Alpha-ketoglutarate protects Streptomyces coelicolor from visible light-induced phototoxicity

It has been known that some Streptomyces species, including the model strain Streptomyces coelicolor, are vulnerable to visible light. Much evidence demonstrated that the phototoxicity induced by visible light is a consequence of the formation of intracellular reactive oxygen species (ROS), which are potentially harmful to cells. In this study, we found that α-ketoglutarate (α-KG) has a protective role against the phototoxicity in S. coelicolor. It could be because that α-KG can detoxify the ROS with the concomitant formation of succinate, which mediates the cells getting into anaerobiosis to produce more NADH and maintain intracellular redox homeostasis, a situation that was demonstrated by overexpressing gdhA in S. coelicolor. This finding, therefore, connects the central metabolites with the bacterial resistance against phototoxicity effect induced by visible light.     

He-Ne laser irradiation protects B-lymphoblasts from UVA-induced DNA damage

The effect of He-Ne laser (632.8 nm) pre-irradiation on UVA (343 nm)-induced DNA damage in the human B-lymphoblast cell line NC37 was investigated using the comet assay. He-Ne laser pre-irradiation was observed to result in a dose-dependent decrease in UVA-induced DNA damage. This effect was also found to be dependent on the incubation period between He-Ne laser pre-irradiation and the UVA exposure. Whereas the control cells with a higher DNA damage point to an initial ability of faster repair, both the control and the He-Ne laser pre-irradiated cells subsequently show the same rate of DNA repair. The results suggest that He-Ne laser irradiation protect the cells from UVA-induced DNA damage primarily through an influence on processes that prevent an initial DNA damage. Received: 10 April 2000 / Accepted: 24 November 2000     

UVA-induced lipid peroxidation in cultured human fibroblasts

The UVA irradiation of cultured human fibroblasts leads to the formation and to the release of thiobarbituric acid-reactive substances in the supernatant. The major thiobarbituric acid-reactive substance is identified by fluorescence spectroscopy and HPLC, as malondialdehyde or malondialdehyde-forming substances under the thiobarbituric acid assay conditions. Malondialdehyde formation strongly suggests a UVA-induced lipid peroxidation. Lipid peroxidation is also supported by the inhibitory effect of D,L-alpha-tocopherol, the well-known chain breaking antioxidant, by the additional malondialdehyde formation in the dark after the photooxidative stress and by membrane damage revealed by lactate dehydrogenase leakage.     

Hydrogen peroxide and catalase in UVA-induced lipid peroxidation in cultured fibroblasts

SUMMARY

UVA-induced lipid peroxidation in cultured human skin fibroblasts, as measured by the release in the supernatant of thiobarbituric acid-reactive substances, is found to be linear with increasing irradiation dose (up to about 250 kJ m^?2). Concomitantly, within this dose range catalase is strongly inactivated by UVA radiation according to an exponential process (k≈0.01 kJ^?1 m²). This suggests that catalase is not involved in modulating the peroxidation process. Inactivation of catalase by 3-amino-1,2,4-triazole can be efficiently achieved prior to irradiation. This inactivation has no consequence on the extent of peroxidation triggered by subsequent exposure to UVA radiation. It may be therefore strongly suggested that catalase is not, via H₂O₂ removal, a key enzyme in the cellular defence equipment towards UV A-peroxidative stress. An alternative interpretation may be formulated which supports the view that H₂O₂ produced upon exposure to UVA has no or very little role in triggering the lipid peroxidation process.     

Metformin-stimulated mannose transport in dermal fibroblasts

The biguanide drug metformin stimulates AMP-activated protein kinase, a master regulator of cellular energy metabolism, and has antihyperglycemic activity due to attenuation of gluconeogenesis in hepatocytes and 2-fold stimulation of glucose transport by skeletal muscle. Here we identify a metformin-stimulated d-mannose transport (MSMT) activity in dermal fibroblasts. MSMT increased mannose uptake 1.8-fold and had greater affinity for mannose than basal mannose transport activity. It was attributed to robust stimulation of a transporter expressed weakly in untreated cells. MSMT was not explained by greater glucose transporter activity because metformin unexpectedly decreased transport of 2-deoxy-d-glucose and 3-O-methyl-d-glucose by fibroblasts. Effective inhibitors of MSMT retained specificity for the 3-, 4-, and 6-OH groups of the mannose ring but not the 2-OH group. Thus, MSMT could be strongly inhibited by glucose and 2-deoxy-d-glucose even though the latter was not a good transport substrate. MSMT was significant because in the presence of 2.5 microm mannose, metformin corrected experimentally induced deficiencies in the synthesis of glucose(3)mannose(9)GlcNAc(2)-P-P-dolichol and N-linked glycosylation. MSMT was also identified in congenital disorder of glycosylation types Ia and Ib fibroblasts, and metformin acted synergistically with 100 microm mannose to correct lipid-linked oligosaccharide synthesis and N-glycosylation in the Ia cells. In conclusion, metformin activates a novel fibroblast mannose-selective transport system. This suggests that AMP-activated protein kinase may be a regulator of mannose metabolism and implies a therapy for congenital disorders of glycosylation-Ia.     

Natural killer cells activate human dermal fibroblasts

Human dermal fibroblasts (HDF) undergo activation and secrete cytokines when cocultured with T cells. Here, we identify potent activators of HDF among human peripheral CD2(+)-lymphocytes. Populations with strong HDF activating capacity consisted essentially of cells with a natural killer (NK) surface marker phenotype (CD3(-), CD4(-), CD8(-), CD56(+)). Addition of these cells to HDF resulted in rapid increase of intracellular free calcium concentrations as an early rapid cell activation signal. Upregulation of mRNA encoding for the inflammatory cytokines IL-1 beta and IL-6 as well as for chemokines IL-8 and MCP-1 was detected after cells were cocultured. Elevated concentrations of IL-6 and IL-8 were found in coculture supernatants of HDF and NK-cells. Skin-homing NK cells leaving the blood-stream during an inflammatory skin reaction might therefore represent potent activators of local inflammatory cytokine and chemokine production.     

Abnormal fibrillin assembly by dermal fibroblasts from two patients with Marfan syndrome

The microfibrillar glycoprotein fibrillin is linked to the Marfan syndrome, an autosomal dominant connective tissue disorder. In this study, fibrillin synthesis, deposition and assembly has been investigated in Marfan dermal fibroblast lines from two unrelated patients for whom distinct mutations in the fibrillin gene FBN1 have been identified. In patient NB, a point mutation has occurred which causes an amino acid substitution and the other patient (GK) has a deletion in one allele. The two cell lines were broadly comparable with respect to de novo fibrillin synthesis and its distribution between medium and cell layer compartments. Electrophoresis of fibrillin immunoprecipitates confirmed the presence of fibrillin in medium and cell layers. GK cells secreted an additional higher relative molecular mass fibrillin-immunoreactive component. The time-course of fibrillin secretion was similar for the two lines, but differences in fibrillin aggregation were apparent. Rotary shadowing electron microscopy of extracted cell layers demonstrated the presence of abundant and extensive microfibrils in NB cell layers. These were abnormal in their gross morphology in comparison to microfibrils isolated from control cultures. No periodic microfibrillar structures were isolated from GK cell layers. These studies underline the need to classify fibrillin defects in terms of biochemical and ultrastructural criteria. Examination of the effects of individual mutations on microfibril organization will be particularly informative in elucidating the relationship between microfibril dysfunction and the complex clinical manifestations of Marfan patients.     

Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation

A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitor L-N5-(1-iminoethyl)-ornithine (0.25 mM). Additionally, preincubation of cells with the NO donor (Z)-1-[N(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-i um-1, 2-diolate at concentrations from 10 to 1000 microM as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection.     

Epicatechin and its methylated metabolite attenuate UVA-induced oxidative damage to human skin fibroblasts

The ultraviolet A component of sunlight causes both acute and chronic damage to human skin. In this study the potential of epicatechin, an abundant dietary flavanol, and 3'-O-methyl epicatechin, one of its major in vivo metabolites, to protect against UVA-induced damage was examined using cultured human skin fibroblasts as an in vitro model. The results obtained clearly show that both epicatechin and its metabolite protect these fibroblasts against UVA damage and cell death. The hydrogen-donating antioxidant properties of these compounds are probably not the mediators of this protective response. The protection is a consequence of induction of resistance to UVA mediated by the compounds and involves newly synthesized proteins. The study provides clear evidence that this dietary flavanol has the potential to protect human skin against the deleterious effects of sunlight.     

MnSOD activity protects mitochondrial morphology of quiescent fibroblasts from age associated abnormalities

Previously, we have shown manganese superoxide dismutase (MnSOD) activity protects quiescent human normal skin fibroblasts (NHFs) from age associated loss in proliferative capacity. The loss in proliferative capacity of aged vs. young quiescent cells is often characterized as the chronological life span, which is clearly distinct from replicative senescence. We investigate the hypothesis that MnSOD activity protects the mitochondrial morphology from age associated damage and preserves the chronological life span of quiescent fibroblasts. Aged quiescent NHFs exhibited abnormalities in mitochondrial morphology including abnormal cristae formation and increased number of vacuoles. These results correlate with the levels of cellular reactive oxygen species (ROS) and mitochondrial morphology in MnSOD homozygous and heterozygous knockout mouse embryonic fibroblasts. The abnormalities in mitochondrial morphology in aged quiescent NHFs cultured in presence of 21% oxygen concentration were more severe than NHFs cultured in 4% oxygen environment. The alteration in mitochondrial morphology was associated with a significant increase in cell population doubling: 54 h in 21% compared to 44 h in 4% oxygen environment. Overexpression of MnSOD decreased ROS levels, and preserved mitochondrial morphology in aged quiescent NHFs. These results demonstrate that MnSOD activity protects mitochondrial morphology and preserves the proliferative capacities of quiescent NHFs from age associated loss.     

Biological characters of human dermal fibroblasts derived from foreskin of male infertile patients

Dermal fibroblasts play a vital role in maintaining skin function. They not only synthesize and secrete extracellular matrix molecules, but also produce a complex mixture of bioactive factors, which both contribute to immune regulation and wound healing. Fibroblasts isolated from skin tissue exhibit wide range of potentials, especially in regenerative medicine. The use of fibroblast cultures for medical purposes requires standardization of cell preparations. To achieve this, we isolated and characterized dermal fibroblasts from human foreskin with a standardized method. The obtained cells grew as typical morphology of fibroblasts, and expressed intermediate filament protein vimentin and nestin. Immunophenotypic analysis indicated that the isolated fibroblasts expressed mesenchymal surface markers CD73, CD90, CD44 and CD105, and were negative for haematopoietic markers CD45 and CD34. Growth kinetics analysis of the cells showed high proliferative properties. Furthermore, cryopreservation had no influence on cell morphology and growth properties. Here, we describe a standardized, repeatable method for isolation of fibroblasts from human foreskin tissues and identify their biological characters according to morphologic, immunohistologic and proliferative criteria, which would be meaningful for future clinical trials and regenerative medicine purposes.     

Differentiation of human dermal fibroblasts towards endothelial cells

The ultimate goal of vascular tissue engineering is the production of functional grafts for clinical use. Difficulties acquiring autologous endothelial cells have motivated the search for alternative cell sources. Differentiation of dermal fibroblasts towards several mesenchymal lineages as well as endothelial cells has been proposed. The aim of the present study was to investigate the endothelial differentiation capacity of human dermal fibroblasts on a gene expression, protein expression and functional physiological level. Endothelial differentiation of fibroblasts was induced by culturing cells in 30% human serum, but not in fetal calf serum. Expression of proteins and genes relevant for endothelial function and differentiation was increased after induction. Furthermore, fibroblasts exposed to 30% human serum displayed increased uptake of low-density lipoprotein and formation of capillary-like networks. The results of this study may have an impact on cell sourcing for vascular tissue engineering, and the development of methods for vascularization of autologous tissue engineered constructs.     

The contraction of collagen matrices by dermal fibroblasts

Floating collagen gel cultures containing human foreskin fibroblasts have been observed to undergo a rapid contraction process. The initial rate of contraction (i.e., within the first 2 hr) was observed to be a linear function of cell number within the concentration range of 10(5)-10(6) cells/gel. Observation of thick, deresined sections of such contracting gels in the SEM, as well as observation of thin sections in the TEM, suggest that the fibroblasts exert a tension upon the surrounding collagen fibers. These observations further indicate that the fibroblasts migrate from the interior regions of the gel matrix and eventually form a monolayer of cells encapsulating the contracted collagen disc. These observations are discussed in terms of the possible mechanisms involved in gel contraction.     

Contractility of single human dermal myofibroblasts and fibroblasts

Human dermal myofibroblasts, characterised by the expression of alpha-smooth muscle actin, are part of the granulation tissue and implicated in the generation of contractile forces during normal wound healing and pathological contractures. We have compared the contractile properties of single human dermal fibroblasts and human dermal myofibroblasts by culturing them on flexible silicone elastomers. The flexibility of the silicone substratum permits the contractile forces exerted by the cells to be measured [Fray et al., 1998: Tissue Eng. 4:273-283], without changing their expression of alpha-smooth muscle actin. The mean contractile force produced by myofibroblasts (2.2 microN per cell) was not significantly different from that generated by fibroblasts (2.0 microN per cell) when cultured on a substrata with a low elastomer stiffness. Forces produced by fibroblasts were unaffected by increases in elastomer stiffness, but forces measured for myofibroblasts increased to a mean value of 4.1 microN/cell. This was associated with a higher proportion of myofibroblasts being able to produce wrinkles on elastomers of high stiffness compared to fibroblasts. We discuss the force measurements at the single cell level, for both fibroblast and myofibroblasts, in relation to the proposed role of myofibroblasts in wound healing and pathological contractures.     

Defining the identity of mouse embryonic dermal fibroblasts

Embryonic dermal fibroblasts in the skin have the exceptional ability to initiate hair follicle morphogenesis and contribute to scarless wound healing. Activation of the Wnt signaling pathway is critical for dermal fibroblast fate selection and hair follicle induction. In humans, mutations in Wnt pathway components and target genes lead to congenital focal dermal hypoplasias with diminished hair. The gene expression signature of embryonic dermal fibroblasts during differentiation and its dependence on Wnt signaling is unknown. Here we applied Shannon entropy analysis to identify the gene expression signature of mouse embryonic dermal fibroblasts. We used available human DNase‐seq and histone modification ChiP‐seq data on various cell‐types to demonstrate that genes in the fibroblast cell identity signature can be epigenetically repressed in other cell‐types. We found a subset of the signature genes whose expression is dependent on Wnt/β‐catenin activity in vivo. With our approach, we have defined and validated a statistically derived gene expression signature that may mediate dermal fibroblast identity and function in development and disease. genesis 54:415–430, 2016. © 2016 Wiley Periodicals, Inc.     

Protective effects of antioxidants against UVA-induced DNA damage in human skin fibroblasts in culture

Ultraviolet A radiation (UVA, 320–400 nm) is mutagenic and induces genomic damage to skin cells. N-acetyl-cysteine (NAC), selenium and zinc have been shown to have antioxidant properties and to exhibit protective effects against UVA cytotoxicity. The present work attempts to delineate the effect of these compounds on genomic integrity of human skin fibroblasts exposed to UVA radiation using the single cell gel electrophoresis (SCGE) or Comet assay. The cells were incubated with NAC (5 mM), sodium selenite (0.6 μM) or zinc chloride (100 μM). Then cells were embedded in low melting point agarose, and immediately submitted to UVA fluences ranging from 1 to 6J/cm². In the Comet assay, the tail moment increased by 45% (1J/cm²) to 89% (6J/cm²) in non-supplemented cells (p<0.01). DNA damage was significantly prevented by NAC, Se and Zn, with a similar efficiency from 1 to 4J/cm² (p<0.05). For the highest UVA dose (6J/cm²), Se and Zn were more effective than NAC (p<0.01).