scholarly journals Phenotypic Differences in Adult and Fetal Dermal Fibroblast Responses to Mechanical Tension

2020 ◽  
Author(s):  
Umang M. Parikh ◽  
Walker D. Short ◽  
Natalie Templeman ◽  
Alexander Blum ◽  
Daniel Colchado ◽  
...  
Keyword(s):  
Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Pericellular Matrix ◽  
Mechanical Tension ◽  
Fetal Fibroblast ◽  
Phenotypic Differences ◽  
Fetal Fibroblasts ◽  
Static Conditions ◽  
Post Hoc

ABSTRACTThe regenerative wound healing phenotype observed in the fetal wounds is characterized by a unique hyaluronan rich wound microenvironment. Fetal fibroblasts produce a pericellular matrix which is abundant in high molecular weight HA. In vivo, fetal skin has negligible resting tension and heals regeneratively when small wounds are made. However, critically large fetal wounds heal with increased scar. We hypothesize that higher mechanical tension will differentially alter fibroblast-mediated HA metabolism in adult and fetal fibroblasts, leading to a profibrotic fetal fibroblast phenotype. C57BL/6J fetal (FFB, E14.5) and adult (AFB, 8 wk) dermal fibroblasts were cultured on silicone membranes +/-10% static strain (1, 3, 6, and 12h). Monolayers were analyzed via PCR for HA-synthesis (HAS1-3), HA-remodeling (HYAL1-2, KIAA1199), and HA-receptor (CD44) genes, normalized to static FFB. Total HA was analyzed by gel electrophoresis at 12h. Data is reported as mean+/-SD (n=3), with p-values calculated by two-way ANOVA (post-hoc Tukey’s test). FFB pericellular matrix was reduced after tension conditions, and HA profile shifted from HMW-HA to LMW-HA. Under static conditions, AFB had increased expression of HAS 1 and 2, as well as increased expression of KIA1199, HYAL1 and 2 when compared to FFB. Overall, tension resulted in an increase in HAS1, HAS3, and HYAL1 expression in FFB, and decreased HAS2 but increased HYAL1 for AFB under tension. Staining for cytoskeletal components demonstrates that tension results in organization of F-actin and aSMA in FFB to resemble AFB. Expression of aSMA is increased in AFB with tension, while CD26 expression is increased in FFB with tension. These insights into the intrinsic differences between regenerative FFB and fibrotic AFB may yield targets to attenuate fibrosis.

scholarly journals In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chee-Wai Wong ◽  
Catherine F. LeGrand ◽  
Beverley F. Kinnear ◽  
Radoslaw M. Sobota ◽  
Rajkumar Ramalingam ◽  
...  
Keyword(s):  
Dermal Fibroblast ◽  
Human Keratinocytes ◽  
Dermal Fibroblasts ◽  
Collagen I ◽  
Matrix Composition ◽  
Major Influence ◽  
Primary Human Keratinocytes ◽  
Matrix Deposition

AbstractThe long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell’s fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.

scholarly journals Human Metabolites of Hamaforton™ (Hamamelis virginiana L. Extract) Modulates Fibroblast Extracellular Matrix Components in Response to UV-A Irradiation

2021 ◽  
Vol 12 ◽  
Author(s):  
Fausta Natella ◽  
Barbara Guantario ◽  
Roberto Ambra ◽  
Giulia Ranaldi ◽  
Federica Intorre ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Gallic Acid ◽  
Culture Media ◽  
Ex Vivo ◽  
Dermal Fibroblast ◽  
Oral Formulation ◽  
Dermal Fibroblasts ◽  
Ultraviolet A ◽  
Extracellular Matrix Components

Hamamelis virginiana L. a rich source of both condensed and hydrolyzable tannins, utilized to treat dermatological disorders. Since no experimental and clinical data is available for its use as oral formulation in skin related disorders, the purpose of this study was to investigate the effects of Hamaforton™ (Hamamelis virginiana extract) metabolites on gene dysregulation induced by ultraviolet A radiation in cultured human dermal fibroblasts. A combination of in vivo and ex vivo experimental designs has been exploited in order to take into account the polyphenol metabolic transformation that occurs in humans. 12 healthy volunteers received either a capsule of Hamaforton™ or a placebo in a randomized, blinded crossover trial. After Hamaforton™ ingestion, the kinetic of appearance of galloyl derivatives was measured in plasma. Then, in the ex vivo experiment, the serum isolated after supplementation was used as a source of Hamaforton™ metabolites to enrich the culture medium of dermal fibroblasts exposed to ultraviolet A radiation. Three different gallic acid metabolites (4-O-methyl gallic acid, 4-O-methyl gallic acid sulphate and trimethyl gallic acid glucuronide) were identified in volunteer plasma. While, ultraviolet A irradiation of dermal fibroblasts affected the expression of extracellular matrix genes, the presence of Hamaforton™ metabolites in the culture media did not affect the expression of most of those genes. However, the activation of the expression of 10 different genes involved in repair processes for the maintenance of skin integrity, suggest that the metabolites can play a role in damage recovery. To our knowledge, this is the first study that demonstrates the bioavailability of Hamaforton™ phenolic compounds, and the effects of its metabolites on cultured dermal fibroblast response to ultraviolet A irradiation.

IL-22 capacitates dermal fibroblast responses to TNF in scleroderma

2015 ◽  
Vol 75 (9) ◽  
pp. 1697-1705 ◽  
Author(s):  
Nicolò Costantino Brembilla ◽  
Aleksandra Maria Dufour ◽  
Montserrat Alvarez ◽  
Stéphanie Hugues ◽  
Elisa Montanari ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Transforming Growth Factor ◽  
Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Th22 Cells ◽  
Matrix Metalloproteinase 1 ◽  
Dermal Thickness ◽  
Extracellular Matrix Components

ObjectivesInterleukin (IL) 22 mRNA in systemic sclerosis (SSc) skin and Th22 cells in SSc peripheral blood are increased, but the role of IL-22 in fibrosis development remains poorly understood.MethodsBiopsies were obtained from the involved skin of 15 SSc, 4 morphea and 8 healthy donors (HD). The presence of IL-22+ cells in the skin was determined by immunostaining. The in vitro response of HD and SSc fibroblasts to IL-22, IL-22 in conjunction with tumour necrosis factor (TNF) or keratinocyte conditioned medium was assessed by ELISA, radioimmunoassay (RIA), real-time PCR and western blot. The in vivo response in mice was assessed by histomorphometry.ResultsIL-22+ cells were over-represented in the dermis and epidermis of morphea and in the epidermis of SSc compared with HD. The majority of dermal IL-22+ cells were T cells. Dermal fibroblasts expressed both IL-22 receptor subunits IL-10RB and IL-22RA, expression of which was enhanced by TNF and reduced by transforming growth factor (TGF)-β. IL-22 induced rapid phosphorylation of p38 and ERK1/2 in fibroblasts, but failed to induce the synthesis of chemokines and extracellular matrix components. However, IL-22 enhanced the production of monocyte chemotactic protein 1, IL-8 and matrix metalloproteinase 1 induced by TNF. Fibroblast responses were maximal in the presence of conditioned medium from keratinocytes activated by IL-22 in conjunction with TNF. Dermal thickness was maximal in mice injected simultaneously with IL-22 and TNF.ConclusionsIL-22 capacitates fibroblast responses to TNF and promotes a proinflammatory fibroblast phenotype by favouring TNF-induced keratinocyte activation. These results define a novel role for keratinocyte–fibroblast interactions in the context of skin fibrosis.

scholarly journals In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

2018 ◽  
Author(s):  
Chee-Wai Wong ◽  
Beverley F. Kinnear ◽  
Radoslaw M. Sobota ◽  
Rajkumar Ramalingam ◽  
Catherine F. LeGrand ◽  
...  
Keyword(s):  
Cell Fate ◽  
Dermal Fibroblast ◽  
Human Keratinocytes ◽  
Human Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Primary Human Keratinocytes ◽  
Matrix Deposition ◽  
Forming Efficiency

SummaryThe long-term expansion of keratinocytes under serum- and feeder free conditions generally results in diminished proliferation and an increased commitment to terminal differentiation. Here we present a serum and xenogeneic feeder free culture system that retains the self-renewal capacity of primary human keratinocytes. In vivo, the tissue microenvironment is a major contributor to determining cell fate and a key component of the microenvironment is the extracellular matrix (ECM). Accordingly, acellular ECMs derived from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, were used as the basis for a xenogeneic-free keratinocyte expansion protocol. A phospholipase A2 decellularisation procedure produced matrices which, by proteomics analysis, resembled in composition the core matrix proteins of skin dermis. On these ECMs keratinocytes proliferated rapidly, retained their small size, expressed p63, did not express keratin 10 and rarely expressed keratin 16. Moreover, the colony forming efficiency of keratinocytes cultured on these acellular matrices was markedly enhanced. Collectively these data indicate that the dermal fibroblast-derived matrices support the in vitro expansion of keratinocytes that maintained stem-like characteristics under serum free conditions.

Analysis of the Dynamics of the Electric Capacitance of a Cell Monolayer at the Late Stages of Caco-2 cell Differentiation

2019 ◽  
Vol 35 (6) ◽  
pp. 87-90
Author(s):  
S.V. Nikulin ◽  
V.A. Petrov ◽  
D.A. Sakharov
Keyword(s):  
Impedance Spectroscopy ◽  
Cell Monolayer ◽  
Microfluidic Chips ◽  
Russian Science ◽  
Electric Capacitance ◽  
A Cell ◽  
Static Conditions ◽  
Late Stages

The real-time monitoring of electric capacitance (impedance spectroscopy) allowed obtaining evidence that structures which look like intestinal villi can be formed during the cultivation under static conditions as well as during the cultivation in microfluidic chips. It was shown in this work via transcriptome analysis that the Hh signaling pathway is involved in the formation of villus-like structures in vitro, which was previously shown for their formation in vivo. impedance spectroscopy, intestine, villi, electric capacitance, Hh The study was funded by the Russian Science Foundation (Project 16-19-10597).

scholarly journals Dopamine adjusts the circadian gene expression of Per2 and Per3 in human dermal fibroblasts from ADHD patients

2021 ◽  
Author(s):  
Frank Faltraco ◽  
Denise Palm ◽  
Adriana Uzoni ◽  
Lena Borchert ◽  
Frederick Simon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dermal Fibroblast ◽  
Sleep Onset ◽  
Dermal Fibroblasts ◽  
Control Group ◽  
Adhd Group ◽  
Healthy Controls ◽  
Circadian Gene ◽  
Significant Difference ◽  
Circadian Preference

AbstractA link between dopamine levels, circadian gene expression, and attention deficit hyperactivity disorder (ADHD) has already been demonstrated. The aim of this study was to investigate the extent of these relationships by measuring circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after dopamine exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different dopamine concentrations in human dermal fibroblast (HDF) cultures, the rhythmicity of circadian gene expression (Clock, Bmal1, Per1-3, Cry1) was analyzed via qRT-PCR. We found no statistical significant effect in the actigraphy of both groups (healthy controls, ADHD group) for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, wake after sleep onset, and total number of wake bouts. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. Dopamine has no effect on Per3 expression in healthy controls, but produces a significant difference in the ADHD group at ZT24 and ZT28. In the ADHD group, incubation with dopamine, either 1 µM or 10 µM, resulted in an adjustment of Per3 expression to control levels. A similar effect also was found in the expression of Per2. Statistical significant differences in the expression of Per2 (ZT4) in the control group compared to the ADHD group were found, following incubation with dopamine. The present study illustrates that dopamine impacts on circadian function. The results lead to the suggestion that dopamine may improve the sleep quality as well as ADHD symptoms by adjustment of the circadian gene expression, especially for Per2 and Per3.

scholarly journals Dendranthema zawadskii var. lucidum (Nakai) J.H. Park Extract Inhibits Cellular Senescence in Human Dermal Fibroblasts and Aging-Related Inflammation in Rats

Processes ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 801
Author(s):  
Jehun Choi ◽  
Gwi-Yeong Jang ◽  
Jeonghoon Lee ◽  
Hae-Young Chung ◽  
Hyung-Jun Noh ◽  
...  
Keyword(s):  
Cellular Senescence ◽  
Cell Cycle Progression ◽  
Inflammatory Responses ◽  
Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Dependent Manner ◽  
Aged Rats ◽  
Age Related ◽  
Dendranthema Zawadskii ◽  
Beta Galactosidase

Senescence is the phenomenon by which physiological functions of organisms degenerate with time. Cellular senescence is marked by an inhibition of cell cycle progression. Beta-galactosidase accumulates in the lysosomes of aged cells. In this study, human dermal fibroblast cells (HDFs) were treated with 0.5 μM doxorubicin for 4 h to induce cellular senescence. Senescence-associated beta-galactosidase (SA-β-gal) activity was then measured 72 h after treatment with aerial parts of Dendranthema zawadskii var. lucidum (Nakai) J.H. Park (DZ) extract. Treatment with DZ extract significantly decreased SA-β-gal activity in a dose-dependent manner in HDFs. Additionally, DZ extract treatment reduced age-related oxidative stress and inflammation in the aortas of aged rats. The reactive oxygen species (ROS) levels in aortas of aged control rats were higher than those in young rats. However, DZ extract-fed aged rats showed significantly lower ROS levels than the aged control rats. When the aged rats were treated with DZ extract at either 0.2 or 1.0 mg∙kg−1∙day−1, NF-κB levels in aorta tissue decreased significantly compared to those in aorta tissue of the aged control rats without DZ treatment. In addition, DZ extract-fed aged rat aortas showed significant reductions in expression of iNOS and COX-2 induced by NF-κB translocation. Therefore, these results suggest that DZ effectively inhibited senescence-related NF-κB activation and inflammation. DZ extract may have a role in the prevention of the vascular inflammatory responses that occur during vascular aging.

The Role of Mechanical Tension in Neurons

2010 ◽  
Vol 1274 ◽  
Author(s):  
Taher Saif ◽  
Jagannathan Rajagopalan ◽  
Alireza Tofangchi
Keyword(s):  
Mechanical Response ◽  
Mechanical Forces ◽  
Active Tension ◽  
Mechanical Tension ◽  
Deformation Response ◽  
Linear Force ◽  
Tension Regulation ◽  
Over Time

AbstractWe used high resolution micromechanical force sensors to study the in vivo mechanical response of embryonic Drosophila neurons. Our experiments show that Drosophila axons have a rest tension of a few nN and respond to mechanical forces in a manner characteristic of viscoelastic solids. In response to fast externally applied stretch they show a linear force-deformation response and when the applied stretch is held constant the force in the axons relaxes to a steady state value over time. More importantly, when the tension in the axons is suddenly reduced by releasing the external force the neurons actively restore the tension, sometimes close to their resting value. Along with the recent findings of Siechen et al (Proc. Natl. Acad. Sci. USA 106, 12611 (2009)) showing a link between mechanical tension and synaptic plasticity, our observation of active tension regulation in neurons suggest an important role for mechanical forces in the functioning of neurons in vivo.

scholarly journals Blockade of LINC01605-enriched exosome generation in M2 macrophages impairs M2 macrophage-induced proliferation, migration, and invasion of human dermal fibroblasts

2021 ◽  
Vol 35 ◽  
pp. 205873842110167
Author(s):  
Zhensen Zhu ◽  
Bo Chen ◽  
Liang Peng ◽  
Songying Gao ◽  
Jingdong Guo ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
M2 Macrophage ◽  
M2 Macrophages ◽  
Luciferase Reporter Gene ◽  
Gene Assay

Activated M2 macrophages are involved in hypertrophic scar (HS) formation via manipulating the differentiation of fibroblasts to myofibroblasts having the proliferative capacity and biological function. However, the function of exosomes derived from M2 macrophages in HS formation is unclear. Thus, this study aims to investigate the role of exosomes derived by M2 in the formation of HS. To understand the effect of exosomes derived from M2 macrophages on formation of HS, M2 macrophages were co-cultured with human dermal fibroblast (HDF) cells. Cell Counting Kit-8 assay was performed to evaluate HDF proliferation. To evaluate the migration and invasion of HDFs, wound-healing and transwell invasion assays were performed, respectively. To investigate the interaction between LINC01605 and miR-493-3p, a dual-luciferase reporter gene assay was adopted; consequently, an interaction between miR-493-3p and AKT1 was detected. Our results demonstrated that exosomes derived from M2 macrophages promoted the proliferation, migration, and invasion of HDFs. Additionally, we found that long noncoding RNA LINC01605, enriched in exosomes derived from M2 macrophages, promoted fibrosis of HDFs and that GW4869, an inhibitor of exosomes, could revert this effect. Mechanistically, LINC01605 promoted fibrosis of HDFs by directly inhibiting the secretion of miR-493-3p, and miR-493-3p down-regulated the expression of AKT1. Exosomes derived from M2 macrophages promote the proliferation and migration of HDFs by transmitting LINC01605, which may activate the AKT signaling pathway by sponging miR-493-3p. Our results provide a novel approach and basis for further investigation of the function of M2 macrophages in HS formation.

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