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1.
Sci Rep ; 12(1): 4795, 2022 03 21.
Article in English | MEDLINE | ID: mdl-35314737

ABSTRACT

Endothelial cells (ECs) lining blood vessels are exposed to mechanical forces, such as shear stress. These forces control many aspects of EC biology, including vascular tone, cell migration and proliferation. Despite a good understanding of the genes responding to shear stress, our insight into the transcriptional regulation of these genes is much more limited. Here, we set out to study alterations in the chromatin landscape of human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress. To do so, we performed ChIP-Seq for H3K27 acetylation, indicative of active enhancer elements and ATAC-Seq to mark regions of open chromatin in addition to RNA-Seq on HUVEC exposed to 6 h of laminar shear stress. Our results show a correlation of gained and lost enhancers with up and downregulated genes, respectively. DNA motif analysis revealed an over-representation of KLF transcription factor (TF) binding sites in gained enhancers, while lost enhancers contained more ETV/ETS motifs. We validated a subset of flow responsive enhancers using luciferase-based reporter constructs and CRISPR-Cas9 mediated genome editing. Lastly, we characterized the shear stress response in ECs of zebrafish embryos using RNA-Seq. Our results lay the groundwork for the exploration of shear stress responsive elements in controlling EC biology.


Subject(s)
Chromatin , Zebrafish , Animals , Binding Sites , Cells, Cultured , Chromatin/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Stress, Mechanical , Transcription Factors/genetics , Transcription Factors/metabolism , Zebrafish/genetics , Zebrafish/metabolism
2.
J Pathol ; 251(2): 123-134, 2020 06.
Article in English | MEDLINE | ID: mdl-32166747

ABSTRACT

Spontaneously regressing infantile haemangiomas and aggressive angiosarcomas are vascular tumours with excessive angiogenesis. When analysing haemangiomas and angiosarcomas immunohistochemically with respect to their chaperone profiles we found that angiosarcomas have significantly elevated protein levels of binding immunoglobulin protein (BIP) and PERK with concomitant attenuated IRE1α levels, whereas haemangioma tissue exhibits the same pattern as embryonal skin tissue. We show that BiP is essential for the maintenance of VEGFR2 protein, which is expressed in the endothelium of both tumour types. When studying the effects of BiP, the IRE1α/Xbp1 -, and PERK/ATF4-signalling pathways on the migration and tube-forming potential of endothelial cells, we show that downregulation of BiP, as well as inhibition of the kinase activity of IRE1α, inhibit in vitro angiogenesis. Downregulation of PERK (PKR-like kinase; PKR = protein kinase R) levels promotes Xbp1 splicing in endoplasmic reticulum (ER)-stressed cells, indicating that in angiosarcoma the elevated PERK levels might result in high levels of unspliced Xbp1, which have been reported to promote cell proliferation and increase tumour malignancy. The data presented in this study revealed that in addition to BiP or PERK, the kinase domains of IRE1α and Xbp1 could be potential targets for the development of novel therapeutic approaches for treating angiosarcomas and to control tumour angiogenesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Subject(s)
Endoribonucleases/metabolism , Endothelial Cells/enzymology , Heat-Shock Proteins/metabolism , Hemangioma/enzymology , Hemangiosarcoma/enzymology , Neovascularization, Pathologic , Protein Serine-Threonine Kinases/metabolism , eIF-2 Kinase/metabolism , Cell Movement , Cell Proliferation , Cells, Cultured , Endoplasmic Reticulum Chaperone BiP , Endoribonucleases/genetics , Endothelial Cells/pathology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Heat-Shock Proteins/genetics , Hemangioma/genetics , Hemangioma/pathology , Hemangiosarcoma/genetics , Hemangiosarcoma/pathology , Human Umbilical Vein Endothelial Cells/enzymology , Humans , Protein Serine-Threonine Kinases/genetics , Signal Transduction , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , X-Box Binding Protein 1/genetics , X-Box Binding Protein 1/metabolism , eIF-2 Kinase/genetics
3.
Cardiovasc Res ; 115(10): 1487-1499, 2019 Aug 01.
Article in English | MEDLINE | ID: mdl-30785199

ABSTRACT

AIMS: Oscillatory shear stress (OSS) is an atheroprone haemodynamic force that occurs in areas of vessel irregularities and is implicated in the pathogenesis of atherosclerosis. Changes in signalling and transcriptional programme in response to OSS have been vigorously studied; however, the underlying changes in the chromatin landscape controlling transcription remain to be elucidated. Here, we investigated the changes in the regulatory element (RE) landscape of endothelial cells under atheroprone OSS conditions in an in vitro model. METHODS AND RESULTS: Analyses of H3K27ac chromatin immunoprecipitation-Seq enrichment and RNA-Seq in primary human umbilical vein endothelial cells 6 h after onset of OSS identified 2806 differential responsive REs and 33 differentially expressed genes compared with control cells kept under static conditions. Furthermore, gene ontology analyses of putative RE-associated genes uncovered enrichment of WNT/HIPPO pathway and cytoskeleton reorganization signatures. Transcription factor (TF) binding motif analysis within RE sequences identified over-representation of ETS, Zinc finger, and activator protein 1 TF families that regulate cell cycle, proliferation, and apoptosis, implicating them in the development of atherosclerosis. Importantly, we confirmed the activation of EGR1 as well as the YAP/TAZ complex early (6 h) after onset of OSS in both cultured human vein and artery endothelial cells and, by undertaking luciferase assays, functionally verified their role in RE activation in response to OSS. CONCLUSIONS: Based on the identification and verification of specific responsive REs early upon OSS exposure, we propose an expanded mechanism of how OSS might contribute to the development of atherosclerosis.


Subject(s)
Atherosclerosis/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Mechanotransduction, Cellular , Response Elements , Transcription Factors/metabolism , Umbilical Arteries/metabolism , Atherosclerosis/genetics , Atherosclerosis/pathology , Atherosclerosis/physiopathology , Cells, Cultured , Gene Expression Regulation , Gene Regulatory Networks , Human Umbilical Vein Endothelial Cells/pathology , Humans , Plaque, Atherosclerotic , Protein Interaction Maps , Regional Blood Flow , Stress, Mechanical , Transcription Factors/genetics , Umbilical Arteries/pathology , Umbilical Arteries/physiopathology
4.
Mater Sci Eng C Mater Biol Appl ; 93: 115-124, 2018 Dec 01.
Article in English | MEDLINE | ID: mdl-30274043

ABSTRACT

Patients diagnosed with osteosarcoma are currently treated with intravenous injections of anticancer agents after tumor resection. However, due to remaining neoplastic cells at the site of tumor removal, cancer recurrence often occurs. Successful bone regeneration combined with the control of residual cancer cells presents a challenge for tissue engineering. Cyclodextrins loaded with chemotherapeutic drugs reversibly release the drugs over time. Hydroxyapatite bone biomaterials coated with doxorubicin-loaded cyclodextrin should release the drug with time after implantation directly at the original tumor site and may be a way to eliminate residual neoplastic cells. In the present study, we have carried out in vitro studies to evaluate such a drug-delivery system and have shown that doxorubicin released from cyclodextrin-coated hydroxyapatite retained biological activity and exhibited longer and higher cytotoxic effects on both cancer (osteosarcoma cells) and healthy cells (primary osteoblasts and endothelial cells) compared to biomaterials without cyclodextrin loaded with doxorubicin. Furthermore, doxorubicin released from biomaterials with cyclodextrin moderately induced the expression of tumor suppressor protein p53 whereas p21 expression was similar to control cells. In addition, hypoxic conditions, which occur after implantation until blood-flow to the area is regenerated, protected endothelial cells and primary osteoblasts from doxorubicin-induced cytotoxicity. This chemo-protective effect was far less prominent for the osteosarcoma cells. These findings indicate that a hydroxyapatite-cyclodextrin-doxorubicin chemotherapeutic strategy may enhance the drug-targeting effect on tumor cells while protecting the more sensitive healthy cells for a period of time after implantation. A successful integration of such a drug delivery system might allow healthy cells to initially survive during the doxorubicin exposure period, while eliminating residual neoplastic cells.


Subject(s)
Antibiotics, Antineoplastic , Bone Neoplasms/drug therapy , Doxorubicin , Drug Delivery Systems/methods , Osteosarcoma/drug therapy , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacokinetics , Antibiotics, Antineoplastic/pharmacology , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cyclodextrins/chemistry , Cyclodextrins/pharmacokinetics , Cyclodextrins/pharmacology , Doxorubicin/chemistry , Doxorubicin/pharmacokinetics , Doxorubicin/pharmacology , Drug Screening Assays, Antitumor , Durapatite/chemistry , Durapatite/pharmacokinetics , Durapatite/pharmacology , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/pathology , Humans , Osteoblasts/metabolism , Osteoblasts/pathology , Osteosarcoma/metabolism , Osteosarcoma/pathology , Postoperative Care/methods
5.
Nat Cell Biol ; 19(8): 928-940, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28714969

ABSTRACT

Angiogenic sprouting needs to be tightly controlled. It has been suggested that the Notch ligand dll4 expressed in leading tip cells restricts angiogenesis by activating Notch signalling in trailing stalk cells. Here, we show using live imaging in zebrafish that activation of Notch signalling is rather required in tip cells. Notch activation initially triggers expression of the chemokine receptor cxcr4a. This allows for proper tip cell migration and connection to the pre-existing arterial circulation, ultimately establishing functional arterial-venous blood flow patterns. Subsequently, Notch signalling reduces cxcr4a expression, thereby preventing excessive blood vessel growth. Finally, we find that Notch signalling is dispensable for limiting blood vessel growth during venous plexus formation that does not generate arteries. Together, these findings link the role of Notch signalling in limiting angiogenesis to its role during artery formation and provide a framework for our understanding of the mechanisms underlying blood vessel network expansion and maturation.


Subject(s)
Arteries/metabolism , Endothelial Cells/metabolism , Homeodomain Proteins/metabolism , Neovascularization, Physiologic , Nerve Tissue Proteins/metabolism , Receptor, Notch1/metabolism , Zebrafish Proteins/metabolism , Animals , Animals, Genetically Modified , Arteries/cytology , Cell Movement , Cells, Cultured , Gene Expression Regulation, Developmental , Genotype , Homeodomain Proteins/genetics , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Fluorescence , Microscopy, Video , Nerve Tissue Proteins/genetics , Phenotype , Receptor, Notch1/genetics , Receptors, CXCR4/genetics , Receptors, CXCR4/metabolism , Signal Transduction , Time Factors , Time-Lapse Imaging , Transfection , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics
6.
Nat Cell Biol ; 19(6): 653-665, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28530658

ABSTRACT

The hierarchical organization of properly sized blood vessels ensures the correct distribution of blood to all organs of the body, and is controlled via haemodynamic cues. In current concepts, an endothelium-dependent shear stress set point causes blood vessel enlargement in response to higher flow rates, while lower flow would lead to blood vessel narrowing, thereby establishing homeostasis. We show that during zebrafish embryonic development increases in flow, after an initial expansion of blood vessel diameters, eventually lead to vessel contraction. This is mediated via endothelial cell shape changes. We identify the transforming growth factor beta co-receptor endoglin as an important player in this process. Endoglin mutant cells and blood vessels continue to enlarge in response to flow increases, thus exacerbating pre-existing embryonic arterial-venous shunts. Together, our data suggest that cell shape changes in response to biophysical cues act as an underlying principle allowing for the ordered patterning of tubular organs.


Subject(s)
Cell Shape , Endoglin/metabolism , Endothelial Cells/metabolism , Hemodynamics , Mechanotransduction, Cellular , Zebrafish Proteins/metabolism , Animals , Arteriovenous Malformations/genetics , Arteriovenous Malformations/metabolism , Arteriovenous Malformations/physiopathology , Endoglin/deficiency , Endoglin/genetics , Genetic Predisposition to Disease , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Mice, Knockout , Mutation , Neovascularization, Physiologic , Phenotype , Regional Blood Flow , Stress, Mechanical , Time Factors , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics
7.
J Tissue Eng Regen Med ; 11(3): 637-648, 2017 03.
Article in English | MEDLINE | ID: mdl-25370800

ABSTRACT

Limitations of current treatments for intervertebral disc (IVD) degeneration have promoted interest in the development of tissue-engineering approaches. Injectable hydrogels loaded with cells can be used as a substitute material for the inner IVD part, the nucleus pulposus (NP), and provide an opportunity for minimally invasive treatment of IVD degeneration. The NP is populated by chondrocyte-like cells; therefore, chondrocytes and mesenchymal stem cells (MSCs), stimulated to differentiate along the chondrogenic lineage, could be used to promote NP regeneration. In this study, the in vitro and in vivo response of human bone marrow-derived MSCs and nasal chondrocytes (NCs) to modified gellan gum-based hydrogels was investigated. Both ionic- (iGG-MA) and photo-crosslinked (phGG-MA) methacrylated gellan gum hydrogels show no cytotoxicity in extraction assays with MSCs and NCs. Furthermore, the materials do not induce pro-inflammatory responses in endothelial cells. Moreover, MSCs and NCs can be encapsulated into the hydrogels and remain viable for at least 2 weeks, although apoptosis is observed in phGG-MA. Importantly, encapsulated MSCs and NCs show signs of in vivo chondrogenesis in a subcutaneous implantation of iGG-MA. Altogether, the data endorse the potential use of modified gellan gum-based hydrogel as a suitable material in NP tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.


Subject(s)
Cells, Immobilized/cytology , Hydrogels/pharmacology , Methacrylates/pharmacology , Nucleus Pulposus/physiology , Polysaccharides, Bacterial/pharmacology , Regeneration/drug effects , Animals , Biocompatible Materials/pharmacology , Cell Survival/drug effects , Cells, Immobilized/drug effects , Cells, Immobilized/metabolism , Chondrogenesis/drug effects , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/pathology , Humans , In Situ Nick-End Labeling , Inflammation/pathology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/ultrastructure , Mice, SCID , Nucleus Pulposus/drug effects , Subcutaneous Tissue/drug effects
8.
Stem Cells Transl Med ; 5(12): 1730-1738, 2016 12.
Article in English | MEDLINE | ID: mdl-27460852

ABSTRACT

: Chondrogenic differentiation of bone marrow-derived mesenchymal stromal/stem cells (MSCs) can be induced by presenting morphogenetic factors or soluble signals but typically suffers from limited efficiency, reproducibility across primary batches, and maintenance of phenotypic stability. Considering the avascular and hypoxic milieu of articular cartilage, we hypothesized that sole inhibition of angiogenesis can provide physiological cues to direct in vivo differentiation of uncommitted MSCs to stable cartilage formation. Human MSCs were retrovirally transduced to express a decoy soluble vascular endothelial growth factor (VEGF) receptor-2 (sFlk1), which efficiently sequesters endogenous VEGF in vivo, seeded on collagen sponges and immediately implanted ectopically in nude mice. Although naïve cells formed vascularized fibrous tissue, sFlk1-MSCs abolished vascular ingrowth into engineered constructs, which efficiently and reproducibly developed into hyaline cartilage. The generated cartilage was phenotypically stable and showed no sign of hypertrophic evolution up to 12 weeks. In vitro analyses indicated that spontaneous chondrogenic differentiation by blockade of angiogenesis was related to the generation of a hypoxic environment, in turn activating the transforming growth factor-ß pathway. These findings suggest that VEGF blockade is a robust strategy to enhance cartilage repair by endogenous or grafted mesenchymal progenitors. This article outlines the general paradigm of controlling the fate of implanted stem/progenitor cells by engineering their ability to establish specific microenvironmental conditions rather than directly providing individual morphogenic cues. SIGNIFICANCE: Chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) is typically targeted by morphogen delivery, which is often associated with limited efficiency, stability, and robustness. This article proposes a strategy to engineer MSCs with the capacity to establish specific microenvironmental conditions, supporting their own targeted differentiation program. Sole blockade of angiogenesis mediated by transduction for sFlk-1, without delivery of additional morphogens, is sufficient for inducing MSC chondrogenic differentiation. The findings represent a relevant step forward in the field because the method allowed reducing interdonor variability in MSC differentiation efficiency and, importantly, onset of a stable, nonhypertrophic chondrocyte phenotype.


Subject(s)
Bone Marrow Cells/cytology , Chondrogenesis , Mesenchymal Stem Cells/cytology , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism , Adult , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Chondrogenesis/drug effects , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Female , Humans , Hypertrophy , Male , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Neovascularization, Physiologic/drug effects , Oxygen/pharmacology , Signal Transduction/drug effects , Transduction, Genetic , Transforming Growth Factor beta/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Young Adult
9.
Acta Biomater ; 20: 10-21, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25861947

ABSTRACT

Intervertebral disc (IVD) degeneration is one of the main causes of low back pain. Current surgical treatments are complex and generally do not fully restore spine mobility. Development of injectable extracellular matrix-based hydrogels offers an opportunity for minimally invasive treatment of IVD degeneration. Here we analyze a specific formulation of collagen-low molecular weight hyaluronic acid (LMW HA) semi-interpenetrating network (semi-IPN) loaded with gelatin microspheres as a potential material for tissue engineering of the inner part of the IVD, the nucleus pulposus (NP). The material displayed a gel-like behavior, it was easily injectable as demonstrated by suitable tests and did not induce cytotoxicity or inflammation. Importantly, it supported the growth and chondrogenic differentiation potential of mesenchymal stem cells (MSC) and nasal chondrocytes (NC) in vitro and in vivo. These properties of the hydrogel were successfully combined with TGF-ß3 delivery by gelatin microspheres, which promoted the chondrogenic phenotype. Altogether, collagen-LMW HA loaded with gelatin microspheres represents a good candidate material for NP tissue engineering as it combines important rheological, functional and biological features.


Subject(s)
Collagen/pharmacology , Gelatin/pharmacology , Hyaluronic Acid/pharmacology , Intervertebral Disc/physiology , Mesenchymal Stem Cell Transplantation , Microspheres , Regeneration/drug effects , Transforming Growth Factor beta3/pharmacology , Animals , Chondrogenesis/drug effects , Elastic Modulus , Humans , Implants, Experimental , Injections , Male , Mesenchymal Stem Cells/cytology , Mice, SCID , Molecular Weight , Rheology/drug effects , Viscosity
10.
Tissue Eng Part A ; 21(11-12): 1869-85, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25760236

ABSTRACT

Cartilage damage and/or aging effects can cause constant pain, which limits the patient's quality of life. Although different strategies have been proposed to enhance the limited regenerative capacity of cartilage tissue, the full production of native and functional cartilaginous extracellular matrix (ECM) has not yet been achieved. Poly(γ-glutamic acid) (γ-PGA), a naturally occurring polyamino acid, biodegradable into glutamate residues, has been explored for tissue regeneration. In this work, γ-PGA's ability to support the production of cartilaginous ECM by human bone marrow mesenchymal stem/stromal cells (MSCs) and nasal chondrocytes (NCs) was investigated. MSC and NC pellets were cultured in basal medium (BM), chondrogenic medium (CM), and CM-γ-PGA-supplemented medium (CM+γ-PGA) over a period of 21 days. Pellet size/shape was monitored with time. At 14 and 21 days of culture, the presence of sulfated glycosaminoglycans (sGAGs), type II collagen (Col II), Sox-9, aggrecan, type XI collagen (Col XI), type X collagen (Col X), calcium deposits, and type I collagen (Col I) was analyzed. After excluding γ-PGA's cytotoxicity, earlier cell condensation, higher sGAG content, Col II, Sox-9 (day 14), aggrecan, and Col X (day 14) production was observed in γ-PGA-supplemented MSC cultures, with no signs of mineralization or Col I. These effects were not evident with NCs. However, Sox-9 (at day 14) and Col X (at days 14 and 21) were increased, decreased, or absent, respectively. Overall, γ-PGA improved chondrogenic differentiation of MSCs, increasing ECM production earlier in culture. It is proposed that γ-PGA incorporation in novel biomaterials has a beneficial impact on future approaches for cartilage regeneration.


Subject(s)
Chondrocytes/drug effects , Chondrogenesis/drug effects , Mesenchymal Stem Cells/drug effects , Polyglutamic Acid/analogs & derivatives , Stromal Cells/drug effects , Aggrecans/metabolism , Calcium/metabolism , Cell Culture Techniques , Cells, Cultured , Chondrocytes/cytology , Collagen/metabolism , Culture Media/pharmacology , Culture Media, Conditioned/chemistry , Extracellular Matrix Proteins/metabolism , Glycosaminoglycans/metabolism , Humans , Mesenchymal Stem Cells/cytology , Nasal Cartilages/cytology , Polyglutamic Acid/pharmacology , Polyglutamic Acid/toxicity , SOX9 Transcription Factor/metabolism , Stromal Cells/cytology , Transforming Growth Factor beta1/metabolism
11.
Biomaterials ; 34(33): 8075-85, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23891083

ABSTRACT

Inflammation is an important step in the early phase of tissue regeneration around an implanted metallic orthopaedic device. However, prolonged inflammation, which can be induced by metallic corrosion products, can lead to aseptic loosening and implant failure. Cells in peri-implant tissue as well as metal corrosion can induce reactive oxygen species (ROS) formation, thus contributing to an oxidative microenvironment around an implant. Understanding cellular reactions to implant-induced oxidative stress and inflammatory activation is important to help prevent an adverse response to metallic materials. In an earlier study we have shown that endothelial cells grown on Ti6Al4V alloy are subjected to oxidative stress. Since endothelial cells play a critical role in inflammation, in this study we examined the role of oxidative stress in their response to pro-inflammatory activation. Therefore, we stimulated endothelial cells in contact with Ti6Al4V with tumour necrosis factor-α (TNF-α) and monitored the expression of inflammation-associated molecules, such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8). The induction of these proteins was lower in endothelial cells on Ti6Al4V compared to control tissue culture conditions. There was, however, a discrepancy in pro-inflammatory activation at protein compared to mRNA level in the cells on Ti6Al4V. To examine the role of oxidative stress in this response we utilized different ROS scavengers and showed that ROS depletion improved cellular response to TNF-α on Ti6Al4V. These results could contribute to developing strategies to improve tissue response to metal implants.


Subject(s)
Endothelial Cells/immunology , Oxidative Stress/physiology , Titanium/immunology , Alloys , Antioxidants/metabolism , Cell Line , Enzyme-Linked Immunosorbent Assay , Humans , Intercellular Adhesion Molecule-1/metabolism , Interleukin-8/metabolism , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/metabolism
12.
Mutat Res ; 670(1-2): 32-41, 2009 Nov 02.
Article in English | MEDLINE | ID: mdl-19616017

ABSTRACT

The present study aimed at elucidating mechanisms dictating cell death triggered by cisplatin-induced DNA damage. We show that CL-V5B hamster mutant cells, a derivative of V79B, are hypersensitive to cisplatin-induced apoptotic death. CL-V5B cells are characterized by attenuated cisplatin-induced early (2-6 h) stress response, such as phosphorylation of stress-activated protein kinases (SAPK/JNK), ATM and Rad3-related (ATR) protein kinase, histone H2AX and checkpoint kinase-1 (Chk-1). Human FANCC cells also showed a reduced phosphorylation of H2AX and SAPK/JNK at early time point after cisplatin treatment. This was not the case for BRCA2-defective VC-8 hamster cells, indicating that the FA core complex, rather than its downstream elements, is involved in early damage response. The alleviated early response of CL-V5B cells is not due to a general dysfunction in ATM/ATR-regulated signaling. It is rather due to a reduced formation of primary cisplatin-DNA adducts in the hypersensitive mutant as shown by analysis of DNA platination, DNA intra- and interstrand crosslink formation and DNA replication blockage. Despite of lower initial DNA damage and attenuated early DNA damage response (DDR), CL-V5B cells are characterized by an excessive G2/M arrest as well as an elevated frequency of DNA double-strand breaks (DSB) and chromosomal aberrations (CA) at late times (16-24h) after cisplatin exposure. This indicates that error-prone processing of cisplatin-induced lesions, notably interstrand crosslinks (ICL), and the formation of secondary DNA lesions (i.e. DSB), results in a powerful delayed DNA damage response and massive pro-apoptotic signaling in CL-V5B cells. The data provide an example that the initial level of cisplatin-DNA adducts and the corresponding early DNA damage response do not necessarily predict the outcome of cisplatin treatment. Rather, the accuracy of DNA damage processing and late checkpoint control mechanisms determine the extent of cell death triggered by cisplatin-induced DNA lesions.


Subject(s)
Apoptosis , Cisplatin/toxicity , DNA Damage , Animals , Cell Line , Chromosome Aberrations , Cricetinae , DNA Adducts/toxicity , Histones/metabolism , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Mutagens , Phosphorylation
13.
Mol Pharmacol ; 76(4): 927-34, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19641035

ABSTRACT

Temozolomide and fotemustine, representing methylating and chloroethylating agents, respectively, are used in the treatment of glioma and malignant melanoma. Because chemoresistance of these tumors is a common phenomenon, identification of the underlying mechanisms is needed. Here we show that Rev3L, the catalytic subunit of the translesion DNA polymerase zeta, mediates resistance to both temozolomide and fotemustine. Rev3L knockout cells are hypersensitive to both agents. It is remarkable that cells heterozygous for Rev3L showed an intermediate sensitivity. Rev3L is not involved in the tolerance of the toxic O6-methylguanine lesion. However, a possible role of Rev3L in the tolerance of O6-chloroethylguanine or the subsequently formed N1-guanine-N3-cytosine interstrand cross-link is shown. Rev3L had no influence on base excision repair (BER) of the N-alkylation lesions but is very likely to be involved in the tolerance of N-alkylations or apurinic/apyrimidinic sites originating from them. We also show that Rev3L exerts its protective effect in replicating cells and that loss of Rev3L leads to a significant increase in DNA double-strand breaks after temozolomide and fotemustine treatment. These data show that Rev3L contributes to temozolomide and fotemustine resistance, thus acting in concert with O6-methylguanine-DNA methyltransferase, BER, mismatch repair, and double-strand break repair in defense against simple alkylating anticancer drugs.


Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , DNA Damage , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Dacarbazine/analogs & derivatives , Nitrosourea Compounds/pharmacology , Organophosphorus Compounds/pharmacology , Animals , Antineoplastic Agents, Alkylating/adverse effects , Apoptosis/drug effects , Cell Line , Dacarbazine/adverse effects , Dacarbazine/pharmacology , Drug Screening Assays, Antitumor , Flow Cytometry , Mice , Mice, Knockout , Microscopy, Fluorescence , Nitrosourea Compounds/adverse effects , Organophosphorus Compounds/adverse effects , Temozolomide
14.
J Mater Sci Mater Med ; 19(4): 1637-44, 2008 Apr.
Article in English | MEDLINE | ID: mdl-17914634

ABSTRACT

Endothelial cells cover the inner surface of blood vessels and form the interface between the blood and the tissues. Endothelial cells are involved in regulating barrier function, which is maintained by the interendothelial cell contacts. These interendothelial cell contacts are established by the interaction of different molecules. The maintenance of the barrier requires an appropriate signalling between these molecules. Thus, a number of different signalling pathways are integrated within interendothelial contacts. Since endothelial cells are important in tissue-implant interactions (especially for stent materials) this study examines the expression pattern of different interendothelial contact molecules to determine the usefulness in the analysis of biocompatibility in vitro. The effects of different pro-inflammatory and toxic stimuli and contact of human microvascular endothelial cells to metallic surfaces were examined for their impact on the pattern of interendothelial contact molecules. Striking modifications in the arrangement of these molecules were induced and the mode of modification was dependent on the tested compound. Thus, examining the pattern of expression of specific interendothelial contact molecules in vitro may be useful for testing the endothelial cell compatibility of biomaterials and their corrosion products.


Subject(s)
Biocompatible Materials/chemistry , Endothelial Cells/cytology , Materials Testing/methods , Cell Adhesion , Cell Communication , Cell Survival , Cells, Cultured , Humans , Inflammation , Microcirculation/cytology , Microscopy, Fluorescence , Microscopy, Video , Models, Biological , Signal Transduction , Stents
15.
Biomaterials ; 28(22): 3263-72, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17466367

ABSTRACT

Nowadays aseptic loosening is the most common cause of orthopaedic implant failure. Some of its reasons have already been described up to now; however, others remain still hypothetical. Besides the inflammatory response to wear particles originating at different sources, the role of reactive oxygen species as products of cellular reactions and/or as a result of the process of corrosion of an implant leading to implant failure has recently been discussed too. In the present study, we used a galvanostatic polarization to simulate the cathodic partial reaction of the corrosion process at a titanium alloy surface. With respect to cells occurring at the interface of a metal implant, the behaviour of osteoblasts and monocytes/macrophages was investigated. It has been found that cathodic polarization of Ti6Al4V induces an increase in the level of intracellular reactive oxygen species as well as suppressing the metabolic activity of cells in a dose-dependent manner. This is in agreement with the results obtained with cells after external addition of hydrogen peroxide as another kind of oxidative stress. In both approaches, monocytes/macrophages show a higher tolerance to oxidative stress than osteoblasts. It could be concluded that the electrochemical setup developed induced intracellular changes occurring during oxidative stress and it could be used for future detailed analysis of the consequences of corrosion processes for cellular reactions.


Subject(s)
Electrochemistry/methods , Macrophages/cytology , Monocytes/cytology , Osteoblasts/metabolism , Reactive Oxygen Species , Titanium/chemistry , Alloys , Animals , Biocompatible Materials/chemistry , Cell Line, Tumor , Corrosion , Electrodes , Humans , Macrophages/metabolism , Mice , Oxidative Stress
16.
Biomaterials ; 28(5): 806-13, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17049373

ABSTRACT

Titanium and its alloys are amongst the most frequently used materials in bone and dental implantology. The good biocompatibility of titanium(-alloys) is attributed to the formation of a titanium oxide layer on the implant surface. However, implant failures do occur and this appears to be due to titanium corrosion. Thus, cells participating in the wound healing processes around an implanted material, among them endothelial cells, might be subjected to reactive oxygen species (ROS) formed by electrochemical processes during titanium corrosion. Therefore, we studied the response of endothelial cells grown on Ti6Al4V alloy to H(2)O(2) and compared this with the response of endothelial cells grown on cell culture polystyrene (PS). We could show that although the cell number was the same on both surfaces, metabolic activity of endothelial cells grown on Ti6Al4V alloy was reduced compared to the cells on PS and further decreased following prototypic oxidative stress (H(2)O(2)-treatment). The analysis of H(2)O(2)-induced oxidative stress showed a higher ROS formation in endothelial cells on Ti6Al4V than on PS. This correlated with the depletion of reduced glutathione (GSH) in endothelial cells grown on Ti6Al4V surfaces and indicated permanent oxidative stress. Thus, endothelial cells in direct contact with Ti6Al4V showed signs of oxidative stress and higher impairment of cell vitality after an additional oxidative stress. However, the exact nature of the agent of oxidative stress generated from Ti6Al4V remains unclear and requires further investigation.


Subject(s)
Endothelial Cells/cytology , Oxidative Stress , Titanium/pharmacology , Alloys , Antioxidants/pharmacology , Cell Culture Techniques/methods , Cells, Cultured , Coated Materials, Biocompatible/pharmacology , Glutathione/metabolism , Humans , Hydrogen Peroxide/pharmacology , Inflammation , Materials Testing , Prostheses and Implants , Reactive Oxygen Species , Superoxide Dismutase
17.
Cancer Lett ; 239(2): 305-13, 2006 Aug 08.
Article in English | MEDLINE | ID: mdl-16298473

ABSTRACT

DNA double-strand breaks (DSBs) are potent killing lesions, and inefficient repair of DSBs does not only lead to cell death but also to genomic instability and tumorigenesis. DSBs are repaired by non-homologous end-joining and homologous recombination (HR). A key player in HR is Xrcc2, a Rad51-like protein. Cells deficient in Xrcc2 are hypersensitive to X-rays and mitomycin C and display increased chromosomal aberration frequencies. In order to elucidate the role of Xrcc2 in resistance to anticancer drugs, we compared Xrcc2 knockout (Xrcc2-/-) mouse embryonic fibroblasts with the corresponding isogenic wild-type and Xrcc2 complemented knockout cells. We show that Xrcc2-/- cells are hypersensitive to the killing effect of the simple methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). They undergo apoptosis after MNNG treatment while necrosis is only marginally enhanced. Complementation of Xrcc2 deficient cells by Xrcc2 cDNA transfection conferred resistance to the cytotoxic and apoptosis-inducing effect of MNNG. The hypersensitivity of Xrcc2-/- cells to MNNG prompted us to investigate their killing and apoptotic response to various methylating, chloroethylating and crosslinking drugs used in anticancer therapy. Xrcc2 deficient cells were found to be hypersensitive to temozolomide, fotemustine and mafosfamide. They were also hypersensitive to cisplatin but not to taxol. The data reveal that Xrcc2 plays a role in the protection against a wide range of anticancer drugs and, therefore, suggest Xrcc2 to be a determinant of anticancer drug resistance. They also indicate that HR is involved in the processing of DNA damage induced by simple alkylating agents.


Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Apoptosis/drug effects , Cyclophosphamide/analogs & derivatives , DNA-Binding Proteins/genetics , Dacarbazine/analogs & derivatives , Methylnitronitrosoguanidine/toxicity , Mutagens/toxicity , Nitrosourea Compounds/pharmacology , Organophosphorus Compounds/pharmacology , Cisplatin/pharmacology , Cyclophosphamide/pharmacology , Dacarbazine/pharmacology , Humans , Mitomycin/pharmacology , Temozolomide
18.
Cell Biol Int ; 29(1): 41-50, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15763498

ABSTRACT

Macrophages attack and kill pathologically changed, transformed and tumor cells. However, in some cases they may also support tumor growth, modulate the action of anticancer drugs, and even facilitate the development of drug resistance in tumor cells. Here we present data that bystander fibroblasts NIH3T3 were not only resistant to murine macrophages J774.2 but also blocked their killing action towards murine transformed fibroblasts L929. Macrophages were isolated from mixed cultures by means of CD11b specific immunomagnetic beads, and changes induced by their former co-culturing were studied using DNA microarray technology and other tests. An expression of candidate genes coding for cytokines and for signal transduction pathway proteins was estimated in macrophages in different variants of their co-culture with target cells. Changes in expression of mRNA for interleukin 1beta, NFkappaB, IkappaBalpha, gadd45, and CD5 were detected as the most prominent in the macrophages co-cultured with the transformed cells. Bystander NIH3T3 fibroblasts abolished these changes in the macrophages J774.2, and the level of expression of the above mentioned genes was close to the level seen in the macrophages which did not exert cytotoxicity towards the target fibroblasts. Potential implications and research perspectives of using the macrophage-target cell co-cultures with different bystander cellular partners are discussed.


Subject(s)
Bystander Effect/physiology , Cell Transformation, Neoplastic , Fibroblasts/physiology , Macrophages/physiology , Animals , Cell Line , Coculture Techniques , Cytokines/biosynthesis , Cytokines/genetics , Enzyme-Linked Immunosorbent Assay , Gene Expression , Mice , NIH 3T3 Cells , Oligonucleotide Array Sequence Analysis
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