The dynamics of the electrotactic reaction of mouse 3T3 fibroblasts.

The molecular mechanisms behind electrotaxis remain largely unknown, with no identified primary direct current electric field (dcEF) sensor. Two leading hypotheses propose mechanisms involving the redistribution of charged components in the cell membrane (driven by electrophoresis or electroosmosis) and the asymmetric activation of ion channels. To investigate these mechanisms, we studied the dynamics of electrotactic behaviour of mouse 3T3 fibroblasts. We observed that 3T3 fibroblasts exhibit cathodal migration within just 1 min when exposed to physiological dcEF. This rapid response suggests the involvement of ion channels in the cell membrane. Our large-scale screening method identified several ion channel genes as potential key players, including the inwardly rectifying potassium channel Kir4.2. Blocking the Kir channel family with Ba2+ or silencing the Kcnj15 gene, encoding Kir4.2, significantly reduced the directional migration of 3T3 cells. Additionally, the levels of the intracellular regulators of Kir channels, spermine (SPM) and spermidine (SPD), had a significant impact on cell directionality. Interestingly, inhibiting Kir4.2 resulted in the temporary cessation of electrotaxis for approximately 1-2 h before its return. This observation suggests a two-phase mechanism for the electrotaxis of mouse 3T3 fibroblasts, where ion channel activation triggers the initial rapid response to dcEF, and the subsequent redistribution of membrane receptors sustains long-term directional movement. In summary, our study unveils the involvement of Kir channels and proposes a biphasic mechanism to explain the electrotactic behaviour of mouse 3T3 fibroblasts, shedding light on the molecular underpinnings of electrotaxis.

Effects of cylindrospermopsin, its decomposition products, and anatoxin-a on human keratinocytes.

Toxins produced by cyanobacteria (cyanotoxins) are among the most dangerous natural compounds. In recent years, there have been many published papers related to the toxic alkaloids cylindrospermopsin (CYN) and anatoxin-a (ANTX-a), which are synthesized by several freshwater species of cyanobacteria (i.e. Raphidiopsis raciborskii and Anabaena flos-aquae) and are some of the most common cyanotoxins in aquatic reservoirs. The harmful properties of CYN are wide and primarily include cytotoxicity. To date, several analogs and decomposition products of CYN have been described, which can potentially increase its toxic effects in living organisms. The mode of action of ANTX-a is different than that observed after CYN exposure and involves structures in the nervous system. One of the most frequent situations in which cyanotoxins are introduced into the human body is by skin contact with contaminated water, i.e., during water sports, fishing or agriculture. Unfortunately, to date, knowledge on the influence of CYN, its decomposition products, and ANTX-a on human skin is limited. In this paper, we investigated the impact of CYN, its decomposition products, and ANTX-a on the proliferation of human keratinocytes, which provide a protective barrier on the skin. Moreover, we described the cytotoxic effects developed in the selected cell type and estimated the ability of the keratinocytes to migrate under the influence of the studied cyanotoxins. The obtained results suggest that CYN and its decomposition products at concentrations corresponding to that determined for CYN in nature (1 µg·mL-1) are strong inhibitors of keratinocyte proliferation (70% inhibition within 24 h for pure CYN). The cytotoxic effects of CYN and the CYN decomposition products on keratinocytes was also significant, and the pure toxin (1 µg·mL-1) was estimated to be 35% after 24 h of exposure. Similarly, harmful effects caused by CYN and its byproducts were observed during keratinocyte migration, and the initial form of the toxin (1 µg·mL-1) showed 40% inhibition within 16 h. Different results were obtained for ANTX-a. The toxic effects of this compound on human keratinocytes estimated by the applied tests was observed only at the highest tested concentration (10 µg·mL-1) and after a long period of exposure. The results presented in this paper are, to the best of our knowledge, the first description of the influence of CYN, CYN decomposition products, and ANTX-a on human epidermal cells. Clearly, CYN and its decomposition products are serious threats not only when acting on internal organs but also during the skin contact stage. Further studies on cyanotoxins should focus on the determination of their decomposition products and ecotoxicology in natural aquatic environments.

Adipose-Derived Stromal Cells Seeded on Integra® Dermal Regeneration Template Improve Post-Burn Wound Reconstruction.

Fibrosis of burn-related wounds remains an unresolved clinical issue that leads to patient disability. The aim of this study was to assess the efficacy of the transplantation of adipose-derived stromal cells seeded onto a collagen-based matrix in the reconstruction of burn-related scars. Here, we characterized an in vitro interaction between adipose-derived stromal cells and a collagen-based matrix, Integra®DRT. Our results show that transcription of pro-angiogenic, remodeling, and immunomodulatory factors was more significant in adipose-derived stromal cells than in fibroblasts. Transcription of metalloproteinases 2 and 9 is positively correlated with the collagenolytic activity of the adipose-derived stromal cells seeded onto Integra®DRT. The increase in the enzymatic activity corresponds to the decrease in the elasticity of the whole construct. Finally, we validated the treatment of a post-excision wound using adipose-derived stromal cells and an Integra®DRT construct in a 25-year-old woman suffering from burn-related scars. Scarless healing was observed in the area treated by adipose-derived stromal cells and the Integra®DRT construct but not in the reference area where Integra®DRT was applied without cells. This clinical observation may be explained by in vitro findings: Enhanced transcription of the vascular endothelial growth factor as well as remodeling of the collagen-based matrix decreased mechanical stress. Our experimental treatment demonstrated that the adipose-derived stromal cells seeded onto Integra®DRT exhibit valuable properties that may improve post-excision wound healing and facilitate skin regeneration without scars.

Fenofibrate Interferes with the Diapedesis of Lung Adenocarcinoma Cells through the Interference with Cx43/EGF-Dependent Intercellular Signaling.

Extravasation of circulating cancer cells is regulated by the intercellular/intracellular signaling pathways that locally impair the endothelial barrier function. Co-cultures of human umbilical vein endothelial cells (HUVECs) with lung adenocarcinoma A549 cells enabled us to identify these pathways and to quantify the effect of fenofibrate (FF) on their activity. A549 cells induced the disruption and local activation of endothelial continuum. These events were accompanied by epidermal growth factor (EGF) up-regulation in endothelial cells. Impaired A549 diapedesis and HUVEC activation were seen upon the chemical inhibition of connexin(Cx)43 functions, EGF/ERK1/2-dependent signaling, and RhoA/Rac1 activity. A total of 25 µM FF exerted corresponding effects on Cx43-mediated gap junctional coupling, EGF production, and ERK1/2 activation in HUVEC/A549 co-cultures. It also directly augmented endothelial barrier function via the interference with focal adhesion kinase (FAK)/RhoA/Rac1-regulated endothelial cell adhesion/contractility/motility and prompted the selective transmigration of epithelioid A549 cells. N-acetyl-L-cysteine abrogated FF effects on HUVEC activation, suggesting the involvement of PPARα-independent mechanism(s) in its action. Our data identify a novel Cx43/EGF/ERK1/2/FAK/RhoA/Rac1-dependent signaling axis, which determines the efficiency of lung cancer cell diapedesis. FF interferes with its activity and reduces the susceptibility of endothelial cells to A549 stimuli. These findings provide the rationale for the implementation of FF in the therapy of malignant lung cancers.

Electrotaxis: Cell Directional Movement in Electric Fields.

Electrotaxis plays an important role during embryogenesis, inflammation, wound healing, and tumour metastasis. However, the mechanisms at play during electrotaxis are still poorly understood. Therefore intensive studies on signaling pathways involved in this phenomenon should be carried out. In this chapter, we described an experimental system for studying electrotaxis of Amoeba proteus, mouse embryonic fibroblasts (MEF), Walker carcinosarcoma cells WC256, and bone marrow adherent cells (BMAC).

[FRET-based biosensors in cell migration research]. / Zastosowanie biosensorów typu FRET w badaniach mikroskopowych procesu migracji komórkowej.

Cell migration is a complicated process, which is crucial for functioning of multicellular organisms. Multiple signalling pathways are deeply involved in the precise control of consecutive cell migration stages based on remodelling of the actin cytoskeleton. Small Rho GTPases (RhoA, Rac1 and Cdc42) as well as multiple protein and lipid kinases, calcium ions and mechanosensors are crucial components in this process. Exploration of those complicated correlations is possible with constant advancement of fluorescence microscopy. A significant progress in this field has been achieved since discovery of fluorescent proteins and subsequently FRET-based biosensors. Such protein constructs react with a change of FRET efficiency in response to the particular protein activity change. Properly designed and regularly improved biosensors offer the possibility of real-time imaging of signalling pathways dynamics in migrating cells. The perception of Rho GTPases involvement and some other signalling pathways connected with cell migration have been clarified with multiple experiments already carried out with such FRET-based biosensors.

Electric field as a potential directional cue in homing of bone marrow-derived mesenchymal stem cells to cutaneous wounds.

Bone marrow-derived cells are thought to participate and enhance the healing process contributing to skin cells or releasing regulatory cytokines. Directional cell migration in a weak direct current electric field (DC-EF), known as electrotaxis, may be a way of cell recruitment to the wound site. Here we examined the influence of electric field on bone marrow adherent cells (BMACs) and its potential role as a factor attracting mesenchymal stem cells to cutaneous wounds. We observed that in an external EF, BMAC movement was accelerated and highly directed with distinction of two cell populations migrating toward opposite poles: mesenchymal stem cells migrated toward the cathode, whereas macrophages toward the anode. Analysis of intracellular pathways revealed that macrophage electrotaxis mostly depended on Rho family small GTPases and calcium ions, but interruption of PI3K and Arp2/3 had the most pronounced effect on electrotaxis of MSCs. However, in all cases we observed only a partial decrease in directionality of cell movement after inhibition of certain proteins. Additionally, although we noticed the accumulation of EGFR at the cathodal side of MSCs, it was not involved in electrotaxis. Moreover, the cell reaction to EF was very dynamic with first symptoms occurring within <1min. In conclusion, the physiological DC-EF may act as a factor positioning bone marrow cells within a wound bed and the opposite direction of MSC and macrophage movement did not result either from utilizing different signalling or redistribution of investigated cell surface receptors.

Lamellipodia and Membrane Blebs Drive Efficient Electrotactic Migration of Rat Walker Carcinosarcoma Cells WC 256.

The endogenous electric field (EF) may provide an important signal for directional cell migration during wound healing, embryonic development and cancer metastasis but the mechanism of cell electrotaxis is poorly understood. Additionally, there is no research addressing the question on the difference in electrotactic motility of cells representing various strategies of cell movement-specifically blebbing vs. lamellipodial migration. In the current study we constructed a unique experimental model which allowed for the investigation of electrotactic movement of cells of the same origin but representing different modes of cell migration: weakly adherent, spontaneously blebbing (BC) and lamellipodia forming (LC) WC256 cells. We report that both BC and LC sublines show robust cathodal migration in a physiological EF (1-3 V/cm). The directionality of cell movement was completely reversible upon reversing the field polarity. However, the full reversal of cell direction after the change of EF polarity was much faster in the case of BC (10 minutes) than LC cells (30 minutes). We also investigated the distinct requirements for Rac, Cdc42 and Rho pathways and intracellular Ca2+ in electrotaxis of WC256 sublines forming different types of cell protrusions. It was found that Rac1 is required for directional movement of LC to a much greater extent than for BC, but Cdc42 and RhoA are more crucial for BC than for LC cells. The inhibition of ROCK did not affect electrotaxis of LC in contrast to BC cells. The results also showed that intracellular Ca2+ is essential only for the electrotactic reaction of BC cells. Moreover, inhibition of MLCK and myosin II did not affect the electrotaxis of LC in contrast to BC cells. In conclusion, our results revealed that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of WC 256 carcinosarcoma cells, however directional migration is mediated by different signalling pathways.

The role of microtubules in electrotaxis of rat Walker carcinosarcoma WC256 cells.

The endogenous electric field may provide an important signal for directional cell migration during cancer metastasis but the mechanism of cell electrotaxis is poorly understood. It was postulated that microtubules play a central role in the polarization and directional migration of several types of cells. In this paper we investigated the role of microtubules in electrotaxis of rat Walker carcinosarcoma WC256 cells. We found that colchicine-stimulated disassembly of microtubules caused the formation of blebs instead of lamellipodia at the front of about 45% of cells. Most of the remaining cells contracted and became rounded or transformed into non-polar cells. Depolymerization of microtubules in both subpopulations of cells reduced the directionality of cell migration to about 50% of the control, but bleb- forming cells migrated much more efficiently than lamellipodia-forming cells. The analysis of microtubules architecture in the presence of an endogenous electric field showed that there is no relationship between the direction of migration and the polarization of microtubules. These results suggest that microtubules are not indispensable for electrotaxis of WC256 cells, however they may improve the directionality of cell migration.

Poly(L-lactide-co-glycolide) thin films can act as autologous cell carriers for skin tissue engineering.

Degradable aliphatic polyesters such as polylactides, polyglycolides and their copolymers are used in several biomedical and pharmaceutical applications. We analyzed the influence of poly(L-lactide-co-glycolide) (PLGA) thin films on the adhesion, proliferation, motility and differentiation of primary human skin keratinocytes and fibroblasts in the context of their potential use as cell carriers for skin tissue engineering. We did not observe visible differences in the morphology, focal contact appearance, or actin cytoskeleton organization of skin cells cultured on PLGA films compared to those cultured under control conditions. Moreover, we did not detect biologically significant differences in proliferative activity, migration parameters, level of differentiation, or expression of vinculin when the cells were cultured on PLGA films and tissue culture polystyrene. Our results indicate that PLGA films do not affect the basic functions of primary human skin keratinocytes and fibroblasts and thus show acceptable biocompatibility in vitro, paving the way for their use as biomaterials for skin tissue engineering.

Microcystin-LR affects properties of human epidermal skin cells crucial for regenerative processes.

The occurrence of cyanobacterial toxic peptides, including microcystins (MCs), is an emerging health issue due to the eutrophication of water bodies. MCs have a strong influence on human cells, predominantly hepatocytes, however, toxicity was also observed in kidney, lung and dermal skin cells. Skin as the most external barrier of the human body is responsible for the maintenance of homeostasis of the whole organism. Simultaneously, skin cells may be the most exposed to MCs during recreational activity. The aim of this study was to examine the impact of MC-LR on processes indispensable for normal skin function and regeneration, namely, viability, migration and actin cytoskeleton organization of human keratinocytes. The results showed that short exposure to MC-LR does not affect proliferation of human skin keratinocytes but it is toxic after longer incubation in dose-dependent manner. Total disruption of the actin cytoskeleton was observed under the same MC-LR concentration. Furthermore, keratinocyte migration was inhibited at MC-LR concentrations of 50 µM after incubation for only 4 h. Some of the negative impacts of MC-LR on the examined cell processes may be partly reversible. The observed effects, regarding the possible high exposition of keratinocytes to toxins including MCs, are severe and may cause diverse health problems.