Reversible Immortalization Enables Seamless Transdifferentiation of Primary Fibroblasts into Other Lineage Cells

AbstractAmong several animal groups (eutherian mammals, birds, reptiles), lifespan positively correlates with body mass over several orders of magnitude. Contradicting this pattern are domesticated dogs, with small dog breeds exhibiting significantly longer lifespans than large dog breeds. The underlying mechanisms of differing aging rates across body masses are unclear, but it is generally agreed that metabolism is a significant regulator of the aging process. Herein, we performed a targeted metabolomics analysis on primary fibroblasts isolated from small and large breed young and old dogs. Regardless of size, older dogs exhibited lower glutathione and ATP, consistent with a role for oxidative stress and bioenergetic decline in aging. Furthermore, several size-specific metabolic patterns were observed with aging, including the following: (i) An apparent defect in the lower half of glycolysis in large old dogs at the level of pyruvate kinase. (ii) Increased glutamine anaplerosis into the TCA cycle in large old dogs. (iii) A potential defect in coenzyme A biosynthesis in large old dogs. (iv) Low nucleotide levels in small young dogs that corrected with age. (v) An age-dependent increase in carnitine in small dogs that was absent in large dogs. Overall, these data support the hypothesis that alterations in metabolism may underlie the different lifespans of small vs. large breed dogs, and further work in this area may afford potential therapeutic strategies to improve the lifespan of large dogs.

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Synthesis and Characterisation of a Boron-Rich Symmetric Triazine Bearing a Hypoxia-Targeting Nitroimidazole Moiety

Symmetry ◽

10.3390/sym13020202 ◽

2021 ◽

Vol 13 (2) ◽

pp. 202

Author(s):

Tobias Hartwig Bünning ◽

Luigi Panza ◽

Abdel Kareem Azab ◽

Barbara Muz ◽

Silvia Fallarini ◽

...

Keyword(s):

Tumor Tissue ◽

High Ratio ◽

Neutron Capture Therapy ◽

Boron Compounds ◽

The Core ◽

Boron Neutron Capture ◽

Starting Point ◽

Primary Fibroblasts ◽

Normal Human ◽

Hypoxia Markers

Boron Neutron Capture Therapy (BNCT) is a binary therapy that promises to be suitable in treating many non-curable cancers. To that, the discovery of new boron compounds able to accumulate selectively in the tumour tissue is still required. Hypoxia, a deficiency of oxygen in tumor tissue, is a great challenge in the conventional treatment of cancer, because hypoxic areas are resistant to conventional anticancer treatments. 2-Nitroimidazole derivatives are known to be hypoxia markers due to their enrichment by bioreduction in hypoxic cells. In the present work, 2-nitroimidazole was chosen as the starting point for the synthesis of a new boron-containing compound based on a 1,3,5-triazine skeleton. Two o-carborane moieties were inserted to achieve a high ratio of boron on the molecular weight, exploiting a short PEG spacer to enhance the polarity of the compound and outdistance the active part from the core. The compound showed no toxicity on normal human primary fibroblasts, while it showed noteworthy toxicity in multiple myeloma cells together with a consistent intracellular boron accumulation.

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Cell Behavior of Primary Fibroblasts and Osteoblasts on Plasma-Treated Fluorinated Polymer Coated with Honeycomb Polystyrene

Materials ◽

10.3390/ma14040889 ◽

2021 ◽

Vol 14 (4) ◽

pp. 889

Author(s):

Klára Fajstavrová ◽

Silvie Rimpelová ◽

Dominik Fajstavr ◽

Václav Švorčík ◽

Petr Slepička

Keyword(s):

Chemical Properties ◽

Plasma Exposure ◽

Research Teams ◽

Polymer Substrates ◽

Substrate Activation ◽

Physico Chemical ◽

Polystyrene Solution ◽

Primary Fibroblasts ◽

Positive Effect ◽

Cell Adhesion And Proliferation

The development of new biocompatible polymer substrates is still of interest to many research teams. We aimed to combine a plasma treatment of fluorinated ethylene propylene (FEP) substrate with a technique of improved phase separation. Plasma exposure served for substrate activation and modification of surface properties, such as roughness, chemistry, and wettability. The treated FEP substrate was applied for the growth of a honeycomb-like pattern from polystyrene solution. The properties of the pattern strongly depended on the primary plasma exposure of the FEP substrate. The physico-chemical properties such as changes of the surface chemistry, wettability, and morphology of the prepared pattern were determined. The cell response of primary fibroblasts and osteoblasts was studied on a honeycomb pattern. The prepared honeycomb-like pattern from polystyrene showed an increase in cell viability and a positive effect on cell adhesion and proliferation for both primary fibroblasts and osteoblasts.

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Involvement of CGRP receptor RAMP1 in cluster headache: A Swedish case-control study

Cephalalgia Reports ◽

10.1177/2515816319879886 ◽

2019 ◽

Vol 2 ◽

pp. 251581631987988 ◽

Cited By ~ 1

Author(s):

Julia M Michalska ◽

Caroline Ran ◽

Carmen Fourier ◽

Anna Steinberg ◽

Christina Sjöstrand ◽

...

Keyword(s):

Cluster Headache ◽

Mrna Expression ◽

Quantitative Polymerase Chain Reaction ◽

Active Phase ◽

Nucleotide Polymorphisms ◽

Cgrp Receptor ◽

Receptor Component ◽

Significant Difference ◽

Potent Vasodilator ◽

Primary Fibroblasts

Background: Increased levels of the potent vasodilator calcitonin gene-related peptide (CGRP) have been found in ipsilateral jugular vein blood during the active phase of cluster headache (CH) and this is hypothesized to cause distinctive vasodilation. The receptor activity-modifying protein 1 (RAMP1) is part of the CGRP receptor complex responsible for ligand binding and specificity and therefore constitutes a promising candidate gene for CH. The aim of this study was to investigate the possible genetic association of RAMP1 with CH in Sweden, with focus on two RAMP1 single nucleotide polymorphisms, rs3754701 and rs7590387, and quantify RAMP1 mRNA expression levels in biological tissue from CH patients and controls. Methods: rs3754701 and rs7590387 were genotyped by quantitative polymerase chain reaction (qPCR) in 542 CH patients and 585 control subjects. RAMP1 mRNA expression was determined by reverse transcription qPCR in tissue from 12 CH patients and 12 controls. Results: We identified a significant difference between the CH patient and control groups for rs3754701 ( p = 0.0088). In addition, RAMP1 mRNA expression was enhanced in primary fibroblasts from CH patients compared to controls ( p = 0.0073). Conclusion: The association between rs3754701 and CH and the enhanced RAMP1 mRNA expression in CH patients support the hypothesis that CGRP and its receptor component RAMP1 are involved in CH pathophysiology.

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HSP70 Induction by ING Proteins Sensitizes Cells to Tumor Necrosis Factor Alpha Receptor-Mediated Apoptosis

Molecular and Cellular Biology ◽

10.1128/mcb.01538-06 ◽

2006 ◽

Vol 26 (24) ◽

pp. 9244-9255 ◽

Cited By ~ 43

Author(s):

Xiaolan Feng ◽

Shirin Bonni ◽

Karl Riabowol

Keyword(s):

Tumor Necrosis Factor ◽

Tumor Necrosis Factor Alpha ◽

Tumor Necrosis ◽

Kinase Inhibitor ◽

Heat Shock Gene ◽

Necrosis Factor Alpha ◽

Tnf Α ◽

Primary Fibroblasts ◽

Factor Alpha ◽

Necrosis Factor

ABSTRACT ING proteins affect apoptosis, growth, and DNA repair by transducing stress signals such as DNA damage, binding histones, and subsequently regulating chromatin structure and p53 activity. p53 target genes, including the p21 cyclin-dependent kinase inhibitor and Bax, an inducer of apoptosis, are regulated by ING proteins. To identify additional targets downstream of p33ING1 and p32ING2, cDNA microarrays were performed on phenotypically normal human primary fibroblasts. The 0.36% of genes affected by ING proteins in primary fibroblasts were distinct from targets seen in established cells and included the HSP70 heat shock gene, whose promoter was specifically induced >10-fold. ING1-induced expression of HSP70 shifted cells from survival to a death pathway in response to tumor necrosis factor alpha (TNF-α), and p33ING1b protein showed synergy with TNF-α in inducing apoptosis, which correlated with reduced NF-κB-dependent transcription. These findings are consistent with previous reports that HSP70 promotes TNF-α-mediated apoptosis by binding I-κΒ kinase gamma and impairing NF-κB survival signaling. Induction of HSP70 required the amino terminus of ING1b but not the plant homeodomain region that was recently identified as a histone binding domain. Regulation of HSP70 gene expression by the ING tumor suppressors provides a novel link between the INGs and the stress-regulated NF-κB survival pathway important in hypoxia and angiogenesis.

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Requirement of ATM in Phosphorylation of the Human p53 Protein at Serine 15 following DNA Double-Strand Breaks

Molecular and Cellular Biology ◽

10.1128/mcb.19.4.2828 ◽

1999 ◽

Vol 19 (4) ◽

pp. 2828-2834 ◽

Cited By ~ 82

Author(s):

Kazumi Nakagawa ◽

Yoichi Taya ◽

Katsuyuki Tamai ◽

Masaru Yamaizumi

Keyword(s):

Heat Shock ◽

Ataxia Telangiectasia ◽

Xeroderma Pigmentosum ◽

P53 Protein ◽

Double Strand Breaks ◽

Nuclear Accumulation ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Absolute Requirement ◽

Primary Fibroblasts

ABSTRACT Microinjection of the restriction endonuclease HaeIII, which causes DNA double-strand breaks with blunt ends, induces nuclear accumulation of p53 protein in normal and xeroderma pigmentosum (XP) primary fibroblasts. In contrast, this induction of p53 accumulation is not observed in ataxia telangiectasia (AT) fibroblasts. HaeIII-induced p53 protein in normal fibroblasts is phosphorylated at serine 15, as determined by immunostaining with an antibody specific for phosphorylated serine 15 of p53. This phosphorylation correlates well with p53 accumulation. Treatment with lactacystin (an inhibitor of the proteasome) or heat shock leads to similar levels of p53 accumulation in normal and AT fibroblasts, but the p53 protein lacks a phosphorylated serine 15. Following microinjection of HaeIII into lactacystin-treated normal fibroblasts, lactacystin-induced p53 protein is phosphorylated at serine 15 and stabilized even in the presence of cycloheximide. However, neither stabilization nor phosphorylation at serine 15 is observed in AT fibroblasts under the same conditions. These results indicate the significance of serine 15 phosphorylation for p53 stabilization after DNA double-strand breaks and an absolute requirement for ATM in this phosphorylation process.

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Application of a Reversible Immortalization System for the Generation of Proliferation-Controlled Cell Lines

Cytotechnology ◽

10.1007/s10616-005-2834-z ◽

2004 ◽

Vol 46 (2-3) ◽

pp. 69-78 ◽

Cited By ~ 4

Author(s):

Tobias May ◽

Werner Lindenmaier ◽

Dagmar Wirth ◽

Peter P. Mueller

Keyword(s):

Cell Lines ◽

Reversible Immortalization

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Evaluation of EPS-PCL Nanofibers as a Nanobiocomposite for Artificial Skin Based on Dermal Fibroblast Culture

Journal of Nanomaterials ◽

10.1155/2013/619893 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 1

Author(s):

Sang-Myung Jung ◽

Dae Seung Kim ◽

Jung Hyeon Ju ◽

Hwa Sung Shin

Keyword(s):

High Cell Density ◽

Undaria Pinnatifida ◽

Dermal Fibroblast ◽

Bioactive Molecules ◽

Artificial Skin ◽

Limited Area ◽

High Cell ◽

Electrospinning Technique ◽

Primary Fibroblasts ◽

Injured Skin

Several natural bioactive molecules have been used in the development of scaffolds to enhance biocompatibility or biodegradability and macroalgae contain many bioactive compounds that regulate the physiological activities of cells. In this study, extrapolymeric substances (EPS) from brown algae,Undaria pinnatifida, were dispersed in poly-ε-caprolactone (PCL) nanofiber, fabricated by electrospinning technique to mimic natural extracellular matrix (ECM), and tested as a scaffold for the production of artificial skin using rat primary fibroblasts. The level of adhesion, viability, and infiltration of cells on the EPS-PCL nanofibers were then assessed. The primary fibroblasts attached well, had good viability, and infiltrated through the nanofiber mat without cytotoxicity. Additionally, fibroblast on EPS-PCL nanofiber overcame the stress derived from high cell density at limited area. These results indicate that EPS-imbedded nanofiber has the potential to be used as scaffolds to develop artificial skin or as wound-healing nanomedicines to regenerate injured skin.

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Abstract 277: Microrna-33 Promotes Cardiac Fibrosis by Maintaining Cellular Lipid Homeostasis

Circulation Research ◽

10.1161/res.119.suppl_1.277 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Masataka Nishiga ◽

Takahiro Horie ◽

Yasuhide Kuwabara ◽

Osamu Baba ◽

Tetsushi Nakao ◽

...

Keyword(s):

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Pressure Overload ◽

Cholesterol Content ◽

Atp Binding Cassette Transporter ◽

Primary Fibroblasts ◽

Proliferation In Vitro ◽

Altered Lipid

Background: A highly conserved microRNA, miR-33 is considered as a potential therapeutic target for atherosclerosis, because recent reports, including ours, indicated miR-33 has atherogenic effects by reducing HDL-C. However, the functions of miR-33 in heart failure remain to be elucidated. Methods and results: To clarify the functions of miR-33 involved in cardiac hypertrophy and fibrosis in vivo, we investigated the responses to pressure overload by transverse aortic constriction (TAC) in miR-33 deficient (KO) mice. When subjected to TAC, miR-33 expression level was significantly up-regulated in wild-type (WT) left ventricles, whereas miR-33 KO hearts displayed no less hypertrophic responses than WT hearts. However, interestingly, histological and gene expression analyses showed ameliorated cardiac fibrosis in miR-33 KO hearts compared to WT hearts. Furthermore, we generated cardiac fibroblast specific miR-33 deficient mice, which also showed ameliorated cardiac fibrosis when they were subjected to TAC. We also found that cardiac fibroblasts were mainly responsible for miR-33 expression in the heart, because its expression was about 4-folds higher in isolated primary cardiac fibroblasts than cardiomyocytes. Deficiency of miR-33 impaired cell proliferation in primary fibroblasts, which was considered due to altered lipid raft cholesterol content by up-regulated ATP-binding cassette transporter A1/G1. Conclusion: Deficiency of miR-33 impaired fibroblast proliferation in vitro, and ameliorated cardiac fibrosis induced by pressure overload in vivo.

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Human primary fibroblasts in vitro express a purinergic P2X7 receptor coupled to ion fluxes, microvesicle formation and IL-6 release

Journal of Cell Science ◽

10.1242/jcs.112.3.297 ◽

1999 ◽

Vol 112 (3) ◽

pp. 297-305

Author(s):

A. Solini ◽

P. Chiozzi ◽

A. Morelli ◽

R. Fellin ◽

F. Di Virgilio

Keyword(s):

Purinergic Receptor ◽

Morphological Changes ◽

Human Fibroblasts ◽

Ion Fluxes ◽

Disulfonic Acid ◽

Triggered Release ◽

Primary Fibroblasts ◽

Purinergic P2x7 Receptor ◽

Skin Biopsies

We have investigated reponses to extracellular ATP in human fibroblasts obtained by skin biopsies. Our data show that these cells express a P2X7 purinergic receptor, as judged by (1) RT-PCR with specific primers, (2) reactivity with a specific anti-P2X7 antiserum, (3) activation by the selective P2X agonist benzoylbenzoylATP and (4) stimulation of transmembrane ion fluxes. Stimulation with benzoylbenzoylATP, and to a lesser extent with ATP, also caused striking morphological changes and increased formation of cytoplasmic microvesicles. These changes were fully reversible upon nucleotide removal. Two known blockers of P2X receptors, oxidised ATP and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, inhibited the morphological changes fully and the ion fluxes partially. The residual rise in intracellular Ca2+ levels and membrane depolarization observed in the presence of the inhibitors were dependent upon activation of a P2Y-type receptor exhibiting a peculiar pharmacological profile, in that CTP was the preferred agonist. ATP stimulation triggered release of the pro-inflammatory cytokine IL-6 in fibroblasts pre-treated with PMA and bacterial endotoxin. These observations reveal a novel pathway for fibroblast activation and for their recruitment in the inflammatory response.

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