Repair replication in cultured normal and transformed human fibroblasts

International Nuclear Information System (INIS)

Smith, C.A.; Hanawalt, P.C.

1976-01-01

Repair replication in response to ultraviolet irradiation has been studied in normal human diploid fibroblast cultures, W138, and an SV40 transformant, VA13. Quantitative comparisons have been made using the combined isotopic and density labelling method for assaying repair replication. No significant difference was found in the amount of repair replication performed, its dose response, or the time course between growing and confluent W138 cells, early passage and senescent cells, or normal W138 cells and the transformed VA13 cells. When [ 3 H]dThd was employed as the isotopic label in the presence of a 30-200 fold excess of unlabelled BrdUrd apparent differences in repair replication were seen between W138 cells shortly after subcultivation and cells which had been allowed to reach confluence. These differences were the same over a wide dose range and regardless of the passage number of the cells, but could be influenced by using different serum lots. The differences were not seen, however, when [ 3 H]BrdUrd provided the isotopic label; thus they reflect either impurities in the [ 3 H]dThd or a slight discrimination by some cellular process

Deficiency of gamma-ray excision repair in skin fibroblasts from patients with Fanconi's anemia

International Nuclear Information System (INIS)

Remsen, J.F.; Cerutti, P.A.

1976-01-01

The capacity of preparations of skin fibroblasts from normal individuals and patients with Fanconi's anemia to excise gamma-ray products of the 5,6-dihydroxydihydrothymine type from exogenous DNA was investigated. The excision capacity of whole-cell homogenates of fibroblasts from two of four patients with Fanconi's anemia was substantially below normal. This repair deficiency was further pronounced in nuclear preparations from cells of the same two patients

Hyperactivation of PARP triggers nonhomologous end-joining in repair-deficient mouse fibroblasts.

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Natalie R Gassman

Full Text Available Regulation of poly(ADP-ribose (PAR synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose polymerase-1 (PARP-1 occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase β (pol β. The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS, or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol β nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol β null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised.

DNA methylation in human fibroblasts following DNA damage and repair

International Nuclear Information System (INIS)

Kastan, M.B.

1984-01-01

Methylation of deoxycytidine (dCyd) incorporated by DNA excision repair synthesis in human diploid fibroblasts following damage with ultraviolet radiation (UV), N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene was studied utilizing [6- 3 H]dCyd to label repaired DNA specifically and high performance liquid chromatographic analysis to quantify the percentage of deoxycytidine converted to 5-methyldeoxycytidine (m 5 dCyd). In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication a level of 3.4% m 5 dCyd is reached in less than 2 hours, following UV-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approx.2.0% m 5 dCyd in the repair patch. This undermethylation of repair patches occurs throughout the genome. In cells from cultures in logarithmic-phase growth, m 5 dCyd formation in UV-induced repair patches occurs faster and to a greater extent, reaching a level of approx.2.7% in 10-20 hours. Pre-existing hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites. The distribution within chromatin of m 5 dCyd in repair patches was also investigated. Over a wide range of extents of digestion by staphylococcal nuclease or deoxyribonuclease I, the level of hypomethylation in repaired DNA in nuclease sensitive and resistant regions of chromatin was constant relative to the genomic level of methylation in these regions. Similar conclusions were reached in experiments with isolated mononucleosomes

Direct conversion of human fibroblasts into functional osteoblasts by defined factors.

Science.gov (United States)

Yamamoto, Kenta; Kishida, Tsunao; Sato, Yoshiki; Nishioka, Keisuke; Ejima, Akika; Fujiwara, Hiroyoshi; Kubo, Toshikazu; Yamamoto, Toshiro; Kanamura, Narisato; Mazda, Osam

2015-05-12

Osteoblasts produce calcified bone matrix and contribute to bone formation and remodeling. In this study, we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some defined factors and culturing in osteogenic medium. Osteoblast-specific transcription factors, Runt-related transcription factor 2 (Runx2), and Osterix, in combination with Octamer-binding transcription factor 3/4 (Oct4) and L-Myc (RXOL) transduction, converted ∼ 80% of the fibroblasts into osteocalcin-producing cells. The directly converted osteoblasts (dOBs) induced by RXOL displayed a similar gene expression profile as normal human osteoblasts and contributed to bone repair after transplantation into immunodeficient mice at artificial bone defect lesions. The dOBs expressed endogenous Runx2 and Osterix, and did not require continuous expression of the exogenous genes to maintain their phenotype. Another combination, Oct4 plus L-Myc (OL), also induced fibroblasts to produce bone matrix, but the OL-transduced cells did not express Osterix and exhibited a more distant gene expression profile to osteoblasts compared with RXOL-transduced cells. These findings strongly suggest successful direct reprogramming of fibroblasts into functional osteoblasts by RXOL, a technology that may provide bone regeneration therapy against bone disorders.

A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts

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Gebhard Daniel

2010-06-01

Full Text Available Abstract Background The Host Cell Reactivation Assay (HCRA is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances. A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing. Results In this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis. The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts. Conclusions The methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity

Involvement of DNA polymerase δ in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

International Nuclear Information System (INIS)

Dresler, S.L.; Gowans, B.J.; Robinson-Hill, R.M.; Hunting, D.J.

1988-01-01

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase δ as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, the authors describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, α or δ. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase α several hundred times more strongly than it inhibits DNA polymerase δ. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase δ. It appears that repair synthesis at late time after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase δ

DNA repair in a Fanconi's anemia fibroblast cell strain

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Little, J.B.; Weichselbaum, R.R.

1979-01-01

DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution. (Auth.)

DNA repair in a Fanconi's anemia fibroblast cell strain

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Fornace, Jr, A J; Little, J B [Harvard School of Public Health, Boston, MA (USA); Weichselbaum, R R [Harvard Medical School, Boston, MA (USA)

1979-01-26

DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution.

Correlation between DNA repair of embryonic fibroblasts and different life span of 3 inbred mouse strains

Energy Technology Data Exchange (ETDEWEB)

Paffenholz, V.

1978-02-01

Primary mouse fibroblast cultures were established from 10 day old embryos of 3 inbred strains with a genetically determined different life expectancy. The capacity for unscheduled DNA synthesis following uv irradiation was studied in these cells at various passage levels of the in vitro ageing process. The mouse fibroblasts show considerable repair synthesis corresponding to the duration of exposure time. The capacity for induction of unscheduled DNA synthesis was different in the cells of each strain and correlated to the natural life span of the animal. In each case, however, the ability to perform repair synthesis was subjected to an age-associated decline, although semiconservative DNA synthesis and proliferative potential of the cell was not changed until the cultures entered phase III passages.

Role of postreplication repair in transformation of human fibroblasts to anchorage independence

International Nuclear Information System (INIS)

Boyer, J.C.; Kaufmann, W.K.; Cordeiro-Stone, M.

1991-01-01

Cellular capacity for postreplication repair (PRR) and sensitivity to transformation to anchorage independence (AI) were quantified in normal foreskin and xeroderma pigmentosum (XP) variant fibroblasts after treatment with UV or benzo(a)pyrene-diol-epoxide I (BPDE-I). PRR is defined here as a collection of pathways that facilitate the replication of DNA damaged by genotoxic agents. It is recognized biochemically as the process by which nascent DNA grows longer than the average distance between two lesions in the DNA template. PRR refers more directly to the elimination of gaps in the daughter-strand DNA by mechanisms which remain to be determined for human cells, but which may include translesion replication and recombination. PRR was measured in diploid human fibroblasts by analysis of the dose kinetics for inhibition of DNA strand growth in carcinogen-treated cells. Logarithmically growing foreskin fibroblasts (NHF1) displayed D0 values of 4.3 J/m 2 and 0.14 microM for the inhibition of DNA synthesis in active replicons by UV and BPDE-I, respectively. XP variant cells (CRL1162) exhibited corresponding D0 values of 1.5 J/m 2 and 0.16 microM. The increased sensitivity to inhibition of DNA replication by UV in these XP variant fibroblasts (2.9-fold greater than normal) was mirrored by an enhanced frequency of transformation to AI. XP variant fibroblasts (CRL1162) were 3.2 times more sensitive to transformation to AI by UV than were the normal foreskin fibroblasts. As predicted by the PRR studies, both cell types exhibited similar frequencies of AI colonies induced by BPDE-I. Apparent thresholds were observed for induction of AI by UV (normal fibroblasts, 2.7 J/m 2 ; XP variant fibroblasts, 0.3 J/m 2 ) and BPDE-I (both, 0.05 microM)

DNA-repair after UV-irradiation in skin fibroblasts from patients with actinic keratosis

International Nuclear Information System (INIS)

Sbano, E.; Andreassi, L.; Fimiani, M.; Valentino, A.; Baiocchi, R.

1978-01-01

Autoradiographic counting technique was utilized to measure the ultraviolet-induced unscheduled DNA synthesis of skin fibroblasts from 12 patients with chronic actinic keratosis and from 12 healthy donors of about the same age. In order to reveal a possible regional difference of DNA repair between the parts of the body ordinarily exposed and those parts unexposed to sunlight, two cell strains were used for each examined subject; one developed from the forehead skin and the other from the abdominal or axillary skin. Unscheduled DNA synthesis appeared depressed in actinic keratosis patients, as compared with controls. In all examined subjects, however, cell strains from exposed skin showed a DNA repair more active than cell strains from unexposed skin. These findings show that skin cancer may be promoted in actinic keratosis patients by a defect of DNA repair. The exalted DNA repair of chronically sun exposed skin is probably the consequence of a defensive process caused by enzymatic induction. (orig.) [de

The inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts

International Nuclear Information System (INIS)

Waters, R.; Crocombe, K.; Mirzayans, R.

1981-01-01

Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of DNA polymerase α, aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase α, this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells. (orig.)

DNA repair after a UV-C irradiation in cultivated human fibroblasts: Studies on patients with actinic keratosis, spinocellular carcinoma and basal cell carcinoma as well as healthy controls

International Nuclear Information System (INIS)

Senner, A.

1984-01-01

Cultivated fibroblasts from 14 healthy control people and 22 patients were irradiated with UV-C light in increasing doses (0-1-2.5-5 mJ/cm 2 ). The incorporation of 3 H-thymidine in the DNA of the fibroblasts which is made visible by the autoradiography technique served as a measure for the unprogrammed DNA synthesis. All of the studied fibroblast types showed with an increase in the UV-C irradiation dose an increase in the repair processes of the DNA. No statistically significant difference could be shown in the repair behaviour of the fibroblasts of controls and patients. (orig./MG) [de

Repair of closely opposed cyclobutyl pyrimidine dimers in UV-sensitive human diploid fibroblasts

International Nuclear Information System (INIS)

Lam, L.H.; Reynolds, R.J.

1986-01-01

An enzyme-sensitive site assay has been used to examine the fate of closely opposed pyrimidine dimers in fibroblasts from individuals afflicted with various genetic disorders that confer increased cellular sensitivity to UV radiation. The disappearance of bifilar enzyme-sensitive sites was found to be normal in cells from individuals with Fanconi's anemia, Cockayne's syndrome, dyskeratosis congenita and the variant form of xeroderma pigmentosum. The rate of bifilar enzyme-sensitive site removal in XP cells assigned to complementation group C was reduced by an amount similar to that observed for the repair of isolated dimers. Our results indicate that the initiation of repair at closely opposed dimers is slow in XP-C cells but normal in all other cells examined. (Auth.)

Impact of matrix stiffness on fibroblast function

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El-Mohri, Hichem; Wu, Yang; Mohanty, Swetaparna; Ghosh, Gargi, E-mail: gargi@umich.edu

2017-05-01

Chronic non-healing wounds, caused by impaired production of growth factors and reduced vascularization, represent a significant burden to patients, health care professionals, and health care system. While several wound dressing biomaterials have been developed, the impact of the mechanical properties of the dressings on the residing cells and consequently on the healing of the wounds is largely overlooked. The primary focus of this study is to explore whether manipulation of the substrate mechanics can regulate the function of fibroblasts, particularly in the context of their angiogenic activity. A photocrosslinkable hydrogel platform with orthogonal control over gel modulus and cell adhesive sites was developed to explore the quantitative relationship between ECM compliance and fibroblast function. Increase in matrix stiffness resulted in enhanced fibroblast proliferation and stress fiber formation. However, the angiogenic activity of fibroblasts was found to be optimum when the cells were seeded on compliant matrices. Thus, the observations suggest that the stiffness of the wound dressing material may play an important role in the progression of wound healing. - Highlights: • Proliferation and stress fiber formation of fibroblasts increase with increasing matrix mechanics. • Cell area correlates with the growth of fibroblasts. • Angiogenic activity of fibroblasts optimum when cells seeded on compliant gels.

Specific sizes of hyaluronan oligosaccharides stimulate fibroblast migration and excisional wound repair.

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Cornelia Tolg

Full Text Available The extracellular matrix polysaccharide hyaluronan (HA plays a key role in both fibrotic and regenerative tissue repair. Accumulation of high molecular weight HA is typical of regenerative repair, which is associated with minimal inflammation and fibrosis, while fragmentation of HA is typical of postnatal wounds, which heal in the presence of inflammation and transient fibrosis. It is generally considered that HA oligosaccharides and fragments of a wide size range support these processes of adult, fibrotic wound repair yet the consequences of sized HA fragments/oligosaccharides to each repair stage is not well characterized. Here, we compared the effects of native HA, HA oligosaccharide mixtures and individual sizes (4-10 mer oligosaccharides, 5 and, 40 kDa of HA oligosaccharides and fragments, on fibroblast migration in scratch wound assays and on excisional skin wound repair in vivo. We confirm that 4-10 mer mixtures significantly stimulated scratch wound repair and further report that only the 6 and 8 mer oligosaccharides in this mixture are responsible for this effect. The HA 6 mer promoted wound closure, accumulation of wound M1 and M2 macrophages and the M2 cytokine TGFβ1, but did not increase myofibroblast differentiation. The effect of 6 mer HA on wound closure required both RHAMM and CD44 expression. In contrast, The 40 kDa HA fragment inhibited wound closure, increased the number of wound macrophages but had no effect on TGFβ1 accumulation or subsequent fibrosis. These results show that specific sizes of HA polymer have unique effects on postnatal wound repair. The ability of 6 mer HA to promote wound closure and inflammation resolution without increased myofibroblast differentiation suggests that this HA oligosaccharide could be useful for treatment of delayed or inefficient wound repair where minimal fibrosis is advantageous.

Enhanced radiosensitivity and defective DNA repair in cultured fibroblasts derived from Rothmund Thomson syndrome patients

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Smith, P J; Paterson, M C [Atomic Energy of Canada Ltd., Chalk River, Ontario. Radiation Biology Branch

1982-01-01

Rothmund Thomson syndrome (RTS) is an oculocutaneous and cancer-prone disorder in which enhanced carcinogen sensitivity, mediated through abnormal DNA metabolism, may be an associated factor. Cultured fibroblasts from 4 RTS patients have been examined for their colony-forming abilities and DNA repair capacities following ..gamma..-irradiation. 2 of the 4 RTS strains showed enhanced sensitivity following hypoxic ..gamma..-irradiation, and 1 of these 2 strains also showed enhanced sensitivity under oxic conditions. Defective DNA repair was implicated in the above abnormal responses to ..gamma..-radiation since both strains displayed reduced levels of repair synthesis and slow removal of radiogenic DNA lesions (assayed by their sensitivity to strand-incising activities present in protein extracts of Micrococcus luteus cells). A hypothesis is presented to rationalize the origin and heterogeneity of these laboratory phenotypes of RTS.

MFG-E8 Reprogramming of Macrophages Promotes Wound Healing by Increased bFGF Production and Fibroblast Functions.

Science.gov (United States)

Laplante, Patrick; Brillant-Marquis, Frédéric; Brissette, Marie-Joëlle; Joannette-Pilon, Benjamin; Cayrol, Romain; Kokta, Victor; Cailhier, Jean-François

2017-09-01

Macrophages are essential for tissue repair. They have a crucial role in cutaneous wound healing, participating actively in the inflammation phase of the process. Unregulated macrophage activation may, however, represent a source of excessive inflammation, leading to abnormal wound healing and hypertrophic scars. Our research group has shown that apoptotic endothelial and epithelial cells secrete MFG-E8, which has the ability to reprogram macrophages from an M1 (proinflammatory) to an M2 (anti-inflammatory, pro-repair) phenotype. Hence, we tested whether modulation of macrophage reprogramming would promote tissue repair. Using a mouse model of wound healing, we showed that the presence and/or addition of MFG-E8 favors wound closure associated with an increase in CD206-positive cells and basic fibroblast growth factor production in healing tissues. More importantly, adoptive transfer of ex vivo MFG-E8-treated macrophages promoted wound closure. We also observed that MFG-E8-treated macrophages produced basic fibroblast growth factor that is responsible for fibroblast migration and proliferation. Taken together, our results strongly suggest that MFG-E8 plays a key role in macrophage reprogramming in tissue healing through induction of an anti-inflammatory M2 phenotype and basic fibroblast growth factor production, leading to fibroblast migration and wound closure. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Effects of an extract from the sea squirt Ecteinascidia turbinata on DNA synthesis and excision repair in human fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Dunn, W.C.; Carrier, W.L.; Regan, J.D.

1982-01-01

An aqueous ethanol extract from the marine tunicate species Ecteinascidia turbinata was studied to determine its effect on semiconservative DNA synthesis in human skin fibroblast cultures as measured by (/sup 3/H) thymidine uptake in acid-insoluble cell fractions. In addition, the effect of this extract on DNA excision repair in ultraviolet light (254 nm) irradiated fibroblasts was measured by the bromodeoxyuridine photolysis assay, thymine dimer chromatography, and DNA single-strand break analysis on alkaline sucrose gradients. Repair inhibition was accompanied by an accumulation of single-strand DNA breaks which was enhanced by the addtion of 2 mM hydroxyurea. These results are discussed with respect to a mechanism of action of the marine tunicate extract at the level of DNA polymerases and are contrasted with previously studied inhibitory mechanisms of arabinofuranosyl nucleosides.

Potentially lethal damage repair in cell lines of radioresistant human tumours and normal skin fibroblasts

International Nuclear Information System (INIS)

Marchese, M.J.; Minarik, L.; Hall, E.J.; Zaider, M.

1985-01-01

Radiation cell survival data were obtained in vitro for three cell lines isolated from human tumours traditionally considered to be radioresistant-two melanomas and one osteosarcoma-as well as from a diploid skin fibroblast cell line. One melanoma cell line was much more radioresistant than the other, while the osteosarcoma and fibroblast cell lines were more radiosensitive than either. For cells growing exponentially, little potentially lethal damage repair (PLDR) could be demonstrated by comparing survival data for cells in which subculture was delayed by 6 h with those sub-cultured immediately after treatment. For the malignant cells in plateau phase, which in these cells might be better termed 'slowed growth phase', since an appreciable fraction of the cells are still cycling, a small amount of PLDR was observed, but not as much as reported by other investigators in the literature. The normal fibroblasts, which achieved a truer plateau phase in terms of noncycling cells, showed a significantly larger amount of PLDR than the tumour cells. (author)

Reduced repair of potentially lethal radiation damage in glutathione synthetase-deficient human fibroblasts after X-irradiation

International Nuclear Information System (INIS)

Midander, J.; Revesz, L.; Deschavanne, P.J.; Debieu, D.; Malaise, E.P.

1986-01-01

Using a human fibroblast strain deficient in glutathione synthetase and a related proficient control strain, the role of glutathione (GSH) in repair of potentially lethal damage (PLD) has been investigated in determining survival by plating cells immediately or 24 h after irradiation. After oxic or hypoxic irradiation, both cell strains repair radiation-induced damage. However, under hypoxic conditions, the proficient cells repair PLD as well as under oxic conditions while the deficient cells repair less PLD after irradiation under hypoxic than under oxic conditions. Therefore, the oxygen enhancement ratio (o.e.r.) for proficient cells is similar whether the cells are plated immediately or 24 h later (2.0 and 2.13, respectively). In contrast, the o.e.r. for deficient cells is lower when the cells are plated 24 h after irradiation than when they are plated immediately thereafter (1.16 as compared to 1.55). The results indicate that GSH is involved in PLD repair and, in particular, in the repair of damage induced by radiation delivered under hypoxic conditions. (author)

The effect of mitotic inhibitors on DNA strand size and radiation-associated break repair in Down syndrome fibroblasts

International Nuclear Information System (INIS)

Woods, W.G.; Steiner, M.E.; Kalvonjian, S.L.

1985-01-01

The effect of mitotic inhibitors on formation and repair of DNA breaks was studied in cultured fibroblasts from patients with Down syndrome in order to investigate the hypothesis that the karyotyping procedure itself may play a role in the increased chromosome breakage seen in these cells after gamma radiation exposure. Using the nondenaturing elution and alkaline elution techniques to examine fibroblasts from Down syndrome patients and from controls, no specific abnormalities in Down syndrome cells could be detected after exposure to mitotic inhibitors, including rate and extent of elution of DNA from filters as well as repair of radiation-induced DNA breaks. In both normal and Down syndrome cell strains, however, exposure to mitotic inhibitors was associated with a decrease in cellular DNA strand size, suggesting the presence of drug-induced DNA strand breaks. The mechanism of increased chromosome sensitivity of Down syndrome cells to gamma radiation remains unknown. (orig.)

Evidence that novobiocin and nalidixic acid do not inhibit excision repair in u.v.-irradiated human skin fibroblasts at a pre-incision

International Nuclear Information System (INIS)

Keyse, S.M.; Tyrrell, R.M.

1985-01-01

The effects of novobiocin and nalidixic acid on the specific toxicity of aphidicolin towards u.v. irradiated arrested human skin fibroblasts have been determined. Contrary to the result expected if either drug were causing inhibition of excision repair at a pre-incision step the sector of toxicity due to a combined treatment of 300 μg ml -1 nalidixic acid and 1.0 μg ml -1 aphidicolin is unchanged when compared with that due to treatment with 1.0 μg ml -1 aphidicolin alone, while that for 150 μg ml -1 novobiocin + 1.0 μg ml -1 aphidicolin was slightly increased. In parallel measurements of the inhibition of u.v.-induced DNA repair synthesis in arrested fibroblasts by these drugs, 150 μg ml -1 novobiocin inhibited repair synthesis by approx.60% over the fluence range employed. Nalidixic acid (300 μg ml -1 ) caused no detectable inhibition of repair synthesis. It was concluded that the mode of action of novobiocin in the inhibition of DNA excision repair is not via the inhibition of a pre-incision step and the data do not support the hypothesis that a type II topoisomerase mediated change in DNA supercoiling is an essential early step in excision repair of u.v.-induced damage. (author)

Fibroblast growth factor 2 and DNA repair involvement in the keratinocyte stem cells response to ionizing radiation

International Nuclear Information System (INIS)

Harfouche, L'Emira Ghida

2010-02-01

Keratinocyte stem cells (KSCs) from the human inter follicular epidermis are regarded as the major target to radiation during radiotherapy. We found herein that KSCs are more resistant to ionizing radiation than their direct progeny, and presented more rapid DNA damage repair kinetics than the progenitors. Furthermore, we provided evidence describing the effect of fibroblast growth factor 2 (FGF2) signaling on the ability of KSCs and progenitors to repair damaged DNA. Despite our knowledge of the fact, that FGF is an anti-apoptotic factor in multiple cell types, the direct link between DNA repair and FGF2 signaling has rarely been shown. Existence of such link is an important issue with implications not only to stem cell field but also to cancer therapy. (author)

Distribution of u.v.-induced repair events in higher-order chromatin loops in human and hamster fibroblasts

International Nuclear Information System (INIS)

Mullenders, L.H.F.; Zeeland, A.A. van; Natarajan, A.T.; Kesteren, A.C. van; Bussmann, C.J.M.

1986-01-01

The repair of u.v.-induced damage in human and rodent cells was investigated at the level of DNA loops attached to the nuclear matrix. After 2 h post-u.v. incubation, DNase I digestion studies revealed a 3- to 4-fold enrichment of repair-labeled DNA at the nuclear matrix in four xeroderma pigmentosum cell strains belonging to complementation group C. Two xeroderma pigmentosum cell strains of complementation group D and Syrian hamster embryonic cells, as well as in HeLa cells and normal human fibroblasts, no enrichment of repair-labeled DNA at the nuclear matrix was observed. Visualization of repair events in DNA loops by autoradiography of DNA halo - matrix structures confirmed the biochemical observations. The presence or absence of preferential repair of nuclear matrix-associated DNA paralleled the presence or absence of inhomogeneity in the distribution of T4 endonuclease-V-sensitive sites. In xeroderma pigmentosum cells of complementation group C showed that after 2 h post-u.v. incubation, repair events were found at both attachment sites in a limited number of loops and that large domains of loops were not subjected to repair. (author)

Evidence that novobiocin and nalidixic acid do not inhibit excision repair in u.v.-irradiated human skin fibroblasts at a pre-incision step

International Nuclear Information System (INIS)

Keyse, S.M.; Tyrrell, R.M.

1985-01-01

The effects of novobiocin and nalidixic acid on the specific toxicity of aphidicolin towards u.v. irradiated arrested (nondividing) human skin fibroblasts have been determined. Contrary to the result expected if either drug were causing inhibition of excision repair at a pre-incision step the sector of toxicity due to a combined treatment of 300 micrograms ml -1 nalidixic acid and 1.0 micrograms ml -1 aphidicolin is unchanged when compared with that due to treatment with 1.0 micrograms ml -1 aphidicolin alone, while that for 150 micrograms ml -1 novobiocin + 1.0 micrograms ml -1 aphidicolin was slightly increased. In parallel measurements of the inhibition of u.v.-induced DNA repair synthesis in arrested fibroblasts by these drugs, 150 micrograms ml -1 novobiocin inhibited repair synthesis by approximately 60% over the fluence range employed. Nalidixic acid at a concentration of 300 micrograms ml -1 caused no detectable inhibition of repair synthesis. The authors conclude that the mode of action of novobiocin in the inhibition of DNA excision repair is not via the inhibition of a pre-incision step and the data do not support the hypothesis that a type II topoisomerase mediated change in DNA supercoiling is an essential early step in excision repair of u.v.-induced damage

Role of DNA repair in repair of cytogenetic damages. Slowly repaired DNA injuries involved in cytogenetic damages repair

International Nuclear Information System (INIS)

Zaichkina, S.I.; Rozanova, O.M.; Aptikaev, G.F.; Ganassi, E.Eh.

1989-01-01

Caffeine was used to study the kinetics of cytogenetic damages repair in Chinese hamster fibroblasts. Its half-time (90 min) was shown to correlate with that of repair of slowly repaired DNA damages. The caffeine-induced increase in the number of irreparable DNA damages, attributed to inhibition of double-strand break repair, is in a quantitative correlation with the effect of the cytogenetic damage modification

Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease

Science.gov (United States)

Gourdie, Robert G.; Dimmeler, Stefanie; Kohl, Peter

2016-01-01

Our understanding of cardiac fibroblast functions has moved beyond their roles in heart structure and extracellular matrix generation, and now includes contributions to paracrine, mechanical and electrical signalling during ontogenesis and normal cardiac activity. Fibroblasts have central roles in pathogenic remodelling during myocardial ischaemia, hypertension and heart failure. As key contributors to scar formation, they are crucial for tissue repair after interventions including surgery and ablation. Novel experimental approaches targeting cardiac fibroblasts are promising potential therapies for heart disease. Indeed, several existing drugs act, at least partially, through effects on cardiac connective tissue. This Review outlines the origins and roles of fibroblasts in cardiac development, homeostasis and disease; illustrates the involvement of fibroblasts in current and emerging clinical interventions; and identifies future targets for research and development. PMID:27339799

Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation

International Nuclear Information System (INIS)

Blanc-Brude, Olivier P.; Archer, Fabienne; Leoni, Patricia; Derian, Claudia; Bolsover, Steven; Laurent, Geoffrey J.; Chambers, Rachel C.

2005-01-01

Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR 1 ). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR 1 -deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR 1 -specific agonists and inhibitors were used to demonstrate that PAR 1 mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR 1 and not PAR 2 . These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis

In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins

DEFF Research Database (Denmark)

Kasten-Pisula, Ulla; Vronskaja, Svetlana; Overgaard, Jens

2008-01-01

in the activity of the DNA-PK complex induced upon irradiation. CONCLUSIONS: For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity. Udgivelsesdato: 2008-Mar......BACKGROUND AND PURPOSE: The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins....... MATERIALS AND METHODS: Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay. RESULTS: The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively...

ANALYSIS OF DNA DAMAGE AND REPAIR IN SKIN FIBROBLASTS OF INFANT AND OLDER CHILDREN USING THE IN VITRO ALKALINE COMET ASSAY

Science.gov (United States)

ANALYSIS OF DNA DAMAGE AND REPAIR IN SKIN FIBROBLASTS OF INFANT AND OLDER CHILDREN USING THE IN VITRO ALKALINE COMET ASSAY, Alan H. Tennant1, Geremy W. Knapp1 and Andrew D. Kligerman1, 1Environmental Carcinogenesis Division, National Health and Environmental Effects Research Lab...

Increased fibroblast functionality on CNN2-loaded titania nanotubes

Directory of Open Access Journals (Sweden)

Wei HB

2012-02-01

Full Text Available Hongbo Wei*, Shuyi Wu*, Zhihong Feng, Wei Zhou, Yan Dong, Guofeng Wu, Shizhu Bai, Yimin Zhao Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, Xi'an, People's Republic of China *These authors contributed equally to this workAbstract: Infection and epithelial downgrowth are major problems associated with maxillofacial percutaneous implants. These complications are mainly due to the improper closure of the implant–skin interface. Therefore, designing a percutaneous implant that better promotes the formation of a stable soft tissue biologic seal around percutaneous sites is highly desirable. Additionally, the fibroblast has been proven to play an important role in the formation of biologic seals. In this study, titania nanotubes were filled with 11.2 kDa C-terminal CCN2 (connective tissue growth factor fragment, which could exert full CCN2 activity to increase the biological functionality of fibroblasts. This drug delivery system was fabricated on a titanium implant surface. CCN2 was loaded into anodized titania nanotubes using a simplified lyophilization method and the loading efficiency was approximately 80%. Then, the release kinetics of CCN2 from these nanotubes was investigated. Furthermore, the influence of CCN2-loaded titania nanotubes on fibroblast functionality was examined. The results revealed increased fibroblast adhesion at 0.25, 0.5, 1, 2, 4, and 24 hours, increased fibroblast viability over the course of 5 days, as well as enhanced actin cytoskeleton organization on CCN2-loaded titania nanotubes surfaces compared to uncoated, unmodified counterparts. Therefore, the results from this in vitro study demonstrate that CCN2-loaded titania nanotubes have the ability to increase fibroblast functionality and should be further studied as a method of promoting the formation of a stable soft tissue biologic seal around percutaneous sites.Keywords: anodization, titania nanotubes, adhesion, connective

LXA4 actions direct fibroblast function and wound closure

International Nuclear Information System (INIS)

Herrera, Bruno S.; Kantarci, Alpdogan; Zarrough, Ahmed; Hasturk, Hatice; Leung, Kai P.; Van Dyke, Thomas E.

2015-01-01

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A 4 (LXA 4 ), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA 4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA 4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA 4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA 4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA 4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA 4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF-β1 up-regulates LXA 4 receptor (ALX

Adaptive response to ionizing radiation in normal human skin fibroblasts. Enhancement of DNA repair rate and modulation of gene expression

International Nuclear Information System (INIS)

Toledo, S.M. de; Mitchel, R.E.J.; Azzam, E.; Ottawa Univ., ON; Raaphorst, G.P.

1994-01-01

Low doses and dose rates of ionizing radiation enhance the rate of DNA repair in human fibroblasts and protect the cells against radiation-induced micronucleus formation. Chronic exposures reduce the mRNA levels of the genes topoisomerase II and FACC-1 (Fanconi's anemia, group C). (authors). 11 refs., 1 tab., 2 figs

Benzo(a)pyrene metabolism, DNA-binding and UV-induced repair of DNA damage in cultured skin fibroblasts from a patient with unilateral multiple basal cell carcinoma

International Nuclear Information System (INIS)

Don, P.S.C.; Mukhtar, H.; Das, M.; Berger, N.A.; Bickers, D.R.

1989-01-01

The metabolism of benzo(a)pyrene (BP), and its subsequent binding to DNA, and the repair of UV-induced DNA damage were studied in fibroblasts cultured from the skin of a 61-year-old male who had multiple basal cell carcinoma (BCC) (>100) on his left upper trunk. Results suggest that BP metabolism and repair of DNA are altered in tumor-bearing site (TSB) cells and that patients with this type of metabolic profile may be at higher risk of the development of cutaneous neoplasms. It is also possible that fibroblasts from tumour bearing skin undergo some as yet unexplained alteration in carcinogen metabolism as a consequence of the induction of neoplasia. (author)

Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

Science.gov (United States)

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

2009-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

OpenAIRE

Wei, Hongbo; Zhao, Lingzhou; Chen, Bangdao; Bai, Shizhu; Zhao, Yimin

2014-01-01

Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; ...

DNA repair in human bronchial epithelial cells

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

1982-01-01

The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

LXA{sub 4} actions direct fibroblast function and wound closure

Energy Technology Data Exchange (ETDEWEB)

Herrera, Bruno S. [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States); Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX (United States); Kantarci, Alpdogan; Zarrough, Ahmed; Hasturk, Hatice [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States); Leung, Kai P., E-mail: kai.p.leung.civ@mail.mil [Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX (United States); Van Dyke, Thomas E., E-mail: tvandyke@forsyth.org [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States)

2015-09-04

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A{sub 4} (LXA{sub 4}), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA{sub 4} on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA{sub 4} receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA{sub 4} receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA{sub 4} slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA{sub 4} tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA{sub 4} in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF

Role of DNA repair in repair of cytogenetic damages. Contribution of repair of single-strand DNA breaks to cytogenetic damages repair

International Nuclear Information System (INIS)

Rozanova, O.M.; Zaichkina, S.I.; Aptikaev, G.F.; Ganassi, E.Eh.

1989-01-01

The comparison was made between the results of the effect of poly(ADP-ribosylation) ingibitors (e.g. nicotinamide and 3-aminobenzamide) and a chromatin proteinase ingibitor, phenylmethylsulfonylfluoride, on the cytogenetic damages repair, by a micronuclear test, and DNA repair in Chinese hamster fibroblasts. The values of the repair half-periods (5-7 min for the cytogenetic damages and 5 min for the rapidly repaired DNA damages) and a similar modyfying effect with regard to radiation cytogenetic damages and kynetics of DNA damages repair were found to be close. This confirms the contribution of repair of DNA single-strand breaks in the initiation of structural damages to chromosomes

Chemosensitivity of primary human fibroblasts with defective unhooking of DNA interstrand cross-links

International Nuclear Information System (INIS)

Clingen, Peter H.; Arlett, Colin F.; Hartley, John A.; Parris, Christopher N.

2007-01-01

Xeroderma pigmentosum (XP) is characterised by defects in nucleotide excision repair, ultraviolet (UV) radiation sensitivity and increased skin carcinoma. Compared to other complementation groups, XP-F patients show relatively mild cutaneous symptoms. DNA interstrand cross-linking agents are a highly cytotoxic class of DNA damage induced by common cancer chemotherapeutics such as cisplatin and nitrogen mustards. Although the XPF-ERCC1 structure-specific endonuclease is required for the repair of ICLs cellular sensitivity of primary human XP-F cells has not been established. In clonogenic survival assays, primary fibroblasts from XP-F patients were moderately sensitive to both UVC and HN2 compared to normal cells (2- to 3-fold and 3- to 5-fold, respectively). XP-A fibroblasts were considerably more sensitive to UVC (10- to 12-fold) but not sensitive to HN2. The sensitivity of XP-F fibroblasts to HN2 correlated with the defective incision or 'unhooking' step of ICL repair. Using the comet assay, XP-F cells exhibited only 20% residual unhooking activity over 24 h. Over the same time, normal and XP-A cells unhooked greater than 95% and 62% of ICLs, respectively. After HN2 treatment, ICL-associated DNA double-strand breaks (DSBs) are detected by pulse field gel electrophoresis in dividing cells. Induction and repair of DNA DSBs was normal in XP-F fibroblasts. These findings demonstrate that in primary human fibroblasts, XPF is required for the unhooking of ICLs and not for the induction or repair of ICL-associated DNA DSBs induced by HN2. In terms of cancer chemotherapy, people with mild DNA repair defects affecting ICL repair may be more prevalent in the general population than expected. Since cellular sensitivity of primary human fibroblasts usually reflects clinical sensitivity such patients with cancer would be at risk of increased toxicity

Mesenchymal stem cells induce dermal fibroblast responses to injury

International Nuclear Information System (INIS)

Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

2010-01-01

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

DNA repair and induction of plasminogen activator in human fetal cells treated with ultraviolet light

International Nuclear Information System (INIS)

Ben-Ishai, R.; Sharon, R.; Rothman, M.; Miskin, R.

1984-01-01

We have tested human fetal fibroblasts for development associated changes in DNA repair by utilizing nucleoid sedimentation as an assay for excision repair. Among skin fibroblasts the rate of excision repair was significantly higher in non-fetal cells than in fibroblasts derived from an 8 week fetus; this was evident by a delay in both the relaxation and the restoration of DNA supercoiling in nucleoids after irradiation. Skin fibroblasts derived at 12 week gestation were more repair proficient than those derived at 8 week gestation. However, they exhibited a somewhat lower rate of repair than non-fetal cells. The same fetal and non-fetal cells were also tested for induction of the protease plasminogen activator (PA) after u.v. irradiation. Enhancement of PA was higher in skin fibroblasts derived at 8 week than in those derived at 12 week gestation and was absent in non-fetal skin fibroblasts. These results are consistent with our previous findings that in human cells u.v. light-induced PA synthesis is correlated with reduced DNA repair capacity. Excision repair and PA inducibility were found to depend on tissue of origin in addition to gestational stage, as shown for skin and lung fibroblasts from the same 12 week fetus. Lung compared to skin fibroblasts exhibited lower repair rates and produced higher levels of PA after irradiation. The sedimentation velocity of nucleoids, prepared from unirradiated fibroblasts, in neutral sucrose gradients with or without ethidium bromide, indicated the presence of DNA strand breaks in fetal cells. It is proposed that reduced DNA repair in fetal cells may result from alterations in DNA supercoiling, and that persistent DNA strand breaks enhance transcription of PA gene(s)

Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

Science.gov (United States)

Wei, Hongbo; Zhao, Lingzhou; Chen, Bangdao; Bai, Shizhu; Zhao, Yimin

2014-01-01

Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; however, those with diameters of 40 and 50 µm improved the spread, actin stress fiber organization, proliferation and fibronectin secretion of the fibroblasts. The microporous structures with micropore diameters of 40–50 µm may be promising for application in the percutaneous part of an implant. PMID:25054322

Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

Directory of Open Access Journals (Sweden)

Hongbo Wei

2014-07-01

Full Text Available Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; however, those with diameters of 40 and 50 µm improved the spread, actin stress fiber organization, proliferation and fibronectin secretion of the fibroblasts. The microporous structures with micropore diameters of 40–50 µm may be promising for application in the percutaneous part of an implant.

Effects of nucleotide pool imbalances on the excision repair of ultraviolet-induced damage in the DNA of human diploid fibroblasts

International Nuclear Information System (INIS)

Snyder, R.D.

1985-01-01

In an attempt to better understand the mechanism of repair inhibition by DNA polymerase inhibitors, and the nature of hydroxyurea enhancement, experiments were initiated in which the effects of a series of ribonucleotide reductase inhibitors on dNTP pools and on the DNA repair process were determined in both quiescent cultures and log-phase cultures of human fibroblasts. It was determined that hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone (TSC), pyrozoloimidazole (IMPY), 3,5-diamino-1,2,4-triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid (THBA) and 3,4-dihydroxy benzohydroxamic acid (DHBA) are all effective inhibitors of the DNA repair process in confluent cells but not in log-phase cells. Moreover, the effects of these inhibitors can be reversed by the addition of certain combinations of deoxynucleosides. These reversal studies and the direct analysis of dNTP pool modulation by these compounds in log phase and confluent cultures support the notion that specific pool depletions rather than general imbalance of pools gives rise to the inhibition of the DNA excision repair process

Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations.

Science.gov (United States)

Philippeos, Christina; Telerman, Stephanie B; Oulès, Bénédicte; Pisco, Angela O; Shaw, Tanya J; Elgueta, Raul; Lombardi, Giovanna; Driskell, Ryan R; Soldin, Mark; Lynch, Magnus D; Watt, Fiona M

2018-04-01

Previous studies have shown that mouse dermis is composed of functionally distinct fibroblast lineages. To explore the extent of fibroblast heterogeneity in human skin, we used a combination of comparative spatial transcriptional profiling of human and mouse dermis and single-cell transcriptional profiling of human dermal fibroblasts. We show that there are at least four distinct fibroblast populations in adult human skin, not all of which are spatially segregated. We define markers permitting their isolation and show that although marker expression is lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signaling, responsiveness to IFN-γ, and ability to support human epidermal reconstitution when introduced into decellularized dermis. These findings suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications in wound healing and diseases characterized by excessive fibrosis. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Evidence of two distinct functionally specialized fibroblast lineages in breast stroma

DEFF Research Database (Denmark)

Morsing, Mikkel; Klitgaard, Marie Christine; Jafari Kermani, Abbas

2016-01-01

Background The terminal duct lobular unit (TDLU) is the most dynamic structure in the human breast and the putative site of origin of human breast cancer. Although stromal cells contribute to a specialized microenvironment in many organs, this component remains largely understudied in the human...... conditions followed by analysis of adipogenic and osteogenic differentiation. To test whether the two fibroblast lineages are functionally imprinted by their site of origin, single cell sorted CD271low/MUC1high normal breast luminal epithelial cells are plated on fibroblast feeders for the observation...... fibroblast lineages exist in the normal human breast, of which the lobular fibroblasts have properties in common with mesenchymal stem cells and support epithelial growth and morphogenesis. We propose that lobular fibroblasts constitute a specialized microenvironment for human breast luminal epithelial...

DNA repair in human fibroblasts, as reflected by host-cell reactivation of a transfected UV-irradiated luciferase gene, is not related to donor age

International Nuclear Information System (INIS)

Merkle, Thomas J.; O'Brien, Katherine; Brooks, Philip J.; Tarone, Robert E.; Robbins, Jay H.

2004-01-01

The effect of donor age on the ability of mammalian cells to repair ultraviolet (UV)-induced DNA damage has been studied using several approaches, most recently via assays that measure the host-cell reactivation (HCR) of UV-irradiated reporter gene-containing plasmid vectors following their transfection into cells. Plasmid HCR assays indirectly quantify a cell line's ability to perform nucleotide excision repair (NER) by measuring the enzyme activity of the repaired reporter gene, e.g., chloramphenical acetyltransferase (cat) or luciferase (luc), and are useful in studies investigating whether increasing age may be a risk factor for the deficient repair of potentially cancer-causing, sunlight-induced, DNA lesions in skin cells. In our study, we quantified the DNA repair ability of cultured, nontransformed, human skin fibroblast lines through their HCR of a transfected UV-C-irradiated plasmid containing luc. HCR was measured at various times after transfection in five lines from normal donors of ages 21-96 years, and from one donor who had xeroderma pigmentosum (XP). The normal lines displayed increasing HCR at successive post-transfection time points and showed no significant correlation between HCR and donor age. The XP-A line, known to be markedly deficient in NER of UV-induced DNA damage, showed minimal evidence of HCR compared to the normal lines. To further assess potential variation in HCR with donor age, fibroblast lines from five old donors, ages 84-94 years, were compared with lines from five young donors, ages 17-26 years. While significant differences in HCR were found between some lines, no significant difference was found between the young and old age groups (P=0.44). Our study provides no indication that the higher incidence of skin cancer observed with increasing age is due to an age-related decrease in the ability to repair UV-induced DNA damage

DNA repair in human fibroblasts, as reflected by host-cell reactivation of a transfected UV-irradiated luciferase gene, is not related to donor age

Energy Technology Data Exchange (ETDEWEB)

Merkle, Thomas J.; O' Brien, Katherine; Brooks, Philip J.; Tarone, Robert E.; Robbins, Jay H

2004-10-04

The effect of donor age on the ability of mammalian cells to repair ultraviolet (UV)-induced DNA damage has been studied using several approaches, most recently via assays that measure the host-cell reactivation (HCR) of UV-irradiated reporter gene-containing plasmid vectors following their transfection into cells. Plasmid HCR assays indirectly quantify a cell line's ability to perform nucleotide excision repair (NER) by measuring the enzyme activity of the repaired reporter gene, e.g., chloramphenical acetyltransferase (cat) or luciferase (luc), and are useful in studies investigating whether increasing age may be a risk factor for the deficient repair of potentially cancer-causing, sunlight-induced, DNA lesions in skin cells. In our study, we quantified the DNA repair ability of cultured, nontransformed, human skin fibroblast lines through their HCR of a transfected UV-C-irradiated plasmid containing luc. HCR was measured at various times after transfection in five lines from normal donors of ages 21-96 years, and from one donor who had xeroderma pigmentosum (XP). The normal lines displayed increasing HCR at successive post-transfection time points and showed no significant correlation between HCR and donor age. The XP-A line, known to be markedly deficient in NER of UV-induced DNA damage, showed minimal evidence of HCR compared to the normal lines. To further assess potential variation in HCR with donor age, fibroblast lines from five old donors, ages 84-94 years, were compared with lines from five young donors, ages 17-26 years. While significant differences in HCR were found between some lines, no significant difference was found between the young and old age groups (P=0.44). Our study provides no indication that the higher incidence of skin cancer observed with increasing age is due to an age-related decrease in the ability to repair UV-induced DNA damage.

Sialylation regulates myofibroblast differentiation of human skin fibroblasts.

Science.gov (United States)

Sasaki, Norihiko; Itakura, Yoko; Toyoda, Masashi

2017-04-18

Fibroblasts are key players in maintaining skin homeostasis and in orchestrating physiological tissue repair and skin regeneration. Dysfunctions in fibroblasts that occur with aging and the senescent process lead to the delayed healing observed in elderly people. The molecular mechanisms leading to fibroblast dysfunction during aging and the senescent process have not yet been clarified. Previously, changes in patterns of glycosylation were observed in fibroblasts in aging and the senescent process, but the effect of these changes on the function of fibroblasts has not been well documented. Here, we investigated whether changes in glycosylation during the process to senescence may have functional effects on fibroblasts. The changes in cell surface glycans on skin fibroblasts during the process to senescence were examined in early-passage (EP) and late-passage (LP) skin fibroblasts by fluorescence-activated cell sorting analysis using lectins. The contributors to the changes in cell surface glycans were examined by real-time polymerase chain reaction or Western blot analysis. The effects of changes in glycosylation on proliferation, migration, induction of cellular senescence, and myofibroblast differentiation induced by transforming growth factor (TGF)-β1 stimulation were examined in EP fibroblasts. The changes in glycosylation were performed by GalNAc-α-O-benzyl or sialidase treatment. A decrease in sialylation of glycoproteins and an increase in sialidase NEU1 were observed in LP fibroblasts. The reduction of sialylation did not have any effect on proliferation, migration, or induction of cellular senescence. On the other hand, myofibroblast differentiation was inhibited by the reduction of sialylation, indicating that sialylation is important for myofibroblast differentiation. The localization of CD44 in lipid rafts, which is required for myofibroblast differentiation, was inhibited by the reduction of sialylation. Furthermore, reduced myofibroblast

Development of a liquid-holding technique for the study of DNA-repair in human diploid fibroblasts

International Nuclear Information System (INIS)

Simons, J.W.I.M.

1979-01-01

Liquid-holding conditions can be obtained for human diploid skin fibro-blasts by keeping confluent cultures stationary over periods of 7 days or longer by means of conditioned medium. Under this condition recovery of radiation damage induced by ultraviolet light or X-rays is observed as an increase in cloning efficiency. The amount of recovery when expressed in a dose-modifying-factor appears higher than in bacteria and yeast. The repair-deficient human cell strains XP25Ro and XP7Be (xeroderma pigmentosum from complementation groups A and D respectively) exhibit less but still discernible recovery after UV-irradiation and the same was observed for AT5Bi (ataxia telangiectasia) after X-irradiation. Experiments on mutation induction indicated that the repair which takes place during liquid holding of UV-irradiated XP7Be cells reduces the mutant frequency considerably while after liquid holding of UV-irradiated wild-type cells the same or lower mutant frequencies were found for the lower exposures and the same or higher mutant frequencies for the higher exposures. (Auth.)

In vivo excision of pyrimidine dimers is mediated by a DNA N-glycosylase in Micrococcus luteus but not in human fibroblasts

International Nuclear Information System (INIS)

La Belle, M.; Linn, S.

1982-01-01

It has been previously shown that Micrococcus luteus possesses a pyrimidine dimer-specific endonuclease which in vitro, functions as both an endonuclease and DNA-glycosylase. To determine if these combined activities function in vivo, the excision products of UV-irradiated M. luteus were isolated and examined. In addition, a procedure was devised to isolate and examine the excision products from UV-irradiated human fibroblasts to determine if an endonuclease/glycosylase activity functions in the excision of UV-induced pyrimidine dimers in human fibroblasts. It was shown that, in vivo, an endonuclease/glycosylase mechanism is utilized extensively in the repair of pyrimidine dimers by M. luteus, but that human fibroblasts do not appear to use this mechanism. (author)

In vivo excision of pyrimidine dimers is mediated by a DNA N-glycosylase in Micrococcus luteus but not in human fibroblasts

Energy Technology Data Exchange (ETDEWEB)

La Belle, M; Linn, S [California Univ., Berkeley (USA). Dept. of Biochemistry

1982-09-01

It has been previously shown that Micrococcus luteus possesses a pyrimidine dimer-specific endonuclease which in vitro, functions as both an endonuclease and DNA-glycosylase. To determine if these combined activities function in vivo, the excision products of UV-irradiated M. luteus were isolated and examined. In addition, a procedure was devised to isolate and examine the excision products from UV-irradiated human fibroblasts to determine if an endonuclease/glycosylase activity functions in the excision of UV-induced pyrimidine dimers in human fibroblasts. It was shown that, in vivo, an endonuclease/glycosylase mechanism is utilized extensively in the repair of pyrimidine dimers by M. luteus, but that human fibroblasts do not appear to use this mechanism.

Satisfaction, function and repair integrity after arthroscopic versus mini-open rotator cuff repair.

Science.gov (United States)

Barnes, L A Fink; Kim, H M; Caldwell, J-M; Buza, J; Ahmad, C S; Bigliani, L U; Levine, W N

2017-02-01

Advances in arthroscopic techniques for rotator cuff repair have made the mini-open approach less popular. However, the mini-open approach remains an important technique for repair for many surgeons. The aims of this study were to compare the integrity of the repair, the function of the shoulder and satisfaction post-operatively using these two techniques in patients aged > 50 years. We identified 22 patients treated with mini-open and 128 patients treated with arthroscopic rotator cuff repair of July 2007 and June 2011. The mean follow-up was two years (1 to 5). Outcome was assessed using the American Shoulder and Elbow Surgeons (ASES) and Simple Shoulder Test (SST) scores, and satisfaction. The integrity of the repair was assessed using ultrasonography. A power analysis ensured sufficient enrolment. There was no statistically significant difference between the age, function, satisfaction, or pain scores (p > 0.05) of the two groups. The integrity of the repair and the mean SST scores were significantly better in the mini-open group (91% of mini-open repairs were intact versus 60% of arthroscopic repairs, p = 0.023; mean SST score 10.9 (standard deviation (sd) 1.3) in the mini-open group; 8.9 (sd 3.5) in arthroscopic group; p = 0.003). The ASES scores were also higher in the mini-open group (mean ASES score 91.0 (sd 10.5) in mini-open group; mean 82.70 (sd 19.8) in the arthroscopic group; p = 0.048). The integrity of the repair and function of the shoulder were better after a mini-open repair than after arthroscopic repair of a rotator cuff tear in these patients. The functional difference did not translate into a difference in satisfaction. Mini-open rotator cuff repair remains a useful technique despite advances in arthroscopy. Cite this article: Bone Joint J 2017;99-B:245-9. ©2017 The British Editorial Society of Bone & Joint Surgery.

DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts.

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Elena K Braithwaite

2010-08-01

Full Text Available Base excision repair (BER is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol beta is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol lambda, was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases lambda and beta in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol beta and pol lambda. Neutral red viability assays demonstrated that pol lambda and pol beta double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol lambda to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol lambda and pol beta interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.

Transfer of Chinese hamster DNA repair gene(s) into repair-deficient human cells (Xeroderma pigmentosum)

International Nuclear Information System (INIS)

Karentz, D.; Cleaver, J.E.

1985-01-01

Transfer of repair genes by DNA transfection into repair-deficient Xeroderma pigmentosum (XP) cells has thus far been unsuccessful, presenting an obstacle to cloning XP genes. The authors chose an indirect route to transfer repair genes in chromosome fragments. DNA repair-competent (UV resistant) hybrid cell lines were established by PEG-mediated fusions of DNA repair-deficient (UV sensitive) human fibroblasts (XP12RO) with wild type Chinese hamster (CHO) cells (AA8). CHO cells were exposed to 5 Krad X-rays prior to fusions, predisposing hybrid cells to lose CHO chromosome fragments preferentially. Repair-competent hybrids were selected by periodic exposures to UV light. Secondary and tertiary hybrid cell lines were developed by fusion of X-irradiated hybrids to XP12RO. The hybrid cell lines exhibit resistance to UV that is comparable to that of CHO cells and they are proficient at repair replication after UV exposure. Whole cell DNA-DNA hybridizations indicate that the hybrids have greater homology to CHO DNA than is evident between XP12RO and CHO. These observations indicate that CHO DNA sequences which can function in repair of UV-damaged DNA in human cells have been transferred into the genome of the repair-deficient XP12RO cells

Radiation-induced chromosome aberrations and cell killing in normal human fibroblasts and ataxia telangiectasia fibroblasts

International Nuclear Information System (INIS)

Kawata, T.; Saito, M.; Uno, T.; Ito, H.; Shigematsu, N.

2003-01-01

Full text: When cells are held in a non-dividing state (G0) after irradiation, an enhanced survival can be observed compared to that of immediate plating. A change of survival depending on post irradiation condition is known to be repair of potentially lethal damage (RPLD). The effects of confluent holding recovery (24-h incubation following irradiation) on chromosome aberrations in normal human fibroblasts (AG1522) and ataxia telangiectasia fibroblasts (GM02052C) were examined. A chemical-induced premature chromosome condensation (PCC) technique with fluorescent in situ hybridization (FISH) was applied to study chromosome aberrations in G2 and M-phase. Results from cell survival showed that the capacity for potentially lethal damage repair was normal in AG1522 cells but very little in GM02052C cells. The frequency of chromosome aberrations in AG1522 cells decreased when cells were allowed to repair for 24-h. Especially complex type exchanges were found to decrease markedly at high doses (4Gy and 6Gy). However, the frequency of chromosome aberrations including complex type exchanges showed little decrease in GM02052C cells. Confluent holding can effectively reduce chromosome aberrations, especially complex type exchanges in normal cells

The role of nuclear hormone receptors in cutaneous wound repair.

Science.gov (United States)

Rieger, Sandra; Zhao, Hengguang; Martin, Paige; Abe, Koichiro; Lisse, Thomas S

2015-01-01

The cutaneous wound repair process involves balancing a dynamic series of events ranging from inflammation, oxidative stress, cell migration, proliferation, survival and differentiation. A complex series of secreted trophic factors, cytokines, surface and intracellular proteins are expressed in a temporospatial manner to restore skin integrity after wounding. Impaired initiation, maintenance or termination of the tissue repair processes can lead to perturbed healing, necrosis, fibrosis or even cancer. Nuclear hormone receptors (NHRs) in the cutaneous environment regulate tissue repair processes such as fibroplasia and angiogenesis. Defects in functional NHRs and their ligands are associated with the clinical phenotypes of chronic non-healing wounds and skin endocrine disorders. The functional relationship between NHRs and skin niche cells such as epidermal keratinocytes and dermal fibroblasts is pivotal for successful wound closure and permanent repair. The aim of this review is to delineate the cutaneous effects and cross-talk of various nuclear receptors upon injury towards functional tissue restoration. Copyright © 2014 John Wiley & Sons, Ltd.

Genetic characterization of cells of homocystinuria patients with disrupted DNA repair system

International Nuclear Information System (INIS)

Sinel'shchikova, T.A.; L'vova, G.N.; Shoniya, N.N.; Zasukhina, G.D.

1986-01-01

Fibroblasts obtained from biopsy material and lymphocytes of patients with homocystinuria were investigated for repair activity according to the following criteria: rejoined DNA breaks, induced by 4-nitroquinoline-1-oxide and γ-radiation; indices of reactivation and induced mutagenesis of smallpox vaccine virus treated with these mutagens. In lymphocytes a defect of DNA repair was observed according to all criteria investigated. During passage of fibroblast cultures, inhibition of repair activity of cells was preserved according to γ-type. Increase in the number of spontaneous and γ-induced mutations of virus was noted according to degree of passage of fibroblasts

DNA repair after a UV-C irradiation in cultivated human fibroblasts: Studies on patients with actinic keratosis, spinocellular carcinoma and basal cell carcinoma as well as healthy controls. DNA-Reparatur nach UV-C-Bestrahlung in kultivierten menschlichen Fibroplasten: Untersuchungen an Patienten mit aktinischen Keratosen, Spinaliomen und Basaliomen sowie gesunden Kontrollen

Energy Technology Data Exchange (ETDEWEB)

Senner, A.

1984-01-11

Cultivated fibroblasts from 14 healthy control people and 22 patients were irradiated with UV-C light in increasing doses (0-1-2.5-5 mJ/cm/sup 2/). The incorporation of /sup 3/H-thymidine in the DNA of the fibroblasts which is made visible by the autoradiography technique served as a measure for the unprogrammed DNA synthesis. All of the studied fibroblast types showed with an increase in the UV-C irradiation dose an increase in the repair processes of the DNA. No statistically significant difference could be shown in the repair behaviour of the fibroblasts of controls and patients.

Cell-based and biomaterial approaches to connective tissue repair

Science.gov (United States)

Stalling, Simone Suzette

Connective tissue injuries of skin, tendon and ligament, heal by a reparative process in adults, filling the wound site with fibrotic, disorganized scar tissue that poorly reflects normal tissue architecture or function. Conversely, fetal skin and tendon have been shown to heal scarlessly. Complete regeneration is not intrinsically ubiquitous to all fetal tissues; fetal diaphragmatic and gastrointestinal injuries form scars. In vivo studies suggest that the presence of fetal fibroblasts is essential for scarless healing. In the orthopaedic setting, adult anterior cruciate ligament (ACL) heals poorly; however, little is known about the regenerative capacity of fetal ACL or fetal ACL fibroblasts. We characterized in vitro wound healing properties of fetal and adult ACL fibroblasts demonstrating that fetal ACL fibroblasts migrate faster and elaborate greater quantities of type I collagen, suggesting the healing potential of the fetal ACL may not be intrinsically poor. Similar to fetal ACL fibroblasts, fetal dermal fibroblasts also exhibit robust cellular properties. We investigated the age-dependent effects of dermal fibroblasts on tendon-to-bone healing in rat supraspinatus tendon injuries, a reparative injury model. We hypothesized delivery of fetal dermal fibroblasts would increase tissue organization and mechanical properties in comparison to adult dermal fibroblasts. However, at 1 and 8 weeks, the presence of dermal fibroblasts, either adult or fetal, had no significant effect on tissue histology or mechanical properties. There was a decreasing trend in cross-sectional area of repaired tendons treated with fetal dermal fibroblasts in comparison to adult, but this finding was not significant in comparison to controls. Finally, we synthesized a novel polysaccharide, methacrylated methylcellulose (MA-MC), and fabricated hydrogels using a well-established photopolymerization technique. We characterized the physical and mechanical properties of MA-MC hydrogels in

Decreased Fibroblast and Increased Osteoblast Functions on Ionic Plasma Deposited Nanostructured Ti Coatings

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Storey Dan

2007-01-01

Full Text Available AbstractBioactive coatings are in high demand to control cellular functions for numerous medical devices. The objective of this in vitro study was to characterize for the first time fibroblast (fibrous scar tissue forming cells adhesion and proliferation on an important polymeric biomaterial (silicone coated with titanium using a novel ionic plasma deposition (IPD process. Fibroblasts are one of the first anchorage-dependent cells to arrive at an implant surface during the wound healing process. Persistent excessive functions of fibroblasts have been linked to detrimental fibrous tissue formation which may cause implant failure. The IPD process creates a surface-engineered nanostructure (with features usually below 100 nm by first using a vacuum to remove all contaminants, then guiding charged metallic ions or plasma to the surface of a medical device at ambient temperature. Results demonstrated that compared to currently used titanium and uncoated silicone, silicone coated with titanium using IPD significantly decreased fibroblast adhesion and proliferation. Results also showed competitively increased osteoblast (bone-forming cells over fibroblast adhesion on silicone coated with titanium; in contrast, osteoblast adhesion was not competitively increased over fibroblast adhesion on uncoated silicone or titanium controls. In this manner, this study strongly suggests that IPD should be further studied for biomaterial applications in which fibrous tissue encapsulation is undesirable (such as for orthopedic implants, cardiovascular components, etc..

Altered Dermal Fibroblasts in Systemic Sclerosis Display Podoplanin and CD90.

Science.gov (United States)

Nazari, Banafsheh; Rice, Lisa M; Stifano, Giuseppina; Barron, Alexander M S; Wang, Yu Mei; Korndorf, Tess; Lee, Jungeun; Bhawan, Jag; Lafyatis, Robert; Browning, Jeffrey L

2016-10-01

Tissue injury triggers the activation and differentiation of multiple cell types to minimize damage and initiate repair processes. In systemic sclerosis, these repair processes appear to run unchecked, leading to aberrant remodeling and fibrosis of the skin and multiple internal organs, yet the fundamental pathological defect remains unknown. We describe herein a transition wherein the abundant CD34(+) dermal fibroblasts present in healthy human skin disappear in the skin of systemic sclerosis patients, and CD34(-), podoplanin(+), and CD90(+) fibroblasts appear. This transition is limited to the upper dermis in several inflammatory skin diseases, yet in systemic sclerosis, it can occur in all regions of the dermis. In vitro, primary dermal fibroblasts readily express podoplanin in response to the inflammatory stimuli tumor necrosis factor and IL-1β. Furthermore, we show that on acute skin injury in both human and murine settings, this transition occurs quickly, consistent with a response to inflammatory signaling. Transitioned fibroblasts partially resemble the cells that form the reticular networks in organized lymphoid tissues, potentially linking two areas of fibroblast research. These results allow for the visualization and quantification of a basic stage of fibroblast differentiation in inflammatory and fibrotic diseases in the skin. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

X ray sensitivity of diploid skin fibroblasts from patients with Fanconi's anemia

Science.gov (United States)

Kale, Ranjini

1989-01-01

Experiments were performed on Fanconi's anemia and normal human fibroblast cell lines growing in culture in an attempt to correlate cell cycle kinetics with genomic damage and determine their bearing on the mechanism of chromosome aberration induction. FA fibroblasts showed a significantly increased susceptibility to chromosomal breakage by x rays in the G2 phase of the cell cycle. No such response was observed in fibroblasts irradiated in the G0 phase. The observed increases in achromatic lesions and in chromatid deletions in FA cells as compared with normal cells appear to indicate that FA cells are deficient in strand break repair and also possibly in base damage excision repair. Experiments are now in progress to further elucidate the mechanisms involved.

Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia.

Science.gov (United States)

Lerman, Oren Z; Galiano, Robert D; Armour, Mary; Levine, Jamie P; Gurtner, Geoffrey C

2003-01-01

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients.

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

International Nuclear Information System (INIS)

Hammond, R.A.; Miller, M.R.; McClung, J.K.

1990-01-01

The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

The CXC chemokine cCAF stimulates precocious deposition of ECM molecules by wound fibroblasts, accelerating development of granulation tissue

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Li Qi-Jing

2002-06-01

Full Text Available Abstract Background During wound repair, fibroblasts orchestrate replacement of the provisional matrix formed during clotting with tenascin, cellular fibronectin and collagen III. These, in turn, are critical for migration of endothelial cells, keratinocytes and additional fibroblasts into the wound site. Fibroblasts are also important in the deposition of collagen I during scar formation. The CXC chemokine chicken Chemotactic and Angiogenic Factor (cCAF, is highly expressed by fibroblasts after wounding and during development of the granulation tissue, especially in areas where extracellular matrix (ECM is abundant. We hypothesized that cCAF stimulates fibroblasts to produce these matrix molecules. Results Here we show that this chemokine can stimulate precocious deposition of tenascin, fibronectin and collagen I, but not collagen III. Studies in culture and in vivo show that tenascin stimulation can also be achieved by the N-terminal 15 aas of the protein and occurs at the level of gene expression. In contrast, stimulation of fibronectin and collagen I both require the entire molecule and do not involve changes in gene expression. Fibronectin accumulation appears to be linked to tenascin production, and collagen I to decreased MMP-1 levels. In addition, cCAF is chemotactic for fibroblasts and accelerates their migration. Conclusions These previously unknown functions for chemokines suggest that cCAF, the chicken orthologue of human IL-8, enhances healing by rapidly chemoattracting fibroblasts into the wound site and stimulating them to produce ECM molecules, leading to precocious development of granulation tissue. This acceleration of the repair process may have important application to healing of impaired wounds.

Different proliferative capacity of lung fibroblasts obtained from control subjects and patients with emphysema

NARCIS (Netherlands)

Noordhoek, JA; Postma, DS; Chong, LL; Vos, JTWM; Kauffman, HF; Timens, W; van Straaten, JFM

2003-01-01

To characterize the possible role of a dysregulated proliferative capacity of pulmonary fibroblasts in insufficient tissue repair in lungs from patients with pulmonary emphysema, the authors undertook in vitro proliferative studies with pulmonary fibroblasts obtained from lung tissue of patients

Repair processes for photochemical damage in mammalian cells

International Nuclear Information System (INIS)

Cleaver, J.E.

1974-01-01

Repair processes for photochemical damage in cells following uv irradiation are reviewed. Cultured fibroblast cells from human patients with xeroderma pigmentosum were used as an example to illustrate aspects of repair of injuries to DNA and proteins. (250 references) (U.S.)

Direct lineage reprogramming of mouse fibroblasts to functional midbrain dopaminergic neuronal progenitors

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Han-Seop Kim

2014-01-01

Full Text Available The direct lineage reprogramming of somatic cells to other lineages by defined factors has led to innovative cell-fate-change approaches for providing patient-specific cells. Recent reports have demonstrated that four pluripotency factors (Oct4, Sox2, Klf4, and c-Myc are sufficient to directly reprogram fibroblasts to other specific cells, including induced neural stem cells (iNSCs. Here, we show that mouse fibroblasts can be directly reprogrammed into midbrain dopaminergic neuronal progenitors (DPs by temporal expression of the pluripotency factors and environment containing sonic hedgehog and fibroblast growth factor 8. Within thirteen days, self-renewing and functional induced DPs (iDPs were generated. Interestingly, the inhibition of both Jak and Gsk3β notably enhanced the iDP reprogramming efficiency. We confirmed the functionality of the iDPs by showing that the dopaminergic neurons generated from iDPs express midbrain markers, release dopamine, and show typical electrophysiological profiles. Our results demonstrate that the pluripotency factors-mediated direct reprogramming is an invaluable strategy for supplying functional and proliferating iDPs and may be useful for other neural progenitors required for disease modeling and cell therapies for neurodegenerative disorders.

Modulation of ROS levels in fibroblasts by altering mitochondria regulates the process of wound healing.

Science.gov (United States)

Janda, Jaroslav; Nfonsam, Valentine; Calienes, Fernanda; Sligh, James E; Jandova, Jana

2016-05-01

Mitochondria are the major source of reactive oxygen species (ROS) in fibroblasts which are thought to be crucial regulators of wound healing with a potential to affect the expression of nuclear genes involved in this process. ROS generated by mitochondria are involved in all stages of tissue repair process but the regulation of ROS-generating system in fibroblasts still remains poorly understood. The purpose of this study was to better understand molecular mechanisms of how the regulation of ROS levels generated by mitochondria may influence the process of wound repair. Cybrid model system of mtDNA variations was used to study the functional consequences of altered ROS levels on wound healing responses in a uniform nuclear background of cultured ρ(0) fibroblasts. Mitochondrial ROS in cybrids were modulated by antioxidants that quench ROS to examine their ability to close the wound. Real-time PCR arrays were used to investigate whether ROS generated by specific mtDNA variants have the ability to alter expression of some key nuclear-encoded genes central to the wound healing response and oxidative stress. Our data suggest levels of mitochondrial ROS affect expression of some nuclear encoded genes central to wound healing response and oxidative stress and modulation of mitochondrial ROS by antioxidants positively affects in vitro process of wound closure. Thus, regulation of mitochondrial ROS-generating system in fibroblasts can be used as effective natural redox-based strategy to help treat non-healing wounds.

Repetitious nature of repaired DNA in mammalian cells. Progress report, June 1, 1976--February 28, 1977

International Nuclear Information System (INIS)

Meltz, M.L.

1977-02-01

Progress is reported on studies of DNA repair in cultured mouse L fibroblasts, human diploid fibroblasts, and cultured human lymphoblastoid cell lines. Data are included on the effects of methyl methanesulfonate treatment, uv light, and age of cell donors on repair replication of DNA

Somatic mosaicism for DNA repair capacity in fibroblasts derived from a group A xeroderma pigmentosum patient

International Nuclear Information System (INIS)

Chang, H.R.; Ishizaki, K.; Sasaki, M.S.; Toguchida, J.; Kato, M.; Nakamura, Y.; Kawamura, S.; Moriguchi, T.; Ikenaga, M.

1989-01-01

A female Japanese xeroderma pigmentosum (XP) patient with severe skin lesions and various neurologic abnormalities was assigned to complementation group A by conventional cell fusion studies. Ultraviolet (UV)-irradiated skin fibroblasts showed a biphasic survival curve, as measured by colony-forming ability. The surviving fraction decreased rapidly up to 2 J/m2 of UV, with a steep slope of D(O) (mean lethal dose) = 0.95 J/m2. At much higher doses it decreased more slowly, with D(O) = 3.5 J/m2. To elucidate the cause of this unique survival response, we isolated a large number of independent clones from single colonies and measured their responses to UV. Of 81 clones analyzed, ten showed a marked resistance to killing by UV, which was only slightly more sensitive than normal cells, and these clones had a rate of unscheduled DNA synthesis (UDS) that was about 45% of normal cells. By contrast, the remaining 71 clones were extremely sensitive to UV, typical of XP group A strains, and had a UDS level 1%-3% of normals. Analysis of restriction fragment length polymorphism using seven polymorphic DNA probes indicated that the UV-resistant clones were derived from the same individual as the UV-sensitive clones. These results clearly demonstrate that this patient's fibroblast cells consist of two types with differing responses to UV, and provide direct evidence of somatic mosaicism for DNA repair capacity in an XP patient

In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

International Nuclear Information System (INIS)

Dresler, S.; Frattini, M.G.; Robinson-Hill, R.M.

1988-01-01

Using permeable diploid human fibroblasts, the authors have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent K m values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 μM. For UV-induced DNA repair synthesis, the apparent K m values were substantially lower, ranging from 0.11 to 0.44 μM for AG1518 cells and from 0.06 to 0.24 μM for IMR-90 cells. Recent data implicate DNA polymerase δ in UV-induced repair synthesis and suggest that DNA polymerases α and δ are both involved in semiconservative replication. They measured K m values for dGTP and dTTP for polymerases α and δ, for comparison with the values for replication and repair synthesis. The deoxyribonucleotide K m values for DNA polymerase δ are much greater than the K m values for UV-induced repair synthesis, suggesting that when polymerase δ functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases. The K m values for UV-induced repair synthesis are 5-80-fold lower than deoxyribonucleotide concentrations that have been reported for intact cultured diploid human fibroblasts. For replication, however, the K m for dGTP is only slightly lower than the average cellular dGTP concentration that has been reported for exponentially growing human fibroblasts. This finding is consistent with the concept that nucleotide compartmentation is required for the attainment of high rates of DNA replication in vivo

Coupling of cytoskeleton functions for fibroblast locomotion

DEFF Research Database (Denmark)

Couchman, J R; Lenn, M; Rees, D A

1985-01-01

caused visible protrusions in projected positions at the leading edge. We conclude that fibroblast locomotion may be driven coordinately by a common set of motility mechanisms and that this coordination may be lost as a result of physical or pharmacological disturbance. Taking our evidence with results...... from other Laboratories, we propose the following cytoskeleton functions. (i) Protrusive activity, probably based on solation--gelation cycles of the actin based cytoskeleton and membrane recycling which provides cellular and membrane components for streaming through the cell body to the leading edge...

Triptonide inhibits the pathological functions of gastric cancer-associated fibroblasts.

Science.gov (United States)

Wang, Zhenfei; Ma, Daguang; Wang, Changshan; Zhu, Zhe; Yang, Yongyan; Zeng, Fenfang; Yuan, Jianlong; Liu, Xia; Gao, Yue; Chen, Yongxia; Jia, Yongfeng

2017-12-01

Direct attacks on tumour cells with chemotherapeutic drugs have the drawbacks of accelerating tumour metastasis and inducing tumour stem cell phenotypes. Inhibition of tumour-associated fibroblasts, which provide nourishment and support to tumour cells, is a novel and promising anti-tumour strategy. However, effective drugs against tumour-associated fibroblasts are currently lacking. In the present study, we explored the possibility of inhibiting the pathological functions of tumour-associated fibroblasts with triptonide. Paired gastric normal fibroblasts (GNFs) and gastric cancer-associated fibroblasts (GCAFs) were obtained from resected tissues. GCAFs showed higher capacities to induce colony formation, migration, and invasion of gastric cancer cells than GNFs. Triptonide treatment strongly inhibited the colony formation-, migration-, and invasion-promoting capacities of GCAFs. The expression of microRNA-301a was higher and that of microRNA-149 was lower in GCAFs than in GNFs. Triptonide treatment significantly down-regulated microRNA-301a expression and up-regulated microRNA-149 expression in GCAFs. Re-establishment of microRNA expression balance increased the production and secretion of tissue inhibitor of metalloproteinase 2, a tumour suppressive factor, and suppressed the production and secretion of IL-6, an oncogenic factor, in GCAFs. Moreover, triptonide treatment abolished the ability of GCAFs to induce epithelial-mesenchymal transition in gastric cancer cells. These results indicate that triptonide inhibits the malignancy-promoting capacity of GCAFs by correcting abnormalities in microRNA expression. Thus, triptonide is a promisingly therapeutic agent for gastric cancer treatment, and traditional herbs may be a valuable source for developing new drugs that can regulate the tumour microenvironment. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Evaluation of the effect of laser radiation on fibroblast proliferation in repair of skin wounds of rats with iron deficiency anemia

Science.gov (United States)

DeCastro, Isabele C. V.; Oliveira-Sampaio, Susana C. P.; Monteiro, Juliana S. de C.; Ferreira, Maria de Fátima L.; Cangussu, Maria T.; N. dos Santos, Jean; Pinheiro, Antonio Luiz B.

2011-03-01

The aim of this study was to assess the effect of low- level laser therapy (LLLT) on fibroblast proliferation on wound repair of rats with Iron deficiency anemia since there is no reports on literature about this subject. Iron deficiency anemia was induced on 36 newborn rats then an excisional wound was created on the dorsum of the animals which were divided into four groups: (I) - non-anemic, (II) - Anemic, (III) - non-anemic + LLLT, (IV) Anemic+ LLLT. The animals in each group were sacrificed at 7, 14 and 21 days. Laser irradiation was performed on each group (λ660nm,40Mw,CW) by contact mode with a dose of 2,5J/ cm2 in four points on the area of the wound and total of 10J/cm2 per session. Data were evaluated by analysis of variance (ANOVA) followed by Paired t-test. The results showed LLLT was able to stimulate fibroblastic proliferation in rats with iron deficiency anemia at the 21st day while at control group (III) no statistically significant differences was found.

Enhanced radiosensitivity of cultured fibroblasts from ataxia telangiectasia heterozygotes manifested by defective colony-forming ability and reduced DNA repair replication after hypoxic γ-irradiation

International Nuclear Information System (INIS)

Paterson, M.C.; Anderson, A.K.; Smith, B.P.; Smith, P.J.

1979-01-01

We have measured the sensitivity to γ-ray inactivation of diploid skin fibroblasts cultured from 10 persons in four families with ataxia telangiectasia (AT). Persons heterozygous for AT, including parents of afflicted patients, are not as yet detectable by any specific clinical or laboratory marker but are believed to constitute a substantial portion of the middle-aged cancer population. In one AT family, fibroblast strains from both parents exhibited a colony-forming ability after hypoxic irradiation which was intermediate between that displayed by five control strains from normal children and that from the affected child. In the remaining three families, cultures from only one parent were available; one parental strain displayed an intermediate survival capacity as above, whereas the other two responded normally. The homozygous recessive strains from the five afflicted children in the four families were all equally hypersensitive to hypoxic γ-ray inactivation. The three presumed AT heterozygous strains that displayed intermediate rayiosensitivity also carried out γ-rad-induced DNA repair replication to an extent intermediate between those in normals and AT homozygotes. These findings suggest that a numerically significant, cancer-prone subpopulation of humans carrying one normal and one abnormal AT gene may also be moderately sensitive to lethal effects of hypoxic γ-rays due to a defect in the enzymatic repair of DNA

Aberrant repair and fibrosis development in skeletal muscle

Directory of Open Access Journals (Sweden)

Mann Christopher J

2011-05-01

Full Text Available Abstract The repair process of damaged tissue involves the coordinated activities of several cell types in response to local and systemic signals. Following acute tissue injury, infiltrating inflammatory cells and resident stem cells orchestrate their activities to restore tissue homeostasis. However, during chronic tissue damage, such as in muscular dystrophies, the inflammatory-cell infiltration and fibroblast activation persists, while the reparative capacity of stem cells (satellite cells is attenuated. Abnormal dystrophic muscle repair and its end stage, fibrosis, represent the final common pathway of virtually all chronic neurodegenerative muscular diseases. As our understanding of the pathogenesis of muscle fibrosis has progressed, it has become evident that the muscle provides a useful model for the regulation of tissue repair by the local microenvironment, showing interplay among muscle-specific stem cells, inflammatory cells, fibroblasts and extracellular matrix components of the mammalian wound-healing response. This article reviews the emerging findings of the mechanisms that underlie normal versus aberrant muscle-tissue repair.

Identification of the DNA repair defects in a case of Dubowitz syndrome.

Directory of Open Access Journals (Sweden)

Jingyin Yue

Full Text Available Dubowitz Syndrome is an autosomal recessive disorder with a unique set of clinical features including microcephaly and susceptibility to tumor formation. Although more than 140 cases of Dubowitz syndrome have been reported since 1965, the genetic defects of this disease has not been identified. In this study, we systematically analyzed the DNA damage response and repair capability of fibroblasts established from a Dubowitz Syndrome patient. Dubowitz syndrome fibroblasts are hypersensitive to ionizing radiation, bleomycin, and doxorubicin. However, they have relatively normal sensitivities to mitomycin-C, cisplatin, and camptothecin. Dubowitz syndrome fibroblasts also have normal DNA damage signaling and cell cycle checkpoint activations after DNA damage. These data implicate a defect in repair of DNA double strand break (DSB likely due to defective non-homologous end joining (NHEJ. We further sequenced several genes involved in NHEJ, and identified a pair of novel compound mutations in the DNA Ligase IV gene. Furthermore, expression of wild type DNA ligase IV completely complement the DNA repair defects in Dubowitz syndrome fibroblasts, suggesting that the DNA ligase IV mutation is solely responsible for the DNA repair defects. These data suggests that at least subset of Dubowitz syndrome can be attributed to DNA ligase IV mutations.

Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

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Benamer, Najate [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France); Fares, Nassim [Laboratoire de Physiologie, Faculte de Medecine, Universite Saint Joseph, Beyrouth (Lebanon); Bois, Patrick [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France); Faivre, Jean-Francois, E-mail: Jean-Francois.Faivre@univ-poitiers.fr [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France)

2011-04-29

Highlights: {yields} In cardiac fibroblasts, SUR2/Kir6.1 channel is activated by S1P via the S1P3R. {yields} S1P increases cell proliferation through SUR2/Kir6.1 activation. {yields} S1P decreases collagen and IL-6 secretion through SUR2/Kir6.1 activation. {yields} S1P stimulates fibroblast migration independently from SUR2/Kir6.1 channel. -- Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac

Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

International Nuclear Information System (INIS)

Benamer, Najate; Fares, Nassim; Bois, Patrick; Faivre, Jean-Francois

2011-01-01

Highlights: → In cardiac fibroblasts, SUR2/Kir6.1 channel is activated by S1P via the S1P3R. → S1P increases cell proliferation through SUR2/Kir6.1 activation. → S1P decreases collagen and IL-6 secretion through SUR2/Kir6.1 activation. → S1P stimulates fibroblast migration independently from SUR2/Kir6.1 channel. -- Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac physiopathological injury.

Repair of damage by ultraviolet radiation in xeroderma pigmentosum cell strains of complementation groups E and F

NARCIS (Netherlands)

Zelle, B.; Berends, F.; Lohman, P.H.M.

1980-01-01

The xeroderma pignemtosum fibroblast strains XP2RO, complementation group E, and XP23OS, group F were compared with normal human primary fibroblasts UV. regard to repair of damage induced by 254-nn UV> In XP2RO cells, repair DNA synthesis, measured by autoradiography (unscheduled DNA synthesis =

DNA repair capacity and rate of excision repair in UV-irradiated mammalian cells

International Nuclear Information System (INIS)

Inoue, Masao; Takebe, Hiraku.

1978-01-01

Repair capacities of five mammalian cell strains were measured by colony-forming ability, HCR of UV-irradiated virus, UDS, pyrimidine dimer excision, and semi-conservative DNA replication. Colony-forming ability of UV-irradiated cells was high for human amnion FL cells and mouse L cells, slightly low for African green monkey CV-1 cells, and extremely low for xeroderma pigmentosum cells. HCR of UV-irradiated Herpes simplex virus was high in CV-1 cells, FL and normal human fibroblast cells, low in both XP and L cells. The amount of UDS was high in FL and normal human fibroblast cells, considerably low in CV-1 cells, and essentially no UDS was observed in XP cells. Rate of UDS after UV-irradiation was slower for CV-1 cells than FL and human fibroblast cells. Rate of the excision of thymine-containing dimers from the acid-insoluble fraction during post-irradiation incubation of the cells was rapid in FL and normal human cells and slow in CV-1 cells, and no excision took place in XP cells. Semi-conservative DNA synthesis was reduced after UV-irradiation in all cell lines, but subsequently recovered in FL, normal human and CV-1 cells. The onset of recovery was 4 h after UV-irradiation for FL and normal human cells, but about 6 h for CV-1 cells. The apparent intermediate repair of CV-1 cells except for HCR may be related to the slow rate of excision repair. ''Patch and cut'' model is more favorable than ''cut and patch'' model to elucidate these results. (auth.)

Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis

International Nuclear Information System (INIS)

Ma, Jane; Bishoff, Bridget; Mercer, R.R.; Barger, Mark; Schwegler-Berry, Diane; Castranova, Vincent

2017-01-01

The emission of cerium oxide nanoparticles (CeO 2 ) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO 2 induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO 2 -induced fibrosis. Male Sprague-Dawley rats were exposed to CeO 2 (0.15 to 7 mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28 days after CeO 2 (3.5 mg/kg) exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO 2 -exposed rats at 28 days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO 2 exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO 2 -exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO 2 exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO 2 nanoparticle exposure. - Highlights: • CeO 2 exposure induced lung fibrosis. • CeO 2 were detected in lung tissue, alveolar type II (ATII) cells and fibroblasts. • CeO 2 caused ATII cell hypertrophy and hyperplasia and altered fibroblast function

Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis

Energy Technology Data Exchange (ETDEWEB)

Ma, Jane [Health Effects Laboratory Division, NIOSH, Morgantown, WV (United States); Bishoff, Bridget [Mylan Pharmaceuticals, Morganntown, WV (United States); Mercer, R.R.; Barger, Mark; Schwegler-Berry, Diane [Health Effects Laboratory Division, NIOSH, Morgantown, WV (United States); Castranova, Vincent, E-mail: vcastran@hsc.wvu.edu [School of Pharmacy, West Virginia University, Morgantown, WV (United States)

2017-05-15

The emission of cerium oxide nanoparticles (CeO{sub 2}) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO{sub 2} induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO{sub 2}-induced fibrosis. Male Sprague-Dawley rats were exposed to CeO{sub 2} (0.15 to 7 mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28 days after CeO{sub 2} (3.5 mg/kg) exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO{sub 2}-exposed rats at 28 days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO{sub 2} exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO{sub 2}-exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO{sub 2} exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO{sub 2} nanoparticle exposure. - Highlights: • CeO{sub 2} exposure induced lung fibrosis. • CeO{sub 2} were detected in lung tissue, alveolar type II (ATII) cells and fibroblasts. • CeO{sub 2} caused ATII

Inhibition of DNA repair in ultraviolet-irradiated human cells by hydroxyurea

International Nuclear Information System (INIS)

Francis, A.A.; Carrier, W.L.; Smith, D.P.; Regan, J.D.; Blevins, R.D.

1979-01-01

The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair: the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thymine-containing dimers, and measurement of specific ultraviolet-endonuclease-sensitive sites in irradiated DNA. Little effect on hydroxyurea was observed at the concentration of 2mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65-70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea incubation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well. (Auth.)

Inhibition of DNA repair in ultraviolet-irradiated human cells by hydroxyurea

Energy Technology Data Exchange (ETDEWEB)

Francis, A.A. (Oak Ridge National Lab., TN); Blevins, R.D.; Carrier, W.L.; Smith, D.P.; Regan, J.D.

1979-01-01

The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair: the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thymine-containing dimers, and measurement of specific ultraviolet-endonuclease-sensitive sites in irradiated DNA. Little effect of hydroxyurea was observed at the concentration of 2 mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65 to 70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea incubation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well.

Change of mitotic cycle and DNA repair in embryonic cells of rat, immortalized by E1 A oncogene and transformated by E1 A and c-Ha-Ras oncogenes under ionizing radiation action

International Nuclear Information System (INIS)

Kirillova, T.V.

1997-01-01

Comparison investigation into the repair of mitotic cycle and the reunion of DN single- and double-strand breaks in gamma-ray irradiated initial E1 A oncogene immortalized and E1 A and c-Ha-Ras oncogene transformed (mutant form) lines of rat embryonic fibroblasts was carried out. Possible involvement of Ras gene product in DNA repair speed governing and absence of tumor suppression function of p 53 protein in the embryonic and E1 A oncogene immortalized cells of rat fibroblast, as well as, presence of the mentioned function of p 53 protein in E1 A and c-Ha-Ras oncogene transformed cells were studied [ru

p53 downregulates the Fanconi anaemia DNA repair pathway.

Science.gov (United States)

Jaber, Sara; Toufektchan, Eléonore; Lejour, Vincent; Bardot, Boris; Toledo, Franck

2016-04-01

Germline mutations affecting telomere maintenance or DNA repair may, respectively, cause dyskeratosis congenita or Fanconi anaemia, two clinically related bone marrow failure syndromes. Mice expressing p53(Δ31), a mutant p53 lacking the C terminus, model dyskeratosis congenita. Accordingly, the increased p53 activity in p53(Δ31/Δ31) fibroblasts correlated with a decreased expression of 4 genes implicated in telomere syndromes. Here we show that these cells exhibit decreased mRNA levels for additional genes contributing to telomere metabolism, but also, surprisingly, for 12 genes mutated in Fanconi anaemia. Furthermore, p53(Δ31/Δ31) fibroblasts exhibit a reduced capacity to repair DNA interstrand crosslinks, a typical feature of Fanconi anaemia cells. Importantly, the p53-dependent downregulation of Fanc genes is largely conserved in human cells. Defective DNA repair is known to activate p53, but our results indicate that, conversely, an increased p53 activity may attenuate the Fanconi anaemia DNA repair pathway, defining a positive regulatory feedback loop.

Localization of ultraviolet-induced excision repair in the nucleus and the distribution of repair events in higher order chromatin loops in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Mullenders, L.H.F.; Zeeland, A.A. van; Natarajan, A.T.

1987-01-01

Several lines of evidence indicate that eukaryotic DNA is arranged in highly supercoiled domains or loops, and that the repeating loops are constrained by attachment to a nuclear skeletal structure termed the nuclear matrix. We have investigated whether the repair of DNA damage occurs in the nuclear matrix compartment. Normal human fibroblasts, ultraviolet (u.v.)-irradiated with 30 J m/sup -2/ and post-u.v. incubated in the presence of hydroxyurea, did not show any evidence for the occurrence of repair synthesis at the nuclear matrix. 5 J m/sup -2/ repair synthesis seems to initiate at the nuclear matrix, although only part of the total repair could be localized there. In u.v.-irradiated (30 J m/sup -2/) normal human fibroblast post-u.v. incubated in the presence of hydroxyurea and arabinsosylcytosine for 2h, multiple single-stranded regions are generated in a DNA loop as a result of the inhibition of the excision repair process. Preferential repair of certain domains in the chromatin was shown to occur in xeroderma pigmentosum cells of complementation group C (XP-C) in contrast to XP-D cells and Syrian hamster embryonic cells.

Adaptive response to ionizing radiation in normal human skin fibroblasts. Enhancement of DNA repair rate and modulation of gene expression. Reponse adaptative au rayonnement ionisant des fibroblastes de peau humaine. Augmentation de la vitesse de reparation de l'ADN et variation de l'expression des genes

Energy Technology Data Exchange (ETDEWEB)

Toledo, S.M. de; Mitchel, R.E.J. (Atomic Energy of Canada Ltd., Chalk River, ON (Canada). Chalk River Nuclear Labs.); Azzam, E. (Atomic Energy of Canada Ltd., Chalk River, ON (Canada). Chalk River Nuclear Labs. Ottawa Univ., ON (Canada). Dept. of Biology); Raaphorst, G.P. (Ottawa Univ., ON (Canada). Dept. of Biology)

Low doses and dose rates of ionizing radiation enhance the rate of DNA repair in human fibroblasts and protect the cells against radiation-induced micronucleus formation. Chronic exposures reduce the mRNA levels of the genes topoisomerase II and FACC-1 (Fanconi's anemia, group C). (authors). 11 refs., 1 tab., 2 figs.

Tryptophan Transport in Human Fibroblast Cells—A Functional Characterization

Directory of Open Access Journals (Sweden)

Ravi Vumma

2011-01-01

Full Text Available There are indications that serotonergic neurotransmission is disturbed in several psychiatric disorders. One explanation may be disturbed transport of tryptophan (precursor for serotonin synthesis across cell membranes. Human fibroblast cells offer an advantageous model to study the transport of amino acids across cell membranes, since they are easy to propagate and the environmental factors can be controlled. The aim of this study was to functionally characterize tryptophan transport and to identify the main transporters of tryptophan in fibroblast cell lines from healthy controls. Tryptophan kinetic parameters ( V max and K m at low and high concentrations were measured in fibroblasts using the cluster tray method. Uptake of 3 H (5-L-tryptophan at different concentrations in the presence and absence of excess concentrations of inhibitors or combinations of inhibitors of amino acid transporters were also measured. Tryptophan transport at high concentration (0.5 mM had low affinity and high V max and the LAT1 isoform of system-L was responsible for approximately 40% of the total uptake of tryptophan. In comparison, tryptophan transport at low concentration (50 nM had higher affinity, lower V max and approximately 80% of tryptophan uptake was transported by system-L with LAT1 as the major isoform. The uptake of tryptophan at the low concentration was mainly sodium (Na + dependent, while uptake at high substrate concentration was mainly Na + independent. A series of different transporter inhibitors had varying inhibitory effects on tryptophan uptake. This study indicates that tryptophan is transported by multiple transporters that are active at different substrate concentrations in human fibroblast cells. The tryptophan transport trough system-L was mainly facilitated by the LAT1 isoform, at both low and high substrate concentrations of tryptophan.

Microarray mRNA expression analysis of Fanconi anemia fibroblasts.

Science.gov (United States)

Galetzka, D; Weis, E; Rittner, G; Schindler, D; Haaf, T

2008-01-01

Fanconi anemia (FA) cells are generally hypersensitive to DNA cross-linking agents, implying that mutations in the different FANC genes cause a similar DNA repair defect(s). By using a customized cDNA microarray chip for DNA repair- and cell cycle-associated genes, we identified three genes, cathepsin B (CTSB), glutaredoxin (GLRX), and polo-like kinase 2 (PLK2), that were misregulated in untreated primary fibroblasts from three unrelated FA-D2 patients, compared to six controls. Quantitative real-time RT PCR was used to validate these results and to study possible molecular links between FA-D2 and other FA subtypes. GLRX was misregulated to opposite directions in a variety of different FA subtypes. Increased CTSB and decreased PLK2 expression was found in all or almost all of the analyzed complementation groups and, therefore, may be related to the defective FA pathway. Transcriptional upregulation of the CTSB proteinase appears to be a secondary phenomenon due to proliferation differences between FA and normal fibroblast cultures. In contrast, PLK2 is known to play a pivotal role in processes that are linked to FA defects and may contribute in multiple ways to the FA phenotype: PLK2 is a target gene for TP53, is likely to function as a tumor suppressor gene in hematologic neoplasia, and Plk2(-/-) mice are small because of defective embryonal development. (c) 2008 S. Karger AG, Basel.

Effect of Basic Fibroblast Growth Factor on Achilles Tendon Healing in Rabbit.

Science.gov (United States)

Najafbeygi, Arash; Fatemi, Mohammad Javad; Lebaschi, Amir Hussein; Mousavi, Seyed Jaber; Husseini, Seyed Abouzar; Niazi, Mitra

2017-01-01

Tendon injuries are common and it takes a long time for an injured tendon to heal. Adverse phenomena such as adhesion and rupture are associated with these injuries. Finding a method to reduce the time required for healing which improves the final outcome, will lead to decreased frequency and intensity of adverse consequences. This study was designed to investigate the effects of basic fibroblast growth factor on the healing of the Achilles tendon in rabbits. In 10 New Zealand white rabbits, Achilles tendon was cut at the intersection of the distal and middle thirds on both hind legs. One microgram of recombinant basic fibroblast growth factor (bFGF) was injected in the proximal and distal stumps of the cut tendon on the right side (study group). Normal saline of equal volume was injected on the left side in the same way (control group). Then the tendons were repaired with 5/0 nylon using modified Kessler technique. A cast was made to immobilize each leg. On day 42, rabbits were euthanized and both hind legs were amputated. Tensometry and histopathologic examination were done on specimens. In tensometric studies, more force was required to rupture the repair site in study group. In histopathologic examination, collagen fibers had significantly better orientation and organization in the study group. No difference was noted regarding number of fibroblast and fibrocytes, and degree of angiogenesis in the two groups. Application of basic fibroblast growth factor at tendon repair site improves the healing process through improvement of collagen fiber orientation and increase in biomechanical resistance.

DNA Repair Defects and Chromosomal Aberrations

Science.gov (United States)

Hada, Megumi; George, K. A.; Huff, J. L.; Pluth, J. M.; Cucinotta, F. A.

2009-01-01

Yields of chromosome aberrations were assessed in cells deficient in DNA doublestrand break (DSB) repair, after exposure to acute or to low-dose-rate (0.018 Gy/hr) gamma rays or acute high LET iron nuclei. We studied several cell lines including fibroblasts deficient in ATM (ataxia telangiectasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. Chromosomes were analyzed using the fluorescence in situ hybridization (FISH) chromosome painting method in cells at the first division post irradiation, and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma irradiation induced greater yields of both simple and complex exchanges in the DSB repair-defective cells than in the normal cells. The quadratic dose-response terms for both simple and complex chromosome exchanges were significantly higher for the ATM- and NBS-deficient lines than for normal fibroblasts. However, in the NBS cells the linear dose-response term was significantly higher only for simple exchanges. The large increases in the quadratic dose-response terms in these repair-defective cell lines points the importance of the functions of ATM and NBS in chromatin modifications to facilitate correct DSB repair and minimize the formation of aberrations. The differences found between ATM- and NBS-deficient cells at low doses suggest that important questions should with regard to applying observations of radiation sensitivity at high dose to low-dose exposures. For aberrations induced by iron nuclei, regression models preferred purely linear dose responses for simple exchanges and quadratic dose responses for complex exchanges. Relative biological effectiveness (RBE) factors of all of

The localization of ultraviolet-induced excision repair in the nucleus and the distribution of repair events in higher order chromatin loops in mammalian cells

International Nuclear Information System (INIS)

Mullenders, L.H.F.; Zeeland, A.A. van; Natarajan, A.T.

1987-01-01

Several lines of evidence indicate that eukaryotic DNA is arranged in highly supercoiled domains or loops, and that the repeating loops are constrained by attachment to a nuclear skeletal structure termed the nuclear matrix. We have investigated whether the repair of DNA damage occurs in the nuclear matrix compartment. Normal human fibroblasts, ultraviolet (u.v.)-irradiated with 30 J m -2 and post-u.v. incubated in the presence of hydroxyurea, did not show any evidence for the occurrence of repair synthesis at the nuclear matrix. 5 J m -2 repair synthesis seems to initiate at the nuclear matrix, although only part of the total repair could be localized there. In u.v.-irradiated (30 J m -2 ) normal human fibroblast post-u.v. incubated in the presence of hydroxyurea and arabinsosylcytosine for 2h, multiple single-stranded regions are generated in a DNA loop as a result of the inhibition of the excision repair process. Preferential repair of certain domains in the chromatin was shown to occur in xeroderma pigmentosum cells of complementation group C (XP-C) in contrast to XP-D cells and Syrian hamster embryonic cells. (author)

Complementing xeroderma pigmentosum fibroblasts restore biological activity to UV-damaged DNA

International Nuclear Information System (INIS)

Day, R.S. III; Kraemer, K.H.; Robbins, J.H.

1975-01-01

UV survival curves of adenovirus 2 using fused complementing xeroderma pigmentosum fibroblast strains as virus hosts showed a component with an inactivation slope identical to that given by normal cells. This component was not observed when the fibroblasts were not fused or when fusions involved strains in the same complementing group. Extrapolation to zero dose indicated that three percent of the viral plaque-forming units had infected cells capable of normal repair; this suggested that three percent of the cells were complementing heterokaryons. Thus, heterokaryons formed from xeroderma pigmentosum fibroblasts belonging to different complementation groups are as capable of restoring biological activity to UV-damaged adenovirus 2 as are normal cells

Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

International Nuclear Information System (INIS)

Kaufmann, W.K.; Cleaver, J.E.

1981-01-01

The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher cytotoxic fluences inhibited DNA synthesis in operating replicons. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences, indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation despite their ability to remove pyrimidine dimers. The analysis suggested that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery. (author)

Magnitude and duration of stretch modulate fibroblast remodeling.

Science.gov (United States)

Balestrini, Jenna L; Billiar, Kristen L

2009-05-01

Mechanical cues modulate fibroblast tractional forces and remodeling of extracellular matrix in healthy tissue, healing wounds, and engineered matrices. The goal of the present study is to establish dose-response relationships between stretch parameters (magnitude and duration per day) and matrix remodeling metrics (compaction, strength, extensibility, collagen content, contraction, and cellularity). Cyclic equibiaxial stretch of 2-16% was applied to fibroblast-populated fibrin gels for either 6 h or 24 h/day for 8 days. Trends in matrix remodeling metrics as a function of stretch magnitude and duration were analyzed using regression analysis. The compaction and ultimate tensile strength of the tissues increased in a dose-dependent manner with increasing stretch magnitude, yet remained unaffected by the duration in which they were cycled (6 h/day versus 24 h/day). Collagen density increased exponentially as a function of both the magnitude and duration of stretch, with samples stretched for the reduced duration per day having the highest levels of collagen accumulation. Cell number and failure tension were also dependent on both the magnitude and duration of stretch, although stretch-induced increases in these metrics were only present in the samples loaded for 6 h/day. Our results indicate that both the magnitude and the duration per day of stretch are critical parameters in modulating fibroblast remodeling of the extracellular matrix, and that these two factors regulate different aspects of this remodeling. These findings move us one step closer to fully characterizing culture conditions for tissue equivalents, developing improved wound healing treatments and understanding tissue responses to changes in mechanical environments during growth, repair, and disease states.

Effect of 3-aminobenzamide on the rate of ligation during repair of alkylated DNA in human fibroblasts

International Nuclear Information System (INIS)

Morgan, W.F.; Cleaver, J.E.

1983-01-01

3-Aminobenzamide, an inhibitor of polyadenosine diphosphoribose polymerase, produced rapid reversible changes in single-strand break frequencies in DNA from primary human fibroblasts damaged by alkylating agents, but it did not cause such changes in the DNA of cells damaged by ultraviolet light. The increase in single-strand peak frequencies was not due to an accumulation of blocked repair sites, such as occurs with DNA polymerase inhibitors, but to a delay in the rejoining of induced breaks. 3-Aminobenzamide increases the net break frequency that results from a dynamic balance between excision and ligation. This balance appears to be regulated at the ligation step by adenosine diphosphate ribosylation, which is rapidly altered by addition or removal of 3-aminobenzamide. The rapidity with which strand break frequencies change in the presence of 3-aminobenzamide implies that individual strand breaks resulting from excision at any time after exposure have a lifetime of no more than about 30 min in the cell

Radiation response in vitro of fibroblasts from a Fanconi anemia patient with marked clinical radiosensitivity

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Djuzenova, C.; Flentje, M. [Dept. of Radiotherapy, Univ. of Wuerzburg, Wuerzburg (Germany); Plowman, P.N. [Radiotherapy/Clinical Oncology, St. Bartholomew' s Hospital, London (United Kingdom)

2004-12-01

Background: fanconi anemia (FA) is an autosomal recessive chromosome instability disorder characterized by progressive pancytopenia and cancer susceptibility. The risks of radiation therapy in FA patients who have cancer remain to be investigated. Recently, Marcou et al. (2001) reported a case of severe clinical radiosensitivity in a female FA patient with a tonsillar squamous cell carcinoma treated by radiotherapy. By contrast, her in vitro irradiated skin fibroblasts revealed nearly normal radiosensitivity as determined by the colony survival assay. Material and methods: in view of this discrepancy, the radiation response of this particular FA fibroblast strain (designated 425BR) was further analyzed in the present study by means of the alkaline single-cell gel electrophoresis (Comet) assay, and also by the cytochalasin-blocked micronuclei (MN) test. In addition, the expression levels of DNA repair proteins, hMre11, Rad50, and Rad51, were investigated using Western blot and foci immunofluorescence staining. Results: the Comet assay revealed that the initial DNA fragmentation in irradiated FA cells was two times higher and the DNA rejoining process was three times slower than that in control (1BR3) fibroblasts. Moreover, although the baseline level of MNs was lower in FA cells than in controls, the FA fibroblasts were more prone (about two times) to MN production than control cells when irradiated with 2-4 Gy. Western blot analysis of the DNA repair proteins (hMre11, Rad50, and Rad51) did not reveal any abnormalities in protein expression levels or their migration patterns in the fibroblasts derived from an FA patient either before or after irradiation. At the same time, in vitro irradiated cells from the FA patient exhibited a significantly reduced number of nuclei with focally concentrated DNA repair Rad51 protein than in control cells. Conclusion: the increased DNA damage and MN induction in irradiated FA fibroblasts, and the reduction of the formation of DNA

Radiation response in vitro of fibroblasts from a Fanconi anemia patient with marked clinical radiosensitivity

International Nuclear Information System (INIS)

Djuzenova, C.; Flentje, M.; Plowman, P.N.

2004-01-01

Background: fanconi anemia (FA) is an autosomal recessive chromosome instability disorder characterized by progressive pancytopenia and cancer susceptibility. The risks of radiation therapy in FA patients who have cancer remain to be investigated. Recently, Marcou et al. (2001) reported a case of severe clinical radiosensitivity in a female FA patient with a tonsillar squamous cell carcinoma treated by radiotherapy. By contrast, her in vitro irradiated skin fibroblasts revealed nearly normal radiosensitivity as determined by the colony survival assay. Material and methods: in view of this discrepancy, the radiation response of this particular FA fibroblast strain (designated 425BR) was further analyzed in the present study by means of the alkaline single-cell gel electrophoresis (Comet) assay, and also by the cytochalasin-blocked micronuclei (MN) test. In addition, the expression levels of DNA repair proteins, hMre11, Rad50, and Rad51, were investigated using Western blot and foci immunofluorescence staining. Results: the Comet assay revealed that the initial DNA fragmentation in irradiated FA cells was two times higher and the DNA rejoining process was three times slower than that in control (1BR3) fibroblasts. Moreover, although the baseline level of MNs was lower in FA cells than in controls, the FA fibroblasts were more prone (about two times) to MN production than control cells when irradiated with 2-4 Gy. Western blot analysis of the DNA repair proteins (hMre11, Rad50, and Rad51) did not reveal any abnormalities in protein expression levels or their migration patterns in the fibroblasts derived from an FA patient either before or after irradiation. At the same time, in vitro irradiated cells from the FA patient exhibited a significantly reduced number of nuclei with focally concentrated DNA repair Rad51 protein than in control cells. Conclusion: the increased DNA damage and MN induction in irradiated FA fibroblasts, and the reduction of the formation of DNA

Transaortic Alfieri Edge-to-Edge Repair for Functional Mitral Regurgitation.

Science.gov (United States)

Imasaka, Ken-Ichi; Tayama, Eiki; Morita, Shigeki; Toriya, Ryohei; Tomita, Yukihiro

2018-03-01

There is controversy about handling functional mitral regurgitation in patients undergoing aortic valve or proximal aortic operations. We describe a transaortic Alfieri edge-to-edge repair for functional mitral regurgitation that reduces operative excessive invasion and prolonged cardiopulmonary bypass time. Between May 2013 and December 2016, 10 patients underwent transaortic Alfieri edge-to-edge mitral repair. There were no operative deaths. The severity of mitral regurgitation immediately after the operation by transesophageal echocardiography was none or trivial in all patients. A transaortic Alfieri edge-to-edge repair for functional mitral regurgitation is a simple and safe approach. Copyright © 2018 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Urinary function after Snodgrass repair of distal hypospadias: comparison with the Mathieu repair.

Science.gov (United States)

Scarpa, Maria Grazia; Castagnetti, Marco; Berrettini, Alfredo; Rigamonti, Waifro; Musi, Luciano

2010-05-01

To evaluate urinary function in patients with distal hypospadias undergoing repair by the tubularized incised-plate urethroplasty (TIP or Snodgrass), compare the results with those in patients treated by the Mathieu technique, and show the potential issues inherent to the evaluation of such results. A cross-sectional assessment was performed of uncomplicated distal hypospadias operated on during a 3-year period, already toilet trained, and able to void volitionally. Evaluation included clinical assessment urinary symptoms and urinary stream, and uroflowmetry. Out of 83 patients operated on during the study period, 10 (12%) developed complication and 32 were not toilet trained or refused to participate in the study. Median follow-up in the remaining 41 patients included in the study was 20 (3-36) months. None of these patients presented voiding symptoms or urinary stream abnormalities. Uroflowmetry was normal in 30 cases and obstructive in 11 (27%). An obstructive flow pattern was more common in patients undergoing TIP versus Mathieu repair, 8 of 19 (42%) versus 3 of 22 (14%), respectively (P = 0.07). Four TIP cases with an obstructive uroflow pattern were managed conservatively. Although both the TIP and the Mathieu repair allow good results in terms of urinary function after distal hypospadias repairs, the TIP technique seems more likely to be associated with urine flow pattern abnormalities. The actual clinical relevance of this finding remains ill defined.

The time course of repair of ultraviolet-induced DNA damage; implications for the structural organization of repair

International Nuclear Information System (INIS)

Collins, A.; Squires, S.

1986-01-01

Alternative molecular mechanisms can be envisaged for the cellular repair of UV-damaged DNA. In the 'random collision' model, DNA damage distributed throughout the genome is recognised and repaired by a process of random collision between DNA damage and repair enzymes. The other model assumes a 'processive' mechanism, whereby DNA is scanned for damage by a repair complex moving steadily along its length. Random collision should result in a declining rate of repair with time as the concentration of lesions in the DNA falls; but the processive model predicts a constant rate until scanning is complete. The authors have examined the time course of DNA repair in human fibroblasts given low doses of UV light. Using 3 distinct assays, the authors find no sign of a constant repair rate after 4 J/m 2 or less, even when the first few hours after irradiation are examined. Thus DNA repair is likely to depend on random collision. (Auth.)

Cytotoxic and mutagenic effects of specific carcinogen-DNA adducts in diploid human fibroblasts

International Nuclear Information System (INIS)

McCormick, J.J.; Maher, V.M.

1985-01-01

A comparison of the cytotoxicity and mutagenicity of a series of carcinogens in normal diploid human fibroblasts and in cells deficient in one or more DNA repair processes has provided insight into the specific DNA adduct(s) responsible for these biological effects. The carcinogens tested include ultraviolet radiation; reactive derivatives of structurally related aromatic amides; metabolites of benzo(a)pyrene; the simple alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea; and aflatoxin B 1 dichloride, a model for the reactive 2,3-epoxide of aflatoxin B 1 . Exponentially growing cells were exposed to agents and assayed for mutations and cell killing. Cells deficient in repair of particular DNA adducts or lesions proved more sensitive to the agent causing those lesions than did normally repairing cells. Many of the carcinogens were compared for their mutagenic and/or cytotoxic effect, not only as a function of dose administered, but also as a function of the initial number of adducts or photoproducts induced in DNA and the number remaining at critical times posttreatment. The results demonstrated a high correlation between the number of DNA lesions remaining unexcised at the time the DNA was replicated and frequency of mutations induced. Comparative studies of the frequency of UV-induced transformation of normal and repair-deficient cells showed this also to be true for transformation

Functional Tricuspid Regurgitation and Ring Annuloplasty Repair

Directory of Open Access Journals (Sweden)

William B. Weir, MD

2018-01-01

Full Text Available Functional tricuspid regurgitation (TR primarily arises from asymmetric dilation of the tricuspid annulus in the setting of right ventricular dysfunction and enlargement in response to left-sided myocardial and valvular abnormalities. Even if the TR is not severe at the time of mitral valve surgery, it can worsen and even appear late after successful mitral valve surgery, which portends a poor prognosis. Despite data demonstrating inferior outcomes in the presence of residual TR, surgical repair for functional TR remains underused. Acceptance of TR, in the presence of tricuspid annular dilation, may be unacceptable. Surgical repair should consist of placement of a rigid or semirigid annular ring, which has been shown to provide superior durability as compared with suture and flexible band techniques. Finally, percutaneous annuloplasty for correction of functional TR may allow treatment of patients with recurrent TR at high risk of reoperation.

Roles of neural stem cells in the repair of peripheral nerve injury.

Science.gov (United States)

Wang, Chong; Lu, Chang-Feng; Peng, Jiang; Hu, Cheng-Dong; Wang, Yu

2017-12-01

Currently, researchers are using neural stem cell transplantation to promote regeneration after peripheral nerve injury, as neural stem cells play an important role in peripheral nerve injury repair. This article reviews recent research progress of the role of neural stem cells in the repair of peripheral nerve injury. Neural stem cells can not only differentiate into neurons, astrocytes and oligodendrocytes, but can also differentiate into Schwann-like cells, which promote neurite outgrowth around the injury. Transplanted neural stem cells can differentiate into motor neurons that innervate muscles and promote the recovery of neurological function. To promote the repair of peripheral nerve injury, neural stem cells secrete various neurotrophic factors, including brain-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, insulin-like growth factor and hepatocyte growth factor. In addition, neural stem cells also promote regeneration of the axonal myelin sheath, angiogenesis, and immune regulation. It can be concluded that neural stem cells promote the repair of peripheral nerve injury through a variety of ways.

Post-UV colony-forming ability of normal fibroblast strains and of the xeroderma pigmentosum group G strain

International Nuclear Information System (INIS)

Barrett, S.F.; Tarone, R.E.; Moshell, A.N.; Ganges, M.B.; Robbins, J.H.

1981-01-01

In xeroderma pigmentosum, an inherited disorder of defective DNA repair, post-uv colony-forming ability of fibroblasts from patients in complementation groups A through F correlates with the patients' neurological status. The first xeroderma pigmentosum patient assigned to the recently discovered group G had the neurological abnormalities of XP. Researchers have determined the post-uv colony-forming ability of cultured fibroblasts from this patient and from 5 more control donors. Log-phase fibroblasts were irradiated with 254 nm uv light from a germicidal lamp, trypsinized, and replated at known densities. After 2 to 4 weeks' incubation the cells were fixed, stained and scored for colony formation. The strains' post-uv colony-forming ability curves were obtained by plotting the log of the percent remaining post-uv colony-forming ability as a function of the uv dose. The post-uv colony-forming ability of 2 of the 5 new normal strains was in the previously defined control donor zone, but that of the other 3 extended down to the level of the most resistant xeroderma pigmentosum strain. The post-uv colony-forming ability curve of the group G fibroblasts was not significantly different from the curves of the group D fibroblast strains from patients with clinical histories similar to that of the group G patient

Asymmetric migration of human keratinocytes under mechanical stretch and cocultured fibroblasts in a wound repair model.

Directory of Open Access Journals (Sweden)

Dongyuan Lü

Full Text Available Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment.

Enhancement of ultraviolet-DNA repair in denV gene transfectants and T4 endonuclease V-liposome recipients

International Nuclear Information System (INIS)

Kibitel, J.T.; Yee, V.; Yarosh, D.B.

1991-01-01

The phage T4 denV gene, coding for the pyrimidine-dimer specific T4 endonuclease V, was transfected into human repair-proficient fibroblasts, repair-deficient xeroderma pigmentosum fibroblasts, and wild type CHO hamster cells. Transfectants maintained denV DNA and expressed denV mRNA. Purified T4 endonuclease V encapsulated in liposomes was also used to treat repair-proficient and -deficient human cells. The denV transfected clones and liposome-treated cells showed increased unscheduled DNA synthesis and enhanced removal of pyrimidine dimers compared to controls. Both denV gene transfection and endonuclease V liposome treatment enhanced post-UV survival in xeroderma pigmentosum cells but had no effect on survival in repair-proficient human or hamster cells. The results demonstrate that an exogenous DNA repair enzyme can correct the DNA repair defect in xeroderma pigmentosum cells and enhance DNA repair in normal cells. (author)

In vitro vitamin K3 effect on conjunctival fibroblast migration and proliferation.

Science.gov (United States)

Pinilla, I; Izaguirre, L B; Gonzalvo, F J; Piazuelo, E; Garcia-Gonzalez, M A; Sanchez-Cano, A I; Sopeña, F

2014-01-01

To evaluate the dose effect of vitamin K3 on wound healing mechanisms. Conjunctival fibroblasts were incubated for 24 hours. An artificial wound was made and the cells were incubated with fresh medium plus doses of vitamin K3 to be tested. Wound repair was monitored at 0, 18, 24, and 48 hours. Proliferation was measured in actively dividing cells by [(3)H]thymidine uptake. Six different groups were tested: group 1/no drugs added, group 2/ethanol 0.1%, group 3/vitamin K3 1 mg/L, group 4/vitamin K3 2 mg/L, group 5/vitamin K3 4 mg/L, and group 6/vitamin K3 6 mg/L. Each experiment was carried out in triplicate and 4 times. There were no differences among groups at the initial time. In vitro wound repair was slower in groups 4, 5, and 6. There were no differences between control and ethanol groups and between control and vitamin K3 1 mg/L groups. Fibroblast mitogenic activity was statistically decreased in all vitamin K groups; statistical differences were found among vitamin K3 1 mg/mL and higher doses too. In groups 5 and 6, cellular toxicity was presented. Vitamin K3 is able to inhibit fibroblast proliferation. Vitamin K3 2 mg/L or higher doses inhibit wound healing repair, exhibiting cellular toxicity at 4 and 6 mg/L.

In Vitro Vitamin K3 Effect on Conjunctival Fibroblast Migration and Proliferation

Directory of Open Access Journals (Sweden)

I. Pinilla

2014-01-01

Full Text Available Purpose. To evaluate the dose effect of vitamin K3 on wound healing mechanisms. Methods. Conjunctival fibroblasts were incubated for 24 hours. An artificial wound was made and the cells were incubated with fresh medium plus doses of vitamin K3 to be tested. Wound repair was monitored at 0, 18, 24, and 48 hours. Proliferation was measured in actively dividing cells by [3H]thymidine uptake. Six different groups were tested: group 1/no drugs added, group 2/ethanol 0.1%, group 3/vitamin K3 1 mg/L, group 4/vitamin K3 2 mg/L, group 5/vitamin K3 4 mg/L, and group 6/vitamin K3 6 mg/L. Each experiment was carried out in triplicate and 4 times. Results. There were no differences among groups at the initial time. In vitro wound repair was slower in groups 4, 5, and 6. There were no differences between control and ethanol groups and between control and vitamin K3 1 mg/L groups. Fibroblast mitogenic activity was statistically decreased in all vitamin K groups; statistical differences were found among vitamin K3 1 mg/mL and higher doses too. In groups 5 and 6, cellular toxicity was presented. Conclusions. Vitamin K3 is able to inhibit fibroblast proliferation. Vitamin K3 2 mg/L or higher doses inhibit wound healing repair, exhibiting cellular toxicity at 4 and 6 mg/L.

Role of DNA lesions and repair in the transformation of human cells

International Nuclear Information System (INIS)

Maher, V.M.; McCormick, J.J.

1987-01-01

Results of studies on the transformation of diploid human fibroblasts in culture into tumor-forming cells by exposure to chemical carcinogens or radiation indicate that such transformation is multi-stepped process that at least one step, acquisition of anchorage independence, occurs as a mutagenic event. Studies comparing normal-repairing human cells with DNA repair-deficient cells, such as those derived from cancer-prone xeroderma pigmentosum patients, indicate that excision repair in human fibroblasts is essentially an error-free process that the ability to excise potentially cytotoxic, mutagenic, or transforming lesions induced DNA by carcinogens determines their ultimate biological consequences. Cells deficient in excision repair are abnormally sensitive to these agents. Studies with cells treated at various times in the cell cycle show that there is a certain limited amount of time available for DNA repair between the initial exposure and the onset of the cellular event responsible for mutation induction and transformation to anchorage independence. The data suggest that DNA replication on a template containing unexcised lesions (photoproducts, adducts) is the critical event

Nucleotide excision repair in differentiated cells

Energy Technology Data Exchange (ETDEWEB)

Wees, Caroline van der [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Jansen, Jacob [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Laarse, Arnoud van der [Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Zeeland, Albert van [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Mullenders, Leon [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands)]. E-mail: l.mullenders@lumc.nl

2007-01-03

Nucleotide excision repair (NER) is the principal pathway for the removal of a wide range of DNA helix-distorting lesions and operates via two NER subpathways, i.e. global genome repair (GGR) and transcription-coupled repair (TCR). Although detailed information is available on expression and efficiency of NER in established mammalian cell lines, little is known about the expression of NER pathways in (terminally) differentiated cells. The majority of studies in differentiated cells have focused on repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4-photoproducts (6-4PP) because of the high frequency of photolesions at low level of toxicity and availability of sensitive technologies to determine photolesions in defined regions of the genome. The picture that emerges from these studies is blurred and rather complex. Fibroblasts and terminally differentiated myocytes of the rat heart display equally efficient GGR of 6-4PP but poor repair of CPD due to the absence of p48 expression. This repair phenotype is clearly different from human terminal differentiated neurons. Furthermore, both cell types were found to carry out TCR of CPD, thus mimicking the repair phenotype of established rodent cell lines. In contrast, in intact rat spermatogenic cells repair was very inefficient at the genome overall level and in transcriptionally active genes indicating that GGR and TCR are non-functional. Also, non-differentiated mouse embryonic stem (ES) cells exhibit low levels of NER after UV irradiation. However, the mechanisms that lead to low NER activity are clearly different: in differentiated spermatogenic cells differences in chromatin compaction and sequestering of NER proteins may underlie the lack of NER activity in pre-meiotic cells, whereas in non-differentiated ES cells NER is impaired by a strong apoptotic response.

A hypothesis: factor VII governs clot formation, tissue repair and apoptosis.

Science.gov (United States)

Coleman, Lewis S

2007-01-01

A hypothesis: thrombin is a "Universal Enzyme of Energy Transduction" that employs ATP energy in flowing blood to activate biochemical reactions and cell effects in both hemostasis and tissue repair. All cells possess PAR-1 (thrombin) receptors and are affected by thrombin elevations, and thrombin effects on individual cell types are determined by their unique complement of PAR-1 receptors. Disruption of the vascular endothelium (VE) activates a tissue repair mechanism (TRM) consisting of the VE, tissue factor (TF), and circulating Factors VII, IX and X that governs localized thrombin elevations to activate clot formation and cellular effects that repair tissue damage. The culmination of the repair process occurs with the restoration of the VE followed by declines in thrombin production that causes Apoptosis ("programmed cell death") in wound-healing fibroblasts, which functions as a mechanism to draw wound edges together. The location and magnitude of TRM activity governs the location and magnitude of Factor VIII activity and clot formation, but the large size of Factor VIII prevents it from penetrating the clot formed by its activity, so that its effects are self-limiting. Factors VII, IX and X function primarily as tissue repair enzymes, while Factor VIII and Factor XIII are the only serine protease enzymes in the "Coagulation Cascade" that are exclusively associated with hemostasis.

Modulation of Wound Healing and Scar Formation by MG53 Protein-mediated Cell Membrane Repair*

Science.gov (United States)

Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M.; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

2015-01-01

Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53−/− mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. PMID:26306047

Functions and Dynamics of DNA Repair Proteins in Mitosis and Meiosis

NARCIS (Netherlands)

E.J. Uringa

2005-01-01

textabstractMy PhD project encompassed studies on the functions of several different proteins, all involved in DNA repair, in somatic and germ-line cells. Hr6b and Rad18Sc are involved in a DNA repair mechanism called ‘Replicative Damage Bypass’ (RDB), and function as ubiquitin conjugating

Analysis of DNA repair in XP-HeLa hybrids; lack of correlation between excision repair of u.v. damage and adenovirus reactivation in an XP(D)-like cell line

International Nuclear Information System (INIS)

Johnson, R.Y.; Squires, S.; Elliott, G.C.

1986-01-01

Hybrids formed between HeLa cells and fibroblasts from xeroderma pigmentosum group D show either HeLa sensitivity or XPD-like hypersensitivity to u.v. radiation and corresponding high or low excision repair capability. Hybrids with low repair are presumed to have lost, via chromosome segregation, the HeLa wild type D alleles. The u.v. sensitivity and excision repair capability of another hybrid, HD1A, derived spontaneously from the normally sensitive hybrid HD1 are analyzed. While HD1A closely resembles the XPD phenotype in terms of u.v. sensitivity and excision repair it differs from XPD because of its ability to reactivate u.v.-irradiated adenovirus 2 to an extent similar to that of its HeLa parent. This capacity functionally dissociates excision repair of chromatin-based damage from damage in a viral environment. Moreover, on the basis of complementation studies the excision repair of genomic damage by HD1A is subtly different from that of a true XPD-like hybrid, HD2. The data are discussed in terms of a second change in the defective D allele of the HD1A cell. (author)

A proteomic analysis of the functional effects of fatty acids in NIH 3T3 fibroblasts

LENUS (Irish Health Repository)

Magdalon, Juliana

2011-11-24

Abstract Previous studies have demonstrated that long chain fatty acids influence fibroblast function at sub-lethal concentrations. This study is the first to assess the effects of oleic, linoleic or palmitic acids on protein expression of fibroblasts, as determined by standard proteomic techniques. The fatty acids were not cytotoxic at the concentration used in this work as assessed by membrane integrity, DNA fragmentation and the MTT assay but significantly increased cell proliferation. Subsequently, a proteomic analysis was performed using two dimensional difference gel electrophoresis (2D-DIGE) and MS based identification. Cells treated with 50 μM oleic, linoleic or palmitic acid for 24 h were associated with 24, 22, 16 spots differentially expressed, respectively. Among the identified proteins, α-enolase and far upstream element binding protein 1 (FBP-1) are of importance due to their function in fibroblast-associated diseases. However, modulation of α-enolase and FBP-1 expression by fatty acids was not validated by the Western blot technique.

Three-dimensional characterization of fibroblast foci in idiopathic pulmonary fibrosis

Science.gov (United States)

Jones, Mark G.; Fabre, Aurélie; Schneider, Philipp; Cinetto, Francesco; Sgalla, Giacomo; Jogai, Sanjay; Alzetani, Aiman; Marshall, Ben G.; O’Reilly, Katherine M.A.; Warner, Jane A.; Lackie, Peter M.; Davies, Donna E.; Hansell, David M.; Nicholson, Andrew G.; Sinclair, Ian; Brown, Kevin K.; Richeldi, Luca

2016-01-01

In idiopathic pulmonary fibrosis (IPF), the fibroblast focus is a key histological feature representing active fibroproliferation. On standard 2D pathologic examination, fibroblast foci are considered small, distinct lesions, although they have been proposed to form a highly interconnected reticulum as the leading edge of a “wave” of fibrosis. Here, we characterized fibroblast focus morphology and interrelationships in 3D using an integrated micro-CT and histological methodology. In 3D, fibroblast foci were morphologically complex structures, with large variations in shape and volume (range, 1.3 × 104 to 9.9 × 107 μm3). Within each tissue sample numerous multiform foci were present, ranging from a minimum of 0.9 per mm3 of lung tissue to a maximum of 11.1 per mm3 of lung tissue. Each focus was an independent structure, and no interconnections were observed. Together, our data indicate that in 3D fibroblast foci form a constellation of heterogeneous structures with large variations in shape and volume, suggesting previously unrecognized plasticity. No evidence of interconnectivity was identified, consistent with the concept that foci represent discrete sites of lung injury and repair. PMID:27275013

Defective recoveryfrom potentially lethal damage in some human fibroblast cell strains

International Nuclear Information System (INIS)

Arlett, C.F.; Priestley, A.

1983-01-01

The repair of potentially lethal damage following treatment by gamma radiation was investigated in human fibroblasts held in a non-cycling state by maintenance in a medium containing 0.5 per cent foetal calf serum. Variation in their capacity to repair PLD was noted between three normal cell strains. A failure to repair PLD in ataxia-telangiectasia cells (AT5BI) was confirmed. In three cell strains which were intermediate between normals and A-T cells in their sensitivity, XP3BR, 46BR and GB1142, a limited capacity for the repair of PLD was observed. Two other cell strains, 47BR and 67BR, which showed little if any hypersensitivity could be clearly distinguished from normals after a 24 hour period for the repair of PLD. Thus the technique might permit better discrimination between cell strains. One other cell strain, H15617, could be distinguished from normals by proving hypersensitive under all conditions. Here, however, the repair of PLD appeared to be normal. (author)

Scaffoldless tissue-engineered nerve conduit promotes peripheral nerve regeneration and functional recovery after tibial nerve injury in rats

Institute of Scientific and Technical Information of China (English)

Aaron M. Adams; Keith W. VanDusen; Tatiana Y. Kostrominova; Jacob P. Mertens; Lisa M. Larkin

2017-01-01

Damage to peripheral nerve tissue may cause loss of function in both the nerve and the targeted muscles it innervates. This study compared the repair capability of engineered nerve conduit (ENC), engineered fibroblast conduit (EFC), and autograft in a 10-mm tibial nerve gap. ENCs were fabricated utilizing primary fibroblasts and the nerve cells of rats on embryonic day 15 (E15). EFCs were fabricated utilizing primary fi-broblasts only. Following a 12-week recovery, nerve repair was assessed by measuring contractile properties in the medial gastrocnemius muscle, distal motor nerve conduction velocity in the lateral gastrocnemius, and histology of muscle and nerve. The autografts, ENCs and EFCs reestablished 96%, 87% and 84% of native distal motor nerve conduction velocity in the lateral gastrocnemius, 100%, 44% and 44% of native specific force of medical gastrocnemius, and 63%, 61% and 67% of native medial gastrocnemius mass, re-spectively. Histology of the repaired nerve revealed large axons in the autograft, larger but fewer axons in the ENC repair, and many smaller axons in the EFC repair. Muscle histology revealed similar muscle fiber cross-sectional areas among autograft, ENC and EFC repairs. In conclusion, both ENCs and EFCs promot-ed nerve regeneration in a 10-mm tibial nerve gap repair, suggesting that the E15 rat nerve cells may not be necessary for nerve regeneration, and EFC alone can suffice for peripheral nerve injury repair.

Are we at risk from level radiation - DNA repair capacity studies

International Nuclear Information System (INIS)

Riklis, E.; Kol, R.; Heimer, Y.M.

1979-01-01

A new biochemical method, based on determination of DNA repair capability, which will enable predetermination of radiation sensitivity and further - an indication of inherent sensitivity which may be expressed only in the future when a cell will be faced with a situation in which its repair capacity will have to function to its full capability was developed. Cells are treated with trioxalen (trimethylpsoralen, TMP) and near ultraviolet light (NUV), bringing about an almost complete cessation of semiconservative DNA synthesis, 99.5 to 99.8 % inhibition. This method enabled the accurate measurement of incorporation of labelled thymidine into DNA following assaults by radiation or chemicals, indicating that repair synthesis is occurring. The method has been found suitable for the following cells: human fibroblasts, human breast cancer cells, chinese hamster V-79 cells, human lymphocytes. Since the method is applicable also for lymphocytes, it will enable carrying out a world-wide interlaboratory comparative study in which the range of 'repair capacity' of 'normal healthy' humans will be established. Individuals showing no repair capacity will not be permitted to be exposed to any level of radiation above natural background. These are the persons 'at risk' from radiation, while the general public, showing normal repair capacity may be considered safe from the effects of low-level radiations. (B.G.)

Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts.

Science.gov (United States)

Wang, Zhen-Fei; Ma, Da-Guang; Zhu, Zhe; Mu, Yong-Ping; Yang, Yong-Yan; Feng, Li; Yang, Hao; Liang, Jun-Qing; Liu, Yong-Yan; Liu, Li; Lu, Hai-Wen

2017-12-28

To investigate the inhibitory effect of astragaloside IV on the pathological functions of cancer-associated fibroblasts, and to explore the underlying mechanism. Paired gastric normal fibroblast (GNF) and gastric cancer-associated fibroblast (GCAF) cultures were established from resected tissues. GCAFs were treated with vehicle control or different concentrations of astragaloside IV. Conditioned media were prepared from GNFs, GCAFs, control-treated GCAFs, and astragaloside IV-treated GCAFs, and used to culture BGC-823 human gastric cancer cells. Proliferation, migration and invasion capacities of BGC-823 cells were determined by MTT, wound healing, and Transwell invasion assays, respectively. The action mechanism of astragaloside IV was investigated by detecting the expression of microRNAs and the expression and secretion of the oncogenic factor, macrophage colony-stimulating factor (M-CSF), and the tumor suppressive factor, tissue inhibitor of metalloproteinase 2 (TIMP2), in different groups of GCAFs. The expression of the oncogenic pluripotency factors SOX2 and NANOG in BGC-823 cells cultured with different conditioned media was also examined. GCAFs displayed higher capacities to induce BGC-823 cell proliferation, migration, and invasion than GNFs ( P GNFs, GCAFs expressed a lower level of microRNA-214 ( P < 0.01) and a higher level of microRNA-301a ( P < 0.01). Astragaloside IV treatment significantly up-regulated microRNA-214 expression ( P < 0.01) and down-regulated microRNA-301a expression ( P < 0.01) in GCAFs. Reestablishing the microRNA expression balance subsequently suppressed M-CSF production ( P < 0.01) and secretion ( P < 0.05), and elevated TIMP2 production ( P < 0.01) and secretion ( P < 0.05). Consequently, the ability of GCAFs to increase SOX2 and NANOG expression in BGC-823 cells was abolished by astragaloside IV. Astragaloside IV can inhibit the pathological functions of GCAFs by correcting their dysregulation of microRNA expression, and it is

Intrinsic radiosensitivity and PLD repair in osteosarcoma cell lines

International Nuclear Information System (INIS)

Sugimoto, M.; Toguchida, J.; Kotoura, Y.; Yamamuro, T.; Utsumi, H.

1992-01-01

The response to radiation of seven osteosarcoma cell lines was analysed by in vitro colony-forming assay and compared with that of eight human fibroblast strains. The values of D 0 , the surviving fraction after 2 Gy (S2Gy), and the mean inactivation dose (D-bar) of osteosarcoma cells in log-phase culture were significantly higher than those of fibroblast strains (p<0.01). PLD (potentially lethal damage) repair of osteosarcoma cells evaluated in the plateau phase of growth showed great variation for enhancement of survival, although all of the values were maximised within 12 h after irradiation. In the osteosarcoma, intrinsic radiosensitivity in vitro reflected the clinical response to radiation. However, the capacity for PLD repair might not be a good indicator for predicting the results of radiation therapy. (author)

Radioresistant DNA synthesis in fibroblasts of a patient with Down's syndrome

International Nuclear Information System (INIS)

Barenfel'd, L.S.; Bil'din, V.N.; Pleskach, N.M.; Prokof'eva, V.V.

1985-01-01

Ionizing radiation effect on DNA replication on fibroblasts of a healthy donor and a patient with Down's syndrome either by direct 3 H-thymidine inclusion into DNA, or by analysis of the sizes of daughter DNA moleculs at the state of stable distribution in acid saccharose, gradients was studied. Gamma-radiation doses (5-10 Gy) suppressing DNA synthesis in normal fibroblasts practically had no effect on DNA synthesisin fibroblasts of a patient with Down's syndrome. Radioresistant DNA synthesis in Down's syndrome is conditioned by a far less supression of replicon initiation as compared with the one in normal cells. So, it is stated that in Down's disease there is no delay in DNA synthesis by ionizing radiation that enables the normal cells to repair DNA damages before replication renewal

Saturation of DNA repair in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Ahmed, F E; Setlow, R B

1979-01-01

Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20 J/m/sup 2/. We measured excision repair in normal human fibroblasts up to 80 J/m/sup 2/. The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus, and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24h following two 20 J/m/sup 2/ doses separated by 24h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the rate limiting steps in repair.

Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway

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Wang Jun

2012-10-01

Full Text Available Abstract Background Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS to imitate inflammatory conditions. Results In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4 antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB, which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1, compared with transforming growth factor-β1 (TGF-β1. Conclusions Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial

Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway.

Science.gov (United States)

Wang, Jun; Si, Yanfang; Wu, Chen; Sun, Lu; Ma, Yudong; Ge, Aili; Li, Baomin

2012-10-17

Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS) to imitate inflammatory conditions. In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP) as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4) antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB), which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1), compared with transforming growth factor-β1 (TGF-β1). Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial infection contributes to the pathological process of

Recovery from UV-induced potentially lethal damage in systemic lupus erythematosus skin fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Zamansky, G B

1986-08-01

The repair of ultraviolet light-induced potentially lethal damage was investigated in density-inhibited skin fibroblast cell strains derived from patients with systemic lupus erythematosus. The effect of exposure to polychromatic ultraviolet light composed of environmentally relevant wavelengths or to the more commonly studied, short wavelength (254 nm) ultraviolet light was studied. Systemic lupus erythematosus cells, which are hypersensitive to ultraviolet light under growth promoting conditions, were able to repair potentially lethal damage as well as normal cells.

Recovery from UV-induced potentially lethal damage in systemic lupus erythematosus skin fibroblasts

International Nuclear Information System (INIS)

Zamansky, G.B.

1986-01-01

The repair of ultraviolet light-induced potentially lethal damage was investigated in density-inhibited skin fibroblast cell strains derived from patients with systemic lupus erythematosus. The effect of exposure to polychromatic ultraviolet light composed of environmentally relevant wavelengths or to the more commonly studied, short wavelength (254 nm) ultraviolet light was studied. Systemic lupus erythematosus cells, which are hypersensitive to ultraviolet light under growth promoting conditions, were able to repair potentially lethal damage as well as normal cells. (author)

Effects of aphidicolin on repair replication and induced chromosomal aberrations in mammalian cells

International Nuclear Information System (INIS)

Zeeland, A.A. van; Filon, A.R.; Natarajan, A.T.; Bussmann, C.J.M.; Degrassi, F.; Kesteren-van Leeuwen, A.C. van; Palitti, F.; Rome Univ.

1982-01-01

The influence of aphidicolin, an inhibitor of polymerase α, on UV-induced repair replication in human skin fibroblasts, as well as in HeLa cells, was determined. In growing fibroblasts and in HeLa cells, aphidicolin had a potentiating effect on UV-induced repair replication, whereas in fibroblasts grown to confluency, aphidicolin had an inhibitory effect. This inhibitory effect was stronger when measured in the presence of hydroxyurea. In HeLa cells the presence of both aphidicolin and hydroxyurea also had an inhibitory effect, but in the presence of hydroxyurea alone, UV-induced repair replication was enhanced. The results of these studies can be explained on the basis of differences in deoxyribonucleotide triphosphate pool sizes in growing and confluent cells. Post-treatment of X-irradiated human lymphocytes in the G 0 and G 1 stages with aphidicolin increased the frequencies of X-ray-induced chromosomal aberrations. Such an increase was not observed in G 1 cells of CHO after similar treatment with X-rays and aphidicolin. However, treatment with aphidicolin, in the G 2 stage, increased the frequencies of induced chromatid breaks. The significance of these results is discussed. (orig.)

N-cadherin is overexpressed in Crohn's stricture fibroblasts and promotes intestinal fibroblast migration.

LENUS (Irish Health Repository)

Burke, John P

2012-02-01

BACKGROUND: Intestinal fibroblasts mediate stricture formation in Crohn\\'s disease (CD). Transforming growth factor-beta (TGF-beta) is important in fibroblast activation, while cell attachment and migration is regulated by the adhesion molecule N-cadherin. The aim of this study was to investigate the expression and function of N-cadherin in intestinal fibroblasts in patients with fibrostenosing CD. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies from patients undergoing resection for terminal ileal fibrostenosing CD (n = 14) or controls patients (n = 8). N-cadherin expression was assessed using Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Fibroblasts were stimulated with TGF-beta and selective pathway inhibitors Y27632, PD98050, and LY294002 were used to examine the Rho\\/ROCK, ERK-1\\/2, and Akt signaling pathways, respectively. Cell migration was assessed using a scratch wound assay. N-cadherin was selectively overexpressed using a plasmid. RESULTS: Fibroblasts from fibrostenosing CD express increased constitutive N-cadherin mRNA and protein and exhibit enhanced basal cell migration relative to those from directly adjacent normal bowel. Control fibroblasts treated with TGF-beta induced N-cadherin in a dose-dependent manner which was inhibited by Rho\\/ROCK and Akt pathway modulation. Control fibroblasts exhibited enhanced cell migration in response to treatment with TGF-beta or transfection with an N-cadherin plasmid. CONCLUSIONS: Fibroblasts from strictures in CD express increased constitutive N-cadherin and exhibit enhanced basal cell migration. TGF-beta is a potent inducer of N-cadherin in intestinal fibroblasts resulting in enhanced cell migration. The TGF-beta-mediated induction of N-cadherin may potentiate Crohn\\'s stricture formation.

Modulation of wound healing and scar formation by MG53 protein-mediated cell membrane repair.

Science.gov (United States)

Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

2015-10-02

Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53(-/-) mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

[Flexor tendon repair: a short story].

Science.gov (United States)

Moutet, F; Corcella, D; Forli, A; Mesquida, V

2014-12-01

This short story of flexor tendon repair aims to illustrate hesitations and wanderings of this surgery. Obviously tendon repair was very early considered, but it developed and diffused rather lately. It became a routine practice only in 20th century. This was due on the one hand, in Occident, to the Galen's dogmatic interdiction, on the other hand, to the repair difficulties of this paradoxical structure. Actually tendon is made of fibroblasts and collagen (sticky substances), and then its only goal is to move. According to this necessity, whatever the used techniques are, gliding is the final purpose. Technical evolutions are illustrated by historical contributions to flexor tendon surgery of several "giants" of hand surgery. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Chemical Conversion of Human Fibroblasts into Functional Schwann Cells

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Eva C. Thoma

2014-10-01

Full Text Available Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

Ultrastructural changes following electron irradiation in three-dimensional culture of normal human dermal fibroblasts

International Nuclear Information System (INIS)

Han, Chunmao; Ishikura, Naotaka; Tsukada, Sadao

1994-01-01

The present study was designed to examine the effect of electron irradiation on fibroblasts and extracellular matrices electron-microscopically. The three-dimensional dermal fibroblast culture was exposed to one, 4 or 10 Gy of electron beams. One day after irradiation, fibroblasts were vacuolated in all irradiated groups and intercellular spaces were increased in a dose-dependent manner. Seven days later, intercellular spaces became dense in both one and 4 Gy groups, although they were still extremely increased in the 10 Gy group. The remaining fibroblasts were still activated in all groups. Thirty days after irradiation, myofibroblastic cells were scarcely observed, but extracellular fine fibrils and collagen fibrils were observed in all irradiated groups. The other ultrastructural findings were similar to those in the control group. In conclusion, electron beams damaged not only cells but also extracellular matrix. The extracellular matrix may be repaired by activated residual fibroblasts, resulting in the mixture of new and old collagen fibrils having different diamters. (N.K.)

Conversational Repair in School-Aged Children with High-Functioning Autism

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Pei-Mei Lu

2015-12-01

Full Text Available The main purpose of this study was to investigate the conversational repair skills of Mandarin Chinese-speaking children with high-functioning autism (HFA as compared with those of typically developing children (TD. Ten school-aged children (age 9 to 12 with HFA were recruited and matched against ten TD children in the control group based on age, gender, and verbal intelligence level. During three different conversation situations (free talk, story picture description, play, an examiner engineered 9 episodes of communicative breakdowns. Each consisted of a stacked series of three prompts for responding to requests for clarification (RQCLs (i.e.‘What?’, ‘I don’t understand’, ‘I still don’t know’. Verbal responses to each RQCL were then coded for further analyses. The results showed that (1 In response to the stacked series RQCLs, children with HFA were similar to the control group children in evidencing repetition, revision, and addition types of repair. Furthermore, children with HFA showed fewer cue type of repair and more inappropriate type of repair than TD group. (2For both groups, the pattern of responding over the series of RQCLs was similar in varying the repetition and revision types of repair strategies. However, the pattern in the addition, cue, and inappropriate types of repair strategies were different. Children with HFA were significantly more likely to respond to an RQCL with an inappropriate response than the language and age-matched controls. It is suggested that teachers and parents could facilitate the conversational repair skills of children with high-functioning autism by offering them opportunities to manage different types of communicative breakdowns.

Impairment of lymphocyte adhesion to cultured fibroblasts and endothelial cells by γ-irradiation

International Nuclear Information System (INIS)

Piela-Smith, T.H.; Aneiro, L.; Nuveen, E.; Korn, J.H.; Aune, T.

1992-01-01

A critical component of immune responsiveness is the localization of effector cells at sites of inflammatory lesions. Adhesive molecules that may play a role in this process have been described on the surfaces of both lymphocytes and connective tissue cells. Adhesive interactions of T lymphocytes with fibroblasts or endothelial cells can be inhibited by preincubation of the fibroblasts or endothelial cells with antibody to intercellular adhesion molecule 1 (CD54) or by preincubation of the T cells with antibody to lymphocyte function-associated Ag 1 (CD11a/CD18), molecules shown to be important in several other cell-cell adhesion interactions. Here the authors show that γ-irradiation of human T lymphocytes impaired their ability to adhere to both fibroblasts and endothelial cells. This impairment was not associated with a loss of cell viability or of cell surface lymphocyte function-associated Ag 1 expression. γ-Irradiation of T cells is known to result in the activation of ADP-ribosyltransferase, an enzyme involved in DNA strand-break repair, causing subsequent depletion of cellular nicotinamide adenine dinucleotide (NAD) pools by increasing NAD consumption for poly(ADP-ribose) formation. Preincubation of T cells with either nicotinamide or 3-aminobenzamide, both known inhibitors of ADP-ribosyltransferase, completely reversed the suppressive effects of γ-irradiation on T cell adhesion. The maintenance of adhesion was accompanied by inhibition of irradiation-induced depletion of cellular NAD. These experiments suggest that the impairment of cellular immune function after irradiation in vivo may be caused, in part, by defective T cell emigration and localization at inflammatory sites. 44 refs., 5 figs., 3 tabs

Fibroblast growth factor receptors in breast cancer.

Science.gov (United States)

Wang, Shuwei; Ding, Zhongyang

2017-05-01

Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of breast cancer. Recent genetic studies have identified some subtypes of fibroblast growth factor receptors as strong genetic loci associated with breast cancer. In this article, we review the recent epidemiological findings and experiment results of fibroblast growth factor receptors in breast cancer. First, we summarized the structure and physiological function of fibroblast growth factor receptors in humans. Then, we discussed the common genetic variations in fibroblast growth factor receptors that affect breast cancer risk. In addition, we also introduced the potential roles of each fibroblast growth factor receptors isoform in breast cancer. Finally, we explored the potential therapeutics targeting fibroblast growth factor receptors for breast cancer. Based on the biological mechanisms of fibroblast growth factor receptors leading to the pathogenesis in breast cancer, targeting fibroblast growth factor receptors may provide new opportunities for breast cancer therapeutic strategies.

Models of mixed irradiation with a 'reciprocal-time' pattern of the repair function

Energy Technology Data Exchange (ETDEWEB)

Suzuki, Shozo; Miura, Yuri; Mizuno, Shoichi [Tokyo Metropolitan Inst. of Gerontology (Japan); Furusawa, Yoshiya [National Inst. of Radiological Sciences, Chiba (Japan)

2002-09-01

Suzuki presented models for mixed irradiation with two and multiple types of radiation by extending the Zaider and Rossi model, which is based on the theory of dual radiation action. In these models, the repair function was simply assumed to be semi-logarithmically linear (i.e., monoexponential), or a first-order process, which has been experimentally contradicted. Fowler, however, suggested that the repair of radiation damage might be largely a second-order process rather than a first-order one, and presented data in support of this hypothesis. In addition, a second-order repair function is preferred to an n-exponential repair function for the reason that only one parameter is used in the former instead of 2n-1 parameters for the latter, although both repair functions show a good fit to the experimental data. However, according to a second-order repair function, the repair rate depends on the dose, which is incompatible with the experimental data. We, therefore, revised the models for mixed irradiation by Zaider and Rossi and by Suzuki, by substituting a 'reciprocal-time' pattern of the repair function, which is derived from the assumption that the repair rate is independent of the dose in a second-order repair function, for a first-order one in reduction and interaction factors of the models, although the underlying mechanism for this assumption cannot be well-explained. The reduction factor, which reduces the contribution of the square of a dose to cell killing in the linear-quadratic model and its derivatives, and the interaction factor, which also reduces the contribution of the interaction of two or more doses of different types of radiation, were formulated by using a 'reciprocal-time' patterns of the repair function. Cell survivals calculated from the older and the newly modified models were compared in terms of the dose-rate by assuming various types of single and mixed irradiation. The result implies that the newly modified models for

The production and repair of double strand breaks in cells from normal humans and patients with ataxia telangiectasia

International Nuclear Information System (INIS)

Lehman, A.R.; Stevens, S.

1977-01-01

The production and repair of double strand breaks induced by γ-rays in the DNA of human fibroblasts have been measured by sedimentation in sucrose gradients under non-denaturing conditions. Unirradiated DNA formed a rapidly sedimenting gel. Low doses of radiation released freely sedimenting DNA molecules from this gel. Higher doses reduced the rate of sedimentation of the free DNA due to the introduction of double strand breaks. The breakage efficiency was 1 break/1.3x10 10 daltons of DNA/krad. Postirradiation incubation after a high dose of radiation resulted in an increase in molecular weight of the free DNA molecules, and after a low dose the rapidly-sedimenting gel was reformed. These data suggest that double strand breaks are repaired in human fibroblasts. No significant differences were found between fibroblasts from two normal donors and four patients with the radiosensitive disorder, ataxia telangiectasia, in either the production or repair of double strand breaks

Impact of radiotherapy on PBMCs DNA repair capacity - Use of a multiplexed functional repair assay

International Nuclear Information System (INIS)

Sauvaigo, S.; Sarrazy, F.; Breton, J.; Caillat, S.; Chapuis, V.

2012-01-01

Radiation therapy is an essential part of cancer treatment as about 50% of patients will receive radiations at least once. Significant broad variation in radiosensitivity has been demonstrated in patients. About 5-10% of patients develop acute toxicity after radiotherapy. Therefore there is a need for the identification of markers able to predict the occurrence of adverse effects and thus adapt the radiotherapy regimen for radiosensitive patients. As a first step toward this goal, and considering the DNA repair defects associated with hypersensitivity radiation syndromes, we investigated the DNA repair phenotype of patients receiving radiotherapy. More precisely, we used a functional repair assay on support to follow the evolution of the glycosylases/AP endonuclease activities of PBMCs extracts of a series of patients during the time course of radiotherapy. For each patient, we collected one PBMCs sample before the first radiotherapy application (S1) and three samples after (S2 to S4) (one day and one week after application 1, and one at the end of the radiotherapy protocol). These four samples have been analysed for 11 donors. Clustering analyses of the results demonstrated a great heterogeneity of responses among the patients. Interestingly, this heterogeneity decreased between S1 and S4 where only 2 classes of patients remained if we except one patient that exhibited an atypical DNA repair phenotype. Furthermore, we showed that repair of several oxidized bases significantly increased between S1 and S3 or S4 (8oxoG, thymine glycol, A paired with 8oxoG), suggesting an adaptation of patients repair systems to the oxidative stress generated by the ionising radiations. Our preliminary results provided evidence that the DNA repair phenotype was impacted by the radiotherapy regimen. Further characterization of patients with known repair defects are needed to determine if atypical repair phenotypes could be associated with radiotherapy complications. Finally

Fibroblast Growth Factors: Biology, Function, and Application for Tissue Regeneration

Directory of Open Access Journals (Sweden)

Ye-Rang Yun

2010-01-01

Full Text Available Fibroblast growth factors (FGFs that signal through FGF receptors (FGFRs regulate a broad spectrum of biological functions, including cellular proliferation, survival, migration, and differentiation. The FGF signal pathways are the RAS/MAP kinase pathway, PI3 kinase/AKT pathway, and PLCγ pathway, among which the RAS/MAP kinase pathway is known to be predominant. Several studies have recently implicated the in vitro biological functions of FGFs for tissue regeneration. However, to obtain optimal outcomes in vivo, it is important to enhance the half-life of FGFs and their biological stability. Future applications of FGFs are expected when the biological functions of FGFs are potentiated through the appropriate use of delivery systems and scaffolds. This review will introduce the biology and cellular functions of FGFs and deal with the biomaterials based delivery systems and their current applications for the regeneration of tissues, including skin, blood vessel, muscle, adipose, tendon/ligament, cartilage, bone, tooth, and nerve tissues.

Excision and crosslink repair of DNA and sister chromatid exchanges in cultured human fibroblasts with different repair capacities

Energy Technology Data Exchange (ETDEWEB)

Fujiwara, Y; Kano, Y; Paul, P; Goto, K; Yamamoto, K [Kobe Univ. (Japan). School of Medicine

1981-01-01

Xeroderma pigmentosum (XP) groups A to G lacked the initial stage of ultraviolet (UV) excision repair in the order of A = G > C > D > E asymptotically equals F, while the XP variant was weakly defective in the later repair steps. Killing sensitivities were in the orders of A >= G > D > C > E asymptotically equals F asymptotically equals variant > normal to UV, A = G > D > F > C = E > variant > normal to 4-nitroquinoline-1-oxide (4NQO), and A > C > D = E = F = variant > G = normal to decarbamoyl mitomycin-C(DCMC). The induced sister chromatid exchange (SCE) frequency was unrelated to the extent of repair deficiency. The SCE induction rate was consistently 3 - 6 fold higher by these UV-like mutagens in XP group A cells than in normal cells. However, repair-proficient Cockayne's syndrome (CS) cells showed a higher SCE induction by UV, which was normalized by NAD/sup +/, suggesting that chromatin lesions as well as DNA damage contribute to SCE. Two-step crosslink repair involves a first rapid half-excision and a second slow nucleotide-excision repair. Fanconi's anemia (FA) cells had an impaired first half-excision and were supersensitive to MC, but not to UV and DCMC. The SCE frequency induced by MC (1 hr) was higher in FA cells than in normal cells despite their normal response to DCMC, and vice versa in XP cells. FA cells lacked the first rapid decline and showed higher remaining SCEs. Thus, part of the crosslink seems to lead to SCE formation. Caffeine synergistically elevated UV-induced SCEs, but not UV induced mutations in V79 cells, implying that SCE may not necessarily involve mutation.

Excision and crosslink repair of DNA and sister chromatid exchanges in cultured human fibroblasts with different repair capacities

International Nuclear Information System (INIS)

Fujiwara, Yoshisada; Kano, Yoshio; Paul, P.; Goto, Kaoru; Yamamoto, Kazuo

1981-01-01

Xeroderma pigmentosum (XP) groups A to G lacked the initial stage of ultraviolet (UV) excision repair in the order of A = G > C > D > E asymptotically equals F, while the XP variant was weakly defective in the later repair steps. Killing sensitivities were in the orders of A >= G > D > C > E asymptotically equals F asymptotically equals variant > normal to UV, A = G > D > F > C = E > variant > normal to 4-nitroquinoline-1-oxide (4NQO), and A > C > D = E = F = variant > G = normal to decarbamoyl mitomycin-C(DCMC). The induced sister chromatid exchange (SCE) frequency was unrelated to the extent of repair deficiency. The SCE induction rate was consistently 3 - 6 fold higher by these UV-like mutagens in XP group A cells than in normal cells. However, repair-proficient Cockayne's syndrome (CS) cells showed a higher SCE induction by UV, which was normalized by NAD + , suggesting that chromatin lesions as well as DNA damage contribute to SCE. Two-step crosslink repair involves a first rapid half-excision and a second slow nucleotide-excision repair. Fanconi's anemia (FA) cells had an impaired first half-excision and were supersensitive to MC, but not to UV and DCMC. The SCE frequency induced by MC (1 hr) was higher in FA cells than in normal cells despite their normal response to DCMC, and vice versa in XP cells. FA cells lacked the first rapid decline and showed higher remaining SCEs. Thus, part of the crosslink seems to lead to SCE formation. Caffeine synergistically elevated UV-induced SCEs, but not UV induced mutations in V79 cells, implying that SCE may not necessarily involve mutation. (J.P.N.)

Excision and crosslink repair of DNA and sister chromatid exchanges in cultured human fibroblasts with different repair capacities

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Fujiwara, Y.; Kano, Y.; Paul, P.; Goto, K.; Yamamoto, K. (Kobe Univ. (Japan). School of Medicine)

1981-01-01

Xeroderma pigmentosum (XP) groups A to G lacked the initial stage of ultraviolet (UV) excision repair in the order of A = G > C > D > E asymptotically equals F, while the XP variant was weakly defective in the later repair steps. Killing sensitivities were in the orders of A >= G > D > C > E asymptotically equals F asymptotically equals variant > normal to UV, A = G > D > F > C = E > variant > normal to 4-nitroquinoline-1-oxide (4NQO), and A > C > D = E = F = variant > G = normal to decarbamoyl mitomycin-C(DCMC). The induced sister chromatid exchange (SCE) frequency was unrelated to the extent of repair deficiency. The SCE induction rate was consistently 3 - 6 fold higher by these UV-like mutagens in XP group A cells than in normal cells. However, repair-proficient Cockayne's syndrome (CS) cells showed a higher SCE induction by UV, which was normalized by NAD/sup +/, suggesting that chromatin lesions as well as DNA damage contribute to SCE. Two-step crosslink repair involves a first rapid half-excision and a second slow nucleotide-excision repair. Fanconi's anemia (FA) cells had an impaired first half-excision and were supersensitive to MC, but not to UV and DCMC. The SCE frequency induced by MC (1 hr) was higher in FA cells than in normal cells despite their normal response to DCMC, and vice versa in XP cells. FA cells lacked the first rapid decline and showed higher remaining SCEs. Thus, part of the crosslink seems to lead to SCE formation. Caffeine synergistically elevated UV-induced SCEs, but not UV induced mutations in V79 cells, implying that SCE may not necessarily involve mutation.

Ionizing radiation-induced phosphorylation of RPA p34 is deficient in ataxia telangiectasia and reduced in aged normal fibroblasts

International Nuclear Information System (INIS)

Xinbo Cheng; Nge Cheong; Ya Wang; Iliakis, George

1996-01-01

Replication protein A (RPA, also called human single stranded DNA binding protein, hSSB) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair and recombination. Phosphorylation of RPA p34 subunit is observed after exposure of cells to radiation and other DNA damaging agents, which implicates the protein not only in repair but also in the regulation of replication on damaged DNA template. Here, we show that the phosphorylation observed in RPA p34 after exposure to ionizing radiation, X- or γ-rays, is reduced and occurs later in primary fibroblasts from patients suffering from ataxia telangiectasia (AT), as compared to normal fibroblasts. We also show that in primary normal human fibroblasts, radiation-induced phosphorylation of RPA p34 is 'age'-dependent and decreases significantly as cultures senesce. Radiation-induced phosphorylation of RPA p34 is nearly absent in non-cycling cells, while the expression of p21 cip1/waf1/sdi1 remains inducible. The results demonstrate a growth-stage and culture-age dependency in radiation-induced RPA p34 phosphorylation, and suggest the operation of a signal transduction pathway that is inactivated in senescing or quiescent fibroblasts and defective in AT cells

Injury induces in vivo expression of platelet-derived growth factor (PDGF) and PDGF receptor mRNAs in skin epithelial cells and PDGF mRNA in connective tissue fibroblasts

International Nuclear Information System (INIS)

Antoniades, H.N.; Galanopoulos, T.; Neville-Golden, J.; Kiritsy, C.P.; Lynch, S.E.

1991-01-01

Platelet-derived growth factor (PDGF) stimulates many of the processes important in tissue repair, including proliferation of fibroblasts and synthesis of extracellular matrices. In this study, the authors have demonstrated with in situ hydridization and immunocytochemistry the reversible expression of 3-sis/PDGF-2 and PDGF receptor (PDGF-R) b mRNAs and their respective protein products in epithelial cells and fibroblasts following cutaneous injury in pigs. Epithelial cells in control, unwounded skin did not express c-sis and PDGF-R mRNAs, and fibroblasts expressed only PDGF-R mRNA. The expression levels in the injured site were correlated with the stage of tissue repair, being highest during the initial stages of the repair process and declining at the time of complete re-epithelialization and tissue remodeling. These studies provide a mulecular basis for understanding the mechanisms contributing to normal tissue repair. They suggest the possibility that a defect in these mechanisms may be associated with defective wound healing. It is also conceivable that chronic injury may induce irreversible gene expression leading to pathologic, unregulated cell growth

Functional Diversity of Fibroblast Growth Factors in Bone Formation

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Yuichiro Takei

2015-01-01

Full Text Available The functional significance of fibroblast growth factor (FGF signaling in bone formation has been demonstrated through genetic loss-of-function and gain-of-function approaches. FGFs, comprising 22 family members, are classified into three subfamilies: canonical, hormone-like, and intracellular. The former two subfamilies activate their signaling pathways through FGF receptors (FGFRs. Currently, intracellular FGFs appear to be primarily involved in the nervous system. Canonical FGFs such as FGF2 play significant roles in bone formation, and precise spatiotemporal control of FGFs and FGFRs at the transcriptional and posttranscriptional levels may allow for the functional diversity of FGFs during bone formation. Recently, several research groups, including ours, have shown that FGF23, a member of the hormone-like FGF subfamily, is primarily expressed in osteocytes/osteoblasts. This polypeptide decreases serum phosphate levels by inhibiting renal phosphate reabsorption and vitamin D3 activation, resulting in mineralization defects in the bone. Thus, FGFs are involved in the positive and negative regulation of bone formation. In this review, we focus on the reciprocal roles of FGFs in bone formation in relation to their local versus systemic effects.

Cellular radiosensitivity and DNA damage in primary human fibroblasts

International Nuclear Information System (INIS)

Wurm, R.; Burnet, N.G.; Duggal, N.

1994-01-01

To evaluate the relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts to decide whether the initial or residual DNA damage levels are more predictive of normal tissue cellular radiosensitivity. Five primary human nonsyndromic and two primary ataxia telangiectasia fibroblast strains grown in monolayer were studied. Cell survival was assessed by clonogenic assay. Irradiation was given at high dose rate (HDR) 1-2 Gy/min. DNA damage was measured in stationary phase cells and expressed as fraction released from the well by pulsed-field gel electrophoresis (PFGE). For initial damage, cells were embedded in agarose and irradiated at HDR on ice. Residual DNA damage was measured in monolayer by allowing a 4-h repair period after HDR irradiation. Following HDR irradiation, cell survival varied between SF 2 0.025 to 0.23. Measurement of initial DNA damage demonstrated linear induction up to 30 Gy, with small differences in the slope of the dose-response curve between strains. No correlation between cell survival and initial damage was found. Residual damage increased linearly up to 80 Gy with a variation in slope by a factor of 3.2. Cell survival correlated with the slope of the dose-response curves for residual damage of the different strains (p = 0.003). The relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts of differing radiosensitivity is closest with the amount of DNA damage remaining after repair. If assays of DNA damage are to be used as predictors of normal tissue response to radiation, residual DNA damage provides the most likely correlation with cell survival. 52 refs., 5 figs., 2 tabs

Toll-like receptor 9 mediated responses in cardiac fibroblasts.

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Ingrid Kristine Ohm

Full Text Available Altered cardiac Toll-like receptor 9 (TLR9 signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β. Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088 or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

Cryopreservation of canine ovarian and testicular fibroblasts.

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Yu, Il-Jeoung; Leibo, S P; Songsasen, Nucharin; Dresser, Betsy L; Kim, In-Shik

2009-01-01

To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5 degree C in a humidified atmosphere of 5 percent CO2 95 percent air, and then their membrane integrity was assayed with a double fluorescent stain, Fertilight. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70 percent and functional survival ranged from 20-40 percent. With frozen-thawed ovarian cells, the average membrane integrity was 55-75 percent and the average functional survival was 35-40 percent. When frozen in ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P less than 0.05). These methods should prove useful to preserve cells collected from canids in the wild.

Fully functional global genome repair of (6-4) photoproducts and compromised transcription-coupled repair of cyclobutane pyrimidine dimers in condensed mitotic chromatin

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Komura, Jun-ichiro, E-mail: junkom@med.tohoku.ac.jp [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Ikehata, Hironobu [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Mori, Toshio [Radioisotope Research Center, Nara Medical University, Kashihara, Nara 634-8521 (Japan); Ono, Tetsuya [Department of Cell Biology, Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan)

2012-03-10

During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cells was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression. -- Highlights: Black-Right-Pointing-Pointer Global genome repair of (6-4) photoproducts is fully active in mitotic cells. Black-Right-Pointing-Pointer DNA in condensed mitotic chromatin does not seem inaccessible or inert. Black-Right-Pointing-Pointer Mitotic transcriptional repression may impair transcription-coupled repair.

Fully functional global genome repair of (6-4) photoproducts and compromised transcription-coupled repair of cyclobutane pyrimidine dimers in condensed mitotic chromatin

International Nuclear Information System (INIS)

Komura, Jun-ichiro; Ikehata, Hironobu; Mori, Toshio; Ono, Tetsuya

2012-01-01

During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cells was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression. -- Highlights: ► Global genome repair of (6-4) photoproducts is fully active in mitotic cells. ► DNA in condensed mitotic chromatin does not seem inaccessible or inert. ► Mitotic transcriptional repression may impair transcription-coupled repair.

Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation

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Konze-Thomas, B.; Hazard, R.M.; Maher, V.M.; McCormick, J.J. (Michigan State Univ., East Lansing (USA). Carcinogenesis Lab.)

1982-01-01

Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G/sub 0/ state. Autoradiography studies of cells released from G/sub 0/ after 72 h and replated at lower densities (3-9 x 10/sup 3/ cells/cm/sup 2/) in fresh medium showed that semiconservative DNA synthesis (S phase) began approx. equal to 24 h after the replating. The task was to determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts.

DNA single strand break in fibroblast from Down syndrome patients

International Nuclear Information System (INIS)

Rozga, B.

1992-01-01

The radiosensitivity of tree trisomic (trisomia +21) strains of human fibroblasts to gamma radiation has been investigated in vitro and the causes of induction and repair of single strand DNA breaks in these cells have been estimated. The single strand breaks in DNA of normal and trisomic cells have been found to be ameliorated with an approximately equal efficiency. Repair has been found to be three times slower in trisomic cells compared to their normal relevant, most likely due to their elevated sensitivity to ionizing radiation and the following mortality of trisomic cells, and/or the potential occurrence of a great number of chromosome aberrations in cells irradiated in vitro. (author). 28 refs, 4 figs, 1 tab

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

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Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

2011-01-01

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

Comparison between the radiosensitivity of human, mouse and chicken fibroblast-like cells using short-term endpoints

International Nuclear Information System (INIS)

Diatloff-Zito, C.; Loria, E.; Maciera-Coelho, A.; Deschavanne, P.J.; Malaise, E.P.

1981-01-01

A comparative study has been made of the radiosensitivity of fibroblastic cell lines from three different animal species: human, mouse and chicken. Endpoints reflecting short term responses were utilized: colony forming ability (CFA), DNA single strand break (SSB) repair and repair of potentially lethal damage (PLD). Regardless of the criterion employed, the response to radiation varies from one species to another. According to survival curves, chicken cells appear to be more radioresistant than those of human and mouse. SSB repair is apparently absent in murine cells, partial in chicken cells and complete in human cells. This lack of correlation between survival curves and SSB repair demonstrates that survival of irradiated cells does not depend only (or at all) on the repair of SSB. The repair of PLD is much more efficient in human and chicken cells than in murine cells. (author)

Resistance of plateau-phase human normal and xeroderma pigmentosum fibroblasts to the cytotoxic effect of ultraviolet light

International Nuclear Information System (INIS)

Chan, G.L.; Little, J.B.

1979-01-01

Clonogenic survival response to 254-nm ultraviolet light was measured in 2 strains of repair-proficient normal human fibroblasts and 4 strains of xeroderma pigmentosum (XP) fibroblasts belonging to complementation groups A, C, D and variant. In all strains except XPA, cells irradiated in plateau phase and subcultured immediately were much more resistant to the lethal effect of UV than cells irradiated in the exponential phase of growth. Typically, 10-20% of plateau-phase cells were extremely resistant. When the cultures were held in plateau phase for 24 h after irradiation and before subculture, there was a further enhancement of survival. By use of a UV-specific endonuclease assay, no difference was found in the number of DNA lesions induced in exponentially growing and plateau cultures by the same dose of UV light. Thus plateau-phase cells appear to be more efficient in their DNA-repair capability than cells in exponential growth. XP group A cells were uniquely found to be deficient in the processes which lead to plateau-phase resistance. Since plateau-phase repair was not lacking in XP groups C, D and variant, it may be related to a DNA-repair process different from that which is responsible for the overall UV sensitivity of these cells. (orig.)

DNA repair in human cells exposed to combinations of carcinogenic agents

International Nuclear Information System (INIS)

Setlow, R.B.; Ahmed, F.E.

1980-01-01

Normal human and XP 2 fibroblasts were treated with uv plus uv-mimetic chemicals. The uv dose used was sufficient to saturate the uv excision repair system. Excision repair after combined treatments was estimated by unscheduled DNA synthesis, BrdUrd photolysis, and the loss of sites sensitive to a uv specific endonuclease. Since the repair of damage from uv and its mimetics is coordinately controlled we expected that there would be similar rate-limiting steps in the repair of uv and chemical damage and that after a combined treatment the total amount of repair would be the same as from uv or the chemicals separately. The expectation was not fulfilled. In normal cells repair after a combined treatment was additive whereas in XP cells repair after a combined treatment was usually less than after either agent separately. The chemicals tested were AAAF, DMBA-epoxide, 4NQO, and ICR-170

Host cell reactivation by fibroblasts from patients with pigmentary degeneration of the retina

International Nuclear Information System (INIS)

Lytle, C.D.; Tarone, R.E.; Barrett, S.F.; Robbins, J.H.; Wirtschafter, J.D.; Dupuy, J.-M.

1983-01-01

Cockayne syndrome (CS) is an autosomal recessive disease characterized by numerous clinical abnormalities including acute sun sensitivity and primary pigmentary degeneration of the retina. Cultured fibroblasts from CS patients are hypersensitive to ultraviolet radiation. Host cell reactivation of irradiated virus was studied in CS and in other diseases with retinal degeneration to evaluate repair. The survival of UV-irradiated Herpes simplex virus type 1 was determined in fibroblast lines from four normal donors, two patients with CS, one with both xeroderma pigmentosum (XP) and CS, and from several other patients with (Usher syndrome, olivopontocerebellar atrophy, retinitis pigmentosa) and without (XP, ataxia telangiectasia) primary pigmentary degeneration of the retina. The viral survival curves in all cell lines showed two components: a very sensitive initial component followed by an exponential, less sensitive component. The exponential component had greater sensitivity than normal in the case of the CS patients, the patient with both XP and CS, and the XP patient. It was proposed that patients with CS have defective repair of DNA which may be the cause of their retinal degeneration. (author)

Host cell reactivation by fibroblasts from patients with pigmentary degeneration of the retina

Energy Technology Data Exchange (ETDEWEB)

Lytle, C.D. (Food and Drug Administration, Rockville, MD (USA)); Tarone, R.E.; Barrett, S.F.; Robbins, J.H. (National Cancer Inst., Bethesda, MD (USA)); Wirtschafter, J.D. (Minnesota Univ., Minneapolis (USA). Hospitals); Dupuy, J.M. (Quebec Univ., Laval-des-Rapides (Canada). Inst. Armand-Frappier)

1983-05-01

Cockayne syndrome (CS) is an autosomal recessive disease characterized by numerous clinical abnormalities including acute sun sensitivity and primary pigmentary degeneration of the retina. Cultured fibroblasts from CS patients are hypersensitive to ultraviolet radiation. Host cell reactivation of irradiated virus was studied in CS and in other diseases with retinal degeneration to evaluate repair. The survival of UV-irradiated Herpes simplex virus type 1 was determined in fibroblast lines from four normal donors, two patients with CS, one with both xeroderma pigmentosum (XP) and CS, and from several other patients with (Usher syndrome, olivopontocerebellar atrophy, retinitis pigmentosa) and without (XP, ataxia telangiectasia) primary pigmentary degeneration of the retina. The viral survival curves in all cell lines showed two components: a very sensitive initial component followed by an exponential, less sensitive component. The exponential component had greater sensitivity than normal in the case of the CS patients, the patient with both XP and CS, and the XP patient. It was proposed that patients with CS have defective repair of DNA which may be the cause of their retinal degeneration.

Inhibition of DNA repair by trifluoperazine

Energy Technology Data Exchange (ETDEWEB)

Charp, P.A.; Regan, J.D.

1985-01-01

The authors examined the possible role of calmodulin in the excision repair of ultraviolet light-induced pyrimidine dimers in damaged DNA by means of specialized assay systems. These assays included bromodeoxyuridine photolysis, dimer chromatography and cytosine arabinoside incorporation in conjunction with hydroxyurea. The calmodulin antagonist, trifluoperazine, and the calcium-chelating agent, EGTA, were employed to ascertain what affect calmodulin played in the repair process. Normal human fibroblast cells are used in all studies described in this report. After exposure to 10 J/m2 of 254 nm light, the authors observed a decrease of about 30% in the number of single-strand breaks produced in the presence of 25 M trifluoperazine (1.9 vs. 3.3) in controls although the numbers of bases re-inserted in the repaired regions were similar (64 vs. 72). Measurement of thymine-containing dimers remaining throughout a 24 h time period indicated a 30% difference decrease in the number of cytosine arabinoside arrested repair sites in the presence of either EGTA or trifluoperazine. The results are discussed with relation to the possibility of calmodulin altering the initial incision by repair endonuclease. 27 references, 3 figures.

Deposition of insoluble elastin by pulmonary fibroblasts from patients with COPD is increased by treatment with versican siRNA.

Science.gov (United States)

Wu, Lian; Zhang, Jing; Qu, Jie Ming; Bai, Chun-Xue; Merrilees, Mervyn J

2017-01-01

A reduced content of alveolar elastic fibers is a key feature of COPD lung. Despite continued elastogenic potential by alveolar fibroblasts in the lung affected by COPD, repair of elastic fibers does not take place, which is due to increased levels of the chondroitin sulfate proteoglycan versican that inhibits the assembly of tropoelastin into fibers. In this study, primary pulmonary fibroblast cell lines from COPD and non-COPD patients were treated with a small interfering RNA (siRNA) against versican to determine if knockdown of versican could restore the deposition of insoluble elastin. Versican siRNA treatment reduced versican expression and secretion by pulmonary fibroblasts from both COPD and non-COPD patients ( P elastin in the COPD cell cultures ( P elastin (tropoelastin) in either the COPD or non-COPD cell cultures, supporting a role for versican in inhibiting assembly but not synthesis of tropoelastin. These results suggest that removal or knockdown of versican may be a possible therapeutic strategy for increasing deposition of insoluble elastin and stimulating repair of elastic fibers in COPD lung.

Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer.

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Julie A Wallace

Full Text Available Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.

Isolation of the functional human excision repair gene ERCC5 by intercosmid recombination

International Nuclear Information System (INIS)

Mudgett, J.S.; MacInnes, M.A.

1990-01-01

The complete human nucleotide exicision repair gene ERCC5 was isolated as a functional gene on overlapping cosmids. ERCC5 corrects the excision repair deficiency of Chinese hamster ovary cell line UV135, of complementation group 5. Cosmids that contained human sequences were obtained from a UV-resistant cell line derived from UV135 cells transformed with human genomic DNA. Individually, none of the cosmids complemented the UV135 repair defect; cosmid groups were formed to represent putative human genomic regions, and specific pairs of cosmids that effectively transformed UV135 cells to UV resistance were identified. Analysis of transformants derived from the active cosmid pairs showed that the functional 32-kbp ERCC5 gene was reconstructed by homologous intercosmid recombination. The cloned human sequences exhibited 100% concordance with the locus designated genetically as ERCC5 located on human chromosome 13q. Cosmid-transformed UV135 host cells repaired cytotoxic damage to levels about 70% of normal and repaired UV-irradiated shuttle vector DNA to levels about 82% of normal

An altered redox balance and increased genetic instability characterize primary fibroblasts derived from xeroderma pigmentosum group A patients.

Science.gov (United States)

Parlanti, Eleonora; Pietraforte, Donatella; Iorio, Egidio; Visentin, Sergio; De Nuccio, Chiara; Zijno, Andrea; D'Errico, Mariarosaria; Simonelli, Valeria; Sanchez, Massimo; Fattibene, Paola; Falchi, Mario; Dogliotti, Eugenia

2015-12-01

Xeroderma pigmentosum (XP)-A patients are characterized by increased solar skin carcinogenesis and present also neurodegeneration. XPA deficiency is associated with defective nucleotide excision repair (NER) and increased basal levels of oxidatively induced DNA damage. In this study we search for the origin of increased levels of oxidatively generated DNA lesions in XP-A cell genome and then address the question of whether increased oxidative stress might drive genetic instability. We show that XP-A human primary fibroblasts present increased levels and different types of intracellular reactive oxygen species (ROS) as compared to normal fibroblasts, with O₂₋• and H₂O₂ being the major reactive species. Moreover, XP-A cells are characterized by decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios as compared to normal fibroblasts. The significant increase of ROS levels and the alteration of the glutathione redox state following silencing of XPA confirmed the causal relationship between a functional XPA and the control of redox balance. Proton nuclear magnetic resonance (¹H NMR) analysis of the metabolic profile revealed a more glycolytic metabolism and higher ATP levels in XP-A than in normal primary fibroblasts. This perturbation of bioenergetics is associated with different morphology and response of mitochondria to targeted toxicants. In line with cancer susceptibility, XP-A primary fibroblasts showed increased spontaneous micronuclei (MN) frequency, a hallmark of cancer risk. The increased MN frequency was not affected by inhibition of ROS to normal levels by N-acetyl-L-cysteine. Copyright © 2015 Elsevier B.V. All rights reserved.

Disentangling the multifactorial contributions of fibronectin, collagen and cyclic strain on MMP expression and extracellular matrix remodeling by fibroblasts.

Science.gov (United States)

Zhang, Yang; Lin, Zhe; Foolen, Jasper; Schoen, Ingmar; Santoro, Alberto; Zenobi-Wong, Marcy; Vogel, Viola

2014-11-01

Early wound healing is associated with fibroblasts assembling a provisional fibronectin-rich extracellular matrix (ECM), which is subsequently remodeled and interlaced by type I collagen. This exposes fibroblasts to time-variant sets of matrices during different stages of wound healing. Our goal was thus to gain insight into the ECM-driven functional regulation of human foreskin fibroblasts (HFFs) being either anchored to a fibronectin (Fn) or to a collagen-decorated matrix, in the absence or presence of cyclic mechanical strain. While the cells reoriented in response to the onset of uniaxial cyclic strain, cells assembled exogenously added Fn with a preferential Fn-fiber alignment along their new orientation. Exposure of HFFs to exogenous Fn resulted in an increase in matrix metalloproteinase (MMP) expression levels, i.e. MMP-15 (RT-qPCR), and MMP-9 activity (zymography), while subsequent exposure to collagen slightly reduced MMP-15 expression and MMP-9 activity compared to Fn-exposure alone. Cyclic strain upregulated Fn fibrillogenesis and actin stress fiber formation, but had comparatively little effect on MMP activity. We thus propose that the appearance of collagen might start to steer HFFs towards homeostasis, as it decreased both MMP secretion and the tension of Fn matrix fibrils as assessed by Fluorescence Resonance Energy Transfer. These results suggest that HFFs might have a high ECM remodeling or repair capacity in contact with Fn alone (early event), which is reduced in the presence of Col1 (later event), thereby down-tuning HFF activity, a processes which would be required in a tissue repair process to finally reach tissue homeostasis. Copyright © 2014. Published by Elsevier B.V.

Specificity and completeness of inhibition of DNA repair by novobiocin and aphidicolin

International Nuclear Information System (INIS)

Cleaver, J.E.

1982-01-01

Novobiocin and aphidicolin were both potent inhibitors of excision repair of u.v.-induced damage to DNA in human embryonic fibroblasts, and both also inhibited semiconservative DNA replication even more strongly. The mechanism of action of these two drugs is, however, different. Novobiocin inhibited repair replication without accumulating single-strand breaks, but aphidicolin inhibited repair replication with the accumulation of numerous single-strand breaks. Novobiocin appears to inhibit repair at an earlier stage than aphidicolin, which may indicate that DNA topoisomerases play a role in eukaryotic DNA repair. Digestion of DNA by exonuclease III indicated that repair patches in novobiocin-treated cells contained no excess 3'OH termini, whereas up to 40% of the repaired DNA in aphidicolin-treated cells had free 3'OH termini. Therefore, although aphidicolin resulted in the accumulation of single-strand breaks, many of the repair events escaped inhibition and the number of breaks is an underestimate of the true number of repair events

Rejoining of DNA double-strand breaks in human fibroblasts and its impairment in one ataxia telangiectasia and two Fanconi strains

International Nuclear Information System (INIS)

Coquerelle, T.M.; Weibezahn, K.F.

1981-01-01

Using the technique of neutral elution through polycarbonate filters as a measure of DNA length, and hence of the number of double-strand breaks incurred as a result of radiation damage, we found that normal human fibroblasts rejoin 50% of all breaks within only 3 min (37 degrees C). This fast rejoining was impaired in fibroblasts from one patient with Ataxia telangiectasia and in fibroblasts from two patients with Fanconi's anemia. Also the number of residual breaks after several hours of repair was higher than in control cells. Other cases with the same diseases were normal in their rejoining of double-strand breaks

Response of human fibroblasts to low dose rate gamma irradiation

International Nuclear Information System (INIS)

Dritschilo, A.; Brennan, T.; Weichselbaum, R.R.; Mossman, K.L.

1984-01-01

Cells from 11 human strains, including fibroblasts from patients with the genetic diseases of ataxia telangiectasia (AT), xeroderma pigmentosum (XP), and Fanconi's anemia (FA), were exposed to γ radiation at high (1.6-2.2 Gy/min) and at low (0.03-0.07 Gy/min) dose rates. Survival curves reveal an increase inthe terminal slope (D 0 ) when cells are irradiated at low dose rates compared to high dose rates. This was true for all cell lines tested, although the AT, FA, and XP cells are reported or postulated to have radiation repair deficiencies. From the response of these cells, it is apparent that radiation sensitivities differ; however, at low dose rate, all tested human cells are able to repair injury

Direct induction of chondrogenic cells from human dermal fibroblast culture by defined factors.

Directory of Open Access Journals (Sweden)

Hidetatsu Outani

Full Text Available The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4 and one chondrogenic factor (SOX9 can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon cells from human dermal fibroblast (HDF culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes.

Science.gov (United States)

Jobert, Laure; Nilsen, Hilde

2014-07-01

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.

Immortalization of Werner syndrome and progeria fibroblasts

International Nuclear Information System (INIS)

Saito, H.; Moses, R.E.

1991-01-01

Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents

Faulty DNA-polymerase δ/ε-mediated excision-repair in response to gamma-radiation or ultraviolet-light in P53-deficient fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome

International Nuclear Information System (INIS)

Mirzayans, R.; Enns, L.; Dietrich, K.; Barley, R.D.C.; Paterson, M.C.; Alberta Univ., Edmonton, AB; Alberta Univ., Edmonton, AB

1996-01-01

Dermal fibroblast strains cultured from affected members of a cancer-prone family with Li-Fraumeni syndrome (LFS) harbor a point mutation in one allele of the p53 tumor suppressor gene, resulting in loss of normal p53-deficient strains to carry out the long-patch mode of excision repair, mediated by DNA polymerases delta and epsilon, after exposure to Co-60 gamma radiation or far ultraviolet (UV) (chiefly 254 mm) light. Repair was monitored by incubation of the irradiated cultures in the presence of aphidicolin (ape) or 1-beta-D-arabinofuranosylcytosine (araC), each a specific inhibitor of long-patch repair, followed by measurement of drug-induced DNA strand breaks (reflecting non-ligated strand incision events) by alkaline surcrose velocity sedimentation. The LFS strains displayed deficient repair capacity in response to both gamma rays and UV light. The repair anomaly in UV-irradiated LFS cultures was manifested not only in the overall genome, but also in the transcriptionally active, preferentially repaired c-myc gene. Using autoradiography we also assessed unscheduled DNA synthesis (UDS) after UV irradiation and found this conventional measure of repair replication to be deficient in LFS strains. Moreover, both ape and araC decreased the level of UV-induced UDS by similar to 75% in normal cells, but each had only a marginal effect on LFS cells. We further demonstrated that the LFS strains are impaired in the recovery of both RNA and replicative DNA syntheses after UV treatment, two molecular anomalies of the DNA repair deficiency disorders xeroderma pigmentosum and Cockayne's syndrome. Together these results imply a critical role for wild-type p53 protein in DNA polymerase delta/epsilon-mediated excision repair, both the mechanism operating on the entire genome and that acting on expressed genes. (Author)

PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts.

Science.gov (United States)

Khamaisi, Mogher; Katagiri, Sayaka; Keenan, Hillary; Park, Kyoungmin; Maeda, Yasutaka; Li, Qian; Qi, Weier; Thomou, Thomas; Eschuk, Danielle; Tellechea, Ana; Veves, Aris; Huang, Chenyu; Orgill, Dennis Paul; Wagers, Amy; King, George L

2016-03-01

Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients.

Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

Directory of Open Access Journals (Sweden)

Sunita Nayak

Full Text Available The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

Defined Engineered Human Myocardium with Advanced Maturation for Applications in Heart Failure Modelling and Repair

Science.gov (United States)

Tiburcy, Malte; Hudson, James E.; Balfanz, Paul; Schlick, Susanne; Meyer, Tim; Liao, Mei-Ling Chang; Levent, Elif; Raad, Farah; Zeidler, Sebastian; Wingender, Edgar; Riegler, Johannes; Wang, Mouer; Gold, Joseph D.; Kehat, Izhak; Wettwer, Erich; Ravens, Ursula; Dierickx, Pieterjan; van Laake, Linda W.; Goumans, Marie Jose; Khadjeh, Sara; Toischer, Karl; Hasenfuss, Gerd; Couture, Larry A.; Unger, Andreas; Linke, Wolfgang A.; Araki, Toshiyuki; Neel, Benjamin; Keller, Gordon; Gepstein, Lior; Wu, Joseph C.; Zimmermann, Wolfram-Hubertus

2017-01-01

Background Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modelling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) towards an adult phenotype under defined conditions. Methods We systematically investigated cell composition, matrix and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We employed morphological, functional, and transcriptome analyses to benchmark maturation of EHM. Results EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M-bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency-response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β1- and β2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and NT-proBNP release; all are classical hallmarks of heart failure. Additionally, we demonstrate scalability of EHM according to anticipated clinical demands for cardiac repair. Conclusions We provide proof-of-concept for a universally applicable technology for the engineering of macro-scale human myocardium for disease modelling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions. PMID:28167635

Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair.

Science.gov (United States)

Tiburcy, Malte; Hudson, James E; Balfanz, Paul; Schlick, Susanne; Meyer, Tim; Chang Liao, Mei-Ling; Levent, Elif; Raad, Farah; Zeidler, Sebastian; Wingender, Edgar; Riegler, Johannes; Wang, Mouer; Gold, Joseph D; Kehat, Izhak; Wettwer, Erich; Ravens, Ursula; Dierickx, Pieterjan; van Laake, Linda W; Goumans, Marie Jose; Khadjeh, Sara; Toischer, Karl; Hasenfuss, Gerd; Couture, Larry A; Unger, Andreas; Linke, Wolfgang A; Araki, Toshiyuki; Neel, Benjamin; Keller, Gordon; Gepstein, Lior; Wu, Joseph C; Zimmermann, Wolfram-Hubertus

2017-05-09

Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions. We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM. EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical β 1 - and β 2 -adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair. We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions. © 2017 American Heart Association, Inc.

Grooved surface topography alters matrix-metalloproteinase production by human fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Brydone, Alistair S; Dominic Meek, R M [Department of Orthopaedics, Southern General Hospital, 1345 Govan Road, Glasgow G51 4TF (United Kingdom); Dalby, Matthew J; Berry, Catherine C; McNamara, Laura E, E-mail: alibrydone@gmail.com [Centre for Cell Engineering, Joseph Black Building, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ (United Kingdom)

2011-06-15

Extracellular matrix (ECM) remodelling is an essential physiological process in which matrix-metalloproteinases (MMPs) have a key role. Manipulating the manner in which cells produce MMPs and ECMs may enable the creation of a desired tissue type, i.e. effect repair, or the prevention of tissue invasion (e.g. metastasis). The aim of this project was to determine if culturing fibroblasts on grooved topography altered collagen deposition or MMP production. Human fibroblasts were seeded on planar or grooved polycaprolactone substrates (grooves were 12.5 {mu}m wide with varying depths of 240 nm, 540 nm or 2300 nm). Cell behaviour and collagen production were studied using fluorescence microscopy and the spent culture medium was assessed using gel zymography to detect MMPs. Total collagen deposition was high on the 240 nm deep grooves, but decreased as the groove depth increased, i.e. as cell contact guidance decreased. There was an increase in gelatinase on the 2300 nm deep grooved topography and there was a difference in the temporal expression of MMP-3 observed on the planar surface compared to the 540 nm and 2300 nm topographies. These results show that topography can alter collagen and MMP production. A fuller understanding of these processes may permit the design of surfaces tailored to tissue regeneration e.g. tendon repair.

Grooved surface topography alters matrix-metalloproteinase production by human fibroblasts

International Nuclear Information System (INIS)

Brydone, Alistair S; Dominic Meek, R M; Dalby, Matthew J; Berry, Catherine C; McNamara, Laura E

2011-01-01

Extracellular matrix (ECM) remodelling is an essential physiological process in which matrix-metalloproteinases (MMPs) have a key role. Manipulating the manner in which cells produce MMPs and ECMs may enable the creation of a desired tissue type, i.e. effect repair, or the prevention of tissue invasion (e.g. metastasis). The aim of this project was to determine if culturing fibroblasts on grooved topography altered collagen deposition or MMP production. Human fibroblasts were seeded on planar or grooved polycaprolactone substrates (grooves were 12.5 μm wide with varying depths of 240 nm, 540 nm or 2300 nm). Cell behaviour and collagen production were studied using fluorescence microscopy and the spent culture medium was assessed using gel zymography to detect MMPs. Total collagen deposition was high on the 240 nm deep grooves, but decreased as the groove depth increased, i.e. as cell contact guidance decreased. There was an increase in gelatinase on the 2300 nm deep grooved topography and there was a difference in the temporal expression of MMP-3 observed on the planar surface compared to the 540 nm and 2300 nm topographies. These results show that topography can alter collagen and MMP production. A fuller understanding of these processes may permit the design of surfaces tailored to tissue regeneration e.g. tendon repair.

Evidence for repair of ultraviolet light-damaged herpes virus in human fibroblasts by a recombination mechanism

International Nuclear Information System (INIS)

Hall, J.D.; Featherston, J.D.; Almy, R.E.

1980-01-01

Human cells were either singly or multiply infected with herpes simplex virus (HSV-1) damaged by ultraviolet (uv) light, and the fraction of cells able to produce infectious virus was measured. The fraction of virus-producing cells was considerably greater for multiply infected cells than for singly infected cells at each uv dose examined. These high survival levels of uv-irradiated virus in multiply infected cells demonstrated that multiplicity-dependent repair, possibly due to genetic exchanges between damaged HSV-1 genomes, was occurring in these cells. To test whether uv light is recombinogenic for HSV-1, the effect of uv irradiation on the yield of temperature-resistant viral recombinants in cells infected with pairs of temperature-sensitive mutants was also investigated. The results of these experiments showed that the defective functions in these mutant host cells are not required for multiplicity-dependent repair or uv-stimulated viral recombination in herpes-infected cells

Methylation of deoxycytidine incorporated by excision-repair synthesis of DNA

International Nuclear Information System (INIS)

Kastan, M.B.; Gowans, B.J.; Lieberman, M.W.

1982-01-01

Methylation of deoxycytidine incorporated by DNA excision-repair was studied in human diploid fibroblasts following damage with ultraviolet radiation, N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene. In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication in logarithmic-phase cultures a steady state level of 3.4% 5-methylcytosine is reached in less than 2 hr after cells are labeled with 6- 3H-deoxycytidine, following ultraviolet-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approximately 2.0% 5-methylcytosine in the repair patch. In cells from cultures in logarithmic-phase growth, 5-methylcytosine formation in ultraviolet-induced repair patches occurs faster and to a greater extent, reaching a level of approximately 2.7% in 10-20 hr. Preexisting hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites

Repair of chromosome damage induced by X-irradiation during G2 phase in a line of normal human fibroblasts and its malignant derivative

International Nuclear Information System (INIS)

Parshad, R.; Gantt, R.; Sanford, K.K.; Jones, G.M.; Tarone, R.E.

1982-01-01

A line of normal human skin fibroblasts (KD) differed from its malignant derivative (HUT-14) in the extent of cytogenetic damage induced by X-irradiation during G2 phase. Malignant cells had significantly more chromatid breaks and gaps after exposure to 25, 50, or 100 rad. The gaps may represent single-strand breaks. Results from alkaline elution of cellular DNA immediately after irradiation showed that the normal and malignant cells in asynchronous population were equally sensitive to DNA single-strand breakage by X-irradiation. Caffeine or beta-cytosine arabinoside (ara-C), inhibitors of DNA repair, when added directly following G2 phase exposure, significantly increased the incidence of radiation-induced chromatid damage in the normal cells. In contrast, similar treatment of the malignant cells had little influence. Ara-C differed from caffeine in its effects; whereas both agents increased the frequency of chromatid breaks and gaps, only ara-C increased the frequency of gaps to the level observed in the irradiated malignant cells. Addition of catalase, a scavenger of the derivative free hydroxyl radical (.OH), to the cultures of malignant cells before, during, and following irradiation significantly reduced the chromatid damage; and catalase prevented formation of chromatid gaps. The DNA damage induced by X-ray during G2 phase in the normal KD cells was apparently repaired by a caffeine- and ara-C-sensitive mechanism(s) that was deficient or absent in their malignant derivatives

Repair of chromosome damage induced by X-irradiation during G2 phase in a line of normal human fibroblasts and its malignant derivative

International Nuclear Information System (INIS)

Parshad, R.; Gantt, R.; Sanford, K.K.; Jones, G.M.; Tarone, R.E.

1982-01-01

A line of normal human skin fibroblasts (KD) differed from its malignant derivative (HUT-14) in the extent of cytogenetic damage induced by X-irradiation during G 2 phase. Malignant cells had significantly more chromatid breaks and gaps after exposure to 25, 50, or 100 rad. Results from alkaline elution of cellular DNA immediately after irradiation showed that the normal and malignant cells in asynchronous population were equally sensitive to DNA single-strand breakage by X-irradiation. Caffeine or #betta#-cytosine arabinoside (ara-C), inhibitors of DNA repair, when added directly following G 2 phase exposure, significantly increased the incidence of radiation-induced chromatid damage in the normal cells. In contrast, similar treatment of the malignant cells had little influence. Ara-C differed from caffeine in its effects; whereas both agents increased the frequency of chromatid breaks and gaps, only ara-C increased the frequency of gaps to the level observed in the irradiated malignant cells. Addition of catalase, which destroys H 2 O 2 , or mannitol, a scavenger of the derivative free hydroxyl radical (.OH), to the cultures of malignant cells before, during, and following irradiation significantly reduced the chromatid damage; and catalase prevented formation of chromatid gaps. The DNA damage induced by X-ray during G 2 phase in the normal KD cells was apparently repaired by a caffeine- and ara-C-sensitive mechanism(s) that was deficient or absent in their malignant derivatives

Functional evaluation of patient after arthroscopic repair of rotator cuff tear.

Science.gov (United States)

Kumar, Rohit; Jadhav, Umesh

2014-06-01

Rotator cuff tear is a common problem either after trauma or after degenerative tear in old age group. Arthroscopic repair is the current concept of rotator cuff repair. Here, we are trying to evaluate the functional outcome after arthroscopic repair of full thickness rotator cuff tear (single row) in Indian population. Twenty five patients (14 males and 11 females) who underwent arthroscopic repair of full thickness rotator cuff tear at a single institution were included in the study. Postoperatively patient's shoulder was rated according to UCLA score, pain was graded according to the visual analog score. The range of motion was analysed and documented. The mean age of the patients were 50.48 years. The preoperative VAS score mode was 7 and post operative VAS was 1 (p value fair in 12% (n = 3), excellent in 8% (n = 2) and poor results were seen in none of the patients. The mean UCLA improved from a score of 15.84 to 30.28 with a p value advantages, hence we used a single row repair considering the Indian population and the cost effectiveness of the surgery with good to excellent results.

Skin fibroblasts from a D-deletion type retinoblastoma patient are abnormally X-ray sensitive

International Nuclear Information System (INIS)

Weichselbaum, R.R.; Nove, J.; Little, J.B.

1977-01-01

Retinoblastoma is a rare malignant eye tumour that appears either spontaneously or in genetically predisposed persons. The latter group is composed of persons who inherit the tumour with a dominant mode of transmission (the familial type) and those who have a deletion in the long arm of chromosome 13 referred to as the D-deletion type. When this deletion is present it is observed in many somatic cells and is often associated with structural defects. Survivors of the genetic forms of retinoblastoma have an increased risk of the development of cancers at other sites. A single genetic locus is unlikely to predispose many somatic cells to tumour formation unless a fundamental molecular defect, possibly related to DNA repair, is present. In order to investigate this hypothesis a study was made of the in vitro X-ray sensitivity of skin fibroblasts derived from three retinoblastoma patients, comprising a pair of twins with the familial type accompanied by no gross chromosome abnormalities, and a patient with the D-deletion type. It was found that fibroblasts derived from the D-deletion patient were significantly more radiosensitive than those from the other two patients. X-ray survival curves are shown. It is concluded that skin fibroblasts derived from a patient with the D-deletion variant of retinoblastoma are abnormally radiosensitive. Future investigations may indicate a specific defect in molecular repair of DNA that will explain the predisposition of these patients to the development of other tumours. (U.K.)

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

International Nuclear Information System (INIS)

Bruzzone, Santina; Battaglia, Florinda; Mannino, Elena; Parodi, Alessia; Fruscione, Floriana; Basile, Giovanna; Salis, Annalisa; Sturla, Laura; Negrini, Simone; Kalli, Francesca; Stringara, Silvia; Filaci, Gilberto

2012-01-01

Highlights: ► ABA is an endogenous hormone in humans, regulating different cell responses. ► ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. ► UV-B irradiation increases ABA content in SSc cultures. ► SSc fibroblasts could benefit from exposure to ABA and/or to UV-B. -- Abstract: The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-β (TGF-β). Conversely, migration toward ABA, but not toward TGF-β, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

Targeting of the pulmonary capillary vascular niche promotes lung alveolar repair and ameliorates fibrosis.

Science.gov (United States)

Cao, Zhongwei; Lis, Raphael; Ginsberg, Michael; Chavez, Deebly; Shido, Koji; Rabbany, Sina Y; Fong, Guo-Hua; Sakmar, Thomas P; Rafii, Shahin; Ding, Bi-Sen

2016-02-01

Although the lung can undergo self-repair after injury, fibrosis in chronically injured or diseased lungs can occur at the expense of regeneration. Here we study how a hematopoietic-vascular niche regulates alveolar repair and lung fibrosis. Using intratracheal injection of bleomycin or hydrochloric acid in mice, we show that repetitive lung injury activates pulmonary capillary endothelial cells (PCECs) and perivascular macrophages, impeding alveolar repair and promoting fibrosis. Whereas the chemokine receptor CXCR7, expressed on PCECs, acts to prevent epithelial damage and ameliorate fibrosis after a single round of treatment with bleomycin or hydrochloric acid, repeated injury leads to suppression of CXCR7 expression and recruitment of vascular endothelial growth factor receptor 1 (VEGFR1)-expressing perivascular macrophages. This recruitment stimulates Wnt/β-catenin-dependent persistent upregulation of the Notch ligand Jagged1 (encoded by Jag1) in PCECs, which in turn stimulates exuberant Notch signaling in perivascular fibroblasts and enhances fibrosis. Administration of a CXCR7 agonist or PCEC-targeted Jag1 shRNA after lung injury promotes alveolar repair and reduces fibrosis. Thus, targeting of a maladapted hematopoietic-vascular niche, in which macrophages, PCECs and perivascular fibroblasts interact, may help to develop therapy to spur lung regeneration and alleviate fibrosis.

Targeting of the pulmonary capillary vascular niche promotes lung alveolar repair and ameliorates fibrosis

Science.gov (United States)

Cao, Zhongwei; Lis, Raphael; Ginsberg, Michael; Chavez, Deebly; Shido, Koji; Rabbany, Sina Y.; Fong, Guo-Hua; Sakmar, Thomas P.; Rafii, Shahin; Ding, Bi-Sen

2016-01-01

Although the lung can undergo self-repair after injury, fibrosis in chronically injured or diseased lungs can occur at the expense of regeneration. Here we study how a hematopoietic-vascular niche regulates alveolar repair and lung fibrosis. Using intratracheal injection of bleomycin or hydrochloric acid in mice, we show that repetitive lung injury activates pulmonary capillary endothelial cells (PCECs) and perivascular macrophages, impeding alveolar repair and promoting fibrosis. Whereas the chemokine receptor CXCR7, expressed on PCECs, acts to prevent epithelial damage and ameliorate fibrosis after a single round of treatment with bleomycin or hydrochloric acid, repeated injury leads to suppression of CXCR7 expression and recruitment of vascular endothelial growth factor receptor 1 (VEGFR1)-expressing perivascular macrophages. This recruitment stimulates Wnt/β-catenin–dependent persistent upregulation of the Notch ligand Jagged1 (encoded by Jag1) in PCECs, which in turn stimulates exuberant Notch signaling in perivascular fibroblasts and enhances fibrosis. Administration of a CXCR7 agonist or PCEC-targeted Jag1 shRNA after lung injury promotes alveolar repair and reduces fibrosis. Thus, targeting of a maladaptbed hematopoietic-vascular niche, in which macrophages, PCECs and perivascular fibroblasts interact, may help to develop therapy to spur lung regeneration and alleviate fibrosis. PMID:26779814

Pulmonary function in automobile repair workers

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Chattopadhyay O

2007-01-01

Full Text Available Background : Automobile repair shop is a place where workers are exposed to harmful chemicals and toxic substances. Objective : To study the occurrence of obstructive and restrictive pulmonary impairment among automobile garage workers. Methods : A cross sectional study involving 151 automobile garage workers from 14 randomly selected garages of urban Kolkata. The study variables were Forced Expiratory Volume in 1 second (FEV 1 , Forced Vital Capacity (FVC, Peak Expiratory Flow Rate (PE FR, age, smoking habit, duration of work, type of work, and respiratory symptoms. The study was analysed using Regression equations, and Chi-square test. Results : All the workers were male. Obstructive impairment was seen in 25.83% of the workers whereas restrictive impairment was seen in 21.19% of the workers. Mixed obstructive and restrictive impairment was seen in 10.6% of the workers. The frequency of obstructive impairment was higher in older workers. In the age group of less than 20 years, 13.6% of the workers had obstructive impairment while 42.86% of workers above 40 years of age had obstructive impairment. Obstructive impairment was more frequently observed in battery repair workers (58.33% and spray painters (37.5% while 16.67% of the body repair workers and 30.19% of the engine mechanics had obstructive impairment. Obstructive impairment was more frequently observed in smokers (53.1 % as compared to ex-smokers (33.3% and non-smokers (6.4%. Obstructive impairment was more frequently observed in workers who had been working for a longer duration. Conclusion: Nearly 36.4% of the automobile garage workers had some form of pulmonary function impairment; obstructive and/or restrictive. The use of personal protective equipment, worker education, and discontinuation of the use of paints containing toxic pigments are recommended.

Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

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Epstein Shochet, Gali; Wollin, Lutz; Shitrit, David

2018-03-12

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. Whether the ECM conditioned by IPF fibroblasts determines the phenotype of naïve fibroblasts is difficult to explore. IPF-derived primary fibroblasts were cultured on Matrigel and then cleared using ammonium hydroxide, creating an IPF-conditioned matrix (CM). Normal fibroblast CM served as control. Normal fibroblasts were cultured on both types of CM, and cell count, cell distribution and markers of myofibroblast differentiation; transforming growth factor beta (TGFβ) signalling; and ECM expression were assessed. The effects of the anti-fibrotic drugs nintedanib and pirfenidone at physiologically relevant concentrations were also explored. Normal fibroblasts cultured on IPF-CM arranged in large aggregates as a result of increased proliferation and migration. Moreover, increased levels of pSmad3, pSTAT3 (phospho signal transducer and activator of transcription 3), alpha smooth muscle actin (αSMA) and Collagen1a were found, suggesting a differentiation towards a myofibroblast-like phenotype. SB505124 (10 μmol/L) partially reversed these alterations, suggesting a TGFβ contribution. Furthermore, nintedanib at 100 nmol/L and, to a lesser extent, pirfenidone at 100 μmol/L prevented the IPF-CM-induced fibroblast phenotype alterations, suggesting an attenuation of the ECM-fibroblast interplay. IPF fibroblasts alter the ECM, thus creating a CM that further propagates an IPF-like phenotype in normal fibroblasts. This assay demonstrated differences in drug activities for approved IPF drugs at clinically relevant concentrations. Thus, the matrix-fibroblast phenotype interplay might be a relevant assay to explore drug candidates for IPF treatment. © 2018 Asian Pacific Society of Respirology.

Annexin A1 is elevated in patients with COPD and affects lung fibroblast function

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Lai TW

2018-02-01

Full Text Available Tianwen Lai,1,* Yanyu Li,1,* Zongjiong Mai,2 Xiaoxia Wen,1 Yingying Lv,1 Zhanqing Xie,3 Quanchao Lv,1 Min Chen,1 Dong Wu,1 Bin Wu1 1Department of Respiratory and Critical Care Medicine, 2Department of Oncology, 3Department of Thoracic Surgery, The Affiliated Hospital of Guangdong Medical University, Zhanjiang, People’s Republic of China *These authors contributed equally to this work Purpose: Fibrosis in peripheral airways is responsible for airflow limitation in chronic obstructive pulmonary disease (COPD. Annexin A1 modulates several key biological events during inflammation. However, little is known about its role in airway fibrosis in COPD. We investigated whether levels of Annexin A1 were upregulated in patients with COPD, and whether it promoted airway fibrosis.Methods: We quantified serum Annexin A1 levels in never-smokers (n=12, smokers without COPD (n=11, and smokers with COPD (n=22. Correlations between Annexin A1 expression and clinical indicators (eg, lung function were assessed. In vitro, human bronchial epithelial (HBE cells were exposed to cigarette smoke extract (CSE and Annexin A1 expression was assessed. Primary human lung fibroblasts were isolated from patients with COPD and effects of Annexin A1 on fibrotic deposition of lung fibroblasts were evaluated.Results: Serum Annexin A1 was significantly higher in patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD guidelines stage III or IV than in those with GOLD stages I or II (12.8±0.8 ng/mL versus 9.8±0.7 ng/mL; p=0.016. Annexin A1 expression was negatively associated with airflow obstruction (forced expiratory volume in one second % predicted; r=−0.72, p<0.001. In vitro, Annexin A1 was significantly increased in CSE-exposed HBE cells in a time- and concentration-dependent manner. Annexin A1 promoted lung fibroblasts proliferation, migration, differentiation, and collagen deposition via the ERK1/2 and p38 mitogen-activated protein kinase pathways

In vivo formation and repair of DNA double-strand breaks after computed tomography examinations.

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Löbrich, Markus; Rief, Nicole; Kühne, Martin; Heckmann, Martina; Fleckenstein, Jochen; Rübe, Christian; Uder, Michael

2005-06-21

Ionizing radiation can lead to a variety of deleterious effects in humans, most importantly to the induction of cancer. DNA double-strand breaks (DSBs) are among the most significant genetic lesions introduced by ionizing radiation that can initiate carcinogenesis. We have enumerated gamma-H2AX foci as a measure for DSBs in lymphocytes from individuals undergoing computed tomography examination of the thorax and/or the abdomen. The number of DSBs induced by computed tomography examination was found to depend linearly on the dose-length product, a radiodiagnostic unit that is proportional to both the local dose delivered and the length of the body exposed. Analysis of lymphocytes sampled up to 1 day postirradiation provided kinetics for the in vivo loss of gamma-H2AX foci that correlated with DSB repair. Interestingly, in contrast to results obtained in vitro, normal individuals repair DSBs to background levels. A patient who had previously shown severe side effects after radiotherapy displayed levels of gamma-H2AX foci at various sampling times postirradiation that were several times higher than those of normal individuals. Gamma-H2AX and pulsed-field gel electrophoresis analysis of fibroblasts obtained from this patient confirmed a substantial DSB repair defect. Additionally, these fibroblasts showed significant in vitro radiosensitivity. These data show that the in vivo induction and repair of DSBs can be assessed in individuals exposed to low radiation doses, adding a further dimension to DSB repair studies and providing the opportunity to identify repair-compromised individuals after diagnostic irradiation procedures.

Response of human corneal fibroblasts on silk film surface patterns.

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Gil, Eun Seok; Park, Sang-Hyug; Marchant, Jeff; Omenetto, Fiorenzo; Kaplan, David L

2010-06-11

Transparent, biodegradable, mechanically robust, and surface-patterned silk films were evaluated for the effect of surface morphology on human corneal fibroblast (hCF) cell proliferation, orientation, and ECM deposition and alignment. A series of dimensionally different surface groove patterns were prepared from optically graded glass substrates followed by casting poly(dimethylsiloxane) (PDMS) replica molds. The features on the patterned silk films showed an array of asymmetric triangles and displayed 37-342 nm depths and 445-3 582 nm widths. hCF DNA content on all patterned films were not significantly different from that on flat silk films after 4 d in culture. However, the depth and width of the grooves influenced cell alignment, while the depth differences affected cell orientation; overall, deeper and narrower grooves induced more hCF orientation. Over 14 d in culture, cell layers and actin filament organization demonstrated that confluent hCFs and their cytoskeletal filaments were oriented along the direction of the silk film patterned groove axis. Collagen type V and proteoglycans (decorin and biglycan), important markers of corneal stromal tissue, were highly expressed with alignment. Understanding corneal stromal fibroblast responses to surface features on a protein-based biomaterial applicable in vivo for corneal repair potential suggests options to improve corneal tissue mimics. Further, the approaches provide fundamental biomaterial designs useful for bioengineering oriented tissue layers, an endemic feature in most biological tissue structures that lead to critical tissue functions.

Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue.

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Sinha, Mithun; Sen, Chandan K; Singh, Kanhaiya; Das, Amitava; Ghatak, Subhadip; Rhea, Brian; Blackstone, Britani; Powell, Heather M; Khanna, Savita; Roy, Sashwati

2018-03-05

Inflammation, following injury, induces cellular plasticity as an inherent component of physiological tissue repair. The dominant fate of wound macrophages is unclear and debated. Here we show that two-thirds of all granulation tissue fibroblasts, otherwise known to be of mesenchymal origin, are derived from myeloid cells which are likely to be wound macrophages. Conversion of myeloid to fibroblast-like cells is impaired in diabetic wounds. In cross-talk between keratinocytes and myeloid cells, miR-21 packaged in extracellular vesicles (EV) is required for cell conversion. EV from wound fluid of healing chronic wound patients is rich in miR-21 and causes cell conversion more effectively compared to that by fluid from non-healing patients. Impaired conversion in diabetic wound tissue is rescued by targeted nanoparticle-based delivery of miR-21 to macrophages. This work introduces a paradigm wherein myeloid cells are recognized as a major source of fibroblast-like cells in the granulation tissue.

Similar distributions of repaired sites in chromatin of normal and xeroderma pigmentosum variant cells damaged by ultraviolet light

International Nuclear Information System (INIS)

Cleaver, J.E.

1979-01-01

Excision repair of damage from ultraviolet light in both normal and xeroderma pigmentosum variant fibroblasts at early times after irradiation occurred preferentially in regions of DNA accessible to micrococcal nuclease digestion. These regions are predominantly the linker regions between nucleosomes in chromatin. The alterations reported at polymerization and ligation steps of excision repair in the variant are therefore not associated with changes in the relative distributions of repair sites in linker and core particle regions of DNA. (Auth.)

Host-cell reactivation of uv-irradiated and chemically treated Herpes simplex virus type 1 strain MP in normal and xeroderma pigmentosum skin fibroblasts

International Nuclear Information System (INIS)

Selsky, C.A.

1976-01-01

The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated herpes simplex virus type 1 strain mp was studied in normal human skin fibroblasts and xeroderma pigmentosum skin fibroblasts from XP genetic complementation groups A-D and in an XP variant. The increasing relative order for the host-cell reactivation of both types of damaged virus in the different complementation groups is A = D < B < C; XP variant = normal controls. XP complementation group D cells, which manifest the most severe inhibition of her ability for both UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus, can reactivate nitrogen mustard treated HSV-1 mp to the same extent as normal cells. Together, these results indicate that (1) Excision repair of UV and N-acetoxy-2-acetylaminofluorene DNA damaged viruses share a common rate limiting enzymatic step and (2) The repair defect in xeroderma pigmentosum cells plays little or no role in the recovery of nitrogen mustard treated virus. The results of studies on the effect of caffeine on the survival of both UV- and N-acetoxy-2-acetylaminofluorene-treated virus in normal and XP cells imply that the reactivation of HSV-1 mp is mediated by an excision repair process with little if any recovery contributed by post-replication repair mechanisms. The host-cell reactivation of N-acetoxy-2-acetylaminofluorene-treated HSV-1 mp was also correlated with the defective UV-induced unscheduled DNA synthesis in two skin fibroblast strains established from a skin biopsy obtained from each of two juvenile females who had been clinically diagnosed as xeroderma pigmentosum. These findings are discussed in relation to the further characterization of the xeroderma pigmentosum phenotype and their possible utilization for the selection and isolation of new mammalian cell DNA repair mutants

Germline stem cell gene PIWIL2 mediates DNA repair through relaxation of chromatin.

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De-Tao Yin

Full Text Available DNA damage response (DDR is an intrinsic barrier of cell to tumorigenesis initiated by genotoxic agents. However, the mechanisms underlying the DDR are not completely understood despite of extensive investigation. Recently, we have reported that ectopic expression of germline stem cell gene PIWIL2 is associated with tumor stem cell development, although the underlying mechanisms are largely unknown. Here we show that PIWIL2 is required for the repair of DNA-damage induced by various types of genotoxic agents. Upon ultraviolet (UV irradiation, silenced PIWIL2 gene in normal human fibroblasts was transiently activated after treatment with UV light. This activation was associated with DNA repair, because Piwil2-deficienct mouse embryonic fibroblasts (mili(-/- MEFs were defective in cyclobutane pyrimidine dimers (CPD repair after UV treatment. As a result, the UV-treated mili(-/- MEFs were more susceptible to apoptosis, as characterized by increased levels of DNA damage-associated apoptotic proteins, such as active caspase-3, cleaved Poly (ADP-ribose polymerase (PARP and Bik. The impaired DNA repair in the mili(-/- MEFs was associated with the reductions of histone H3 acetylation and chromatin relaxation, although the DDR pathway downstream chromatin relaxation appeared not to be directly affected by Piwil2. Moreover, guanine-guanine (Pt-[GG] and double strand break (DSB repair were also defective in the mili(-/- MEFs treated by genotoxic chemicals Cisplatin and ionizing radiation (IR, respectively. The results indicate that Piwil2 can mediate DNA repair through an axis of Piwil2 → histone acetylation → chromatin relaxation upstream DDR pathways. The findings reveal a new role for Piwil2 in DNA repair and suggest that Piwil2 may act as a gatekeeper against DNA damage-mediated tumorigenesis.

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

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Bruzzone, Santina, E-mail: santina.bruzzone@unige.it [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Battaglia, Florinda [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Mannino, Elena [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Parodi, Alessia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Fruscione, Floriana [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Basile, Giovanna [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Salis, Annalisa; Sturla, Laura [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Negrini, Simone; Kalli, Francesca; Stringara, Silvia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Filaci, Gilberto [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Department of Internal Medicine, Viale Benedetto XV 6, 16132 Genova (Italy); and others

2012-05-25

Highlights: Black-Right-Pointing-Pointer ABA is an endogenous hormone in humans, regulating different cell responses. Black-Right-Pointing-Pointer ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. Black-Right-Pointing-Pointer UV-B irradiation increases ABA content in SSc cultures. Black-Right-Pointing-Pointer SSc fibroblasts could benefit from exposure to ABA and/or to UV-B. -- Abstract: The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-{beta} (TGF-{beta}). Conversely, migration toward ABA, but not toward TGF-{beta}, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

DNA-repair measurements by use of the modified comet assay

DEFF Research Database (Denmark)

Godschalk, Roger W L; Ersson, Clara; Riso, Patrizia

2013-01-01

The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity...... on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating...... laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell...

Preferential repair of nuclear matrix associated DNA in xeroderma pigmentosum complementation group C

International Nuclear Information System (INIS)

Mullenders, L.H.F.; Kesteren, A.C. van; Bussmann, C.J.M.; Zeeland, A.A. van; Natarajan, A.T.

1984-01-01

The distribution of ultraviolet-induced DNA repair patches in the genome of xeroderma pigmentosum cells of complementation group C was investigated by determining the molecular weight distribution of repair labeled DNA and prelabeled DNA in alkaline sucrose gradients after treatment with the dimer-specific endonuclease V of bacteriophage T 4 . The results suggest that DNA-repair synthesis in xeroderma pigmentosum cells of complementation group C occurs in localized regions of the genome. Analysis of the spatial distribution of ultraviolet-induced repair patches in DNA loops attached to the nuclear matrix revealed that in xeroderma pigmentosum cells of complementation group C repair patches are preferentially situated near the attachment sites of DNA loops at the nuclear matrix. In normal human fibroblasts the authors observed no enrichment of repair-labeled DNA at the nuclear matrix and repair patches appeared to be distributed randomly along the DNA loops. The enrichment of repair-labeled DNA at the nuclear matrix in xeroderma pigmentosum cells of complementation group C may indicate that the residual DNA-repair synthesis in these cells occurs preferentially in regions of the genome. (Auth.)

Swallowing function after laryngeal cleft repair: more than just fixing the cleft.

Science.gov (United States)

Osborn, Alexander J; de Alarcon, Alessandro; Tabangin, Meredith E; Miller, Claire K; Cotton, Robin T; Rutter, Michael J

2014-08-01

To evaluate and describe the swallowing function in children after laryngeal cleft repair. Ten-year (2002-2012) retrospective chart review. Academic tertiary care pediatric otolaryngology practice. Records of 60 children who had surgical repair of laryngeal cleft (ages 2 weeks-14 years) and postoperative functional endoscopic evaluation of swallowing or videofluoroscopic swallow studies were examined retrospectively. Twenty-nine children had one postoperative swallow evaluation, 19 children had two, 4 children had three, 5 children had four, and 3 children had five. Median time to the first evaluation was 10.8 weeks (interquartile range [IQR]: 36.5, 231). On the final swallow evaluation, 34 (57%) children demonstrated normal swallowing parameters, 12 (20%) children showed penetration, and 14 (23%) children showed aspiration. Forty-three (72%) children were able to take everything by mouth normally or with minor behavioral modifications, 11 (18%) children required thickened fluids, and six (10%) children were kept nil per os (NPO). Mean improvement on the penetration-aspiration (pen-asp) scale was 2.13. On multivariable analysis, neurodevelopmental issues and gastronomy tube use were associated with the need for NPO status. Despite a high rate of surgical success, a substantial minority of children have persistent swallowing dysfunction after laryngeal cleft repair. Swallowing dysfunction after repair is multifactorial and arises from concomitant neurologic, anatomic, or other comorbidities that contribute to oropharyngeal and pharyngeal dysphagia. Based on our results, we recommend a testing schedule for postoperative swallowing evaluations after cleft repair. © 2014 The American Laryngological, Rhinological and Otological Society, Inc.

Atrioventricular valve repair in patients with functional single-ventricle physiology: impact of ventricular and valve function and morphology on survival and reintervention.

Science.gov (United States)

Honjo, Osami; Atlin, Cori R; Mertens, Luc; Al-Radi, Osman O; Redington, Andrew N; Caldarone, Christopher A; Van Arsdell, Glen S

2011-08-01

This study was to determine whether atrioventricular valve repair modifies natural history of single-ventricle patients with atrioventricular valve insufficiency and to identify factors predicting survival and reintervention. Fifty-seven (13.5%) of 422 single-ventricle patients underwent atrioventricular valve repair. Valve morphology, regurgitation mechanism, and ventricular morphology and function were analyzed for effect on survival, transplant, and reintervention with multivariate logistic and Cox regression models. Comparative analysis used case-matched controls. Atrioventricular valve was tricuspid in 67% and common in 28%. Ventricular morphology was right in 83%. Regurgitation mechanisms were prolapse (n = 24, 46%), dysplasia (n = 18, 35%), annular dilatation (n = 8, 15%), and restriction or cleft (n = 2, 4%). Postrepair insufficiency was none or trivial in 14 (26%), mild in 33 (61%), and moderate in 7 (13%). Survival in repair group was lower than in matched controls (78.9% vs 92.7% at 1 year, 68.7% vs 90.6% at 3 years, P = .015). Patients with successful repair and normal ventricular function had equivalent survival to matched controls (P = .36). Independent predictors for death or transplant included increased indexed annular size (P = .05), increased cardiopulmonary bypass time (P = .04), and decreased postrepair ventricular function (P = .01). Ventricular dilation was a time-related factor for all events, including failed repair. Survival was lower in single-ventricle patients operated on for atrioventricular valve insufficiency than in case-matched controls. Patients with little postoperative residual regurgitation and preserved ventricular function had equivalent survival to controls. Lower grade ventricular function and ventricular dilation correlated with death and repair failure, suggesting that timing of intervention may affect outcome. Copyright © 2011 The American Association for Thoracic Surgery. Published by Mosby, Inc. All

Typical xeroderma pigmentosum complementation group A fibroblasts have detectable ultraviolet light-induced unscheduled DNA synthesis

International Nuclear Information System (INIS)

Petinga, R.A.; Andrews, A.D.; Robbins, J.H.; Tarone, R.E.

1977-01-01

Ultraviolet-induced nuclear uptake of tritiated thymidine [ 3 H]dThd demonstrable by autoradiography in non-synthesis phases of the cell cycle is known as unscheduled DNA synthesis and reflects repair replication of ultraviolet-damaged DNA. We have reported that the rate of any such unscheduled DNA synthesis in typical group A xeroderma pigmentosum fibroblasts, if present, is less than 2% of the normal rate. We have now performed experiments to determine whether these fibroblasts have any unscheduled DNA synthesis. Fibroblast coverslip cultures of four xeroderma pigmentosum group A strains were prepared. Irradiated (254 nm ultraviolet light) and unirradiated cultures from each strain were incubated with [ 3 H]dThd at 37degC, and autoradiograms were prepared using NTB-3 emulsion. A nuclear grain count was made of 100 consecutive nuclei of non-S-phase irradiated and unirradiated cells. A slide background grain count was simultaneously made from an acellular area adjacent to each cell analyzed. When a strain's irradiated and unirradiated autoradiograms having similar slide background grain count averages were compared, the nuclear grain count average of the irradiated cells was always higher than that of the unirradiated cells. This ultraviolet-induced increase in the mean nuclear grain count ranged from 0.4 to 1.3% of that given by normal non-xeroderma pigmentosum fibroblasts and was not reduced by 10 -2 M hydroxyurea. Planimetric studies showed that the ultraviolet-induced increase in nuclear grain count is not due to an increased nuclear area in irradiated cells. We conclude that these typical group A xeroderma pigmentosum strains perform very low, but detectable, ultraviolet-induced unscheduled DNA synthesis which probably reflects repair replication. We cannot, however, determine if there are significantly different rates of ultraviolet-induced unscheduled DNA synthesis among these ultraviolet strains

Noncanonical WNT-5A signaling impairs endogenous lung repair in COPD

Science.gov (United States)

Baarsma, Hoeke A.; John-Schuster, Gerrit; Heinzelmann, Katharina; Dagouassat, Maylis; Boczkowski, Jorge; Brusselle, Guy G.; Smits, Ron; Yildirim, Ali Ö.

2017-01-01

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. One main pathological feature of COPD is the loss of functional alveolar tissue without adequate repair (emphysema), yet the underlying mechanisms are poorly defined. Reduced WNT–β-catenin signaling is linked to impaired lung repair in COPD; however, the factors responsible for attenuating this pathway remain to be elucidated. Here, we identify a canonical to noncanonical WNT signaling shift contributing to COPD pathogenesis. We demonstrate enhanced expression of noncanonical WNT-5A in two experimental models of COPD and increased posttranslationally modified WNT-5A in human COPD tissue specimens. WNT-5A was increased in primary lung fibroblasts from COPD patients and induced by COPD-related stimuli, such as TGF-β, cigarette smoke (CS), and cellular senescence. Functionally, mature WNT-5A attenuated canonical WNT-driven alveolar epithelial cell wound healing and transdifferentiation in vitro. Lung-specific WNT-5A overexpression exacerbated airspace enlargement in elastase-induced emphysema in vivo. Accordingly, inhibition of WNT-5A in vivo attenuated lung tissue destruction, improved lung function, and restored expression of β-catenin–driven target genes and alveolar epithelial cell markers in the elastase, as well as in CS-induced models of COPD. We thus identify a novel essential mechanism involved in impaired mesenchymal–epithelial cross talk in COPD pathogenesis, which is amenable to therapy. PMID:27979969

Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

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Kyle J Hewitt

2011-02-01

Full Text Available Human induced pluripotent stem (hiPS cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK and their hES-derived counterparts (EDK showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM-production (COL1A1 by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ, revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

Does double-row rotator cuff repair improve functional outcome of patients compared with single-row technique? A systematic review.

Science.gov (United States)

DeHaan, Alexander M; Axelrad, Thomas W; Kaye, Elizabeth; Silvestri, Lorenzo; Puskas, Brian; Foster, Timothy E

2012-05-01

The advantage of single-row versus double-row arthroscopic rotator cuff repair techniques has been a controversial issue in sports medicine and shoulder surgery. There is biomechanical evidence that double-row techniques are superior to single-row techniques; however, there is no clinical evidence that the double-row technique provides an improved functional outcome. When compared with single-row rotator cuff repair, double-row fixation, although biomechanically superior, has no clinical benefit with respect to retear rate or improved functional outcome. Systematic review. The authors reviewed prospective studies of level I or II clinical evidence that compared the efficacy of single- and double-row rotator cuff repairs. Functional outcome scores included the American Shoulder and Elbow Surgeons (ASES) shoulder scale, the Constant shoulder score, and the University of California, Los Angeles (UCLA) shoulder rating scale. Radiographic failures and complications were also analyzed. A test of heterogeneity for patient demographics was also performed to determine if there were differences in the patient profiles across the included studies. Seven studies fulfilled our inclusion criteria. The test of heterogeneity across these studies showed no differences. The functional ASES, Constant, and UCLA outcome scores revealed no difference between single- and double-row rotator cuff repairs. The total retear rate, which included both complete and partial retears, was 43.1% for the single-row repair and 27.2% for the double-row repair (P = .057), representing a trend toward higher failures in the single-row group. Through a comprehensive literature search and meta-analysis of current arthroscopic rotator cuff repairs, we found that the single-row repairs did not differ from the double-row repairs in functional outcome scores. The double-row repairs revealed a trend toward a lower radiographic proven retear rate, although the data did not reach statistical significance. There

Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts.

Directory of Open Access Journals (Sweden)

Jon M Carthy

Full Text Available Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts.The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2.Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

Directional cell migration and chemotaxis in wound healing response to PDGF-AA are coordinated by the primary cilium in fibroblasts

DEFF Research Database (Denmark)

Schneider, Linda; Cammer, Michael; Lehman, Jonathan

2010-01-01

Cell motility and migration play pivotal roles in numerous physiological and pathophysiological processes including development and tissue repair. Cell migration is regulated through external stimuli such as platelet-derived growth factor-AA (PDGF-AA), a key regulator in directional cell migration....... Here we used micropipette analysis to show that a normal chemosensory response to PDGF-AA in fibroblasts requires the primary cilium. In vitro and in vivo wound healing assays revealed that in ORPK mouse (IFT88(Tg737Rpw)) fibroblasts, where ciliary assembly is defective, chemotaxis towards PDGF-AA...

Repair of damaged DNA in vivo: Final technical report

International Nuclear Information System (INIS)

Hanawalt, P.C.

1987-09-01

This contract was initiated in 1962 with the US Atomic Energy Commission to carry out basic research on the effects of radiation on the process of DNA replication in bacteria. Within the first contract year we discovered repair replication at the same time that Setlow and Carrier discovered pyrimidine dimer excision. These discoveries led to the elucidation of the process of excision-repair, one of the most important mechanisms by which living systems, including humans, respond to structural damage in their genetic material. We improved methodology for distinguishing repair replication from semiconservative replication and instructed others in these techniques. Painter then was the first to demonstrate repair replication in ultraviolet irradiated human cells. He, in turn, instructed James Cleaver who discovered that skin fibroblasts from patients with xeroderma pigmentosum were defective in excision-repair. People with this genetic defect are extremely sensitive to sunlight and they develop carcinomas and melanomas of the skin with high frequency. The existence of this hereditary disease attests to the importance of DNA repair in man. We certainly could not survive in the normal ultraviolet flux from the sun if our DNA were not continuously monitored for damage and repaired. Other hereditary diseases such as ataxia telangiectasia, Cockayne's syndrome, Blooms syndrome and Fanconi's anemia also involve deficiencies in DNA damage processing. The field of DNA repair has developed rapidly as we have learned that most environmental chemical carcinogens as well as radiation produce repairable damage in DNA. 251 refs

The Fanconi anemia group A protein modulates homologous repair of DNA double-strand breaks in mammalian cells.

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Yang, Yun-Gui; Herceg, Zdenko; Nakanishi, Koji; Demuth, Ilja; Piccoli, Colette; Michelon, Jocelyne; Hildebrand, Gabriele; Jasin, Maria; Digweed, Martin; Wang, Zhao-Qi

2005-10-01

Fanconi anemia (FA) cells exhibit hypersensitivity to DNA interstrand cross-links (ICLs) and high levels of chromosome instability. FA gene products have been shown to functionally or physically interact with BRCA1, RAD51 and the MRE11/RAD50/NBS1 complex, suggesting that the FA complex may be involved in the repair of DNA double-strand breaks (DSBs). Here, we have investigated specifically the function of the FA group A protein (FANCA) in the repair of DSBs in mammalian cells. We show that the targeted deletion of Fanca exons 37-39 generates a null for Fanca in mice and abolishes ubiquitination of Fancd2, the downstream effector of the FA complex. Cells lacking Fanca exhibit increased chromosomal aberrations and attenuated accumulation of Brca1 and Rad51 foci in response to DNA damage. The absence of Fanca greatly reduces gene-targeting efficiency in mouse embryonic stem (ES) cells and compromises the survival of fibroblast cells in response to ICL agent treatment. Fanca-null cells exhibit compromised homology-directed repair (HDR) of DSBs, particularly affecting the single-strand annealing pathway. These data identify the Fanca protein as an integral component in the early step of HDR of DSBs and thereby minimizing the genomic instability.

Differences in the levels of UV repair and in clinical symptoms in two sibs affected by xeroderma pigmentosum

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Stefanini, M; Nuzzo, F [Consiglio Nazionale delle Ricerche, Pavia (Italy). Lab. di Genetica Biochimica ed Evoluzionistica; Keijzer, W [Erasmus Universiteit, Rotterdam (Netherlands). Dept. of Cell Biology and Genetics; Dalpra, L [Milan Univ. (Italy). Istituto di Biologia; Elli, R; Nicoletti, B [Rome Univ. (Italy). Ist. di Biologia Generale 1; Nazzaro Porro, M [Ospedale Dermatologico S. Gallicano, Rome (Italy)

1980-01-01

UV-repair activity was studied in two sibs affected by XP showing different clinical symptoms. Complementation studies indicated that both patients fit into complementation group A. The levels of UV-induced /sup 3/H-thymidine incorporation, in fibroblasts and in lymphocytes, are different in the two patients: residual level of repair DNA synthesis in the sister is higher than in the brother. In one of the cell samples analyzed UDS analysis showed that in the sister a low proportion of cells with normal repair synthesis is present.

Fibrocytes and the tissue niche in lung repair

Directory of Open Access Journals (Sweden)

Bjermer Leif

2011-06-01

Full Text Available Abstract Human fibrocytes are bone marrow-derived mesenchymal progenitor cells that express a variety of markers related to leukocytes, hematopoietic stem cells and a diverse set of fibroblast phenotypes. Fibrocytes can be recruited from the circulation to the tissue where they further can differentiate and proliferate into various mesenchymal cell types depending on the tissue niche. This local tissue niche is important because it modulates the fibrocytes and coordinates their role in tissue behaviour and repair. However, plasticity of a niche may be co-opted in chronic airway diseases such as asthma, idiopathic pulmonary fibrosis and obliterative bronchiolitis. This review will therefore focus on a possible role of fibrocytes in pathological tissue repair processes in those diseases.

Fibroblast Cluster Formation on 3D Collagen Matrices Requires Cell Contraction-Dependent Fibronectin Matrix Organization

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da Rocha-Azevedo, Bruno; Ho, Chin-Han; Grinnell, Frederick

2012-01-01

Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy. PMID:23117111

Fibroblast cluster formation on 3D collagen matrices requires cell contraction dependent fibronectin matrix organization.

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da Rocha-Azevedo, Bruno; Ho, Chin-Han; Grinnell, Frederick

2013-02-15

Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin (FN) fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy. Copyright © 2012 Elsevier Inc. All rights reserved.

Connective tissue fibroblasts and Tcf4 regulate myogenesis

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Mathew, Sam J.; Hansen, Jody M.; Merrell, Allyson J.; Murphy, Malea M.; Lawson, Jennifer A.; Hutcheson, David A.; Hansen, Mark S.; Angus-Hill, Melinda; Kardon, Gabrielle

2011-01-01

Muscle and its connective tissue are intimately linked in the embryo and in the adult, suggesting that interactions between these tissues are crucial for their development. However, the study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. Here, we show that the transcription factor Tcf4 (transcription factor 7-like 2; Tcf7l2) is strongly expressed in connective tissue fibroblasts and that Tcf4GFPCre mice allow genetic manipulation of these fibroblasts. Using this new reagent, we find that connective tissue fibroblasts critically regulate two aspects of myogenesis: muscle fiber type development and maturation. Fibroblasts promote (via Tcf4-dependent signals) slow myogenesis by stimulating the expression of slow myosin heavy chain. Also, fibroblasts promote the switch from fetal to adult muscle by repressing (via Tcf4-dependent signals) the expression of developmental embryonic myosin and promoting (via a Tcf4-independent mechanism) the formation of large multinucleate myofibers. In addition, our analysis of Tcf4 function unexpectedly reveals a novel mechanism of intrinsic regulation of muscle fiber type development. Unlike other intrinsic regulators of fiber type, low levels of Tcf4 in myogenic cells promote both slow and fast myogenesis, thereby promoting overall maturation of muscle fiber type. Thus, we have identified novel extrinsic and intrinsic mechanisms regulating myogenesis. Most significantly, our data demonstrate for the first time that connective tissue is important not only for adult muscle structure and function, but is a vital component of the niche within which muscle progenitors reside and is a critical regulator of myogenesis. PMID:21177349

Repair of DNA in replicated and unreplicated portions of the human genome

International Nuclear Information System (INIS)

Waters, R.

1979-01-01

Portions of the human genome that have replicated after ultraviolet light irradiation and those that remain unreplicated have both been examined for the distribution of pyrimidine dimers and the extent of repair replication following their removal. The data indicate that the number of unrepaired dimers and the extent of repair replication seen after their excision are equal in the replicated and unreplicated DNA. Furthermore, the daughter strand of replicated DNA is larger than the average interdimer distance found in the parental strand. Hence, DNA replication in normal human fibroblasts is clearly capable of getting past pyrimidine dimers, and a preferential repair of such lesions in DNA that is about to be or has been replicated does not operate to any visible extent in these cells. (author)

[Clinical effect of functional repair of bilateral cleft lip in 66 patients].

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Li, Ming; Jiang, Hong-bing; Yuan, Hua; DU, Yi-fei; Wu, Yu-nong; Wan, Lin-zhong

2014-10-01

To explore the clinical effect of functional repair for bilateral cleft lip using modified Mulliken method. Sixty-six patients with bilateral cleft lip were selected and assigned to receive modified Mulliken method. During the operation, the prolabium was kept as narrow "tie" shape, the orbicularis oris was anatomically repositioned, and the orbicularis oris ring was re-built. Vermilion tubercle was reconstructed with the lateral red vermillion. The nasal deformity was preliminarily repaired. and the nasal columella was elongated at the same time. All the patients were followed-up for 0.5-2 years, there was no "trisection upper lip". The symmetry and natural shape of Cupid's bow were obtained in more than 80% patients. The width of philtrum was similar to normal children. Full vermilion of the lips, moderate-size vermilion tubercles and good dynamic and static shape were obtained without whistle deformities. Normal width of nasal base and nostril symmetry were gained. The columella was elongated. Satisfactory contour of the nasal tip was achieved. Modified Mulliken method could functionally repair bilateral cleft lip and effectively correct nasolabial deformities. It is worthy of wide clinical application.

Radiation studies on sensitivity and repair of human mammary epithelial cells

International Nuclear Information System (INIS)

Tracy Chuihsu Yang; Stampfer, M.R.; Tobias, C.A.

1989-01-01

The authors present results indicating that normal breast epithelial cells and fibroblasts respond to X-rays similarly, lacking significant repair of sublethal damage when 2 Gy was used as the conditioning dose. Epithelial cells from tumor and from parenchymal tissue peripheral to the tumor, however, did show an efficient repair of sublethal damage. The reasons for this difference is unknown. Heavy-ion studies suggest energetic carbon and neon particles can be more effective in killing normal and tumour cells. The RBE for normal cells, however, appeared to be slightly less than for tumor cells. The repair of sublethal damage in tumor cells was less for neon particles than for X-rays. These findings suggest that heavy ions might be more advantageous than X-rays in treating breast tumors. (author)

Simvastatin induces apoptosis by a Rho-dependent mechanism in cultured cardiac fibroblasts and myofibroblasts

International Nuclear Information System (INIS)

Copaja, Miguel; Venegas, Daniel; Aranguiz, Pablo; Canales, Jimena; Vivar, Raul; Catalan, Mabel; Olmedo, Ivonne; Rodriguez, Andrea E.; Chiong, Mario; Leyton, Lisette; Lavandero, Sergio; Diaz-Araya, Guillermo

2011-01-01

Several clinical trials have shown the beneficial effects of statins in the prevention of coronary heart disease. Additionally, statins promote apoptosis in vascular smooth muscle cells, in renal tubular epithelial cells and also in a variety of cell lines; yet, the effects of statins on cardiac fibroblast and myofibroblast, primarily responsible for cardiac tissue healing are almost unknown. Here, we investigated the effects of simvastatin on cardiac fibroblast and myofibroblast viability and studied the molecular cell death mechanism triggered by simvastatin in both cell types. Methods: Rat neonatal cardiac fibroblasts and myofibroblasts were treated with simvastatin (0.1-10 μM) up to 72 h. Cell viability and apoptosis were evaluated by trypan blue exclusion method and by flow cytometry, respectively. Caspase-3 activation and Rho protein levels and activity were also determined by Western blot and pull-down assay, respectively. Results: Simvastatin induces caspase-dependent apoptosis of cardiac fibroblasts and myofibroblasts in a concentration- and time-dependent manner, with greater effects on fibroblasts than myofibroblasts. These effects were prevented by mevalonate, farnesylpyrophosphate and geranylgeranylpyrophosphate, but not squalene. These last results suggest that apoptosis was dependent on small GTPases of the Rho family rather than Ras. Conclusion: Simvastatin triggered apoptosis of cardiac fibroblasts and myofibroblasts by a mechanism independent of cholesterol synthesis, but dependent of isoprenilation of Rho protein. Additionally, cardiac fibroblasts were more susceptible to simvastatin-induced apoptosis than cardiac myofibroblasts. Thus simvastatin could avoid adverse cardiac remodeling leading to a less fibrotic repair of the damaged tissues. - Research Highlights: → Simvastatin decreases CF and CMF viability independent of cholesterol synthesis. → Simvastatin induces CF and CMF apoptosis in a caspase-dependent manner being CMF more resistant

Neurotrophic regulation of fibroblast dedifferentiation during limb skeletal regeneration in the axolotl (Ambystoma mexicanum).

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Satoh, Akira; Cummings, Gillian M C; Bryant, Susan V; Gardiner, David M

2010-01-15

The ability of animals to repair tissue damage is widespread and impressive. Among tissues, the repair and remodeling of bone occurs during growth and in response to injury; however, loss of bone above a threshold amount is not regenerated, resulting in a "critical-size defect" (CSD). The development of therapies to replace or regenerate a CSD is a major focus of research in regenerative medicine and tissue engineering. Adult urodeles (salamanders) are unique in their ability to regenerate complex tissues perfectly, yet like mammals do not regenerate a CSD. We report on an experimental model for the regeneration of a CSD in the axolotl (the Excisional Regeneration Model) that allows for the identification of signals to induce fibroblast dedifferentiation and skeletal regeneration. This regenerative response is mediated in part by BMP signaling, as is the case in mammals; however, a complete regenerative response requires the induction of a population of undifferentiated, regeneration-competent cells. These cells can be induced by signaling from limb amputation to generate blastema cells that can be grafted to the wound, as well as by signaling from a nerve and a wound epithelium to induce blastema cells from fibroblasts within the wound environment. Copyright 2009 Elsevier Inc. All rights reserved.

Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

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Zhu, Zhong Xin [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Sun, Cong Cong [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Zheng, Jia Yong [Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Zhou, Xuan [Ningbo First Hospital, Ningbo, Zhejiang (China); Cong, Wei Tao [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Li, Xiao Kun, E-mail: proflxk@163.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Jin, Li Tai, E-mail: jin_litai@126.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China)

2017-06-15

Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

International Nuclear Information System (INIS)

Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

2017-01-01

Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

Renal Tubule Repair: Is Wnt/β-Catenin a Friend or Foe?

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Gewin, Leslie S

2018-01-24

Wnt/β-catenin signaling is extremely important for proper kidney development. This pathway is also upregulated in injured renal tubular epithelia, both in acute kidney injury and chronic kidney disease. The renal tubular epithelium is an important target of kidney injury, and its response (repair versus persistent injury) is critical for determining whether tubulointerstitial fibrosis, the hallmark of chronic kidney disease, develops. This review discusses how Wnt/β-catenin signaling in the injured tubular epithelia promotes either repair or fibrosis after kidney injury. There is data suggesting that epithelial Wnt/β-catenin signaling is beneficial in acute kidney injury and important in tubular progenitors responsible for epithelial repair. The role of Wnt/β-catenin signaling in chronically injured epithelia is less clear. There is convincing data that Wnt/β-catenin signaling in interstitial fibroblasts and pericytes contributes to the extracellular matrix accumulation that defines fibrosis. However, some recent studies question whether Wnt/β-catenin signaling in chronically injured epithelia actually promotes fibrosis or repair.

Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.

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Morales, Julio C; Richard, Patricia; Rommel, Amy; Fattah, Farjana J; Motea, Edward A; Patidar, Praveen L; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N; Chiang, Cheng-Ming; Manley, James L; Boothman, David A

2014-04-01

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.

Platelets stimulate fibroblast-mediated contraction of collagen gels

Directory of Open Access Journals (Sweden)

Lundahl Joachim

2003-10-01

Full Text Available Abstract Background Platelets are thought to play a role in a variety of inflammatory conditions in the lung, some of which may lead to fibrosis. In the current study we tested the hypothesis that whole platelets and platelet lysate can mediate remodelling of extracellular matrix in vitro by affecting fibroblast-mediated contraction of a collagen gel. We also sought to determine to what extent platelet-derived growth factor (PDGF and transforming growth factor-β (TGF-β contribute to this effect. Methods Washed platelets, isolated from healthy blood donors, and platelet lysate (freezing and thawing, were cast together with human lung fibroblasts in three-dimensional collagen gels. The gels were then released and cultured for four days. PDGF and TGF-β1 concentrations were measured in culture supernatants by ELISA. Results Both platelets and platelet lysate augmented fibroblast-mediated gel contraction in a time and concentration dependent manner (19.9% ± 0.1 (mean ± SEM of initial area vs. 48.0% ± 0.4 at 48 hours; P 1 and PDGF-AA/AB were released in co-culture. PDGF-AA/AB had a maximum release at 24 hours whereas TGF-β1 release increased with longer culture periods. Neutralising antibodies to these mediators partially inhibited platelet-induced gel contraction. Conclusion We conclude that platelets may promote remodelling of extracellular matrix in vitro and that PDGF and TGF-β partially mediate this effect, also indicating a role for other mediators. The findings may be an important mechanism in regulating repair processes after injury.

Host-cell reactivation of UV-irradiated and chemically-treated herpes simplex virus-1 by xeroderma pigmentosum, xp heterozygotes and normal skin fibroblasts

International Nuclear Information System (INIS)

Selsky, C.A.

1978-01-01

The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylamino-fluorene-treated herpes simplex virus type 1 strain MP was studied in normal and xeroderma pigmentosum human skin fibroblasts. Virus treated with either agent demonstrated lower survival in XP cells from complementation groups A, B, C and D than in normal fibroblasts. The relative reactivation ability of XP cells from the different genetic complementation groups was found to be the same for both irradiated and chemically treated virus. In addition, the inactivation kinetics for virus treated with either agent in the XP variant were comparable to that seen in normal skin fibroblasts. The addition of 2 or 4 mmoles caffeine to the post-infection assay medium had no effect on the inactivation kinetics of virus treated by either agent in the XP variant or in XP cells from the different genetic complementation groups. Treatment of the virus with nitrogen mustard resulted in equivalent survival in normal and XP genetic complementation group D cells. No apparent defect was observed in the ability of XP heterozygous skin fibroblasts to repair virus damaged with up to 100 μg N-acetoxy-2-acetylaminofluorene per ml. These findings indicate that the repair of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus is accomplished by the same pathway or different pathways sharing a common intermediate step and that the excision defect of XP cells plays little if any role in the reactivation of nitrogen mustard treated virus. (Auth.)

Repair and replication of DNA in hereditary (bilateral) retinoblastoma cells after X-irradiation

International Nuclear Information System (INIS)

Cleaver, J.E.; Char, D.; Charles, W.C.; Rand, N.

1982-01-01

Fibroblasts from patients with hereditary retinoblastoma reportedly exhibit increased sensitivity to killing by X-rays. Although some human syndromes with similar or greater hypersensitivity to DNA-damaging agents (e.g., X-rays, ultraviolet light, and chemical carcinogens), such as xeroderma pigmentosum, are deficient in DNA repair, most do not have such clearly demonstrable defects in repair. Retinoblastoma cells appear to be normal in repairing single-strand breaks and performing repair replication after X-irradiation and also in synthesizing poly(adenosine diphosphoribose). Semiconservative DNA replication in these cells, however, is slightly more resistant than normal after X-irradiation, suggesting that continued replication of damaged parental DNA could contribute to the pathogenesis of the disease. This effect is small, however, and may be a consequence rather than a cause of the fundamental enzymatic abnormality in retinoblastoma that causes the tumorigenesis

Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

International Nuclear Information System (INIS)

Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A.

1989-01-01

A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3',4,4'-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4'-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4'-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers

Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

Energy Technology Data Exchange (ETDEWEB)

Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A. (National Center for Toxicological Research, Jefferson, AK (USA) Univ. of Arkansas for Medical Sciences, Little Rock (USA))

1989-01-01

A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3{prime},4,4{prime}-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4{prime}-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4{prime}-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers.

Defective repair of UV-damaged DNA in human tumor and SV40-transformed human cells but not in adenovirus-transformed human cells

International Nuclear Information System (INIS)

Rainbow, A.J.

1989-01-01

The DNA repair capacities of five human tumor cell lines, one SV40-transformed human cell line and one adenovirus-transformed human cell line were compared with that of normal human fibroblasts using a sensitive host cell reactivation (HCR) technique. Unirradiated and UV-irradiated suspensions of adenovirus type 2 (Ad 2) were assayed for their ability to form viral structural antigens (Vag) in the various cell types using immunofluorescent staining. The survival of Vag formation for UV-irradiated Ad 2 was significantly reduced in all the human tumor cell lines and the SV40-transformed human line compared to the normal human fibroblasts, but was apparently normal in the adenovirus-transformed human cells. D 0 values for the UV survival of Ad 2 Vag synthesis in the tumor and virally transformed lines expressed as a percentage of that obtained on normal fibroblast strains were used as a measure of DNA repair capacity. Percent HCR values ranged from 26 to 53% in the tumor cells. These results indicate a deficiency in the repair of UV-induced DNA damage associated with human tumorigenesis and the transformation of human cells by SV40 but not the transformation of human cells by adenovirus. (author)

Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

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Paola Brun

Full Text Available Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and

Extracellular Matrix Modulates Morphology, Growth, Oxidative Stress Response and Functionality of Human Skin Fibroblasts during Aging In Vitro

DEFF Research Database (Denmark)

Jørgensen, Peter; Rattan, Suresh

2014-01-01

recent observations indicate that replicative lifespan, senescence and functionality of cells in vitro can be significantly affected by the quality of the extra cellular matrix (ECM). Following up on those reports, here we show that using the ECM prepared from early passage young cells, partial...... rejuvenation of serially passaged human facial skin fibroblasts was possible in pre-senescent middle-aged cells, but not in fully senescent late passage cells. ECM from young cells improved the appearance, viability, stress tolerance and wound healing ability of skin fibroblasts. Furthermore, young ECM...... modulated the oxidative stress response transcription factor Nrf-2 and its downstream effector haem-oxygenase (HO-1), possibly through the amelioration of the environmental stress induced by the plastic surface of the culturing flasks. Therefore, it is important to consider the role of ECM in modulating...

Silymarin protects epidermal keratinocytes from ultraviolet radiation-induced apoptosis and DNA damage by nucleotide excision repair mechanism.

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Santosh K Katiyar

Full Text Available Solar ultraviolet (UV radiation is a well recognized epidemiologic risk factor for melanoma and non-melanoma skin cancers. This observation has been linked to the accumulation of UVB radiation-induced DNA lesions in cells, and that finally lead to the development of skin cancers. Earlier, we have shown that topical treatment of skin with silymarin, a plant flavanoid from milk thistle (Silybum marianum, inhibits photocarcinogenesis in mice; however it is less understood whether chemopreventive effect of silymarin is mediated through the repair of DNA lesions in skin cells and that protect the cells from apoptosis. Here, we show that treatment of normal human epidermal keratinocytes (NHEK with silymarin blocks UVB-induced apoptosis of NHEK in vitro. Silymarin reduces the amount of UVB radiation-induced DNA damage as demonstrated by reduced amounts of cyclobutane pyrimidine dimers (CPDs and as measured by comet assay, and that ultimately may lead to reduced apoptosis of NHEK. The reduction of UV radiation-induced DNA damage by silymarin appears to be related with induction of nucleotide excision repair (NER genes, because UV radiation-induced apoptosis was not blocked by silymarin in NER-deficient human fibroblasts. Cytostaining and dot-blot analysis revealed that silymarin repaired UV-induced CPDs in NER-proficient fibroblasts from a healthy individual but did not repair UV-induced CPD-positive cells in NER-deficient fibroblasts from patients suffering from xeroderma pigmentosum complementation-A disease. Similarly, immunohistochemical analysis revealed that silymarin did not reduce the number of UVB-induced sunburn/apoptotic cells in the skin of NER-deficient mice, but reduced the number of sunburn cells in their wild-type counterparts. Together, these results suggest that silymarin exert the capacity to reduce UV radiation-induced DNA damage and, thus, prevent the harmful effects of UV radiation on the genomic stability of epidermal cells.

Repair of human DNA in molecules that replicate or remain unreplicated following ultraviolet irradiation

International Nuclear Information System (INIS)

Waters, R.

1980-01-01

The extent of DNA replication, the incidence of uv induced pyrimidine dimers and the repair replication observed after their excision was monitored in human fibroblasts uv irradiated with single or split uv doses. The excision repair processes were measured in molecules that remained unreplicated or in those that replicated after the latter uv irradiation. Less DNA replication was observed after a split as opposed to single uv irradiation. Furthermore, a split dose did not modify the excision parameters measured after a single irradiation, regardless of whether the DNA had replicated or not

Stromal fibroblasts derived from mammary gland of bovine with mastitis display inflammation-specific changes.

Science.gov (United States)

Chen, Qing; He, Guiliang; Zhang, Wenyao; Xu, Tong; Qi, Hongliang; Li, Jing; Zhang, Yong; Gao, Ming-Qing

2016-06-07

Fibroblasts are predominant components of mammary stromal cells and play crucial roles in the development and involution of bovine mammary gland; however, whether these cells contribute to mastitis has not been demonstrated. Thus, we have undertaken biological and molecular characterization of inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis and normal fibroblasts (NFs) from slaughtered dairy cows because of fractured legs during lactation. The functional contributions of INFs to normal epithelial cells were also investigated by using an in vitro co-culture model. We present evidence that the INFs were activated fibroblasts and showed inflammation-related features. Moreover, INFs significantly inhibited the proliferation and β-casein secretion of epithelial cells, as well as upregulated the expression of tumor necrosis factor-α and interleukin-8 in epithelial cells. These findings indicate that functional alterations can occur in stromal fibroblasts within the bovine mammary gland during mastitis, demonstrating the importance of stromal fibroblasts in bovine mastitis and its treatment.

PAI1 mediates fibroblast-mast cell interactions in skin fibrosis.

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Pincha, Neha; Hajam, Edries Yousaf; Badarinath, Krithika; Batta, Surya Prakash Rao; Masudi, Tafheem; Dey, Rakesh; Andreasen, Peter; Kawakami, Toshiaki; Samuel, Rekha; George, Renu; Danda, Debashish; Jacob, Paul Mazhuvanchary; Jamora, Colin

2018-05-01

Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast-mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.

Analysis of Chromosomal Aberrations after Low and High Dose Rate Gamma Irradiation in ATM or NBS Suppressed Human Fibroblast Cells

Science.gov (United States)

Hada, M.; Huff, J. L.; Patel, Z.; Pluth, J. M.; George, K. A.; Cucinotta, F. A.

2009-01-01

A detailed understanding of the biological effects of heavy nuclei is needed for space radiation protection and for cancer therapy. High-LET radiation produces more complex DNA lesions that may be non-repairable or that may require additional processing steps compared to endogenous DSBs, increasing the possibility of misrepair. Interplay between radiation sensitivity, dose, and radiation quality has not been studied extensively. Previously we studied chromosome aberrations induced by low- and high- LET radiation in several cell lines deficient in ATM (ataxia telangactasia mutated; product of the gene that is mutated in ataxia telangiectasia patients) or NBS (nibrin; product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase (DNA-PK) activity. We found that the yields of both simple and complex chromosomal aberrations were significantly increased in the DSB repair defective cells compared to normal cells. The increased aberrations observed for the ATM and NBS defective lines was due to a significantly larger quadratic dose-response term compared to normal fibroblasts for both simple and complex aberrations, while the linear dose-response term was significantly higher in NBS cells only for simple exchanges. These results point to the importance of the functions of ATM and NBS in chromatin modifications that function to facilitate correct DSB repair and minimize aberration formation. To further understand the sensitivity differences that were observed in ATM and NBS deficient cells, in this study, chromosomal aberration analysis was performed in normal lung fibroblast cells treated with KU-55933, a specific ATM kinase inhibitor, or Mirin, an MRN complex inhibitor involved in activation of ATM. We are also testing siRNA knockdown of these proteins. Normal and ATM or NBS suppressed cells were irradiated with gamma-rays and chromosomes were collected with a premature chromosome

The effect of caffeine on X-ray-induced mitotic delay in normal human and ataxia-telangiectasia fibroblasts

International Nuclear Information System (INIS)

Zampetti-Bosseler, F.; Scott, D.

1985-01-01

The authors previously showed that radiation-sensitive fibroblasts from ataxia-telangiectasia (A-T) patients sustain less G 2 delay after X-irradiation than normal fibroblasts. Caffeine is known to reduce the amount of X-ray-induced delay in various mammalian cell types. It is proposed that A-T cells have an altered chromatin structure, similar to that of caffeine-treated normal cells and that this results in a failure of A-T cells to delay their progression through the cell cycle to allow time for DNA repair. The authors now show that caffeine treatment after X-irradiation reduces G 2 delay in both A-T and normal cells. The authors confirm the results previously obtained on lymphocytes that caffeine potentiates the chromosome-damaging effects of X-rays in both A-T and normal fibroblasts. These and other data suggest that the radiation responses of A-T cells and of caffeine-treated normal cells are caused by different mechanisms. (Auth.)

Lack of co-ordinate expression of the alpha1(I) and alpha1(III) procollagen genes in fibroblast clonal cultures.

Science.gov (United States)

Yamaguchi, Y; Crane, S; Zhou, L; Ochoa, S M; Falanga, V

2000-12-01

Several extracellular matrix genes, most notably alpha1(I) and alpha1(III) procollagen, are reported to be co-ordinately expressed in cultures of dermal fibroblasts. However, it remains unclear whether the expression of these genes is truly co-ordinate or whether it may be the result of averaging the phenotypic expression of different fibroblast subpopulations present within each culture. Objectives To determine by Northern analysis the correlation between alpha1(I) and alpha1(III) procollagen mRNA levels in clonal populations of human dermal fibroblasts. As previously described, clonal cultures were derived from parent strains of human dermal fibroblasts by a microscopically controlled dilution technique and by stimulation of single cells with low oxygen tension in the early phases of clonal growth. In agreement with previous reports, we found that baseline steady-state levels of alpha1(I) procollagen mRNA were co-ordinately regulated with the alpha1(III) procollagen mRNA in 26 parent strains (r = 0. 9003; P ordinate regulation observed in non-clonal cultures, suggesting that these two genes operate under different sets of regulatory controls. This clonal heterogeneity may provide additional flexibility to the process of tissue repair and fibroblast clonal expansion.

Anti-fibrotic effects of theophylline on lung fibroblasts

International Nuclear Information System (INIS)

Yano, Yukihiro; Yoshida, Mitsuhiro; Hoshino, Shigenori; Inoue, Koji; Kida, Hiroshi; Yanagita, Masahiko; Takimoto, Takayuki; Hirata, Haruhiko; Kijima, Takashi; Kumagai, Toru; Osaki, Tadashi; Tachibana, Isao; Kawase, Ichiro

2006-01-01

Theophylline has been used in the management of bronchial asthma and chronic obstructive pulmonary disease for over 50 years. It has not only a bronchodilating effect, but also an anti-inflammatory one conducive to the inhibition of airway remodeling, including subepithelial fibrosis. To date however, whether theophylline has a direct inhibitory effect on airway fibrosis has not been established. To clarify this question, we examined whether theophylline affected the function of lung fibroblasts. Theophylline suppressed TGF-β-induced type I collagen (COL1) mRNA expression in lung fibroblasts and also inhibited fibroblast proliferation stimulated by FBS and TGF-β-induced α-SMA protein. A cAMP analog also inhibited TGF-β-induced COL1 mRNA expression in lung fibroblasts. A PKA inhibitor reduced the inhibitory effect of theophylline on TGF-β-induced COL1 mRNA expression. These results indicate that theophylline exerts anti-fibrotic effects, at least partly, through the cAMP-PKA pathway

Fibroblastic rheumatism

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Jyoti Ranjan Parida

2017-01-01

Full Text Available Fibroblastic rheumatism (FR is a rare dermoarthopathy reported from different parts of the world since 1980. Although the exact cause is unknown, few reports implicate infection may be a triggering event. Patients usually present with multiple skin nodules and polyarthropathy with progressive skin contractures. Laboratory parameters including acute phase reactants are usually normal. The confirmatory diagnosis is based on histopathologic study of skin nodules, which demonstrate fibroblastic proliferation, thickened collagen fibers, dermal fibrosis, and decreased number of elastic fibers. Immunoreactivity for b-catenin, smooth muscle actin, and the monoclonal antibody HHF35 show myofibroblastic differentiation. Treatments with oral prednisolone and other disease-modifying drugs such as methotrexate, infliximab, and interferon have been tried with variable success. In general, skin lesions respond more aptly than joint symptoms indicating that skin fibroblast is more amenable to treatment than synovial fibroblasts. Awareness regarding this orphan disease among clinicians and pathologists will help in more reporting of such cases and finding out optimal treatment regimen.

Pyrimidine dimer sites associated with the daughter DNA strands in uv-irradiated human fibroblasts

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Lehmann, A R; Kirk-Bell, S [Sussex Univ., Brighton (UK)

1978-03-01

Pyrimidine dimer sites associated with the newly-synthesized DNA were detected during post-replication repair of DNA in uv-irradiated human fibroblasts. These pyrimidine dimer sites were inferred from a decrease in the molecular weight of pulse-labelled DNA after treatment with an extract of Micrococcus luteus containing uv-specific endonuclease activity. In DNA synthesized immediately after irradiation, the frequency of these daughter strand dimer sites was 7 to 20% of that in the parental DNA. Such sites were found in fibroblasts from normal donors and from xeroderma pigmentosum patients (with defects in excision-repair or post-replication repair). They were excised from the DNA of normal cells. As the time between uv irradiation and pulse-labelling was increased, the frequency of dimer sites associated with the labelled DNA decreased. If the pulse-label was delivered 6 h after irradiation of normal cells or excision-defective xeroderma pigmentosum cells, no dimer sites were detected in the labelled DNA. It has usually been assumed that daughter-strand dimer sites were the result of recombinational exchanges. The assay procedure used in these experiments and in similar experiments of others did not distinguish between labelled DNA containing pyrimidine dimers within the labelled section, and labelled DNA which did not contain pyrimidine dimers but was attached to unlabelled DNA which did contain dimers. The latter structures would arise during normal replication immediately following uv irradiation of mammalian cells. Calculations are presented which suggest that a significant proportion and conceivably all of the dimer sites associated with the daughter strands may have arisen in this way, rather than from recombinational exchanges as has been generally assumed.

Pyrimidine dimer sites associated with the daughter DNA strands in UV-irradiated human fibroblasts

International Nuclear Information System (INIS)

Lehmann, A.R.; Kirk-Bell, S.

1978-01-01

Pyrimidine dimer sites associated with the newly-synthesized DNA were detected during post-replication repair of DNA in UV-irradiated human fibroblasts. These pyrimidine dimer sites were inferred from a decrease in the molecular weight of pulse-labelled DNA after treatment with an extract of Micrococcus luteus containing UV-specific endonuclease activity. In DNA synthesized immediately after irradiation the frequency of these daughter strand dimer sites was 7-20% of that in the parental DNA. Such sites were found in fibroblasts from normal donors and from xeroderma pigmentosum patients (with defects in excision-repair or post-replication repair). They were excised from the DNA of normal cells. As the time between UV-irradiation and pulse-labelling was increased, the frequency of dimer sites associated with the labelled DNA decreased. If the pulse-label was delivered 6 h after irradiation of normal cells or excision-defective xeroderma pigmentosum cells, no dimer sites were detected in the labelled DNA. It has usually been assumed that daughter-strand dimer sites were the result of recombinational exchanges. The assay procedure used in these experiments and in similar experiments of others did not distinguish between labelled DNA containing pyrimidine dimers within the labelled section, and labelled DNA which did not contain pyrimidine dimers but was attached to unlabelled DNA which did contain dimers. The latter structures would arise during normal replication immediately following UV-irradiation of mammalian cells. Calculations are presented which suggest that a significant proportion and conceivably all of the dimer sites associated with the daughter strands may have arisen in this way, rather than from recombinational exchanges as has been generally assumed. (author)

Repair of radiation-induced DNA damage in rat epidermis as a function of age

International Nuclear Information System (INIS)

Sargent, E.V.; Burns, F.J.

1985-01-01

The rate of repair of radiation-induced DNA damage in proliferating rat epidermal cells diminished progressively with increasing age of the animal. The dorsal skin was irradiated with 1200 rad of 0.8 MeV electrons at various ages, and the amount of DNA damage was determined as a function of time after irradiation by the method of alkaline unwinding followed by S 1 nuclease digestion. The amount of DNA damage immediately after irradiation was not age dependent, while the rate of damage removal from the DNA decreased with increasing age. By fitting an exponential function to the relative amount of undamaged DNA as a function of time after irradiation, DNA repair halftimes of 20, 27, 69, and 107 min were obtained for 28, 100-, 200-, and 400-day-old animals, respectively

Thy-1 attenuates TNF-alpha-activated gene expression in mouse embryonic fibroblasts via Src family kinase.

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Bin Shan

Full Text Available Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+ and negative (Thy-1- subsets of mouse embryonic fibroblasts (MEF. TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

Clinical symptoms and DNA repair characteristics of xeroderma pigmentosum patients from Germany

International Nuclear Information System (INIS)

Thielmann, H.W.; Popanda, O.; Edler, L.; Jung, E.G.

1991-01-01

Sixty-one xeroderma pigmentosum (XP) patients living in the Federal Republic of Germany were investigated. Clinical symptoms were correlated with DNA repair parameters measured in fibroblasts grown from skin biopsies. Classification according to the international complementation groups revealed that of the 61 patients 3 belonged to group A, 26 to group C, 16 to group D, 3 to group E, and 2 to group F; 11 were of the XP variant type. A striking clinical aspect was the frequency of histogenetically different skin tumors varying from one XP complementation group to the other: squamous and basal cell carcinomas predominated in XP group C; lentigo maligna melanomas were most frequent in group D; basal cell carcinomas occurred preferentially in group E and XP variants. Three DNA repair parameters were determined for 46 fibroblast strains: colony-forming ability (D0); DNA repair synthesis (G0); and DNA-incising capacity (E0). Dose-response experiments with up to 13 dose levels were performed throughout to achieve sufficient experimental accuracy. DNA-damaging treatments included UV light, the 'UV-like' carcinogen N-acetoxy-2-acetylaminofluorene, and the alkylating carcinogens methyl methanesulfonate and N-methyl-N-nitrosourea. Comparison of clinical signs and repair data was made on the basis of D0, G0, and E0 values of both individual cell strains and weighted means of XP complementation groups. Despite considerable clinical and biochemical heterogeneity within complementation groups distinctive features emerged. In general, D0, G0, and E0 values of all XP strains investigated, including XP variants, were found to be reduced upon treatment with UV light or N-acetoxy-2-acetylaminofluorene

An altered redox balance and increased genetic instability characterize primary fibroblasts derived from xeroderma pigmentosum group A patients

International Nuclear Information System (INIS)

Parlanti, Eleonora; Pietraforte, Donatella; Iorio, Egidio; Visentin, Sergio; De Nuccio, Chiara; Zijno, Andrea; D’Errico, Mariarosaria; Simonelli, Valeria; Sanchez, Massimo; Fattibene, Paola; Falchi, Mario; Dogliotti, Eugenia

2015-01-01

Highlights: • Increased levels and different types of intracellular radical species as well as an altered glutathione redox state characterize XP-A human cells when compared to normal. • A more glycolytic metabolism and higher ATP levels are associated with alteration of mitochondrial morphology and response to mitochondrial toxicants when XPA is defective. • XP-A human cells show increased spontaneous micronuclei frequency, a hallmark of cancer risk. - Abstract: Xeroderma pigmentosum (XP)-A patients are characterized by increased solar skin carcinogenesis and present also neurodegeneration. XPA deficiency is associated with defective nucleotide excision repair (NER) and increased basal levels of oxidatively induced DNA damage. In this study we search for the origin of increased levels of oxidatively generated DNA lesions in XP-A cell genome and then address the question of whether increased oxidative stress might drive genetic instability. We show that XP-A human primary fibroblasts present increased levels and different types of intracellular reactive oxygen species (ROS) as compared to normal fibroblasts, with O_2_−· and H_2O_2 being the major reactive species. Moreover, XP-A cells are characterized by decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios as compared to normal fibroblasts. The significant increase of ROS levels and the alteration of the glutathione redox state following silencing of XPA confirmed the causal relationship between a functional XPA and the control of redox balance. Proton nuclear magnetic resonance ("1H NMR) analysis of the metabolic profile revealed a more glycolytic metabolism and higher ATP levels in XP-A than in normal primary fibroblasts. This perturbation of bioenergetics is associated with different morphology and response of mitochondria to targeted toxicants. In line with cancer susceptibility, XP-A primary fibroblasts showed increased spontaneous micronuclei (MN) frequency, a hallmark of cancer

An altered redox balance and increased genetic instability characterize primary fibroblasts derived from xeroderma pigmentosum group A patients

Energy Technology Data Exchange (ETDEWEB)

Parlanti, Eleonora [Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Pietraforte, Donatella; Iorio, Egidio; Visentin, Sergio; De Nuccio, Chiara [Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Zijno, Andrea; D’Errico, Mariarosaria; Simonelli, Valeria [Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Sanchez, Massimo [Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Fattibene, Paola [Department of Technology and Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Falchi, Mario [National AIDS Center, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Dogliotti, Eugenia, E-mail: dogliotti@iss.it [Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy)

2015-12-15

Highlights: • Increased levels and different types of intracellular radical species as well as an altered glutathione redox state characterize XP-A human cells when compared to normal. • A more glycolytic metabolism and higher ATP levels are associated with alteration of mitochondrial morphology and response to mitochondrial toxicants when XPA is defective. • XP-A human cells show increased spontaneous micronuclei frequency, a hallmark of cancer risk. - Abstract: Xeroderma pigmentosum (XP)-A patients are characterized by increased solar skin carcinogenesis and present also neurodegeneration. XPA deficiency is associated with defective nucleotide excision repair (NER) and increased basal levels of oxidatively induced DNA damage. In this study we search for the origin of increased levels of oxidatively generated DNA lesions in XP-A cell genome and then address the question of whether increased oxidative stress might drive genetic instability. We show that XP-A human primary fibroblasts present increased levels and different types of intracellular reactive oxygen species (ROS) as compared to normal fibroblasts, with O{sub 2−}· and H{sub 2}O{sub 2} being the major reactive species. Moreover, XP-A cells are characterized by decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios as compared to normal fibroblasts. The significant increase of ROS levels and the alteration of the glutathione redox state following silencing of XPA confirmed the causal relationship between a functional XPA and the control of redox balance. Proton nuclear magnetic resonance ({sup 1}H NMR) analysis of the metabolic profile revealed a more glycolytic metabolism and higher ATP levels in XP-A than in normal primary fibroblasts. This perturbation of bioenergetics is associated with different morphology and response of mitochondria to targeted toxicants. In line with cancer susceptibility, XP-A primary fibroblasts showed increased spontaneous micronuclei (MN) frequency, a

NAXE Mutations Disrupt the Cellular NAD(P)HX Repair System and Cause a Lethal Neurometabolic Disorder of Early Childhood.

Science.gov (United States)

Kremer, Laura S; Danhauser, Katharina; Herebian, Diran; Petkovic Ramadža, Danijela; Piekutowska-Abramczuk, Dorota; Seibt, Annette; Müller-Felber, Wolfgang; Haack, Tobias B; Płoski, Rafał; Lohmeier, Klaus; Schneider, Dominik; Klee, Dirk; Rokicki, Dariusz; Mayatepek, Ertan; Strom, Tim M; Meitinger, Thomas; Klopstock, Thomas; Pronicka, Ewa; Mayr, Johannes A; Baric, Ivo; Distelmaier, Felix; Prokisch, Holger

2016-10-06

To safeguard the cell from the accumulation of potentially harmful metabolic intermediates, specific repair mechanisms have evolved. APOA1BP, now renamed NAXE, encodes an epimerase essential in the cellular metabolite repair for NADHX and NADPHX. The enzyme catalyzes the epimerization of NAD(P)HX, thereby avoiding the accumulation of toxic metabolites. The clinical importance of the NAD(P)HX repair system has been unknown. Exome sequencing revealed pathogenic biallelic mutations in NAXE in children from four families with (sub-) acute-onset ataxia, cerebellar edema, spinal myelopathy, and skin lesions. Lactate was elevated in cerebrospinal fluid of all affected individuals. Disease onset was during the second year of life and clinical signs as well as episodes of deterioration were triggered by febrile infections. Disease course was rapidly progressive, leading to coma, global brain atrophy, and finally to death in all affected individuals. NAXE levels were undetectable in fibroblasts from affected individuals of two families. In these fibroblasts we measured highly elevated concentrations of the toxic metabolite cyclic-NADHX, confirming a deficiency of the mitochondrial NAD(P)HX repair system. Finally, NAD or nicotinic acid (vitamin B3) supplementation might have therapeutic implications for this fatal disorder. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Genetic manipulation in Sulfolobus islandicus and functional analysis of DNA repair genes

DEFF Research Database (Denmark)

Zhang, Changyi; Tian, Bin; Li, Suming

2013-01-01

Recently, a novel gene-deletion method was developed for the crenarchaeal model Sulfolobus islandicus, which is a suitable tool for addressing gene essentiality in depth. Using this technique, we have investigated functions of putative DNA repair genes by constructing deletion mutants and studying...

Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

Energy Technology Data Exchange (ETDEWEB)

Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

2004-07-14

Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

Cardiosphere-Derived Cells Facilitate Heart Repair by Modulating M1/M2 Macrophage Polarization and Neutrophil Recruitment

Science.gov (United States)

Hasan, Al Shaimaa; Luo, Lan; Yan, Chen; Zhang, Tian-Xia; Urata, Yoshishige; Goto, Shinji; Mangoura, Safwat A.; Abdel-Raheem, Mahmoud H.; Zhang, Shouhua; Li, Tao-Sheng

2016-01-01

Cardiosphere-derived cells (CDCs), one of the promising stem cell sources for myocardial repair, have been tested in clinical trials and resulted in beneficial effects; however, the relevant mechanisms are not fully understood. In this study, we examined the hypothesis that CDCs favor heart repair by switching the macrophages from a pro-inflammatory phenotype (M1) into a regulatory anti-inflammatory phenotype (M2). Macrophages from mice were cultured with CDCs-conditioned medium or with fibroblasts-conditioned medium as a control. Immunostaining showed that CDCs-conditioned medium significantly enhanced the expression of CD206 (a marker for M2 macrophages), but decreased the expression of CD86 (a marker for M1 macrophages) 3 days after culture. For animal studies, we used an acute myocardial infarction model of mice. We injected CDCs, fibroblasts, or saline only into the border zone of infarction. Then we collected the heart tissues for histological analysis 5 and 14 days after treatment. Compared with control animals, CDCs treatment significantly decreased M1 macrophages and neutrophils but increased M2 macrophages in the infarcted heart. Furthermore, CDCs-treated mice had reduced infarct size and fewer apoptotic cells compared to the controls. Our data suggest that CDCs facilitate heart repair by modulating M1/M2 macrophage polarization and neutrophil recruitment, which may provide a new insight into the mechanisms of stem cell-based myocardial repair. PMID:27764217

Repair of 8-methoxypsoralen + UVA-induced damage in specific sequences in chromosomal and episomal DNA in human cells

Energy Technology Data Exchange (ETDEWEB)

Dean, S.W.

1989-07-01

A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, approximately 35% of cross-links in an episomally maintained Epstein--Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA.

Repair of 8-methoxypsoralen + UVA-induced damage in specific sequences in chromosomal and episomal DNA in human cells

International Nuclear Information System (INIS)

Dean, S.W.

1989-01-01

A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, ∼35% of cross-links in an episomally maintained Epstein-Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA. (author)

In Vitro Vitamin K 3 Effect on Conjunctival Fibroblast Migration and Proliferation

OpenAIRE

Pinilla, I.; Izaguirre, L. B.; Gonzalvo, F. J.; Piazuelo, E.; Garcia-Gonzalez, M. A.; Sanchez-Cano, A. I.; Sopeña, F.

2014-01-01

Purpose. To evaluate the dose effect of vitamin K3 on wound healing mechanisms. Methods. Conjunctival fibroblasts were incubated for 24 hours. An artificial wound was made and the cells were incubated with fresh medium plus doses of vitamin K3 to be tested. Wound repair was monitored at 0, 18, 24, and 48 hours. Proliferation was measured in actively dividing cells by [3H]thymidine uptake. Six different groups were tested: group 1/no drugs added, group 2/ethanol 0.1%, group 3/vitamin K3 1 mg/L...

Microporous dermal-mimetic electrospun scaffolds pre-seeded with fibroblasts promote tissue regeneration in full-thickness skin wounds.

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Paul P Bonvallet

Full Text Available Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone electrospun scaffold (70:30 col/PCL containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM, and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344 rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14% over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold. Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration

Fibroblasts maintained in 3 dimensions show a better differentiation state and higher sensitivity to estrogens

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Montani, Claudia [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy); Steimberg, Nathalie; Boniotti, Jennifer [Laboratory of Tissue Engineering, Anatomy and Physiopathology Unit, Department of Clinical and Experimental Sciences, School of Medicine, University of Brescia (Italy); Biasiotto, Giorgio; Zanella, Isabella [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy); Department of Molecular and Translational Medicine, University of Brescia, Brescia (Italy); Diafera, Giuseppe [Integrated Systems Engineering (ISE), Milan (Italy); Biunno, Ida [IRGB-CNR, Milan (Italy); IRCCS-Multimedica, Milan (Italy); Caimi, Luigi [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy); Department of Molecular and Translational Medicine, University of Brescia, Brescia (Italy); Mazzoleni, Giovanna [Laboratory of Tissue Engineering, Anatomy and Physiopathology Unit, Department of Clinical and Experimental Sciences, School of Medicine, University of Brescia (Italy); Di Lorenzo, Diego, E-mail: diego.dilorenzo@yahoo.it [Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia (Italy)

2014-11-01

Cell differentiation and response to hormonal signals were studied in a 3D environment on an in-house generated mouse fibroblast cell line expressing a reporter gene under the control of estrogen responsive sequences (EREs). 3D cell culture conditions were obtained in a Rotary Cell Culture System; (RCCS™), a microgravity based bioreactor that promotes the aggregation of cells into multicellular spheroids (MCS). In this bioreactor the cells maintained a better differentiated phenotype and more closely resembled in vivo tissue. The RCCS™ cultured fibroblasts showed higher expression of genes regulating cell assembly, differentiation and hormonal functions. Microarray analysis showed that genes related to cell cycle, proliferation, cytoskeleton, migration, adhesion and motility were all down-regulated in 3D as compared to 2D conditions, as well as oncogene expression and inflammatory cytokines. Controlled remodeling of ECM, which is an essential aspect of cell organization, homeostasis and tissue was affected by the culture method as assessed by immunolocalization of β-tubulin. Markers of cell organization, homeostasis and tissue repair, metalloproteinase 2 (MMP2) and its physiological inhibitor (TIMP4) changed expression in association with the relative formation of cell aggregates. The fibroblasts cultured in the RCCS™ maintain a better responsiveness to estrogens, measured as expression of ERα and regulation of an ERE-dependent reporter and of the endogenous target genes CBP, Rarb, MMP1 and Dbp. Our data highlight the interest of this 3D culture model for its potential application in the field of cell response to hormonal signals and the pharmaco-toxicological analyses of chemicals and natural molecules endowed of estrogenic potential. - Highlights: • We here characterized the first cell line derived from an estrogen reporter mouse. • In the RCCS cells express an immortalized behavior but not a transformed phenotype. • The RCCS provides a system for

High fat diet accelerates cartilage repair in DBA/1 mice.

Science.gov (United States)

Wei, Wu; Bastiaansen-Jenniskens, Yvonne M; Suijkerbuijk, Mathijs; Kops, Nicole; Bos, Pieter K; Verhaar, Jan A N; Zuurmond, Anne-Marie; Dell'Accio, Francesco; van Osch, Gerjo J V M

2017-06-01

Obesity is a well-known risk factor for osteoarthritis, but it is unknown what it does on cartilage repair. Here we investigated whether a high fat diet (HFD) influences cartilage repair in a mouse model of cartilage repair. We fed DBA/1 mice control or HFD (60% energy from fat). After 2 weeks, a full thickness cartilage defect was made in the trochlear groove. Mice were sacrificed, 1, 8, and 24 weeks after operation. Cartilage repair was evaluated on histology. Serum glucose, insulin and amyloid A were measured 24 h before operation and at endpoints. Immunohistochemical staining was performed on synovium and adipose tissue to evaluate macrophage infiltration and phenotype. One week after operation, mice on HFD had defect filling with fibroblast-like cells and more cartilage repair as indicated by a lower Pineda score. After 8 weeks, mice on a HFD still had a lower Pineda score. After 24 weeks, no mice had complete cartilage repair and we did not detect a significant difference in cartilage repair between diets. Bodyweight was increased by HFD, whereas serum glucose, amyloid A and insulin were not influenced. Macrophage infiltration and phenotype in adipose tissue and synovium were not influenced by HFD. In contrast to common wisdom, HFD accelerated intrinsic cartilage repair in DBA/1 mice on the short term. Resistance to HFD induced inflammatory and metabolic changes could be associated with accelerated cartilage repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1258-1264, 2017. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Abnormal responses to mid-ultraviolet light of cultured fibroblasts from patients with disorders featuring sunlight sensitivity

International Nuclear Information System (INIS)

Smith, P.J.; Paterson, M.C.

1982-01-01

The postultraviolet light (UV) colony-forming ability and DNA repair properties were examined of human skin fibroblasts derived from two groups of donors at high risk of cancer; (a) persons exhibiting sensitivity to sunlight; and (b) persons with conditions possibly associated with an underlying defect in the repair of radiogenic DNA damage. A comparison was made between the effects of far UV (254 nm) and mid UV (313 nm). Biochemical studies performed on strains sensitive to 313-nm UV alone suggest that their unusual photoresponse is not attributable to defective repair of cyclobutyl pyrimidine dimers. Furthermore, our data imply that thymine glycols are unlikely candidates for the critical lethal lesions in 313-nm UV-sensitive strains. Evidence is presented in support of the contention that mid UV may be partially radiomimetic, thus extending our understanding of the deleterious biological effects of this ubiquitous environmental carcinogen

The Fanconi anaemia components UBE2T and FANCM are functionally linked to nucleotide excision repair.

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Ian R Kelsall

Full Text Available The many proteins that function in the Fanconi anaemia (FA monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes--UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage. Comprehensive genetic dissection experiments indicate that both of these FA genes collaborate to promote nucleotide excision repair rather than translesion bypass to protect cells form UV genotoxicity. Furthermore, UBE2T deficiency impacts on the efficient removal of the UV-induced photolesion cyclobutane pyrimidine dimer. Therefore, this work reveals that the FA pathway shares two components with nucleotide excision repair, intimating not only crosstalk between the two major repair pathways, but also potentially identifying a UBE2T-mediated ubiquitin-signalling response pathway that contributes to nucleotide excision repair.

FGF2 mediates DNA repair in epidermoid carcinoma cells exposed to ionizing radiation

International Nuclear Information System (INIS)

Marie, Melanie; Hafner, Sophie; Moratille, Sandra; Vaigot, Pierre; Rigaud, Odile; Martin, Michele T.; Mine, Solene

2012-01-01

Fibroblast growth factor 2 (FGF2) is a well-known survival factor. However, its role in DNA repair is poorly documented. The present study was designed to investigate in epidermoid carcinoma cells the potential role of FGF2 in DNA repair. The side population (SP) with cancer stem cell-like properties and the main population (MP) were isolated from human A431 squamous carcinoma cells. Radiation-induced DNA damage and repair were assessed using the alkaline comet assay. FGF2 expression was quantified by enzyme linked immunosorbent assay (ELISA). SP cells exhibited rapid repair of radiation induced DNA damage and a high constitutive level of nuclear FGF2. Blocking FGF2 signaling abrogated the rapid DNA repair. In contrast, in MP cells, a slower repair of damage was associated with low basal expression of FGF2. Moreover, the addition of exogenous FGF2 accelerated DNA repair in MP cells. When irradiated, SP cells secreted FGF2, whereas MP cells did not. FGF2 was found to mediate DNA repair in epidermoid carcinoma cells. We postulate that carcinoma stem cells would be intrinsically primed to rapidly repair DNA damage by a high constitutive level of nuclear FGF2. In contrast, the main population with a low FGF2 content exhibits a lower repair rate which can be increased by exogenous FGF2. (authors)

Estimation of low-dose radiation-responsive proteins in the absence of genomic instability in normal human fibroblast cells.

Science.gov (United States)

Yim, Ji-Hye; Yun, Jung Mi; Kim, Ji Young; Nam, Seon Young; Kim, Cha Soon

2017-11-01

Low-dose radiation has various biological effects such as adaptive responses, low-dose hypersensitivity, as well as beneficial effects. However, little is known about the particular proteins involved in these effects. Here, we sought to identify low-dose radiation-responsive phosphoproteins in normal fibroblast cells. We assessed genomic instability and proliferation of fibroblast cells after γ-irradiation by γ-H2AX foci and micronucleus formation analyses and BrdU incorporation assay, respectively. We screened fibroblast cells 8 h after low-dose (0.05 Gy) γ-irradiation using Phospho Explorer Antibody Microarray and validated two differentially expressed phosphoproteins using Western blotting. Cell proliferation proceeded normally in the absence of genomic instability after low-dose γ-irradiation. Phospho antibody microarray analysis and Western blotting revealed increased expression of two phosphoproteins, phospho-NFκB (Ser536) and phospho-P70S6K (Ser418), 8 h after low-dose radiation. Our findings suggest that low-dose radiation of normal fibroblast cells activates the expression of phospho-NFκB (Ser536) and phospho-P70S6K (Ser418) in the absence of genomic instability. Therefore, these proteins may be involved in DNA damage repair processes.

The dependence of fibroblast radiosensitivity on cell pH

International Nuclear Information System (INIS)

Veksler, A.M.; Kublik, L.N.; Degtyareva, O.V.; Ehjdus, L.Kh.

1983-01-01

The problem of the change of radiosensitivity of Chinese hamster fibroblasts, irradiated under aerobic and hypoxic conditions in the course of intracellular pH (pHsub(intr.)) change by means of a phosphate buffer has been studied. It has been found that pHsub(intr.) reduction considerably increases the radiosensitivity, the effect being more pronounced on hypoxic cells which is essential for radiotherapy of tumors. The survival rate of cell irradiated under hypoxia conditions does not depend on season while cell resistance in case of irradiation in open air in spring and autumn is different. The effect discovery in case of pHsub(intr.) reduction upon irradiation shows up the influence of the studied factor on repair processes

Induction of growth and proliferation of fibroblast cells in magnetic field

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Naghmeh Ezatti

2015-02-01

Full Text Available Background: Tissue engineering is generally defined as developing and changing the laboratory growth of molecules and cells in tissues or organs to replace and repair the damaged part of body. This study was carried out to stimulate the growth of cultured fibroblast cells by a physical electromagnetic method. Methods: First, an air-core coil was prepared and the cell culture plate was placed comfortably into the mold, then the plate containing the culture medium and human fibroblast cell along with air-core coil were placed in an incubator and then connected to the power supply. Thus, the sample underwent electromagnetic field at different times, and cell proliferation was studied by MTTassay. Results: Microscopic images indicated that the cells undergoing electromagnetic field (0.35 amps had a significant growth compared to the cells in control group in a definit range of stimulation. Conclusion: In conclusion, electromagnetic stimulation in a definite range led to cell proliferation and could be used as a positive factor in tissue engineering.

Deposition of insoluble elastin by pulmonary fibroblasts from patients with COPD is increased by treatment with versican siRNA

Directory of Open Access Journals (Sweden)

Wu L

2017-01-01

Full Text Available Lian Wu,1,2 Jing Zhang,3 Jie Ming Qu,4 Chun-xue Bai,3 Mervyn J Merrilees5 1Department of Community and Health Services, Unitec, 2Department of Pharmacology & Clinical Pharmacology, University of Auckland, Auckland, New Zealand; 3Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, 4Department of Pulmonary Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China; 5Department of Anatomy and Medical Imaging, University of Auckland, Auckland, New Zealand Abstract: A reduced content of alveolar elastic fibers is a key feature of COPD lung. Despite continued elastogenic potential by alveolar fibroblasts in the lung affected by COPD, repair of elastic fibers does not take place, which is due to increased levels of the chondroitin sulfate proteoglycan versican that inhibits the assembly of tropoelastin into fibers. In this study, primary pulmonary fibroblast cell lines from COPD and non-COPD patients were treated with a small interfering RNA (siRNA against versican to determine if knockdown of versican could restore the deposition of insoluble elastin. Versican siRNA treatment reduced versican expression and secretion by pulmonary fibroblasts from both COPD and non-COPD patients (P<0.01 and significantly increased deposition of insoluble elastin in the COPD cell cultures (P<0.05. The treatment, however, did not significantly affect production of soluble elastin (tropoelastin in either the COPD or non-COPD cell cultures, supporting a role for versican in inhibiting assembly but not synthesis of tropoelastin. These results suggest that removal or knockdown of versican may be a possible therapeutic strategy for increasing deposition of insoluble elastin and stimulating repair of elastic fibers in COPD lung. Keywords: pulmonary fibroblasts, COPD, elastin, versican

Sensitization to radiation and alkylating agents by inhibitors of poly(ADP-ribose) polymerase is enhanced in cells deficient in DNA double-strand break repair.

Science.gov (United States)

Löser, Dana A; Shibata, Atsushi; Shibata, Akiko K; Woodbine, Lisa J; Jeggo, Penny A; Chalmers, Anthony J

2010-06-01

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents.

Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

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Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Karhemo, Piia-Riitta [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Räsänen, Kati [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Laakkonen, Pirjo [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Vaheri, Antti [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland)

2016-07-01

Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.

Capacity of ultraviolet-induced DNA repair in human glioma cells

Energy Technology Data Exchange (ETDEWEB)

Itoh, Hiroji

1987-04-01

A DNA repair abnormality is likely related to an increased incidence of neoplasms in several autosomal recessive diseases such as xeroderma pigmentosum, Fanconi's anemia, Bloom's syndrome and ataxia telangiectasia. In human glioma cells, however, there are only a few reports on DNA repair. In this study, an ultraviolet (UV)-induced DNA repair was examined systematically in many human glioma cells. Two human malignant glioma cell lines (MMG-851, U-251-MG) and 7 human glioma cell strains (4, benign; 3, malignant) of short term culture, in which glial fibrillary acidic protein (GFAP) staining were positive, were used. To investigate the capacity of DNA repair, UV sensitivity was determined by colony formation; excision repair by autoradiography and Cytosine Arabinoside (Ara-C) assay; and post-replication repair by the joining rate of newly synthesized DNA. As a result, the colony-forming abilities of malignant glioma cell lines were lower than those of normal human fibroblasts, but no difference was found between two malignant glioma cell lines. The excision repair of the malignant group (2 cell lines and 3 cell strains) was apparently lower than that of the benign group (4 cell strains). In two malignant glioma cell lines, the excision repair of MMG-851 was lower than that of U-251-MG, and the post-replication repair of MMG-851 was higher than that of U-251-MG. These results were considered to correspond well with colony-forming ability. The results indicate that there are some differences in each human malignant glioma cell in its UV-induced DNA repair mechanism, and that the excision repair of the malignant glioma cells is apparently lower than that of the benign glioma cells. These findings may be useful for diagnosis and treatment.

Fibroblasts and myofibroblasts in wound healing

Directory of Open Access Journals (Sweden)

Darby IA

2014-11-01

Full Text Available Ian A Darby,1 Betty Laverdet,2 Frédéric Bonté3, Alexis Desmoulière2 1School of Medical Sciences, RMIT University, Melbourne, VIC, Australia; 2Department of Physiology and EA 6309, FR 3503, Faculties of Medicine and Pharmacy, University of Limoges, Limoges, France; 3LVMH Recherche, Saint Jean de Braye, France Abstract: (Myofibroblasts are key players for maintaining skin homeostasis and for orchestrating physiological tissue repair. (Myofibroblasts are embedded in a sophisticated extracellular matrix (ECM that they secrete, and a complex and interactive dialogue exists between (myofibroblasts and their microenvironment. In addition to the secretion of the ECM, (myofibroblasts, by secreting matrix metalloproteinases and tissue inhibitors of metalloproteinases, are able to remodel this ECM. (Myofibroblasts and their microenvironment form an evolving network during tissue repair, with reciprocal actions leading to cell differentiation, proliferation, quiescence, or apoptosis, and actions on growth factor bioavailability by binding, sequestration, and activation. In addition, the (myofibroblast phenotype is regulated by mechanical stresses to which they are subjected and thus by mechanical signaling. In pathological situations (excessive scarring or fibrosis, or during aging, this dialogue between the (myofibroblasts and their microenvironment may be altered or disrupted, leading to repair defects or to injuries with damaged and/or cosmetic skin alterations such as wrinkle development. The intimate dialogue between the (myofibroblasts and their microenvironment therefore represents a fascinating domain that must be better understood in order not only to characterize new therapeutic targets and drugs able to prevent or treat pathological developments but also to interfere with skin alterations observed during normal aging or premature aging induced by a deleterious environment. Keywords: myofibroblast, fibroblast, α-smooth muscle actin

Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

International Nuclear Information System (INIS)

Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

1982-01-01

The metabolism of [1,2,6,7-3H]testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC

Platelet-rich plasma (PRP) and adipose-derived mesenchymal stem cells: stimulatory effects on proliferation and migration of fibroblasts and keratinocytes in vitro.

Science.gov (United States)

Stessuk, Talita; Puzzi, Maria Beatriz; Chaim, Elinton Adami; Alves, Paulo César Martins; de Paula, Erich Vinicius; Forte, Andresa; Izumizawa, Juliana Massae; Oliveira, Carolina Caliári; Frei, Fernando; Ribeiro-Paes, João Tadeu

2016-09-01

The clinical use of tissue engineering associated with cell therapy is considered a new alternative therapy for the repair of chronic lesions with potential application in different medical areas, mostly in orthopedic and dermatological diseases. Platelet-rich plasma (PRP) is a rich source of growth factors and cytokines important for wound healing. Adipose-derived mesenchymal stem cells (ADSCs) have shown potential to accelerate the resolution of ulcers, to stimulate cell proliferation, and to benefit the quality of skin repair. This study aims to determine the effect of PRP and conditioned medium (CM) from ADSC on fibroblast and keratinocyte proliferation in vitro. Migration and proliferation assays were performed to evaluate the growth of fibroblasts and keratinocytes in the presence of PRP, CM, and CM + PRP. Significant proliferative stimulation was observed after 48 h of culture (p PRP, 100 % CM, and 25 % PRP + 25 % CM, if compared with control. Keratinocyte proliferation was stimulated after 48 h in cultures with 25, 50, and 100 % CM, and growth was compared with controls. The migration assay detected a significant migratory stimulus in fibroblasts cultured with 10 % PRP + 10 % CM after 48 h. These in vitro results suggest that PRP and ADSC have therapeutic potential for healing and re-epithelialization of chronic wounds in vivo.

A cell-free scaffold-based cartilage repair provides improved function hyaline-like repair at one year.

Science.gov (United States)

Siclari, Alberto; Mascaro, Gennaro; Gentili, Chiara; Cancedda, Ranieri; Boux, Eugenio

2012-03-01

Bone marrow stimulation techniques in cartilage repair such as drilling are limited by the formation of fibrous to hyaline-like repair tissue. It has been suggested such techniques can be enhanced by covering the defect with scaffolds. We present an innovative approach using a polyglycolic acid (PGA)-hyaluronan scaffold with platelet-rich-plasma (PRP) in drilling. We asked whether (1) PRP immersed in a cell-free PGA-hyaluronan scaffold improves patient-reported 1-year outcomes for the Knee injury and Osteoarthritis Score (KOOS), and (2) implantation of the scaffold in combination with bone marrow stimulation leads to the formation of hyaline-like cartilage repair tissue. We reviewed 52 patients who had arthroscopic implantation of the PGA-hyaluronan scaffold immersed with PRP in articular cartilage defects of the knee pretreated with Pridie drilling. Patients were assessed by KOOS. At 9 months followup, histologic staining was performed in specimens obtained from five patients to assess the repair tissue quality. The KOOS subscores improved for pain (55 to 91), symptoms (57 to 88), activities of daily living (69 to 86), sports and recreation (36 to 70), and quality of life (38 to 73). The histologic evaluation showed a homogeneous hyaline-like cartilage repair tissue. The cell-free PGA-hyaluronan scaffold combined with PRP leads to cartilage repair and improved patient-reported outcomes (KOOS) during 12 months of followup. Histologic sections showed morphologic features of hyaline-like repair tissue. Long-term followup is needed to determine if the cartilage repair tissue is durable. Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks.

Science.gov (United States)

Tichy, Elisia D; Pillai, Resmi; Deng, Li; Liang, Li; Tischfield, Jay; Schwemberger, Sandy J; Babcock, George F; Stambrook, Peter J

2010-11-01

Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells. Of the 2 major pathways that repair double-strand breaks, error-prone nonhomologous end joining (NHEJ) predominated in mouse embryonic fibroblasts, whereas the high fidelity homologous recombinational repair (HRR) predominated in ES cells. Microhomology-mediated end joining, an emerging repair pathway, persisted at low levels in all cell types examined. The levels of proteins involved in HRR and microhomology-mediated end joining were highly elevated in ES cells compared with mouse embryonic fibroblasts, whereas those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV protein was also low in ES cells. Attempts to increase the abundance of DNA Ligase IV protein by overexpression or inhibition of its degradation, and thereby elevate NHEJ in ES cells, were unsuccessful. When ES cells were induced to differentiate, however, the level of DNA Ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than NHEJ may lend ES cells an additional layer of genomic protection and that the limited levels of DNA Ligase IV may account for the low level of NHEJ activity.

Cardiosphere-Derived Cells Facilitate Heart Repair by Modulating M1/M2 Macrophage Polarization and Neutrophil Recruitment.

Directory of Open Access Journals (Sweden)

Al Shaimaa Hasan

Full Text Available Cardiosphere-derived cells (CDCs, one of the promising stem cell sources for myocardial repair, have been tested in clinical trials and resulted in beneficial effects; however, the relevant mechanisms are not fully understood. In this study, we examined the hypothesis that CDCs favor heart repair by switching the macrophages from a pro-inflammatory phenotype (M1 into a regulatory anti-inflammatory phenotype (M2. Macrophages from mice were cultured with CDCs-conditioned medium or with fibroblasts-conditioned medium as a control. Immunostaining showed that CDCs-conditioned medium significantly enhanced the expression of CD206 (a marker for M2 macrophages, but decreased the expression of CD86 (a marker for M1 macrophages 3 days after culture. For animal studies, we used an acute myocardial infarction model of mice. We injected CDCs, fibroblasts, or saline only into the border zone of infarction. Then we collected the heart tissues for histological analysis 5 and 14 days after treatment. Compared with control animals, CDCs treatment significantly decreased M1 macrophages and neutrophils but increased M2 macrophages in the infarcted heart. Furthermore, CDCs-treated mice had reduced infarct size and fewer apoptotic cells compared to the controls. Our data suggest that CDCs facilitate heart repair by modulating M1/M2 macrophage polarization and neutrophil recruitment, which may provide a new insight into the mechanisms of stem cell-based myocardial repair.

Unusual Glycosaminoglycans from a Deep Sea Hydrothermal Bacterium Improve Fibrillar Collagen Structuring and Fibroblast Activities in Engineered Connective Tissues

Directory of Open Access Journals (Sweden)

Jean Guezennec

2013-04-01

Full Text Available Biopolymers produced by marine organisms can offer useful tools for regenerative medicine. Particularly, HE800 exopolysaccharide (HE800 EPS secreted by a deep-sea hydrothermal bacterium displays an interesting glycosaminoglycan-like feature resembling hyaluronan. Previous studies demonstrated its effectiveness to enhance in vivo bone regeneration and to support osteoblastic cell metabolism in culture. Thus, in order to assess the usefulness of this high-molecular weight polymer in tissue engineering and tissue repair, in vitro reconstructed connective tissues containing HE800 EPS were performed. We showed that this polysaccharide promotes both collagen structuring and extracellular matrix settle by dermal fibroblasts. Furthermore, from the native HE800 EPS, a low-molecular weight sulfated derivative (HE800 DROS displaying chemical analogy with heparan-sulfate, was designed. Thus, it was demonstrated that HE800 DROS mimics some properties of heparan-sulfate, such as promotion of fibroblast proliferation and inhibition of matrix metalloproteinase (MMP secretion. Therefore, we suggest that the HE800EPS family can be considered as an innovative biotechnological source of glycosaminoglycan-like compounds useful to design biomaterials and drugs for tissue engineering and repair.

Unusual Glycosaminoglycans from a Deep Sea Hydrothermal Bacterium Improve Fibrillar Collagen Structuring and Fibroblast Activities in Engineered Connective Tissues

Science.gov (United States)

Senni, Karim; Gueniche, Farida; Changotade, Sylvie; Septier, Dominique; Sinquin, Corinne; Ratiskol, Jacqueline; Lutomski, Didier; Godeau, Gaston; Guezennec, Jean; Colliec-Jouault, Sylvia

2013-01-01

Biopolymers produced by marine organisms can offer useful tools for regenerative medicine. Particularly, HE800 exopolysaccharide (HE800 EPS) secreted by a deep-sea hydrothermal bacterium displays an interesting glycosaminoglycan-like feature resembling hyaluronan. Previous studies demonstrated its effectiveness to enhance in vivo bone regeneration and to support osteoblastic cell metabolism in culture. Thus, in order to assess the usefulness of this high-molecular weight polymer in tissue engineering and tissue repair, in vitro reconstructed connective tissues containing HE800 EPS were performed. We showed that this polysaccharide promotes both collagen structuring and extracellular matrix settle by dermal fibroblasts. Furthermore, from the native HE800 EPS, a low-molecular weight sulfated derivative (HE800 DROS) displaying chemical analogy with heparan-sulfate, was designed. Thus, it was demonstrated that HE800 DROS mimics some properties of heparan-sulfate, such as promotion of fibroblast proliferation and inhibition of matrix metalloproteinase (MMP) secretion. Therefore, we suggest that the HE800EPS family can be considered as an innovative biotechnological source of glycosaminoglycan-like compounds useful to design biomaterials and drugs for tissue engineering and repair. PMID:23612369

Studies on the relationship between the cancer chemotherapeutic agent, hydroxyurea, and DNA repair in mammalian cells

International Nuclear Information System (INIS)

Katz, E.J.

1988-01-01

To examine the possibility that manipulating DNA repair might lessen drug resistance, we investigated whether depletion of the thymidine triphosphate (TTP) pool or administration of hydroxyurea could interfere with the ability of confluent normal human skin fibroblasts to repair ultraviolet irradiation-induced DNA damage. A method was developed for the quantitation of cellular TTP pools by labeling them with [ 3 H]thymidine. The addition of hydroxyurea, either simultaneously with [ 3 H]thymidine or two hours later, resulted in a dose- and time-dependent increase in the [ 3 H]TTP pool. The capacity of these cells to carry out DNA repair was quantitated by their ability to perform repair replication synthesis of DNA after exposure to ultraviolet irradiation. This radiation produces thymine dimers in DNA, which are repaired by the nucleotide excision repair pathway. The experimental protocol resulted in an 8-10-fold reduction in the [ 3 H]TTP pool. Saturating levels of DNA repair synthesis were observed under these conditions, with no further diminution of the already reduced [ 3 H]TTP pool. Repair replication and [ 3 H]TTP pool measurements were identical in cultures treated with 10 mM hydroxyurea and in those not exposed to the drug

Entrainment of Spontaneously Hypertensive Rat Fibroblasts by Temperature Cycles

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Sládek, Martin; Sumová, Alena

2013-01-01

The functional state of the circadian system of spontaneously hypertensive rats (SHR) differs in several characteristics from the functional state of normotensive Wistar rats. Some of these changes might be due to the compromised ability of the central pacemaker to entrain the peripheral clocks. Daily body temperature cycles represent one of the important cues responsible for the integrity of the circadian system, because these cycles are driven by the central pacemaker and are able to entrain the peripheral clocks. This study tested the hypothesis that the aberrant peripheral clock entrainment of SHR results from a compromised peripheral clock sensitivity to the daily temperature cycle resetting. Using cultured Wistar rat and SHR fibroblasts transfected with the circadian luminescence reporter Bmal1-dLuc, we demonstrated that two consecutive square-wave temperature cycles with amplitudes of 2.5°C are necessary and sufficient to restart the dampened oscillations and entrain the circadian clocks in both Wistar rat and SHR fibroblasts. We also generated a phase response curve to temperature cycles for fibroblasts of both rat strains. Although some of the data suggested a slight resistance of SHR fibroblasts to temperature entrainment, we concluded that the overall effect it too weak to be responsible for the differences between the SHR and Wistar in vivo circadian phenotype. PMID:24116198

Bladder function after sacrospinous hysteropexy and vaginal wall repair in women with uterovaginal prolapse.

Science.gov (United States)

Sayed Ahmed, Waleed A; Taha, Omima T; Hamdy, Mostafa A; Ibrahim, Zakia M; Elshahat, Amal M

2018-05-01

To evaluate the impact of repair of uterovaginal prolapse using sacrospinous hysteropexy and vaginal wall repair on the bladder function. The study was conducted at the urogynecology clinic of Suez Canal University Hospitals, Ismailia from January 2014 to March 2016. This study included women with a diagnosis of uterovaginal prolapse and wishing to preserve their uteri. Bladder function was evaluated through assessment of urological symptoms using a standardized questionnaire - the urogenital distress inventory (UDI-6) - in addition to urodynamic studies just before and six-months after the sacrospinous hysteropexy ± associated vaginal wall repair operation. Twenty-seven patients completed the study with a mean age of 36.5 ± 4 years. Only 3 women had sacrospinous hysteropexy with no additional procedures. Other procedures included anterior colporrhaphy (12), posterior colporrhaphy (9) and perineorrhaphy (15). Based on UDI-6, there was no significant difference between the pre- and post-operative symptoms of stress urinary incontinence (SUI) [8/27 (29.6%) vs. 9/27 (33.3%) respectively; p value = 0.7]. The pre- and post-operative symptoms of urge urinary incontinence were also insignificantly different [13/27 (48.1%) vs. 15/27 (55.5%); p value = 0.5]. The total score of UDI-6 increased from 24.5 ± (14.2) to 32.8 ± (29.4) which was not statistically significant (p value = 0.12). Urodynamically, voiding dysfunction was found less frequently after the operation, however the difference was statistically insignificant [9/27 (33.3%) vs. 8/27 (29.6%); p value = 0.7]. Sacrospinous hysteropexy and associated vaginal wall repair do not affect the bladder function either subjectively or objectively. Copyright © 2018. Published by Elsevier B.V.

Long-term successful arthroscopic repair of large and massive rotator cuff tears with a functional and degradable reinforcement device.

Science.gov (United States)

Proctor, Christopher S

2014-10-01

Rotator cuff repair is a procedure with varying outcomes, and there has been subsequent interest in devices that reinforce the repair and enhance structural and functional outcomes. The objective of this study was to determine these outcomes for arthroscopic repair of large and massive rotator cuff tears augmented with a synthetic absorbable mesh designed specifically for reinforcement of tendon repair by imaging and clinical assessments. Consecutive arthroscopic repairs were performed on 18 patients with large to massive rotator cuff tears by use of a poly-l-lactic acid synthetic patch as a reinforcement device and fixation with 4 sutures. Patients were assessed preoperatively and at 6 months, 12 months, and a mean of 42 months after surgery by the American Shoulder and Elbow Surgeons (ASES) shoulder score to evaluate clinical performance and at 12 months by ultrasound to assess structural repair. Ultrasound showed that 15 of 18 patients had intact rotator cuff repair at 12 months; at 42 months, an additional patient had a failed repair. Patients showed improvement in the ASES shoulder score from 25 preoperatively to 71 at 12 months and 70 at 42 months after surgery. Patients with intact rotator cuff (n = 14) at 42 months had an ASES shoulder score of 82. The poly-l-lactic acid bioabsorbable patch designed specifically to reinforce the surgical repair of tendons supported successful repair of large to massive rotator cuff tears in 83% of patients at 12 months after surgery and 78% of patients at 42 months after surgery, with substantial functional improvement. Copyright © 2014 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

Rescue of mitochondrial function in parkin-mutant fibroblasts using drug loaded PMPC-PDPA polymersomes and tubular polymersomes.

Science.gov (United States)

Yealland, G; Battaglia, G; Bandmann, O; Mortiboys, H

2016-09-06

Mutations in parkin cause autosomal recessive Parkinsonism and mitochondrial defects. A recent drug screen identified a class of steroid-like hydrophobic compounds able to rescue mitochondrial function in parkin-mutant fibroblasts. Whilst these possess therapeutic potential, the size and high hydrophobicity of some may limit their ability to penetrate the blood-brain barrier from systemic circulation, something that could be improved by novel drug formulations. In the present study, the steroid-like compounds Ursolic Acid (UA) and Ursocholanic Acid (UCA) were successfully encapsulated within nanoscopic polymersomes formed by poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-di-isopropylamino)ethyl methacrylate) (PMPC-PDPA) and separated into spherical and tubular morphologies to assess the effects of nanoparticle mediated delivery on drug efficacy. Following incubation with either morphology, parkin-mutant fibroblasts demonstrated time and concentration dependent increases in intracellular ATP levels, resembling those resulting from treatment with nascent UA and UCA formulated in 0.1% DMSO, as used in the original drug screen. Empty PMPC-PDPA polymersomes did not alter physiological measures related to mitochondrial function or induce cytotoxicity. In combination with other techniques such as ligand functionalisation, PMPC-PDPA nanoparticles of well-defined morphology may prove a promising platform for tailoring the pharmacokinetic profile and organ specific bio-distribution of highly hydrophobic compounds. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Intraoperative Assessment of Tricuspid Valve Function After Conservative Repair

OpenAIRE

Revuelta, J.M.; Gomez-Duran, C.; Garcia-Rinaldi, R.; Gallagher, M.W.

1982-01-01

It is desirable to repair coexistent tricuspid valve pathology at the time of mitral valve corrections. Conservative tricuspid repair may consist of commissurotomy, annuloplasty, or both. It is important that the repair be appropriate or tricuspid valve replacement may be necessary. A simple reproducible method of intraoperative testing for tricuspid valve insufficiency has been developed and used in 25 patients. Fifteen patients have been recatheterized, and the correlation between the intra...

Growth Factor Supplementation Improves Native and Engineered Meniscus Repair in Vitro

Science.gov (United States)

Ionescu, Lara C.; Lee, Gregory C.; Huang, Kevin L.; Mauck, Robert L.

2012-01-01

Few therapeutic options exist for meniscus repair after injury. Local delivery of growth factors may stimulate repair and create a favorable environment for engineered replacement materials. In this study, we assessed the effect of basic fibroblast growth factor (bFGF) (a pro-mitotic agent) and transforming growth factor beta 3 (TGF-β3) (a pro-matrix formation agent) on meniscus repair and the integration/maturation of electrospun poly(ε-caprolactone) (PCL) scaffolds for meniscus tissue engineering. Circular meniscus repair constructs were formed and refilled with either native tissue or scaffolds. Repair constructs were cultured in serum-containing media for 4 and 8 weeks with various growth factor formulations, and assessed for mechanical strength, biochemical content, and histological appearance. Results showed that either short-term delivery of bFGF or sustained delivery of TGF-β3 increased integration strength for both juvenile and adult bovine tissue, with similar findings for engineered materials. While TGF-β3 increased proteoglycan content in the explants, bFGF did not increase DNA content after 8 weeks. This work suggests that in vivo delivery of bFGF or TGF-β3 may stimulate meniscus repair, but that the time course of delivery will strongly influence success. Further, this study demonstrates that electrospun scaffolds are a promising material for meniscus tissue engineering, achieving comparable or superior integration compared to native tissue. PMID:22698946

Mitochondrial Bioenergetics Is Altered in Fibroblasts from Patients with Sporadic Alzheimer's Disease

Science.gov (United States)

Pérez, María J.; Ponce, Daniela P.; Osorio-Fuentealba, Cesar; Behrens, Maria I.; Quintanilla, Rodrigo A.

2017-01-01

The identification of an early biomarker to diagnose Alzheimer's disease (AD) remains a challenge. Neuropathological studies in animal and AD patients have shown that mitochondrial dysfunction is a hallmark of the development of the disease. Current studies suggest the use of peripheral tissues, like skin fibroblasts as a possibility to detect the early pathological alterations present in the AD brain. In this context, we studied mitochondrial function properties (bioenergetics and morphology) in cultured fibroblasts obtained from AD, aged-match and young healthy patients. We observed that AD fibroblasts presented a significant reduction in mitochondrial length with important changes in the expression of proteins that control mitochondrial fusion. Moreover, AD fibroblasts showed a distinct alteration in proteolytic processing of OPA1, a master regulator of mitochondrial fusion, compared to control fibroblasts. Complementary to these changes AD fibroblasts showed a dysfunctional mitochondrial bioenergetics profile that differentiates these cells from aged-matched and young patient fibroblasts. Our findings suggest that the human skin fibroblasts obtained from AD patients could replicate mitochondrial impairment observed in the AD brain. These promising observations suggest that the analysis of mitochondrial bioenergetics could represent a promising strategy to develop new diagnostic methods in peripheral tissues of AD patients. PMID:29056898

Non-homologous end joining is the responsible pathway for the repair of fludarabine-induced DNA double strand breaks in mammalian cells

International Nuclear Information System (INIS)

Campos-Nebel, Marcelo de; Larripa, Irene; Gonzalez-Cid, Marcela

2008-01-01

Fludarabine (FLU), an analogue of adenosine, interferes with DNA synthesis and inhibits the chain elongation leading to replication arrest and DNA double strand break (DSB) formation. Mammalian cells use two main pathways of DSB repair to maintain genomic stability: homologous recombination (HR) and non-homologous end joining (NHEJ). The aim of the present work was to evaluate the repair pathways employed in the restoration of DSB formed following replication arrest induced by FLU in mammalian cells. Replication inhibition was induced in human lymphocytes and fibroblasts by FLU. DSB occurred in a dose-dependent manner on early/middle S-phase cells, as detected by γH2AX foci formation. To test whether conservative HR participates in FLU-induced DSB repair, we measured the kinetics of Rad51 nuclear foci formation in human fibroblasts. There was no significant induction of Rad51 foci after FLU treatment. To further confirm these results, we analyzed the frequency of sister chromatid exchanges (SCE) in both human cells. We did not find increased frequencies of SCE after FLU treatment. To assess the participation of NHEJ pathway in the repair of FLU-induced damage, we used two chemical inhibitors of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), vanillin and wortmannin. Human fibroblasts pretreated with DNA-PKcs inhibitors showed increased levels of chromosome breakages and became more sensitive to cell death. An active role of NHEJ pathway was also suggested from the analysis of Chinese hamster cell lines. XR-C1 (DNA-PKcs-deficient) and XR-V15B (Ku80-deficient) cells showed hypersensitivity to FLU as evidenced by the increased frequency of chromosome aberrations, decreased mitotic index and impaired survival rates. In contrast, CL-V4B (Rad51C-deficient) and V-C8 (Brca2-deficient) cell lines displayed a FLU-resistant phenotype. Together, our results suggest a major role for NHEJ repair in the preservation of genome integrity against FLU-induced DSB

Non-homologous end joining is the responsible pathway for the repair of fludarabine-induced DNA double strand breaks in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Campos-Nebel, Marcelo de [Departamento de Genetica, Instituto de Investigaciones Hematologicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires (Argentina)], E-mail: mnebel@hematologia.anm.edu.ar; Larripa, Irene; Gonzalez-Cid, Marcela [Departamento de Genetica, Instituto de Investigaciones Hematologicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires (Argentina)

2008-11-10

Fludarabine (FLU), an analogue of adenosine, interferes with DNA synthesis and inhibits the chain elongation leading to replication arrest and DNA double strand break (DSB) formation. Mammalian cells use two main pathways of DSB repair to maintain genomic stability: homologous recombination (HR) and non-homologous end joining (NHEJ). The aim of the present work was to evaluate the repair pathways employed in the restoration of DSB formed following replication arrest induced by FLU in mammalian cells. Replication inhibition was induced in human lymphocytes and fibroblasts by FLU. DSB occurred in a dose-dependent manner on early/middle S-phase cells, as detected by {gamma}H2AX foci formation. To test whether conservative HR participates in FLU-induced DSB repair, we measured the kinetics of Rad51 nuclear foci formation in human fibroblasts. There was no significant induction of Rad51 foci after FLU treatment. To further confirm these results, we analyzed the frequency of sister chromatid exchanges (SCE) in both human cells. We did not find increased frequencies of SCE after FLU treatment. To assess the participation of NHEJ pathway in the repair of FLU-induced damage, we used two chemical inhibitors of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), vanillin and wortmannin. Human fibroblasts pretreated with DNA-PKcs inhibitors showed increased levels of chromosome breakages and became more sensitive to cell death. An active role of NHEJ pathway was also suggested from the analysis of Chinese hamster cell lines. XR-C1 (DNA-PKcs-deficient) and XR-V15B (Ku80-deficient) cells showed hypersensitivity to FLU as evidenced by the increased frequency of chromosome aberrations, decreased mitotic index and impaired survival rates. In contrast, CL-V4B (Rad51C-deficient) and V-C8 (Brca2-deficient) cell lines displayed a FLU-resistant phenotype. Together, our results suggest a major role for NHEJ repair in the preservation of genome integrity against FLU

Cytoglobin inhibits migration through PI3K/AKT/mTOR pathway in fibroblast cells.

Science.gov (United States)

Demirci, Selami; Doğan, Ayşegül; Apdik, Hüseyin; Tuysuz, Emre Can; Gulluoglu, Sukru; Bayrak, Omer Faruk; Şahin, Fikrettin

2018-01-01

Cell proliferation and migration are crucial in many physiological processes including development, cancer, tissue repair, and wound healing. Cell migration is regulated by several signaling molecules. Identification of genes related to cell migration is required to understand molecular mechanism of non-healing chronic wounds which is a major concern in clinics. In the current study, the role of cytoglobin (CYGB) gene in fıbroblast cell migration and proliferation was described. L929 mouse fibroblast cells were transduced with lentiviral particles for CYGB and GFP, and analyzed for cell proliferation and migration ability. Fibroblast cells overexpressing CYGB displayed decreased cell proliferation, colony formation capacity, and cell migration. Phosphorylation levels of mTOR and two downstream effectors S6 and 4E-BP1 which take part in PI3K/AKT/mTOR signaling declined in CYGB-overexpressing cells. Microarray analysis indicated that CYGB overexpression leads to downregulation of cell proliferation, migration, and tumor growth associated genes in L929 cell line. This study demonstrated the role of CYGB in fibroblast cell motility and proliferation. CYGB could be a promising candidate for further studies as a potential target for diseases related to cell migration such as cancer and chronic wound treatment.

Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François, E-mail: fberthia@rci.rutgers.edu

2015-02-27

During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

International Nuclear Information System (INIS)

Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François

2015-01-01

During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β 1 (TGF-β 1 )-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β 1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β 1 is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β 1

Effects of Transplanted Heparin-Poloxamer Hydrogel Combining Dental Pulp Stem Cells and bFGF on Spinal Cord Injury Repair

Directory of Open Access Journals (Sweden)

Lihua Luo

2018-01-01

Full Text Available Spinal cord injury (SCI is one of serious traumatic diseases of the central nervous system and has no effective treatment because of its complicated pathophysiology. Tissue engineering strategy which contains scaffolds, cells, and growth factors can provide a promising treatment for SCI. Hydrogel that has 3D network structure and biomimetic microenvironment can support cellular growth and embed biological macromolecules for sustaining release. Dental pulp stem cells (DPSCs, derived from cranial neural crest, possess mesenchymal stem cell (MSC characteristics and have an ability to provide neuroprotective and neurotrophic properties for SCI treatment. Basic fibroblast growth factor (bFGF is able to promote cell survival and proliferation and also has beneficial effect on neural regeneration and functional recovery after SCI. Herein, a thermosensitive heparin-poloxamer (HP hydrogel containing DPSCs and bFGF was prepared, and the effects of HP-bFGF-DPSCs on neuron restoration after SCI were evaluated by functional recovery tests, western blotting, magnetic resonance imaging (MRI, histology evaluation, and immunohistochemistry. The results suggested that transplanted HP hydrogel containing DPSCs and bFGF had a significant impact on spinal cord repair and regeneration and may provide a promising strategy for neuron repair, functional recovery, and tissue regeneration after SCI.

Fibroblast Growth Factor 23 and Kidney Disease Progression in Autosomal Dominant Polycystic Kidney Disease.

Science.gov (United States)

Chonchol, Michel; Gitomer, Berenice; Isakova, Tamara; Cai, Xuan; Salusky, Isidro; Pereira, Renata; Abebe, Kaleab; Torres, Vicente; Steinman, Theodor I; Grantham, Jared J; Chapman, Arlene B; Schrier, Robert W; Wolf, Myles

2017-09-07

Increases in fibroblast growth factor 23 precede kidney function decline in autosomal dominant polycystic kidney disease; however, the role of fibroblast growth factor 23 in autosomal dominant polycystic kidney disease has not been well characterized. We measured intact fibroblast growth factor 23 levels in baseline serum samples from 1002 participants in the HALT-PKD Study A ( n =540; mean eGFR =91±17 ml/min per 1.73 m 2 ) and B ( n =462; mean eGFR =48±12 ml/min per 1.73 m 2 ). We used linear mixed and Cox proportional hazards models to test associations between fibroblast growth factor 23 and eGFR decline, percentage change in height-adjusted total kidney volume, and composite of time to 50% reduction in eGFR, onset of ESRD, or death. Median (interquartile range) intact fibroblast growth factor 23 was 44 (33-56) pg/ml in HALT-PKD Study A and 69 (50-93) pg/ml in Study B. In adjusted models, annualized eGFR decline was significantly faster in the upper fibroblast growth factor 23 quartile (Study A: quartile 4, -3.62; 95% confidence interval, -4.12 to -3.12 versus quartile 1, -2.51; 95% confidence interval, -2.71 to -2.30 ml/min per 1.73 m 2 ; P for trend kidney volume in adjusted models (quartile 4, 6.76; 95% confidence interval, 5.57 to 7.96 versus quartile 1, 6.04; 95% confidence interval, 5.55 to 6.54; P for trend =0.03). In Study B, compared with the lowest quartile, the highest fibroblast growth factor 23 quartile was associated with elevated risk for the composite outcome (hazard ratio, 3.11; 95% confidence interval, 1.84 to 5.25). Addition of fibroblast growth factor 23 to a model of annualized decline in eGFR≥3.0 ml/min per 1.73 m 2 did not improve risk prediction. Higher serum fibroblast growth factor 23 concentration was associated with kidney function decline, height-adjusted total kidney volume percentage increase, and death in patients with autosomal dominant polycystic kidney disease. However, fibroblast growth factor 23 did not substantially

The Effects of Age at Cleft Palate Repair on Middle Ear Function and Hearing Level.

Science.gov (United States)

Lou, Qun; Zhu, Hongping; Luo, Yi; Zhou, Zhibo; Ma, Lian; Ma, Xiaoran; Fu, Yuan

2018-05-01

To investigate the age effects of cleft palate repair on middle ear function and hearing level in patients who underwent cleft palate repair at different ages by audiologic examination. Medical histories were gathered in detail, and audiologic tests (ie, tympanometry and pure tone hearing threshold) were conducted in 126 patients after palatoplasty. The patients were divided into the following 4 groups according to their ages when they underwent cleft palate repair: group I (0-3 years, 73 patients), group II (4-7 years, 29 patients), group III (8-11 years, 16 patients), and group IV (12 years and older, 8 patients). The data regarding tympanograms, hearing levels, and the average hearing thresholds of each group were analyzed using chi-square tests. The prevalence of middle ear dysfunction and hearing loss in the patients who underwent palatoplasty before 3 years old (27.4% and 2.0% respectively) was significantly lower than that in patients who underwent palatopalsty at 12 years or older (75.0% and 43.7%, respectively). Linear-by-linear association revealed that the prevalences of middle ear dysfunction and hearing loss among the 4 groups were significantly different ( P cleft palate repair. From an audiologist's perspective, palatoplasty at an early age is very beneficial in helping children with cleft palates acquire better middle ear function and hearing level.

Mutagenesis and lethality following S phase irradiation of xeroderma pigmentosum and normal human diploid fibroblasts with ultraviolet light

International Nuclear Information System (INIS)

Grosovsky, A.J.; Little, J.B.

1983-01-01

The mutagenic and lethal effects of u.v. light exposure in the DNA synthetic phase of the cell cycle were determined in xeroderma pigmentosum complementation group A (XP-A), hereditary adenomatosis of the colon and rectum (ACR), and a normal, foreskin derived cell strain (AG1522). For AG1522, an increased sensitivity to the cytotoxic effects of u.v. light was observed as compared to previous findings for confluent, non-proliferating cultures. XP-A fibroblasts were markedly hypersensitive and ACR fibroblasts exhibited an intermediate response. The mutagenic response of ACR fibroblasts, however, was similar to normal fibroblasts. A threshold of 1.5-2 J/m 2 was observed for u.v. induced mutagenesis in normal and ACR fibroblasts. XP fibroblasts, on the other hand, were strikingly hypermutable and demonstrated little or no threshold. When S phase mutagenesis was considered as a function of survival level rather than u.v. light dose, XP fibroblasts remained significantly hypermutable as compared with normal fibroblasts at all survival levels. Previous mutagenesis results with confluent, non-proliferating cultures of XP and normal fibroblasts were reanalyzed as a function of cytotoxicity; XP hypermutability at all survival levels was also observed. (author)

Non-functional genes repaired at the RNA level.

Science.gov (United States)

Burger, Gertraud

2016-01-01

Genomes and genes continuously evolve. Gene sequences undergo substitutions, deletions or nucleotide insertions; mobile genetic elements invade genomes and interleave in genes; chromosomes break, even within genes, and pieces reseal in reshuffled order. To maintain functional gene products and assure an organism's survival, two principal strategies are used - either repair of the gene itself or of its product. I will introduce common types of gene aberrations and how gene function is restored secondarily, and then focus on systematically fragmented genes found in a poorly studied protist group, the diplonemids. Expression of their broken genes involves restitching of pieces at the RNA-level, and substantial RNA editing, to compensate for point mutations. I will conclude with thoughts on how such a grotesquely unorthodox system may have evolved, and why this group of organisms persists and thrives since tens of millions of years. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Caspase 3 activity in isolated fetal rat lung fibroblasts and rat periodontal ligament fibroblasts: cigarette smoke-induced alterations

Directory of Open Access Journals (Sweden)

James Elliot Scott

2016-03-01

Full Text Available Background Cigarette smoking is the leading cause of preventable death in the world. It has been implicated in the pathogenesis of pulmonary, oral and systemic diseases. Smoking during pregnancy is clearly a risk factor for the developing fetus and may be a major cause of infant mortality. Moreover, the oral cavity is the first site of exposure to cigarette smoke and may be a possible source for the spread of toxins to other organs of the body. Fibroblasts in general are morphologically heterogeneous connective tissue cells with diverse functions. Apoptosis or programmed cell death is a crucial process during embryogenesis and for the maintenance of homeostasis throughout life. Deregulation of apoptosis has been implicated in abnormal lung development in the fetus and disease progression in adults. Caspases, are proteases which belong to the family of cysteine aspartic acid proteases and are the key components for the downstream amplification of intra-cellular apoptotic signals. Of the 14 caspases known, caspase-3 is the key executioner of apoptosis. Fetal rat lung fibroblasts but not PDL viability is reduced by exposure to CSE. In addition Caspase 3 activity is elevated after CSE exposure in fetal lung fibroblasts but not in PDLs. Expression of caspase 3 is induced in CSE exposed lung fibroblasts but not in PDLs. Caspase 3 was localized to the cytoplasm in both cell types.

Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis

NARCIS (Netherlands)

Scheres, N; Laine, M L; de Vries, T J; Everts, V; van Winkelhoff, A J

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth-supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can

The radiation response of human dermal fibroblasts

Science.gov (United States)

Mitchell, Stephen Andrew

A clinically reliable predictive assay based on normal-tissue radiosensitivity may lead to improved tumour control through individualised dose prescriptions. In-vitro fibroblast radiosensitivity has been shown, in several studies, to correlate with late radiation morbidity. The aim of this study was to investigate some of the cellular mechanisms underlying the normal-tissue response. In this study, seventeen primary fibroblast strains were established by enzymatic disaggregation of skin biopsies obtained from patients. These comprised seven who experienced acute tissue reactions to radiotherapy, four patients with a normal response and six non-cancer volunteers. An AT cell line was included as a positive control for radiosensitivity. In-vitro radiosensitivity was measured using a clonogenic assay at both high (HDR: 1.6 Gymin-1) and low dose rate (LDR: 0.01 Gymin-1). The radiation parameter HDR SF2 was the most sensitive in discriminating the seven sensitive patients from the remaining ten normal patients (range 0.11-0.19 sensitive patients compared with 0.17-0.34 control patients: puse of an internal control or LDR radiation protocol increased this discrimination. Pulsed-field gel electrophoresis (PFGE) was used to measure the level of initial and residual double-strand breaks following irradiation. No correlation was found between HDR SF2 and initial DNA damage. However, a strong correlation was found between clonogenic survival and both residual DNA damage (measured over 10-70 Gy, allowing 4 h repair, correlation coefficient: 0.90, <0.0001) and the ratio of residual/initial DNA damage, with the sensitive cell lines generally showing a higher level of residual DNA damage. Cell-cycle delays were found in all 18 cell strains in response to 2 Gy irradiation, but were not found to discriminate between sensitive and normal patients. Associated studies found no mutations of the ATM gene in the five radiosensitive patients studied. However, a coding sequence alteration

scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair

International Nuclear Information System (INIS)

Biedermann, K.A.; Sun, J.R.; Giaccia, A.J.; Tosto, L.M.; Brown, J.M.

1991-01-01

C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair

The influence of platelet- derived products on angiogenesis and tissue repair: a concise update

Directory of Open Access Journals (Sweden)

Constanza E Martínez

2015-10-01

Full Text Available Platelet degranulation allows the release of a large amount of soluble mediators, is an essential step for wound healing initiation, and stimulates clotting and angiogenesis. The latter process is one of the most critical biological events observed during tissue repair,increasing the growth of blood vessels in the maturing wound. Angiogenesis requires the action of a variety of growth factors that act in an appropriate physiological ratio to assure functional blood vessel restoration. Platelets release main regulators of angiogenesis: Vascular Endothelial Growth Factors (VEGFs, basic fibroblast growth factor (FGF-2, and Platelet derived growth factors (PDGFs, among others. In order to stimulate tissue repair, platelet derived fractions have been used as an autologous source of growth factors and biomolecules, namely Platelet Rich Plasma (PRP, Platelet Poor Plasma (PPP and Platelet Rich Fibrin(PRF. The continuous release of these growth factors has been proposed to promote angiogenesis both in vitro and in vivo. Considering the existence of clinical trials currently evaluating the efficacy of autologous PRP, the present review analyses fundamental questions regarding the putative role of platelet derived fractions as regulators of angiogenesis and evaluates the possible clinical implications of these formulations.

Intraoperative Assessment of Tricuspid Valve Function After Conservative Repair

Science.gov (United States)

Revuelta, J.M.; Gomez-Duran, C.; Garcia-Rinaldi, R.; Gallagher, M.W.

1982-01-01

It is desirable to repair coexistent tricuspid valve pathology at the time of mitral valve corrections. Conservative tricuspid repair may consist of commissurotomy, annuloplasty, or both. It is important that the repair be appropriate or tricuspid valve replacement may be necessary. A simple reproducible method of intraoperative testing for tricuspid valve insufficiency has been developed and used in 25 patients. Fifteen patients have been recatheterized, and the correlation between the intraoperative and postoperative findings has been consistent. PMID:15226931

Progranulin Overproduction Due to Fli-1 Deficiency Contributes to the Resistance of Dermal Fibroblasts to Tumor Necrosis Factor in Systemic Sclerosis.

Science.gov (United States)

Ichimura, Yohei; Asano, Yoshihide; Akamata, Kaname; Noda, Shinji; Taniguchi, Takashi; Takahashi, Takehiro; Toyama, Tetsuo; Tada, Yayoi; Sugaya, Makoto; Sato, Shinichi; Kadono, Takafumi

2015-12-01

Progranulin is a growth factor that is active in wound repair and is an antagonist of tumor necrosis factor (TNF) receptors, regulating fibroblast activation, angiogenesis, and inflammation. Because long-standing activation of gene programs related to wound healing is a hallmark of systemic sclerosis (SSc), we sought to investigate the role of progranulin in SSc. Progranulin expression levels in human and murine skin samples were determined by immunohistochemical analysis and quantitative reverse transcription-polymerase chain reaction. The role of progranulin in fibroblast activation was examined using a gene-silencing technique. Progranulin levels in serum obtained from 60 patients with SSc and 16 healthy control subjects were determined by enzyme-linked immunosorbent assay. Progranulin expression was increased in SSc dermal fibroblasts compared with normal dermal fibroblasts, both in vivo and in vitro. Transcription factor Fli-1, a deficiency of which is involved in the activation of SSc dermal fibroblasts, served as a potent repressor of the progranulin gene, and Fli-1(+/-) mice and bleomycin-treated wild-type mice exhibited up-regulated expression of progranulin in dermal fibroblasts. SSc dermal fibroblasts were resistant to the antifibrotic effect of TNF, but this resistance was reversed by gene silencing of progranulin. Serum progranulin levels were elevated in patients with early diffuse cutaneous SSc (dcSSc), especially in those with inflammatory skin symptoms, and were positively correlated with the C-reactive protein level. Progranulin overproduction due to Fli-1 deficiency may contribute to the constitutive activation of SSc dermal fibroblasts by antagonizing the antifibrotic effect of TNF. Progranulin may also be involved in the inflammatory process associated with progressive skin sclerosis in early dcSSc. © 2015, American College of Rheumatology.

In vitro study of RRS HA injectable mesotherapy/biorevitalization product on human skin fibroblasts and its clinical utilization.

Science.gov (United States)

Deglesne, Pierre-Antoine; Arroyo, Rodrigo; Ranneva, Evgeniya; Deprez, Philippe

2016-01-01

Mesotherapy/biorevitalization with hyaluronic acid (HA) is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS(®) (Repairs, Refills, Stimulates) HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15%) and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts.

Inherited DNA repair defects in H. sapiens: their relation to uv-associated processes in xeroderma pigmentosum

International Nuclear Information System (INIS)

Robbins, J.H.; Kraemer, K.H.; Andrews, A.D.

1976-01-01

Xeroderma pigmentosum (XP) is an autosomal recessive disease in which patients develop pigmentation abnormalities and numerous malignancies on areas of skin exposed to sunlight. Some XP patients have neurological abnormalities in addition to their cutaneous pathology. Genetic defects in DNA repair have now been found in all studied XP patients. Here, we shall review and present studies relating the different inherited DNA repair defects of XP to several uv-associated processes. Peripheral blood lymphocytes and skin fibroblasts obtained from patients were cultured and the uv-induced thymidine incorporation in DNA was measured by autoradiography or by scintillation spectroscopy

Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

Science.gov (United States)

Sakai, Yusuke; Koike, Makiko; Kawahara, Daisuke; Hasegawa, Hideko; Murai, Tomomi; Yamanouchi, Kosho; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Fujita, Fumihiko; Kuroki, Tamotsu; Eguchi, Susumu

2018-03-05

Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×10 3  cells/cm 2 . Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

CTRP6 inhibits fibrogenesis in TGF-β1-stimulated human dermal fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Fan, Rong-hui, E-mail: fan_ronghuixa@163.com [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China); Zhu, Xiu-mei; Sun, Yao-wen [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China); Peng, Hui-zi [Department of Cosmetology Plastic Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061 (China); Wu, Hang-li; Gao, Wen-jie [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China)

2016-07-08

Skin fibrosis is characterized by excessive proliferation of fibroblasts and overproduction of extracellular matrix (ECM). C1q/tumor necrosis factor-related protein 6 (CTRP6), a member of CTRPs, has been involved in the development of cardiac fibrosis. However, the function and detailed regulatory mechanism of CTRP6 in skin fibrosis remain unclear. The aim of this study was to investigate the effect of CTRP6 on the activation of human dermal fibroblasts. Our results showed that CTRP6 was lowly expressed in scar tissues and transforming growth factor-β1 (TGF-β1)-treated dermal fibroblasts. CTRP6 overexpression significantly inhibited the proliferation of dermal fibroblasts, as well as suppressed the expression of ECM in TGF-β1-treated dermal fibroblasts. Furthermore, CTRP6 overexpression markedly inhibited TGF-β1-induced phosphorylation of Smad3 in dermal fibroblasts. In conclusion, the data reported here demonstrate that CTRP6 is able to inhibit the proliferation and ECM expression in human dermal fibroblasts through suppressing the TGF-β1/Smad3 signaling pathway. These findings suggest that CTRP6 may be a potential therapeutic target for the prevention of skin fibrosis. -- Highlights: •CTRP6 expression was decreased in scar tissues and TGF-β1-treated dermal fibroblasts. •CTRP6 inhibits TGF-β1-induced the proliferation of dermal fibroblasts. •CTRP6 inhibits expression of collagen type I and α-SMA. •CTRP6 inhibits the activation of TGF-β1/Smad3 signaling pathway in dermal fibroblasts.

CTRP6 inhibits fibrogenesis in TGF-β1-stimulated human dermal fibroblasts

International Nuclear Information System (INIS)

Fan, Rong-hui; Zhu, Xiu-mei; Sun, Yao-wen; Peng, Hui-zi; Wu, Hang-li; Gao, Wen-jie

2016-01-01

Skin fibrosis is characterized by excessive proliferation of fibroblasts and overproduction of extracellular matrix (ECM). C1q/tumor necrosis factor-related protein 6 (CTRP6), a member of CTRPs, has been involved in the development of cardiac fibrosis. However, the function and detailed regulatory mechanism of CTRP6 in skin fibrosis remain unclear. The aim of this study was to investigate the effect of CTRP6 on the activation of human dermal fibroblasts. Our results showed that CTRP6 was lowly expressed in scar tissues and transforming growth factor-β1 (TGF-β1)-treated dermal fibroblasts. CTRP6 overexpression significantly inhibited the proliferation of dermal fibroblasts, as well as suppressed the expression of ECM in TGF-β1-treated dermal fibroblasts. Furthermore, CTRP6 overexpression markedly inhibited TGF-β1-induced phosphorylation of Smad3 in dermal fibroblasts. In conclusion, the data reported here demonstrate that CTRP6 is able to inhibit the proliferation and ECM expression in human dermal fibroblasts through suppressing the TGF-β1/Smad3 signaling pathway. These findings suggest that CTRP6 may be a potential therapeutic target for the prevention of skin fibrosis. -- Highlights: •CTRP6 expression was decreased in scar tissues and TGF-β1-treated dermal fibroblasts. •CTRP6 inhibits TGF-β1-induced the proliferation of dermal fibroblasts. •CTRP6 inhibits expression of collagen type I and α-SMA. •CTRP6 inhibits the activation of TGF-β1/Smad3 signaling pathway in dermal fibroblasts.

Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

DEFF Research Database (Denmark)

Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders

2002-01-01

The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations...... days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When...... cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether...

Preclinical study of mouse pluripotent parthenogenetic embryonic stem cell derivatives for the construction of tissue-engineered skin equivalent.

Science.gov (United States)

Rao, Yang; Cui, Jihong; Yin, Lu; Liu, Wei; Liu, Wenguang; Sun, Mei; Yan, Xingrong; Wang, Ling; Chen, Fulin

2016-10-22

Embryonic stem cell (ESC) derivatives hold great promise for the construction of tissue-engineered skin equivalents (TESE). However, harvesting of ESCs destroys viable embryos and may lead to political and ethical concerns over their application. In the current study, we directed mouse parthenogenetic embryonic stem cells (pESCs) to differentiate into fibroblasts, constructed TESE, and evaluated its function in vivo. The stemness marker expression and the pluripotent differentiation ability of pESCs were tested. After embryoid body (EB) formation and adherence culture, mesenchymal stem cells (MSCs) were enriched and directed to differentiate into fibroblastic lineage. Characteristics of derived fibroblasts were assessed by quantitative real-time PCR and ELISA. Functional ability of the constructed TESE was tested by a mouse skin defects repair model. Mouse pESCs expressed stemness marker and could form teratoma containing three germ layers. MSCs could be enriched from outgrowths of EBs and directed to differentiate into fibroblastic lineage. These cells express a high level of growth factors including FGF, EGF, VEGF, TGF, PDGF, and IGF1, similar to those of ESC-derived fibroblasts and mouse fibroblasts. Seeded into collagen gels, the fibroblasts derived from pESCs could form TESE. Mouse skin defects could be successfully repaired 15 days after transplantation of TESE constructed by fibroblasts derived from pESCs. pESCs could be induced to differentiate into fibroblastic lineage, which could be applied to the construction of TESE and skin defect repair. Particularly, pESC derivatives avoid the limitations of political and ethical concerns, and provide a promising source for regenerative medicine.

Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells.

Science.gov (United States)

Poletto, Mattia; Yang, Di; Fletcher, Sally C; Vendrell, Iolanda; Fischer, Roman; Legrand, Arnaud J; Dianov, Grigory L

2017-09-29

Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM protein function. Neurodegeneration and cancer predisposition, both hallmarks of A-T, are likely to emerge as a consequence of the persistent oxidative stress and DNA damage observed in this disease. Surprisingly however, despite these severe features, a lack of functional ATM is still compatible with early life, suggesting that adaptation mechanisms contributing to cell survival must be in place. Here we address this gap in our knowledge by analysing the process of human fibroblast adaptation to the lack of ATM. We identify profound rearrangement in cellular proteostasis occurring very early on after loss of ATM in order to counter protein damage originating from oxidative stress. Change in proteostasis, however, is not without repercussions. Modulating protein turnover in ATM-depleted cells also has an adverse effect on the DNA base excision repair pathway, the major DNA repair system that deals with oxidative DNA damage. As a consequence, the burden of unrepaired endogenous DNA lesions intensifies, progressively leading to genomic instability. Our study provides a glimpse at the cellular consequences of loss of ATM and highlights a previously overlooked role for proteostasis in maintaining cell survival in the absence of ATM function. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Aging and DNA repair capability. [Review

Energy Technology Data Exchange (ETDEWEB)

Tice, R R

1977-01-01

A review of the literature on DNA repair processes in relation to aging is presented under the following headings: DNA repair processes; age-related occurrence of unrepaired DNA lesions; DNA repair capability as a function of age; tissue-specific DNA repair capability; acceleration of the aging process by exposure to DNA damaging agents; human genetic syndromes; and longevity and DNA repair processes. (HLW)

Radiosensitivity of fibroblasts obtained from a cafe-au-lait spot and normal-appearing skin of a patient with neurofibromatosis (NF-6)

International Nuclear Information System (INIS)

Hannan, M.A.; Smith, B.P.; Sigut, D.; Sackey, K.

1990-01-01

Fibroblast cells derived from a cafe-au-lait spot and normal-appearing skin of a neurofibromatosis (NF-6) patient were studied for radiosensitivity in comparison with two normal cell lines used as controls. No difference in radiosensitivity was observed between the patient's cell lines and the controls using acute gamma-irradiation. However, a markedly increased radiosensitivity of the fibroblasts obtained from the patient's skin of normal appearance was demonstrated after chronic gamma-irradiation. The cells from the cafe-au-lait spot showed intermediate sensitivity to chronic irradiation as compared with the control cell lines and the fibroblasts derived from the normal skin of the patient. These results showed the usefulness of chronic irradiation in detecting increased cellular radiosensitivity which may result from a unique DNA repair defect in an NF patient. We suggest that enhanced genetic changes in radiosensitive NF patients may lead to formation of cafe-au-lait lesions and certain tumors. Such a transformation may be associated with production of radiotolerant cells

Effects of ascorbate and B-aminopropionitrile on tendon fibroblast migration in vitro

International Nuclear Information System (INIS)

Nelson, J.M.; Cohen, I.K.; Diegelmann, R.F.

1986-01-01

Ascorbate (Asc) stimulates collagen synthesis and hydroxylation whereas beta-aminopropionitrile (BAPN) inhibits collagen cross-link formation. In this study, an in vitro model employing isolated chicken tendon biopsies (2 mm in dia.) in a fibrin clot has been used to examine the effect of Asc and BAPN on tendon fibroblast migration and proliferation. After 5 days in culture with either Asc (0.1 mM), or BAPN (0.5, 1.0, 2.0 mM), cell migration was measured using an automatic planimeter and cell proliferation was quantitated by 125 IUDR incorporation into DNA. In cultures treated with Asc alone, cell migration was enhanced by approximately 33% compared to controls without ascorbate (5.9 mm 2 vs. 4.4 mm 2 ; p < 0.05) with no significant effect on cell proliferation. In contrast, cultures incubated with increasing concentrations of BAPN (0.5 - 2.0 mM) displayed a dose-dependent decrease (up to 9.6-fold at 2 mM) in fibroblast migration into the clot. The inhibitory effect of BAPN on cell migration was not due to a corresponding inhibition of fibroblast proliferation. These observations suggest that Asc enhanced collagen formation and thus allowed greater cell migration into the fibrin matrix. In contrast, in the absence of a mature, cross-linked collagen matrix following exposure to BAPN, cell migration was sub-optimal. These in vitro studies support the hypothesis that modulation of the collagen matrix may be a useful means of regulating tissue repair in vivo

The hallmarks of fibroblast ageing.

Science.gov (United States)

Tigges, Julia; Krutmann, Jean; Fritsche, Ellen; Haendeler, Judith; Schaal, Heiner; Fischer, Jens W; Kalfalah, Faiza; Reinke, Hans; Reifenberger, Guido; Stühler, Kai; Ventura, Natascia; Gundermann, Sabrina; Boukamp, Petra; Boege, Fritz

2014-06-01

Ageing is influenced by the intrinsic disposition delineating what is maximally possible and extrinsic factors determining how that frame is individually exploited. Intrinsic and extrinsic ageing processes act on the dermis, a post-mitotic skin compartment mainly consisting of extracellular matrix and fibroblasts. Dermal fibroblasts are long-lived cells constantly undergoing damage accumulation and (mal-)adaptation, thus constituting a powerful indicator system for human ageing. Here, we use the systematic of ubiquitous hallmarks of ageing (Lopez-Otin et al., 2013, Cell 153) to categorise the available knowledge regarding dermal fibroblast ageing. We discriminate processes inducible in culture from phenomena apparent in skin biopsies or primary cells from old donors, coming to the following conclusions: (i) Fibroblasts aged in culture exhibit most of the established, ubiquitous hallmarks of ageing. (ii) Not all of these hallmarks have been detected or investigated in fibroblasts aged in situ (in the skin). (iii) Dermal fibroblasts aged in vitro and in vivo exhibit additional features currently not considered ubiquitous hallmarks of ageing. (iv) The ageing process of dermal fibroblasts in their physiological tissue environment has only been partially elucidated, although these cells have been a preferred model of cell ageing in vitro for decades. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Fibronectin potentiates topical erythropoietin-induced wound repair in diabetic mice.

Science.gov (United States)

Hamed, Saher; Ullmann, Yehuda; Egozi, Dana; Daod, Essam; Hellou, Elias; Ashkar, Manal; Gilhar, Amos; Teot, Luc

2011-06-01

Diabetes mellitus disrupts all phases of the wound repair cascade and leads to development of chronic wounds. We previously showed that topical erythropoietin (EPO) can promote wound repair in diabetic rats. Fibronectin (FN) has a critical role throughout the process of wound healing, yet it is deficient in wound tissues of diabetic patients. Therefore, we investigated the effect of topical treatment of both EPO and FN (EPO/FN) on wound repair in diabetic mice. Full-thickness excisional skin wounds in diabetic and nondiabetic mice were treated with a cream containing vehicle, EPO, FN, or EPO/FN. We assessed the rate of wound closure, angiogenesis, apoptosis, and expression of inflammatory cytokines, endothelial nitric oxide synthase (eNOS) and β1-integrin, in the wound tissues. We also investigated the effect of EPO, FN, and EPO/FN on human dermal microvascular endothelial cells and fibroblasts cultured on fibrin-coated plates, or in high glucose concentrations. EPO/FN treatment significantly increased the rate of wound closure and this effect was associated with increased angiogenesis, increased eNOS and β1-integrin expression, and reduced expression of inflammatory cytokines and apoptosis. Our findings show that EPO and FN have an additive effect on wound repair in diabetic mice.

Excision repair in ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome, and Bloom's syndrome after treatment with ultraviolet radiation and N-acetoxy-2-acetylaminofluorene

International Nuclear Information System (INIS)

Ahmed, F.E.; Setlow, R.B.

1978-01-01

Excision repair of damage due to ultraviolet radiation, N-acetoxy-2-acetylaminofluorene and a combination of both agents was studied in normal human fibroblasts and various cells from cancer prone patients (ataxia telangiectasia, Fanconi's anemia, Cockayne syndrome and Bloom's syndrome). Three methods giving similar results were used: unscheduled DNA synthesis by radioautography, photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and loss of sites sensitive to an ultraviolet endonuclease. All cell lines were proficient in repair of ultraviolet and acetoxy acetylaminofluorene damage and at saturation doses of both agents repair was additive. We interpret these data as indicating that the rate limiting step in excision repair of ultraviolet and acetoxy acetylaminofluorene is different and that there are different enzyme(s) working on incision of both types of damages. (Auth.)

Fluorescent-light-induced lethality and DNA repair in normal and xeroderma pigmentosum fibroblasts

International Nuclear Information System (INIS)

Ritter, M.A.; Williams, J.R.

1981-01-01

Cell survival and induction of endonuclease-sensitive sites in DNA were measured in human fibroblast cells exposed to fluorescent light or germicidal ultraviolet light. Cells from a xeroderma pigmentosum patient were hypersensitive to cell killing by fluorescent light, although less so than for germicidal ultraviolet light. Xeroderma pigmentosum cells were deficient in the removal of fluorescent light-induced endonuclease sites that are probably pyrimidine dimers, and both the xeroderma pigmentosum and normal cells removed these sites with kinetics indistinguishable from those for ultraviolet light-induced sites. A comparison of fluorescent with ultraviolet light data demonstrates that there are markedly fewer pyrimidine dimers per lethal event for fluorescent than for ultraviolet light, suggesting a major role for non-dimer damage in fluorescent lethality. (Auth.)

Wound healing properties of ethyl acetate fraction of Moringa oleifera in normal human dermal fibroblasts

Directory of Open Access Journals (Sweden)

Sivapragasam Gothai

2016-03-01

Full Text Available Background/Aim: Wounds are the outcome of injuries to the skin that interrupt the soft tissue. Healing of a wound is a complex and long-drawn-out process of tissue repair and remodeling in response to injury. A large number of plants are used by folklore traditions for treatment of cuts, wounds and burns. Moringa oleifera is an herb used as traditional folk medicine for the treatment of various skin wounds and associated diseases. The underlying mechanisms of wound healing activity of ethyl acetate fraction of M. oleifera leaves extract are completely unknown. Methods: In the current study, ethyl acetate fraction of Moringa oleifera leaves was investigated for its efficacy on cell viability, proliferation and migration (wound closure rate in human normal dermal fibroblast cells. Results: Results revealed that lower concentration (12.5 µg/ml, 25 µg/ml, and 50 µg/ml of ethyl acetate fraction of M. oleifera leaves showed remarkable proliferative and migratory effect on normal human dermal fibroblasts. Conclusion: The present study suggested that ethyl acetate fraction of M. oleifera leaves might be a potential therapeutic agent for skin wound healing by promoting fibroblast proliferation and migration through increasing the wound closure rate corroborating its traditional use. [J Complement Med Res 2016; 5(1.000: 1-6

Functions and Mechanisms of Fibroblast Growth Factor (FGF Signalling in Drosophila melanogaster

Directory of Open Access Journals (Sweden)

Hans-Arno J. Müller

2013-03-01

Full Text Available Intercellular signalling via growth factors plays an important role in controlling cell differentiation and cell movements during the development of multicellular animals. Fibroblast Growth Factor (FGF signalling induces changes in cellular behaviour allowing cells in the embryo to move, to survive, to divide or to differentiate. Several examples argue that FGF signalling is used in multi-step morphogenetic processes to achieve and maintain a transitional state of the cells required for the control of cell fate. In the genetic model Drosophila melanogaster, FGF signalling via the receptor tyrosine kinases Heartless (Htl and Breathless (Btl is particularly well studied. These FGF receptors affect gene expression, cell shape and cell–cell interactions during mesoderm layer formation, caudal visceral muscle (CVM formation, tracheal morphogenesis and glia differentiation. Here, we will address the current knowledge of the biological functions of FGF signalling in the fly on the tissue, at a cellular and molecular level.

Production of ascorbic acid releasing biomaterials for pelvic floor repair.

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Mangır, Naşide; Bullock, Anthony J; Roman, Sabiniano; Osman, Nadir; Chapple, Christopher; MacNeil, Sheila

2016-01-01

An underlying abnormality in collagen turnover is implied in the occurrence of complications and recurrences after mesh augmented pelvic floor repair surgeries. Ascorbic acid is a potent stimulant of collagen synthesis. The aim of this study is to produce ascorbic acid releasing poly-lactic acid (PLA) scaffolds and evaluate them for their effects on extracellular matrix production and the strength of the materials. Scaffolds which contained either l-ascorbic acid (AA) and Ascorbate-2-Phosphate (A2P) were produced with emulsion electrospinning. The release of both drugs was measured by UV spectrophotometry. Human dermal fibroblasts were seeded on scaffolds and cultured for 2weeks. Cell attachment, viability and total collagen production were evaluated as well as mechanical properties. No significant differences were observed between AA, A2P, Vehicle and PLA scaffolds in terms of fibre diameter and pore size. The encapsulation efficiency and successful release of both AA and A2P were demonstrated. Both AA and A2P containing scaffolds were significantly more hydrophilic and stronger in both dry and wet states compared to PLA scaffolds. Fibroblasts produced more collagen on scaffolds containing either AA or A2P compared to cells grown on control scaffolds. This study is the first to directly compare the two ascorbic acid derivatives in a tissue engineered scaffold and shows that both AA and A2P releasing electrospun PLA scaffolds increased collagen production of fibroblasts to similar extents but AA scaffolds seemed to be more hydrophilic and stronger compared to A2P scaffolds. Mesh augmented surgical repair of the pelvic floor currently relies on non-degradable materials which results in severe complications in some patients. There is an unmet and urgent need for better pelvic floor repair materials. Our current understanding suggests that the ideal material should be able to better integrate into sites of implantation both biologically and mechanically. The impact of

The effect of tranilast on fibroblast activation protein α (FAP-α expression in normal and keloid fibroblasts in vitro

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Paweł P. Antończak

2017-07-01

Full Text Available Introduction . Tranilast (N-(3’,4’-demethoxycinnamoyl-anthranilic acid is an anti-allergic drug. Its mechanism of action is based on the inhibition of antigen-induced release of chemical mediators from mast cells and basophils. It also reveals antifibroproliferative activities. These properties of tranilast are used in the treatment of hypertrophic scars and keloids. Keloids are characterized by incorrect extracellular matrix components turnover. Fibroblasts derived from keloids reveal overproduction of collagen type I and decreased degradation of extracellular matrix in comparison with normal fibroblasts. Fibroblast activation protein α (FAP-α may play an important role in remodeling of extracellular matrix and the invasive properties of keloids. Objective . In the present study, the effect of tranilast on expression of FAP-α gene and its protein was evaluated in normal human dermal fibroblasts and fibroblasts derived from keloids cultured in vitro . Materials and methods. In the first stage of the study, the influence of tranilast on cell viability was estimated. The second stage of the study included the quantitative evaluation of FAP-α mRNA expression in normal and keloid fibroblasts treated with tranilast. The third stage of the study comprised fibroblast activation protein α expression analysis in the examined cells treated with tranilast. Results and conclusions . The expression of FAP-α gene and fibroblast activation protein α is higher in keloid fibroblasts. Tranilast at concentrations of 3 μM and 30 μM up-regulated mRNA FAP-α expression in normal fibroblasts but did not influence keloid fibroblasts. The drug, at concentrations of 30 μM and 300 μM up-regulated fibroblast activation protein α expression in normal fibroblasts and did not influence keloid fibroblasts. Tranilast antiproliferative effect is not associated with FAP-α expression in keloid fibroblasts.

Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

International Nuclear Information System (INIS)

Walter, M.N.M.; Wright, K.T.; Fuller, H.R.; MacNeil, S.; Johnson, W.E.B.

2010-01-01

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

International Nuclear Information System (INIS)

Smith, C.A.; Hanawalt, P.C.

1978-01-01

The repair mode of DNA replication has been demonstrated in isolated nuclei from uv-irradiated human cells. Nuclei are incubated in a mixture containing [ 3 H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The 3 H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is uv-dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg 2+ -dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote uv-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms

Polyphenol-rich strawberry extract protects human dermal fibroblasts against hydrogen peroxide oxidative damage and improves mitochondrial functionality.

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Giampieri, Francesca; Alvarez-Suarez, José M; Mazzoni, Luca; Forbes-Hernandez, Tamara Y; Gasparrini, Massimiliano; Gonzàlez-Paramàs, Ana M; Santos-Buelga, Celestino; Quiles, José L; Bompadre, Stefano; Mezzetti, Bruno; Battino, Maurizio

2014-06-11

Strawberry bioactive compounds are widely known to be powerful antioxidants. In this study, the antioxidant and anti-aging activities of a polyphenol-rich strawberry extract were evaluated using human dermal fibroblasts exposed to H2O2. Firstly, the phenol and flavonoid contents of strawberry extract were studied, as well as the antioxidant capacity. HPLC-DAD analysis was performed to determine the vitamin C and β-carotene concentration, while HPLC-DAD/ESI-MS analysis was used for anthocyanin identification. Strawberry extract presented a high antioxidant capacity, and a relevant concentration of vitamins and phenolics. Pelargonidin- and cyanidin-glycosides were the most representative anthocyanin components of the fruits. Fibroblasts incubated with strawberry extract and stressed with H2O2 showed an increase in cell viability, a smaller intracellular amount of ROS, and a reduction of membrane lipid peroxidation and DNA damage. Strawberry extract was also able to improve mitochondrial functionality, increasing the basal respiration of mitochondria and to promote a regenerative capacity of cells after exposure to pro-oxidant stimuli. These findings confirm that strawberries possess antioxidant properties and provide new insights into the beneficial role of strawberry bioactive compounds on protecting skin from oxidative stress and aging.

Clinical results and functional outcomes after direct intralaminar screw repair of spondylolysis.

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Menga, Emmanuel N; Kebaish, Khaled M; Jain, Amit; Carrino, John A; Sponseller, Paul D

2014-01-01

Prospective analysis. Our objective was to analyze clinical and functional results of patients with spondylolysis treated via direct intralaminar screw fixation and autograft, a minimally invasive and motion-preserving surgery. Spondylolysis is usually treated nonoperatively; multiple surgical techniques are available when nonoperative measures fail. No studies evaluate the clinical and functional outcomes and their correlation with pars defect size and disc morphology on magnetic resonance imaging. We reviewed patients with spondylolysis treated with intralaminar screw fixation and bone grafting from 2000 through 2010. Of the 31 patients (mean age, 16 yr; range, 10-37 yr), 25 (81%) were competitive athletes. Preoperative computed tomographic scans were used to measure the pars defect size, and preoperative magnetic resonance images were graded using Pfirrmann classification for correlation with postoperative outcomes. Student t test was used for analysis (significance, P spondylolysis repair for persistent pain starting 18 months after intralaminar screw fixation, 2 patients sustained unilateral intralaminar screw fractures at L5, and 1 patient required irrigation and debridement for a superficial postoperative infection. There was no correlation among preoperative magnetic resonance imaging disc morphology, defect size on computed tomography, patient age, and clinical outcomes. Direct repair of spondylolysis with intralaminar screws offers a low profile, reliable treatment with good functional outcome and a low complication rate in active patients. 4.

Chemical Cocktails Enable Hepatic Reprogramming of Mouse Fibroblasts with a Single Transcription Factor

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Ren Guo

2017-08-01

Full Text Available Liver or hepatocytes transplantation is limited by the availability of donor organs. Functional hepatocytes independent of the donor sources may have wide applications in regenerative medicine and the drug industry. Recent studies have demonstrated that chemical cocktails may induce reprogramming of fibroblasts into a range of functional somatic cells. Here, we show that mouse fibroblasts can be transdifferentiated into the hepatocyte-like cells (iHeps using only one transcription factor (TF (Foxa1, Foxa2, or Foxa3 plus a chemical cocktail. These iHeps show typical epithelial morphology, express multiple hepatocyte-specific genes, and acquire hepatocyte functions. Genetic lineage tracing confirms the fibroblast origin of these iHeps. More interestingly, these iHeps are expandable in vitro and can reconstitute the damaged hepatic tissues of the fumarylacetoacetate hydrolase-deficient (Fah−/− mice. Our study provides a strategy to generate functional hepatocyte-like cells by using a single TF plus a chemical cocktail and is one step closer to generate the full-chemical iHeps.

The Fanconi anemia DNA repair pathway: structural and functional insights into a complex disorder.

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Walden, Helen; Deans, Andrew J

2014-01-01

Mutations in any of at least sixteen FANC genes (FANCA-Q) cause Fanconi anemia, a disorder characterized by sensitivity to DNA interstrand crosslinking agents. The clinical features of cytopenia, developmental defects, and tumor predisposition are similar in each group, suggesting that the gene products participate in a common pathway. The Fanconi anemia DNA repair pathway consists of an anchor complex that recognizes damage caused by interstrand crosslinks, a multisubunit ubiquitin ligase that monoubiquitinates two substrates, and several downstream repair proteins including nucleases and homologous recombination enzymes. We review progress in the use of structural and biochemical approaches to understanding how each FANC protein functions in this pathway.

p53 regulates the repair of DNA double-strand breaks by both homologous and non-homologous recombination

International Nuclear Information System (INIS)

Willers, H.; Powell, S.N.; Dahm-Daphi, J.

2003-01-01

Full text: p53 is known to suppress spontaneous homologous recombination (HR), while its role in non-homologous recombination (NHR) remains to be clarified. Here, we sought to determine the influence of p53 on the repair of chromosomal double-strand breaks (DSBs) by HR or NHR using specially designed recombination substrates that integrate into the genome. Isogenic mouse fibroblast pairs with or without expression of exogenous p53 protein were utilized. A reporter plasmid carrying a mutated XGPRT gene was chromosomally integrated and DSBs were generated within the plasmid by the I-SceI endonuclease. Subsequent homology-mediated repair from an episomal donor resulted in XGPRT reconstitution and cellular resistance to a selection antibiotic. Analogously, the repair of chromosomal I-SceI breaks by NHR using another novel reporter plasmid restored XGPRT translation. For p53-null cells, the mean frequency of I-SceI break repair via HR was 5.5 x 10 -4 . The p53-Val135 mutant, which previously has been shown to suppress spontaneous HR by 14-fold employing the same cell system and reporter gene, only caused a 2- to 3-fold suppression of break-induced HR. In contrast, a dramatic effect of p53 on repair via NHR was found. Preliminary sequence analysis indicated that there was at least a 1000-fold reduction of illegitimate repair events resulting in loss of sequence at the break sites. The observed effects were mediated by p53 mutants defective in regulation of the cell-cycle and apoptosis. The main findings were: (1) p53 virtually blocked illegitimate rejoining of chromosomal ends. (2) The suppression of homologous DSB repair was less pronounced than the inhibition of spontaneous HR. We hypothesize that p53 allows to a certain extent error-free homology-dependent repair to proceed, while blocking error-prone NHR. The data support and extent a previous model, in which p53 maintains genomic stability by regulating recombination independently of its transactivation function

High inorganic phosphate causes DNMT1 phosphorylation and subsequent fibrotic fibroblast activation

Energy Technology Data Exchange (ETDEWEB)

Tan, Xiaoying [Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Xu, Xingbo [Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Zeisberg, Elisabeth M. [Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); German Center for Cardiovascular Research (DZHK), Göttingen (Germany); Zeisberg, Michael, E-mail: mzeisberg@med.uni-goettingen.de [Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); German Center for Cardiovascular Research (DZHK), Göttingen (Germany)

2016-04-08

Phosphate is an essential constituent of critical cellular functions including energy metabolism, nucleic acid synthesis and phosphorylation-dependent cell signaling. Increased plasma phosphate levels are an independent risk factor for lowered life-expectancy as well as for heart and kidney failure. Nevertheless, direct cellular effects of elevated phosphate concentrations within the microenvironment are poorly understood and have been largely neglected in favor of phosphor-regulatory hormones. Because interstitial fibrosis is the common determinant of chronic progressive kidney disease, and because fibroblasts are major mediators of fibrogenesis, we here explored the effect of high extracellular phosphate levels on renal fibroblasts. We demonstrate that high inorganic phosphate directly induces fibrotic fibroblast activation associated with increased proliferative activity, increased expression of α-smooth muscle actin and increased synthesis of type I collagen. We further demonstrate that such fibroblast activation is dependent on phosphate influx, aberrant phosphorylation of DNA methyltransferase DNMT1 and aberrant CpG island promoter methylation. In summary, our studies demonstrate that elevated phosphate concentrations induce pro-fibrotic fibroblast activation independent of phospho-regulatory hormones. - Highlights: • We exposed human kidney fibroblasts to media containing 1 mM or 3 mM phosphate. • Increased phosphate influx causes phosphorylation of DNA methyltransferase Dnmt1. • Phosphorylated Dnmt1 causes promoter methylation and transcriptional silencing of RASAL1. • Depletion of RASAL1 causes increased intrinsic Ras-GTP activity and fibroblast activation. • Inorganic phosphate causes fibroblast activation independent of phospho-regulatory hormones.

Monophasic Pulsed 200-?A Current Promotes Galvanotaxis With Polarization of Actin Filament and Integrin ?2?1 in Human Dermal Fibroblasts

OpenAIRE

Uemura, Mikiko; Maeshige, Noriaki; Koga, Yuka; Ishikawa-Aoyama, Michiko; Miyoshi, Makoto; Sugimoto, Masaharu; Terashi, Hiroto; Usami, Makoto

2016-01-01

Objective: The monophasic pulsed microcurrent is used to promote wound healing, and galvanotaxis regulation has been reported as one of the active mechanisms in the promotion of tissue repair with monophasic pulsed microcurrent. However, the optimum monophasic pulsed microcurrent parameters and intracellular changes caused by the monophasic pulsed microcurrent have not been elucidated in human dermal fibroblasts. The purpose of this study was to investigate the optimum intensity for promoting...

Differential response of human and rodent cell lines to chemical inhibition of the repair of potentially lethal damage

Energy Technology Data Exchange (ETDEWEB)

Little, J.B.; Ueno, A.M.; Dahlberg, W.K.

1989-07-01

We have examined the effects of several classes of metabolic inhibitors on the repair of potentially lethal damage in density-inhibited cultures of two rodent and two human cell systems which differ in their growth characteristics. Aphidicolin, 1-..beta..-D-arabinofuranosylcytosine (ara-C) and hydroxyurea showed no effect on PLD repair, whereas the effects of 9-..beta..-D-arabinofuranosyladenine (ara-A) and 3-aminobenzamide (3-AB) were cell line dependent. For example, 3-AB suppressed PLD repair almost completely in CHO cells, but showed no inhibitory effects in human diploid fibroblasts. These results indicate that inhibitors of DNA replication and poly(ADP-ribose) synthesis are not efficient inhibitors of cellular recovery in irradiated cells and, moreover, that such effects may be cell line dependent.

Curcumin induces differential expression of cytoprotective enzymes but similar apoptotic responses in fibroblasts and myofibroblasts

NARCIS (Netherlands)

Lundvig, D.M.S.; Pennings, S.W.C.; Brouwer, K.M.; Mtaya-Mlangwa, M.; Mugonzibwa, E.A.; Kuijpers-Jagtman, A.M.; Hoff, J.W. Von den; Wagener, F.A.D.T.G.

2015-01-01

Excessive extracellular matrix (ECM) deposition and tissue contraction after injury can lead to esthetic and functional problems. Fibroblasts and myofibroblasts activated by transforming growth factor (TGF)-beta1 play a key role in these processes. The persistence of (myo)fibroblasts and their

Dysregulated proinflammatory and fibrogenic phenotype of fibroblasts in cystic fibrosis.

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François Huaux

Full Text Available Morbi-mortality in cystic fibrosis (CF is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy.

Binding, uptake, and release of nicotine by human gingival fibroblasts

International Nuclear Information System (INIS)

Hanes, P.J.; Schuster, G.S.; Lubas, S.

1991-01-01

Previous studies of the effects of nicotine on fibroblasts have reported an altered morphology and attachment of fibroblasts to substrates and disturbances in protein synthesis and secretion. This altered functional and attachment response may be associated with changes in the cell membrane resulting from binding of the nicotine, or to disturbances in cell metabolism as a result of high intracellular levels of nicotine. The purpose of the present study, therefore, was to (1) determine whether gingival fibroblasts bound nicotine and if any binding observed was specific or non-specific in nature; (2) determine whether gingival fibroblasts internalized nicotine, and if so, at what rate; (3) determine whether gingival fibroblasts also released nicotine back into the extracellular environment; and (4) if gingival fibroblasts release nicotine intact or as a metabolite. Cultures of gingival fibroblasts were prepared from gingival connective tissue biopsies. Binding was evaluated at 4 degree C using a mixture of 3 H-nicotine and unlabeled nicotine. Specific binding was calculated as the difference between 3 H-nicotine bound in the presence and absence of unlabeled nicotine. The cells bound 1.44 (+/- 0.42) pmols/10(6) cells in the presence of unlabeled nicotine and 1.66 (+/- 0.55) pmols/10(6) cells in the absence of unlabeled nicotine. The difference was not significant. Uptake of nicotine was measured at 37 degree C after treating cells with 3 H-nicotine for time periods up to 4 hours. Uptake in pmols/10(6) cells was 4.90 (+/- 0.34) at 15 minutes, 8.30 (+/- 0.75) at 30 minutes, 12.28 (+/- 2.62) at 1 hour and 26.31 (+/- 1.15) at 4 hours

DNA damage in cultured human skin fibroblasts exposed to excimer laser radiation

Energy Technology Data Exchange (ETDEWEB)

Rimoldi, D.; Miller, A.C.; Freeman, S.E.; Samid, D. (Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD (USA))

1991-06-01

Ultraviolet excimer lasers are being considered for use in a variety of refractive and therapeutic procedures, the long-term biologic consequences of which are unknown. The effect of sublethal doses of 193-nm laser radiation on cellular DNA was examined in cultured human skin fibroblasts. In contrast to 248 nm, treatments with the 193-nm laser radiation below 70 J/m2 did not cause significant pyrimidine dimer formation in the skin cells. This was indicated by the lack of excision repair activities (unscheduled DNA synthesis assay), and further demonstrated by direct analysis of pyrimidine dimers in DNA from irradiated cells. However, a low level of unscheduled DNA synthesis could be detected following irradiation at 193 nm with 70 J/m2. Both the 193-nm and 248-nm radiation were able to induce chromosomal aberrations, as indicated by a micronucleus assay. A dose-dependent increase in micronuclei frequency was observed 48 and 72 h after laser irradiation. These results indicate that exposure of actively replicating human skin fibroblasts to sublethal doses of either 193- or 248-nm laser radiation can result in genotoxicity.

Role of nuclear hexokinase II in DNA repair

International Nuclear Information System (INIS)

Khanna, S.; Bhatt, A.N.; Dwarakanath, B.S.; Kalaiarasan, P.; Brahmachari, V.

2012-01-01

A common signature of many cancer cells is a high glucose catabolic rate primarily due to the over expression of Type II hexokinase (HKII; responsible for the phosphorylation of glucose), generally known as cytosolic and mitochondrial bound enzyme that also suppresses cell death. Although, nuclear localization and transcriptional regulation of HKII has been reported in yeast; we and few others have recently demonstrated its nuclear localization in malignant cell lines. Interestingly, modification of a human glioma cell line (BMG-1) for enhancing glycolysis through mitochondrial respiration (OPMBMG cells) resulted in a higher nuclear localization of HKII as compared to the parental cells with concomitant increase in DNA repair and radio-resistance. Further, the glucose phosphorylation activity of the nuclear HKII was nearly 2 folds higher in the relatively more radioresistant HeLa cells (human cervical cancer cell line) as compared to MRC-5 cells (human normal lung fibroblast cell line). Therefore, we hypothesize that nuclear HKII facilitates DNA repair, in a hither to unknown mechanism, that may partly contribute to the enhanced resistance of highly glycolytic cells to radiation. Sequence alignment studies suggest that the isoenzymes, HKI and HKII share strong homology in the kinase active site, which is also found in few protein kinases. Interestingly HKI has been shown to phosphorylate H2A in-vitro. Further, in-silico protein-protein interaction data suggest that HKII can interact with several DNA repair proteins including ATM. Taken together; available experimental evidences as well as in-silico predictions strongly suggest that HKII may play a role in DNA repair by phosphorylation of certain DNA repair proteins. (author)

Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and fibroblasts.

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Nayak, Alok Ranjan; Shajahan, T K; Panfilov, A V; Pandit, Rahul

2013-01-01

Cardiac fibroblasts, when coupled functionally with myocytes, can modulate the electrophysiological properties of cardiac tissue. We present systematic numerical studies of such modulation of electrophysiological properties in mathematical models for (a) single myocyte-fibroblast (MF) units and (b) two-dimensional (2D) arrays of such units; our models build on earlier ones and allow for zero-, one-, and two-sided MF couplings. Our studies of MF units elucidate the dependence of the action-potential (AP) morphology on parameters such as [Formula: see text], the fibroblast resting-membrane potential, the fibroblast conductance [Formula: see text], and the MF gap-junctional coupling [Formula: see text]. Furthermore, we find that our MF composite can show autorhythmic and oscillatory behaviors in addition to an excitable response. Our 2D studies use (a) both homogeneous and inhomogeneous distributions of fibroblasts, (b) various ranges for parameters such as [Formula: see text], and [Formula: see text], and (c) intercellular couplings that can be zero-sided, one-sided, and two-sided connections of fibroblasts with myocytes. We show, in particular, that the plane-wave conduction velocity [Formula: see text] decreases as a function of [Formula: see text], for zero-sided and one-sided couplings; however, for two-sided coupling, [Formula: see text] decreases initially and then increases as a function of [Formula: see text], and, eventually, we observe that conduction failure occurs for low values of [Formula: see text]. In our homogeneous studies, we find that the rotation speed and stability of a spiral wave can be controlled either by controlling [Formula: see text] or [Formula: see text]. Our studies with fibroblast inhomogeneities show that a spiral wave can get anchored to a local fibroblast inhomogeneity. We also study the efficacy of a low-amplitude control scheme, which has been suggested for the control of spiral-wave turbulence in mathematical models for cardiac

Alcohol consumption negates estrogen-mediated myocardial repair in ovariectomized mice by inhibiting endothelial progenitor cell mobilization and function.

Science.gov (United States)

Mackie, Alexander R; Krishnamurthy, Prasanna; Verma, Suresh K; Thorne, Tina; Ramirez, Veronica; Qin, Gangjian; Abramova, Tatiana; Hamada, Hiromichi; Losordo, Douglas W; Kishore, Raj

2013-06-21

We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17β-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.

Low-Dose Hyper-Radiosensitivity Is Not a Common Effect in Normal Asynchronous and G2-Phase Fibroblasts of Cancer Patients

International Nuclear Information System (INIS)

Słonina, Dorota; Biesaga, Beata; Janecka, Anna; Kabat, Damian; Bukowska-Strakova, Karolina; Gasińska, Anna

2014-01-01

Purpose: In our previous study, using the micronucleus assay, a low-dose hyper-radiosensitivity (HRS)-like phenomenon was observed for normal fibroblasts of 2 of the 40 cancer patients investigated. In this article we report, for the first time, the survival response of primary fibroblasts from 25 of these patients to low-dose irradiation and answer the question regarding the effect of G2-phase enrichment on HRS elicitation. Methods and Materials: The clonogenic survival of asynchronous as well as G2-phase enriched fibroblast populations was measured. Separation of G2-phase cells and precise cell counting was performed using a fluorescence-activated cell sorter. Sorted and plated cells were irradiated with single doses (0.1-4 Gy) of 6-MV x-rays. For each patient, at least 4 independent experiments were performed, and the induced-repair model was fitted over the whole data set to confirm the presence of HRS effect. Results: The HRS response was demonstrated for the asynchronous and G2-phase enriched cell populations of 4 patients. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, G2-phase enrichment had no effect on HRS elicitation. Conclusions: The fact that low-dose hyper-radiosensitivity is not a common effect in normal human fibroblasts implies that HRS may be of little consequence in late-responding connective tissues with regard to radiation fibrosis

Merkel Cell Polyomavirus Infection of Animal Dermal Fibroblasts.

Science.gov (United States)

Liu, Wei; Krump, Nathan A; MacDonald, Margo; You, Jianxin

2018-02-15

Merkel cell polyomavirus (MCPyV) is the first polyomavirus to be associated with human cancer. Mechanistic studies attempting to fully elucidate MCPyV's oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. In this study, we examined the ability of MCPyV-GFP pseudovirus (containing a green fluorescent protein [GFP] reporter construct), MCPyV recombinant virions, and several MCPyV chimeric viruses to infect dermal fibroblasts isolated from various model animals, including mouse ( Mus musculus ), rabbit ( Oryctolagus cuniculus ), rat ( Rattus norvegicus ), chimpanzee ( Pan troglodytes ), rhesus macaque ( Macaca mulatta ), patas monkey ( Erythrocebus patas ), common woolly monkey ( Lagothrix lagotricha ), red-chested mustached tamarin ( Saguinus labiatus ), and tree shrew ( Tupaia belangeri ). We found that MCPyV-GFP pseudovirus was able to enter the dermal fibroblasts of all species tested. Chimpanzee dermal fibroblasts were the only type that supported vigorous MCPyV gene expression and viral replication, and they did so to a level beyond that of human dermal fibroblasts. We further demonstrated that both human and chimpanzee dermal fibroblasts produce infectious MCPyV virions that can successfully infect new cells. In addition, rat dermal fibroblasts supported robust MCPyV large T antigen expression after infection with an MCPyV chimeric virus in which the entire enhancer region of the MCPyV early promoter has been replaced with the simian virus 40 (SV40) analog. Our results suggest that viral transcription and/or replication events represent the major hurdle for MCPyV cross-species transmission. The capacity of rat dermal fibroblasts to support MCPyV early gene expression suggests that the rat is a candidate model organism for studying viral oncogene function during Merkel cell carcinoma (MCC) oncogenic progression. IMPORTANCE MCPyV plays an important role in the development of a highly aggressive form of skin cancer, Merkel

Phenomenology of an inducible mutagenic DNA repair pathway in Escherichia coli: SOS repair hypothesis

International Nuclear Information System (INIS)

Radman, M.

1974-01-01

A hypothesis is proposed according to which E. coli possesses an inducible DNA repair system. This hypothetical repair, which we call SOS repair, is manifested only following damage to DNA, and requires de novo protein synthesis. SOS repair in E. coli requires some known genetic elements: recA + , lex + and probably zab + . Mutagenesis by ultraviolet light is observed only under conditions of functional SOS repair: we therefore suspect that this is a mutation-prone repair. A number of phenomena and experiments is reviewed which at this point can best be interpreted in terms of an inducible mutagenic DNA repair system. Two recently discovered phenomena support the proposed hypothesis: existence of a mutant (tif) which, after a shift to elevated temperature, mimicks the effect of uv irradiation in regard to repair of phage lambda and uv mutagenesis, apparent activation of SOS repair by introduction into the recipient cell of damaged plasmid or Hfr DNA. Several specific predictions based on SOS repair hypothesis are presented in order to stimulate further experimental tests. (U.S.)

Functional and structural outcomes of single-row versus double-row versus combined double-row and suture-bridge repair for rotator cuff tears.

Science.gov (United States)

Mihata, Teruhisa; Watanabe, Chisato; Fukunishi, Kunimoto; Ohue, Mutsumi; Tsujimura, Tomoyuki; Fujiwara, Kenta; Kinoshita, Mitsuo

2011-10-01

Although previous biomechanical research has demonstrated the superiority of the suture-bridge rotator cuff repair over double-row repair from a mechanical point of view, no articles have described the structural and functional outcomes of this type of procedure. The structural and functional outcomes after arthroscopic rotator cuff repair may be different between the single-row, double-row, and combined double-row and suture-bridge (compression double-row) techniques. Cohort study; Level of evidence, 3. There were 206 shoulders in 201 patients with full-thickness rotator cuff tears that underwent arthroscopic rotator cuff repair. Eleven patients were lost to follow-up. Sixty-five shoulders were repaired using the single-row, 23 shoulders using the double-row, and 107 shoulders using the compression double-row techniques. Clinical outcomes were evaluated at an average of 38.5 months (range, 24-74 months) after rotator cuff repair. Postoperative cuff integrity was determined using Sugaya's classification of magnetic resonance imaging (MRI). The retear rates after arthroscopic rotator cuff repair were 10.8%, 26.1%, and 4.7%, respectively, for the single-row, double-row, and compression double-row techniques. In the subcategory of large and massive rotator cuff tears, the retear rate in the compression double-row group (3 of 40 shoulders, 7.5%) was significantly less than those in the single-row group (5 of 8 shoulders, 62.5%, P row group (5 of 12 shoulders, 41.7%, P row and suture-bridge techniques, which had the lowest rate of postoperative retear, is an effective option for arthroscopic repair of the rotator cuff tendons because the postoperative functional outcome in patients with a retear is inferior to that without retear.

Contrast enema findings in patients presenting with poor functional outcome after primary repair for Hirschsprung disease.

Science.gov (United States)

Garrett, Kevin M; Levitt, Marc A; Peña, Alberto; Kraus, Steven J

2012-09-01

The radiologic evaluation of Hirschsprung disease is well described in the literature. However, there is a paucity of literature describing the appearance of the neo-rectum and colon after repair, specifically describing findings in patients with poor functional outcome, which would suggest the need for reoperation. We describe findings on contrast enema and correlate them with surgical findings at reoperation in children with poor functional outcome after primary repair for Hirschsprung disease who suffer from bowel dysfunction that can manifest with either soiling or obstructive symptoms such as enterocolitis. Children were identified from our colorectal surgery database. At the time of abstract submission, 35 children had contrast enemas prior to reoperation. Additional children continue to present for evaluation. The majority of children included in the study had their primary repair performed elsewhere. The initial procedures included: Duhamel (n = 11), Soave (n = 20) or Swenson (n = 3). One child had undergone a primary Soave repair and subsequently had a Swenson-type reoperation but continued to have a poor outcome. One child's initial surgical repair could not be determined. Images were reviewed by a staff pediatric radiologist and a pediatric radiology fellow. Findings encountered on contrast enema in these children include a distal narrowed segment due to stricture or aganglionic/transitional zone segment (8), dilated/hypomotile distal segment (7), thickened presacral space due to compressing Soave cuff (11), dilated Duhamel pouch (8), active enterocolitis (3) and partially obstructing twist of the pull-through segment (1). Multiple anatomical and pathological complications exist that can lead to bowel dysfunction in children after repair of Hirschsprung disease. Little recent literature exists regarding the radiographic findings in children. We had the opportunity to review a substantial series of these children, describe the contrast enema

Formation and repair of physically and chemically induced DNA damage in human cells. Final report, September 1, 1976-November 30, 1978

International Nuclear Information System (INIS)

Cerutti, P.A.

1979-01-01

The major topic was the study of the formation and repair of DNA damage by energy related physical and chemical agents in cultured human cells. Two pathways of damage production were distinguished: (1) indirect action, i.e., attack of DNA by active oxygen species which are formed by the reaction of the primary agent with a non-DNA target; and (2) direct action, i.e., reaction of the primary agent or a chemical derivative of the primary agent with DNA usually resulting in the formation of a covalent adduct. Near-ultraviolet light and ionizing radiation were studied as agents which operate at least in part via indirect action and benzo(a)pyrene as chemical carcinogen operating mostly by direct action. The formation of monomeric thymine damage of the 5,6-dihydroxy-dihydrothymine type by γ-rays and ultraviolet light was investigated. Indirect action of near-ultraviolet light is also responsible for the induction of DNA single strand breaks. Their formation and repair following exposure to 313 nm light was studied in skin fibroblasts from patients with the hereditary disease Xeroderma pigmentosum (XP). Excision repair of γ-ray induced 5,6-dihydroxy-dihydrothymine type lesions was studied in fibroblasts from Ataxia telangiectasia (AT) patients. The formation and repair of covalent purine adducts was studied in actively metabolizing rodent and human cells following treatment with the procarcinogen benzo(a)pyrene and with the ultimate metabolite benzo(a)pyrene-diol-epoxide I

Enhanced capacity of DNA repair in human cytomegalovirus-infected cells

International Nuclear Information System (INIS)

Nishiyama, Y.; Rapp, F.

1981-01-01

Plaque formation in Vero cells by UV-irradiated herpes simplex virus was enhanced by infection with human cytomegalovirus (HCMV), UV irradiation, or treatment with methylmethanesulfonate. Preinfection of Vero cells with HCMV enhanced reactivation of UV-irradiated herpes simplex virus more significantly than did treatment with UV or methylmethanesulfonate alone. A similar enhancement by HCMV was observed in human embryonic fibroblasts, but not in xeroderma pigmentosum (XP12BE) cells. It was also found that HCMV infection enhanced hydroxyurea-resistant DNA synthesis induced by UV light or methylmethanesulfonate. Alkaline sucrose gradient sedimentation analysis revealed an enhanced rate of synthesis of all size classes of DNA in UV-irradiated HCMV-infected Vero cells. However, HCMV infection did not induce repairable lesions in cellular DNA and did not significantly inhibit host cell DNA synthesis, unlike UV or methylmethanesulfonate. These results indicate that HCMV enhanced DNA repair capacity in the host cells without producing detectable lesions in cellular DNA and without inhibiting DNA synthesis. This repair appeared to be error proof for UV-damaged herpes simplex virus DNA when tested with herpes simplex virus thymidine kinase-negative mutants

Congestive heart failure effects on atrial fibroblast phenotype: differences between freshly-isolated and cultured cells.

Directory of Open Access Journals (Sweden)

Kristin Dawson

Full Text Available Fibroblasts are important in the atrial fibrillation (AF substrate resulting from congestive heart failure (CHF. We previously noted changes in in vivo indices of fibroblast function in a CHF dog model, but could not detect changes in isolated cells. This study assessed CHF-induced changes in the phenotype of fibroblasts freshly isolated from control versus CHF dogs, and examined effects of cell culture on these differences.Left-atrial fibroblasts were isolated from control and CHF dogs (ventricular tachypacing 240 bpm × 2 weeks. Freshly-isolated fibroblasts were compared to fibroblasts in primary culture. Extracellular-matrix (ECM gene-expression was assessed by qPCR, protein by Western blot, fibroblast morphology with immunocytochemistry, and K(+-current with patch-clamp. Freshly-isolated CHF fibroblasts had increased expression-levels of collagen-1 (10-fold, collagen-3 (5-fold, and fibronectin-1 (3-fold vs. control, along with increased cell diameter (13.4 ± 0.4 µm vs control 8.4 ± 0.3 µm and cell spreading (shape factor 0.81 ± 0.02 vs. control 0.87 ± 0.02, consistent with an activated phenotype. Freshly-isolated control fibroblasts displayed robust tetraethylammonium (TEA-sensitive K(+-currents that were strongly downregulated in CHF. The TEA-sensitive K(+-current differences between control and CHF fibroblasts were attenuated after 2-day culture and eliminated after 7 days. Similarly, cell-culture eliminated the ECM protein-expression and shape differences between control and CHF fibroblasts.Freshly-isolated CHF and control atrial fibroblasts display distinct ECM-gene and morphological differences consistent with in vivo pathology. Culture for as little as 48 hours activates fibroblasts and obscures the effects of CHF. These results demonstrate potentially-important atrial-fibroblast phenotype changes in CHF and emphasize the need for caution in relating properties of cultured fibroblasts to in vivo systems.

Turbine repair process, repaired coating, and repaired turbine component

Science.gov (United States)

Das, Rupak; Delvaux, John McConnell; Garcia-Crespo, Andres Jose

2015-11-03

A turbine repair process, a repaired coating, and a repaired turbine component are disclosed. The turbine repair process includes providing a turbine component having a higher-pressure region and a lower-pressure region, introducing particles into the higher-pressure region, and at least partially repairing an opening between the higher-pressure region and the lower-pressure region with at least one of the particles to form a repaired turbine component. The repaired coating includes a silicon material, a ceramic matrix composite material, and a repaired region having the silicon material deposited on and surrounded by the ceramic matrix composite material. The repaired turbine component a ceramic matrix composite layer and a repaired region having silicon material deposited on and surrounded by the ceramic matrix composite material.

Microinjection of Escherichia coli UvrA, B, C and D proteins into fibroblasts of xeroderma pigmentosum complementation groups A and C does not result in restoration of UV-induced DNA synthesis.

NARCIS (Netherlands)

J.C.M. Zwetsloot; A.P. Barbeiro; W. Vermeulen (Wim); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude)

1986-01-01

textabstractThe UV-induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts of repair-deficient xeroderma pigmentosum complementation groups A and C was assayed after injection of identical activities of either Uvr excinuclease (UvrA, B, C and D) from Escherichia coli or endonuclease V

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

International Nuclear Information System (INIS)

Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G.

2012-01-01

Highlights: ► We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. ► YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. ► There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. ► The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. ► The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929–933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

Curcumin-induced fibroblast apoptosis and in vitro wound contraction are regulated by antioxidants and heme oxygenase: implications for scar formation.

NARCIS (Netherlands)

Scharstuhl, A.; Mutsaers, H.A.M.; Pennings, S.W.C.; Szarek, W.A.; Russel, F.G.M.; Wagener, F.A.D.T.G.

2009-01-01

Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis-inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25-microM curcumin causes fibroblast apoptosis and

BIDIRECTIONAL FUNCTION OF SHENGHE POWDER ON REPAIR ...

African Journals Online (AJOL)

USER

Keywords: DNA repair, radiation, glioma, astrocyte, Chinese herbs. Introduction .... Wet transfer was performed for 4 hr at constant voltage (40 V) using polyvinylidene difluoride membrane ..... Mitochondrial survivin inhibits apoptosis and.

Polyphenol-Rich Strawberry Extract Protects Human Dermal Fibroblasts against Hydrogen Peroxide Oxidative Damage and Improves Mitochondrial Functionality

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Francesca Giampieri

2014-06-01

Full Text Available Strawberry bioactive compounds are widely known to be powerful antioxidants. In this study, the antioxidant and anti-aging activities of a polyphenol-rich strawberry extract were evaluated using human dermal fibroblasts exposed to H2O2. Firstly, the phenol and flavonoid contents of strawberry extract were studied, as well as the antioxidant capacity. HPLC-DAD analysis was performed to determine the vitamin C and β-carotene concentration, while HPLC-DAD/ESI-MS analysis was used for anthocyanin identification. Strawberry extract presented a high antioxidant capacity, and a relevant concentration of vitamins and phenolics. Pelargonidin- and cyanidin-glycosides were the most representative anthocyanin components of the fruits. Fibroblasts incubated with strawberry extract and stressed with H2O2 showed an increase in cell viability, a smaller intracellular amount of ROS, and a reduction of membrane lipid peroxidation and DNA damage. Strawberry extract was also able to improve mitochondrial functionality, increasing the basal respiration of mitochondria and to promote a regenerative capacity of cells after exposure to pro-oxidant stimuli. These findings confirm that strawberries possess antioxidant properties and provide new insights into the beneficial role of strawberry bioactive compounds on protecting skin from oxidative stress and aging.

Cultured Human Fibroblast Biostimulation Using a 940 nm Diode Laser

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Illescas-Montes, Rebeca; Melguizo-Rodríguez, Lucía; Manzano-Moreno, Francisco Javier; García-Martínez, Olga; Ruiz, Concepción

2017-01-01

Background: Fibroblasts are the main cells involved in regeneration during wound healing. The objective was to determine the effect of 940 nm diode laser on cultured human fibroblasts using different irradiation regimens. Methods: The CCD-1064Sk human epithelial fibroblast cell line was treated with a 940 nm diode laser at different energy doses (power: 0.2–1 W and energy density: 1–7 J/cm2) using different transmission modes (continuous or pulsed). The effect on cell growth at 24 and 72 h post-treatment was examined by measuring the proliferative capacity, the impact on the cell cycle, and the effect on cell differentiation. Results: fibroblast proliferative capacity was increased at 24 and 72 h post-treatment as a function of the energy dose. The greatest increase was observed with a power of 0.2 or 0.5 W and energy density between 1 and 4 J/cm2; no difference was observed between continuous and pulsed modes. There were no significant differences in cell cycle between treated groups and controls. α-actin expression was increased by treatment, indicating enhanced cell differentiation. Conclusion: The 940 nm diode laser has biostimulating effects on fibroblasts, stimulating proliferative capacity and cell differentiation without altering the cell cycle. Further researches are necessary to explore its potential clinical usefulness in wound healing. PMID:28773152

Fibrosis of Two: Epithelial Cell-Fibroblast Interactions in Pulmonary Fibrosis

Science.gov (United States)

Sakai, Norihiko; Tager, Andrew M.

2013-01-01

Idiopathic pulmonary fibrosis (IPF) is characterized by the progressive and ultimately fatal accumulation of fibroblasts and extracellular matrix in the lung that distorts its architecture and compromises its function. IPF is now thought to result from wound-healing processes that, although initiated to protect the host from injurious environmental stimuli, lead to pathological fibrosis due to these processes becoming aberrant or over-exuberant. Although the environmental stimuli that trigger IPF remain to be identified, recent evidence suggests that they initially injure the alveolar epithelium. Repetitive cycles of epithelial injury and resultant alveolar epithelial cell death provoke the migration, proliferation, activation and myofibroblast differentiation of fibroblasts, causing the accumulation of these cells and the extracellular matrix that they synthesize. In turn, these activated fibroblasts induce further alveolar epithelial cell injury and death, thereby creating a vicious cycle of pro-fibrotic epithelial cell-fibroblast interactions. Though other cell types certainly make important contributions, we focus here on the “pas de deux” (steps of two), or perhaps more appropriate to IPF pathogenesis, the “folie à deux” (madness of two) of epithelial cells and fibroblasts that drives the progression of pulmonary fibrosis. We describe the signaling molecules that mediate the interactions of these cell types in their “fibrosis of two”, including transforming growth factor-β, connective tissue growth factor, sonic hedgehog, prostaglandin E2, angiotensin II and reactive oxygen species. PMID:23499992

Cultured Human Fibroblast Biostimulation Using a 940 nm Diode Laser

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Rebeca Illescas-Montes

2017-07-01

Full Text Available Background: Fibroblasts are the main cells involved in regeneration during wound healing. The objective was to determine the effect of 940 nm diode laser on cultured human fibroblasts using different irradiation regimens. Methods: The CCD-1064Sk human epithelial fibroblast cell line was treated with a 940 nm diode laser at different energy doses (power: 0.2–1 W and energy density: 1–7 J/cm2 using different transmission modes (continuous or pulsed. The effect on cell growth at 24 and 72 h post-treatment was examined by measuring the proliferative capacity, the impact on the cell cycle, and the effect on cell differentiation. Results: fibroblast proliferative capacity was increased at 24 and 72 h post-treatment as a function of the energy dose. The greatest increase was observed with a power of 0.2 or 0.5 W and energy density between 1 and 4 J/cm2; no difference was observed between continuous and pulsed modes. There were no significant differences in cell cycle between treated groups and controls. α-actin expression was increased by treatment, indicating enhanced cell differentiation. Conclusion: The 940 nm diode laser has biostimulating effects on fibroblasts, stimulating proliferative capacity and cell differentiation without altering the cell cycle. Further researches are necessary to explore its potential clinical usefulness in wound healing.

Transfection of Primary Human Skin Fibroblasts for Peroxisomal Studies

NARCIS (Netherlands)

Koster, Janet; Waterham, Hans R.

2017-01-01

Functional studies with primary human skin fibroblasts from patients with a peroxisomal disorder often require efficient transfection with plasmids to correct the genetic defect or to express heterologous reporter proteins. Here, we describe a protocol we commonly use for efficient nonviral

Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

International Nuclear Information System (INIS)

Kukat, Alexandra; Edgar, Daniel; Bratic, Ivana; Maiti, Priyanka; Trifunovic, Aleksandra

2011-01-01

Highlights: → Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. → This process is independent of endogenous ROS production. → Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O 2 ) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells.

Science.gov (United States)

Liu, Zhiyong; Hartman, Yolanda E; Warram, Jason M; Knowles, Joseph A; Sweeny, Larissa; Zhou, Tong; Rosenthal, Eben L

2011-08-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN depleted head and neck cancer tumor cells

Science.gov (United States)

Liu, Zhiyong; Hartman, Yolanda E.; Warram, Jason M.; Knowles, Joseph A.; Sweeny, Larrisa; Zhou, Tong; Rosenthal, Eben L.

2011-01-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were co-cultured with fibroblasts or inoculated with fibroblasts into SCID mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Co-culture experiments demonstrated fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN silenced cells compared to control vector transfected cells, while inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast co-culture, suggesting the importance of FGFR2 signaling in fibroblast mediated tumor growth. Analysis of xenografted tumors revealed EMMPRIN silenced tumors had a larger stromal compartment compared to control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast independent tumor growth. PMID:21665938

Human mismatch repair protein hMutLα is required to repair short slipped-DNAs of trinucleotide repeats.

Science.gov (United States)

Panigrahi, Gagan B; Slean, Meghan M; Simard, Jodie P; Pearson, Christopher E

2012-12-07

Mismatch repair (MMR) is required for proper maintenance of the genome by protecting against mutations. The mismatch repair system has also been implicated as a driver of certain mutations, including disease-associated trinucleotide repeat instability. We recently revealed a requirement of hMutSβ in the repair of short slip-outs containing a single CTG repeat unit (1). The involvement of other MMR proteins in short trinucleotide repeat slip-out repair is unknown. Here we show that hMutLα is required for the highly efficient in vitro repair of single CTG repeat slip-outs, to the same degree as hMutSβ. HEK293T cell extracts, deficient in hMLH1, are unable to process single-repeat slip-outs, but are functional when complemented with hMutLα. The MMR-deficient hMLH1 mutant, T117M, which has a point mutation proximal to the ATP-binding domain, is defective in slip-out repair, further supporting a requirement for hMLH1 in the processing of short slip-outs and possibly the involvement of hMHL1 ATPase activity. Extracts of hPMS2-deficient HEC-1-A cells, which express hMLH1, hMLH3, and hPMS1, are only functional when complemented with hMutLα, indicating that neither hMutLβ nor hMutLγ is sufficient to repair short slip-outs. The resolution of clustered short slip-outs, which are poorly repaired, was partially dependent upon a functional hMutLα. The joint involvement of hMutSβ and hMutLα suggests that repeat instability may be the result of aberrant outcomes of repair attempts.

Biological Differences between Hanwoo longissimus dorsi and semimembranosus Muscles in Collagen Synthesis of Fibroblasts.

Science.gov (United States)

Subramaniyan, Sivakumar Allur; Hwang, Inho

2017-01-01

Variations in physical toughness between muscles and animals are a function of growth rate and extend of collagen type I and III. The current study was designed to investigate the ability of growth rate, collagen concentration, collagen synthesizing and degrading genes on two different fibroblast cells derived from Hanwoo m. longissimus dorsi (LD) and semimembranosus (SM) muscles. Fibroblast cell survival time was determined for understanding about the characteristics of proliferation rate between the two fibroblasts. We examined the collagen concentration and protein expression of collagen type I and III between the two fibroblasts. The mRNA expression of collagen synthesis and collagen degrading genes to elucidate the molecular mechanisms on toughness and tenderness through collagen production between the two fibroblast cells. From our results the growth rate, collagen content and protein expression of collagen type I and III were significantly higher in SM than LD muscle fibroblast. The mRNA expressions of collagen synthesized genes were increased whereas the collagen degrading genes were decreased in SM than LD muscle. Results from confocal microscopical investigation showed increased fluorescence of collagen type I and III appearing stronger in SM than LD muscle fibroblast. These results implied that the locomotion muscle had higher fibroblast growth rate, leads to produce more collagen, and cause tougher than positional muscle. This in vitro study mirrored that background toughness of various muscles in live animal is likely associated with fibroblast growth pattern, collagen synthesis and its gene expression.

circHIPK2-mediated σ-1R promotes endoplasmic reticulum stress in human pulmonary fibroblasts exposed to silica.

Science.gov (United States)

Cao, Zhouli; Xiao, Qingling; Dai, Xiaoniu; Zhou, Zewei; Jiang, Rong; Cheng, Yusi; Yang, Xiyue; Guo, Huifang; Wang, Jing; Xi, Zhaoqing; Yao, Honghong; Chao, Jie

2017-12-13

Silicosis is characterized by fibroblast accumulation and excessive deposition of extracellular matrix. Although the roles of SiO 2 -induced chemokines and cytokines released from alveolar macrophages have received significant attention, the direct effects of SiO 2 on protein production and functional changes in pulmonary fibroblasts have been less extensively studied. Sigma-1 receptor, which has been associated with cell proliferation and migration in the central nervous system, is expressed in the lung, but its role in silicosis remains unknown. To elucidate the role of sigma-1 receptor in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Both molecular biological assays and pharmacological techniques, combined with functional experiments, such as migration and proliferation, were applied in human pulmonary fibroblasts from adults to analyze the molecular and functional changes induced by SiO 2 . SiO 2 induced endoplasmic reticulum stress in association with enhanced expression of sigma-1 receptor. Endoplasmic reticulum stress promoted migration and proliferation of human pulmonary fibroblasts-adult exposed to SiO 2 , inducing the development of silicosis. Inhibition of sigma-1 receptor ameliorated endoplasmic reticulum stress and fibroblast functional changes induced by SiO 2 . circHIPK2 is involved in the regulation of sigma-1 receptor in human pulmonary fibroblasts-adult exposed to SiO 2 . Our study elucidated a link between SiO 2 -induced fibrosis and sigma-1 receptor signaling, thereby providing novel insight into the potential use of sigma-1 receptor/endoplasmic reticulum stress in the development of novel therapeutic strategies for silicosis treatment.

Increased levels of unscheduled DNA synthesis in UV-irradiated human fibroblasts pretreated with sodium butyrate

International Nuclear Information System (INIS)

Williams, J.I.; Friedberg, E.C.

1982-01-01

Pretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5-20 mM results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0-25 J/m 2 . The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV-induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV-induced repair synthesis by reducing endogenous levels of thymidine. (author)

Increased Functional Half-life of Fibroblast Growth Factor-1 by Recovering a Vestigial Disulfide Bond

Directory of Open Access Journals (Sweden)

Jihun Lee

2010-12-01

Full Text Available The fibroblast growth factor (FGF family of proteins contains an absolutely conserved Cys residue at position 83 that is present as a buried free cysteine. We have previously shown that mutation of the structurally adjacent residue, Ala66, to cysteine results in the formation of a stabilizing disulfide bond in FGF-1. This result suggests that the conserved free cysteine residue at position 83 in the FGF family of proteins represents a vestigial half-cystine. Here, we characterize the functional half-life and mitogenic activity of the oxidized form of the Ala66Cys mutation to identify the effect of the recovered vestigial disulfide bond between Cys83 and Cys66 upon the cellular function of FGF-1. The results show that the mitogenic activity of this mutant is significantly increased and that its functional half-life is greatly extended. These favorable effects are conferred by the formation of a disulfide bond that simultaneously increases thermodynamic stability of the protein and removes a reactive buried thiol at position 83. Recovering this vestigial disulfide by introducing a cysteine at position 66 is a potentially useful protein engineering strategy to improve the functional half-life of other FGF family members.

In vitro study of RRS HA injectable mesotherapy/biorevitalization product on human skin fibroblasts and its clinical utilization

Directory of Open Access Journals (Sweden)

Deglesne PA

2016-02-01

Full Text Available Pierre-Antoine Deglesne,* Rodrigo Arroyo,* Evgeniya Ranneva, Philippe Deprez Research and Development, SKIN TECH PHARMA GROUP, Castelló d'Empúries, Spain  *These authors contributed equally to this work Abstract: Mesotherapy/biorevitalization with hyaluronic acid (HA is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS® (Repairs, Refills, Stimulates HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15% and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts.Keywords: mesotherapy, medical device, RRS, collagen, elastin, extracellular matrix

Functional evaluation of arthroscopic repair of rotator cuff injuries in patients with pseudoparalysis,

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Alberto Naoki Miyazaki

2014-04-01

Full Text Available OBJECTIVE: to evaluate the functional result from arthroscopic repair of rotator cuff injuries in patients with pseudoparalysis, defined as incapacity to actively raise the arm above 90◦ , while complete passive elevation was possible.METHODS: we reevaluated 38 patients with a mean follow-up of 51 months (minimum of 24. We analyzed the pseudoparalysis reversion rate and the functional result obtained.RESULTS: according to the assessment criteria of the University of California in Los Angeles (UCLA, 31 (82% patients had good and excellent results, two (5% had fair results and five (13% had poor results. The mean active elevation went from 39◦ before the operation to 139◦ after the operation (p < 0.05; the mean active lateral rotation went from 30◦ to 48◦ (p < 0.05 and the mean active medial rotation went from level L3 to T12 (p < 0.05.CONCLUSION: arthroscopic repair of rotator cuff injuries produced good and excellent results in 82% of the cases and a statistically significant improvement of active range of motion, with reversion of the pseudoparalysis in 97.4% of the cases. It is therefore a good treatment option.

Post-irradiation replication and repair in UV-irradiated cells of Proteus mirabilis depends on protein synthesis and a functioning rec+ gene

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Hofemeister, J.

1977-01-01

The amount of and the molecular weight of newly synthesized DNA (piDNA) as well as its repair after UV irradiation in excision-proficient strains of P.mirabilis and E.coli K12 have been compared. A fraction of post-replication repair (PRR) in P.mirabilis is found to be dependent on de novo protein synthesis after UV irradiation. Pre-irradiation by UV and pre-treatment with nalidixic acid increase the efficiency of post-irradiation replication and PRR even in the presence of chloramphenicol. An inducible repair function in P.mirabilis is supposed to stimulate post-irradiation replication and repair. (author)

Functional Outcomes and Return to Sports After Acute Repair, Chronic Repair, and Allograft Reconstruction for Proximal Hamstring Ruptures.

Science.gov (United States)

Rust, David A; Giveans, M Russell; Stone, Rebecca M; Samuelson, Kathryn M; Larson, Christopher M

2014-06-01

There are limited data regarding outcomes and return to sports after surgery for acute versus chronic proximal hamstring ruptures. Surgery for chronic proximal hamstring ruptures leads to improved outcomes and return to sports but at a lower level than with acute repair. Proximal hamstring reconstruction with an Achilles allograft for chronic ruptures is successful when direct repair is not possible. Cohort study; Level of evidence, 3. Between 2002 and 2012, a total of 72 patients with a traumatic proximal hamstring rupture (51 acute, 21 chronic) underwent either direct tendon repair with suture anchors (n = 58) or Achilles allograft tendon reconstruction (n = 14). Results from the Single Assessment Numeric Evaluation (SANE) for activities of daily living (ADL) and sports-related activities, Short Form-12 (SF-12), visual analog scale (VAS), and a patient satisfaction questionnaire were obtained. The mean time to surgery in the chronic group was 441.4 days versus 17.8 days in the acute group. At a mean follow-up of 45 months, patients with chronic tears had inferior sports activity scores (70.2% vs 80.3%, respectively; P = .026) and a trend for decreased ADL scores (86.5% vs 93.3%, respectively; P = .085) compared with those with acute tears. Patients with chronic tears, however, reported significant improvements postoperatively for both sports activity scores (30.3% to 70.2%; P sports activity scores equal to those of chronic repair (P = .507 and P = .904, respectively). There were no significant differences between groups in SF-12, VAS, or patient satisfaction outcomes (mean, 85.2% satisfaction overall). Acute repair was superior to chronic surgery with regard to return to sports. Acute and chronic proximal hamstring repair and allograft reconstruction had favorable results for ADL. For low-demand patients or those with medical comorbidities, delayed repair or reconstruction might be considered with an expected 87% return to normal ADL. For patients who desire to

Identification of pulmonary PDGFRalpha-positive fibroblast specific miRNA and mRNA expression profiles during postnatal lung development

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Dontireddy, Daria Agnieszka

2015-01-01

BACKGROUND AND AIM The process of alveolarization is tightly regulated and requires the contribution of different subpopulations of fibroblasts such as myofibroblasts, lipofibroblasts and platelet-derived growth factor receptor alpha (PDGFRalpha)-positive fibroblasts. Each of this fibroblasts subset fulfills certain functions during lung development in a time-dependent manner. In particular PDGFRalpha-positive cells are crucial for alveolar septation and myofibroblasts differentiation. PDG...

Chromosomal radiosensitivity during the G2 cell-cycle period of skin fibroblasts from individuals with familial cancer

International Nuclear Information System (INIS)

Parshad, R.; Sanford, K.K.; Jones, G.M.

1985-01-01

The authors reported previously that human cells after neoplastic transformation in culture had acquired an increased susceptibility to chromatid damage induced by x-irradiation during the G2 phase of the cell cycle. Evidence suggested that this results from deficient DNA repair during G2 phase. Cells derived from human tumors also showed enhanced G2-phase chromosomal radiosensitivity. Furthermore, skin fibroblasts from individuals with genetic diseases predisposing to a high risk of cancer, including ataxia-telangiectasia, Bloom syndrome, Fanconi anemia, and xeroderma pigmentosum exhibited enhanced G2-phase chromosomal radiosensitivity. The present study shows that apparently normal skin fibroblasts from individuals with familial cancer--i.e., from families with a history of neoplastic disease--also exhibit enhanced G2-phase chromosomal radiosensitivity. This radiosensitivity appears, therefore, to be associated with both a genetic predisposition to cancer and a malignant neoplastic state. Furthermore, enhanced G2-phase chromosomal radiosensitivity may provide the basis for an assay to detect genetic susceptibility to cancer

Arthroscopic double row cuff repair with suture-bridging and autologous conditioned plasma injection: Functional and structural results.

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Werthel, Jean-David; Pelissier, Alexandre; Massin, Philippe; Boyer, Patrick; Valenti, Philippe

2014-10-01

The double row cuff repair with suture bridging is commonly used for arthroscopic rotator cuff repair (RCR). Despite its biomechanical qualities, the rate of iterative tears with this technique is important. The aim of our study was to evaluate the effect of autologous conditioned plasma (ACP) on functional results and on the rate of iterative tears after RCR by suture bridging. A consecutive series of 65 patients who underwent arthroscopic double-row suture bridge (Speed-Bridge, Arthrex) primary cuff repair of symptomatic full-thickness supraspinatus tear (retraction 4 were considered as iterative tears. Mean follow-up was 19 months (+/-42) in the 2 groups. The mean quantity of ACP injected was 6ml. (+/-1.5) and no specific complication of the injection was found. Mean preoperative Constant-Murley scores were 41,2 (±7,7) and 38 (±11)in group B. Mean normalized Constant-Murley score increased from 41 points (±7) pre-operatively to 70 points (±8) post-operatively in group A and from 38 points (±11) to 73 points (±11) in group B. There were no significative differences between the two groups (P > 0.05). In group A, 31 repairs were Sugaya 1-3 (94%), vs. 30 in group B (93%), and 1 was type 4 in group A (5%) vs. 2 in group B (8%). In both groups, RCR with suture bridging gave successful functional outcomes, with a low rate of iterative tear. In this preliminary study, the adjuvant effect of ACP injections could not be showed on both functional and structural results. Longer follow-up is needed to evaluate potential differences.

Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis.

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Yang, Zhizhou; Sun, Zhaorui; Liu, Hongmei; Ren, Yi; Shao, Danbing; Zhang, Wei; Lin, Jinfeng; Wolfram, Joy; Wang, Feng; Nie, Shinan

2015-07-01

It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson's trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury.

Effect of ventricular function and volumes on exercise capacity in adults with repaired Tetralogy of Fallot

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Natalia Dłużniewska

2018-01-01

Conclusion: Exercise intolerance in adults with repaired ToF is markedly depressed. The decreased exercise capacity is correlated with impaired RV function and may be associated also with LV dysfunction, which suggests right-to-left ventricular interaction.

Right ventricular function late after total repair of tetralogy of Fallot

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Straten, Alexander van; Roos, Albert de [Leiden University Medical Center, Department of Radiology, Leiden (Netherlands); Vliegen, Hubert W. [Leiden University Medical Center, Department of Cardiology, Leiden (Netherlands); Hazekamp, Mark G. [Leiden University Medical Center, Department of Cardiothoracic Surgery, Leiden (Netherlands)

2005-04-01

Over the past decades, life expectancy in patients with congenital heart disease has increased dramatically. However, serious complications may develop late after total repair in infancy. These complications are usually the result of longstanding pulmonary regurgitation, which leads to dilatation of the right ventricle and an increased risk for severe arrhythmias. Therefore lifelong follow-up in these patients is required. Cardiac magnetic resonance imaging is the current imaging tool of choice because it offers superior imaging quality and enables accurate quantification of functional parameters such as flow volumes and systolic and diastolic performance. (orig.)

Right ventricular function late after total repair of tetralogy of Fallot

International Nuclear Information System (INIS)

Straten, Alexander van; Roos, Albert de; Vliegen, Hubert W.; Hazekamp, Mark G.

2005-01-01

Over the past decades, life expectancy in patients with congenital heart disease has increased dramatically. However, serious complications may develop late after total repair in infancy. These complications are usually the result of longstanding pulmonary regurgitation, which leads to dilatation of the right ventricle and an increased risk for severe arrhythmias. Therefore lifelong follow-up in these patients is required. Cardiac magnetic resonance imaging is the current imaging tool of choice because it offers superior imaging quality and enables accurate quantification of functional parameters such as flow volumes and systolic and diastolic performance. (orig.)

Aging and photo-aging DNA repair phenotype of skin cells-Evidence toward an effect of chronic sun-exposure

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Prunier, Chloe; Masson-Genteuil, Gwenaeelle [Laboratoire Lesions des Acides Nucleiques, CEA, DSM, INAC, SCIB, UMR-E CEA/UJF-Grenoble 1, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9 (France); Ugolin, Nicolas [Laboratoire de Cancerologie Experimentale, CEA, DSV, IRCM, SREIT, BP6, Fontenay-aux-Roses Cedex F-92265 (France); Sarrazy, Fanny [Laboratoire Lesions des Acides Nucleiques, CEA, DSM, INAC, SCIB, UMR-E CEA/UJF-Grenoble 1, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9 (France); Sauvaigo, Sylvie, E-mail: sylvie.sauvaigo@cea.fr [Laboratoire Lesions des Acides Nucleiques, CEA, DSM, INAC, SCIB, UMR-E CEA/UJF-Grenoble 1, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9 (France)

2012-08-01

Several studies have demonstrated the deleterious effect of aging on the capacity of cells to repair their DNA. However, current existing assays aimed at measuring DNA repair address only a specific repair step dedicated to the correction of a specific DNA lesion type. Consequently they provide no information regarding the repair pathways that handle other types of lesions. In addition to aging, consequences of photo-exposure on these repair processes remain elusive. In this study we evaluated the consequence of aging and of chronic and/or acute photo-exposure on DNA repair in human skin fibroblasts using a multiplexed approach, which provided detailed information on several repair pathways at the same time. The resulting data were analyzed with adapted statistics/bioinformatics tools. We showed that, irrespective of the repair pathway considered, excision/synthesis was less efficient in non-exposed cells from elderly compared to cells from young adults and that photo-exposure disrupted this very clear pattern. Moreover, it was evidenced that chronic sun-exposure induced changes in DNA repair properties. Finally, the identification of a specific signature at the level of the NER pathway in cells repeatedly exposed to sun revealed a cumulative effect of UVB exposure and chronic sun irradiation. The uses of bioinformatics tools in this study was essential to fully take advantage of the large sum of data obtained with our multiplexed DNA repair assay and unravel the effects of environmental exposure on DNA repair pathways.

Evaluating the integrity of the reinforced concrete structure repaired by epoxy injection using simulated transfer function of impact-echo response

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Cheng, Chia-Chi; Yu, Chih-peng; Wu, Jiunn-Hong; Hsu, Keng-Tsan; Ke, Ying-Tsu [Chaoyang University of Technology, Department of Construction Engineering, Taichung, Taiwan (China)

2014-02-18

Cracks and honeycombs are often found inside reinforced concrete (RC) structure caused by excessive external force, or improper casting of concrete. The repairing method usually involves epoxy injection. The impact-echo method, which is a sensitive for detecting of the interior voids, may not be applicable to assess the integrity of the repaired member as both air and epoxy are less in acoustic impedances. In this study, the repaired RC structure was evaluated by the simulated transfer function of the IE displacement waveform where the R-wave displacement waveform is used as a base of a simulated force-time function. The effect of different thickness of the epoxy layer to the amplitude corresponding to the interface is studied by testing on specimen containing repaired naturally delaminated cracks with crack widths about 1 mm, 3 mm and 5 mm. The impact-echo responses were compared with the drilling cores at the test positions. The results showed the cracks were not fully filled with epoxy when the peak amplitude corresponding to the interface dropped less than 20%. The peak corresponding to the thicker epoxy layer tends to be larger in amplitude. A field study was also performed on a column damaged by earthquake before and after repairing.

KL-6, a human MUC1 mucin, promotes proliferation and survival of lung fibroblasts

International Nuclear Information System (INIS)

Ohshimo, Shinichiro; Yokoyama, Akihito; Hattori, Noboru; Ishikawa, Nobuhisa; Hirasawa, Yutaka; Kohno, Nobuoki

2005-01-01

The serum level of KL-6, a MUC1 mucin, is a clinically useful marker for various interstitial lung diseases. Previous studies demonstrated that KL-6 promotes chemotaxis of human fibroblasts. However, the pathophysiological role of KL-6 remains poorly understood. Here, we further investigate the functional aspects of KL-6 in proliferation and apoptosis of lung fibroblasts. KL-6 accelerated the proliferation and inhibited the apoptosis of all human lung fibroblasts examined. An anti-KL-6 monoclonal antibody counteracted both of these effects induced by KL-6 on human lung fibroblasts. The pro-fibroproliferative and anti-apoptotic effects of KL-6 are greater than and additive to those of the maximum effective concentrations of platelet-derived growth factor, basic fibroblast growth factor, and transforming growth factor-β. These findings indicate that increased levels of KL-6 in the epithelial lining fluid may stimulate fibrotic processes in interstitial lung diseases and raise the possibility of applying an anti-KL-6 antibody to treat interstitial lung diseases

Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

International Nuclear Information System (INIS)

Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

2011-01-01

Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

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Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

2011-07-01

Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage.

Science.gov (United States)

Hill, Sarah J; Mordes, Daniel A; Cameron, Lisa A; Neuberg, Donna S; Landini, Serena; Eggan, Kevin; Livingston, David M

2016-11-29

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.

MicroRNA-124 controls the proliferative, migratory, and inflammatory phenotype of pulmonary vascular fibroblasts.

Science.gov (United States)

Wang, Daren; Zhang, Hui; Li, Min; Frid, Maria G; Flockton, Amanda R; McKeon, B Alexandre; Yeager, Michael E; Fini, Mehdi A; Morrell, Nicholas W; Pullamsetti, Soni S; Velegala, Sivareddy; Seeger, Werner; McKinsey, Timothy A; Sucharov, Carmen C; Stenmark, Kurt R

2014-01-03

proliferative, migratory, and inflammatory phenotype of hypertensive pulmonary adventitial fibroblasts. Thus, therapies directed at restoring miR-124 function, including histone deacetylase inhibitors, should be investigated.

Exonuclease 1 is a critical mediator of survival during DNA double strand break repair in nonquiescent hematopoietic stem and progenitor cells.

Science.gov (United States)

Desai, Amar; Qing, Yulan; Gerson, Stanton L

2014-02-01

Hematopoietic stem cell (HSC) populations require DNA repair pathways to maintain their long-term survival and reconstitution capabilities, but mediators of these processes are still being elucidated. Exonuclease 1 (Exo1) participates in homologous recombination (HR) and Exo1 loss results in impaired 5' HR end resection. We use cultured Exo1(mut) fibroblasts and bone marrow to demonstrate that loss of Exo1 function results in defective HR in cycling cells. Conversely, in Exo1(mut) mice HR is not required for maintenance of quiescent HSCs at steady state, confirming the steady state HSC reliance on nonhomologous end joining (NHEJ). Exo1(mut) mice sustained serial repopulation, displayed no defect in competitive repopulation or niche occupancy, and exhibited no increased sensitivity to whole body ionizing radiation. However, when Exo1(mut) HSCs were pushed into cell cycle in vivo with 5-fluorouracil or poly IC, the hematopoietic population became hypersensitive to IR, resulting in HSC defects and animal death. We propose Exo1-mediated HR is dispensable for stem cell function in quiescent HSC, whereas it is essential to HSC response to DNA damage processing after cell cycle entry, and its loss is not compensated by intact NHEJ. In HSCs, the maintenance of stem cell function after DNA damage is dependent on the DNA repair capacity, segregated by active versus quiescent points in cell cycle. © AlphaMed Press.

The mutation studies of mutagen-sensitive and DNA repair mutants of Chinese hamster fibroblasts

International Nuclear Information System (INIS)

Schultz, R.A.; Chang, C.C.; Trosko, J.E.

1981-01-01

We have previously reported the isolation and partial characterization of DNA repair and/or mutagen-sensitive mutant Chinese hamster cell strains. Here we present the results of a detailed study of the ultraviolet light (UV)-induced mutability of one of these strains, UVs-7, and provide preliminary mutability data on two additional lines, UVr-23 and UVs-40. UVs-7 in extremely deficient in unscheduled DNA synthesis (UDS) but only slightly more sensitive to UV than the parental line. When examined for the UV-inducibility of mutants resistant to ouabain, 6-thioguanine, or diphtheria toxin, UVs-7 was found to be hypermutable at all three loci as compared to the parental line. The degree of hypermutability was not the same for any two loci. UVs-40, a highly UV-sensitive strain, was also found to be hypermutable at the ouabain-resistant (ouar) locus. UVr-23, which is UV-resistant and more proficient at UDS than the parental line, appeared to exhibit a tendency toward hypomutability at both the ouabain(ouar) and 6-thioguanine--resistant (6TGr) loci. Further characterization of all these lines should aid in delineating mammalian mechanisms of DNA repair and mutagenesis

A novel role of EMMPRIN/CD147 in transformation of quiescent fibroblasts to cancer-associated fibroblasts by breast cancer cells

Science.gov (United States)

Xu, Jing; Lu, Yang; Qiu, Songbo; Chen, Zhi-Nan; Fan, Zhen

2013-01-01

We tested the novel hypothesis that EMMPRIN/CD147, a transmembrane glycoprotein overexpressed in breast cancer cells, has a previously unknown role in transforming fibroblasts to cancer-associated fibroblasts, and that cancer-associated fibroblasts in turn induce epithelial-to-mesenchymal transition of breast cancer cells. Co-culture of fibroblasts with breast cancer cells or treatment of fibroblasts with breast cancer cell conditioned culture medium or recombinant EMMPRIN/CD147 induced expression of α-SMA in the fibroblasts in an EMMPRIN/CD147-dependent manner and promoted epithelial-to-mesenchymal transition of breast cancer cells and enhanced cell migration potential. These findings support a novel role of EMMPRIN/CD147 in regulating the interaction between cancer and stroma. PMID:23474495

Pre-operative Tei Index does not predict left ventricular function immediately after mitral valve repair

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Chirojit Mukherjee

2012-01-01

Full Text Available Echocardiographic assessment of systolic left ventricular (LV function in patients with severe mitral regurgitation (MR undergoing mitral valve (MV repair can be challenging because the measurement of ejection fraction (EF or fractional area change (FAC in pathological states is of questionable value. The aim of our study was to evaluate the usefulness of the pre-operative Tei Index in predicting left ventricular EF or FAC immediately after MV repair. One hundred and thirty patients undergoing MV repair with sinus rhythm pre- and post-operatively were enrolled in this prospective study. Twenty-six patients were excluded due to absence of sinus rhythm post-operatively. Standard transesophageal examination(IE 33,Philips,Netherlands was performed before and after cardiopulmonary bypass according to the guidelines of the ASE/SCA. FAC was determined in the transgastric midpapillary short-axis view. LV EF was measured in the midesophageal four- and two-chamber view. For calculation of the Tei Index, the deep transgastric and the midesophageal four-chamber view were used. Statistical analysis was performed with SPSS 17.0. values are expressed as mean with standard deviation. LV FAC and EF decreased significantly after MV repair (FAC: 56±12% vs. 50±14%, P<0.001; EF: 58±11 vs. 50±12Έ P<0.001. The Tei Index decreased from 0.66±0.23 before MV repair to 0.41±0.19 afterwards (P<0.001. No relationship between pre-operative Tei Index and post-operative FAC or post-operative EF were found (FAC: r=−0.061, P=0.554; EF: r=−0.29, P=0.771. Conclusion: Pre-operative Tei Index is not a good predictor for post-operative FAC and EF in patients undergoing MV repair.

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

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Zhi-yuan Guo

2015-01-01

Full Text Available Human umbilical cord-derived mesenchymal stem cells (hUCMSCs represent a promising young-state stem cell source for cell-based therapy. hUCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of hUCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that hUCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with hUCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of hUCMSCs in peripheral nerve repair.

CDCP1 identifies a CD146 negative subset of marrow fibroblasts involved with cytokine production.

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Mineo Iwata

Full Text Available In vitro expanded bone marrow stromal cells contain at least two populations of fibroblasts, a CD146/MCAM positive population, previously reported to be critical for establishing the stem cell niche and a CD146-negative population that expresses CUB domain-containing protein 1 (CDCP1/CD318. Immunohistochemistry of marrow biopsies shows that clusters of CDCP1+ cells are present in discrete areas distinct from areas of fibroblasts expressing CD146. Using a stromal cell line, HS5, which approximates primary CDCP1+ stromal cells, we show that binding of an activating antibody against CDCP1 results in tyrosine-phosphorylation of CDCP1, paralleled by phosphorylation of Src Family Kinases (SFKs Protein Kinase C delta (PKC-δ. When CDCP1 expression is knocked-down by siRNA, the expression and secretion of myelopoietic cytokines is increased. These data suggest CDCP1 expression can be used to identify a subset of marrow fibroblasts functionally distinct from CD146+ fibroblasts. Furthermore the CDCP1 protein may contribute to the defining function of these cells by regulating cytokine expression.

Development and validation of a skin fibroblast biomarker profile for schizophrenic patients

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Marianthi Logotheti

2016-12-01

Full Text Available Gene expression profiles of non-neural tissues through microarray technology could be used in schizophrenia studies, adding more information to the results from similar studies on postmortem brain tissue. The ultimate goal of such studies is to develop accessible biomarkers. Supervised machine learning methodologies were used, in order to examine if the gene expression from skin fibroblast cells could be exploited for the classification of schizophrenic subjects. A dataset of skin fibroblasts gene expression of schizophrenia patients was obtained from Gene Expression Omnibus database. After applying statistical criteria, we concluded to genes that present a differential expression between the schizophrenic patients and the healthy controls. Based on those genes, functional profiling was performed with the BioInfoMiner web tool. After the statistical analysis, 63 genes were identified as differentially expressed. The functional profiling revealed interesting terms and pathways, such as mitogen activated protein kinase and cyclic adenosine monophosphate signaling pathways, as well as immune-related mechanisms. A subset of 16 differentially expressed genes from fibroblast gene expression profiling that occurred after Support Vector Machines Recursive Feature Elimination could efficiently separate schizophrenic from healthy controls subjects. These findings suggest that through the analysis of fibroblast based gene expression signature and with the application of machine learning methodologies we might conclude to a diagnostic classification model in schizophrenia.

Human uracil DNA N-glycosidase: studies in normal and repair defective cultured fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Kuhnlein, U; Lee, B; Linn, S

1978-01-01

Uracil DNA N-glycosidase, an enzyme which participates in the excision of uracil from DNA, was measured in extracts from fibroblast lines cultured from normal subjects, from several subjects with the genetic disease xeroderma pigmentosum, and from a subject with ataxia telangiectasia. The cell lines representative of complementation groups A and D of xeroderma pigmentosum and of ataxia telangiectasia had roughly the same level of activity as did the normal cells. On the other hand, cells from two xeroderma pigmentosum variants (XP4BE and XP13BE) had roughly half the normal level of activity, and cells from the heterozygous mother of XP4BE had an intermediate level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent K/sub m/ for substrate, or purification characteristics were noted for enzyme from any of the lines. Thus a causal relationship, if any, between levels of activity and the disease symptoms is equivocal.

Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

International Nuclear Information System (INIS)

Story, M.T.

1989-01-01

To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue

Randomised trial of mitral valve repair with leaflet resection versus leaflet preservation on functional mitral stenosis (The CAMRA CardioLink-2 Trial).

Science.gov (United States)

Chan, Vincent; Chu, Michael W A; Leong-Poi, Howard; Latter, David A; Hall, Judith; Thorpe, Kevin E; de Varennes, Benoit E; Quan, Adrian; Tsang, Wendy; Dhingra, Natasha; Yared, Kibar; Teoh, Hwee; Chu, F Victor; Chan, Kwan-Leung; Mesana, Thierry G; Connelly, Kim A; Ruel, Marc; Jüni, Peter; Mazer, C David; Verma, Subodh

2017-05-30

The gold-standard treatment of severe mitral regurgitation (MR) due to degenerative disease is valve repair, which is surgically performed with either a leaflet resection or leaflet preservation approach. Recent data suggest that functional mitral stenosis (MS) may occur following valve repair using a leaflet resection strategy, which adversely affects patient prognosis. A randomised comparison of these two approaches to mitral repair on functional MS has not been conducted. This is a prospective, multicentre randomised controlled trial designed to test the hypothesis that leaflet preservation leads to better preservation of mitral valve geometry, and therefore, will be superior to leaflet resection for the primary outcome of functional MS as assessed by 12-month mean mitral valve gradient at peak exercise. Eighty-eight patients with posterior leaflet prolapse will be randomised intraoperatively once deemed by the operating surgeon to feasibly undergo mitral repair using either a leaflet resection or leaflet preservation approach. Secondary end points include comparison of repair strategies with regard to mitral valve orifice area, leaflet coaptation height, 6 min walk test and a composite major adverse event end point consisting of recurrent MR ≥2+, death or hospital readmission for congestive heart failure within 12 months of surgery. Institutional ethics approval has been obtained from all enrolling sites. Overall, there remains clinical equipoise regarding the mitral valve repair strategy that is associated with the least likelihood of functional MS. This trial hopes to introduce high-quality evidence to help surgical decision making in this context. NCT02552771. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

The chalcone butein from Rhus verniciflua Stokes inhibits clonogenic growth of human breast cancer cells co-cultured with fibroblasts

Directory of Open Access Journals (Sweden)

Tan Jenny

2005-03-01

Full Text Available Abstract Background Butein (3,4,2',4'-tetrahydroxychalone, a plant polyphenol, is a major biologically active component of the stems of Rhus verniciflua Stokes. It has long been used as a food additive in Korea and as an herbal medicine throughout Asia. Recently, butein has been shown to suppress the functions of fibroblasts. Because fibroblasts are believed to play an important role in promoting the growth of breast cancer cells, we investigated the ability of butein to inhibit the clonogenic growth of small numbers of breast cancer cells co-cultured with fibroblasts in vitro. Methods We first measured the clonogenic growth of small numbers of the UACC-812 human breast cancer cell line co-cultured on monolayers of serum-activated, human fibroblasts in the presence of butein (2 μg/mL or various other modulators of fibroblast function (troglitazone-1 μg/mL; GW9662-1 μM; meloxican-1 μM; and 3,4 dehydroproline-10 μg/mL. In a subsequent experiment, we measured the dose-response effect on the clonogenic growth of UACC-812 breast cancer cells by pre-incubating the fibroblasts with varying concentrations of butein (10 μg/ml-1.25 μg/mL. Finally, we measured the clonogenic growth of primary breast cancer cells obtained from 5 clinical specimens with normal fibroblasts and with fibroblasts that had been pre-treated with a fixed dose of butein (2.5 μg/mL. Results Of the five modulators of fibroblast function that we tested, butein was by far the most potent inhibitor of clonogenic growth of UACC-812 breast cancer cells co-cultured with fibroblasts. Pre-treatment of fibroblasts with concentrations of butein as low as 2.5 μg/mL nearly abolished subsequent clonogenic growth of UACC-812 breast cancer cells co-cultured with the fibroblasts. A similar dose of butein had no effect on the clonogenic growth of breast cancer cells cultured in the absence of fibroblasts. Significantly, clonogenic growth of the primary breast cancer cells was also

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene.

Science.gov (United States)

Gerlach, Max; Kraft, Theresia; Brenner, Bernhard; Petersen, Björn; Niemann, Heiner; Montag, Judith

2018-06-13

During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7 -gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene ( GMNN ). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9- GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9- GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.