Response Of Human Fibroblasts Research Articles

Mitochondrial reactive oxygen species impact human fibroblast responses to protracted γ-ray exposures

Purpose: Continuous exposure to ionizing radiation at a low dose rate poses significant health risks to humans on deep space missions, prompting the need for mechanistic studies to identify countermeasures against its deleterious effects. Mitochondria are a major subcellular locus of radiogenic injury, and may trigger secondary cellular responses through the production of reactive oxygen species (mtROS) with broader biological implications. Methods and Materials: To determine the contribution of mtROS to radiation-induced cellular responses, we investigated the impacts of protracted γ-ray exposures (IR; 1.1 Gy delivered at 0.16 mGy/min continuously over 5 days) on mitochondrial function, gene expression, and the protein secretome of human HCA2-hTERT fibroblasts in the presence and absence of a mitochondria-specific antioxidant mitoTEMPO (MT; 5 µM). Results: IR increased fibroblast mitochondrial oxygen consumption (JO2) and H2O2 release rates (JH2O2) under energized conditions, which corresponded to higher protein expression of NADPH Oxidase (NOX) 1, NOX4, and nuclear DNA-encoded subunits of respiratory chain Complexes I and III, but depleted mtDNA transcripts encoding subunits of the same complexes. This was associated with activation of gene programs related to DNA repair, oxidative stress, and protein ubiquination, all of which were attenuated by MT treatment along with radiation-induced increases in JO2 and JH2O2. IR also increased secreted levels of interleukin-8 and Type I collagens, while decreasing Type VI collagens and enzymes that coordinate assembly and remodeling of the extracellular matrix. MT treatment attenuated many of these effects while augmenting others, revealing complex effects of mtROS in fibroblast responses to IR. Conclusion: These results implicate mtROS production in fibroblast responses to protracted radiation exposure, and suggest potentially protective effects of mitochondrial-targeted antioxidants against radiogenic tissue injury in vivo.

Combined inhibition of IL-1, IL-33 and IL-36 signalling by targeting IL1RAP ameliorates skin and lung fibrosis in preclinical models of systemic sclerosis

BackgroundThe interleukin (IL)-1 receptor accessory protein (IL1RAP) is an essential coreceptor required for signalling through the IL-1, IL-33 and IL-36 receptors. Here, we investigate the antifibrotic potential of the combined...

The Remodulation of Actin Bundles during the Stimulation of Mitochondria in Adult Human Fibroblasts in Response to Light.

β-actin belongs to cytoskeletal structures that change dynamically in cells according to various stimuli. Human skin can be considered as an organ that is very frequently exposed to various stress factors, of which light plays an important role. The present study focuses on adult human fibroblasts exposed to two types of light stress. Orange light with a wavelength of 590 nm was used here to stimulate the photosensitizer localized in the cells as a residual dose of photodynamic therapy (PDT). On the other hand, near-infrared light with a wavelength of 808 nm was considered for photobiomodulation (PBM), which is often used in healing processes. Confocal fluorescence microscopy was used to observe changes in intercellular communication, mitochondrial structures, and cytoskeletal dynamics defined by the remodulation of β-actin of fibroblasts. The number of β-actin bundles forming spherical structures was detected after light exposure. These structures as β-actin oligomers were confirmed with super-resolution microscopy. While PDT led to the disintegration of actin oligomers, PBM increased their number. The interaction of β-actin with mitochondria was observed. The combination of PDT and PBM treatments is important to minimize the side effects of cancer treatment with PDT on healthy cells, as shown by the cell metabolism assay in this work. In this work, β-actin is presented as an important parameter that changes and is involved in the response of cells to PDT and PBM.

Decoding the role of m6A Regulators in identifying and characterizing molecular subtypes of rosacea

Rosacea is a common skin disease that predominantly affects individuals aged between 30 and 50 years. While the exact cause of the disease remains unclear, various factors have been shown to trigger or exacerbate its symptoms. N6-methyladenosine (m6A) modification is one of the most abundant epigenetic methylation modification in messenger RNA (mRNA) and non-coding RNA (ncRNA), plays a crucial role in RNA splicing, export, stability, and translation. In this study, we aimed to characterize m6A genes in rosacea, identify molecular subtypes based on m6A gene expression, characterize the immune features among subtypes, explore key molecules based on co-expression analysis, and identify potential targets and drugs. To achieve our objectives, we first compared the expression pattern and immune regulation of m6A genes between healthy and diseased groups. Then, we performed clustering to stratify disease samples into different subtypes and analyzed immune regulation and functional enrichment among the subtypes. Then, we conducted differential analysis between subtypes and applied weighted gene co-expression network analysis (WGCNA) in three subtypes. We found hub differential expression analysis (DEG) genes and their potential drug based on the WGCNA results and the drug-gene interaction database (DGIdb). Finally, in vivo and in vitro studies showed significant differences in m6A methyltransferase METTL3 levels in rosacea mice and control mice, as well as in the skin of rosacea patients and healthy people, while reducing METTL3 significantly inhibited the inflammatory response of human fibroblasts (HDFs) stimulated by LL37, suggesting that METTL3 may be associated with changes in overall m6A levels in rosacea. Taken together, our findings provide valuable insights into therapeutic targets and drug predictions for rosacea.

Mapping interindividual dynamics of innate immune response at single-cell resolution

Common genetic variants across individuals modulate the cellular response to pathogens and are implicated in diverse immune pathologies, yet how they dynamically alter the response upon infection is not well understood. Here, we triggered antiviral responses in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-sequencing. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), a statistical approach designed to identify nonlinear dynamic genetic effects across transcriptional trajectories of cells. This approach identified 1,275 expression quantitative trait loci (local false discovery rate 10%) that manifested during the responses, many of which were colocalized with susceptibility loci identified by genome-wide association studies of infectious and autoimmune diseases, including the OAS1 splicing quantitative trait locus in a COVID-19 susceptibility locus. In summary, our analytical approach provides a unique framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

In-Vitro Study of Indium (III) Sulfate-Containing Medium on the Viability and Adhesion Behaviors of Human Dermal Fibroblast on Engineered Surfaces.

Tissues and organs consist of cells organized in specified patterns that support their function, as exemplified by tissues such as skin, muscle, and cornea. It is, therefore, important to understand how external cues, such as engineered surfaces or chemical contaminants, can influence the organization and morphology of cells. In this work, we studied the impact of indium sulfate on human dermal fibroblast (GM5565) viability, production of reactive oxygen species (ROS), morphology, and alignment behavior on tantalum/silicon oxide parallel line/trench surface structures. The viability of cells was measured using the alamarBlue™ Cell Viability Reagent probe, while the ROS levels in cells were quantified using cell-permeant 2',7'-dichlorodihydrofluorescein diacetate. Cell morphology and orientation on the engineered surfaces were characterized using fluorescence confocal and scanning electron microscopy. When cells were cultured in media containing indium (III) sulfate, the average cell viability decreased by as much as ~32% and the concentration of cellular ROS increased. Cell geometry became more circular and compact in the presence of indium sulfate. Even though actin microfilaments continue to preferentially adhere to tantalum-coated trenches in the presence of indium sulfate, the cells are less able to orient along the line axes of the chips. Interestingly, the indium sulfate-induced changes in cell alignment behavior are pattern dependent-a larger proportion of adherent cells on structures with line/trench widths in the range of 1 μm and 10 μm lose the ability to orient themselves, compared to those grown on structures with line widths smaller than 0.5 μm. Our results show that indium sulfate impacts the response of human fibroblasts to the surface structure to which they adhere and underscores the importance of evaluating cell behaviors on textured surfaces, especially in the presence of potential chemical contaminants.

A Review on the Heightened Mechanical Features of Nanosilica-Based Concrete and the Response of Human Fibroblasts to Nanosilica

Cement is utilized extensively in the manufacturing of concrete, which makes it the most common material used in building construction. However, the usage of a great deal of cement results in a great deal of CO2 emissions, which leads to the greenhouse effect. Numerous studies have developed the use of nano-SiO2 in concrete materials to lower the cement content of concrete mixtures while improving mechanical properties. Additionally, a number of studies have demonstrated that silica NPs trigger an inflammatory response in pulmonary fibroblasts. The main cells that produce and maintain the extracellular matrix (ECM) in the connection of the tissue are fibroblasts. Fibroblasts are involved in processes including tissue regeneration and wound healing. Similar to angiogenesis, inflammation, cancer, and pathological and physiological tissue fibrosis, fibroblasts act as intermediaries. The effect of silica nanoparticles on the mechanical properties of concrete (compressive strength, split tensile strength, and flexural strength) was succinctly presented in this paper. Likewise, a number of studies on the reaction of human fibroblasts to silica nanoparticles were evaluated. Numerous research on the addition of silica nanoparticles to concrete revealed that doing so significantly enhanced the material's mechanical properties. The controlled interaction of silica nanoparticles with human fibroblast cells was demonstrated to have potential in a number of applications, including aesthetics, intracellular drug release systems, improving scar tissue, determining the fate of biomaterials in vivo, and designing potential prosthetics and implant surfaces to reduce bacterial adhesion.

Cellular fractionation reveals transcriptome responses of human fibroblasts to UV-C irradiation

While cells activate a multifaceted DNA damage response to remove transcription-blocking DNA lesions, mechanisms to regulate genome-wide reduction of RNA synthesis and the paradoxical continuous loading of RNAP II at initiation sites are still poorly understood. Uncovering how dramatic changes to the transcriptional program contribute to TC-NER (transcription-coupled nucleotide excision repair) is important in DNA repair research. However, the functional significance of transcriptome dynamics and the mechanisms of chromatin attachment for thousands of unstudied human lncRNAs remain unclear. To address these questions, we examined UV-induced gene expression regulation in human fibroblasts by performing RNA-seq with fractionated chromatin-associated and cytoplasmic transcripts. This approach allowed us to separate the synthesis of nascent transcripts from the accumulation of mature RNAs. In addition to documenting the subcellular locations of coding transcripts, our results also provide a high-resolution view of the transcription activities of noncoding RNAs in response to cellular stress. At the same time, the data showed that vast majority of genes exhibit large changes in chromatin-associated nascent transcripts without corresponding changes in cytoplasmic mRNA levels. Distinct from protein-coding genes that transcripts with shorter length prefer to be recovered first, repression of lncRNA transcription after UV exposure is inactivated first on noncoding transcripts with longer length. This work provides an updated framework for cellular RNA organization in response to stress and may provide useful information in understanding how cells respond to transcription-blocking DNA damage.

ATM-Mediated Mitochondrial Radiation Responses of Human Fibroblasts.

Ataxia telangiectasia (AT) is characterized by extreme sensitivity to ionizing radiation. The gene mutated in AT, Ataxia Telangiectasia Mutated (ATM), has serine/threonine protein kinase activity and mediates the activation of multiple signal transduction pathways involved in the processing of DNA double-strand breaks. Reactive oxygen species (ROS) created as a byproduct of the mitochondria’s oxidative phosphorylation (OXPHOS) has been proposed to be the source of intracellular ROS. Mitochondria are uniquely vulnerable to ROS because they are the sites of ROS generation. ROS-induced mitochondrial mutations lead to impaired mitochondrial respiration and further increase the likelihood of ROS generation, establishing a vicious cycle of further ROS production and mitochondrial damage. AT patients and ATM-deficient mice display intrinsic mitochondrial dysfunction and exhibit constitutive elevations in ROS levels. ATM plays a critical role in maintaining cellular redox homeostasis. However, the precise mechanism of ATM-mediated mitochondrial antioxidants remains unclear. The aim of this review paper is to introduce our current research surrounding the role of ATM on maintaining cellular redox control in human fibroblasts. ATM-mediated signal transduction is important in the mitochondrial radiation response. Perturbation of mitochondrial redox control elevates ROS which are key mediators in the development of cancer by many mechanisms, including ROS-mediated genomic instability, tumor microenvironment formation, and chronic inflammation.

The C0-C1f Region of Cardiac Myosin Binding Protein-C Induces Pro-Inflammatory Responses in Fibroblasts via TLR4 Signaling.

Myocardial injury is associated with inflammation and fibrosis. Cardiac myosin-binding protein-C (cMyBP-C) is cleaved by µ-calpain upon myocardial injury, releasing C0-C1f, an N-terminal peptide of cMyBP-C. Previously, we reported that the presence of C0-C1f is pathogenic within cardiac tissue and is able to activate macrophages. Fibroblasts also play a crucial role in cardiac remodeling arising from ischemic events, as they contribute to both inflammation and scar formation. To understand whether C0-C1f directly modulates fibroblast phenotype, we analyzed the impact of C0-C1f on a human fibroblast cell line in vitro by performing mRNA microarray screening, immunofluorescence staining, and quantitative real-time PCR. The underlying signaling pathways were investigated by KEGG analysis and determined more precisely by targeted inhibition of the potential signaling cascades in vitro. C0-C1f induced pro-inflammatory responses that might delay TGFβ-mediated myofibroblast conversion. TGFβ also counteracted C0-C1f-mediated fibroblast activation. Inhibition of TLR4 or NFκB as well as the delivery of miR-146 significantly reduced C0-C1f-mediated effects. In conclusion, C0-C1f induces inflammatory responses in human fibroblasts that are mediated via TRL4 signaling, which is decreased in the presence of TGFβ. Specific targeting of TLR4 signaling could be an innovative strategy to modulate C0-C1f-mediated inflammation.

In Vitro Evidences of Different Fibroblast Morpho-Functional Responses to Red, Near-Infrared and Violet-Blue Photobiomodulation: Clues for Addressing Wound Healing

Although photobiomodulation (PBM) has proven promising to treat wounds, the lack of univocal guidelines and of a thorough understanding of light–tissue interactions hampers its mainstream adoption for wound healing promotion. This study compared murine and human fibroblast responses to PBM by red (635 ± 5 nm), near-infrared (NIR, 808 ± 1 nm), and violet-blue (405 ± 5 nm) light (0.4 J/cm2 energy density, 13 mW/cm2 power density). Cell viability was not altered by PBM treatments. Light and confocal laser scanning microscopy and biochemical analyses showed, in red PBM irradiated cells: F-actin assembly reduction, up-regulated expression of Ki67 proliferation marker and of vinculin in focal adhesions, type-1 collagen down-regulation, matrix metalloproteinase-2 and metalloproteinase-9 expression/functionality increase concomitant to their inhibitors (TIMP-1 and TIMP-2) decrease. Violet-blue and even more NIR PBM stimulated collagen expression/deposition and, likely, cell differentiation towards (proto)myofibroblast phenotype. Indeed, these cells exhibited a higher polygonal surface area, stress fiber-like structures, increased vinculin- and phospho-focal adhesion kinase-rich clusters and α-smooth muscle actin. This study may provide the experimental groundwork to support red, NIR, and violet-blue PBM as potential options to promote proliferative and matrix remodeling/maturation phases of wound healing, targeting fibroblasts, and to suggest the use of combined PBM treatments in the wound management setting.

Effects of the signal modulation on the response of human fibroblasts to in vitro stimulation with subthermal RF currents

ABSTRACT Capacitive-Resistive Electric Transfer (CRET) thermotherapies aim at tissue repair and regeneration through non-invasive application of RF currents. We have reported that the cellular response to subthermal CRET currents is non-linearly dependent on the signal frequency, and that in vitro exposure to a 448-kHz CRET signal promotes ADSC proliferation, as well as collagen and glycosaminoglycan synthesis in prechondrocytic cells. The present work investigates the response of neonatal fibroblasts to subthermal exposure (100 µA/mm2) to two CRET signals: a 448-kHz, non-modulated sinusoidal wave vs. a 20-kHz amplitude-modulation of the 448-kHz carrier. To that end, cell proliferation and expression of the biomarkers Hsp47, Hsp27 and decorin were assessed by cell count, PCNA and Western blotting. The results revealed that while both signals significantly and equivalently increased early (4 h) expression of Hsp47, the modulated signal was more efficient in inducing Hsp27 and decorin overexpression and promoting cell proliferation. These data indicate that the cellular response is dependent on the RF signal modulation and suggest that the therapeutic effects of CRET could be mediated by promotion of fibroblastic proliferation and overexpression of biomarkers that are essential in skin regeneration.

Human fibroblast response to Crotalus atrox venom and model wound healing

Human fibroblast response to Crotalus atrox venom and model wound healing

Novel Xenografting Model to Explore the Mechanisms Mediating Acute and Chronic Fibrosis in Human Skin Fibroblasts

INTRODUCTION: Human scar formation and fibrosis are difficult to accurately recapitulate using mouse models, given the significant anatomical and physiologic differences between mouse and human skin. Xenografts of human skin on immunodeficient mice provide an accessible means of assessing human skin’s physiology and response to wounding or fibrosis-inducing conditions in vivo. However, current xenograft models are limited by poor engraftment rates and inability to specifically explore the mechanisms mediating fibrosis in human fibroblasts. OBJECTIVE: We describe a novel skin xenografting model to investigate the response of human dermal fibroblasts to different fibrosis-promoting conditions. METHODS: Full-thickness circular 8-mm human foreskin samples were sutured into the dorsum of P2 immunocompromised (NSG) mice as subcutaneous grafts (n = 30), and surgically exposed after 7 days to produce cutaneous grafts. Successful engraftment and preservation of normal skin physiology were confirmed by histology. Machine learning–based assessment of collagen fiber networks from stained skin histology specimens was achieved using a novel computational algorithm developed by our laboratory. To study the acute fibrotic response, 4-mm partial-thickness wounds were created within the xenografts using a biopsy punch; wounds were monitored until closure. To explore the chronic fibrotic response, xenografted skin was irradiated with 30 Gy fractionated into six 5 Gy doses delivered every other day for a total of 12 days. Following radiation, chronic fibrotic changes were allowed to develop over an interval of 4 weeks. At the respective endpoints, xenografted skin was harvested for histology. Human fibroblasts were isolated using flow cytometry with a negative gating strategy to exclude mouse and human hematopoietic cells (CD45−/Ter-119−/CD235a−), endothelial cells (CD31−), and epithelial cells (CD326−[mEpCam]/mCD324−[E-Cadherin]), and a positive gate to include only human fibroblasts (CD90+). Microarray analyses were used to compare gene expression of human fibroblasts isolated from scarred/irradiated xenografts to those from unwounded/nonirradiated xenografts. RESULTS: Xenografted foreskin was structurally similar to native (ungrafted) foreskin on histology. Collagen fiber network analysis confirmed that xenografted skin was more similar to foreskin than to scarred adult human skin. Wounds created in xenografted skin exhibited slower wound closure compared to stented mouse wounds, indicating healing primarily via formation of granulation tissue (akin to human skin) rather than contraction (typical of mouse skin). Irradiated skin was significantly indurated on histologic assessment, consistent with chronic irradiation damage/fibrosis. Immunofluorescence staining confirmed successful xenograft vascularization and survival of human skin cells and human origin of granulation tissue. Gene expression analysis of fibroblasts isolated from acutely and chronically fibrosed xenografts revealed upregulation of the Wnt and FAK pathways and increased expression of the CD26 surface antigen. CONCLUSIONS: We present a novel foreskin xenografting model and demonstrate its utility in specifically investigating the in vivo human fibroblast response in acute and chronic fibrosis. This model provides an accessible and informative tool to aid in elucidation of fibroblast-driven mechanisms responsible for scarring and fibrosis. Ultimately, this work may enable the discovery of novel cellular and molecular targets to reduce skin scarring and fibrosis.

Controlling fibroblast adhesion and proliferation by 1D Al2O3 nanostructures.

The fibrotic encapsulation, which is mainly accompanied by an excessive proliferation of fibroblasts, is an undesired phenomenon after the implantation of various medical devices. Beside the surface chemistry, the topography plays also a major role in the fibroblast-surface interaction. In the present study, one-dimensional aluminium oxide (1D Al2O3) nanostructures with different distribution densities were prepared to reveal the response of human fibroblasts to the surface topography. The cell size, the cell number and the ability to form well-defined actin fibres and focal adhesions were significantly impaired with increasing distribution density of the 1D Al2O3 nanostructures on the substratum.

The impact of clay-based hypoxia mimetic hydrogel on human fibroblasts of the periodontal soft tissue

Thixotropic clays have favorable properties for tissue regeneration. Hypoxia mimetic agents showed promising results in pre-clinical models for hard and soft tissue regeneration. It is unclear if clays can be used as carrier for hypoxia mimetic agent in a periodontal regenerative setting. Here, we tested the response of human fibroblasts of the periodontal soft tissue to synthetic clay hydrogels and assessed hypoxia mimetic agent release. Cells were cultured on synthetic clay hydrogels (5.00%-0.15%). We assessed viability and differentiation capacity with resazurin-based toxicity assays, MTT staining, Live-Dead staining, and alkaline phosphatase staining. To reveal the response of fibroblasts to hypoxia mimetic agent-loaded clay hydrogels, cells were exposed to clay supplemented with dimethyloxalylglycine, deferoxamine, l-mimosine, and CoCl2. Supernatants from hypoxia mimetic agent-loaded clay hydrogels were harvested and replaced with medium at hour 1, 3, 6, 24, 48, and 72. To reveal the hypoxia mimetic capacity of supernatants, vascular endothelial growth factor production in the fibroblasts was assessed in the culture medium. Our data show that clay did not induce relevant toxic effects in the fibroblasts which remained capable to differentiate into alkaline phosphatase-positive cells at the relevant concentrations. Fibroblasts cultured on clay hydrogel loaded with dimethyloxalylglycine, deferoxamine, l-mimosine, and CoCl2 remained vital, however, no significant increase in vascular endothelial growth factor levels was found in the culture medium. Only dimethyloxalylglycine-loaded clay supernatants taken in the first hours stimulated vascular endothelial growth factor production in fibroblasts. In conclusion no pronounced toxic effects of synthetic clay were observed. Supplementation with dimethyloxalylglycine leads to hypoxia mimetic activity. This pilot study provides first insights into the impact of synthetic clay on periodontal tissue.

Surface Nanocoating with Plant‐Derived Pectins Improves Fibroblast Response In Vitro

Pectins, plant‐derived polysaccharides, are novel candidates for biomaterial nanocoatings. Their chemical structure imitates the polysaccharides from the extracellular matrix of mammals, providing bio‐specific cell adhesion. It is possible to control the structure of pectin rhamnogalacturonan‐I (RG‐I) by enzymatic modification. RG‐Is has been proposed for surface nanocoating of orthopedic and dental titanium implants due to its effect on osseointegration with stimulation of bone and connective tissue healing. The aim of the current study is to evaluate in vitro, the impact of plant‐derived pectin RG‐I nanocoating on the cellular response of human fibroblasts. In this study, unmodified RG‐I and enzymatically modified RG‐I with low amount of arabinose and high amount of galactose are examined. All in vitro studies are performed on tissue culture polystyrene plates, whose surfaces are coated with unmodified and modified RG‐I. The results show that the nanocoating with pectin RG‐Is increas proliferation, cell metabolic activity, and gene expression of COL1A1, FN1, FGFR1, BMP7, and MMP2 as compared to control surface, without pectin coatings. The results show that nanocoating with RG‐Is has the capacity to improve fibroblast proliferation, differentiation, and extracellular matrix protein production, and may therefore be considered as a potential candidate for further improvement of bone and connective tissue regeneration.

A simple way of modulating in vitro angiogenic response using Cu and Co-doped bioactive glasses

The copper and cobalt-doped bioactive glasses (BGs) from the basic SiO2–CaO–P2O5 system, differing in the CaO/SiO2 molar ratios, were produced using sol-gel method. The in vitro angiogenic response of human fibroblasts and umbilical vein endothelial cells (HUVECs) on Cu-BGs and Co-BGs was investigated. The incorporation of Cu and Co into BGs significantly enhanced vascular endothelial growth factor secretion from fibroblasts. Cu-BGs and Co-BGs extracts might facilitate the migration of HUVECs and the self-assembled network formation after 3 days of culture without basement membrane matrix, demonstrating strong angiogenic activity. The results show that cellular response was correlated with Cu and Co release rate which, in turn, was affected by the CaO/SiO2 molar ratios, indicating new opportunities for controlling the release rate of therapeutic ions from BGs.

PD50-10 GENE EXPRESSION AND PATTERNS OF SCARRING RESPONSE IN HUMAN FIBROBLASTS IN RESPONSE TO MESH AND CATHETER MATERIALS USING A NOVEL 3-D COLLAGEN MODEL

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Female Incontinence: Therapy II1 Apr 2017PD50-10 GENE EXPRESSION AND PATTERNS OF SCARRING RESPONSE IN HUMAN FIBROBLASTS IN RESPONSE TO MESH AND CATHETER MATERIALS USING A NOVEL 3-D COLLAGEN MODEL Li Yunyuan, Lynn Stothers, and Aziz Ghahary Li YunyuanLi Yunyuan More articles by this author , Lynn StothersLynn Stothers More articles by this author , and Aziz GhaharyAziz Ghahary More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2222AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Scarring secondary to mesh and prosthetic materials is a serious clinical problem within the GU tract. Fibrotic matrices contain fibronectin; and alpha-smooth muscle actin contributes contraction. Metalloproteinases (MMPs) such as MMP-1 and -3 can modulate matrix protein accumulation through degradation. Results from our institution have shown that silica materials can directly induce scarring through the interaction with tissue fibroblasts in vitro. By extension, we hypothesized that other materials may induce fibrotic changes through cellular matrix gene expression. Objectives: 1) to establish a 3D model of human fibroblasts to study patterns of fibroblast response to materials and 2) measure gene expression in human fibroblasts exposed to prosthetic and mesh materials compared to a control. METHODS Collagen gel was prepared by using 3 mg/ml in final concentration with 0.5% of polyvinyl alcohol (PVA). Mesh or catheter materials and human dermal fibroblasts (70,000 /ml) were added to a collagen gel and seeded in a 24-well plate (0.5 ml of gel in each well) to create a 3D environment for fibroblast response. After polymerization of collagen, another 250,000 cells in 0.5 ml medium were added on the top of gel. Cells were cultured at 37 0C, 5% CO2 for indicated time point. Images of cells were taken under reverse microscopy to determine the pattern of the scarring contraction. Gel cell matrix was harvested and digested with 1 mg/ml of collagenase for 15 minutes, pelleted by centrifugation and RNA was extracted. RT-PCR was performed for 32 cycles to analyze gene expression. RESULTS After 5 days, fibroblast contractions were identified surrounding prosthetic materials but not within the control. There were increases in type 1 collagen, a-smooth muscle actin and fibronectin expression in fibroblasts exposed to prosthetic materials compared to fibroblasts grown in collagen gel alone. MMP-1 and MMP-3 were also detected. CONCLUSIONS Fibroblasts exposed to mesh and catheter materials responded with an increase in fibronectin, alpha smooth muscle actin and type 1 collagen that is increased compared to controls. This may indicate why in vivo these materials induce fibrosis. Because fibronectin, type 1 collagen and alpha smooth muscle actin are main components of scarring and their gene expression is elevated, future directions include development of medical devices that could induce downregulation of these genes. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e983 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Li Yunyuan More articles by this author Lynn Stothers More articles by this author Aziz Ghahary More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

BK polyomavirus infection activates the type I interferon response in human fibroblasts, depending on the cGAS-STING pathway

BK polyomavirus infection activates the type I interferon response in human fibroblasts, depending on the cGAS-STING pathway