accelerated the process of corneal wound healing while knockdown of ΔNp63α impaired the process. ΔNp63α positively up-regulated several cell growth promoter genes and could be referred as a positive regulator of limbal epithelial cell proliferation. It might also inhibit cell differentiation and cell death by differential target gene regulation.     

ENaC contribution to epithelial wound healing is independent of the healing mode and of any increased expression in the channel

Previous work from our laboratory and others has shown that, in some epithelia, the epithelial sodium channel (ENaC) increases its expression during wound healing. In these cases, inhibition of the channel determines a decrease in the healing rate, a result suggesting a role for ENaC in the overall healing process. To understand further this role of ENaC in epithelia, we explored the participation of ENaC in wound healing in four cultured epithelial cell lines selected on the basis of their different embryonic origins, function and modality of healing, i.e., by lamellipodial cell crawling or by actin cable formation. Three of the cell lines (bovine corneal endothelial cells, rabbit corneal epithelial cells and Madin-Darby canine kidney cells) exhibited an increase in ENaC expression and consequent membrane potential depolarization and an increase in cytosolic sodium and calcium, whereas one line (bovine aortal endothelial cells, BAEC) did not exhibit any of these changes. In all of the cell lines, however, ENaC inhibition determined a similar decrease in the rate of wound healing. In BAEC monolayers, the increase in ENaC activity produced plasma membrane depolarization, increased cytosolic sodium and calcium, and augmented the velocity of healing. These novel findings contribute to the idea that ENaC plays a critical role in wound healing in various epithelia, independently of the modality of healing and of any increase in the expression of the channel.     

Bmx tyrosine kinase transgene induces skin hyperplasia, inflammatory angiogenesis, and accelerated wound healing

The Bmx gene, a member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in arterial endothelium and in certain hematopoietic and epithelial cells. Previous in vitro studies have implicated Bmx signaling in cell migration and survival and suggested that it contributes to the progression of prostate carcinomas. However, the function of Bmx in normal tissues in vivo is unknown. We show here that Bmx expression is induced in skin keratinocytes during wound healing. To analyze the role of Bmx in epidermal keratinocytes in vivo, we generated transgenic mice overexpressing Bmx in the skin. We show that Bmx overexpression accelerates keratinocyte proliferation and wound reepithelialization. Bmx expression also induces chronic inflammation and angiogenesis in the skin, and gene expression profiling suggests that this occurs via cytokine-mediated recruitment of inflammatory cells. Our studies provide the first data on Bmx function in vivo and form the basis of evaluation of its role in epithelial neoplasia.     

Quantitative Stain-Free and Continuous Multimodal Monitoring of Wound Healing In Vitro with Digital Holographic Microscopy

Impaired epithelial wound healing has significant pathophysiological implications in several conditions including gastrointestinal ulcers, anastomotic leakage and venous or diabetic skin ulcers. Promising drug candidates for accelerating wound closure are commonly evaluated in in vitro wound assays. However, staining procedures and discontinuous monitoring are major drawbacks hampering accurate assessment of wound assays. We therefore investigated digital holographic microscopy (DHM) to appropriately monitor wound healing in vitro and secondly, to provide multimodal quantitative information on morphological and functional cell alterations as well as on motility changes upon cytokine stimulation. Wound closure as reflected by proliferation and migration of Caco-2 cells in wound healing assays was studied and assessed in time-lapse series for 40 h in the presence of stimulating epidermal growth factor (EGF) and inhibiting mitomycin c. Therefore, digital holograms were recorded continuously every thirty minutes. Morphological changes including cell thickness, dry mass and tissue density were analyzed by data from quantitative digital holographic phase microscopy. Stimulation of Caco-2 cells with EGF or mitomycin c resulted in significant morphological changes during wound healing compared to control cells. In conclusion, DHM allows accurate, stain-free and continuous multimodal quantitative monitoring of wound healing in vitro and could be a promising new technique for assessment of wound healing.     

Gene expression analysis in burn wounds of rats

The events occurring early in the burn wound trigger a sequence of local and systemic responses that influence cell and tissue survival and, consequently, wound healing and recovery. Using high-density oligonucleotide arrays we identified gene expression patterns in skin samples taken from a region of injury in the burn rat model. The associated genomic events include the differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation, and immune response. The functional gene cluster detected and their time appearance matched the time sequence known to occur in burn wound healing.     

Stanniocalcin-1 is induced by hypoxia inducible factor in rat alveolar epithelial cells

Alveolar type II (ATII) cells remain differentiated and express surfactant proteins when cultured at an air–liquid (A/L) interface. When cultured under submerged conditions, ATII cells dedifferentiate and change their gene expression profile. We have previously shown that gene expression under submerged conditions is regulated by hypoxia inducible factor (HIF) signaling due to focal hypoxia resulting from ATII cell metabolism. Herein, we sought to further define gene expression changes in ATII cells cultured under submerged conditions. We performed a genome wide microarray on RNA extracted from rat ATII cells cultured under submerged conditions for 24–48 h after switching from an A/L interface. We found significant alterations in gene expression, including upregulation of the HIF target genes stanniocalcin-1 (STC1), tyrosine hydroxylase (Th), enolase (Eno) 2, and matrix metalloproteinase (MMP) 13, and we verified upregulation of these genes by RT-PCR. Because STC1, a highly evolutionarily conserved glycoprotein with anti-inflammatory, anti-apoptotic, anti-oxidant, and wound healing properties, is widely expressed in the lung, we further explored the potential functions of STC1 in the alveolar epithelium. We found that STC1 was induced by hypoxia and HIF in rat ATII cells, and this induction occurred rapidly and reversibly. We also showed that recombinant human STC1 (rhSTC1) enhanced cell motility with extended lamellipodia formation in alveolar epithelial cell (AEC) monolayers but did not inhibit the oxidative damage induced by LPS. We also confirmed that STC1 was upregulated by hypoxia and HIF in human lung epithelial cells. In this study, we have found that several HIF target genes including STC1 are upregulated in AECs by a submerged condition, that STC1 is regulated by hypoxia and HIF, that this regulation is rapidly and reversibly, and that STC1 enhances wound healing moderately in AEC monolayers. However, STC1 did not inhibit oxidative damage in rat AECs stimulated by LPS in vitro. Therefore, alterations in gene expression by ATII cells under submerged conditions including STC1 were largely induced by hypoxia and HIF, which may be relevant to our understanding of the pathogenesis of various lung diseases in which the alveolar epithelium is exposed to relative hypoxia.     

Select de novo Gene and Protein Expression During Renal Epithelial Cell Culture in Rotating Wall Vessels is Shear Stress Dependent

The rotating wall vessel has gained popularity as a clinical cell culture tool to produce hormonal implants. It is desirable to understand the mechanisms by which the rotating wall vessel induces genetic changes, if we are to prolong the useful life of implants. During rotating wall vessel culture gravity is balanced by equal and opposite hydrodynamic forces including shear stress. The current study provides the first evidence that shear stress response elements, which modulate gene expression in endothelial cells, are also active in epithelial cells. Rotating wall culture of renal cells changes expression of select gene products including the giant glycoprotein scavenger receptors cubulin and megalin, the structural microvillar protein villin, and classic shear stress response genes ICAM, VCAM and MnSOD. Using a putative endothelial cell shear stress response element binding site as a decoy, we demonstrate the role of this sequence in the regulation of selected genes in epithelial cells. However, many of the changes observed in the rotating wall vessel are independent of this response element. It remains to define other genetic response elements modulated during rotating wall vessel culture, including the role of hemodynamics characterized by 3-dimensionality, low shear and turbulence, and cospatial relation of dissimilar cell types. Received: 30 June 1998/Revised: 30 November 1998     

Sequential cultivation of human epidermal keratinocytes and dermal mesenchymal like stromal cells in vitro

Human skin has continuous self-renewal potential throughout adult life and serves as first line of defence. Its cellular components such as human epidermal keratinocytes (HEKs) and dermal mesenchymal stromal cells (DMSCs) are valuable resources for wound healing applications and cell based therapies. Here we show a simple, scalable and cost-effective method for sequential isolation and propagation of HEKs and DMSCs under defined culture conditions. Human skin biopsy samples obtained surgically were cut into fine pieces and cultured employing explant technique. Plated skin samples attached and showed outgrowth of HEKs. Gross microscopic examination displayed polygonal cells with a granular cytoplasm and H&E staining revealed archetypal HEK morphology. RT–PCR and immunocytochemistry authenticated the presence of key HEK markers including trans-membrane protein epithelial cadherin (E-cadherin), keratins and cytokeratin. After collection of HEKs by trypsin–EDTA treatment, mother explants were left intact and cultured further. Interestingly, we observed the appearance of another cell type with fibroblastic or stromal morphology which were able to grow up to 15 passages in vitro. Growth pattern, expression of cytoskeletal protein vimentin, surface proteins such as CD44, CD73, CD90, CD166 and mesodermal differentiation potential into osteocytes, adipocytes and chondrocytes confirmed their bonafide mesenchymal stem cell like status. These findings albeit preliminary may open up significant opportunities for novel applications in wound healing.     

Surface molecules on HaCaT keratinocytes after interaction with non‐thermal atmospheric pressure plasma

Non‐thermal atmospheric‐pressure plasmas have been developed that will be used in future for several purposes, e.g. medicine. Living tissues and cells are at the focus of plasma treatment, e.g. to improve wound healing, or induce apoptosis and growth arrest in tumour cells. Detailed investigations of plasma‐cell interactions are needed. Cell surface adhesion molecules as integrins, cadherins or the EGFR (epidermal growth factor receptor) are of importance in wound healing and also for development of cancer metastasis. This study has focused on measurement of cell surface molecules on human HaCaT keratinocytes (human adult low calcium temperature keratinocytes) promoting adhesion, migration and proliferation as one important feature of plasma‐cell interactions. HaCaT keratinocytes were treated with plasma by a surface dielectric barrier discharge in air. Cell surface molecules and induction of intracellular ROS (reactive oxygen species) were analysed by flow cytometry 24 h after plasma treatment. Besides a reduction of cell viability a significant down‐regulation of E‐cadherin and the EGFR expression occurred. The influence on α₂‐ and β₁‐integrins was less pronounced, and expression of ICAM‐1 (intercellular adhesion molecule 1) was unaffected. The extent of effects depended on the exposure time of cells to the plasma and the treatment regimen. Intracellular level of ROS detected by the fluorescent dye H₂DCFDA (2′,7′‐dichlorodihydrofluorescein diacetate) increased by plasma treatment, but it was neither dependent on the treatment time nor related to the different treatment regimens. Two‐dimensional cultures of HaCaT keratinocytes appear to be a suitable method of investigating plasma‐cell interactions.