Acute and chronic wound fluids influence keratinocyte function differently.
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ABSTRACT:
SUBMITTER: Thamm OC
PROVIDER: S-EPMC7950806 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Wound healing is a complex biological process that requires a well-orchestrated interaction of mediators as well as resident and infiltrating cells. In this context, mesenchymal stem cells play a crucial role as they are attracted to the wound site and influence tissue regeneration by various mechanisms. In chronic wounds, these processes are disturbed. In a comparative approach, adipose-derived stem cells (ASC) were treated with acute and chronic wound fluids (AWF and CWF, respectively). Proliferation and migration were investigated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and transwell migration assay. Gene expression changes were analysed using quantitative real time-polymerase chain reaction. AWF had a significantly stronger chemotactic impact on ASC than CWF (77·5% versus 59·8% migrated cells). While proliferation was stimulated by AWF up to 136·3%, CWF had a negative effect on proliferation over time (80·3%). Expression of b-FGF, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 was strongly induced by CWF compared with a mild induction by AWF. These results give an insight into impaired ASC function in chronic wounds. The detected effect of CWF on proliferation and migration of ASC might be one reason for an insufficient healing process in chronic wounds.
| S-EPMC7950656 | biostudies-literature
Project description:BackgroundThe process of wound healing consists of several well distinguishable and finely tuned phases. For most of these phases specific proteins have been characterized, although the underlying mechanisms of regulation are not yet fully understood. It is an open question as to whether deficits in wound healing can be traced back to chronic illnesses such as diabetes mellitus. Previous research efforts in this field focus largely on a restricted set of marker proteins due to the limitations detection by antibodies imposes. For mechanistic purposes the elucidation of differences in acute and chronic wounds can be addressed by a less restricted proteome study. Mass spectrometric (MS) methods, e.g. multi dimensional protein identification technology (MudPIT), are well suitable for this complex theme of interest. The human wound fluid proteome is extremely complex, as is human plasma. Therefore, high-abundant proteins often mask the mass spectrometric detection of lower-abundant ones, which makes a depletion step of such predominant proteins inevitable.FindingsIn this study a commercially available immunodepletion kit was evaluated for the detection of low-abundant proteins from wound fluids. The dynamic range of the entire workflow was significantly increased to 5-6 orders of magnitude, which makes low-abundant regulatory proteins involved in wound healing accessible for MS detection.ConclusionThe depletion of abundant proteins is absolutely necessary in order to analyze highly complex protein mixtures such as wound fluids using mass spectrometry. For this the used immunodepletion kit is a first but important step in order to represent the entire dynamic range of highly complex protein mixtures in the future.
| S-EPMC3020179 | biostudies-literature
Project description:Wound healing is a complex multiphase process which can be hampered by many factors including impaired local circulation, hypoxia, infection, malnutrition, immunosuppression, and metabolic dysregulation in diabetes. Redox dysregulation is a common feature of many skin diseases demonstrated by virtually all cell types in the skin with overproduction of reactive oxygen and nitrogen species. The objective of this study was to characterize the redox environment in wound fluids and sera from patients suffering from chronic leg ulcers (n = 19) and acute wounds (bulla fluids from second degree burns; n = 11) with serum data also compared to those from healthy volunteers (n = 7). Significantly higher concentrations of TNF-?, interleukine-8, vascular endothelial growth factor, and lactate dehydrogenase (measure of cell damage) were found in fluids from chronic wounds compared to acute ones. The extent of protein carbonylation (measure of protein oxidation), lipid peroxidation, and tyrosine nitration (indicator of peroxynitrite production) was similar in acute and chronic wound fluids, while radical scavenging activity and glutathione (GSH) levels were elevated in chronic wound fluids compared to acute wounds. Sera were also assessed for the same set of parameters with no significant differences detected. Nitrotyrosine (the footprint of the potent oxidant peroxynitrite) and poly(ADP-ribose) (the product of the DNA damage sensor enzyme PARP-1) could be detected in wound biopsies. Our data identify multiple signs of redox stress in chronic wounds with notable differences. In chronic wounds, elevations in antioxidant levels/activities may indicate compensatory mechanisms against inflammation. The presence of nitrotyrosine and poly(ADP-ribose) in tissues from venous leg ulcers indicate peroxynitrite production and PARP activation in chronic wounds.
| S-EPMC6276414 | biostudies-literature
Project description:The clinical assessment of wound infection and related complications is challenging and the development of objective methods to measure wound status and predict healing outcomes is therefore essential. As bacterial protease activities play important roles in infection, analysis of changes in the wound peptidome by individual bacterial enzymes could provide insight into proteolytic events occurring in infected wounds, and may aid in the discovery of biomarkers. Wound infection was mimicked by incubating sterile acute wound fluids ex vivo with the bacterial proteases Pseudomonas aeruginosa LasB and Staphyloccocus aureus V8. Using a peptidomics approach, we characterized the low-molecular-weight peptidome of the digested samples and identified over 100 protein targets for each enzyme. Additionally, we found enzyme-specific peptides and digestion patterns.
2024-03-07 | PXD037047 | Pride
Project description:Diabetes patients are at risk of having chronic wounds, which would take months to years to resolve naturally. Chronic wounds can be countered using the electrical stimulation technique (EST) by dielectrophoresis (DEP), which is label-free, highly sensitive, and selective for particle trajectory. In this study, we focus on the validation of polystyrene particles of 3.2 and 4.8 μm to predict the behavior of keratinocytes to estimate their crossover frequency (fXO) using the DEP force (FDEP) for particle manipulation. MyDEP is a piece of java-based stand-alone software used to consider the dielectric particle response to AC electric fields and analyzes the electrical properties of biological cells. The prototypic 3.2 and 4.8 μm polystyrene particles have fXO values from MyDEP of 425.02 and 275.37 kHz, respectively. Fibroblast cells were also subjected to numerical analysis because the interaction of keratinocytes and fibroblast cells is essential for wound healing. Consequently, the predicted fXO from the MyDEP plot for keratinocyte and fibroblast cells are 510.53 and 28.10 MHz, respectively. The finite element method (FEM) is utilized to compute the electric field intensity and particle trajectory based on DEP and drag forces. Moreover, the particle trajectories are quantified in a high and low conductive medium. To justify the simulation, further DEP experiments are carried out by applying a non-uniform electric field to a mixture of different sizes of polystyrene particles and keratinocyte cells, and these results are well agreed. The alive keratinocyte cells exhibit NDEP force in a highly conductive medium from 100 kHz to 25 MHz. 2D/3D motion analysis software (DIPP-MotionV) can also perform image analysis of keratinocyte cells and evaluate the average speed, acceleration, and trajectory position. The resultant NDEP force can align the keratinocyte cells in the wound site upon suitable applied frequency. Thus, MyDEP estimates the Clausius-Mossotti factors (CMF), FEM computes the cell trajectory, and the experimental results of prototypic polystyrene particles are well correlated and provide an optimistic response towards keratinocyte cells for rapid wound healing applications.
| S-EPMC8123363 | biostudies-literature
Project description:Unsuccessful wound closure in chronic wounds can be linked to altered keratinocyte activation and their inability to re-epithelize. Suggested mechanisms driving this impairment involve unbalanced cytokine signaling. However, the molecular events leading to these aberrant responses are poorly understood. Among cytokines affecting keratinocyte responses, Transforming Growth Factor-β (TFG-β) is thought to have a great impact. In this study, we have used a previously characterized skin epidermal in vitro model, HaCaT cells continuously exposed to TGF-β1, to study the wound recovery capabilities of chronified/senescent keratinocytes. In this setting, chronified keratinocytes show decreased migration and reduced activation in response to injury. Amniotic membrane (AM) has been used successfully to manage unresponsive complicated wounds. In our in vitro setting, AM treatment of chronified keratinocytes re-enabled migration in the early stages of wound healing, also promoting proliferation at later stages. Interestingly, when checking the gene expression of markers known to be altered in TGF-β chronified cells and involved in cell cycle regulation, early migratory responses, senescence, and chronic inflammation, we discovered that AM treatment seemed to reset back to keratinocyte status. The analysis of the evolution of both the levels of keratinocyte activation marker cytokeratin 17 and the spatial-temporal expression pattern of the proliferation marker Ki-67 in human in vivo biopsy samples suggests that responses to AM recorded in TGF-β chronified HaCaT cells would be homologous to those of resident keratinocytes in chronic wounds. All these results provide further evidence that sustained TGF-β might play a key role in wound chronification and postulate the validity of our TGF-β chronified HaCaT in vitro model for the study of chronic wound physiology.
| S-EPMC10094701 | biostudies-literature
Project description:p63 is a transcriptional regulator of ectodermal development that is required for basal cell proliferation and stem cell maintenance. p73 is a closely related p53 family member that is expressed in select p63-positive basal cells and can heterodimerize with p63. p73-/- mice lack multiciliated cells and have reduced numbers of basal epithelial cells in select tissues; however, the role of p73 in basal epithelial cells is unknown. Herein, we show that p73-deficient mice exhibit delayed wound healing despite morphologically normal-appearing skin. The delay in wound healing is accompanied by decreased proliferation and increased levels of biomarkers of the DNA damage response in basal keratinocytes at the epidermal wound edge. In wild-type mice, this same cell population exhibited increased p73 expression after wounding. Analyzing single-cell transcriptomic data, we found that p73 was expressed by epidermal and hair follicle stem cells, cell types required for wound healing. Moreover, we discovered that p73 isoforms expressed in the skin (ΔNp73) enhance p63-mediated expression of keratinocyte genes during cellular reprogramming from a mesenchymal to basal keratinocyte-like cell. We identified a set of 44 genes directly or indirectly regulated by ΔNp73 that are involved in skin development, cell junctions, cornification, proliferation, and wound healing. Our results establish a role for p73 in cutaneous wound healing through regulation of basal keratinocyte function.
| S-EPMC6583996 | biostudies-literature
Project description:A number of Rho-kinase inhibitors have been developed for various clinical applications. We examined the effects of the Rho-kinase inhibitor Y27632 on keratinocyte proliferation and migration, and found that it promoted primary human keratinocyte proliferation and migration in both monolayer and skin explant cultures. In addition, topical application of Y27632 enhanced cutaneous wound closure in the majority of wounds in mice. The growth and migration effects of Y27632 appeared to be specific to keratinocytes compared with dermal fibroblasts, and required intact Jun kinase function. Y27632 seems to be a promising agent for keratinocyte procurement and wounding healing.
| S-EPMC3748215 | biostudies-literature
Project description:BackgroundWound assessment relies on visual evaluation by physicians. Such assessment is largely subjective and presents the opportunity to explore the use of emergent technologies.MethodsEmergent and powerful noninvasive imaging technologies applicable to assess burn and chronic wounds are reviewed.ResultsThe need to estimate wound depth is critical in both chronic wound and burn injury settings. Harmonic ultrasound technology is powerful to study wound depth. It addresses the limitations of optical imaging with limited depth of penetration. What if a wound appears epithelialized by visual inspection, which shows no discharge yet is covered by repaired skin that lacks barrier function? In this case although the wound is closed as defined by current standards, it remains functionally open, presenting the risk of infection and other postclosure complications. Thus, assessment of skin barrier function is valuable in the context of assessing wound closure. Options for the study of tissue vascularization are many. If noncontact and noninvasive criteria are of importance, laser speckle imaging is powerful. Fluorescence imaging is standard in several clinical settings and is likely to serve the wound clinics well as long as indocyanine green injection is not of concern. A major advantage of harmonic ultrasound imaging of wound depth is that the same system is capable of providing information on blood flow dynamics in arterial perforators.ConclusionWith many productive imaging platforms to choose from, wound care is about to be transformed by technology that would help assess wound severity.
| S-EPMC5207795 | biostudies-literature
Project description:Chronic ulcers represent a major health burden in our society. Despite many available therapies, a large number of ulcers do not heal. Protein based therapies fail in part due to proteolytic activity in the chronic wound bed. The aim of this in vitro study was to determine whether typical inflammatory cytokines and human salivary histatins remain stable when incubated with chronic wound extracts. Furthermore we determined whether a short exposure of histatins or cytokines was sufficient to exert long term effects on fibroblast migration. Stability of human recombinant cytokines IL-6 and CXCL8, and histatin variants (Hst1, Hst2, cyclic Hst1, minimal active domain of Hst1) in the presence of chronic wound extracts isolated from non-healing ulcers, was monitored by capillary zone electrophoresis. Migration-stimulating activity was assessed using a dermal fibroblast wound healing scratch assay. Histatins and cytokines stayed stable in saline for > 24 h at 37°C, making them ideal as an off-the-shelf product. However, incubation with chronic wound extracts resulted in serious breakdown of Hst1 and Hst2 (~50% in 8 h) and to lesser extent cyclic Hst1 and the minimal active domain of Hst1 (~20% in 8 h). The cytokines IL-6 and CXCL8 were more stable in chronic wound extracts (~40% degradation in 96 h). An initial 8-hour pulse of histatins or cytokines during a 96-hour study period was sufficient to stimulate fibroblast migration equally well as a continuous 96-hour exposure, indicating that they may possibly be used as novel bioactive therapeutics, exerting their activity for up to four days after a single exposure.
| S-EPMC4809600 | biostudies-literature