Human Fibroblast Strains Research Articles

Analysis of genetic variant associated with heart failure mortality implicates thymic stromal lymphopoietin as mediator of strain-induced myocardial fibroblast-mast cell crosstalk and fibrosis.

Heart failure (HF) is a leading cause of death and disability globally. Heritable factors and the extent and pattern of myocardial fibrosis are important determinants of outcomes in patients with HF. In a genome-wide association study of mortality in HF, we recently identified a genetic polymorphism on chromosome 5q22 associated with HF mortality. Here, we sought to study the mechanisms by which this variant may influence myocardial disease processes. We find that the risk allele is located in an enhancer motif upstream of the TSLP gene (encoding thymic stromal lymphopoietin), conferring increased binding of the transcription factor nescient helix-loop helix 1 (NHLH1) and increased TSLP expression in human heart. Further, we find that increased strain of primary human myocardial fibroblasts results in increased TSLP expression and that the TSLP receptor is expressed in myocardial mast cells in human single nuclei RNA sequence data. Finally, we show that TSLP overexpression induces increased transforming growth factor β expression in myocardial mast cells and tissue fibrosis. Collectively, our findings based on follow-up of a human genetic finding implicate a novel pathway in myocardial tissue homeostasis and remodeling.

Synthesis, cytotoxic activity evaluation and mechanistic investigation of novel 3,7-diarylsubstituted 6-azaindoles

A number of new disubstituted 6-azaindoles have been designed and synthesized bearing a crucial structural modification in respect to an analogous antiproliferative hit compound. The synthesis was performed using 2-amino-3-nitro-4-picoline, that was suitably modified and converted to 7-chloro-3-iodo-6-azaindole and this central scaffold was used for successive Suzuki-type couplings, to result in the target compounds. The evaluation of the cytotoxic activity was performed against four human cancer cell lines, as well as a normal human fibroblast strain. Certain compounds possessed strong anticancer activity without affecting normal cells. At subcytotoxic concentrations for cancer cells, these compounds displayed an anti-proliferative effect by arresting the cells at the G2/M phase of the cell cycle, which could be associated with the observed decrease in the phosphorylation levels of the MEK1- ERK1/2 pathway and/or the activation of the p53-p21WAF1 axis.

Antimicrobial Activity of Aspergillus sp. from the Amazon Biome: Isolation of Kojic Acid.

The antimicrobial potential of Aspergillus sp., isolated from the Amazon biome, which is stored at the Amazon Fungi Collection-CFAM at ILMD/FIOCRUZ, was evaluated. The fungal culture was cultivated in yeast extract agar and sucrose (YES) for cold extraction of the biocompounds in ethyl acetate at 28 °C for 7 days in a BOD type incubator. The obtained extract was evaluated for its antimicrobial activity against Candida albicans and Gram-positive and negative bacteria by the “cup plate” method and the determination of the minimum inhibitory concentration (MIC) by the broth microdilution method. The extract was subjected to thin layer chromatography (TLC) and fractionated by open and semipreparative column chromatography. The fractions of interest had their chemical constituents elucidated by nuclear magnetic resonance and mass spectrometry. The elucidated molecule was evaluated for cytotoxicity against the human fibroblast strain (MRC5). The extract presented inhibitory activity against both Gram-positive and negative bacteria, with the range of inhibition halos from 5.3 to 14 mm in diameter and an MIC ranging from 500 to 15.6 μg/mL. Seventy-one fractions were collected and TLC analysis suggested the presence of substances with double bond groups: coumarins, flavonoids, phenolic, alkaloids, and terpenes. NMR and MS analyses demonstrated that the isolated molecule was kojic acid. The results of the cytotoxicity test showed that MRC5 cells presented viability at concentrations from 500 to 7.81 μg/mL. The kojic acid molecule of Aspergillus sp., with antibacterial activity and moderate toxicity at the concentrations tested, is a promising prototype of an alternative active principle of an antimicrobial drug.

In silico stress fibre content affects peak strain in cytoplasm and nucleus but not in the membrane for uniaxial substrate stretch.

Existing in silico models for single cell mechanics feature limited representations of cytoskeletal structures that contribute substantially to the mechanics of a cell. We propose a micromechanical hierarchical approach to capture the mechanical contribution of actin stress fibres. For a cell-specific fibroblast geometry with membrane, cytoplasm and nucleus, the Mori-Tanaka homogenization method was employed to describe cytoplasmic inhomogeneities and constitutive contribution of actin stress fibres. The homogenization was implemented in a finite element model of the fibroblast attached to a substrate through focal adhesions. Strain in cell membrane, cytoplasm and nucleus due to uniaxial substrate stretch was assessed for different stress fibre volume fractions and different elastic modulus of the substrate. A considerable decrease of the peak strain with increasing stress fibre content was observed in cytoplasm and nucleus but not the membrane, whereas the peak strain in cytoplasm, nucleus and membrane increased for increasing elastic modulus of the substrate. Finite element mesh of reconstructed human fibroblast and intracellular strain distribution in cell subjected to substrate stretch.

Gene Expression Profiles Reveal Extracellular Matrix and Inflammatory Signaling in Radiation-Induced Premature Differentiation of Human Fibroblast in vitro.

PurposeFibroblasts are considered to play a major role in the development of fibrotic reaction after radiotherapy and premature radiation-induced differentiation has been proposed as a cellular basis. The purpose was to relate gene expression profiles to radiation-induced phenotypic changes of human skin fibroblasts relevant for radiogenic fibrosis.Materials and MethodsExponentially growing or confluent human skin fibroblast strains were irradiated in vitro with 1–3 fractions of 4 Gy X-rays. The differentiated phenotype was detected by cytomorphological scoring and immunofluorescence microscopy. Microarray analysis was performed on Human Genome U133 plus2.0 microarrays (Affymetrix) with JMP Genomics software, and pathway analysis with Reactome R-package. The expression levels and kinetics of selected genes were validated with quantitative real-time PCR (qPCR) and Western blotting.ResultsIrradiation of exponentially growing fibroblast with 1 × 4 Gy resulted in phenotypic differentiation over a 5-day period. This was accompanied by downregulation of cell cycle-related genes and upregulation of collagen and other extracellular matrix (ECM)-related genes. Pathway analysis confirmed inactivation of proliferation and upregulation of ECM- and glycosaminoglycan (GAG)-related pathways. Furthermore, pathways related to inflammatory reactions were upregulated, and potential induction and signaling mechanisms were identified. Fractionated irradiation (3 × 4 Gy) of confluent cultures according to a previously published protocol for predicting the risk of fibrosis after radiotherapy showed similar downregulation but differences in upregulated genes and pathways.ConclusionGene expression profiles after irradiation of exponentially growing cells were related to radiation-induced differentiation and inflammatory reactions, and potential signaling mechanisms. Upregulated pathways by different irradiation protocols may reflect different aspects of the fibrogenic process thus providing a model system for further hypothesis-based studies of radiation-induced fibrogenesis.

Novel resveratrol derivatives have diverse effects on the survival, proliferation and senescence of primary human fibroblasts.

Resveratrol alters the cytokinetics of mammalian cell populations in a dose dependent manner. Concentrations above 25-50µM typically trigger growth arrest, senescence and/or apoptosis in multiple different cell types. In contrast, concentrations below 10µM enhance the growth of log phase cell cultures and can rescue senescence in multiple strains of human fibroblasts. To better understand the structural features that regulate these effects, a panel of 24 structurally-related resveralogues were synthesised and evaluated for their capacity to activate SIRT1, as determined by an ex-vivo SIRT1 assay, their toxicity, as measured by lactate dehydrogenase release, and their effects on replicative senescence in MRC5 human fibroblasts as measured by their effects on Ki67 immunoreactivity and senescence-associated β galactosidase activity.Minor modifications to the parent stilbene, resveratrol, significantly alter the biological activities of the molecules. Replacement of the 3,5-dihydroxy substituents with 3,5-dimethoxy groups significantly enhances SIRT1 activity, and reduces toxicity. Minimising other strong conjugative effects also reduces toxicity, but negatively impacts SIRT1 activation.At 100µM many of the compounds, including resveratrol, induce senescence in primary MRC5 cells in culture. Modifications that reduce or remove this effect match those that reduce toxicity leading to a correlation between reduction in labelling index and increase in LDH release.At 10µM, the majority of our compounds significantly enhance the growth fraction of log phase cultures of MRC5 cells, consistent with the rescue of a subpopulation of cells within the culture from senescence. SIRT1 activation is not required for rescue to occur but enhances the size of the effect.

The prognostic significance of fibroblast activation protein-α in human lung adenocarcinoma.

BackgroundFibroblast activation protein (FAP) is a type II cell surface-bound integral serine protease, which is an important biomarker of cancer-associated fibroblasts. FAP-α performs several biological activities, including remolding extracellular matrix and acting as an immunosuppressor in the tumor microenvironment. However, the proliferation role of FAP-α in human lung adenocarcinoma has not been fully elucidated.MethodsThe expression of FAP-α in 94-paired human lung adenocarcinoma tissues was identified by immunohistochemistry test. The effect of FAP on cell proliferation was examined by CCK-8 assay. RNA-sequencing and bioinformatics analysis were utilized to investigate the underlying mechanism. Western blot analysis, quantitative polymerase chain reaction (qPCR), and nude mice experiments, were also conducted for further validation.ResultsThe proliferation rates of human fibroblast strains FAP-HFF and FAP-BJ, and human lung adenocarcinoma cell line FAP-SPC-A-1 were higher than those of controls. The nude mice experiment also showed that FAP could promote the proliferation of SPC-A-1 cell line in vivo. qPCR and Western blot analysis indicated that CCNB1 was upregulated by the overexpression of FAP in the lung adenocarcinoma cell line. The expression of FAP-α was higher in both the cytoplasm and stroma of lung adenocarcinoma than in adjacent normal tissues. Survival analysis indicated that patients with higher expression of FAP-α in tumor stroma had a poor prognosis (P=0.019). The Cancer Genome Atlas Program (TCGA) data also showed that the expression of FAP within tumor tissues was higher (in both cytoplasm and stroma) compared with that in normal tissues (P<0.05).ConclusionsOur study indicates that FAP-α could facilitate the proliferation of lung adenocarcinoma cells and can be a prognostic marker in human lung adenocarcinoma.

Assessment of the anti-snakebite properties of extracts of Aniba fragrans Ducke (Lauraceae) used in folk medicine as complementary treatment in cases of envenomation by Bothrops atrox

Ethnopharmacological relevanceExtracts of leaves and bark of Aniba fragrans are used as tea (decoction) to treat snakebites in communities in the Brazilian Amazon. The aqueous extract of the leaves of A. fragrans has been proven to be effective against Bothrops venom, but only when pre-incubated with the venom. This study sought to assess the potential of different types of extract of this species to inhibit the biological activities of Bothrops atrox venom (BaV) when used the same way as in folk medicine. The main classes of secondary metabolites and the concentrations of phenolics in the extracts were also determined. Materials and methodsFour types of extract of A. fragrans were prepared: aqueous extract of the leaf (AEL), aqueous extract of the bark (AEB), hydroalcoholic leaf extract (HLE) and extract of the residue from hydrodistillation of the leaf (ERHL). The phytochemical profiles of the aqueous extracts were determined using thin layer chromatography (TLC), and the concentrations of phenolics were measured by colorimetric assays. To investigate the potential of the extracts to inhibit the biological activities of BaV, in vitro tests for antiphospholipase and antifibrinolytic activities were performed. In vivo tests for antihemorrhagic and antidefibrinating activities were also carried out, as well as antimicrobial tests for activity against the main bacteria found in the oral cavity of snakes. Interaction between the extracts and the proteins in BaV was assessed by electrophoresis (SDS-PAGE) and Western blot (WB). The cytotoxicity of the extracts was assessed in a strain of MRC-5 human fibroblasts. ResultsTerpenoids, flavonoids and condensed and hydrolysable tannins were detected in all the extracts. Metabolites such as coumarins, fatty acids and alkaloids were present in some extracts but not in others, indicating different phytochemical profiles. Phenolics content varied between extracts, and there were more tannins in AEB and HLE. In the in vitro tests, the extracts inhibited the phospholipase and fibrinolytic activities of BaV in the two ratios of venom to extract used. HLE exhibited effective antimicrobial action as it inhibited growth of 11 of the 15 bacteria investigated, including Morganella morganii, the main bacteria described in the oral cavity of snakes. The extracts failed to inhibit the defibrinating activity of BaV, and only the Bothrops antivenom had a significant effect (96.1%) on this activity. BaV-induced hemorrhage was completely inhibited by AEL and AEB when the pre-incubation (venom:extract) protocol was used. When administered orally, as in folk medicine, both AEB and AEL produced significant inhibition of hemorrhagic activity (maximum inhibition 46.5% and 39.2%, respectively). SDS-PAGE and WB of the extracts pre-incubated with BaV showed that the main proteins in the venom had been precipitated by the extracts. None of the four extracts showed cytotoxic effects in the tests carried out with a human fibroblast cell line. ConclusionIn addition to being effective in reducing hemorrhage when administered orally, the extracts displayed a high antimicrobial potential against microorganisms involved in secondary infections at the site of the snakebite. Once the extracts have been tested in accordance with the appropriate regulations, this species could potentially be used to produce a phytomedicine for complementary treatment of the secondary infections due to bacteria that aggravate the local signs and symptoms after snakebite envenomation.

Apigenin suppresses the senescence-associated secretory phenotype and paracrine effects on breast cancer cells.

Apigenin (4',5,7,-trihydroxyflavone) is a flavonoid found in certain herbs, fruits, and vegetables. Apigenin can attenuate inflammation, which is associated with many chronic diseases of aging. Senescent cells-stressed cells that accumulate with age in mammals-display a pro-inflammatory senescence-associated secretory phenotype (SASP) that can drive or exacerbate several age-related pathologies, including cancer. Flavonoids, including apigenin, were recently shown to reduce the SASP of a human fibroblast strain induced to senesce by bleomycin. Here, we confirm that apigenin suppresses the SASP in three human fibroblast strains induced to senesce by ionizing radiation, constitutive MAPK (mitogen-activated protein kinase) signaling, oncogenic RAS, or replicative exhaustion. Apigenin suppressed the SASP in part by suppressing IL-1α signaling through IRAK1 and IRAK4, p38-MAPK, and NF-κB. Apigenin was particularly potent at suppressing the expression and secretion of CXCL10 (IP10), a newly identified SASP factor. Further, apigenin-mediated suppression of the SASP substantially reduced the aggressive phenotype of human breast cancer cells, as determined by cell proliferation, extracellular matrix invasion, and epithelial-mesenchymal transition. Our results support the idea that apigenin is a promising natural product for reducing the impact of senescent cells on age-related diseases such as cancer.

New agents that target senescent cells: the flavone, fisetin, and the BCL-XL inhibitors, A1331852 and A1155463.

Senescent cells accumulate with aging and at sites of pathology in multiple chronic diseases. Senolytics are drugs that selectively promote apoptosis of senescent cells by temporarily disabling the pro-survival pathways that enable senescent cells to resist the pro-apoptotic, pro-inflammatory factors that they themselves secrete. Reducing senescent cell burden by genetic approaches or by administering senolytics delays or alleviates multiple age- and disease-related adverse phenotypes in preclinical models. Reported senolytics include dasatinib, quercetin, navitoclax (ABT263), and piperlongumine. Here we report that fisetin, a naturally-occurring flavone with low toxicity, and A1331852 and A1155463, selective BCL-XL inhibitors that may have less hematological toxicity than the less specific BCL-2 family inhibitor navitoclax, are senolytic. Fisetin selectively induces apoptosis in senescent but not proliferating human umbilical vein endothelial cells (HUVECs). It is not senolytic in senescent IMR90 cells, a human lung fibroblast strain, or primary human preadipocytes. A1331852 and A1155463 are senolytic in HUVECs and IMR90 cells, but not preadipocytes. These agents may be better candidates for eventual translation into clinical interventions than some existing senolytics, such as navitoclax, which is associated with hematological toxicity.

Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence.

BackgroundCellular senescence is induced either internally, for example by replication exhaustion and cell division, or externally, for example by irradiation. In both cases, cellular damages accumulate which, if not successfully repaired, can result in senescence induction. Recently, we determined the transcriptional changes combined with the transition into replicative senescence in primary human fibroblast strains. Here, by γ-irradiation we induced premature cellular senescence in the fibroblast cell strains (HFF and MRC-5) and determined the corresponding transcriptional changes by high-throughput RNA sequencing.ResultsComparing the transcriptomes, we found a high degree of similarity in differential gene expression in replicative as well as in irradiation induced senescence for both cell strains suggesting, in each cell strain, a common cellular response to error accumulation. On the functional pathway level, “Cell cycle” was the only pathway commonly down-regulated in replicative and irradiation-induced senescence in both fibroblast strains, confirming the tight link between DNA repair and cell cycle regulation. However, “DNA repair” and “replication” pathways were down-regulated more strongly in fibroblasts undergoing replicative exhaustion. We also retrieved genes and pathways in each of the cell strains specific for irradiation induced senescence.ConclusionWe found the pathways associated with “DNA repair” and “replication” less stringently regulated in irradiation induced compared to replicative senescence. The strong regulation of these pathways in replicative senescence highlights the importance of replication errors for its induction.Electronic supplementary materialThe online version of this article (doi:10.1186/s40659-016-0095-2) contains supplementary material, which is available to authorized users.

Conserved Senescence Associated Genes and Pathways in Primary Human Fibroblasts Detected by RNA-Seq.

Cellular senescence correlates with changes in the transcriptome. To obtain a complete view on senescence-associated transcription networks and pathways, we assessed by deep RNA sequencing the transcriptomes of five of the most commonly used laboratory strains of human fibroblasts during their transition into senescence. In a number of cases, we verified the RNA-seq data by real-time PCR. By determining cellular protein levels we observed that the age-related expression of most but not all genes is regulated at the transcriptional level. We found that 78% of the age-affected differentially expressed genes were commonly regulated in the same direction (either up- or down-regulated) in all five fibroblast strains, indicating a strong conservation of age-associated changes in the transcriptome. KEGG pathway analyses confirmed up-regulation of the senescence-associated secretory phenotype and down-regulation of DNA synthesis/repair and most cell cycle pathways common in all five cell strains. Newly identified senescence-induced pathways include up-regulation of endocytotic/phagocytic pathways and down-regulation of the mRNA metabolism and the mRNA splicing pathways. Our results provide an unprecedented comprehensive and deep view into the individual and common transcriptome and pathway changes during the transition into of senescence of five human fibroblast cell strains.

Joint use of developed collagen-containing complexes and cell cultures in creating new tissue equivalents

The purpose of the study is to assess the possibility of applying the integrated module as the basis of a celltissue equivalent for treatment of wounds of skin and soft tissues. In the frame of the set task the following problems were being solved: research of the spatial structure and architectonics of the surface of the developed base collagen-containing materials and their biocompatibility with cell cultures. Materials and methods. The study of a material which is a two-layer complex film, consisting of collagen and polysaccharide components was carried out. The collagen was separated from the dermis and was then impregnated with particulate demineralized bone matrix (DCM) according to the original methodology. For the purposes of the study the dehydrated material was created in the form of a film. Electron microscopic examination of surfaces was performed on scanning electron microscope JEOL JSM-IT300LV in high vacuum and at low values of probe current (< 0,1 nА). Studies to assess the viability of the cells cultivated on films of collagen material (tested for cytotoxicity and the adhesive capacity) were performed in vitro using strains of diploid human fibroblasts 4–6 passage. The culture condition was visually assessed using an inverted Leica microscope DM IL (Carl Zeiss, Austria), equipped with a computerizes program of control of culture growth (Leica IM 1000). Results. The data obtained in the study of the surface structure of the developed complex module showed that it seems to be promising as a basic component of the cellular-tissue system with its large number of structural formations for fixation of the cells and a well-organized barrier layer capable of vapor - permeability. Experiments in vitro confirmed the absence of toxicity of the material being studied in relation to the culture of dermal human fibroblasts, suggesting the possibility of creation on its basis of cell-tissue complex and further experimental studies in vivo. Conclusion. Thus it was experimentally confirmed that the physical characteristics of the developed integrated module satisfy the requirements for the materials for cultivation of cells. The absence of cytotoxicity on the model of a culture of dermal human fibroblasts allows to make a conclusion about a possibility of its use as the basis of cell-tissue equivalent. Preliminary results indicate the advisability of further experiments in vivo aimed at improving complex collagen-containing materials, the development of different ways of their application and clinical evaluation of the effectiveness in the treatment of wounds of various genesis.

Chitosan Treatment Delays the Induction of Senescence in Human Foreskin Fibroblast Strains.

Fibroblasts have been extensively used as a model to study cellular senescence. The purpose of this study was to investigate whether the human foreskin fibroblast aging process could be regulated by using the biomaterial chitosan. Fibroblasts cultured on commercial tissue culture polystyrene (TCPS) entered senescence after 55–60 population doublings (PDs), and were accompanied by larger cell shape, higher senescence-associated β-galactosidase (SA β-gal) activity, lower proliferation capacity, and upregulation of senescence-associated molecular markers p21, p53, retinoblastoma (pRB), and p16. Before senescence was reached, PD48 cells were collected from TCPS and seeded on chitosan for three days (PD48-Cd3) to form multicellular spheroids. The protein expression of senescence-associated secretory phenotypes (SASPs) and senescence-associated molecular markers of these cells in PD48-Cd3 spheroids were downregulated significantly. Following chitosan treatment, fibroblasts reseeded on TCPS showed lower SA β-gal activity, increased cellular motility, and a higher proliferation ability of 70–75 PDs. These phenotypic changes were not accompanied by colonies forming in soft agar and a continuous decrease in the senescence-associated proteins p53 and pRB which act as a barrier to tumorigenesis. These results demonstrate that chitosan treatment could delay the induction of senescence which may be useful and safe for future tissue engineering applications.

Hormetic effect of rotenone in primary human fibroblasts.

BackgroundRotenone inhibits the electron transfer from complex I to ubiquinone, in this way interfering with the electron transport chain in mitochondria. This chain of events induces increased levels of intracellular reactive oxygen species, which in turn can contribute to acceleration of telomere shortening and induction of DNA damage, ultimately resulting in aging. In this study, we investigated the effect of rotenone treatment in human fibroblast strains.ResultsFor the first time we here describe that rotenone treatment induced a hormetic effect in human fibroblast strains. We identified a number of genes which were commonly differentially regulated due to low dose rotenone treatment in fibroblasts independent of their cell origin. However, these genes were not among the most strongly differentially regulated genes in the fibroblast strains on treatment with rotenone. Thus, if there is a common hormesis regulation, it is superimposed by cell strain specific individual responses. We found the rotenone induced differential regulation of pathways common between the two fibroblast strains, being weaker than the pathways individually regulated in the single fibroblast cell strains. Furthermore, within the common pathways different genes were responsible for this different regulation. Thus, rotenone induced hormesis was related to a weak pathway signal, superimposed by a stronger individual cellular response, a situation as found for the differentially expressed genes.ConclusionWe found that the concept of hormesis also applies to in vitro aging of primary human fibroblasts. However, in depth analysis of the genes as well as the pathways differentially regulated due to rotenone treatment revealed cellular hormesis being related to weak signals which are superimposed by stronger individual cell-internal responses. This would explain that in general hormesis is a small effect. Our data indicate that the observed hormetic phenotype does not result from a specific strong well-defined gene or pathway regulation but from weak common cellular processes induced by low levels of reactive oxygen species. This conclusion also holds when comparing our results with those obtained for C. elegans in which the same low dose rotenone level induced a life span extending, thus hormetic effect.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-015-0038-8) contains supplementary material, which is available to authorized users.

Effects of ebselen and N-acetyl cysteine on replicative aging of primary human fibroblast strains.

Administration of selected concentrations of ebselen and N-acetyl cysteine have been proven to display an antioxidant potential based on their effect on markers of T cell integrity and function in human peripheral blood mononuclear cells and CD4+ T cell clones. Here we assessed the impact of various antioxidant concentrations on replicative aging of primary human fibroblast strains derived from embryonic lung (MRC-5) and foreskin (HFF). None of the antioxidant concentrations affected the cumulative population doublings, levels of oxidative DNA damage, intracellular GSH:GSSG ratio, potency of heat shock responses and the induction of senescence in both fibroblast strains. Our results showed no effect of both antioxidants on primary fibroblast strains and reveal their cell type specific antioxidant potential.Electronic supplementary materialThe online version of this article (doi:10.1186/s12979-015-0035-y) contains supplementary material, which is available to authorized users.

Identification of novel mechanisms involved in generating localized vulvodynia pain

Our goal was to gain a better understanding of the inflammatory pathways affected during localized vulvodynia, a poorly understood, common, and debilitating condition characterized by chronic pain of the vulvar vestibule. In a control matched study, primary human fibroblast strains were generated from biopsies collected from localized provoked vulvodynia (LPV) cases and from age- and race-matched controls. We then examined intracellular mechanisms by which these fibroblasts recognize pathogenic Candida albicans; >70% of vulvodynia patients report the occurrence of prior chronic Candida infections, which is accompanied by localized inflammation and elevated production of proinflammatory/pain-associated interleukin (IL)-6 and prostaglandin E2 (PGE2). We focused on examining the signaling pathways involved in recognition of yeast components that are present and abundant during chronic infection. Dectin-1, a surface receptor that binds C albicans cell wall glucan, was significantly elevated in vestibular vs external vulvar cells (from areas without pain) in both cases and controls, while its abundance was highest in LPV cases. Blocking Dectin-1 signaling significantly reduced pain-associated IL-6 and PGE2 production during the response to C albicans. Furthermore, LPV patient vestibular cells produced inflammatory mediators in response to low numbers of Calbicans cells, while external vulvar fibroblasts were nonresponsive. Inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (proinflammatory transcription factor) nearly abrogated IL-6 and PGE2 production induced by C albicans, in keeping with observations that Dectin-1 signals through the nuclear factor kappa-light-chain-enhancer of activated B cells pathway. These findings implicate that a fibroblast-mediated proinflammatory response to C albicans contributes to the induction of pain in LPV cases. Targeting this response may be an ideal strategy for the development of new vulvodynia therapies.

Resolvin D1 inhibits multiple pro-inflammatory signaling pathways in primary human lung cells exposed to diverse inflammatory stimuli (IRM9P.721)

Abstract Resolution of inflammation is a bioactive process involving a new genus of lipid molecules called “specialized pro-resolving lipid mediators” (SPMs), mainly derived from dietary ω-3 polyunsaturated fatty acids, which actively antagonize inflammatory signaling pathways and promote resolution. Although it is broadly known that SPMs inhibit inflammation, the specific pathways and targets of many SPMs remained to be discovered. Here, we investigated the mechanism by which the SPM resolvin D1 (RvD1) inhibits pro-inflammatory signaling pathways using two distinct stimuli; IL-1β, which acts primarily via its own receptor, and poly(I:C), a double-stranded viral RNA mimetic that acts primarily through toll-like receptor (TLR)3. RvD1 inhibited production of pro-inflammatory mediators including IL-6 and IL-8, both primary human lung fibroblast and epithelial cell strains, when stimulated with either IL-1β or poly(I:C). RvD1 also blocked phosphorylation of ERK1/2 and translocation of NF-κB p65 in cells treated with both stimuli. Importantly, we found that RvD1 inhibited phosphorylation of TAK1, an upstream effector protein that regulates both the MAP-kinase and NF-κB pathways. These results demonstrate that RvD1 targets multiple pro-inflammatory signaling pathways and may have significant therapeutic potential in complex inflammatory diseases including pulmonary fibrosis, asthma and chronic obstructive pulmonary disease.

MicroRNA-663 induction upon oxidative stress in cultured human fibroblasts depends on the chronological age of the donor

MicroRNAs, regulators of messenger RNA translation, have been observed to influence many physiological processes, amongst them the process of aging. Higher levels of microRNA-663 (miR-663) have previously been observed in human dermal fibroblasts subject to both replicative and stress-induced senescence compared to early passage cells. Also, higher levels of miR-663 have been found in memory T-cells and in human fibroblasts derived from older donors compared to younger donors. In previous studies we observed that dermal fibroblasts from donors of different chronological and biological age respond differentially to oxidative stress measured by markers of cellular senescence and apoptosis. In the present study we set out to study the association between miR-663 levels and chronological and biological age. Therefore we tested in a total of 92 human dermal fibroblast strains whether the levels of miR-663 in non-stressed and stressed conditions (fibroblasts were treated with 0.6μM rotenone in stressed conditions) were different in young, middle aged and old donors and whether they were different in middle aged donors dependent on their biological age, as indicated by the propensity for familial longevity. In non-stressed conditions the level of miR-663 did not differ between donors of different age categories and was not dependent on biological age. Levels of miR-663 did not differ dependent on biological age in stressed conditions either. However, for different age categories the level of miR-663 in stressed conditions did differ: the level of miR-663 was higher at higher age categories. Also, the ratio of miR-663 induction upon stress was significantly higher in donors from older age categories. In conclusion, we present evidence for an association of miR-663 upon stress and chronological age.

Nuclear accumulation of cyclin D1 following long-term fractionated exposures to low-dose ionizing radiation in normal human diploid cells

Cyclin D1 is a mitogenic sensor that responds to growth signals from the extracellular environment and regulates the G1-to-S cell cycle transition. When cells are acutely irradiated with a single dose of 10 Gy, cyclin D1 is degraded, causing cell cycle arrest at the G1/S checkpoint. In contrast, cyclin D1 accumulates in human tumor cells that are exposed to long-term fractionated radiation (0.5 Gy/fraction of X-rays). In this study we investigated the effect of fractionated low-dose radiation exposure on cyclin D1 localization in 3 strains of normal human fibroblasts. To specifically examine the nuclear accumulation of cyclin D1, cells were treated with a hypotonic buffer containing detergent to remove cytoplasmic cyclin D1. Proliferating cell nuclear antigen (PCNA) immunofluorescence was used to identify cells in S phase. With this approach, we observed S-phase nuclear retention of cyclin D1 following low-dose fractionated exposures, and found that cyclin D1 nuclear retention increased with exposure time. Cells that retained nuclear cyclin D1 were more likely to have micronuclei than non-retaining cells, indicating that the accumulation of nuclear cyclin D1 was associated with genomic instability. Moreover, inhibition of the v-akt murine thymoma viral oncogene homolog (AKT) pathway facilitated cyclin D1 degradation and eliminated cyclin D1 nuclear retention in cells exposed to fractionated radiation. Thus, cyclin D1 may represent a useful marker for monitoring long-term effects associated with exposure to low levels of radiation.