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Sample records for invading endothelial cells

  1. Treponema pallidum Invades Intercellular Junctions of Endothelial Cell Monolayers

    Science.gov (United States)

    Thomas, D. Denee; Navab, Mahamad; Haake, David A.; Fogelman, Alan M.; Miller, James N.; Lovett, Michael A.

    1988-05-01

    The pathogenesis of syphilis reflects invasive properties of Treponema pallidum, but the actual mode of tissue invasion is unknown. We have found two in vitro parallels of treponemal invasiveness. We tested whether motile T. pallidum could invade host cells by determining the fate of radiolabeled motile organisms added to a HeLa cell monolayer; 26% of treponemes associated with the monolayer in a trypsin-resistant niche, presumably between the monolayer and the surface to which it adhered, but did not attain intracellularity. Attachment of T. pallidum to cultured human and rabbit aortic and human umbilical vein endothelial cells was 2-fold greater than to HeLa cells. We added T. pallidum to aortic endothelial cells grown on membrane filters under conditions in which tight intercellular junctions had formed. T. pallidum was able to pass through the endothelial cell monolayers without altering tight junctions, as measured by electrical resistance. In contrast, heat-killed T. pallidum and the nonpathogen Treponema phagedenis biotype Reiter failed to penetrate the monolayer. Transmission electron micrographs of sections of the monolayer showed T. pallidum in intercellular junctions. Our in vitro observations suggest that these highly motile spirochetes may leave the circulation by invading the junctions between endothelial cells.

  2. Infections and endothelial cells

    NARCIS (Netherlands)

    Keller, Tymen T.; Mairuhu, Albert T. A.; de Kruif, Martijn D.; Klein, Saskia K.; Gerdes, Victor E. A.; ten Cate, Hugo; Brandjes, Dees P. M.; Levi, Marcel; van Gorp, Eric C. M.

    2003-01-01

    Systemic infection by various pathogens interacts with the endothelium and may result in altered coagulation, vasculitis and atherosclerosis. Endothelium plays a role in the initiation and regulation of both coagulation and fibrinolysis. Exposure of endothelial cells may lead to rapid activation of

  3. Streptococcus equi subsp zooepidemicus Invades and Survives in Epithelial Cells

    DEFF Research Database (Denmark)

    Skive, Bolette; Rohde, Manfred; Molinari, Gabriella

    2017-01-01

    showed three morphologically different types of invasion for both bacterial strains. The main port of entry was through large invaginations in the epithelial cell membrane. Pili-like bacterial appendages were observed when the S. zooepidemicus cells were in close proximity to the epithelial cells...... protection assays. Both S. zooepidemicus strains investigated were able to invade epithelial cells although at different magnitudes. The immunofluorescence data showed significantly higher adhesion and invasion rates for strain 1-4a when compared to strain S31A1. S. zooepidemicus was able to survive...

  4. Streptococcus equi subsp. zooepidemicus Invades and Survives in Epithelial Cells

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    Bolette Skive

    2017-11-01

    Full Text Available Streptococcus equi subsp. zooepidemicus (S. zooepidemicus is an opportunistic pathogen of several species including humans. S. zooepidemicus is found on mucus membranes of healthy horses, but can cause acute and chronic endometritis. Recently S. zooepidemicus was found able to reside in the endometrium for prolonged periods of time. Thus, we hypothesized that an intracellular phase may be part of the S. zooepidemicus pathogenesis and investigated if S. zooepidemicus was able to invade and survive inside epithelial cells. HEp-2 and HeLa cell lines were co-cultured with two S. zooepidemicus strains (1-4a and S31A1 both originating from the uterus of mares suffering from endometritis. Cells were fixed at different time points during the 23 h infection assay and field emission scanning electron microscopy (FESEM was used to characterize adhesion and invasion mechanisms. The FESEM images showed three morphologically different types of invasion for both bacterial strains. The main port of entry was through large invaginations in the epithelial cell membrane. Pili-like bacterial appendages were observed when the S. zooepidemicus cells were in close proximity to the epithelial cells indicating that attachment and invasion were active processes. Adherent and intracellular S. zooepidemicus, and bacteria in association with lysosomes was determined by immunofluorescence staining techniques and fluorescence microscopy. Quantification of intracellular bacteria was determined in penicillin protection assays. Both S. zooepidemicus strains investigated were able to invade epithelial cells although at different magnitudes. The immunofluorescence data showed significantly higher adhesion and invasion rates for strain 1-4a when compared to strain S31A1. S. zooepidemicus was able to survive intracellularly, but the survival rate decreased over time in the cell culture system. Phagosome-like compartments containing S. zooepidemicus at some stages fused with

  5. Streptococcus equi subsp. zooepidemicus Invades and Survives in Epithelial Cells.

    Science.gov (United States)

    Skive, Bolette; Rohde, Manfred; Molinari, Gabriella; Braunstein, Thomas Hartig; Bojesen, Anders M

    2017-01-01

    Streptococcus equi subsp. zooepidemicus ( S. zooepidemicus ) is an opportunistic pathogen of several species including humans. S. zooepidemicus is found on mucus membranes of healthy horses, but can cause acute and chronic endometritis. Recently S. zooepidemicus was found able to reside in the endometrium for prolonged periods of time. Thus, we hypothesized that an intracellular phase may be part of the S. zooepidemicus pathogenesis and investigated if S. zooepidemicus was able to invade and survive inside epithelial cells. HEp-2 and HeLa cell lines were co-cultured with two S. zooepidemicus strains (1-4a and S31A1) both originating from the uterus of mares suffering from endometritis. Cells were fixed at different time points during the 23 h infection assay and field emission scanning electron microscopy (FESEM) was used to characterize adhesion and invasion mechanisms. The FESEM images showed three morphologically different types of invasion for both bacterial strains. The main port of entry was through large invaginations in the epithelial cell membrane. Pili-like bacterial appendages were observed when the S. zooepidemicus cells were in close proximity to the epithelial cells indicating that attachment and invasion were active processes. Adherent and intracellular S. zooepidemicus , and bacteria in association with lysosomes was determined by immunofluorescence staining techniques and fluorescence microscopy. Quantification of intracellular bacteria was determined in penicillin protection assays. Both S. zooepidemicus strains investigated were able to invade epithelial cells although at different magnitudes. The immunofluorescence data showed significantly higher adhesion and invasion rates for strain 1-4a when compared to strain S31A1. S. zooepidemicus was able to survive intracellularly, but the survival rate decreased over time in the cell culture system. Phagosome-like compartments containing S. zooepidemicus at some stages fused with lysosomes to form a

  6. Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

    International Nuclear Information System (INIS)

    Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.; Haudenschild, C.C.; Diamond, R.D.

    1987-01-01

    Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of 51 Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of 51 Cr release from radiolabeled monolayers

  7. In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Ken Kono

    Full Text Available Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

  8. Candida albicans: The Ability to Invade Epithelial Cells and Survive under Oxidative Stress Is Unlinked to Hyphal Length

    Directory of Open Access Journals (Sweden)

    Paloma K. Maza

    2017-07-01

    Full Text Available In its hyphal form, Candida albicans invades epithelial and endothelial cells by two distinct mechanisms: active penetration and induced endocytosis. The latter is dependent on a reorganization of the host cytoskeleton (actin/cortactin recruitment, whilst active penetration does not rely on the host's cellular machinery. The first obstacle for the fungus to reach deep tissues is the epithelial barrier and this interaction is crucial for commensal growth, fungal pathogenicity and host defense. This study aimed to characterize in vitro epithelial HeLa cell invasion by four different isolates of C. albicans with distinct clinical backgrounds, including a C. albicans SC5314 reference strain. All isolates invaded HeLa cells, recruited actin and cortactin, and induced the phosphorylation of both Src-family kinases (SFK and cortactin. Curiously, L3881 isolated from blood culture of a patient exhibited the highest resistance to oxidative stress, although this isolate showed reduced hyphal length and displayed the lowest cell damage and invasion rates. Collectively, these data suggest that the ability of C. albicans to invade HeLa cells, and to reach and adapt to the host's blood, including resistance to oxidative stress, may be independent of hyphal length.

  9. Isolation and culture of pulmonary endothelial cells.

    OpenAIRE

    Ryan, U S

    1984-01-01

    Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vas...

  10. Endothelial cells in dengue hemorrhagic fever.

    Science.gov (United States)

    Srikiatkhachorn, Anon; Kelley, James F

    2014-09-01

    Therapies to prevent or reverse endothelial dysfunction and vascular leak found in dengue hemorrhagic fever (DHF) have not been identified. In this review we summarize dengue viruses and the spectrum of human disease and highlight evidence of endothelial cell dysfunction in DHF based on studies in patients and mouse and tissue culture models. Evidence suggests that both virus antigen and host immune response, can cause endothelial cell dysfunction and weaken endothelial barrier integrity. We suggest possible therapeutic interventions and highlight how therapies targeting altered endothelial function might be evaluated in animal models and in patients with DHF. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells

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    Park Tae

    2008-12-01

    Full Text Available Abstract Background Acinetobacter baumannii is a nosocomial pathogen of increasing importance, but the pathogenic mechanism of this microorganism has not been fully explored. This study investigated the potential of A. baumannii to invade epithelial cells and determined the role of A. baumannii outer membrane protein A (AbOmpA in interactions with epithelial cells. Results A. baumannii invaded epithelial cells by a zipper-like mechanism, which is associated with microfilament- and microtubule-dependent uptake mechanisms. Internalized bacteria were located in the membrane-bound vacuoles. Pretreatment of recombinant AbOmpA significantly inhibited the adherence to and invasion of A. baumannii in epithelial cells. Cell invasion of isogenic AbOmpA- mutant significantly decreased as compared with wild-type bacteria. In a murine pneumonia model, wild-type bacteria exhibited a severe lung pathology and induced a high bacterial burden in blood, whereas AbOmpA- mutant was rarely detected in blood. Conclusion A. baumannii adheres to and invades epithelial cells. AbOmpA plays a major role in the interactions with epithelial cells. These findings contribute to the understanding of A. baumannii pathogenesis in the early stage of bacterial infection.

  12. Mitochondria, endothelial cell function, and vascular diseases.

    Science.gov (United States)

    Tang, Xiaoqiang; Luo, Yu-Xuan; Chen, Hou-Zao; Liu, De-Pei

    2014-01-01

    Mitochondria are perhaps the most sophisticated and dynamic responsive sensing systems in eukaryotic cells. The role of mitochondria goes beyond their capacity to create molecular fuel and includes the generation of reactive oxygen species, the regulation of calcium, and the activation of cell death. In endothelial cells, mitochondria have a profound impact on cellular function under both healthy and diseased conditions. In this review, we summarize the basic functions of mitochondria in endothelial cells and discuss the roles of mitochondria in endothelial dysfunction and vascular diseases, including atherosclerosis, diabetic vascular dysfunction, pulmonary artery hypertension, and hypertension. Finally, the potential therapeutic strategies to improve mitochondrial function in endothelial cells and vascular diseases are also discussed, with a focus on mitochondrial-targeted antioxidants and calorie restriction.

  13. Mitochondria, Endothelial Cell Function and Vascular Diseases

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    Xiaoqiang eTang

    2014-05-01

    Full Text Available Mitochondria are perhaps the most sophisticated and dynamic responsive sensing systems in eukaryotic cells. The role of mitochondria goes beyond their capacity to create molecular fuel and includes the generation of reactive oxygen species, the regulation of calcium, and the activation of cell death. In endothelial cells, mitochondria have a profound impact on cellular function under both healthy and diseased conditions. In this review, we summarize the basic functions of mitochondria in endothelial cells and discuss the roles of mitochondria in endothelial dysfunction and vascular diseases, including atherosclerosis, diabetic vascular dysfunction, pulmonary artery hypertension and hypertension. Finally, the potential therapeutic strategies to improve mitochondrial function in endothelial cells and vascular diseases are also discussed, with a focus on mitochondrial-targeted antioxidants and calorie restriction.

  14. Dysfunctional Endothelial Progenitor Cells in Metabolic Syndrome

    Science.gov (United States)

    Devaraj, Sridevi; Jialal, Ishwarlal

    2012-01-01

    The metabolic syndrome (MetS) is highly prevalent and confers an increased risk of diabetes and cardiovascular disease. A key early event in atherosclerosis is endothelial dysfunction. Numerous groups have reported endothelial dysfunction in MetS. However, the measurement of endothelial function is far from optimum. There has been much interest recently in a subtype of progenitor cells, termed endothelial progenitor cells (EPCs), that can circulate, proliferate, and dfferentiate into mature endothelial cells. EPCs can be characterized by the assessment of surface markers, CD34 and vascular endothelial growth factor receptor-2, VEGFR-2 (KDR). The CD34+KDR+ phenotype has been demonstrated to be an independent predictor of cardiovascular outcomes. MetS patients without diabetes or cardiovascular diseases have decreased EPC number and functionality as evidenced by decreased numbers of colony forming units, decreased adhesion and migration, and decreased tubule formation. Strategies that have been shown to upregulate and enhance EPC number and functionality include statins, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and peroxisome-proliferator-activating-receptor gamma agonists. Mechanisms by which they affect EPC number and functionality need to be studied. Thus, EPC number and/or functionality could emerge as novel cellular biomarkers of endothelial dysfunction and cardiovascular disease risk in MetS. PMID:21941528

  15. Isolation and culture of pulmonary endothelial cells.

    Science.gov (United States)

    Ryan, U S

    1984-06-01

    Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan.

  16. Endothelial Cell Response to Fusobacterium nucleatum.

    Science.gov (United States)

    Mendes, Reila Tainá; Nguyen, Daniel; Stephens, Danielle; Pamuk, Ferda; Fernandes, Daniel; Van Dyke, Thomas E; Kantarci, Alpdogan

    2016-07-01

    Vascular response is an essential aspect of an effective immune response to periodontal disease pathogens, as new blood vessel formation contributes to wound healing and inflammation. Gaining a greater understanding of the factors that affect vascular response may then contribute to future breakthroughs in dental medicine. In this study, we have characterized the endothelial cell response to the common bacterium Fusobacterium nucleatum, an important bridging species that facilitates the activity of late colonizers of the dental biofilm. Endothelial cells were infected with Fusobacterium nucleatum (strain 25586) for periods of 4, 12, 24, or 48 h. Cell proliferation and tube formation were analyzed, and expression of adhesion molecules (CD31 and CD34) and vascular endothelial growth factor (VEGF) receptors 1 and 2 was measured by fluorescence-activated cell sorter (FACS) analysis. Data indicate that F. nucleatum impaired endothelial cell proliferation and tube formation. The findings suggest that the modified endothelial cell response acts as a mechanism promoting the pathogenic progression of periodontal diseases and may potentially suggest the involvement of periodontopathogens in systemic diseases associated with periodontal inflammation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. Reduced Ang2 expression in aging endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

    2016-06-03

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  18. Reduced Ang2 expression in aging endothelial cells

    International Nuclear Information System (INIS)

    Hohensinner, P.J.; Ebenbauer, B.; Kaun, C.; Maurer, G.; Huber, K.; Wojta, J.

    2016-01-01

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  19. Endothelial cells, endoplasmic reticulum stress and oxysterols

    Directory of Open Access Journals (Sweden)

    F. Luchetti

    2017-10-01

    Full Text Available Oxysterols are bioactive lipids that act as regulators of lipid metabolism, inflammation, cell viability and are involved in several diseases, including atherosclerosis. Mounting evidence linked the atherosclerosis to endothelium dysfunction; in fact, the endothelium regulates the vascular system with roles in processes such as hemostasis, cell cholesterol, hormone trafficking, signal transduction and inflammation. Several papers shed light the ability of oxysterols to induce apoptosis in different cell lines including endothelial cells. Apoptotic endothelial cell and endothelial denudation may constitute a critical step in the transition to plaque erosion and vessel thrombosis, so preventing the endothelial damaged has garnered considerable attention as a novel means of treating atherosclerosis. Endoplasmic reticulum (ER is the site where the proteins are synthetized and folded and is necessary for most cellular activity; perturbations of ER homeostasis leads to a condition known as endoplasmic reticulum stress. This condition evokes the unfolded protein response (UPR an adaptive pathway that aims to restore ER homeostasis. Mounting evidence suggests that chronic activation of UPR leads to cell dysfunction and death and recently has been implicated in pathogenesis of endothelial dysfunction. Autophagy is an essential catabolic mechanism that delivers misfolded proteins and damaged organelles to the lysosome for degradation, maintaining basal levels of autophagic activity it is critical for cell survival. Several evidence suggests that persistent ER stress often results in stimulation of autophagic activities, likely as a compensatory mechanism to relieve ER stress and consequently cell death. In this review, we summarize evidence for the effect of oxysterols on endothelial cells, especially focusing on oxysterols-mediated induction of endoplasmic reticulum stress.

  20. Lipoprotein receptors in cultured bovine endothelial cells

    International Nuclear Information System (INIS)

    Struempfer, A.E.M.

    1983-07-01

    In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

  1. Identification of epigenetically silenced genes in tumor endothelial cells

    NARCIS (Netherlands)

    Hellebrekers, Debby M. E. I.; Melotte, Veerle; Vire, Emmanuelle; Langenkamp, Elise; Molema, Grietje; Fuks, Francois; Herman, James G.; Van Criekinge, Wim; Griffioen, Arjan W.; van Engeland, Manon

    2007-01-01

    Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells. We recently showed that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors directly repress endothelial cell growth and tumor angiogenesis, suggesting that epigenetic modifications mediated

  2. Qidantongmai Protects Endothelial Cells Against Hypoxia-Induced ...

    African Journals Online (AJOL)

    induced damage. The ability of QDTM to modulate the serum VEGF-A level may play an important role in its effects on endothelial cells. Key words: Traditional Chinese Medicine, human umbilical vein endothelial cells, hypoxia, VEGF ...

  3. Endothelial cell transfection of ex vivo arteries

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Alexander Lohman, Adam Straub & Brant Isakson ### Abstract The vascular endothelium plays an essential role in regulating blood vessel tone, blood flow and blood pressure. Current vascular model systems for examination of endothelial cell biology and blood vessel physiology and pathology rely on cell culture and the generation of genetically modified animals. While these systems are advantageous for studying the endothelium, many cell culture models omit the contribution of o...

  4. Endothelial cell seeding on crosslinked collagen : Effects of crosslinking on endothelial cell proliferation and functional parameters

    NARCIS (Netherlands)

    Wissink, MJB; van Luyn, MJA; Dijk, F; Poot, AA; Engbers, GHM; Beugeling, T; van Aken, WG; Feijen, J

    Endothelial cell seeding, a promising method to improve the performance of small-diameter vascular grafts, requires a suitable substrate, such as crosslinked collagen. Commonly used crosslinking agents such as glutaraldehyde and formaldehyde cause, however, cytotoxic reactions and thereby hamper

  5. Valvular interstitial cells suppress calcification of valvular endothelial cells

    NARCIS (Netherlands)

    Hjortnaes, Jesper; Shapero, Kayle; Goettsch, Claudia; Hutcheson, Joshua D; Keegan, Joshua; Kluin, J; Mayer, John E; Bischoff, Joyce; Aikawa, Elena

    BACKGROUND: Calcific aortic valve disease (CAVD) is the most common heart valve disease in the Western world. We previously proposed that valvular endothelial cells (VECs) replenish injured adult valve leaflets via endothelial-to-mesenchymal transformation (EndMT); however, whether EndMT contributes

  6. Endothelial progenitor cell biology in ankylosing spondylitis.

    Science.gov (United States)

    Verma, Inderjeet; Syngle, Ashit; Krishan, Pawan

    2015-03-01

    Endothelial progenitor cells (EPCs) are unique populations which have reparative potential in overcoming endothelial damage and reducing cardiovascular risk. Patients with ankylosing spondylitis (AS) have increased risk of cardiovascular morbidity and mortality. The aim of this study was to investigate the endothelial progenitor cell population in AS patients and its potential relationships with disease variables. Endothelial progenitor cells were measured in peripheral blood samples from 20 AS and 20 healthy controls by flow cytometry on the basis of CD34 and CD133 expression. Disease activity was evaluated by using Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Functional ability was monitored by using Bath Ankylosing Spondylitis Functional Index (BASFI). EPCs were depleted in AS patients as compared to healthy controls (CD34(+) /CD133(+) : 0.027 ± 0.010% vs. 0.044 ± 0.011%, P < 0.001). EPC depletions were significantly associated with disease duration (r = -0.52, P = 0.01), BASDAI (r = -0.45, P = 0.04) and C-reactive protein (r = -0.5, P = 0.01). This is the first study to demonstrate endothelial progenitor cell depletion in AS patients. EPC depletions inversely correlate with disease duration, disease activity and inflammation, suggesting the pivotal role of inflammation in depletion of EPCs. EPC would possibly also serve as a therapeutic target for preventing cardiovascular disease in AS. © 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

  7. Endothelial cell oxidative stress and signal transduction

    Directory of Open Access Journals (Sweden)

    ROCIO FONCEA

    2000-01-01

    Full Text Available Endothelial dysfunction (ED is an early event in atherosclerotic disease, preceding clinical manifestations and complications. Increased reactive oxygen species (ROS have been implicated as important mechanisms that contribute to ED, and ROS’s may function as intracellular messengers that modulate signaling pathways. Several intracellular signal events stimulated by ROS have been defined, including the identification of two members of the mitogen activated protein kinase family (ERK1/2 and big MAP kinase, BMK1, tyrosine kinases (Src and Syk and different isoenzymes of PKC as redox-sensitive kinases. ROS regulation of signal transduction components include the modification in the activity of transcriptional factors such as NFkB and others that result in changes in gene expression and modifications in cellular responses. In order to understand the intracellular mechanisms induced by ROS in endothelial cells (EC, we are studying the response of human umbilical cord vein endothelial cells to increased ROS generation by different pro-atherogenic stimuli. Our results show that Homocysteine (Hcy and oxidized LDL (oxLDL enhance the activity and expression of oxidative stress markers, such as NFkB and heme oxygenase 1. These results suggest that these pro-atherogenic stimuli increase oxidative stress in EC, and thus explain the loss of endothelial function associated with the atherogenic process

  8. Endothelial progenitor cells in diabetes complications

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    Marina Sergeevna Michurova

    2014-12-01

    Full Text Available Patients with diabetes mellitus (DM have a 2- to 4-times higher risk of developing cardiovascular complications compared with non-diabetic controls. Hyperglycemia activates pathophysiological mechanisms that damage the endothelium. According to the current views, circulating progenitor cells derived from bone marrow repair the damage. These cells, known as endothelial progenitor cells (EPCs, maintain endothelial homeostasis and contribute to the formation of new vessels. Many clinical studies have reported that EPC population is dysfunctional and declines in numbers in patients with type 1 and type 2 DM. In addition, bone marrow doesn’t respond adequately to mobilizing stimuli in DM. Therefore, EPC alterations might have a pathogenic role in the complications of DM. In this review, EPC alterations will be examined in the context of macrovascular and microvascular complications of DM, highlighting their roles and functions in the progression of the disease.

  9. Endothelial juxtaposition of distinct adult stem cells activates angiogenesis signaling molecules in endothelial cells.

    Science.gov (United States)

    Mohammadi, Elham; Nassiri, Seyed Mahdi; Rahbarghazi, Reza; Siavashi, Vahid; Araghi, Atefeh

    2015-12-01

    Efficacy of therapeutic angiogenesis needs a comprehensive understanding of endothelial cell (EC) function and biological factors and cells that interplay with ECs. Stem cells are considered the key components of pro- and anti-angiogenic milieu in a wide variety of physiopathological states, and interactions of EC-stem cells have been the subject of controversy in recent years. In this study, the potential effects of three tissue-specific adult stem cells, namely rat marrow-derived mesenchymal stem cells (rBMSCs), rat adipose-derived stem cells (rADSCs) and rat muscle-derived satellite cells (rSCs), on the endothelial activation of key angiogenic signaling molecules, including VEGF, Ang-2, VEGFR-2, Tie-2, and Tie2-pho, were investigated. Human umbilical vein endothelial cells (HUVECs) and rat lung microvascular endothelial cells (RLMECs) were cocultured with the stem cells or incubated with the stem cell-derived conditioned media on Matrigel. Following HUVEC-stem cell coculture, CD31-positive ECs were flow sorted and subjected to western blotting to analyze potential changes in the expression of the pro-angiogenic signaling molecules. Elongation and co-alignment of the stem cells were seen along the EC tubes in the EC-stem cell cocultures on Matrigel, with cell-to-cell dye communication in the EC-rBMSC cocultures. Moreover, rBMSCs and rADSCs significantly improved endothelial tubulogenesis in both juxtacrine and paracrine manners. These two latter stem cells dynamically up-regulated VEGF, Ang-2, VREGR-2, and Tie-2 but down-regulated Tie2-pho and the Tie2-pho/Tie-2 ratio in HUVECs. Induction of pro-angiogenic signaling in ECs by marrow- and adipose-derived MSCs further indicates the significance of stem cell milieu in angiogenesis dynamics.

  10. Bone invading NSCLC cells produce IL-7: mice model and human histologic data

    International Nuclear Information System (INIS)

    Roato, Ilaria; Mussa, Antonio; Ferracini, Riccardo; Caldo, Davide; Godio, Laura; D'Amico, Lucia; Giannoni, Paolo; Morello, Emanuela; Quarto, Rodolfo; Molfetta, Luigi; Buracco, Paolo

    2010-01-01

    Bone metastases are a common and dismal consequence of lung cancer that is a leading cause of death. The role of IL-7 in promoting bone metastases has been previously investigated in NSCLC, but many aspects remain to be disclosed. To further study IL-7 function in bone metastasis, we developed a human-in-mice model of bone aggression by NSCLC and analyzed human bone metastasis biopsies. We used NOD/SCID mice implanted with human bone. After bone engraftment, two groups of mice were injected subcutaneously with A549, a human NSCLC cell line, either close or at the contralateral flank to the human bone implant, while a third control group did not receive cancer cells. Tumor and bone vitality and IL-7 expression were assessed in implanted bone, affected or not by A549. Serum IL-7 levels were evaluated by ELISA. IL-7 immunohistochemistry was performed on 10 human bone NSCLC metastasis biopsies for comparison. At 12 weeks after bone implant, we observed osteogenic activity and neovascularization, confirming bone vitality. Tumor aggressive cells implanted close to human bone invaded the bone tissue. The bone-aggressive cancer cells were positive for IL-7 staining both in the mice model and in human biopsies. Higher IL-7 serum levels were found in mice injected with A549 cells close to the bone implant compared to mice injected with A549 cells in the flank opposite to the bone implant. We demonstrated that bone-invading cells express and produce IL-7, which is known to promote osteoclast activation and osteolytic lesions. Tumor-bone interaction increases IL-7 production, with an increase in IL-7 serum levels. The presented mice model of bone invasion by contiguous tumor is suitable to study bone-tumor cell interaction. IL-7 plays a role in the first steps of metastatic process

  11. Oxidative stress induced pulmonary endothelial cell proliferation is ...

    African Journals Online (AJOL)

    Cellular hyper-proliferation, endothelial dysfunction and oxidative stress are hallmarks of the pathobiology of pulmonary hypertension. Indeed, pulmonary endothelial cells proliferation is susceptible to redox state modulation. Some studies suggest that superoxide stimulates endothelial cell proliferation while others have ...

  12. Human liver endothelial cells, but not macrovascular or microvascular endothelial cells, engraft in the mouse liver

    NARCIS (Netherlands)

    Filali, Ebtisam El; Hiralall, Johan K.; van Veen, Henk A.; Stolz, Donna B.; Seppen, Jurgen

    2013-01-01

    Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and

  13. Arecoline is cytotoxic for human endothelial cells.

    Science.gov (United States)

    Ullah, Mafaz; Cox, Stephen; Kelly, Elizabeth; Boadle, Ross; Zoellner, Hans

    2014-11-01

    Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Endothelial progenitor cells in coronary artery disease.

    Science.gov (United States)

    Donahue, Michael; Quintavalle, Cristina; Chiariello, Giovanni Alfonso; Condorelli, Gerolama; Briguori, Carlo

    2013-10-01

    In the last two decades a great deal of evidence has been collected on the key role of endothelial progenitor cells (EPC) in the mechanisms of vascular healing. The role of EPC as a marker of vascular health and prognosis of cardiovascular disease is already consolidated. This review aims to examine and evaluate recent data regarding EPC, as biomarkers, prognostic factor and potential therapy in cardiovascular disease.

  15. Do endothelial cells dream of eclectic shape?

    Science.gov (United States)

    Bentley, Katie; Philippides, Andrew; Ravasz Regan, Erzsébet

    2014-04-28

    Endothelial cells (ECs) exhibit dramatic plasticity of form at the single- and collective-cell level during new vessel growth, adult vascular homeostasis, and pathology. Understanding how, when, and why individual ECs coordinate decisions to change shape, in relation to the myriad of dynamic environmental signals, is key to understanding normal and pathological blood vessel behavior. However, this is a complex spatial and temporal problem. In this review we show that the multidisciplinary field of Adaptive Systems offers a refreshing perspective, common biological language, and straightforward toolkit that cell biologists can use to untangle the complexity of dynamic, morphogenetic systems. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Syncytin is involved in breast cancer-endothelial cell fusions

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Holck, S.; Christensen, I.J.

    2006-01-01

    Cancer cells can fuse spontaneously with normal host cells, including endothelial cells, and such fusions may strongly modulate the biological behaviour of tumors. However, the underlying mechanisms are unknown. We now show that human breast cancer cell lines and 63 out of 165 (38%) breast cancer...... specimens express syncytin, an endogenous retroviral envelope protein, previously implicated in fusions between placental trophoblast cells. Additionally, endothelial and cancer cells are shown to express ASCT-2, a receptor for syncytin. Syncytin antisense treatment decreases syncytin expression...... and inhibits fusions between breast cancer cells and endothelial cells. Moreover, a syncytin inhibitory peptide also inhibits fusions between cancer and endothelial cells. These results are the first to show that syncytin is expressed by human cancer cells and is involved in cancer-endothelial cell fusions....

  17. Endoderm Generates Endothelial Cells during Liver Development

    Directory of Open Access Journals (Sweden)

    Orit Goldman

    2014-10-01

    Full Text Available Organogenesis requires expansion of the embryonic vascular plexus that migrates into developing organs through a process called angiogenesis. Mesodermal progenitors are thought to derive endothelial cells (ECs that contribute to both embryonic vasculogenesis and the subsequent organ angiogenesis. Here, we demonstrate that during development of the liver, which is an endoderm derivative, a subset of ECs is generated from FOXA2+ endoderm-derived fetal hepatoblast progenitor cells expressing KDR (VEGFR2/FLK-1. Using human and mouse embryonic stem cell models, we demonstrate that KDR+FOXA2+ endoderm cells developing in hepatic differentiation cultures generate functional ECs. This introduces the concept that ECs originate not exclusively from mesoderm but also from endoderm, supported in Foxa2 lineage-tracing mouse embryos by the identification of FOXA2+ cell-derived CD31+ ECs that integrate the vascular network of developing fetal livers.

  18. Production of soluble Neprilysin by endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@monash.edu [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Rajapakse, Niwanthi W. [Department of Physiology, Building 13F, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Minond, Dmitriy [Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, FL 34987 (United States); Smith, A. Ian [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia)

    2014-04-04

    Highlights: • A soluble full-length form of Neprilysin exists in media of endothelial cells. • Exosomal release is the key mechanism for the production of soluble Neprilysin. • Inhibition of ADAM-17 by specific inhibitors reduce Neprilysin release. • Exosome mediated release of Neprilysin is dependent on ADAM-17 activity. - Abstract: A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC{sub 50} values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.

  19. Endothelial Progenitor Cells Enter the Aging Arena.

    Directory of Open Access Journals (Sweden)

    Kate eWilliamson

    2012-02-01

    Full Text Available Age is a significant risk factor for the development of vascular diseases, such as atherosclerosis. Although pharmacological treatments, including statins and anti-hypertensive drugs, have improved the prognosis for patients with cardiovascular disease, it remains a leading cause of mortality in those aged 65 years and over. Furthermore, given the increased life expectancy of the population in developed countries, there is a clear need for alternative treatment strategies. Consequently, the relationship between aging and progenitor cell-mediated repair is of great interest. Endothelial progenitor cells (EPCs play an integral role in the cellular repair mechanisms for endothelial regeneration and maintenance. However, EPCs are subject to age-associated changes that diminish their number in circulation and function, thereby enhancing vascular disease risk. A great deal of research is aimed at developing strategies to harness the regenerative capacity of these cells.In this review, we discuss the current understanding of the cells termed ‘EPCs’, examine the impact of age on EPC-mediated repair and identify therapeutic targets with potential for attenuating the age-related decline in vascular health via beneficial actions on EPCs.

  20. Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

    Science.gov (United States)

    Yano, Shuya; Miwa, Shinji; Mii, Sumiyuki; Hiroshima, Yukihiko; Uehara, Fuminari; Yamamoto, Mako; Kishimoto, Hiroyuki; Tazawa, Hiroshi; Bouvet, Michael; Fujiwara, Toshiyoshi; Hoffman, Robert M

    2014-01-01

    Invasive cancer cells are a critical target in order to prevent metastasis. In the present report, we demonstrate real-time visualization of cell cycle kinetics of invading cancer cells in 3-dimensional (3D) Gelfoam® histoculture, which is in vivo-like. A fluorescence ubiquitination cell cycle indicator (FUCCI) whereby G0/G1 cells express a red fluorescent protein and S/G2/M cells express a green fluorescent protein was used to determine the cell cycle position of invading and non-invading cells. With FUCCI 3D confocal imaging, we observed that cancer cells in G0/G1 phase in Gelfoam® histoculture migrated more rapidly and further than cancer cells in S/G2/M phases. Cancer cells ceased migrating when they entered S/G2/M phases and restarted migrating after cell division when the cells re-entered G0/G1. Migrating cancer cells also were resistant to cytotoxic chemotherapy, since they were preponderantly in G0/G1, where cytotoxic chemotherapy is not effective. The results of the present report suggest that novel therapy targeting G0/G1 cancer cells should be developed to prevent metastasis. PMID:24552821

  1. Endothelial cells promote the proliferation of lymphocytes partly through the Wnt pathway via LEF-1

    International Nuclear Information System (INIS)

    Wang, Shu-Hong; Nan, Ke-Jun; Wang, Yao-Chun

    2009-01-01

    The function of T cells and B cells is to recognize specific 'non-self' antigens, during a process known as antigen presentation. Once they have identified an invader, the cells generate specific responses that are tailored to maximally eliminate specific pathogens or pathogen-infected cells. Endothelial cells (ECs) can trigger the activation of T cells through their class I and class II MHC molecules. In this study, we examined the effect of ECs on the proliferation of lymphocytes. We report that the proliferation of T and B cells can be improved by interaction with ECs. LEF-1 is one of the main molecular mediators in this process, and the inhibition of LEF-1 induces apoptosis. These results suggest that LEF-1 modulates positively the proliferation of lymphocytes induced by their interaction with ECs.

  2. Endothelial Cell-Targeted Adenoviral Vector for Suppressing Breast Tumors

    National Research Council Canada - National Science Library

    Huang, Shuang

    2003-01-01

    .... We incorporated five previously published endothelial cell-specific peptide sequences into adenovirus capsid fiber sequence and the modified fibers were added to Beta-galactosidase- containing...

  3. Endothelial progenitor cells and revascularization following stroke.

    Science.gov (United States)

    Ma, Feifei; Morancho, Anna; Montaner, Joan; Rosell, Anna

    2015-10-14

    Brain injury after ischemia induces the mobilization of endothelial progenitor cells (EPCs), a population of bone marrow-derived cells with angio-vasculogenic capabilities. These cells have been also tested in pre-clinical models and proposed for neurorepair therapy aiming to treat patients in the delayed phases of stroke disease. Promising results in the pre-clinical field encourage the translation into a clinical therapeutic approach. In this review, we will describe EPCs actions for enhanced revascularization and neurorepair, which on one hand are by their direct incorporation into new vascular networks/structures or by direct cell-cell interactions with other brain cells, but also to indirect cell-cell communication thorough EPCs secreted growth factors. All these actions contribute to potentiate neurovascular remodeling and neurorepair. The data presented in this review encourages for a deep understanding of the mechanisms of the cross-talks between EPCs and other brain and progenitor cells, which deserves additional investigations and efforts that may lead to new EPCs-based therapies for stroke patients. This article is part of a Special Issue entitled SI: Cell Interactions In Stroke. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Effects of helium on inflammatory and oxidative stress-induced endothelial cell damage

    NARCIS (Netherlands)

    Smit, Kirsten F.; Kerindongo, Raphaela P.; Böing, Anita; Nieuwland, Rienk; Hollmann, Markus W.; Preckel, Benedikt; Weber, Nina C.

    2015-01-01

    Helium induces preconditioning in human endothelium protecting against postischemic endothelial dysfunction. Circulating endothelial microparticles are markers of endothelial dysfunction derived in response to injury. Another noble gas, xenon, protected human umbilical vein endothelial cells (HUVEC)

  5. Carcinoma cells misuse the host tissue damage response to invade the brain.

    Science.gov (United States)

    Chuang, Han-Ning; van Rossum, Denise; Sieger, Dirk; Siam, Laila; Klemm, Florian; Bleckmann, Annalen; Bayerlová, Michaela; Farhat, Katja; Scheffel, Jörg; Schulz, Matthias; Dehghani, Faramarz; Stadelmann, Christine; Hanisch, Uwe-Karsten; Binder, Claudia; Pukrop, Tobias

    2013-08-01

    The metastatic colonization of the brain by carcinoma cells is still barely understood, in particular when considering interactions with the host tissue. The colonization comes with a substantial destruction of the surrounding host tissue. This leads to activation of damage responses by resident innate immune cells to protect, repair, and organize the wound healing, but may distract from tumoricidal actions. We recently demonstrated that microglia, innate immune cells of the CNS, assist carcinoma cell invasion. Here we report that this is a fatal side effect of a physiological damage response of the brain tissue. In a brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia and astrocytes comparable to that seen at the interface of human cerebral metastases. While the glial damage response intended to protect the brain from intrusion of benign epithelial cells by inducing apoptosis, it proved ineffective against various malignant cell types. They did not undergo apoptosis and actually exploited the local tissue reaction to invade instead. Gene expression and functional analyses revealed that the C-X-C chemokine receptor type 4 (CXCR4) and WNT signaling were involved in this process. Furthermore, CXCR4-regulated microglia were recruited to sites of brain injury in a zebrafish model and CXCR4 was expressed in human stroke patients, suggesting a conserved role in damage responses to various types of brain injuries. Together, our findings point to a detrimental misuse of the glial damage response program by carcinoma cells resistant to glia-induced apoptosis. Copyright © 2013 Wiley Periodicals, Inc.

  6. Differentiation state determines neural effects on microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Muffley, Lara A., E-mail: muffley@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Pan, Shin-Chen, E-mail: pansc@mail.ncku.edu.tw [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Smith, Andria N., E-mail: gnaunderwater@gmail.com [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Ga, Maricar, E-mail: marga16@uw.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Hocking, Anne M., E-mail: ahocking@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Gibran, Nicole S., E-mail: nicoleg@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States)

    2012-10-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  7. Invasion of human aortic endothelial cells by oral viridans group streptococci and induction of inflammatory cytokine production.

    Science.gov (United States)

    Nagata, E; de Toledo, A; Oho, T

    2011-02-01

    Oral viridans group streptococci are the major commensal bacteria of the supragingival oral biofilm and have been detected in human atheromatous plaque. Atherosclerosis involves an ongoing inflammatory response, reportedly involving chronic infection caused by multiple pathogens. The aim of this study was to examine the invasion of human aortic endothelial cells (HAECs) by oral viridans group streptococci and the subsequent cytokine production by viable invaded HAECs. The invasion of HAECs by bacteria was examined using antibiotic protection assays and was visualized by confocal scanning laser microscopy. The inhibitory effects of catalase and cytochalasin D on the invasion of HAECs were also examined. The production of cytokines by invaded or infected HAECs was determined using enzyme-linked immunosorbent assays, and a real-time polymerase chain reaction method was used to evaluate the expression of cytokine messenger RNA. The oral streptococci tested were capable of invading HAECs. The number of invasive bacteria increased with the length of the co-culture period. After a certain co-culture period, some organisms were cytotoxic to the HAECs. Catalase and cytochalasin D inhibited the invasion of HAECs by the organism. HAECs invaded by Streptococcus mutans Xc, Streptococcus gordonii DL1 (Challis), Streptococcus gordonii ATCC 10558 and Streptococcus salivarius ATCC 13419 produced more cytokine(s) (interleukin-6, interleukin-8, monocyte chemoattractant protein-1) than non-invaded HAECs. The HAECs invaded by S. mutans Xc produced the largest amounts of cytokines, and the messenger RNA expression of cytokines by invaded HAECs increased markedly compared with that by non-invaded HAECs. These results suggest that oral streptococci may participate in the pathogenesis of atherosclerosis. © 2010 John Wiley & Sons A/S.

  8. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    International Nuclear Information System (INIS)

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K.

    1990-01-01

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling

  9. Listeria monocytogenes efficiently invades caco-2 cells after low-temperature storage in broth and on deli meat

    DEFF Research Database (Denmark)

    Larsen, Marianne Halberg; Koch, Anette Granly; Ingmer, Hanne

    2010-01-01

    The objective of this study was to investigate how various growth conditions influence the virulence of Listeria monocytogenes monitored by its ability to invade the epithelial cell lines Caco-2 and INT-407. The growth conditions examined were modified atmosphere-packaged deli meat and brain heart...

  10. Effects of high glucose on human cavernous endothelial cells.

    Science.gov (United States)

    Ning, Hongxiu; Qiu, Xuefeng; Baine, Lia; Lin, Guiting; Lue, Tom F; Lin, Ching-Shwun

    2012-11-01

    To obtain experimental evidence for a causal effect of high glucose (HG) on cavernous endothelial dysfunction. Cavernous tissues were obtained from patients undergoing surgery for penile prosthesis implantation. Endothelial cells were isolated by binding to anti-CD31 antibody, followed by magnetic capture. Their endothelial identity was verified by flow cytometry and immunofluorescence staining for endothelial markers CD31, von Willebrand factor, and endothelial nitric oxide synthase, and by their ability to form tube-like structures in matrigel (tube formation) and to endocytose acetylated low-density lipoprotein (low-density lipoprotein uptake). The cells were then cultured under normal glucose (NG) (5 mM) or HG (25 mM) conditions, followed by analysis for endothelial gene expression, function, proliferation, apoptosis, and mitochondrial fragmentation. Human cavernous endothelial cell (HCEC) strains were established and determined to be nearly 100% pure endothelial cells. In the HG culture condition, HCECs expressed approximately 50% less CD31, von Willebrand factor, and endothelial nitric oxide synthase, but nearly twice as much collagen IV compared with HCECs grown in NG medium. HG also suppressed low-density lipoprotein uptake and tube formation by approximately 50%. HCECs grew significantly slower in the high-glucose medium than in the NG medium. Approximately 3 times as many cells exhibited apoptosis in the HG medium as in the NG medium. Approximately 4 times as many cells contained fragmented mitochondria in the HG medium as in the NG medium. HG caused a decrease in endothelial proliferation, function, and marker expression. It also caused an increase in endothelial collagen IV expression, apoptosis, and mitochondrial fragmentation. Together, these HG-induced changes in cavernous endothelial cells provide an explanation for hyperglycemia's detrimental effects on erectile function. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Heterogeneity of type III secretion system (T3SS)-1-independent entry mechanisms used by Salmonella Enteritidis to invade different cell types.

    Science.gov (United States)

    Rosselin, Manon; Abed, Nadia; Virlogeux-Payant, Isabelle; Bottreau, Elisabeth; Sizaret, Pierre-Yves; Velge, Philippe; Wiedemann, Agnès

    2011-03-01

    Salmonella causes a wide range of diseases from acute gastroenteritis to systemic typhoid fever, depending on the host. To invade non-phagocytic cells, Salmonella has developed different mechanisms. The main invasion system requires a type III secretion system (T3SS) known as T3SS-1, which promotes a Trigger entry mechanism. However, other invasion factors have recently been described in Salmonella, including Rck and PagN, which were not expressed under our bacterial culture conditions. Based on these observations, we used adhesion and invasion assays to analyse the respective roles of Salmonella Enteritidis T3SS-1-dependent and -independent invasion processes at different times of infection. Diverse cell lines and cell types were tested, including endothelial, epithelial and fibroblast cells. We demonstrated that cell susceptibility to the T3SS-1-independent entry differs by a factor of nine between the most and the least permissive cell lines tested. In addition, using scanning electron and confocal microscopy, we showed that T3SS-1-independent entry into cells was characterized by a Trigger-like alteration, as for the T3SS-1-dependent entry, and also by Zipper-like cellular alteration. Our results demonstrate for what is believed to be the first time that Salmonella can induce Trigger-like entry independently of T3SS-1 and can induce Zipper-like entry independently of Rck. Overall, these data open new avenues for discovering new invasion mechanisms in Salmonella.

  12. Towards clinical application of microvascular endothelial cell seeding

    NARCIS (Netherlands)

    Arts, C.H.P. (Catharina Henrica Paulina)

    2002-01-01

    The central question in this thesis is whether microvascular endothelial cells (MVEC) from subcutaneous fat tissue are suitable for the seeding of prosthetic vascular grafts and deendothelialized surfaces. The aim of the application of endothelial cells (EC) is the inhibition of thrombogenicity and

  13. Comparison of Endothelial Cell Loss by Specular Microscopy ...

    African Journals Online (AJOL)

    Group A had undergone phacoemulsification and Group B had under gone manual SICS. In Group A 50 cases were performed by temporal CCI and remaining 50 cases were performed by superior SI technique. Endothelial cell count was evaluated by using a noncontact specular microscope. Results: Mean endothelial cell ...

  14. Endothelial cell cultures as a tool in biomaterial research

    NARCIS (Netherlands)

    Kirkpatrick, CJ; Otto, M; van Kooten, T; Krump, [No Value; Kriegsmann, J; Bittinger, F

    1999-01-01

    Progress in biocompatibility and tissue engineering would today be inconceivable without the aid of in vitro techniques. Endothelial cell cultures represent a valuable tool not just in haemocompatibility testing, but also in the concept of designing hybrid organs. In the past endothelial cells (EC)

  15. Obstructive sleep apnea and endothelial progenitor cells

    Directory of Open Access Journals (Sweden)

    Wang Q

    2013-10-01

    Full Text Available Qing Wang,1,* Qi Wu,2,* Jing Feng,3,4 Xin Sun5 1The Second Respiratory Department of the First People's Hospital of Kunming, Yunnan, People's Republic of China; 2Tianjin Haihe Hospital, Tianjin, People's Republic of China; 3Respiratory Department of Tianjin Medical University General Hospital, Tianjin, People's Republic of China; 4Division of Pulmonary and Critical Care Medicine, Duke University Medical Center, Durham, NC, USA; 5Respiratory Department of Tianjin Haihe Hospital, Tianjin, People's Republic of China *These authors contributed equally to this work Background: Obstructive sleep apnea (OSA occurs in 4% of middle-aged men and 2% of middle-aged women in the general population, and the prevalence is even higher in specific patient groups. OSA is an independent risk factor for a variety of cardiovascular diseases. Endothelial injury could be the pivotal determinant in the development of cardiovascular pathology in OSA. Endothelial damage ultimately represents a dynamic balance between the magnitude of injury and the capacity for repair. Bone marrow–derived endothelial progenitor cells (EPCs within adult peripheral blood present a possible means of vascular maintenance that could home to sites of injury and restore endothelial integrity and normal function. Methods: We summarized pathogenetic mechanisms of OSA and searched for available studies on numbers and functions of EPCs in patients with OSA to explore the potential links between the numbers and functions of EPCs and OSA. In particular, we tried to elucidate the molecular mechanisms of the effects of OSA on EPCs. Conclusion: Intermittent hypoxia cycles and sleep fragmentation are major pathophysiologic characters of OSA. Intermittent hypoxia acts as a trigger of oxidative stress, systemic inflammation, and sympathetic activation. Sleep fragmentation is associated with a burst of sympathetic activation and systemic inflammation. In most studies, a reduction in circulating EPCs has

  16. Ionizing radiation activates vascular endothelial growth factor-A transcription in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyounji; Kim, Kwang Seok; Jeong, Jae Hoon; Lim, Young Bin [Radiation Cancer Biology Team, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2016-12-15

    Vascular endothelial growth factor (VEGF) is an essential paracrine factor for developmental and pathological angiogenesis. VEGF also exerts its effects in an autocrine manner in VEGF-producing cells. For instance, autocrine VEGF signaling occurs in tumor cells and contributes to key aspects of tumorigenesis, such as in the function of cancer stem cells and tumor initiation, which are independent of angiogenesis. In addition to tumors cells, non-transformed cells also express VEGF. For example, a VEGF dependent intracellular autocrine mechanism is crucial for the survival of hematopoietic stem cells and hematopoiesis. Stereotactic body radiation therapy (SBRT) is a novel treatment modality for early primary cancer and oligometastatic disease. SBRT delivers high-dose hypofractionated radiation, such as 20-60 Gy, to tumors in a single fraction or 2-5 fractions. As VEGF is a critical regulator of functional integrity and viability of vascular endothelial cells, we examined whether high-dose irradiation alters VEGF signaling by measuring the expression levels of VEGFA transcript. It is generally believed that endothelial cells do not produce VEGF in response to radiation. In present study, however, we provide the first demonstration of transcriptional regulation of VEGFA in human vascular endothelial cells by IR treatment. Irradiation with doses higher than 10 Gy in a single exposure triggers up-regulation of VEGFA transcription within 2 hours in HUVECs, whereas irradiation with 10 Gy does not alter VEGFA levels. Our data have shown that high-dose irradiation triggers immediate transactivation of VEGFA in human vascular endothelial cells.

  17. Human Endothelial Cell Models in Biomaterial Research.

    Science.gov (United States)

    Hauser, Sandra; Jung, Friedrich; Pietzsch, Jens

    2017-03-01

    Endothelial cell (EC) models have evolved as important tools in biomaterial research due to ubiquitously occurring interactions between implanted materials and the endothelium. However, screening the available literature has revealed a gap between material scientists and physiologists in terms of their understanding of these biomaterial-endothelium interactions and their relative importance. Consequently, EC models are often applied in nonphysiological experimental setups, or too extensive conclusions are drawn from their results. The question arises whether this might be one reason why, among the many potential biomaterials, only a few have found their way into the clinic. In this review, we provide an overview of established EC models and possible selection criteria to enable researchers to determine the most reliable and relevant EC model to use. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Propranolol treatment of infantile hemangioma endothelial cells: A molecular analysis

    Science.gov (United States)

    STILES, JESSICA; AMAYA, CLARISSA; PHAM, ROBERT; ROWNTREE, REBECCA K.; LACAZE, MARY; MULNE, ARLYNN; BISCHOFF, JOYCE; KOKTA, VICTOR; BOUCHERON, LAURA E.; MITCHELL, DIANNE C.; BRYAN, BRAD A.

    2012-01-01

    Infantile hemangiomas (IHs) are non-malignant, largely cutaneous vascular tumors affecting approximately 5–10% of children to varying degrees. During the first year of life, these tumors are strongly proliferative, reaching an average size ranging from 2 to 20 cm. These lesions subsequently stabilize, undergo a spontaneous slow involution and are fully regressed by 5 to 10 years of age. Systemic treatment of infants with the non-selective β-adrenergic receptor blocker, propranolol, has demonstrated remarkable efficacy in reducing the size and appearance of IHs. However, the mechanism by which this occurs is largely unknown. In this study, we sought to understand the molecular mechanisms underlying the effectiveness of β blocker treatment in IHs. Our data reveal that propranolol treatment of IH endothelial cells, as well as a panel of normal primary endothelial cells, blocks endothelial cell proliferation, migration, and formation of the actin cytoskeleton coincident with alterations in vascular endothelial growth factor receptor-2 (VEGFR-2), p38 and cofilin signaling. Moreover, propranolol induces major alterations in the protein levels of key cyclins and cyclin-dependent kinase inhibitors, and modulates global gene expression patterns with a particular affect on genes involved in lipid/sterol metabolism, cell cycle regulation, angiogenesis and ubiquitination. Interestingly, the effects of propranolol were endothelial cell-type independent, affecting the properties of IH endothelial cells at similar levels to that observed in neonatal dermal microvascular and coronary artery endothelial cells. This data suggests that while propranolol markedly inhibits hemangioma and normal endothelial cell function, its lack of endothelial cell specificity hints that the efficacy of this drug in the treatment of IHs may be more complex than simply blockage of endothelial function as previously believed. PMID:23170111

  19. Targeting Endothelial Cells with Multifunctional GaN/Fe Nanoparticles.

    Science.gov (United States)

    Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Andrée, Birgit; Cebotari, Serghei; Boyle, Erin C; Haverich, Axel; Hilfiker, Andres

    2017-08-10

    In this paper, we report on the interaction of multifunctional nanoparticles with living endothelial cells. The nanoparticles were synthesized using direct growth of gallium nitride on zinc oxide nanoparticles alloyed with iron oxide followed by core decomposition in hydrogen flow at high temperature. Using transmission electron microscopy, we demonstrate that porcine aortic endothelial cells take up GaN-based nanoparticles suspended in the growth medium. The nanoparticles are deposited in vesicles and the endothelial cells show no sign of cellular damage. Intracellular inert nanoparticles are used as guiding elements for controlled transportation or designed spatial distribution of cells in external magnetic fields.

  20. Targeting Endothelial Cells with Multifunctional GaN/Fe Nanoparticles

    Science.gov (United States)

    Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Andrée, Birgit; Cebotari, Serghei; Boyle, Erin C.; Haverich, Axel; Hilfiker, Andres

    2017-08-01

    In this paper, we report on the interaction of multifunctional nanoparticles with living endothelial cells. The nanoparticles were synthesized using direct growth of gallium nitride on zinc oxide nanoparticles alloyed with iron oxide followed by core decomposition in hydrogen flow at high temperature. Using transmission electron microscopy, we demonstrate that porcine aortic endothelial cells take up GaN-based nanoparticles suspended in the growth medium. The nanoparticles are deposited in vesicles and the endothelial cells show no sign of cellular damage. Intracellular inert nanoparticles are used as guiding elements for controlled transportation or designed spatial distribution of cells in external magnetic fields.

  1. Endothelial cell energy metabolism, proliferation, and apoptosis in pulmonary hypertension.

    Science.gov (United States)

    Xu, Weiling; Erzurum, Serpil C

    2011-01-01

    Pulmonary arterial hypertension (PAH) is a fatal disease characterized by impaired regulation of pulmonary hemodynamics and excessive growth and dysfunction of the endothelial cells that line the arteries in PAH lungs. Establishment of methods for culture of pulmonary artery endothelial cells from PAH lungs has provided the groundwork for mechanistic translational studies that confirm and extend findings from model systems and spontaneous pulmonary hypertension in animals. Endothelial cell hyperproliferation, survival, and alterations of biochemical-metabolic pathways are the unifying endothelial pathobiology of the disease. The hyperproliferative and apoptosis-resistant phenotype of PAH endothelial cells is dependent upon the activation of signal transducer and activator of transcription (STAT) 3, a fundamental regulator of cell survival and angiogenesis. Animal models of PAH, patients with PAH, and human PAH endothelial cells produce low nitric oxide (NO). In association with the low level of NO, endothelial cells have reduced mitochondrial numbers and cellular respiration, which is associated with more than a threefold increase in glycolysis for energy production. The shift to glycolysis is related to low levels of NO and likely to the pathologic expression of the prosurvival and proangiogenic signal transducer, hypoxia-inducible factor (HIF)-1, and the reduced mitochondrial antioxidant manganese superoxide dismutase (MnSOD). In this article, we review the phenotypic changes of the endothelium in PAH and the biochemical mechanisms accounting for the proliferative, glycolytic, and strongly proangiogenic phenotype of these dysfunctional cells, which consequently foster the panvascular progressive pulmonary remodeling in PAH. © 2011 American Physiological Society.

  2. Autocrine VEGF isoforms differentially regulate endothelial cell behavior

    Directory of Open Access Journals (Sweden)

    Hideki Yamamoto

    2016-09-01

    Full Text Available Vascular endothelial growth factor A (VEGF is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2. We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell

  3. Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

    Science.gov (United States)

    Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

    2017-09-15

    Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by

  4. Animal study on transplantation of human umbilical vein endothelial cells for corneal endothelial decompensation

    Directory of Open Access Journals (Sweden)

    Li Cui

    2014-06-01

    Full Text Available AIM: To explore the feasibility of culturing human umbilical vein endothelial cells(HUVECon acellular corneal stroma and performing the posterior lamellar endothelial keratoplasty(PLEKtreating corneal endothelial decompensation.METHODS: Thirty New-Zealand rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma group and 10 for control group. Corneal endothelial cells were removed to establish animal model of corneal endothelial failure. PLEK was performed on the rabbits of experimental group and stroma group, and nothing was transplantated onto the rabbits of control group with the deep layer excised only. Postoperative observation was taken for 3mo. The degree of corneal edema and central corneal thickness were recorded for statistical analysis.RESULTS: Corneas in experimental group were relieved in edema obviously compared with that in stroma group and the control group, and showed increased transparency 7d after the operation. The average density of endothelial cells was 2 026.4±129.3cells/mm2, and average central corneal thickness was 505.2±25.4μm in experimental group, while 1 535.6±114.5μm in stroma group and 1 493.5±70.2μm in control group 3mo after operation.CONCLUSION:We achieved preliminary success in our study that culturing HUVEC on acellular corneal stroma and performing PLEK for corneal endothelial decompensation. HUVEC transplanted could survive in vivo, and have normal biological function of keeping cornea transparent. This study provides a new idea and a new way clinically for the treatment of corneal endothelial diseases.

  5. The effect of uric acid on homocysteine-induced endothelial dysfunction in bovine aortic endothelial cells.

    Science.gov (United States)

    Papezikova, Ivana; Pekarova, Michaela; Lojek, Antonin; Kubala, Lukas

    2009-01-01

    Elevated plasma uric acid indicates an increased risk of cardiovascular diseases associated with endothelial dysfunction. However, the role of uric acid in the pathogenesis of endothelial dysfunction is still a matter of debate. It is not clear whether uric acid is a real causative risk factor, an inert marker, or even a protective molecule with respect to its antioxidant properties. We have studied the effect of uric acid on intact endothelial cells as well as cells with homocysteine-induced endothelial dysfunction. Bovine aortic endothelial cells were treated with uric acid (100 - 600 muM) and homocysteine (100 muM) or with uric acid only. After 24 hours, the cells were stimulated with 1 mug/ml of calcium ionophore A23187, and nitric oxide (NO) production was measured electrochemically with the use of a NO-sensitive microelectrode. The expression of endothelial nitric oxide synthase (eNOS) and eNOS phosphorylation at Ser1179 was estimated with the use of Western blotting. Interaction between NO and uric acid was measured with a NO electrode. Superoxide generation was measured with the use of the fluorescence dye MitoSox Red. Homocysteine strongly diminished A23187-induced NO release. 100 muM uric acid slightly restored NO production; higher concentrations were ineffective. Interestingly, a dose-dependent decrease of NO release was observed in the cells treated only with uric acid. Uric acid did not scavenge NO and did not change eNOS protein expression or phosphorylation at Ser1179, but dose-dependently increased superoxide production in A23187-stimulated cells. In conclusion, uric acid decreased NO bioavailability and enhanced superoxide generation in A23187-stimulated bovine aortic endothelial cells.

  6. Physical View on the Interactions Between Cancer Cells and the Endothelial Cell Lining During Cancer Cell Transmigration and Invasion

    Science.gov (United States)

    Mierke, Claudia T.

    There exist many reviews on the biological and biochemical interactions of cancer cells and endothelial cells during the transmigration and tissue invasion of cancer cells. For the malignant progression of cancer, the ability to metastasize is a prerequisite. In particular, this means that certain cancer cells possess the property to migrate through the endothelial lining into blood or lymph vessels, and are possibly able to transmigrate through the endothelial lining into the connective tissue and follow up their invasion path in the targeted tissue. On the molecular and biochemical level the transmigration and invasion steps are well-defined, but these signal transduction pathways are not yet clear and less understood in regards to the biophysical aspects of these processes. To functionally characterize the malignant transformation of neoplasms and subsequently reveal the underlying pathway(s) and cellular properties, which help cancer cells to facilitate cancer progression, the biomechanical properties of cancer cells and their microenvironment come into focus in the physics-of-cancer driven view on the metastasis process of cancers. Hallmarks for cancer progression have been proposed, but they still lack the inclusion of specific biomechanical properties of cancer cells and interacting surrounding endothelial cells of blood or lymph vessels. As a cancer cell is embedded in a special environment, the mechanical properties of the extracellular matrix also cannot be neglected. Therefore, in this review it is proposed that a novel hallmark of cancer that is still elusive in classical tumor biological reviews should be included, dealing with the aspect of physics in cancer disease such as the natural selection of an aggressive (highly invasive) subtype of cancer cells displaying a certain adhesion or chemokine receptor on their cell surface. Today, the physical aspects can be analyzed by using state-of-the-art biophysical methods. Thus, this review will present

  7. Brain Tumor Tropism of Transplanted Human Neural Stem Cells Is Induced by Vascular Endothelial Growth Factor

    Directory of Open Access Journals (Sweden)

    Nils Ole Schmidt

    2005-06-01

    Full Text Available The transplantation of neural stem cells (NSCs offers a new potential therapeutic approach as a cell-based delivery system for gene therapy in brain tumors. This is based on the unique capacity of NSCs to migrate throughout the brain and to target invading tumor cells. However, the signals controlling the targeted migration of transplanted NSCs are poorly defined. We analyzed the in vitro and in vivo effects of angiogenic growth factors and protein extracts from surgical specimens of brain tumor patients on NSC migration. Here, we demonstrate that vascular endothelial growth factor (VEGF is able to induce a long-range attraction of transplanted human NSCs from distant sites in the adult brain. Our results indicate that tumorupregulated VEGF and angiogenic-activated microvasculature are relevant guidance signals for NSC tropism toward brain tumors.

  8. Listeria monocytogenes efficiently invades Caco-2 cells after low-temperature storage in broth and on deli meat.

    Science.gov (United States)

    Larsen, Marianne Halberg; Koch, Anette Granly; Ingmer, Hanne

    2010-09-01

    The objective of this study was to investigate how various growth conditions influence the virulence of Listeria monocytogenes monitored by its ability to invade the epithelial cell lines Caco-2 and INT-407. The growth conditions examined were modified atmosphere-packaged deli meat and brain heart infusion broth (BHI) with and without salt. Five strains of L. monocytogenes were selected to investigate their invasiveness and all strains invaded Caco-2 cells at higher levels than INT-407 cells. Further, the clinical strains (3443 and 3734) were more invasive (p 0.05) in invasiveness after 7 days at 10 degrees C in BHI broth or on sausage, whereas a slight increase (p < 0.05) was observed after incubation on ham for 2 and 4 weeks compared to that in BHI broth. Most importantly, our results show that L. monocytogenes efficiently invade Caco-2 cells even after 4 weeks of storage at chilled temperature. This is highly relevant for safety assessment of this organism in food as these conditions reflect storage of ready-to-eat food products in domestic refrigerators.

  9. Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases

    OpenAIRE

    Angelot, Fanny; Seillès, Estelle; Biichlé, Sabeha; Berda, Yael; Gaugler, Béatrice; Plumas, Joel; Chaperot, Laurence; Dignat-George, Françoise; Tiberghien, Pierre; Saas, Philippe; Garnache-Ottou, Francine

    2009-01-01

    International audience; BACKGROUND: Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. DESIGN AND METHODS: Microparticles generated from endothelial cell lines, platelets or activated T ...

  10. Suprabasin as a novel tumor endothelial cell marker

    OpenAIRE

    Alam, Mohammad T.; Nagao-Kitamoto, Hiroko; Ohga, Noritaka; Akiyama, Kosuke; Maishi, Nako; Kawamoto, Taisuke; Shinohara, Nobuo; Taketomi, Akinobu; Shindoh, Masanobu; Hida, Yasuhiro; Hida, Kyoko

    2014-01-01

    Recent studies have reported that stromal cells contribute to tumor progression. We previously demonstrated that tumor endothelial cells (TEC) characteristics were different from those of normal endothelial cells (NEC). Furthermore, we performed gene profile analysis in TEC and NEC, revealing that suprabasin (SBSN) was upregulated in TEC compared with NEC. However, its role in TEC is still unknown. Here we showed that SBSN expression was higher in isolated human and mouse TEC than in NEC. SBS...

  11. Endothelial cell loss secondary to two different phacoemulsification techniques.

    Science.gov (United States)

    Kohlhaas, M; Klemm, M; Kammann, J; Richard, G

    1998-11-01

    The endothelial cell count after phacoemulsification serves as a sensitive indicator for the level of corneal damage caused by different phacoemulsification techniques. In a prospective and randomized study, the "Reversed Tip and Snip" technique and the "Divide and Conquer" technique were performed in groups of 30 patients each. The corneal endothelial cell count was measured preoperatively as well as 4 weeks and 3 months postoperatively. The endothelial cell count showed significant (P < .001) reduction by approximately 10% after the "Reversed Tip and Snip" technique and by approximately 15% (P < .001) after the "Divide and Conquer" technique. The latter produced a significantly (P < .001) greater cell loss. The "Reversed Tip and Snip" phacoemulsification technique produces less endothelial cell loss than the "Divide and Conquer" technique.

  12. Trypanosoma cruzi infection: a continuous invader-host cell cross talk with participation of extracellular matrix and adhesion and chemoattractant molecules

    Directory of Open Access Journals (Sweden)

    Marino A.P.M.P.

    2003-01-01

    Full Text Available Several lines of evidence have shown that Trypanosoma cruzi interacts with host extracellular matrix (ECM components producing breakdown products that play an important role in parasite mobilization and infectivity. Parasite-released antigens also modulate ECM expression that could participate in cell-cell and/or cell-parasite interactions. Increased expression of ECM components has been described in the cardiac tissue of chronic chagasic patients and diverse target tissues including heart, thymus, central nervous system and skeletal muscle of experimentally T. cruzi-infected mice. ECM components may adsorb parasite antigens and cytokines that could contribute to the establishment and perpetuation of inflammation. Furthermore, T. cruzi-infected mammalian cells produce cytokines and chemokines that not only participate in the control of parasitism but also contribute to the establishment of chronic inflammatory lesions in several target tissues and most frequently lead to severe myocarditis. T. cruzi-driven cytokines and chemokines may also modulate VCAM-1 and ICAM-1 adhesion molecules on endothelial cells of target tissues and play a key role in cell recruitment, especially of activated VLA-4+LFA-1+CD8+ T lymphocytes, resulting in a predominance of this cell population in the inflamed heart, central nervous system and skeletal muscle. The VLA-4+-invading cells are surrounded by a fine network of fibronectin that could contribute to cell anchorage, activation and effector functions. Since persistent "danger signals" triggered by the parasite and its antigens are required for the establishment of inflammation and ECM alterations, therapeutic interventions that control parasitism and selectively modulate cell migration improve ECM abnormalities, paving the way for the development of new therapeutic strategies improving the prognosis of T. cruzi-infected individuals.

  13. PECAM-1 polymorphism affects monocyte adhesion to endothelial cells.

    Science.gov (United States)

    Goodman, Reyna S; Kirton, Christopher M; Oostingh, Gertie J; Schön, Michael P; Clark, Michael R; Bradley, J Andrew; Taylor, Craig J

    2008-02-15

    Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) plays an important role in leukocyte-endothelial cell adhesion and transmigration. Single nucleotide polymorphisms of PECAM-1 encoding amino acid substitutions at positions 98 leucine/valine (L/V), 536 serine/asparagine (S/N), and 643 arginine/glycine (R/G) occur in strong genetic linkage resulting in two common haplotypes (LSR and VNG). These PECAM-1 polymorphisms are associated with graft-versus-host disease after hematopoietic stem cell transplantation and with cardiovascular disease, but whether they influence PECAM-1 function is unknown. We examined the effect of homozygous and heterozygous expression of the PECAM-1 LSR and VNG genotypes on the adhesive interactions of peripheral blood monocytes and activated endothelial cell monolayers under shear stress in a flow-based cell adhesion assay. There was no difference in monocyte adhesion between the two homozygous genotypes of PECAM-1 but when monocytes expressed both alleles in heterozygous form, firm adhesion of monocytes to endothelial cells was markedly increased. PECAM-1 polymorphism expressed in homozygous or heterozygous form by endothelial cells did not influence monocyte adhesion. This is, to our knowledge, the first demonstration that PECAM-1 genotype can alter the level of monocyte binding to endothelial cells and a demonstration that heterozygous expression of a polymorphic protein may lead to altered function.

  14. Elevated PTH induces endothelial-to-chondrogenic transition in aortic endothelial cells.

    Science.gov (United States)

    Wu, Min; Zhang, Jian-Dong; Tang, Ri-Ning; Crowley, Steven D; Liu, Hong; Lv, Lin-Li; Ma, Kun-Ling; Liu, Bi-Cheng

    2017-03-01

    Previous studies have shown that increased parathyroid hormone (PTH) attributable to secondary hyperparathyroidism in chronic kidney disease accelerates the arteriosclerotic fibrosis and calcification. Although the underlying mechanisms remain largely unknown, endothelial cells (ECs) have recently been demonstrated to participate in calcification in part by providing chondrogenic cells via the endothelial-to-mesenchymal transition (EndMT). Therefore, this study aimed to investigate whether elevated PTH could induce endothelial-to-chondrogenic transition in aortic ECs and to determine the possible underlying signaling pathway. We found that treatment of ECs with PTH significantly upregulated the expression of EndMT-related markers. Accordingly, ECs treated with PTH exhibited chondrogenic potential. In vivo, lineage-tracing model-subjected mice with endothelial-specific green fluorescent protein fluorescence to chronic PTH infusion showed a marked increase in the aortic expression of chondrocyte markers, and confocal microscopy revealed the endothelial origin of cells expressing chondrocyte markers in the aorta after PTH infusion. Furthermore, this in vitro study showed that PTH enhanced the nuclear localization of β-catenin in ECs, whereas β-catenin siRNA or DKK1, an inhibitor of β-catenin nuclear translocation, attenuated the upregulation of EndMT-associated and chondrogenic markers induced by PTH. In summary, our study demonstrated that elevated PTH could induce the transition of ECs to chondrogenic cells via EndMT, possibly mediated by the nuclear translocation of β-catenin. Copyright © 2017 the American Physiological Society.

  15. Activation of Endothelial Nitric Oxide (eNOS Occurs through Different Membrane Domains in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Jason Tran

    Full Text Available Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC with cholesterol and the oxysterol 7-ketocholesterol (7KC. Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1 colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

  16. Magnetizable stent-grafts enable endothelial cell capture

    Energy Technology Data Exchange (ETDEWEB)

    Tefft, Brandon J. [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States); Uthamaraj, Susheil [Division of Engineering, Mayo Clinic, Rochester, MN (United States); Harburn, J. Jonathan [School of Medicine, Pharmacy and Health, Durham University, Stockton-on-Tees (United Kingdom); Hlinomaz, Ota [Department of Cardioangiology, St. Anne' s University Hospital, Brno (Czech Republic); Lerman, Amir [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States); Dragomir-Daescu, Dan [Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN (United States); Sandhu, Gurpreet S., E-mail: sandhu.gurpreet@mayo.edu [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States)

    2017-04-01

    Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance. - Highlights: • Magnetic stent-grafts were made from 2205 steel stents and polyurethane nanofibers. • Stent-grafts remained patent and formed a thin and uniform neointima when implanted. • Stent-grafts captured endothelial cells labeled with magnetic nanoparticles.

  17. Growth of fibroblasts and endothelial cells on wettability gradient surfaces

    NARCIS (Netherlands)

    Ruardy, TG; Moorlag, HE; Schakenraad, JM; VanderMei, HC; Busscher, HJ

    1997-01-01

    The growth, spreading, and shape of human skin fibroblasts (PK 84) and human umbilical cord endothelial cells on dichlorodimethylsilane (DDS) and dimethyloctadecylchlorosilane (DOGS) gradient surfaces were investigated in the presence of serum proteins. Gradient surfaces were prepared on glass using

  18. Tumor-derived circulating endothelial cell clusters in colorectal cancer.

    KAUST Repository

    Cima, Igor

    2016-06-29

    Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

  19. Uptake of gold nanoparticles in primary human endothelial cells

    DEFF Research Database (Denmark)

    Klingberg, Henrik; Oddershede, Lene B.; Löschner, Katrin

    2015-01-01

    Gold nanoparticles (AuNPs) are relevant in nanomedicine for drug delivery in the vascular system, where endothelial cells are the first point of contact. We investigated the uptake of 80 nm AuNPs in primary human umbilical vein endothelial cells (HUVECs) by flow cytometry, 3D confocal microscopy....... Uptake of AuNPs in HUVECs occurred mainly by clathrin-mediated endocytosis and trafficking to membrane enclosures in the form of single particles and agglomerates of 2–3 particles....

  20. Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture

    Energy Technology Data Exchange (ETDEWEB)

    Conceição, R.A. [Departamento de Genética, Evolução e Bioagentes, Universidade Estadual de Campinas, Campinas, SP (Brazil); Ludovico, M.S. [Departamento de Microbiologia, Universidade Estadual de Londrina, Londrina, PR (Brazil); Andrade, C.G.T.J. [Departamento de Biologia Geral, Universidade Estadual de Londrina, Londrina, PR (Brazil); Yano, T. [Departamento de Genética, Evolução e Bioagentes, Universidade Estadual de Campinas, Campinas, SP (Brazil)

    2012-04-13

    The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 × 10{sup 7} CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.

  1. Expansion and cryopreservation of porcine and human corneal endothelial cells.

    Science.gov (United States)

    Marquez-Curtis, Leah A; McGann, Locksley E; Elliott, Janet A W

    2017-08-01

    Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me 2 SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me 2 SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Endothelial cell confluence regulates Weibel-Palade body formation.

    Science.gov (United States)

    Howell, Gareth J; Herbert, Shane P; Smith, Jennifer M; Mittar, Shweta; Ewan, Lorna C; Mohammed, Mudassir; Hunter, Alison R; Simpson, Nigel; Turner, Anthony J; Zachary, Ian; Walker, John H; Ponnambalam, Sreenivasan

    2004-01-01

    Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (VWF) are characteristic of the mammalian endothelium. We hypothesized that vascular-specific antigens such as VWF are linked to endothelial identity and proliferation in vitro. To test this idea, the cellular accumulation of VWF in WPBs was monitored as a function of cell proliferation, confluence and passage number in human umbilical vein endothelial cells (HUVECs). We found that as passage number increased the percentage of cells containing VWF in WPBs was reduced significantly, whilst the protein was still detected within the secretory pathway at all times. However, the endothelial-specific marker protein, PECAM-1, is present on all cells even when WPBs are absent, indicating partial maintenance of endothelial identity. Biochemical studies show that a significant pool of immature pro-VWF can be detected in sub-confluent HUVECs; however, a larger pool of mature, processed VWF is detected in confluent cells. Newly synthesized VWF must thus be differentially sorted and packaged along the secretory pathway in semi-confluent versus confluent endothelial cells. Our studies thus show that WPB formation is linked to the formation of a confluent endothelial monolayer.

  3. VEGF masks BNIP3-mediated apoptosis of hypoxic endothelial cells.

    Science.gov (United States)

    Jurasz, Paul; Yurkova, Natasha; Kirshenbaum, Lorrie; Stewart, Duncan J

    2011-05-01

    Hypoxia results in the apoptotic death of myocytes, neurons, and epithelial cells, through the actions of Bcl-2 and Nineteen kilodalton Interacting Protein-3 (BNIP3). On the contrary, endothelial cells are especially adept at surviving conditions of oxygen deprivation via up-regulation of vascular endothelial growth factor (VEGF) the most potent endothelial survival factor. Both VEGF and BNIP3 expression are transcriptionally regulated by hypoxia inducible factor and may antagonize each other's affects in endothelial cells (ECs). Since factors that promote and inhibit apoptosis may be expressed at the same time in endothelial cells under hypoxic conditions, we decided to investigate whether VEGF and BNIP3 have opposing actions in endothelial cells. Human microvascular endothelial cells were exposed to hypoxic conditions in a Billups-Rothenburg chamber. Under hypoxic conditions BNIP3 expression by endothelial cells increased as measured by real-time PCR and immunoblot. After 48 h of hypoxia, EC apoptosis was assessed by flow cytometry and was lower than in corresponding normoxia serum starved controls. The increase in EC survival under hypoxic conditions corresponded with an increase in the expression of VEGF. Under normoxic conditions adenoviral BNIP3 over-expression promoted apoptosis of ECs; however, recombinant VEGF (100 pg/ml) antagonized the BNIP3 apoptosis promoting affects. SiRNA knockdown of VEGF expression by hypoxic ECs resulted in increased apoptosis with a concomitant increase in BNIP3 expression. SiRNA knockdown of BNIP3 expression by hypoxic ECs reduced the increase in EC apoptosis as a result of VEGF knockdown. We conclude that under hypoxic conditions VEGF counteracts and masks the apoptosis promoting affects of BNIP3.

  4. Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability

    International Nuclear Information System (INIS)

    Gabrys, Dorota; Greco, Olga; Patel, Gaurang; Prise, Kevin M.; Tozer, Gillian M.; Kanthou, Chryso

    2007-01-01

    Purpose: To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation. Methods and Materials: Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays. Results: Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability. Conclusion: This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain

  5. Circulating endothelial progenitor cells in obese children and adolescents.

    Science.gov (United States)

    Pires, António; Martins, Paula; Paiva, Artur; Pereira, Ana Margarida; Marques, Margarida; Castela, Eduardo; Sena, Cristina; Seiça, Raquel

    2015-01-01

    This study aimed to investigate the relationship between circulating endothelial progenitor cell count and endothelial activation in a pediatric population with obesity. Observational and transversal study, including 120 children and adolescents with primary obesity of both sexes, aged 6-17 years, who were recruited at this Cardiovascular Risk Clinic. The control group was made up of 41 children and adolescents with normal body mass index. The variables analyzed were: age, gender, body mass index, systolic and diastolic blood pressure, high-sensitivity C-reactive protein, lipid profile, leptin, adiponectin, homeostasis model assessment-insulin resistance, monocyte chemoattractant protein-1, E-selectin, asymmetric dimethylarginine and circulating progenitor endothelial cell count. Insulin resistance was correlated to asymmetric dimethylarginine (ρ=0.340; p=0.003), which was directly, but weakly correlated to E-selectin (ρ=0.252; p=0.046). High sensitivity C-reactive protein was not found to be correlated to markers of endothelial activation. Systolic blood pressure was directly correlated to body mass index (ρ=0.471; p<0.001) and the homeostasis model assessment-insulin resistance (ρ=0.230; p=0.012), and inversely correlated to adiponectin (ρ=-0.331; p<0.001) and high-density lipoprotein cholesterol (ρ=-0.319; p<0.001). Circulating endothelial progenitor cell count was directly, but weakly correlated, to body mass index (r=0.211; p=0.016), leptin (ρ=0.245; p=0.006), triglyceride levels (r=0.241; p=0.031), and E-selectin (ρ=0.297; p=0.004). Circulating endothelial progenitor cell count is elevated in obese children and adolescents with evidence of endothelial activation, suggesting that, during infancy, endothelial repairing mechanisms are present in the context of endothelial activation. Copyright © 2015 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  6. Varicose veins as a source of adult human endothelial cells.

    Science.gov (United States)

    Ryan, U S; White, L A

    1985-01-01

    Endothelial cells can be harvested from segments of adult human saphenous vein in a varicose condition removed from patients having single or bilateral vein ligation and stripping. The cells are harvested by scraping with a scalpel, seeded on to gelatin coated or Primaria flasks and are passaged by removal with a rubber policeman. The cells cultured in this manner are maintained in a growth medium that is not supplemented with growth factors. The cells grow with a cobblestone monolayer morphology, possess angiotensin converting enzyme activity and react with antibodies to Factor VIII antigen. The cells fluoresce brightly after reaction with monoclonal antibodies specific for human endothelial cells. Thus, stripped varicose vein segments provide a readily available source of endothelial cells.

  7. Effect of sunitinib combined with ionizing radiation on endothelial cells

    International Nuclear Information System (INIS)

    Zhang Haiping; Jiao Xiaodong; Li Rui; Wang Jiejun; Takayama, Koichi; Su Bo

    2011-01-01

    The aims of present study were to evaluate the efficacy of combining sunitinib with ionizing radiation (IR) on endothelial cells in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) were exposed to IR with or without sunitinib pretreatment. Apoptosis assay and cell cycle distribution were analyzed by flow cytometry. Clonogenic survival assay at 3 Gy dose with or without sunitinib was performed. The activity of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway was detected by Western immunoblot. Lewis lung carcinoma mouse model was built to examine the effect of combination therapy on endothelial cells in vivo. Microvasculature changes were detected by immunohistochemistry using anti-CD31 antibody. Our results showed combination therapy of sunitinib and IR significantly increased apoptosis of endothelial cells and inhibited colony formation compared to sunitinib or radiotherapy alone. It also resulted in cell cycle redistribution (decreasing cells in S phase and increasing cells in G2/M phase). The activity of PI3K/Akt signal pathway was inhibited, which could be the potential mechanisms that account for the enhanced radiation response induced by sunitinib. In vivo analysis showed that combination therapy significantly decreased microvasculature formation. The results demonstrated that combination therapy of sunitinib and IR has the potential to increase the cytotoxic effects on endothelial cells. (author)

  8. Endothelialization of Artificial Surfaces: Does Surface Tension Determine in vitro Growth of Human Saphenous Vein Endothelial Cells?

    OpenAIRE

    Fasol, Roland; Zilla, Peter; Deutsch, Manfred; Fischlein, Teddy; Kadletz, Margit; Griesmacher, Andrea; Müller, Mathias M.

    1987-01-01

    To evaluate the possibility of providing, in vitro, an endothelial lining for artificial hearts, we cultivated adult autologous endothelial cells on two polyurethane and two silicone rubber surfaces. Over the ensuing 11-day period, we investigated the resulting cell proliferation and morphology by means of scanning electron and light microscopy. On the silicone rubber surfaces, seeding of 200,000 human saphenous vein endothelial cells per cm2 produced an ideal cobblestone monolayer within a s...

  9. Corneal endothelial cell density and morphology in healthy Turkish eyes.

    Science.gov (United States)

    Arıcı, Ceyhun; Arslan, Osman Sevki; Dikkaya, Funda

    2014-01-01

    Purpose. To describe the normative values of corneal endothelial cell density, morphology, and central corneal thickness in healthy Turkish eyes. Methods. Specular microscopy was performed in 252 eyes of 126 healthy volunteers (M : F, 42 : 84). Parameters studied included mean endothelial cell density (MCD), mean cell area (MCA), coefficient of variation (CV) in cell size, percentage of hexagonal cells, and central corneal thickness (CCT). Results. The mean age of volunteers was 44.3 ± 13.5 (range, 20 to 70) years. There was a statistically significant decrease in MCD (P Filipino eyes and higher than that described in Indian, Thai, and Iranian eyes.

  10. Long-term outcome after resection of non-small cell lung cancer invading the thoracic inlet.

    Science.gov (United States)

    Collaud, Stéphane; Machuca, Tiago; Mercier, Olaf; Waddell, Thomas K; Yasufuku, Kazuhiro; Pierre, Andrew F; Darling, Gail E; Cypel, Marcelo; Rampersaud, Yoga R; Lewis, Stephen J; Shepherd, Frances A; Leighl, Natasha B; Cho, John B C; Bezjak, Andrea; Keshavjee, Shaf; de Perrot, Marc

    2014-09-01

    The aim of this study was to update our previous experience and describe long-term results after resection of non-small-cell lung cancer (NSCLC) invading the thoracic inlet. Patients from a single center undergoing resection of NSCLC invading the thoracic inlet were reviewed with data retrieved retrospectively from their charts. Sixty-five consecutive patients with a median age of 61 (32-76) years underwent resection of NSCLC invading the thoracic inlet from 1991 to 2011. Tumor location was divided into 5 anatomic zones from anterior to posterior. Fifty-two (80%) patients had induction therapy, mostly with 2 cycles of cisplatin-etoposide and 45 Gy of concurrent irradiation. All patients underwent at least first rib resection. Lobectomy was performed in 60 patients (92%). Twenty-four patients (37%) had vertebral resection. Arterial resections were performed in 7 patients (11%). Postoperative morbidity and mortality were 46% and 6%, respectively. Pathologic response to induction was complete (pCR) (n = 19) or nearly complete (pNR) (n = 12) in 31 patients (48%). Adjuvant treatment was administered in 14 (25%) patients. After a median follow-up of 20 (0-193) months, 34 patients are alive without recurrence. The overall 5-year survival reached 69%. Univariate analysis identified site of tumor within the thoracic inlet (p = 0.050), response to induction (p = 0.004), and presence of adjuvant treatment (p = 0.028) as survival predictors. Survival after resection of NSCLC invading the thoracic inlet in highly selected patients reached 69% at 5 years. Tumor location within the thoracic inlet, pathologic response to induction therapy, and adjuvant treatments were significant survival predictors. Copyright © 2014 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

  11. Endothelial cells present antigens in vivo

    Directory of Open Access Journals (Sweden)

    Tellides George

    2004-03-01

    Full Text Available Abstract Background Immune recognition of vascular endothelial cells (EC has been implicated in allograft rejection, protection against pathogens, and lymphocyte recruitment. However, EC pervade nearly all tissues and predominate in none, complicating any direct test of immune recognition. Here, we examined antigen presentation by EC in vivo by testing immune responses against E. coli β-galactosidase (β-gal in two lines of transgenic mice that express β-gal exclusively in their EC. TIE2-lacZ mice express β-gal in all EC and VWF-lacZ mice express β-gal in heart and brain microvascular EC. Results Transgenic and congenic wild type FVB mice immunized with β-gal expression vector DNA or β-gal protein generated high titer, high affinity antisera containing comparable levels of antigen-specific IgG1 and IgG2a isotypes, suggesting equivalent activation of T helper cell subsets. The immunized transgenic mice remained healthy, their EC continued to express β-gal, and their blood vessels showed no histological abnormalities. In response to β-gal in vitro, CD4+ and CD8+ T cells from immunized transgenic and FVB mice proliferated, expressed CD25, and secreted IFN-γ. Infection with recombinant vaccinia virus encoding β-gal raised equivalent responses in transgenic and FVB mice. Hearts transplanted from transgenic mice into FVB mice continued to beat and the graft EC continued to express β-gal. These results suggested immunological ignorance of the transgene encoded EC protein. However, skin transplanted from TIE2-lacZ onto FVB mice lost β-gal+ EC and the hosts developed β-gal-specific antisera, demonstrating activation of host immune effector mechanisms. In contrast, skin grafted from TIE2-lacZ onto VWF-lacZ mice retained β-gal+ EC and no antisera developed, suggesting a tolerant host immune system. Conclusion Resting, β-gal+ EC in transgenic mice tolerize specific lymphocytes that would otherwise respond against β-gal expressed by EC within

  12. The influence of biomaterials on endothelial cell thrombogenicity

    Science.gov (United States)

    McGuigan, Alison P.; Sefton, Michael V.

    2007-01-01

    Driven by tissue engineering and regenerative medicine, endothelial cells are being used in combination with biomaterials in a number of applications for the purpose of improving blood compatibility and host integration. Endothelialized vascular grafts are beginning to be used clinically with some success in some centers, while endothelial seeding is being explored as a means of creating a vasculature within engineered tissues. The underlying assumption of this strategy is that when cultured on artificial biomaterials, a confluent layer of endothelial cells maintain their non-thrombogenic phenotype. In this review the existing knowledge base of endothelial cell thrombogenicity cultured on a number of different biomaterials is summarized. The importance of selecting appropriate endpoint measures that are most reflective of overall surface thrombogenicity is the focus of this review. Endothelial cells inhibit thrombosis through three interconnected regulatory systems (1) the coagulation cascade (2) the cellular components of the blood such as leukocytes and platelets and (3) the complement cascade, and also through effects on fibrinolysis and vascular tone, the latter which influences blood flow. Thus, in order to demonstrate the thromobgenic benefit of seeding a biomaterial with EC, the conditions under which EC surfaces are more likely to exhibit lower thrombogenicity than unseeded biomaterial surfaces need to be consistent with the experimental context. The endpoints selected should be appropriate for the dominant thrombotic process that occurs under the given experimental conditions. PMID:17316788

  13. An endothelial cell genetic screen identifies the GTPase Rem2 as a suppressor of p19ARF expression that promotes endothelial cell proliferation and angiogenesis

    NARCIS (Netherlands)

    Bierings, Ruben; Beato, Miguel; Edel, Michael J.

    2008-01-01

    Angiogenesis requires an increase in endothelial cell proliferation to support an increase in mass of blood vessels. We designed an in vitro endothelial cell model to functionally screen for genes that regulate endothelial cell proliferation. A gain of function screen for genes that bypass p53

  14. Endothelial glycocalyx on brain endothelial cells is lost in experimental cerebral malaria

    DEFF Research Database (Denmark)

    Hempel, Casper; Hyttel, Poul; Kurtzhals, Jørgen Al

    2014-01-01

    We hypothesized that the glycocalyx, which is important for endothelial integrity, is lost in severe malaria. C57BL/6 mice were infected with Plasmodium berghei ANKA, resulting in cerebral malaria, or P. chabaudi AS, resulting in uncomplicated malaria. We visualized the glycocalyx with transmission...... electron microscopy and measured circulating glycosaminoglycans by dot blot and ELISA. The glycocalyx was degraded in brain vasculature in cerebral and to a lesser degree uncomplicated malaria. It was affected on both intact and apoptotic endothelial cells. Circulating glycosaminoglycan levels suggested...

  15. Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Zhong-Xiu-Zi [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China); Huang, Da-Yong [Department of Oncology, The Second Clinical Hospital, Harbin Medical University, Harbin (China); Li, Hai-Xia; Zhang, Li-Na; Lv, Yan-Hong; Cui, Hai-Dong [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China); Zheng, Jin-Hua, E-mail: jhzhenghrbmu@yahoo.cn [Department of Anatomy, Basic Medical Science College, Harbin Medical University, Harbin (China)

    2010-09-10

    Research highlights: {yields} It has been shown that scutellarin exhibits a variety of pharmacological actions, including anti-oxidative, anti-inflammatory, vasodilator as well as cardiovascular and cerebrovascular ischemia protective effects, indicating beneficial vascular effects of scutellarin. Therefore, it is speculated that scutellarin may be able to stimulate angiogenesis, which could be beneficial in the treatment of ischemic disease, wound healing and tissue regeneration. {yields} The purpose of the present study was to elucidate the direct angiogenic actions of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. {yields} Our results showed that scutellarin to directly induce in vitro angiogenesis, which is closely correlated with upregulated MMP-2 expression, suggesting a potential for increasing angiogenesis. -- Abstract: Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin's effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin's angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly

  16. Development of Cell Analysis Software for Cultivated Corneal Endothelial Cells.

    Science.gov (United States)

    Okumura, Naoki; Ishida, Naoya; Kakutani, Kazuya; Hongo, Akane; Hiwa, Satoru; Hiroyasu, Tomoyuki; Koizumi, Noriko

    2017-11-01

    To develop analysis software for cultured human corneal endothelial cells (HCECs). Software was designed to recognize cell borders and to provide parameters such as cell density, coefficient of variation, and polygonality of cultured HCECs based on phase contrast images. Cultured HCECs with high or low cell density were incubated with Ca-free and Mg-free phosphate-buffered saline for 10 minutes to reveal the cell borders and were then analyzed with software (n = 50). Phase contrast images showed that cell borders were not distinctly outlined, but these borders became more distinctly outlined after phosphate-buffered saline treatment and were recognized by cell analysis software. The cell density value provided by software was similar to that obtained using manual cell counting by an experienced researcher. Morphometric parameters, such as the coefficient of variation and polygonality, were also produced by software, and these values were significantly correlated with cell density (Pearson correlation coefficients -0.62 and 0.63, respectively). The software described here provides morphometric information from phase contrast images, and it enables subjective and noninvasive quality assessment for tissue engineering therapy of the corneal endothelium.

  17. Characterization and comparison of embryonic stem cell-derived KDR+ cells with endothelial cells.

    Science.gov (United States)

    Sun, Xuan; Cheng, Lamei; Duan, Huaxin; Lin, Ge; Lu, Guangxiu

    2012-09-01

    Growing interest in utilizing endothelial cells (ECs) for therapeutic purposes has led to the exploration of human embryonic stem cells (hESCs) as a potential source for endothelial progenitors. In this study, ECs were induced from hESC lines and their biological characteristics were analyzed and compared with both cord blood endothelial progenitor cells (CBEPCs) and human umbilical vein endothelial cells (HUVECs) in vitro. The results showed that isolated embryonic KDR+ cells (EC-KDR+) display characteristics that were similar to CBEPCs and HUVECs. EC-KDR+, CBEPCs and HUVECs all expressed CD31 and CD144, incorporated DiI-Ac-LDL, bound UEA1 lectin, and were able to form tube-like structures on Matrigel. Compared with CBEPCs and HUVECs, the expression level of endothelial progenitor cell markers such as CD133 and KDR in EC-KDR+ was significantly higher, while the mature endothelial marker vWF was lowly expressed in EC-KDR+. In summary, the study showed that EC-KDR+ are primitive endothelial-like progenitors and might be a potential source for therapeutic vascular regeneration and tissue engineering. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Characterization of vascular endothelial progenitor cells from chicken bone marrow

    Directory of Open Access Journals (Sweden)

    Bai Chunyu

    2012-05-01

    Full Text Available Abstract Background Endothelial progenitor cells (EPC are a type of stem cell used in the treatment of atherosclerosis, vascular injury and regeneration. At present, most of the EPCs studied are from human and mouse, whereas the study of poultry-derived EPCs has rarely been reported. In the present study, chicken bone marrow-derived EPCs were isolated and studied at the cellular level using immunofluorescence and RT-PCR. Results We found that the majority of chicken EPCs were spindle shaped. The growth-curves of chicken EPCs at passages (P 1, -5 and -9 were typically “S”-shaped. The viability of chicken EPCs, before and after cryopreservation was 92.2% and 81.1%, respectively. Thus, cryopreservation had no obvious effects on the viability of chicken EPCs. Dil-ac-LDL and FITC-UAE-1 uptake assays and immunofluorescent detection of the cell surface markers CD34, CD133, VEGFR-2 confirmed that the cells obtained in vitro were EPCs. Observation of endothelial-specific Weibel-Palade bodies using transmission electron microscopy further confirmed that the cells were of endothelial lineage. In addition, chicken EPCs differentiated into endothelial cells and smooth muscle cells upon induction with VEGF and PDGF-BB, respectively, suggesting that the chicken EPCs retained multipotency in vitro. Conclusions These results suggest that chicken EPCs not only have strong self-renewal capacity, but also the potential to differentiate into endothelial and smooth muscle cells. This research provides theoretical basis and experimental evidence for potential therapeutic application of endothelial progenitor cells in the treatment of atherosclerosis, vascular injury and diabetic complications.

  19. Rapid flow-induced responses in endothelial cells

    Science.gov (United States)

    Stamatas, G. N.; McIntire, L. V.

    2001-01-01

    Endothelial cells alter their morphology, growth rate, and metabolism in response to fluid shear stress. To study rapid flow-induced responses in the 3D endothelial cell morphology and calcium distribution, coupled fluorescence microscopy with optical sectioning, digital imaging, and numerical deconvolution techniques have been utilized. Results demonstrate that within the first minutes of flow application nuclear calcium is increasing. In the same time frame whole cell height and nuclear height are reduced by about 1 microm. Whole cell height changes may facilitate reduction of shear stress gradients on the luminal surface, whereas nuclear structural changes may be important for modulating endothelial growth rate and metabolism. To study the role of the cytoskeleton in these responses, endothelial cells have been treated with specific disrupters (acrylamide, cytochalasin D, and colchicine) of each of the cytoskeleton elements (intermediate filaments, microfilaments, and microtubules, respectively). None of these compounds had any effect on the shear-induced calcium response. Cytochalasin D and acrylamide did not affect the shear-induced nuclear morphology changes. Colchicine, however, completely abrogated the response, indicating that microtubules may be implicated in force transmission from the plasma membrane to the nucleus. A pedagogical model based on tensegrity theory principles is presented that is consistent with the results on the 3D endothelial morphology.

  20. Nanofiber density determines endothelial cell behavior on hydrogel matrix

    Energy Technology Data Exchange (ETDEWEB)

    Berti, Fernanda V., E-mail: fernanda@intelab.ufsc.br [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Rambo, Carlos R. [Department of Electrical Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Dias, Paulo F. [Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Porto, Luismar M. [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil)

    2013-12-01

    When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography.

  1. Nanofiber density determines endothelial cell behavior on hydrogel matrix

    International Nuclear Information System (INIS)

    Berti, Fernanda V.; Rambo, Carlos R.; Dias, Paulo F.; Porto, Luismar M.

    2013-01-01

    When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography

  2. 5-Hydroxytryptamine 4 Receptor in the Endothelial Cells

    DEFF Research Database (Denmark)

    Profirovic, Jasmina; Vardya, Irina; Voyno-Yasenetskaya, Tatyana

    2006-01-01

    gap formation in HUVECs. We are currently investigating the mechanism underlying 5-HT4 receptor-induced actin cytoskeleton changes in the endothelial cells. These data suggest that by activating 5-HT4 receptor, serotonin could be involved in regulation of actin cytoskeleton dynamics in the endothelial......39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events...... in the central nervous system (CNS). We have recently demonstrated that 5-HT4 receptor couples to G13 protein to induce RhoA-dependent gene transcription, neurite retraction, and neuronal cell rounding (Ponimaskin et al, 2002). Although multiple studies were focused on the function of the 5-HT4 receptor...

  3. The ultrastructural localization of von Willebrand factor in endothelial cells.

    Science.gov (United States)

    Warhol, M J; Sweet, J M

    1984-11-01

    Factor VIII-related antigen was localized ultrastructurally in a variety of human tissues (smooth muscle, skeletal muscle, breast, capillary hemangioma) with the use of a low-temperature embedding protein A-gold technique with both polyclonal and monoclonal antisera directed against von Willebrand factor. All endothelial cells examined localized the anti-von Willebrand factor to Weibel-Palade bodies. Cisternae of the endoplasmic reticulum, and cytoplasmic vacuoles were also labeled. These results establish the distribution of factor VIII-related antigen at the subcellular level. The observed distribution suggests that the endothelial cells synthesize von Willebrand factor, store it in Weibel-Palade bodies, and release it by exocytosis. These observations provide in vivo confirmation for previous biochemical and immunocytochemical data obtained from studies on cultured endothelial cells.

  4. Endothelial cell division in angiogenic sprouts of differing cellular architecture

    OpenAIRE

    Aydogan, Vahap; Lenard, Anna; Denes, Alexandru Stefan; Sauteur, Loic; Belting, Heinz-Georg; Affolter, Markus

    2015-01-01

    ABSTRACT The vasculature of the zebrafish trunk is composed of tubes with different cellular architectures. Unicellular tubes form their lumen through membrane invagination and transcellular cell hollowing, whereas multicellular vessels become lumenized through a chord hollowing process. Endothelial cell proliferation is essential for the subsequent growth and maturation of the blood vessels. However, how cell division, lumen formation and cell rearrangement are coordinated during angiogenic ...

  5. High glucose induced endothelial to mesenchymal transition in human umbilical vein endothelial cell.

    Science.gov (United States)

    Yu, Chun-Hong; Suriguga; Gong, Meng; Liu, Wen-Juan; Cui, Ning-Xuan; Wang, Ying; Du, Xin; Yi, Zong-Chun

    2017-06-01

    Studies have shown that endothelial-to-mesenchymal transition (EndMT) could contribute to the progression of diabetic nephropathy, diabetic renal fibrosis, and cardiac fibrosis. The aim of this study was to investigate the influence of high glucose and related mechanism of MAPK inhibitor or specific antioxidant on the EndMT. In vitro human umbilical vein endothelial cells (HUVEC) were cultured with 11mM, 30mM, 60mM and 120mM glucose for 0, 24, 48, 72 and 168h. Endothelial cell morphology was observed with microscope, and RT-PCR was used to detect mRNA expression of endothelial markers VE-cadherin and CD31, mesenchymal markers α-SMA and collagen I, and transforming growth factor TGF-β1. Immunofluorescence staining was performed to detect the expression of CD31 and α-SMA. The concentration of TGF-β1 in the supernatant was detected by ELISA. ERK1/2 phosphorylation level was detected by Western blot analysis. High glucose induced EndMT and increased the TGF-β1 level in HUVEC cells. Cells in high glucose for 7 days showed a significant decrease in mRNA expression of CD31 and VE-cadherin, and a significant increase in that of α-SMA and collagen I, while lost CD31 staining and acquired α-SMA staining. ERK signaling pathway blocker PD98059 significantly attenuated the high glucose-induced increase in the ERK1/2 phosphorylation level. PD98059 and NAC both inhibited high glucose-induced TGF-β1 expression and attenuated EndMT marker protein synthesis. High glucose could induce HUVEC cells to undergo EndMT. NAC and ERK signaling pathway may play important role in the regulation of the TGF-β1 biosynthesis during high glucose-induced EndMT. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases.

    Science.gov (United States)

    Angelot, Fanny; Seillès, Estelle; Biichlé, Sabeha; Berda, Yael; Gaugler, Béatrice; Plumas, Joel; Chaperot, Laurence; Dignat-George, Françoise; Tiberghien, Pierre; Saas, Philippe; Garnache-Ottou, Francine

    2009-11-01

    Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions. Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-alpha) and also induced allogeneic naive CD4(+) T cells to proliferate and to produce type 1 cytokines such as interferon-gamma and tumor necrosis factor-alpha. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation. Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and

  7. Research advances on tissue-engineered corneal endothelial cells transplantation

    Directory of Open Access Journals (Sweden)

    Si-Jie Zhao

    2015-02-01

    Full Text Available Due to the serious shortage of donor cornea materials and the donor limit, clinical popularization of penetrating keratoplasty is severely restricted. It is a hot spot of current research that applying tissue engineering in vitro to culture corneal endothelial cells(CECwith high density, regular hexagonal shape and healthy endothelial function. In this article, we reviewed the latest progress in the study of source of CEC seeder cells, selection of cultivating carries, type of CEC transplantation and immune mechanism that summarized the current research problems and made a prospect to the future.

  8. Mechanotransduction in Endothelial Cells Studied with Fluorescence Imaging

    International Nuclear Information System (INIS)

    Chien Shu

    2011-01-01

    Mechanotransduction involves the conversion of mechanical stimuli to intracellular signaling to modulate gene and protein expressions and hence cellular functions in endothelial cells, thus playing importance roles in the regulation of homeostasis in health and disease. The aim of this paper is to investigate the dynamics of mechanotransduction in endothelial cells by the use of fluorescent resonance energy transfer (FRET) to study the temporal and spatial activation of Src kinase and focal adhesion kinase, both of which play critical roles in many cellular processes. The results have contributed to the elucidation of the roles of these two important signaling molecules and their interactions in mediating mechanotransduction.

  9. Karyotype changes in cultured human corneal endothelial cells

    OpenAIRE

    Miyai, Takashi; Maruyama, Yoko; Osakabe, Yasuhiro; Nejima, Ryohei; Miyata, Kazunori; Amano, Shiro

    2008-01-01

    Purpose To examine karyotype changes in cultured human corneal endothelial cells (HCECs). Methods HCECs with Descemet’s membrane were removed from 20 donors of various ages (range, 2–77 years; average, 43.7±26.4 years) and cultured on dishes coated with extracellular matrix produced by bovine corneal endothelial cells (BCECs). Karyotype changes were examined by G-band karyotyping of HCECs at the third passage from 12 donors and the fifth passage from 16 donors. The number of chromosomes was a...

  10. Decellularized extracellular matrix of human umbilical vein endothelial cells promotes endothelial differentiation of stem cells from exfoliated deciduous teeth.

    Science.gov (United States)

    Gong, Ting; Heng, Boon Chin; Xu, Jianguang; Zhu, Shaoyue; Yuan, Changyong; Lo, Edward Chin Man; Zhang, Chengfei

    2017-04-01

    Dental stem cells can serve as a potential source of functional endothelial cells for tissue engineering applications, but the endothelial-lineage differentiation efficiency is rather low even with growth factors and mechanical stimuli, which greatly limits their clinical applications. This is partly due to the deficiency of standard two-dimensional (2-D) culture systems, which is unable to recapitulate the three-dimensional (3-D) in vivo milieu that is rich in extracellular matrix. Hence, we extracted decellularized extracellular matrix from human umbilical vein endothelial cells (HUVECs-DECM) to provide a bioactive substratum conducive to the endothelial differentiation of dental stem cells. Compared to cells plated on tissue culture polystyrene (TCP), stem cells from exfoliated deciduous teeth (SHED) cultured on the HUVECs-DECM demonstrated more regular arrangement and elongated morphology. HUVECs-DECM significantly enhanced the rapid adhesion and proliferation rates of SHED, as demonstrated by WST-8 assay and immunocytochemistry indicating higher expression levels of vinculin by newly adherent SHED on HUVECs-DECM versus TCP. In addition, there was twofold to fivefold higher mRNA expression levels of endothelial-specific markers CD31 and VEGFR-2 in SHED after seven days of culture on DECM versus TCP. Functional testing with in vitro matrigel angiogenesis assay identified more capillary-like structure formation with significantly higher tubule length in SHED induced by DECM versus TCP. Hence, the results of this study provide a better understanding of the unique characteristics of cell-specific ECM and demonstrated the potential use of HUVECs-DECM as a culture substratum conducive for stimulating the endothelial differentiation of SHED for therapeutic angiogenic applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1083-1093, 2017. © 2017 Wiley Periodicals, Inc.

  11. Effects of irradiated biodegradable polymer in endothelial cell monolayer formation

    Energy Technology Data Exchange (ETDEWEB)

    Arbeitman, Claudia R.; Grosso, Mariela F. del [CONICET – Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina); Gerencia de Investigación y Aplicaciones, TANDAR-CNEA (Argentina); Behar, Moni [Instituto de Física, UFRGS, Porto Alegre, RS (Brazil); García Bermúdez, Gerardo, E-mail: ggb@tandar.cnea.gov.ar [CONICET – Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina); Gerencia de Investigación y Aplicaciones, TANDAR-CNEA (Argentina); Escuela de Ciencia y Tecnología, UNSAM (Argentina)

    2013-11-01

    In this work we study cell adhesion, proliferation and cell morphology of endothelial cell cultured on poly-L-lactide acid (PLLA) modified by heavy ion irradiation. Thin films of PLLA samples were irradiated with sulfur (S) at energies of 75 MeV and gold (Au) at 18 MeV ion-beams. Ion beams were provided by the Tandar (Buenos Aires, Argentina) and Tandetron (Porto Alegre, Brazil) accelerators, respectively. The growth of a monolayer of bovine aortic endothelial cells (BAEC) onto unirradiated and irradiated surfaces has been studied by in vitro techniques in static culture. Cell viability and proliferation increased on modified substrates. But the results on unirradiated samples, indicate cell death (necrosis/apoptosis) with the consequent decrease in proliferation. We analyzed the correlation between irradiation parameters and cell metabolism and morphology.

  12. Detection and Quantification of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases in Primary Human Endothelial Cells.

    Science.gov (United States)

    Fearnley, Gareth W; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

    2015-01-01

    Proteins differ widely in their pattern of expression depending on organism, tissue, and regulation in response to changing conditions. In the mammalian vasculature, the endothelium responds to vascular endothelial growth factors (VEGFs) via membrane-bound receptor tyrosine kinases (VEGFRs) to modulate many aspects of vascular physiology including vasculogenesis, angiogenesis, and blood pressure. Studies on VEGFR biology are thus dependent on detecting expression levels in different cell types and evaluating how changes in protein levels correlate with changing conditions including circulating VEGF levels. Here, we present a robust immunoblot-based protocol for detecting and quantifying VEGFRs in human endothelial cells. Using internal and external standards, we can rapidly evaluate receptor copy number and assess how this is altered in response to the cellular environment.

  13. Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation

    Science.gov (United States)

    Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.

    1983-11-01

    Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

  14. Caveolae, caveolins, cavins and endothelial cell function: new insights

    Directory of Open Access Journals (Sweden)

    Grzegorz eSowa

    2012-01-01

    Full Text Available Caveolae are cholesterol and glycosphingolipid-rich flask-shaped invaginations of the plasma membrane which are particularly abundant in vascular endothelium and present in all other cell types of the cardiovascular system, including vascular smooth muscle cells, macrophages, cardiac myocytes, and fibroblasts. Caveolins and the more recently discovered cavins are the major protein components of caveolae. When caveolae were discovered, their functional role was believed to be limited to transport across the endothelial cell barrier. Since then, however, a large body of evidence has accumulated, suggesting that these microdomains are very important in regulating many other important endothelial cell functions, mostly due to their ability to concentrate and compartmentalize various signaling molecules. Over the course of several years, multiple studies involving knockout mouse and small interfering RNA approaches have considerably enhanced our understanding of the role of caveolae and caveolin-1 in regulating many cardiovascular functions. New findings have been reported implicating other caveolar protein components in endothelial cell signaling and function, such as the understudied caveolin-2 and newly discovered cavin proteins. The aim of this review is to focus primarily on molecular and cellular aspects of the role of caveolae, caveolins, and cavins in endothelial cell signaling and function. In addition, where appropriate, the possible implications for the cardiovascular and pulmonary physiology and pathophysiology will be discussed.

  15. Tumor-Endothelium Cross Talk Blocks Recruitment of Neutrophils to Endothelial Cells: A Novel Mechanism of Endothelial Cell Anergy

    Directory of Open Access Journals (Sweden)

    Roman A. Blaheta

    2009-10-01

    Full Text Available Tumor cells have evolved effective strategies to escape the host immune response. The objective of this study was to determine whether tumor cells can condition endothelial cells in a specific manner to prevent subsequent adhesion of polymorphonuclear neutrophils (PMNs and/or peripheral blood lymphocytes (PBLs. Human umbilical vein endothelial cells (HUVECs and UKF-NB-4 neuroblastoma tumor cells were established in coculture on opposite sides of porous transwell filters. After 24 hours with and without HUVEC conditioning, PMNs or PBLs were added to the HUVEC monolayer. Adhesion to conditioned HUVEC versus adhesion to nonconditioned HUVEC was compared. Effects on endothelial CD44v4, CD44v5, CD44v7, intercellular adhesion molecule 1 (ICAM-1, E-selectin, and vascular cell adhesion molecule 1 (VCAM-1 adhesion receptor expression were analyzed by flow cytometry, intracellular signaling proteins of the mitogen-activated protein kinase pathway and protein kinase C (PKC subtypes quantified by Western blot analysis. Endothelial conditioning led to a distinct reduction in PMN but not in PBL adhesion to HUVEC. CD44 was significantly reduced, whereas ICAM-1, E-selectin, and VCAM-1 were not altered during HUVEC conditioning. Antibody blockade against CD44v4, CD44v5, and CD44v7 inhibited PMN but not PBL binding. The observed effects were caused by direct tumor cell-HUVEC contact because addition of isolated tumor cell membrane fragments but not of soluble cell culture supernatant to HUVEC induced the CD44 receptor loss. PKCα activity was strongly enhanced in conditioned HUVEC. Blocking PKC prevented the reduction in PMN binding, indicating that this protein is involved in PMN adhesion regulation. A novel tumor escape strategy is presented here. Cell contact-dependent adhesion of tumor cells to the vascular wall promotes down-regulation of endothelial CD44 receptor expression, impairing an effective neutrophil attack.

  16. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

    Directory of Open Access Journals (Sweden)

    Monica Salamone

    Full Text Available In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4 and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs or Serine Integral Membrane Peptidases (SIMPs caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

  17. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

    Science.gov (United States)

    Salamone, Monica; Carfì Pavia, Francesco; Ghersi, Giulio

    2016-01-01

    In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

  18. Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells.

    Science.gov (United States)

    Fearnley, Gareth W; Smith, Gina A; Odell, Adam F; Latham, Antony M; Wheatcroft, Stephen B; Harrison, Michael A; Tomlinson, Darren C; Ponnambalam, Sreenivasan

    2014-01-01

    The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs. Here, we describe a series of assays to evaluate the effects of VEGF-A-stimulated intracellular signaling from intracellular compartments such as the endosome-lysosome system. These assays include the initial isolation and characterization of primary human endothelial cells, performing reverse genetics for analyzing protein function; methods used to study receptor trafficking, signaling, and proteolysis; and assays used to measure changes in cell migration, proliferation, and tubulogenesis. Each of these assays has been exemplified with studies performed in our laboratories. In conclusion, we describe necessary techniques for studying the role of VEGF-A in endothelial cell function. © 2014 Elsevier Inc. All rights reserved.

  19. In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

    Science.gov (United States)

    Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

    2015-01-01

    Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue. © 2014 International Federation for Cell Biology.

  20. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  1. PMab-48 Recognizes Dog Podoplanin of Lymphatic Endothelial Cells.

    Science.gov (United States)

    Yamada, Shinji; Itai, Shunsuke; Kaneko, Mika K; Kato, Yukinari

    2018-02-01

    Podoplanin, a type I transmembrane glycoprotein, is a specific marker of lymphatic endothelial cells (LECs). Recently, we developed PMab-38, an anti-dog podoplanin monoclonal antibody that did not stain canine LECs. In this study, we newly developed PMab-48 against dog podoplanin. Immunohistochemical analysis revealed that PMab-48 reacts not only with canine squamous cell carcinoma cells but also with LECs of the normal colon. Therefore, PMab-48 may be useful in investigating the function of dog podoplanin in LECs.

  2. VUV modification promotes endothelial cell proliferation on PTFE vascular grafts

    Science.gov (United States)

    Cezeaux, J. L.; Romoser, C. E.; Benson, R. S.; Buck, C. K.; Sackman, J. E.

    1998-05-01

    Small diameter (⩽6 mm ID ) synthetic vascular grafts, used as lower-limb vessel replacements in patients without suitable autologous saphenous veins, have a failure rate of 53% after 4 yr. Graft failure is due to thrombosis and intimal hyperplasia, an increase in smooth muscle cells in the lumen of the vessel which leads to progressive closing and ultimate occlusion of the vessel. In an effort to increase patency rates of synthetic grafts, investigators have seeded vascular grafts with endothelial cells prior to implantation in an attempt to control both thrombosis and smooth muscle proliferation. This technique has been successful for the development of an endothelial monolayer in animal trials, but has met with limited success in humans. The hydrophobicity, low surface energy, and weak electrical charge of expanded polytetrafluoroethylene (ePTFE) provides conditions which are not optimal for endothelial cell attachment. The purpose of this study is to evaluate the effect of vacuum ultraviolet (VUV) modification of ePTFE on endothelial cell adhesion and proliferation. Pieces of ePTFE graft material were exposed to 10, 20 or 40 W VUV radiation for 10, 20 or 40 min using a UV excimer lamp. Prior to cell adhesion and proliferation experiments, the grafts pieces were autoclaved and cut into pledgets. Half of the pledgets were precoated with fibronectin ( 20 μg/ml). Cell adhesion was measured by seeding 3H-thymidine labeled human umbilical vein endothelial cells (HUVEC) onto the pledgets for 60 min. The pledgets were then washed and the remaining radioactivity assayed using scintillation counting. For the cell proliferation experiments, pledgets were seeded with unlabeled HUVEC which were allowed to adhere to the graft material for 18 h. The cells were then exposed to 3H-thymidine ( 1 μCi/ml) for approximately 48 h and then washed to remove any unincorporated 3H-thymidine. Incorporation of 3H-thymidine was measured using scintillation counting. Four replicate

  3. Cell fusion-independent differentiation of neural stem cells to the endothelial lineage.

    Science.gov (United States)

    Wurmser, Andrew E; Nakashima, Kinichi; Summers, Robert G; Toni, Nicolas; D'Amour, Kevin A; Lie, Dieter C; Gage, Fred H

    2004-07-15

    Somatic stem cells have been claimed to possess an unexpectedly broad differentiation potential (referred to here as plasticity) that could be induced by exposing stem cells to the extracellular developmental signals of other lineages in mixed-cell cultures. Recently, this and other experimental evidence supporting the existence of stem-cell plasticity have been refuted because stem cells have been shown to adopt the functional features of other lineages by means of cell-fusion-mediated acquisition of lineage-specific determinants (chromosomal DNA) rather than by signal-mediated differentiation. In this study we co-cultured mouse neural stem cells (NSCs), which are committed to become neurons and glial cells, with human endothelial cells, which form the lining of blood vessels. We show that in the presence of endothelial cells six per cent of the NSC population converted to cells that did not express neuronal or glial markers, but instead showed the stable expression of multiple endothelial markers and the capacity to form capillary networks. This was surprising because NSCs and endothelial cells are believed to develop from the ectoderm and mesoderm, respectively. Experiments in which endothelial cells were killed by fixation before co-culture with live NSCs (to prevent cell fusion) and karyotyping analyses, revealed that NSCs had differentiated into endothelial-like cells independently of cell fusion. We conclude that stem-cell plasticity is a true characteristic of NSCs and that the conversion of NSCs to unanticipated cell types can be accomplished without cell fusion.

  4. Blood on the tracks: hematopoietic stem cell-endothelial cell interactions in homing and engraftment.

    Science.gov (United States)

    Perlin, Julie R; Sporrij, Audrey; Zon, Leonard I

    2017-08-01

    Cells of the hematopoietic system undergo rapid turnover. Each day, humans require the production of about one hundred billion new blood cells for proper function. Hematopoietic stem cells (HSCs) are rare cells that reside in specialized niches and are required throughout life to produce specific progenitor cells that will replenish all blood lineages. There is, however, an incomplete understanding of the molecular and physical properties that regulate HSC migration, homing, engraftment, and maintenance in the niche. Endothelial cells (ECs) are intimately associated with HSCs throughout the life of the stem cell, from the specialized endothelial cells that give rise to HSCs, to the perivascular niche endothelial cells that regulate HSC homeostasis. Recent studies have dissected the unique molecular and physical properties of the endothelial cells in the HSC vascular niche and their role in HSC biology, which may be manipulated to enhance hematopoietic stem cell transplantation therapies.

  5. Corneal Endothelial Cell Density and Morphology in Healthy Turkish Eyes

    Directory of Open Access Journals (Sweden)

    Ceyhun Arıcı

    2014-01-01

    Full Text Available Purpose. To describe the normative values of corneal endothelial cell density, morphology, and central corneal thickness in healthy Turkish eyes. Methods. Specular microscopy was performed in 252 eyes of 126 healthy volunteers (M : F, 42 : 84. Parameters studied included mean endothelial cell density (MCD, mean cell area (MCA, coefficient of variation (CV in cell size, percentage of hexagonal cells, and central corneal thickness (CCT. Results. The mean age of volunteers was 44.3±13.5 (range, 20 to 70 years. There was a statistically significant decrease in MCD (P<0.001; correlation, −0.388 and percentage of hexagonal cells, (P<0.001; correlation, −0.199 with age. There was also a statistically significant increase in MCA (P<0.001; correlation, 0.363 with increasing age. There was no statistically significant difference in MCD, MCA, CV in cell size, percentage of hexagonal cells, and CCT between genders and there was also no significant difference in these parameters between fellow eyes of subjects. Conclusions. Normotive data for the endothelium in the Turkish population are reported. Endothelial cell density in the Turkish eyes is less than that described in the Japanese, American, Chinese, and Filipino eyes and higher than that described in Indian, Thai, and Iranian eyes.

  6. NAP reduces murine microvascular endothelial cells proliferation induced by hyperglycemia.

    Science.gov (United States)

    D'Amico, Agata Grazia; Scuderi, Soraya; Maugeri, Grazia; Cavallaro, Sebastiano; Drago, Filippo; D'Agata, Velia

    2014-11-01

    Hyperglycemia has been identified as a risk factor responsible for micro- and macrovascular complications in diabetes. NAP (Davunetide) is a peptide whose neuroprotective actions are widely demonstrated, although its biological role on endothelial dysfunctions induced by hyperglycemia remains uninvestigated. In the present study we hypothesized that NAP could play a protective role on hyperglycemia-induced endothelial cell proliferation. To this end we investigated the effects of NAP on an in vitro model of murine microvascular endothelial cells grown in high glucose for 7 days. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and cyclin D1 protein expression analysis revealed that NAP treatment significantly reduces viability and proliferation of the cells. Hyperglycemia induced the activation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase and/or phosphatidylinositol-3 kinase/Akt pathways in a time-dependent manner. NAP treatment reduced the phosphorylation levels of ERK and AKT in cells grown in high glucose. These evidences suggest that NAP might be effective in the regulation of endothelial dysfunction induced by hyperglycemia.

  7. Endothelial cell density after deep anterior lamellar keratoplasty (Melles technique)

    NARCIS (Netherlands)

    van Dooren, Bart T. H.; Mulder, Paul G. H.; Nieuwendaal, Carla P.; Beekhuis, W. Houdijn; Melles, Gerrit R. J.

    2004-01-01

    To measure the recipient endothelial cell loss after the Melles technique for deep anterior lamellar keratoplasty. In 21 eyes of 21 patients, a deep anterior lamellar keratoplasty procedure was performed. Before surgery and at 6, 12, and 24 months after surgery, specular microscopy was performed to

  8. Endothelial progenitor cell-based neovascularization : implications for therapy

    NARCIS (Netherlands)

    Krenning, Guido; van Luyn, Marja J. A.; Harmsen, Martin C.

    Ischemic cardiovascular events are a major cause of death globally. Endothelial progenitor cell (EPC)-based approaches can result in improvement of vascular perfusion and might offer clinical benefit. However, although functional improvement is observed, the lack of long-term engraftment of EPCs

  9. Donor-derived circulating endothelial cells after kidney transplantation

    NARCIS (Netherlands)

    Popa, ER; Kas-Deelen, AM; Hepkema, BG; van Son, WJ; The, TH; Harmsen, MC

    2002-01-01

    Background. In solid-organ transplantation, the allograft vasculature, in particular the endothelium, is prone to injury inflicted by peritransplantational and posttransplantational factors. Previously, we have shown that circulating endothelial cells (cEC) can be detected in the peripheral blood of

  10. Endothelial cell chimerism after renal transplantation and vascular rejection.

    NARCIS (Netherlands)

    Lagaaij, E.L.; Cramer-Knijnenburg, G.F.; Kemenade, F.J. van; Es, L.A. van; Bruijn, J.A.; Krieken, J.H.J.M. van

    2001-01-01

    BACKGROUND: The blood vessels of a transplanted organ are the interface between donor and recipient. The endothelium in the blood vessels is thought to be the major target for graft rejection. Endothelial cells of a transplanted organ are believed to remain of donor origin after transplantation. We

  11. Effects of hypergravity on the angiogenic potential of endothelial cells

    NARCIS (Netherlands)

    Costa-Almeida, R. (Raquel); Carvalho, D.T.O. (Daniel T.O.); Ferreira, M.J.S. (Miguel J.S.); Aresta, G. (Guilherme); Gomes, M.E. (Manuela E.); Van Loon, J.J.W.A. (Jack J.W.A.); K. van der Heiden (Kim); Granja, P.L. (Pedro L.)

    2016-01-01

    textabstractAngiogenesis, the formation of blood vessels from pre-existing ones, is a key event in pathology, including cancer progression, but also in homeostasis and regeneration. As the phenotype of endothelial cells (ECs) is continuously regulated by local biomechanical forces, studying

  12. Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation.

    Science.gov (United States)

    Regazzetti, Claire; De Donatis, Gian Marco; Ghorbel, Houda Hammami; Cardot-Leccia, Nathalie; Ambrosetti, Damien; Bahadoran, Philippe; Chignon-Sicard, Bérengère; Lacour, Jean-Philippe; Ballotti, Robert; Mahns, Andre; Passeron, Thierry

    2015-12-01

    Findings of increased vascularization in melasma lesions and hyperpigmentation in acquired bilateral telangiectatic macules suggested a link between pigmentation and vascularization. Using high-magnification digital epiluminescence dermatoscopy, laser confocal microscopy, and histological examination, we showed that benign vascular lesions of the skin have restricted but significant hyperpigmentation compared with the surrounding skin. We then studied the role of microvascular endothelial cells in regulating skin pigmentation using an in vitro co-culture model using endothelial cells and melanocytes. These experiments showed that endothelin 1 released by microvascular endothelial cells induces increased melanogenesis signaling, characterized by microphthalmia-associated transcription factor phosphorylation, and increased tyrosinase and dopachrome tautomerase levels. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared with perilesional skin. Endothelin acts through the activation of endothelin receptor B and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK)1/2, and p38, to induce melanogenesis. Finally, culturing of reconstructed skin with microvascular endothelial cells led to increased skin pigmentation that could be prevented by inhibiting EDNRB. Taken together these results demonstrated the role of underlying microvascularization in skin pigmentation, a finding that could open new fields of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma.

  13. Surface determinants of low density lipoprotein uptake by endothelial cells

    International Nuclear Information System (INIS)

    Goeroeg, P.; Pearson, J.D.

    1984-01-01

    The surface sialic acid content of aortic endothelial cells in vitro was substantially lower in sparse cultures than at confluence. Binding of LDL to endothelial cells did not change at different culture densities and was unaffected by brief pretreatment with neuraminidase to partially remove surface sialic acid residues. In contrast, internalisation of LDL declined by a factor of 3 between low density cell cultures and confluent monolayers; neuraminidase pretreatment increased LDL uptake and the effect was most marked (>10-fold) at confluence. Pretreatment with cationised ferritin, which removed most of the surface sialic acid residues as well as glycosaminoglycans, increased LDL internalisation by up to 20-fold, again with most effect on confluent monolayers. Thus LDL uptake is inversely correlated with sialic acid content. We conclude that changes in the surface density of sialic acid (and possibly other charged) residues significantly modulate endothelial LDL uptake, and suggest that focal increases in LDL accumulation during atherogenesis may be related to alterations in endothelial endocytic properties at sites of increased cell turnover or damage. (author)

  14. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... nuclear factor-kappa B (NF-KB). This will result in the inhibition of the expression of cell surface adhesion molecules and the reduction in the synthesis and release of inflammatory cytokine, inhibiting neutrophil activation and extravasation in damaged tissue parts. Endothelial protein C receptor (EPCR) was ...

  15. Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Masahiro Myojo

    Full Text Available Because endothelial nitric oxide synthase (eNOS has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177 in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172 and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP. Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

  16. Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

    Science.gov (United States)

    Gonzalez-Rodriguez, David; Barakat, Abdul I.

    2015-01-01

    Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process. PMID:25901833

  17. DMPD: Lipopolysaccharide signaling in endothelial cells. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16357866 Lipopolysaccharide signaling in endothelial cells. Dauphinee SM, Karsan A.... Lab Invest. 2006 Jan;86(1):9-22. (.png) (.svg) (.html) (.csml) Show Lipopolysaccharide signaling in endothe...lial cells. PubmedID 16357866 Title Lipopolysaccharide signaling in endothelial cells. Authors Dauphinee SM,

  18. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones.

    Directory of Open Access Journals (Sweden)

    Mona Saffarzadeh

    Full Text Available Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET. These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction.

  19. In Vitro Endothelialization of Biodegradable Vascular Grafts Via Endothelial Progenitor Cell Seeding and Maturation in a Tubular Perfusion System Bioreactor

    OpenAIRE

    Melchiorri, Anthony J.; Bracaglia, Laura G.; Kimerer, Lucas K.; Hibino, Narutoshi; Fisher, John P.

    2016-01-01

    A critical challenge to the success of biodegradable vascular grafts is the establishment of a healthy endothelium. To establish this monolayer of endothelial cells (ECs), a variety of techniques have been developed, including cell seeding. Vascular grafts may be seeded with relevant cell types and allowed to mature before implantation. Due to the low proliferative ability of adult ECs and issues with donor site morbidity, there has been increasing interest in using endothelial progenitor cel...

  20. Sildenafil Reduces Insulin-Resistance in Human Endothelial Cells

    Science.gov (United States)

    Mammi, Caterina; Pastore, Donatella; Lombardo, Marco F.; Ferrelli, Francesca; Caprio, Massimiliano; Consoli, Claudia; Tesauro, Manfredi; Gatta, Lucia; Fini, Massimo; Federici, Massimo; Sbraccia, Paolo; Donadel, Giulia; Bellia, Alfonso; Rosano, Giuseppe M.; Fabbri, Andrea; Lauro, Davide

    2011-01-01

    Background The efficacy of Phosphodiesterase 5 (PDE5) inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS) proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro. Methodology/Principal Findings Human umbilical vein endothelial cells (HUVECs) were treated with insulin in presence of glucose 30 mM (HG) and glucosamine 10 mM (Gluc-N) with or without sildenafil. Insulin increased the expression of PDE5 and eNOS mRNA assayed by Real time-PCR. Cytofluorimetric analysis showed that sildenafil significantly increased NO production in basal condition. This effect was partially inhibited by the PI3K inhibitor LY 294002 and completely inhibited by the NOS inhibitor L-NAME. Akt-1 and eNOS activation was reduced in conditions mimicking insulin resistance and completely restored by sildenafil treatment. Conversely sildenafil treatment can counteract this noxious effect by increasing NO production through eNOS activation and reducing oxidative stress induced by hyperglycaemia and glucosamine. Conclusions/Significance These data indicate that sildenafil might improve NOS activity of endothelial cells in insulin resistance conditions and suggest the potential therapeutic use of sildenafil for improving vascular function in diabetic patients. PMID:21297971

  1. Sildenafil reduces insulin-resistance in human endothelial cells.

    Directory of Open Access Journals (Sweden)

    Caterina Mammi

    Full Text Available BACKGROUND: The efficacy of Phosphodiesterase 5 (PDE5 inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Human umbilical vein endothelial cells (HUVECs were treated with insulin in presence of glucose 30 mM (HG and glucosamine 10 mM (Gluc-N with or without sildenafil. Insulin increased the expression of PDE5 and eNOS mRNA assayed by Real time-PCR. Cytofluorimetric analysis showed that sildenafil significantly increased NO production in basal condition. This effect was partially inhibited by the PI3K inhibitor LY 294002 and completely inhibited by the NOS inhibitor L-NAME. Akt-1 and eNOS activation was reduced in conditions mimicking insulin resistance and completely restored by sildenafil treatment. Conversely sildenafil treatment can counteract this noxious effect by increasing NO production through eNOS activation and reducing oxidative stress induced by hyperglycaemia and glucosamine. CONCLUSIONS/SIGNIFICANCE: These data indicate that sildenafil might improve NOS activity of endothelial cells in insulin resistance conditions and suggest the potential therapeutic use of sildenafil for improving vascular function in diabetic patients.

  2. Organizational behavior of human umbilical vein endothelial cells.

    Science.gov (United States)

    Maciag, T; Kadish, J; Wilkins, L; Stemerman, M B; Weinstein, R

    1982-09-01

    Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization.

  3. Nylon-3 polymers that enable selective culture of endothelial cells.

    Science.gov (United States)

    Liu, Runhui; Chen, Xinyu; Gellman, Samuel H; Masters, Kristyn S

    2013-11-06

    Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications.

  4. Fungal invasion of normally non-phagocytic host cells.

    Directory of Open Access Journals (Sweden)

    Scott G Filler

    2006-12-01

    Full Text Available Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research.

  5. The effect of nicotine on aortic endothelial cell turnover

    International Nuclear Information System (INIS)

    Zimmerman, Matthew; McGeachie, John

    1985-01-01

    Endothelial injury and increased mitotic activity are early features in the pathogenesis of intimal thickening in arteries. This study examines the effect of systemic nicotine on mitotic activity in endothelial cells. Nine adult mice were given nicotine in their drinking water for 5 weeks. The dose (5 mg/kg body wt/day) was equivalent to a human smoking 50-100 cigarettes/day. A group of 8 similar mice, not exposed to nicotine, was the control. At the end of the exposure period all mice were injected with ( 3 H)thymidine (1uCi/g body wt) and were killed 24 h later. After perfusion fixation, en-face preparations of aortic endothelium were processed for autoradiography. In nicotine-affected endothelium 0.46.+-0.11% (SEM) of cells were labeled, which was significantly higher (P<0.01) than in controls (0.14+-0.06). However, there was no difference in cell density between the groups. On this evidence it was concluded that the rate of cell loss, or cell turnover, was greater in nicotine-affected endothelium. Because other studies have shown that increased mitotic acitivity and cell loss are established features of endothelial injury, the present findings provide evidence in support of the hypothesis that nicotine contributes to the pathogenesis of arterial disease in smokers. (author)

  6. An Important Method in the Investigation of Vascular Pathologies: Endothelial Cell Culture

    Directory of Open Access Journals (Sweden)

    Yusufhan Yazır

    2012-12-01

    Full Text Available Endothelial cells line the interior surface of blood vessels and form an interface between circulating blood in the lumen and the rest of the vessel wall. Endothelial cells are involved in many aspects of vascular biology, including barrier function, vasoconstriction, coagulation and inflamation. The endothelial cells in different organs have different functions and surface phenotype. These cells express prostoglandin-I2, platelet activating factor, collagen, endothelin-1, laminin, fibronectin and growth factors including platelet derived growth factor, fibroblast growth factor. İn the cell culture, cells can be isolated, maintened and proliferate in the laboratory conditions. The techniques of the cell culture have allowed scientists to use the cells in vitro for experimental studies, such as the production of vaccine, antibody and enzime, drug research, cell-cell interactions. Human umbilical vein endothelial cell is a good source for endothelial cell, because it is cheaper, easy to find and has the basic features of the normal endothelial cells.

  7. Lmo0171, a novel internalin-like protein, determines cell morphology of Listeria monocytogenes and its ability to invade human cell lines.

    Science.gov (United States)

    Stachowiak, Radosław; Jagielski, Tomasz; Roeske, Katarzyna; Osińska, Olga; Gunerka, Paweł; Wiśniewski, Jarosław; Bielecki, Jacek

    2015-02-01

    Internalins comprise a class of Listeria monocytogenes proteins responsible for activation of signalling pathways leading to phagocytic uptake of the bacterium by the host cell. In this paper, a possible role of Lmo0171-a new member of the internalin family was investigated. Disruption of the lmo0171 gene resulted in important cell morphology alterations along with a decrease in the ability to invade three eukaryotic cell lines, that is Int407, Hep-2 and HeLa and diminished adhesion efficiency to int407, thereby suggesting bifunctionality of the newly characterised Lmo0171 internalin.

  8. XIAP reverses various functional activities of FRNK in endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Sunyoung; Kim, Hyun Jeong [Department of Molecular Biology and Institute of Nanosensor and Biotechnology, BK21 Graduate Program for RNA Biology, Dankook Univiersity, 126, Jukjeon-dong, Suji-gu, Yongin-si, Gyeonggi-do 448-701 (Korea, Republic of); Chi, Sung-Gil [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Park, Heonyong, E-mail: heonyong@dankook.ac.kr [Department of Molecular Biology and Institute of Nanosensor and Biotechnology, BK21 Graduate Program for RNA Biology, Dankook Univiersity, 126, Jukjeon-dong, Suji-gu, Yongin-si, Gyeonggi-do 448-701 (Korea, Republic of)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer FRNK domain is recruited into focal adhesion (FA), controlling endothelial cell adhesion. Black-Right-Pointing-Pointer XIAP binds the FRNK domain of FAK. Black-Right-Pointing-Pointer XIAP inhibits recruitment of FRNK into Fas and FRNK-promoted cell adhesion. Black-Right-Pointing-Pointer XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK. -- Abstract: In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.

  9. Initial contact of glioblastoma cells with existing normal brain endothelial cells strengthen the barrier function via fibroblast growth factor 2 secretion: a new in vitro blood-brain barrier model.

    Science.gov (United States)

    Toyoda, Keisuke; Tanaka, Kunihiko; Nakagawa, Shinsuke; Thuy, Dinh Ha Duy; Ujifuku, Kenta; Kamada, Kensaku; Hayashi, Kentaro; Matsuo, Takayuki; Nagata, Izumi; Niwa, Masami

    2013-05-01

    Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood-brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.

  10. Suprabasin as a novel tumor endothelial cell marker.

    Science.gov (United States)

    Alam, Mohammad T; Nagao-Kitamoto, Hiroko; Ohga, Noritaka; Akiyama, Kosuke; Maishi, Nako; Kawamoto, Taisuke; Shinohara, Nobuo; Taketomi, Akinobu; Shindoh, Masanobu; Hida, Yasuhiro; Hida, Kyoko

    2014-12-01

    Recent studies have reported that stromal cells contribute to tumor progression. We previously demonstrated that tumor endothelial cells (TEC) characteristics were different from those of normal endothelial cells (NEC). Furthermore, we performed gene profile analysis in TEC and NEC, revealing that suprabasin (SBSN) was upregulated in TEC compared with NEC. However, its role in TEC is still unknown. Here we showed that SBSN expression was higher in isolated human and mouse TEC than in NEC. SBSN knockdown inhibited the migration and tube formation ability of TEC. We also showed that the AKT pathway was a downstream factor of SBSN. These findings suggest that SBSN is involved in the angiogenic potential of TEC and may be a novel TEC marker. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  11. Endothelial cell senescence with aging in healthy humans: prevention by habitual exercise and relation to vascular endothelial function.

    Science.gov (United States)

    Rossman, Matthew J; Kaplon, Rachelle E; Hill, Sierra D; McNamara, Molly N; Santos-Parker, Jessica R; Pierce, Gary L; Seals, Douglas R; Donato, Anthony J

    2017-11-01

    Cellular senescence is emerging as a key mechanism of age-related vascular endothelial dysfunction, but evidence in healthy humans is lacking. Moreover, the influence of lifestyle factors such as habitual exercise on endothelial cell (EC) senescence is unknown. We tested the hypothesis that EC senescence increases with sedentary, but not physically active, aging and is associated with vascular endothelial dysfunction. Protein expression (quantitative immunofluorescence) of p53, a transcription factor related to increased cellular senescence, and the cyclin-dependent kinase inhibitors p21 and p16 were 116%, 119%, and 128% greater (all P 0.05) in venous ECs from older exercising adults (57 ± 1 yr, n = 13). Furthermore, venous EC protein levels of p53 ( r  = -0.49, P = 0.003), p21 ( r  = -0.38, P = 0.03), and p16 ( r  = -0.58, P = 0.002) were inversely associated with vascular endothelial function (brachial artery flow-mediated dilation). Similarly, protein expression of p53 and p21 was 26% and 23% higher (both P 0.05) in older habitually exercising adults (59 ± 1 yr, n = 14). These data indicate that EC senescence is associated with sedentary aging and is linked to endothelial dysfunction. Moreover, these data suggest that prevention of EC senescence may be one mechanism by which aerobic exercise protects against endothelial dysfunction with age. NEW & NOTEWORTHY Our study provides novel evidence in humans of increased endothelial cell senescence with sedentary aging, which is associated with impaired vascular endothelial function. Furthermore, our data suggest an absence of age-related increases in endothelial cell senescence in older exercising adults, which is linked with preserved vascular endothelial function. Copyright © 2017 the American Physiological Society.

  12. Synthesis of an endothelial cell mimicking surface containing thrombomodulin and endothelial protein C receptor

    Science.gov (United States)

    Kador, Karl Erich

    Synthetic materials for use in blood contacting applications have been studied for many years with limited success. One of the main areas of need for these materials is the design of synthetic vascular grafts for use in the hundreds of thousands of patients who have coronary artery bypass grafting, many without suitable veins for autologous grafts. The design of these grafts is constrained by two common modes of failure, the formation of intimal hyperplasia (IH) and thrombosis. IH formation has been previously linked to a mismatching of the mechanical properties of the graft and has been overcome by creating grafts using materials whose compliance mimics that of the native artery. Several techniques and surface modification have been designed to limit thrombosis on the surface of synthetic materials. One which has shown the greatest promise is the immobilization of Thrombomodulin (TM), a protein found on the endothelial cell membrane lining native blood vessels involved in the activation of the anticoagulant Protein C (PC). While TM immobilization has been shown to arrest thrombin formation and limit fibrous formations in in-vitro and in-vivo experiments, it has shown to be transport limiting under arterial flow. On the endothelial cell surface, TM is co-localized with Endothelial Protein C Receptor (EPCR), which increases PC transport onto the cell surface and increases PC activation via TM between 20-100 fold. This dissertation will describe the chemical modification of medical grade polyurethane (PU), whose compliance has been shown to match that of native arteries. This modification will enable the immobilization of two proteins on an enzymatically relevant scale estimated at less than 10 nm. This dissertation will further describe the immobilization of the proteins TM and EPCR, and analyze the ability of a surface co-immobilized with these proteins to activate the anticoagulant PC. Finally, it will compare the ability of this co-immobilized surface to delay

  13. Coniferyl Aldehyde Ameliorates Radiation Intestine Injury via Endothelial Cell Survival

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Ye Ji; Jung, Myung Gu; Lee, Yoonjin; Lee, Haejune [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Yunsil [Ewha Woman' s Univ., Seoul (Korea, Republic of); Ko, Younggyu [Korea Univ., Seoul (Korea, Republic of)

    2014-05-15

    Cancer treatments related gastrointestinal toxicity has also been recognized as a significant economic burden. Especially, extensive apoptosis of microvascular endothelial cell of the lamina propria is the primary lesion initiating intestinal radiation damage after abdominal radiation therapy. Coniferyl aldehyde (CA) is phenolic compounds isolated from cork stoppers, and one of the major pyrolysis products of lignin. Shi H. was support for the empirical use of CA as a medicinal food for cardiovascular diseases. CA has positive effect in broad way but there is no consequence in radiation induced intestine damage. Here, we investigate effect of CA on small intestine after abdominal IR to mice in this study. In this study, CA increased the survival rate in C3H mice against 13.5 Gy abdominal IR. We found CA protects small intestine via preventing endothelial cell apoptosis and enhancing their angiogenic activity. CA also showed protective effect on crypt cell survival. Endothelial cell survival may affect crypt cell protection against IR. From this data, we concluded that CA is effective for protection against abdominal radiation injury. CA could ameliorate side-effect of radiation therapy.

  14. A microarray analysis of two distinct lymphatic endothelial cell populations

    Directory of Open Access Journals (Sweden)

    Bernhard Schweighofer

    2015-06-01

    Full Text Available We have recently identified lymphatic endothelial cells (LECs to form two morphologically different populations, exhibiting significantly different surface protein expression levels of podoplanin, a major surface marker for this cell type. In vitro shockwave treatment (IVSWT of LECs resulted in enrichment of the podoplaninhigh cell population and was accompanied by markedly increased cell proliferation, as well as 2D and 3D migration. Gene expression profiles of these distinct populations were established using Affymetrix microarray analyses. Here we provide additional details about our dataset (NCBI GEO accession number GSE62510 and describe how we analyzed the data to identify differently expressed genes in these two LEC populations.

  15. Protection of Candida parapsilosis from neutrophil killing through internalization by human endothelial cells.

    Science.gov (United States)

    Glass, Kyle A; Longley, Sarah J; Bliss, Joseph M; Shaw, Sunil K

    2015-01-01

    Candida parapsilosis is a fungal pathogen that is associated with hematogenously disseminated disease in premature neonates, acutely ill or immunocompromised patients. In cell culture, C. parapsilosis cells are actively and avidly endocytosed by endothelial cells via actin polymerization mediated by N-WASP. Here we present evidence that C. parapsilosis that were internalized by endothelial cells remained alive, and avoided being acidified or otherwise damaged via the host cell. Internalized fungal cells reproduced intracellularly and eventually burst out of the host endothelial cell. When neutrophils were added to endothelium and C. parapsilosis, they patrolled the endothelial surface and efficiently killed most adherent fungal cells prior to endocytosis. But after endocytosis by endothelial cells, internalized fungal cells evaded neutrophil killing. Silencing endothelial N-WASP blocked endocytosis of C. parapsilosis and left fungal cells stranded on the cell surface, where they were susceptible to neutrophil killing. These observations suggest that for C. parapsilosis to escape from the bloodstream, fungi may adhere to and be internalized by endothelial cells before being confronted and phagocytosed by a patrolling leukocyte. Once internalized by endothelial cells, C. parapsilosis may safely replicate to cause further rounds of infection. Immunosurveillance of the intravascular lumen by leukocytes crawling on the endothelial surface and rapid killing of adherent yeast may play a major role in controlling C. parapsilosis dissemination and infected endothelial cells may be a significant reservoir for fungal persistence.

  16. Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Jingshan Zhao

    Full Text Available The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL, a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF signaling and angiogenesis in endothelial cells (ECs. We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis.

  17. Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells.

    Science.gov (United States)

    Zhao, Jingshan; Niu, Honglin; Li, Aiying; Nie, Lei

    2016-01-01

    The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL), a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF) signaling and angiogenesis in endothelial cells (ECs). We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day) recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis.

  18. The Arf GTPase-activating protein family is exploited by Salmonella enterica serovar Typhimurium to invade nonphagocytic host cells.

    Science.gov (United States)

    Davidson, Anthony C; Humphreys, Daniel; Brooks, Andrew B E; Hume, Peter J; Koronakis, Vassilis

    2015-02-10

    To establish intracellular infections, Salmonella bacteria trigger host cell membrane ruffling and invasion by subverting cellular Arf guanine nucleotide exchange factors (GEFs) that activate Arf1 and Arf6 GTPases by promoting GTP binding. A family of cellular Arf GTPase-activating proteins (GAPs) can downregulate Arf signaling by stimulating GTP hydrolysis, but whether they do this during infection is unknown. Here, we uncovered a remarkable role for distinct Arf GAP family members in Salmonella invasion. The Arf6 GAPs ACAP1 and ADAP1 and the Arf1 GAP ASAP1 localized at Salmonella-induced ruffles, which was not the case for the plasma membrane-localized Arf6 GAPs ARAP3 and GIT1 or the Golgi-associated Arf1 GAP1. Surprisingly, we found that loss of ACAP1, ADAP1, or ASAP1 impaired Salmonella invasion, revealing that GAPs cannot be considered mere terminators of cytoskeleton remodeling. Salmonella invasion was restored in Arf GAP-depleted cells by expressing fast-cycling Arf derivatives, demonstrating that Arf GTP/GDP cycles facilitate Salmonella invasion. Consistent with this view, both constitutively active and dominant-negative Arf derivatives that cannot undergo GTP/GDP cycles inhibited invasion. Furthermore, we demonstrated that Arf GEFs and GAPs colocalize at invading Salmonella and collaborate to drive Arf1-dependent pathogen invasion. This study revealed that Salmonella bacteria exploit a remarkable interplay between Arf GEFs and GAPs to direct cycles of Arf GTPase activation and inactivation. These cycles drive Salmonella cytoskeleton remodeling and enable intracellular infections. To initiate infections, the Salmonella bacterial pathogen remodels the mammalian actin cytoskeleton and invades host cells by subverting host Arf GEFs that activate Arf1 and Arf6 GTPases. Cellular Arf GAPs deactivate Arf GTPases and negatively regulate cell processes, but whether they target Arfs during infection is unknown. Here, we uncovered an important role for the Arf GAP

  19. [Expression and function of receptors for advanced glycation end products in bovine corneal endothelial cells].

    Science.gov (United States)

    Kaji, Yuichi

    2005-11-01

    Corneal endothelial cell loss is a change that occurs with age, but its mechanism is still unclear. We postulated that interaction between advanced glycation end product(AGE) and its receptors is implicated in the corneal endothelial cell loss with age. We investigated the expression of AGE receptors: receptors for AGE(RAGE) and galectin-3 in bovine corneal endothelial cells by reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemistry. In addition, we investigated the effect of AGE on the cultured corneal endothelial cells. Expression of RAGE and galectin-3 was detected in bovine corneal endothelial cells. Galectin-3 was important in the internalization of AGE. In contrast, RAGE was important in the generation of reactive oxygen species and induction of apoptosis. Based on these data, the interaction of AGE in aqueous humor and AGE receptors expressed on the corneal endothelial cells was speculated to have a role in the corneal endothelial cell loss with age.

  20. Induced thyme product prevents VEGF-induced migration in human umbilical vein endothelial cells.

    Science.gov (United States)

    Krill, Diane; Madden, John; Huncik, Kevin; Moeller, Peter D

    2010-12-17

    Compounds with anti-angiogenic properties are useful in combating cancer by preventing new blood vessel formation to support the tumor. In this report we introduce a rapid method for screening potential anti-angiogenic compounds in a model system that stimulates the production of secondary defense chemicals in plants. This methodology identified an inducible vascular factor (IVF3), which was found to be inhibitory in all of the model systems tested. Thyme plants were exposed to highly vascular mint plants and the methanol extracts were analyzed by reverse phase HPLC. The thyme compounds induced by the invading mint tissue, and not present in the thyme plants grown alone, were tested in a vertical plate assay measuring root length as a quantitative assay for drug sensitivity. The HPLC-purified extract, referred to as IVF3, reduced the growth of root vascular tissue compared to the control and vehicle control, and 50% as well as known angiogenesis inhibitors, VEGF receptor tyrosine kinase inhibitor and amiloride hydrochloride. Extracted compounds that were effective inhibitors of plant roots were assayed in Madin Darby canine kidney epithelial cells (MDCK) for toxicity, and in human umbilical vein endothelial cells (HUVEC) for their effect on migration. IVF3 was effective at limiting HUVEC migration in VEGF-stimulated cultures. In vivo video capture of intersegmental vessel circulation between 48 and 72 h post fertilization in the developing vasculature of zebrafish embryos showed IVF3 also significantly reduced ISV functional circulation. This report demonstrates the anti-angiogenic effects of IVF3 extract in endothelial cells and in an intact vertebrate model for angiogenesis. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. Glioblastoma stem cell differentiation into endothelial cells evidenced through live-cell imaging.

    Science.gov (United States)

    Mei, Xin; Chen, Yin-Sheng; Chen, Fu-Rong; Xi, Shao-Yan; Chen, Zhong-Ping

    2017-08-01

    Glioblastoma cell-initiated vascularization is an alternative angiogenesis called vasculogenic mimicry. However, current knowledge on the mechanism of de novo vessel formation from glioblastoma stem cells (GSCs) is limited. Sixty-four glioblastoma samples from patients and 10 fluorescent glioma xenograft samples were examined by immunofluorescence staining for endothelial marker (CD34 and CD31) and glial cell marker (glial fibrillary acidic protein [GFAP]) expression. GSCs were then isolated from human glioblastoma tissue and CD133+/Sox2+ red fluorescent protein-containing (RFP)-GSC-1 cells were established. The ability of these cells to form vascular structures was examined by live-cell imaging of 3D cultures. CD34-GFAP or CD31-GFAP coexpressing glioblastoma-derived endothelial cells (GDEC) were found in 30 of 64 (46.9%) of clinical glioblastoma samples. In those 30 samples, GDEC were found to form vessel structures in 21 (70%) samples. Among 21 samples with GDEC vessels, the CD34+ GDEC vessels and CD31+ GDEC vessels accounted for about 14.16% and 18.08% of total vessels, respectively. In the xenograft samples, CD34+ GDEC were found in 7 out of 10 mice, and 4 out of 7 mice had CD34+ GDEC vessels. CD31+ GDEC were also found in 7 mice, and 4 mice had CD31+ GDEC vessels (10 mice in total). Through live-cell imaging, we observed gradual CD34 expression when cultured with vascular endothelial growth factor in some glioma cells, and a dynamic increase in endothelial marker expression in RFP-GSC-1 in vitro was recorded. Cells expressed CD34 (9.46%) after 6 hours in culture. The results demonstrated that GSCs may differentiate into endothelial cells and promote angiogenesis in glioblastomas. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  2. Endothelialization of artificial surfaces: does surface tension determine in vitro growth of human saphenous vein endothelial cells?

    Science.gov (United States)

    Fasol, R; Zilla, P; Deutsch, M; Fischlein, T; Kadletz, M; Griesmacher, A; Müller, M M

    1987-06-01

    To evaluate the possibility of providing, in vitro, an endothelial lining for artificial hearts, we cultivated adult autologous endothelial cells on two polyurethane and two silicone rubber surfaces. Over the ensuing 11-day period, we investigated the resulting cell proliferation and morphology by means of scanning electron and light microscopy. On the silicone rubber surfaces, seeding of 200,000 human saphenous vein endothelial cells per cm(2) produced an ideal cobblestone monolayer within a single day. In contrast, the polyurethane surfaces displayed an uneven, patchy distribution of endothelial cells. Scanning electron microscopy revealed microvilli and marginal overlapping in both groups. After the first day, the cell count on the polyurethane surfaces increased, whereas the count on the silicone rubber surfaces decreased. Morphologic investigations revealed that the ideally shaped cells initially on the silicone rubber had begun to overspread and subsequently to become detached, leaving denuded spheroid areas. Moreover, cultivation for 11 days on the polyurethane surfaces resulted in an unevenness of cell distribution that far exceeded the unevenness seen on the first day. Thus, despite the fact that materials with a high surface tension (such as silicone rubbers) seem to be ideal for initial cell spreading, subsequent cultivation results in cell detachment and death. On materials with a lower surface tension (such as polyurethanes), the less differentiated monolayers do at least proliferate, although their morphology remains unsatisfactory. Even if adult human endothelial cells should prove shear-stress-resistant, a minimum of 6 to 8 weeks would be required to establish autologous endothelial cell monolayers on the inner surface. Therefore, the endothelialization of artificial hearts is not possible when such hearts are used for urgent "bridging" before cardiac transplantation.

  3. Testosterone modulates endothelial progenitor cells in rat corpus cavernosum.

    Science.gov (United States)

    Hwang, Insang; Lee, Hyun-Suk; Yu, Ho Song; Kim, Mi Eun; Lee, Jun Sik; Park, Kwangsung

    2016-06-01

    To investigate the effects of testosterone on cavernosal endothelial progenitor cells (EPCs) in a castrated rat model. In all, 45 male Sprague-Dawley rats (12-weeks old) were divided into control, surgical castration, and castration with testosterone replacement groups. The rats were castrated under ketamine anaesthesia, and testosterone was administered by daily subcutaneous injection of 3 mg/kg testosterone propionate. The corpus cavernosum was obtained after perfusion with 10 mL saline via the abdominal aorta 4 weeks later. The expression of EPC-specific markers [cluster of differentiation 34 (CD34), fetal liver kinase 1 (Flk1), and vascular endothelial (VE)-cadherin] was evaluated by flow cytometry analysis and immunofluorescence staining. CD34+/Flk1+ and CD34+/VE-cadherin+ cells were detected in the cavernosal sinusoidal endothelial space. Flow cytometry analysis showed that CD34 and Flk1 double positive cells (EPCs) comprised ≈3.79% of the corpus cavernosum in normal rats. The percentage of EPC marker-positive cells decreased significantly in the castration group (2.8%; P testosterone supplementation. Confocal microscopy revealed that the numbers of CD34+/Flk1+ and CD34+/VE-cadherin+ cells decreased in castrated rats compared with controls, but were similar to control levels in rats receiving testosterone replacement. The EPC markers were expressed in the cavernosal sinusoidal endothelial space, and the numbers of resident EPCs were regulated by testosterone. These results suggest that testosterone replacement therapy may improve erectile function by modulating EPCs in patients with hypogonadism. © 2016 The Authors BJU International © 2016 BJU International Published by John Wiley & Sons Ltd.

  4. The Glycoprofile Patterns of Endothelial Cells in Usual Interstitial Pneumonia

    Directory of Open Access Journals (Sweden)

    A Barkhordari

    2014-09-01

    Full Text Available [THIS ARTICLE HAS BEEN RETRACTED FOR DUPLICATE PUBLICATION] Background: The pathological classification of cryptogenic fibrosing alveolitis has been a matter of debate and controversy for histopathologists. Objective: To identify and specify the glycotypes of capillary endothelial cells in usual interstitial pneumonia (UIP compared to those found in normal tissue. Methods: Sections of formalin-fixed, paraffin-embedded blocks from 16 cases of UIP were studied by lectin histochemistry with a panel of 27 biotinylated lectins and an avidin-peroxidase revealing system. Results: High expression of several classes of glycan was seen de novo in capillary endothelial cells from patients with UIP including small complex and bi/tri-antennary bisected complex N-linked sequences bolund by Concanavalin A and erythro-phytohemagglutinin, respectively, GalNAca1 residues bound by Helix pomatia and Maclura pomifera agglutinins, and L-fucosylated derivatives of type II glycan chains recognized by Ulex europaeus agglutinin-I. Glycans bound by agglutinins from Lycopersicon esculentum (β1,4GlcNAc and Wisteria floribunda (GalNAc as well as GlcNAc oligomers bound by Phytolacca americana and succinylated Wheat Germ agglutinin were also seen in the capillary endothelial cells of UIP. In contrast, L-fucosylated derivatives of type I glycan chains were absent in cells from cases of UIP when Anguilla anguilla agglutinin was applied, unlike the situation in normal tissue. Conclusion: These results may indicate existence of two distinct populations of endothelial cell in UIP with markedly different patterns of glycosylation, reflecting a pattern of differentiation and angiogenesis, which is not detectable morphologically.

  5. A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

    DEFF Research Database (Denmark)

    Fens, Marcel H A M; van Wijk, Richard; Andringa, Grietje

    2012-01-01

    Background Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells gener...... cells play a role in red blood cell clearance in vivo. Significant erythrophagocytosis can induce endothelial cell loss, which may contribute to vasopathological effects as seen, for instance, in sickle cell disease.......Background Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells...... generally occurs by macrophages in the spleen and liver. Previously, however, we have shown that endothelial cells are also capable of erythrophagocytosis. Key players in the erythrophagocytosis by endothelial cells appeared to be lactadherin and αv-integrin. Phagocytosis via the phosphatidylserine...

  6. Endothelial progenitor cell subsets and preeclampsia: Findings and controversies

    Directory of Open Access Journals (Sweden)

    Armin Attar

    2017-10-01

    Full Text Available Vascular remodeling is an essential component of gestation. Endothelial progenitor cells (EPCs play an important role in the regulation of vascular homeostasis. The results of studies measuring the number of EPCs in normal pregnancies and in preeclampsia have been highly controversial or even contradictory because of some variations in technical issues and different methodologies enumerating three distinct subsets of EPCs: circulating angiogenic cells (CAC, colony forming unit endothelial cells (CFU-ECs, and endothelial colony-forming cells (ECFCs. In general, most studies have shown an increase in the number of CACs in the maternal circulation with a progression in the gestational age in normal pregnancies, while functional capacities measured by CFU-ECs and ECFCs remain intact. In the case of preeclampsia, mobilization of CACs and ECFCs occurs in the peripheral blood of pregnant women, but the functional capacities shown by culture of the derived colony-forming assays (CFU-EC and ECFC assays are altered. Furthermore, the number of all EPC subsets will be reduced in umbilical cord blood in the case of preeclampsia. As EPCs play an important role in the homeostasis of vascular networks, the difference in their frequency and functionality in normal pregnancies and those with preeclampsia can be expected. In this review, there was an attempt to provide a justification for these controversies.

  7. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

    Science.gov (United States)

    Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

  8. Endothelial cell chimerism associated with graft rejection after human lung transplantation.

    OpenAIRE

    Ratajczak , Philippe; Murata , Hideyuki; Meignin , Véronique; Groussard , Odile; Fournier , Michel; Socié , Gérard; Mal , Hervé; Janin , Anne

    2008-01-01

    International audience; Endotheliitis is a major sign of graft rejection. Recipient-derived endothelial cells found in two series of liver and kidney transplants were related to graft rejection. Here, we assessed the presence and the number of chimeric endothelial cells in lung transplants, and their relation with graft rejection. In six males grafted with female lungs out of 193 lung transplantations, endothelial chimerism was studied by combined XY-fluorescent in situ hybridization with CD3...

  9. Endothelial cell-initiated extravasation of cancer cells visualized in zebrafish

    Directory of Open Access Journals (Sweden)

    Masamitsu Kanada

    2014-12-01

    Full Text Available The extravasation of cancer cells, a key step for distant metastasis, is thought to be initiated by disruption of the endothelial barrier by malignant cancer cells. An endothelial covering-type extravasation of cancer cells in addition to conventional cancer cell invasion-type extravasation was dynamically visualized in a zebrafish hematogenous metastasis model. The inhibition of VEGF-signaling impaired the invasion-type extravasation via inhibition of cancer cell polarization and motility. Paradoxically, the anti-angiogenic treatment showed the promotion, rather than the inhibition, of the endothelial covering-type extravasation of cancer cells, with structural changes in the endothelial walls. These findings may be a set of clues to the full understanding of the metastatic process as well as the metastatic acceleration by anti-angiogenic reagents observed in preclinical studies.

  10. Endothelial cell-initiated extravasation of cancer cells visualized in zebrafish.

    Science.gov (United States)

    Kanada, Masamitsu; Zhang, Jinyan; Yan, Libo; Sakurai, Takashi; Terakawa, Susumu

    2014-01-01

    The extravasation of cancer cells, a key step for distant metastasis, is thought to be initiated by disruption of the endothelial barrier by malignant cancer cells. An endothelial covering-type extravasation of cancer cells in addition to conventional cancer cell invasion-type extravasation was dynamically visualized in a zebrafish hematogenous metastasis model. The inhibition of VEGF-signaling impaired the invasion-type extravasation via inhibition of cancer cell polarization and motility. Paradoxically, the anti-angiogenic treatment showed the promotion, rather than the inhibition, of the endothelial covering-type extravasation of cancer cells, with structural changes in the endothelial walls. These findings may be a set of clues to the full understanding of the metastatic process as well as the metastatic acceleration by anti-angiogenic reagents observed in preclinical studies.

  11. Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

    Directory of Open Access Journals (Sweden)

    Shumei Man

    2008-01-01

    Full Text Available Endothelial cells that functionally express blood brain barrier (BBB properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs and human umbilical vein endothelial cells (HUVECs. With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

  12. Conversion of vascular endothelial cells into multipotent stem-like cells

    Science.gov (United States)

    Medici, Damian; Shore, Eileen M.; Lounev, Vitali Y.; Kaplan, Frederick S.; Kalluri, Raghu; Olsen, Bjorn R.

    2011-01-01

    Mesenchymal stem cells can give rise to several cell types, but variations depending on isolation method and tissue source have led to controversies about their usefulness in clinical medicine. Here we show that vascular endothelial cells can transform into multipotent stem-like cells by an ALK2 receptor-dependent mechanism. In lesions from patients with Fibrodysplasia Ossificans Progressiva, a disease where heterotopic ossification occurs as a result of activating ALK2 mutations, or from a mutant ALK2 transgenic mouse model, chondrocytes and osteoblasts express endothelial markers. Tie2-Cre lineage tracing also suggests an endothelial origin of these cells. Expressing mutant ALK2 in endothelial cells, or treatment with the ALK2 ligands TGF-β2 or BMP4, causes endothelial-mesenchymal transition and acquisition of a stem cell-like phenotype. In selective media, these cells differentiate into osteoblasts, chondrocytes, or adipocytes. The process is inhibited by ALK2-specific siRNA. Conversion of endothelial cells to stem-like cells may provide a novel approach to tissue engineering. PMID:21102460

  13. Glatiramer acetate (GA) prevents TNF-α-induced monocyte adhesion to primary endothelial cells through interfering with the NF-κB pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Guoqian; Zhang, Xueyan; Su, Zhendong; Li, Xueqi, E-mail: xueqili075@yeah.net

    2015-01-30

    Highlights: • GA inhibited TNF-α-induced binding of monocytes to endothelial cells. • GA inhibited the induction of adhesion molecules MCP-1, VCAM-1 and E-selectin. • GA inhibits NF-κB p65 nuclear translocation and transcriptional activity. • GA inhibits TNF-α-induced IκBα degradation. - Abstract: Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) is considered to be the major one contributing to the process of development of endothelial dysfunction. Exposure to TNF-α induces the expression of a number of proinflammatory chemokines, such as monocyte chemotactic protein-1 (MCP-1), and adhesion molecules, including vascular adhesion molecule-1 (VCAM-1) and E-selectin, which mediate the interaction of invading monocytes with vascular endothelial cells. Glatiramer acetate (GA) is a licensed clinical drug for treating patients suffering from multiple sclerosis (MS). The effects of GA in vascular disease have not shown before. In this study, we found that GA significantly inhibited TNF-α-induced binding of monocytes to endothelial cells. Mechanistically, we found that GA ameliorated the upregulation of MCP-1, VCAM-1, and E-selectin induced by TNF-α. Notably, this process is mediated by inhibiting the nuclear translocation and activation of NF-κB. Our results also indicate that GA pretreatment attenuates the up-regulation of COX-2 and iNOS. These data suggest that GA might have a potential benefit in therapeutic endothelial dysfunction related diseases.

  14. The fundamental role of endothelial cells in hantavirus pathogenesis

    Directory of Open Access Journals (Sweden)

    Jussi eHepojoki

    2014-12-01

    Full Text Available Hantavirus, a genus of rodent- and insectivore-borne viruses in the family Bunyaviridae, is a group of emerging zoonotic pathogens. Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS and hantavirus cardiopulmonary syndrome (HCPS in man, often with severe consequences. Vascular leakage is evident in severe hantavirus infections, and increased permeability contributes to the pathogenesis. This review summarizes the current knowledge on hantavirus interactions with endothelial cells, and their effects on the increased vascular permeability.

  15. Corneal endothelial cell density and morphology in Phramongkutklao Hospital

    Directory of Open Access Journals (Sweden)

    Narumon Sopapornamorn

    2008-03-01

    Full Text Available Narumon Sopapornamorn1, Manapon Lekskul1, Suthee Panichkul21Department of Ophthalmology, Phramongkutklao Hospital, Bangkok, Thailand; 2Department of Obstetrics and Gynecology, Phramongkutklao College of Medicine, Bangkok, ThailandObjective: To describe the corneal endothelial density and morphology in patients of Phramongkutklao Hospital and the relationship between endothelial cell parameters and other factors.Methods: Four hundred and four eyes of 202 volunteers were included. Noncontact specular microscopy was performed after taking a history and testing the visual acuity, intraocular pressure measurement, Schirmer’s test and routine eye examination by slit lamp microscope. The studied parameters included mean endothelial cell density (MCD, coefficient of variation (CV, and percentage of hexagonality.Results: The mean age of volunteers was 45.73 years; the range being 20 to 80 years old. Their MCD (SD, mean percentage of CV (SD and mean (SD percentage of hexagonality were 2623.49(325 cell/mm2, 39.43(8.23% and 51.50(10.99%, respectively. Statistically, MCD decreased significantly with age (p < 0.01. There was a significant difference in the percentage of CV between genders. There was no statistical significance between parameters and other factors.Conclusion: The normative data of the corneal endothelium of Thai eyes indicated that, statistically, MCD decreased significantly with age. Previous studies have reported no difference in MCD, percentage of CV, and percentage of hexagonality between gender. Nevertheless, significantly different percentages of CV between genders were presented in this study.Keywords: Corneal endothelial cell, parameters, age, gender, smoking, Thailand

  16. Differential adhesion of tumor cells to capillary endothelial cells in vitro.

    OpenAIRE

    Alby, L; Auerbach, R

    1984-01-01

    Adhesion studies were carried out to determine the relative ability of glioma cells and ovary-derived teratoma cells to adhere to endothelial cells obtained from mouse brain capillaries (designated MBE cell line) or mouse ovaries (designated MOE cell line). The teratoma cells showed preferential adhesion to MOE cells, whereas the glioma cells showed preferential adhesion to the MBE cell line. In contrast, the glioma and teratoma cells adhered equally to L929 and 3T3 fibroblasts. A testicular ...

  17. Single-cell analysis of endothelial morphogenesis in vivo

    Science.gov (United States)

    Yu, Jianxin A.; Castranova, Daniel; Pham, Van N.; Weinstein, Brant M.

    2015-01-01

    Vessel formation has been extensively studied at the tissue level, but the difficulty in imaging the endothelium with cellular resolution has hampered study of the morphogenesis and behavior of endothelial cells (ECs) in vivo. We are using endothelial-specific transgenes and high-resolution imaging to examine single ECs in zebrafish. By generating mosaics with transgenes that simultaneously mark endothelial nuclei and membranes we are able to definitively identify and study the morphology and behavior of individual ECs during vessel sprouting and lumen formation. Using these methods, we show that developing trunk vessels are composed of ECs of varying morphology, and that single-cell analysis can be used to quantitate alterations in morphology and dynamics in ECs that are defective in proper guidance and patterning. Finally, we use single-cell analysis of intersegmental vessels undergoing lumen formation to demonstrate the coexistence of seamless transcellular lumens and single or multicellular enclosed lumens with autocellular or intercellular junctions, suggesting that heterogeneous mechanisms contribute to vascular lumen formation in vivo. The tools that we have developed for single EC analysis should facilitate further rigorous qualitative and quantitative analysis of EC morphology and behavior in vivo. PMID:26253401

  18. SSeCKS/AKAP12 induces repulsion between human prostate cancer and microvessel endothelial cells through the activation of Semaphorin 3F.

    Science.gov (United States)

    Xie, Wen; Su, Wei; Zhang, Lijuan; Shang, Qingkun; Su, Bing

    2017-09-02

    Metastasis remains the primary cause of prostate cancer related death. Cancer cells need to contact endothelial cells and disrupt endothelial junctions to cross the endothelium for invasion and metastasis. The suppression of heterotypic repulsion between cancer and endothelial cells allows cancer cells to invade into the surrounding tissue. Here, we demonstrate that SSeCKS/AKAP12 induced repulsion between human prostate cancer and microvessel endothelial cells, which was mediated by an angiogenesis inhibitor Semaphorin 3F. Moreover, we examined AKAP12 and Semaphorin 3F mRNA expression in 42 prostate cancer and 30 benign prostatic hyperplasia tissue samples, and found that the expression of AKAP12 and Semaphorin 3F mRNA was inversely associated with the degree of aggressiveness of prostate cancer cells and tissues. An ordinal logistic regression analysis indicates that there is a positive association between the expression of AKAP12 and Semaphorin 3F in prostate cancer, suggesting that the activation of Semaphorin 3F by SSeCKS/AKAP12 may be involved in prostate cancer progression and metastasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. In vitro analysis of human periodontal microvascular endothelial cells.

    Science.gov (United States)

    Tsubokawa, Mizuki; Sato, Soh

    2014-08-01

    Endothelial cells (ECs) participate in key aspects of vascular biology, such as maintenance of capillary permeability, initiation of coagulation, and regulation of inflammation. According to previous reports, ECs have revealed highly specific characteristics depending on the organs and tissues. However, some reports have described the characteristics of the capillaries formed by human periodontal ECs. Therefore, the aim of the present study is to examine the functional characteristics of the periodontal microvascular ECs in vitro. Human periodontal ligament-endothelial cells (HPDL-ECs) and human gingiva-endothelial cells (HG-ECs) were isolated by immunoprecipitation with magnetic beads conjugated to a monoclonal anti-CD31 antibody. The isolated HPDL-ECs and HG-ECs were characterized to definitively demonstrate that these cell cultures represented pure ECs. Human umbilical-vein ECs and human dermal microvascular ECs were used for comparison. These cells were compared according to the proliferation potential, the formation of capillary-like tubes, the transendothelial electric resistance (TEER), and the expression of tight junction proteins. HPDL-ECs and HG-ECs with characteristic cobblestone monolayer morphology were obtained, as determined by light microscopy at confluence. Furthermore, the HPDL-ECs and HG-ECs expressed the EC markers platelet endothelial cell adhesion molecule-1 (also known as CD31), von Willebrand factor, and Ulex europaeus agglutinin 1, and the cells stained strongly positive for CD31 and CD309. In addition, the HPDL-ECs and HG-ECs were observed to form capillary-like tubes, and they demonstrated uptake of acetylated low-density lipoprotein. Functional analyses of the HPDL-ECs and HG-ECs showed that, compared to the control cells, tube formation persisted for only a brief period of time, and TEER was substantially reduced at confluence. Furthermore, the cells exhibited delocalization of zonula occludens-1 and occludin at cell-cell contact sites

  20. Pulmonary artery endothelial cell dysfunction and decreased populations of highly proliferative endothelial cells in experimental congenital diaphragmatic hernia

    Science.gov (United States)

    Seedorf, Gregory J.; Abman, Steven H.; Nozik-Grayck, Eva; Partrick, David A.; Gien, Jason

    2013-01-01

    Decreased lung vascular growth and pulmonary hypertension contribute to poor outcomes in congenital diaphragmatic hernia (CDH). Mechanisms that impair angiogenesis in CDH are poorly understood. We hypothesize that decreased vessel growth in CDH is caused by pulmonary artery endothelial cell (PAEC) dysfunction with loss of a highly proliferative population of PAECs (HP-PAEC). PAECs were harvested from near-term fetal sheep that underwent surgical disruption of the diaphragm at 60–70 days gestational age. Highly proliferative potential was measured via single cell assay. PAEC function was assessed by assays of growth and tube formation and response to known proangiogenic stimuli, vascular endothelial growth factor (VEGF), and nitric oxide (NO). Western blot analysis was used to measure content of angiogenic proteins, and superoxide production was assessed. By single cell assay, the proportion of HP-PAEC with growth of >1,000 cells was markedly reduced in the CDH PAEC, from 29% (controls) to 1% (CDH) (P CDH PAEC growth and tube formation were decreased by 31% (P = 0.012) and 54% (P CDH PAEC growth and tube formation. VEGF and VEGF-R2 proteins were increased in CDH PAEC; however, eNOS and extracellular superoxide dismutase proteins were decreased by 29 and 88%, respectively. We conclude that surgically induced CDH in fetal sheep causes endothelial dysfunction and marked reduction of the HP-PAEC population. We speculate that this CDH PAEC phenotype contributes to impaired vascular growth in CDH. PMID:24124189

  1. Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

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    Kathryn L McCabe

    Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

  2. Endothelial Cell Toxicity of Vancomycin Infusion Combined with Other Antibiotics

    Science.gov (United States)

    Drouet, Maryline; Chai, Feng; Barthélémy, Christine; Lebuffe, Gilles; Debaene, Bertrand; Odou, Pascal

    2015-01-01

    French guidelines recommend central intravenous (i.v.) infusion for high concentrations of vancomycin, but peripheral intravenous (p.i.v.) infusion is often preferred in intensive care units. Vancomycin infusion has been implicated in cases of phlebitis, with endothelial toxicity depending on the drug concentration and the duration of the infusion. Vancomycin is frequently infused in combination with other i.v. antibiotics through the same administrative Y site, but the local toxicity of such combinations has been poorly evaluated. Such an assessment could improve vancomycin infusion procedures in hospitals. Human umbilical vein endothelial cells (HUVEC) were challenged with clinical doses of vancomycin over 24 h with or without other i.v. antibiotics. Cell death was measured with the alamarBlue test. We observed an excess cellular death rate without any synergistic effect but dependent on the numbers of combined infusions when vancomycin and erythromycin or gentamicin were infused through the same Y site. Incompatibility between vancomycin and piperacillin-tazobactam was not observed in our study, and rinsing the cells between the two antibiotic infusions did not reduce endothelial toxicity. No endothelial toxicity of imipenem-cilastatin was observed when combined with vancomycin. p.i.v. vancomycin infusion in combination with other medications requires new recommendations to prevent phlebitis, including limiting coinfusion on the same line, reducing the infusion rate, and choosing an intermittent infusion method. Further studies need to be carried out to explore other drug combinations in long-term vancomycin p.i.v. therapy so as to gain insight into the mechanisms of drug incompatibility under multidrug infusion conditions. PMID:26055373

  3. Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention.

    Science.gov (United States)

    Uthamaraj, Susheil; Tefft, Brandon J; Hlinomaz, Ota; Sandhu, Gurpreet S; Dragomir-Daescu, Dan

    2015-09-18

    Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

  4. Differential Adhesion of Tumor Cells to Capillary Endothelial Cells in vitro

    Science.gov (United States)

    Alby, Laverna; Auerbach, Robert

    1984-09-01

    Adhesion studies were carried out to determine the relative ability of glioma cells and ovary-derived teratoma cells to adhere to endothelial cells obtained from mouse brain capillaries (designated MBE cell line) or mouse ovaries (designated MOE cell line). The teratoma cells showed preferential adhesion to MOE cells, whereas the glioma cells showed preferential adhesion to the MBE cell line. In contrast, the glioma and teratoma cells adhered equally to L929 and 3T3 fibroblasts. A testicular teratoma with ovary-seeking properties in vivo also adhered preferentially to MOE cells, while the preference for MBE cells was shared by glioma cells with an endothelioma and a bladder tumor line. The endothelioma, interestingly, showed a marked preferential adhesion to 3T3 cells, thus distinguishing it from the glioma. The experiments demonstrate that capillary endothelial cells derived from different sources are not alike and that differences expressed at the cell surface of these cells can be distinguished by tumor cells.

  5. Fate of cerium dioxide nanoparticles in endothelial cells: exocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Strobel, Claudia, E-mail: Claudia.Strobel@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany); Oehring, Hartmut [Jena University Hospital – Friedrich Schiller University Jena, Institute of Anatomy II (Germany); Herrmann, Rudolf [University of Augsburg, Department of Physics (Germany); Förster, Martin [Jena University Hospital – Friedrich Schiller University Jena, Department of Internal Medicine I, Division of Pulmonary Medicine and Allergy/Immunology (Germany); Reller, Armin [University of Augsburg, Department of Physics (Germany); Hilger, Ingrid, E-mail: ingrid.hilger@med.uni-jena.de [Jena University Hospital – Friedrich Schiller University Jena, Department of Experimental Radiology, Institute of Diagnostic and Interventional Radiology (Germany)

    2015-05-15

    Although cytotoxicity and endocytosis of nanoparticles have been the subject of numerous studies, investigations regarding exocytosis as an important mechanism to reduce intracellular nanoparticle accumulation are rather rare and there is a distinct lack of knowledge. The current study investigated the behavior of human microvascular endothelial cells to exocytose cerium dioxide (CeO{sub 2}) nanoparticles (18.8 nm) by utilization of specific inhibitors [brefeldin A; nocodazole; methyl-β-cyclodextrin (MβcD)] and different analytical methods (flow cytometry, transmission electron microscopy, inductively coupled plasma mass spectrometry). Overall, it was found that endothelial cells were able to release CeO{sub 2} nanoparticles via exocytosis after the migration of nanoparticle containing endosomes toward the plasma membrane. The exocytosis process occurred mainly by fusion of vesicular membranes with plasma membrane resulting in the discharge of vesicular content to extracellular environment. Nevertheless, it seems to be likely that nanoparticles present in the cytosol could leave the cells in a direct manner. MβcD treatment led to the strongest inhibition of the nanoparticle exocytosis indicating a significant role of the plasma membrane cholesterol content in the exocytosis process. Brefeldin A (inhibitor of Golgi-to-cell-surface-transport) caused a higher inhibitory effect on exocytosis than nocodazole (inhibitor of microtubules). Thus, the transfer from distal Golgi compartments to the cell surface influenced the exocytosis process of the CeO{sub 2} nanoparticles more than the microtubule-associated transport. In conclusion, endothelial cells, which came in contact with nanoparticles, e.g., after intravenously applied nano-based drugs, can regulate their intracellular nanoparticle amount, which is necessary to avoid adverse nanoparticle effects on cells.

  6. Static and dynamic light scattering of healthy and malaria-parasite invaded red blood cells

    Science.gov (United States)

    Park, Yongkeun; Diez-Silva, Monica; Fu, Dan; Popescu, Gabriel; Choi, Wonshik; Barman, Ishan; Suresh, Subra; Feld, Michael S.

    2010-03-01

    We present the light scattering of individual Plasmodium falciparum-parasitized human red blood cells (Pf-RBCs), and demonstrate progressive alterations to the scattering signal arising from the development of malaria-inducing parasites. By selectively imaging the electric fields using quantitative phase microscopy and a Fourier transform light scattering technique, we calculate the light scattering maps of individual Pf-RBCs. We show that the onset and progression of pathological states of the Pf-RBCs can be clearly identified by the static scattering maps. Progressive changes to the biophysical properties of the Pf-RBC membrane are captured from dynamic light scattering.

  7. Regulation of vascular endothelial cell polarization and migration by Hsp70/Hsp90-organizing protein.

    Science.gov (United States)

    Li, Jingyu; Sun, Xiaodong; Wang, Zaizhu; Chen, Li; Li, Dengwen; Zhou, Jun; Liu, Min

    2012-01-01

    Hsp70/Hsp90-organizing protein (HOP) is a member of the co-chaperone family, which directly binds to chaperones to regulate their activities. The participation of HOP in cell motility and endothelial cell functions remains largely unknown. In this study, we demonstrate that HOP is critically involved in endothelial cell migration and angiogenesis. Tube formation and capillary sprouting experiments reveal that depletion of HOP expression significantly inhibits vessel formation from endothelial cells. Wound healing and transwell migration assays show that HOP is important for endothelial cell migration. By examination of centrosome reorientation and membrane ruffle dynamics, we find that HOP plays a crucial role in the establishment of cell polarity in response to migratory stimulus. Furthermore, our data show that HOP interacts with tubulin and colocalizes with microtubules in endothelial cells. These findings indicate HOP as a novel regulator of angiogenesis that functions through promoting vascular endothelial cell polarization and migration.

  8. Antioxidant Effects of Sheep Whey Protein on Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Efthalia Kerasioti

    2016-01-01

    Full Text Available Excessive production of reactive oxygen species (ROS may cause endothelial dysfunction and consequently vascular disease. In the present study, the possible protective effects of sheep whey protein (SWP from tert-butyl hydroperoxide- (tBHP- induced oxidative stress in endothelial cells (EA.hy926 were assessed using oxidative stress biomarkers. These oxidative stress biomarkers were glutathione (GSH and ROS levels determined by flow cytometry. Moreover, thiobarbituric acid-reactive substances (TBARS, protein carbonyls (CARB, and oxidized glutathione (GSSG were determined spectrophotometrically. The results showed that SWP at 0.78, 1.56, 3.12, and 6.24 mg of protein mL−1 increased GSH up to 141%, while it decreased GSSG to 46.7%, ROS to 58.5%, TBARS to 52.5%, and CARB to 49.0%. In conclusion, the present study demonstrated for the first time that SWP protected endothelial cells from oxidative stress. Thus, SWP may be used for developing food supplements or biofunctional foods to attenuate vascular disturbances associated with oxidative stress.

  9. Pulmonary endothelial cell DNA methylation signature in pulmonary arterial hypertension.

    Science.gov (United States)

    Hautefort, Aurélie; Chesné, Julie; Preussner, Jens; Pullamsetti, Soni S; Tost, Jorg; Looso, Mario; Antigny, Fabrice; Girerd, Barbara; Riou, Marianne; Eddahibi, Saadia; Deleuze, Jean-François; Seeger, Werner; Fadel, Elie; Simonneau, Gerald; Montani, David; Humbert, Marc; Perros, Frédéric

    2017-08-08

    Pulmonary arterial hypertension (PAH) is a severe and incurable pulmonary vascular disease. One of the primary origins of PAH is pulmonary endothelial dysfunction leading to vasoconstriction, aberrant angiogenesis and smooth muscle cell proliferation, endothelial-to-mesenchymal transition, thrombosis and inflammation. Our objective was to study the epigenetic variations in pulmonary endothelial cells (PEC) through a specific pattern of DNA methylation. DNA was extracted from cultured PEC from idiopathic PAH ( n = 11), heritable PAH ( n = 10) and controls ( n = 18). DNA methylation was assessed using the Illumina HumanMethylation450 Assay. After normalization, samples and probes were clustered according to their methylation profile. Differential clusters were functionally analyzed using bioinformatics tools. Unsupervised hierarchical clustering allowed the identification of two clusters of probes that discriminates controls and PAH patients. Among 147 differential methylated promoters, 46 promoters coding for proteins or miRNAs were related to lipid metabolism. Top 10 up and down-regulated genes were involved in lipid transport including ABCA1, ABCB4, ADIPOQ, miR-26A, BCL2L11. NextBio meta-analysis suggested a contribution of ABCA1 in PAH. We confirmed ABCA1 mRNA and protein downregulation specifically in PAH PEC by qPCR and immunohistochemistry and made the proof-of-concept in an experimental model of the disease that its targeting may offer novel therapeutic options. In conclusion, DNA methylation analysis identifies a set of genes mainly involved in lipid transport pathway which could be relevant to PAH pathophysiology.

  10. Enhanced adhesion of early endothelial progenitor cells to radiation-induced senescence-like vascular endothelial cells in vitro

    International Nuclear Information System (INIS)

    Sermsathanasawadi, N.; Inoue, Yoshinori; Iwai, Takehisa; Ishii, Hideto; Yoshida, Masayuki; Igarashi, Kaori; Miura, Masahiko

    2009-01-01

    The effects of ionizing radiation (IR) on tumor neovascularization are still unclear. We previously reported that vascular endothelial cells (ECs) expressing the IR-induced senescence-like (IRSL) phenotype exhibit a significant decrease in angiogenic activity in vitro. In this study, we examined the effects of the IRSL phenotype on adhesion to early endothelial progenitor cells (early EPCs). Adhesion of human peripheral blood-derived early EPCs to human umbilical vein endothelial cells (HUVECs) expressing the IRSL phenotype was evaluated by an adhesion assay under static conditions. It was revealed that the IRSL HUVECs supported significantly more adhesion of early EPCs than normal HUVECs. Expressions of ICAM-1, VCAM-1 and E-selectin were up-regulated in IRSL HUVECs. Pre-treatment of IRSL HUVECs with adhesion-blocking monoclonal antibodies against E-selectin and VCAM-1 significantly reduced early EPC adhesion to IRSL HUVECs, suggesting a potential role for the E-selectin and VCAM-1 in the adhesion between IRSL ECs and early EPCs. Therefore, the IRSL phenotype expressed in ECs may enhance neovascularization via increased homing of early EPCs. Our findings are first to implicate the complex effects of this phenotype on tumor neovascularization following irradiation. (author)

  11. Heparin: Effects upon the Glycocalyx and Endothelial Cells.

    Science.gov (United States)

    Spiess, Bruce D

    2017-09-01

    Unfractionated heparin (UFH) is the most widely used injectable medication in the United States. UFH is a poly-dispersed, relatively impure combination of many polysaccharides known as a glycosaminoglycan. It is used as the primary anticoagulant for heart surgery as well as for active treatment of deep venous thrombosis, vascular thrombosis, stroke, and many other potentially catastrophic clotting syndromes. Many perfusionists and cardiac team members know little of the biology of UFH other than its use for cardiopulmonary bypass. UFH is very similar to heparin sulfate, found on the surface of endothelial cells. Heparan sulfate protects endothelial surfaces from inflammatory attack and serves as a mechano-transducer for vascular shear. UFH and all glycosaminoglycans have far reaching pleotropic actions. This review elaborates on some of fascinating unique biology of these polysaccharides. Perhaps a number of the complex complications attributed to CPB are either caused by, or set up to occur by the complicated biology of UFH?

  12. Modeling human endothelial cell transformation in vascular neoplasias.

    Science.gov (United States)

    Wen, Victoria W; MacKenzie, Karen L

    2013-09-01

    Endothelial cell (EC)-derived neoplasias range from benign hemangioma to aggressive metastatic angiosarcoma, which responds poorly to current treatments and has a very high mortality rate. The development of treatments that are more effective for these disorders will be expedited by insight into the processes that promote abnormal proliferation and malignant transformation of human ECs. The study of primary endothelial malignancy has been limited by the rarity of the disease; however, there is potential for carefully characterized EC lines and animal models to play a central role in the discovery, development and testing of molecular targeted therapies for vascular neoplasias. This review describes molecular alterations that have been identified in EC-derived neoplasias, as well as the processes that underpin the immortalization and tumorigenic conversion of ECs. Human EC lines, established through the introduction of defined genetic elements or by culture of primary tumor tissue, are catalogued and discussed in relation to their relevance as models of vascular neoplasia.

  13. In-vivo cell tracking to quantify endothelial cell migration during zebrafish angiogenesis

    Science.gov (United States)

    Menon, Prahlad G.; Rochon, Elizabeth R.; Roman, Beth L.

    2016-03-01

    The mechanism of endothelial cell migration as individual cells or collectively while remaining an integral component of a functional blood vessel has not been well characterized. In this study, our overarching goal is to define an image processing workflow to facilitate quantification of how endothelial cells within the first aortic arch and are proximal to the zebrafish heart behave in response to the onset of flow (i.e. onset of heart beating). Endothelial cell imaging was conducted at this developmental time-point i.e. ~24-28 hours post fertilization (hpf) when flow first begins, using 3D+time two-photon confocal microscopy of a live, wild-type, transgenic, zebrafish expressing green fluorescent protein (GFP) in endothelial cell nuclei. An image processing pipeline comprised of image signal enhancement, median filtering for speckle noise reduction, automated identification of the nuclei positions, extraction of the relative movement of nuclei between consecutive time instances, and finally tracking of nuclei, was designed for achieving the tracking of endothelial cell nuclei and the identification of their movement towards or away from the heart. Pilot results lead to a hypothesis that upon the onset of heart beat and blood flow, endothelial cells migrate collectively towards the heart (by 21.51+/-10.35 μm) in opposition to blood flow (i.e. subtending 142.170+/-21.170 with the flow direction).

  14. Changes of junctions of endothelial cells in coronary sclerosis: A review

    Directory of Open Access Journals (Sweden)

    Li-Zi Zhang

    2016-03-01

    Full Text Available Atherosclerosis, the major cause of cardiovascular diseases, has been a leading contributor to morbidity and mortality in the United States and it has been on the rise globally. Endothelial cell–cell junctions are critical for vascular integrity and maintenance of vascular function. Endothelial cell junctions dysfunction is the onset step of future coronary events and coronary artery disease. Keywords: Coronary atherosclerosis, Junctions, Endothelial cells

  15. Angiocrine functions of organ-specific endothelial cells.

    Science.gov (United States)

    Rafii, Shahin; Butler, Jason M; Ding, Bi-Sen

    2016-01-21

    Endothelial cells that line capillaries are not just passive conduits for delivering blood. Tissue-specific endothelium establishes specialized vascular niches that deploy sets of growth factors, known as angiocrine factors. These cues participate actively in the induction, specification, patterning and guidance of organ regeneration, as well as in the maintainance of homeostasis and metabolism. When upregulated following injury, they orchestrate self-renewal and differentiation of tissue-specific resident stem and progenitor cells into functional organs. Uncovering the mechanisms by which organotypic endothelium distributes physiological levels of angiocrine factors both spatially and temporally will lay the foundation for clinical trials that promote organ repair without scarring.

  16. An angiogenin-binding protein from endothelial cells

    International Nuclear Information System (INIS)

    Hu, Guofu; Chang, Sooik; Riordan, J.F.; Vallee, B.L.

    1991-01-01

    A 42-kDa bovine protein that binds bovine angiogenin [angiogenin binding protein (AngBP)] has been identified as a dissociable cell-surface component of calf pulmonary artery endothelial cells and a transformed bovine endothelial cell line, GM7373. 125 I-Ang can be crosslinked efficiently to AngBP by a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide. Bovine ribonuclease A competes with the binding of 125 I-Ang to AngBP, but lysozyme does not. Direct binding to AngBP of 125 I-labeled bovine ribonuclease A is, however, much weaker than that of 125 I-Ang. Two enzymatically active derivatives of angiogenin cleaved at residues 60-61 and 67-68, respectively, fail to induce angiogenesis and also bind to AngBP only weakly. AngBP has been isolated by treatment of cells with heparan sulfate, affinity chromatography on angiogenin-Sepharose of the material dissociated from the cell surface, and gel filtration HPLC. The results suggest that AngBP has the characteristics of a receptor that may likely function in angiogenesis

  17. Differentiation of Umbilical Cord Lining Membrane-Derived Mesenchymal Stem Cells into Endothelial-Like Cells

    Science.gov (United States)

    Chung Doan, Chinh; Long Le, Thanh; Son Hoang, Nghia; Trung Doan, Ngoc; Dong Le, Van; Si Do, Minh

    2014-01-01

    Background: Stem cell therapy for the treatment of vascular-related diseases through functional revascularization is one of the most important research areas in tissue engineering. The aim of this study was to investigate the in vitro differentiation of umbilical CL-MSC into endothelial lineage cells. Methods: In this study, isolated cells were characterized for expression of MSC-specific markers and osteogenic and adipogenic differentiation. They were induced to differentiate into endothelial-like cells and then examined for expression of the endothelial-specific markers, karyotype, and functional behavior of cells. Results: Isolated cells expressed MSC-specific markers and differentiated into adipocytes and osteoblasts. After endothelial differentiation, they expressed CD31, vWF, VE-cadherin, VEGFR1, and VEGFR2 at both mRNA and protein level, but their morphological changes were not apparent when compared with those of undifferentiated cells. There were no significant changes in karyotype of differentiated cells. Furthermore, angiogenesis assay and LDL uptake assay showed that differentiated cells were able to form the capillary-like structures and uptake LDL, respectively. Conclusion: The results indicated that umbilical CL-MSC could differentiate into functional endothelial-like cells. Also, they are suitable for basic and clinical studies to cure several vascular-related diseases. PMID:24518546

  18. Liver sinusoidal endothelial cells induce immunosuppressive IL-10-producing Th1 cells via the Notch pathway

    NARCIS (Netherlands)

    Neumann, Katrin; Rudolph, Christine; Neumann, Christian; Janke, Marko; Amsen, Derk; Scheffold, Alexander

    2015-01-01

    Under homeostasis, liver sinusoidal endothelial cells (LSECs) shift intrahepatic T-cell responses towards tolerance. However, the role of LSECs in the regulation of T-cell-induced liver inflammation is less clear. Here, we studied the capacity of LSECs to modulate pro-inflammatory Th1-cell

  19. Recombinant Treponema pallidum protein Tp0965 activates endothelial cells and increases the permeability of endothelial cell monolayer.

    Directory of Open Access Journals (Sweden)

    Rui-Li Zhang

    Full Text Available The recombinant Treponema pallidum protein Tp0965 (rTp0965, one of the many proteins derived from the genome of T. pallidum subsp. pallidum, shows strong immunogenicity and immunoreactivity. In this study, we investigated the effects of rTp0965 on the endothelial barrier. Treatment of human umbilical vein endothelial cells (HUVECs with rTp0965 resulted in increased levels of ICAM-1, E-selectin, and MCP-1 mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1 cells to HUVECs preincubated with rTp0965. In addition, rTp0965 induced reorganization of F-actin and decreased expression of claudin-1 in HUVECs. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced higher endothelial permeability as well as transendothelial migration of monocytes. These data indicate that Tp0965 protein may play an important role in the immunopathogenesis of syphilis.

  20. Endogenous Vascular Endothelial Growth Factor-A (VEGF-A) Maintains Endothelial Cell Homeostasis by Regulating VEGF Receptor-2 Transcription*

    Science.gov (United States)

    E, Guangqi; Cao, Ying; Bhattacharya, Santanu; Dutta, Shamit; Wang, Enfeng; Mukhopadhyay, Debabrata

    2012-01-01

    Vascular endothelial growth factor A (VEGF-A) is one of the most important factors controlling angiogenesis. Although the functions of exogenous VEGF-A have been widely studied, the roles of endogenous VEGF-A remain unclear. Here we focused on the mechanistic functions of endogenous VEGF-A in endothelial cells. We found that it is complexed with VEGF receptor 2 (VEGFR-2) and maintains a basal expression level for VEGFR-2 and its downstream signaling activation. Endogenous VEGF-A also controls expression of key endothelial specific genes including VEGFR-2, Tie-2, and vascular endothelial cadherin. Of importance, endogenous VEGF-A differs from exogenous VEGF-A by regulating VEGFR-2 transcription through mediation of FoxC2 binding to the FOX:ETS motif, and the complex formed by endogenous VEGF-A with VEGFR-2 is localized within the EEA1 (early endosome antigen 1) endosomal compartment. Taken together, our results emphasize the importance of endogenous VEGF-A in endothelial cells by regulating key vascular proteins and maintaining the endothelial homeostasis. PMID:22167188

  1. Maslinic acid inhibits proliferation of renal cell carcinoma cell lines and suppresses angiogenesis of endothelial cells

    Directory of Open Access Journals (Sweden)

    Parth Thakor

    2017-03-01

    Full Text Available Despite the introduction of many novel therapeutics in clinical practice, metastatic renal cell carcinoma (RCC remains a treatment-re-sistant cancer. As red and processed meat are considered risk factors for RCC, and a vegetable-rich diet is thought to reduce this risk, research into plant-based therapeutics may provide valuable complementary or alternative therapeutics for the management of RCC. Herein, we present the antiproliferative and antiangiogenic effects of maslinic acid, which occurs naturally in edible plants, particularly in olive fruits, and also in a variety of medicinal plants. Human RCC cell lines (ACHN, Caki-1, and SN12K1, endothelial cells (human umbilical vein endothelial cell line [HUVEC], and primary cultures of kidney proximal tubular epithelial cells (PTEC were treated with maslinic acid. Maslinic acid was relatively less toxic to PTEC when compared with RCC under similar experimental conditions. In RCC cell lines, maslinic acid induced a significant reduction in proliferation, proliferating cell nuclear antigen, and colony formation. In HUVEC, maslinic acid induced a significant reduction in capillary tube formation in vitro and vascular endothelial growth factor. This study provides a rationale for incorporating a maslinic acid–rich diet either to reduce the risk of developing kidney cancer or as an adjunct to existing antiangiogenic therapy to improve efficacy.

  2. Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

    International Nuclear Information System (INIS)

    Rubin, D.B.; Drab, E.A.; Bauer, K.D.

    1989-01-01

    Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: (1) cell volume; (2) cell cycle position; and (3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings (1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and (2) demonstrate EC subpopulations in culture

  3. Interactions between endothelial progenitor cells (EPC) and titanium implant surfaces.

    Science.gov (United States)

    Ziebart, Thomas; Schnell, Anne; Walter, Christian; Kämmerer, Peer W; Pabst, Andreas; Lehmann, Karl M; Ziebart, Johanna; Klein, Marc O; Al-Nawas, Bilal

    2013-01-01

    Endothelial cells play an important role in peri-implant angiogenesis during early bone formation. Therefore, interactions between endothelial progenitor cells (EPCs) and titanium dental implant surfaces are of crucial interest. The aim of our in vitro study was to investigate the reactions of EPCs in contact with different commercially available implant surfaces. EPCs from buffy coats were isolated by Ficoll density gradient separation. After cell differentiation, EPC were cultured for a period of 7 days on different titanium surfaces. The test surfaces varied in roughness and hydrophilicity: acid-etched (A), sand-blasted-blasted and acid-etched (SLA), hydrophilic A (modA), and hydrophilic SLA (modSLA). Plastic and fibronectin-coated plastic surfaces served as controls. Cell numbers and morphology were analyzed by confocal laser scanning microscopy. Secretion of vascular endothelial growth factor (VEGF)-A was measured by enzyme-linked immunosorbent assay and expressions of iNOS and eNOS were investigated by real-time polymerase chain reaction. Cell numbers were higher in the control groups compared to the cells of titanium surfaces. Initially, hydrophilic titanium surfaces (modA and modSLA) showed lower cell numbers than hydrophobic surfaces (A and SLA). After 7 days smoother surfaces (A and modA) showed increased cell numbers compared to rougher surfaces (SLA and modSLA). Cell morphology of A, modA, and control surfaces was characterized by a multitude of pseudopodia and planar cell soma architecture. SLA and modSLA promoted small and plump cell soma with little quantity of pseudopodia. The lowest VEGF level was measured on A, the highest on modSLA. The highest eNOS and iNOS expressions were found on modA surfaces. The results of this study demonstrate that biological behaviors of EPCs can be influenced by different surfaces. The modSLA surface promotes an undifferentiated phenotype of EPCs that has the ability to secrete growth factors in great quantities. In

  4. A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells.

    Science.gov (United States)

    Ware, Brenton R; Durham, Mitchell J; Monckton, Chase P; Khetani, Salman R

    2018-03-01

    Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs. Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously. LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures. PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.

  5. Oleic acid increases mitochondrial reactive oxygen species production and decreases endothelial nitric oxide synthase activity in cultured endothelial cells.

    Science.gov (United States)

    Gremmels, Hendrik; Bevers, Lonneke M; Fledderus, Joost O; Braam, Branko; van Zonneveld, Anton Jan; Verhaar, Marianne C; Joles, Jaap A

    2015-03-15

    Elevated plasma levels of free fatty acids (FFA) are associated with increased cardiovascular risk. This may be related to FFA-induced elevation of oxidative stress in endothelial cells. We hypothesized that, in addition to mitochondrial production of reactive oxygen species, endothelial nitric oxide synthase (eNOS)-mediated reactive oxygen species production contributes to oleic acid (OA)-induced oxidative stress in endothelial cells, due to eNOS uncoupling. We measured reactive oxygen species production and eNOS activity in cultured endothelial cells (bEnd.3) in the presence of OA bound to bovine serum albumin, using the CM-H2DCFDA assay and the L-arginine/citrulline conversion assay, respectively. OA induced a concentration-dependent increase in reactive oxygen species production, which was inhibited by the mitochondrial complex II inhibitor thenoyltrifluoroacetone (TTFA). OA had little effect on eNOS activity when stimulated by a calcium-ionophore, but decreased both basal and insulin-induced eNOS activity, which was restored by TTFA. Pretreatment of bEnd.3 cells with tetrahydrobiopterin (BH4) prevented OA-induced reactive oxygen species production and restored inhibition of eNOS activity by OA. Elevation of OA levels leads to both impairment in receptor-mediated stimulation of eNOS and to production of mitochondrial-derived reactive oxygen species and hence endothelial dysfunction. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Functional and gene expression analysis of hTERT overexpressed endothelial cells

    Directory of Open Access Journals (Sweden)

    Haruna Takano

    2008-09-01

    Full Text Available Haruna Takano1, Satoshi Murasawa1,2, Takayuki Asahara1,2,31Institute of Biomedical Research and Innovation, Kobe, Japan; 2RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; 3Tokai University of School of Medicine, Tokai, JapanAbstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT overexpressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt pathway

  7. Barrier Functionality of Porcine and Bovine Brain Capillary Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ailar Nakhlband

    2011-09-01

    Full Text Available Introduction: To date, isolated cell based blood-brain barrier (BBB models have been widely used for brain drug delivery and targeting, due to their relatively proper bioelectrical and permeability properties. However, primary cultures of brain capillary endothelial cells (BCECs isolated from different species vary in terms of bioelectrical and permeability properties. Methods: To pursue this, in the current investigation, primary porcine and bovine BCECs (PBCECs and BBCECs, respectively were isolated and used as an in vitro BBB model. The bioelectrical and permeability properties were assessed in BCECs co-cultured with C6 cells with/without hydrocortisone (550 nM. The bioelectrical properties were further validated by means of the permeability coefficients of transcellular and paracellular markers. Results: The primary PBCECs displayed significantly higher trans-endothelial electrical resistance (~900 W.cm2 than BBCECs (~700 W.cm2 - both co-cultured with C6 cells in presence of hydrocortisone. Permeability coefficients of propranolol/diazepam and mannitol/sucrose in PBCECs were ~21 and ~2 (×10-6 cm.sec-1, where these values for BBCECs were ~25 and ~5 (×10-6 cm.sec-1. Conclusion: Upon our bioelectrical and permeability findings, both models display discriminative barrier functionality but porcine BCECs seem to provide a better platform than bovine BCECs for drug screening and brain targeting.

  8. A novel minimally-invasive method to sample human endothelial cells for molecular profiling.

    Directory of Open Access Journals (Sweden)

    Stephen W Waldo

    Full Text Available The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity.Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34/CD105/CD146 with the concomitant absence of leukocyte and platelet specific markers (CD11b/CD45. Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR.A median of 4,212 (IQR: 2161-6583 endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001, nitric oxide synthase 3 (NOS3, P<0.001 and vascular cell adhesion molecule 1 (VCAM-1, P<0.003 in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001.This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.

  9. Exogenous endothelial cells as accelerators of hematopoietic reconstitution

    Directory of Open Access Journals (Sweden)

    Mizer J

    2012-11-01

    Full Text Available Abstract Despite the successes of recombinant hematopoietic-stimulatory factors at accelerating bone marrow reconstitution and shortening the neutropenic period post-transplantation, significant challenges remain such as cost, inability to reconstitute thrombocytic lineages, and lack of efficacy in conditions such as aplastic anemia. A possible means of accelerating hematopoietic reconstitution would be administration of cells capable of secreting hematopoietic growth factors. Advantages of this approach would include: a ability to regulate secretion of cytokines based on biological need; b long term, localized production of growth factors, alleviating need for systemic administration of factors that possess unintended adverse effects; and c potential to actively repair the hematopoietic stem cell niche. Here we overview the field of hematopoietic growth factors, discuss previous experiences with mesenchymal stem cells (MSC in accelerating hematopoiesis, and conclude by putting forth the rationale of utilizing exogenous endothelial cells as a novel cellular therapy for acceleration of hematopoietic recovery.

  10. Jolkinolide A and Jolkinolide B Inhibit Proliferation of A549 Cells and Activity of Human Umbilical Vein Endothelial Cells.

    Science.gov (United States)

    Shen, Lei; Zhang, Shan-Qiang; Liu, Lei; Sun, Yu; Wu, Yu-Xuan; Xie, Li-Ping; Liu, Ji-Cheng

    2017-01-14

    BACKGROUND Jolkinolide A (JA) and Jolkinolide B (JB) are diterpenoids extracted from the roots of Euphorbia fischeriana Steud and have been shown to have anti-tumor activity. However, their effects on the ability of tumor cells to invade blood vessels and metastasize remain largely unknown. Investigations into the effects of JA and JB on the angiogenesis of tumor tissues may facilitate the identification of new natural drugs with anti-tumor growth and metastasis activities. MATERIAL AND METHODS We used different concentrations of JA and JB (20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml) to stimulate A549 cells and then studied the effects on the growth and metastasis of lung cancers. In addition, we used conditional media from A549 cells (A549-CM) stimulated by either JA or JB in different concentrations to culture human umbilical vein endothelial cells (HUVECs). RESULTS We found that both JA and JB significantly inhibited the Akt-STAT3-mTOR signaling pathway and reduced the expression of VEGF in A549 cells, but JB exhibited more significant inhibitory effects than JA. The JB-stimulated A549 cell conditional media had a greater inhibitory effect on the proliferation and migration of HUVECs than did the conditional media of JA-stimulated A549 cells. This effect gradually increased with increasing concentrations of either type of Jolkinolide. CONCLUSIONS Our results suggest that JA and JB inhibited VEGF expression in A549 cells through the inhibition of the Akt-STAT3-mTOR signaling pathway, and directly inhibited the proliferation and migration of HUVECs. These findings are of great significance for the development of new plant-derived chemotherapy agents for the treatment of cancer.

  11. Metabolic and structural integrity of magnetic nanoparticle-loaded primary endothelial cells for targeted cell therapy.

    Science.gov (United States)

    Orynbayeva, Zulfiya; Sensenig, Richard; Polyak, Boris

    2015-05-01

    To successfully translate magnetically mediated cell targeting from bench to bedside, there is a need to systematically assess the potential adverse effects of magnetic nanoparticles (MNPs) interacting with 'therapeutic' cells. Here, we examined in detail the effects of internalized polymeric MNPs on primary rat endothelial cells' structural intactness, metabolic integrity and proliferation potential. The intactness of cytoskeleton and organelles was studied by fluorescent confocal microscopy, flow cytometry and high-resolution respirometry. MNP-loaded primary endothelial cells preserve intact cytoskeleton and organelles, maintain normal rate of proliferation, calcium signaling and mitochondria energy metabolism. This study provides supportive evidence that MNPs at doses necessary for targeting did not induce significant adverse effects on structural integrity and functionality of primary endothelial cells - potential cell therapy vectors.

  12. Effects of blood products on inflammatory response in endothelial cells in vitro.

    Directory of Open Access Journals (Sweden)

    Martin Urner

    Full Text Available BACKGROUND: Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products. METHODS: The inflammatory response from pre-activated (endotoxin-stimulated and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC, platelet concentrates (PC and fresh frozen plasma (FFP was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated. RESULTS: Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product. CONCLUSION: Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells.

  13. Brassinosteroids inhibit in vitro angiogenesis in human endothelial cells

    Czech Academy of Sciences Publication Activity Database

    Rárová, L.; Zahler, S.; Liebl, J.; Kryštof, Vladimír; Sedlák, David; Bartůněk, Petr; Kohout, Ladislav; Strnad, Miroslav

    2012-01-01

    Roč. 77, č. 13 (2012), s. 1502-1509 ISSN 0039-128X R&D Projects: GA MŠk(CZ) LC06077 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z50520514; CEZ:AV0Z40550506 Keywords : Angiogenesis * Human umbilical vein endothelial cells * Migration Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.803, year: 2012

  14. Brief secondhand smoke exposure depresses endothelial progenitor cells activity and endothelial function: sustained vascular injury and blunted nitric oxide production.

    Science.gov (United States)

    Heiss, Christian; Amabile, Nicolas; Lee, Andrew C; Real, Wendy May; Schick, Suzaynn F; Lao, David; Wong, Maelene L; Jahn, Sarah; Angeli, Franca S; Minasi, Petros; Springer, Matthew L; Hammond, S Katharine; Glantz, Stanton A; Grossman, William; Balmes, John R; Yeghiazarians, Yerem

    2008-05-06

    This study sought to analyze the effects of acute secondhand smoke (SHS) exposure on the number and function of endothelial progenitor cells (EPCs) over 24 h. Secondhand smoke increases the risk of vascular disease and is a major public health concern, but the mechanism(s) of action are not fully understood. Healthy nonsmokers (age SEM 30.3 +/- 1.3 years, n = 10) were exposed to 30 min of SHS yielding cotinine levels commonly observed in passive smokers and to smokefree air on 2 separate days. Measurements were taken before exposure (baseline), immediately after (0 h), and at 1 h, 2.5 h, and 24 h after. The EPCs (CD133(+)/KDR(+), CD34(+)/KDR(+)) and endothelial microparticles (EMPs: CD31(+)/CD41(-), CD144(+), CD62e(+)) were determined in blood using flow cytometry. The EPC chemotaxis toward vascular endothelial growth factor was measured. Endothelial function was assessed as flow-mediated dilation (FMD) using ultrasound. Secondhand smoke exposure increased EPCs and plasma vascular endothelial growth factor and completely abolished EPC chemotaxis during 24 h after exposure. Secondhand smoke increased EMPs and decreased FMD. Although FMD returned to baseline at 2.5 h, EMPs and vascular endothelial growth factor levels remained elevated at 24 h, suggesting endothelial activation and injury with functional impairment of the vascular endothelium. Exposure to smokefree air had no effect. Incubation of EPCs from nonexposed subjects with plasma isolated from SHS-exposed subjects in vitro decreased chemotaxis by blockade of vascular endothelial growth factor-stimulated nitric oxide production. Brief exposure to real-world levels of SHS leads to sustained vascular injury characterized by mobilization of dysfunctional EPCs with blocked nitric oxide production. Our results suggest that SHS not only affects the vascular endothelium, but also the function of EPCs.

  15. Expression of glycoprotein VI in vascular endothelial cells.

    Science.gov (United States)

    Sun, Bing; Tao, Lian; Lin, Shihua; Calingasan, Noel Y; Li, Jess; Tandon, Narendra N; Yoshitake, Masuhiro; Kambayashi, Jun-ichi

    2003-06-01

    Glycoprotein (GP) VI, a collagen receptor, plays a important role in collagen-mediated platelet aggregation and adhesion. To date, GPVI expression has been found only in platelets and megakaryocytes. In the present studies, we have demonstrated that GPVI was also expressed in cultured human umbilical vein endothelial cells (HUVEC) at both transcript and protein levels. Using a GPVI-specific probe, a approximately 6-kb band was detected in HUVEC as well as in platelets and megakaryoblastic cell lines by Northern blotting. Using polyclonal antibodies raised against platelet GPVI peptides, the same size band (57 kDa) was labeled with convulxin (CVX) after immuo-precipitation in both HUVEC and platelet lysates. In addition, a approximately 70-kDa band was also labeled in HUVEC. Surface expression of GPVI in HUVEC was confirmed by flow cytometry with GPVI-specific IgG or by direct labeling with FITC-conjugated CVX. Since HUVEC lack FcRgamma chain that forms complex with GPVI in platelets for signaling process, the function of GPVI in vascular endothelial cells remains to be determined.

  16. Endothelial Progenitor Cells for Diagnosis and Prognosis in Cardiovascular Disease

    Directory of Open Access Journals (Sweden)

    Caterina Oriana Aragona

    2016-01-01

    Full Text Available Objective. To identify, evaluate, and synthesize evidence on the predictive power of circulating endothelial progenitor cells (EPCs in cardiovascular disease, through a systematic review of quantitative studies. Data Sources. MEDLINE was searched using keywords related to “endothelial progenitor cells” and “endothelium” and, for the different categories, respectively, “smoking”; “blood pressure”; “diabetes mellitus” or “insulin resistance”; “dyslipidemia”; “aging” or “elderly”; “angina pectoris” or “myocardial infarction”; “stroke” or “cerebrovascular disease”; “homocysteine”; “C-reactive protein”; “vitamin D”. Study Selection. Database hits were evaluated against explicit inclusion criteria. From 927 database hits, 43 quantitative studies were included. Data Syntheses. EPC count has been suggested for cardiovascular risk estimation in the clinical practice, since it is currently accepted that EPCs can work as proangiogenic support cells, maintaining their importance as regenerative/reparative potential, and also as prognostic markers. Conclusions. EPCs showed an important role in identifying cardiovascular risk conditions, and to suggest their evaluation as predictor of outcomes appears to be reasonable in different defined clinical settings. Due to their capability of proliferation, circulation, and the development of functional progeny, great interest has been directed to therapeutic use of progenitor cells in atherosclerotic diseases. This trial is registered with registration number: Prospero CRD42015023717.

  17. Behaviour of endothelial cells in a tridimensional in vitro environment.

    Science.gov (United States)

    Ayata, Raif Eren; Chabaud, Stéphane; Auger, Michèle; Pouliot, Roxane

    2015-01-01

    Angiogenesis is a fundamental process in healing, tumor growth, and a variety of medical conditions. For this reason, in vitro angiogenesis is an area of interest for researchers. Additionally, in vitro angiogenesis is important for the survival of prevascularized tissue-engineering models. The aim of this study was to observe the self-tubular organization behaviour of endothelial cells in the self-assembly method. In this study, bilayered and dermal substitutes were prepared using the self-assembly method. Histological, immunostaining, and biochemical tests were performed. The behavioural dynamics of endothelial cells in this biological environment of supportive cells were observed, as were the steps of the in vitro angiogenic cascade with self-organizing capillary-like structures formation. The epidermal component of the substitutes was seen to promote network expansion and density. It also increased the quantity of angiogenic factors (VEGF and Ang-1) without increasing the proinflammatory factor (IL-8). In addition, the increased MMP activity contributed to matrix degradation, which facilitated capillary formation.

  18. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik

    1997-01-01

    1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase...

  19. Circulating endothelial cells: a potential parameter of organ damage in sickle cell anemia?

    NARCIS (Netherlands)

    Strijbos, Michiel H.; Landburg, Precious P.; Nur, Erfan; Teerlink, Tom; Leebeek, Frank W. G.; Rijneveld, Anita W.; Biemond, Bart J.; Sleijfer, Stefan; Gratama, Jan W.; Duits, Ashley J.; Schnog, John-John B.

    2009-01-01

    Objective laboratory tools are needed to monitor developing organ damage in sickle cell disease (SCD). Circulating endothelial cells (CECs) are indicative of vascular injury. We determined whether elevated CEC can be detected in asymptomatic SCD with the CellSearch system and whether the CEC count

  20. [Circulating endothelial progenitor cell levels in treated hypertensive patients].

    Science.gov (United States)

    Maroun-Eid, C; Ortega-Hernández, A; Abad, M; García-Donaire, J A; Barbero, A; Reinares, L; Martell-Claros, N; Gómez-Garre, D

    2015-01-01

    Most optimally treated hypertensive patients still have an around 50% increased risk of any cardiovascular event, suggesting the possible existence of unidentified risk factors. In the last years there has been evidence of the essential role of circulating endothelial progenitor cells (EPCs) in the maintenance of endothelial integrity and function, increasing the interest in their involvement in cardiovascular disease. In this study, the circulating levels of EPCs and vascular endothelial growth factor (VEGF) are investigated in treated hypertensive patients with adequate control of blood pressure (BP). Blood samples were collected from treated hypertensive patients with controlled BP. Plasma levels of EPCs CD34+/KDR+ and CD34+/VE-cadherin+ were quantified by flow cytometry. Plasma concentration of VEGF was determined by ELISA. A group of healthy subjects without cardiovascular risk factors was included as controls. A total of 108 hypertensive patients were included (61±12 years, 47.2% men) of which 82.4% showed BP<140/90 mmHg, 91.7% and 81.5% controlled diabetes (HbA1c <7%) and cLDL (<130 or 100 mg/dL), respectively, and 85.2% were non-smokers. Around 45% of them were obese. Although patients had cardiovascular parameters within normal ranges, they showed significantly lower levels of CD34+/KDR+ and CD34+/VE-cadherin+ compared with healthy control group, although plasma VEGF concentration was higher in patients than in controls. Despite an optimal treatment, hypertensive patients show a decreased number of circulating EPCs that could be, at least in part, responsible for their residual cardiovascular risk, suggesting that these cells could be a therapeutic target. Copyright © 2015 SEHLELHA. Published by Elsevier España, S.L.U. All rights reserved.

  1. Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Vallon, Mario, E-mail: m.vallon@arcor.de [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany); Rohde, Franziska; Janssen, Klaus-Peter [Chirurgische Klinik und Poliklinik, Technische Universitaet Muenchen, Munich (Germany); Essler, Markus [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany)

    2010-02-01

    Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

  2. Radiation-induced inhibition of human endothelial cells replicating in culture

    International Nuclear Information System (INIS)

    DeGowin, R.L.; Lewis, L.J.; Mason, R.E.; Borke, M.K.; Hoak, J.C.

    1976-01-01

    The radiosensitivity of some tumors may depend upon the sensitivity of their microvasculature to radiation. Heretofore, the dose-response of human endothelial cells replicating in tissue culture has not been published. In studies reported here, we exposed flasks containing 4 to 7 x 10 4 genetically identical human endothelial cells to doses of x irradiation from 125 to 1000 rad. During the phase of logarithmic growth, cell counts were compared to those of an unirradiated control to construct a dose--response curve. Similar studies were performed with normal fibroblasts. We found that 160 rad suppressed endothelial cell replication by 37 percent. Although recovery was evident with doses of 500 rad, no net increase in cell number occurred in 3 weeks in flasks of endothelial cells that received 750 or 1000 rad. Fibroblasts were slightly less sensitive under these conditions. To our knowledge, this is the first report of a radiation dose--response curve for human endothelial cells replicating in culture

  3. Tumor cells secrete an angiogenic factor that stimulates basic fibroblast growth factor and urokinase expression in vascular endothelial cells

    NARCIS (Netherlands)

    Peverali, F.A.; Mandriota, S.J.; Ciana, P.; Marelli, R.; Quax, P.; Rifkin, D.B.; Della Valle, G.; Mignatti, P.

    1994-01-01

    Culture medium conditioned by human SK-Hep1 hepatoma cells or mouse S180 sarcoma cells rapidly up-regulates endothelial cell expression of basic fibroblast growth factor (bFGF) and induces formation of capillary-like structures by vascular endothelial cells grown on three-dimensional fibrin gels (in

  4. Endothelial-mesenchymal transition and its contribution to the emergence of stem cell phenotype

    Science.gov (United States)

    Medici, Damian; Kalluri, Raghu

    2012-01-01

    Vascular endothelial cells can demonstrate considerable plasticity to generate other cell types during embryonic development and disease progression. This process occurs through a cell differentiation mechanism known as endothelial-mesenchymal transition (EndMT). The generation of mesenchymal cells from endothelium is a crucial step in endothelial cell differentiation to several lineages including fibroblasts, myofibroblasts, mural cells, osteoblasts, chondrocytes, and adipocytes. Such differentiation patterns have been observed in systems of cardiac development, fibrosis, diabetic nephropathy, heterotopic ossification and cancer. Here we describe the EndMT program and discuss the current evidence of EndMT-mediated acquisition of stem cell characteristics and multipotent differentiation capabilities. PMID:22554794

  5. SNEV overexpression extends the life span of human endothelial cells

    International Nuclear Information System (INIS)

    Voglauer, Regina; Chang, Martina Wei-Fen; Dampier, Brigitta; Wieser, Matthias; Baumann, Kristin; Sterovsky, Thomas; Schreiber, Martin; Katinger, Hermann; Grillari, Johannes

    2006-01-01

    In a recent screening for genes downregulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients. Since these mRNA profiles suggested a role of SNEV in the regulation of cell proliferation, the effect of its overexpression was tested. Thereby, a significant extension of the cellular life span was observed, which was not caused by altered telomerase activity or telomere dynamics but rather by enhanced stress resistance. When SNEV overexpressing cells were treated with bleomycin or bleomycin combined with BSO, inducing DNA damage as well as reactive oxygen species, a significantly lower fraction of apoptotic cells was found in comparison to vector control cells. These data suggest that high levels of SNEV might extend the cellular life span by increasing the resistance to stress or by improving the DNA repair capacity of the cells

  6. BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

    Science.gov (United States)

    Mondragon, Albert A; Betts-Obregon, Brandi S; Moritz, Robert J; Parvathaneni, Kalpana; Navarro, Mary M; Kim, Hong Seok; Lee, Chi Fung; LeBaron, Richard G; Asmis, Reto; Tsin, Andrew T

    2015-01-01

    Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFβ to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFβ, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFβ-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFβ1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFβ1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFβ showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFβ, as well as TGFβ receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFβ or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFβ released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.

  7. Effect of vitamin D on endothelial progenitor cells function.

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    Yoav Hammer

    Full Text Available Endothelial progenitor cells (EPCs are a population of bone marrow-derived cells, which have an important role in the process of endothelialization and vascular repair following injury. Impairment of EPCs, which occurs in patients with diabetes, was shown to be related to endothelial dysfunction, coronary artery disease (CAD and adverse clinical outcomes. Recent evidence has shown that calcitriol, the active hormone of vitamin D, has a favorable impact on the endothelium and cardiovascular system. There is limited data on the effect of vitamin D on EPCs function.To examine the in vitro effects of Calcitriol on EPCs from healthy subjects and patients with diabetes.Fifty-one patients with type 2 diabetes (60±11 years, 40% women, HbA1C: 9.1±0.8% and 23 healthy volunteers were recruited. EPCs were isolated and cultured with and without calcitriol. The capacity of the cells to form colony-forming units (CFUs, their viability (measured by MTT assay, KLF-10 levels and angiogenic markers were evaluated after 1 week of culture.In diabetic patients, EPC CFUs and cell viability were higher in EPCs exposed to calcitriol vs. EPCs not exposed to calcitriol [EPC CFUs: 1.25 (IQR 1.0-2.0 vs. 0.5 (IQR 0.5-1.9, p < 0.001; MTT:0.62 (IQR 0.44-0.93 vs. 0.52 (IQR 0.31-0.62, p = 0.001]. KLF-10 levels tended to be higher in EPCs exposed to vitamin D, with no differences in angiopoietic markers. In healthy subjects, calcitriol supplementation also resulted in higher cell viability [MTT: 0.23 (IQR 0.11-0.46 vs. 0.19 (0.09-0.39, p = 0.04], but without differences in CFU count or angiopoietic markers.In patients with diabetes mellitus, in vitro vitamin D supplementation improved EPCs capacity to form colonies and viability. Further studies regarding the mechanisms by which vitamin D exerts its effect are required.

  8. Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

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    Hannah J Levis

    Full Text Available Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.

  9. Double suicide genes selectively kill human umbilical vein endothelial cells

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    Liu Lunxu

    2011-02-01

    Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful

  10. LPS-mediated endothelial activation in pulmonary endothelial cells: role of Nox2-dependent IKK-β phosphorylation

    Science.gov (United States)

    Menden, Heather; Tate, Everett; Hogg, Neil

    2013-01-01

    Lipopolysaccharide (LPS)-mediated endothelial activation contributes to lung inflammation and alveolar remodeling seen in premature infants with bronchopulmonary dysplasia (BPD). The mechanisms underlying LPS-mediated oxidative stress and proinflammatory signaling in human pulmonary microvascular endothelial cells (HPMEC) remain unclear. We hypothesized that NADPH oxidase (Nox) mediates LPS-induced endothelial activation in HPMEC by regulating phosphorylation of Toll-like receptor (TLR) pathway proteins. LPS-induced expression of intercellular adhesion molecule 1 (ICAM-1) was associated with increased 2-OH-E+ (marker for superoxide formation) levels and was attenuated by apocynin and the Nox inhibitor, VAS2870. LPS triggered membrane translocation of p67phox, suggesting activation of Nox2. Silencing Nox2, but not Nox4, suppressed LPS-induced ICAM-1 expression in HPMEC. Immunoprecipitation studies showed that inhibitor of κ-B kinase-β (IKK-β) serine phosphorylation induced by LPS was inhibited by Nox2 silencing. We examined whether Nox2-dependent, LPS-mediated IKK-β phosphorylation was regulated by protein phosphatase 2A (PP2A) or TGF-β associated kinase-1 (TAK1) in HPMEC. LPS increased PP2A activity in HPMEC, and inhibition of PP2A did not alter LPS-mediated ICAM-1 expression but attenuated IKK-β phosphorylation. TAK1 inhibition decreased LPS-induced ICAM-1 expression in HPMEC, and Nox2 silencing attenuated LPS-mediated TAK1 phosphorylation (Thr184/187). We demonstrate that Nox2 regulates LPS-mediated endothelial activation in pulmonary endothelial cells by modulating phosphorylation of key kinases in the TLR signaling cascade. Our data support a novel mechanism by which Nox-dependent signaling regulates proinflammatory signaling in pulmonary endothelial cells. Inhibition of vascular Nox may potentially limit lung injury and alveolar remodeling caused by infections in BPD. PMID:23333803

  11. The Impact of Microgravity and Hypergravity on Endothelial Cells

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    Jeanette A. M. Maier

    2015-01-01

    Full Text Available The endothelial cells (ECs, which line the inner surface of vessels, play a fundamental role in maintaining vascular integrity and tissue homeostasis, since they regulate local blood flow and other physiological processes. ECs are highly sensitive to mechanical stress, including hypergravity and microgravity. Indeed, they undergo morphological and functional changes in response to alterations of gravity. In particular microgravity leads to changes in the production and expression of vasoactive and inflammatory mediators and adhesion molecules, which mainly result from changes in the remodelling of the cytoskeleton and the distribution of caveolae. These molecular modifications finely control cell survival, proliferation, apoptosis, migration, and angiogenesis. This review summarizes the state of the art on how microgravity and hypergravity affect cultured ECs functions and discusses some controversial issues reported in the literature.

  12. Integrin Targeting and Toxicological Assessment of Peptide-Conjugated Liposome Delivery Systems to Activated Endothelial Cells

    DEFF Research Database (Denmark)

    Kermanizadeh, Ali; Villadsen, Klaus; Østrem, Ragnhild Garborg

    2017-01-01

    constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide-conjugated liposomes induced only cytotoxicity at the highest concentration in non-activated or activated endothelial cells, as well as in co-culture of endothelial cells...... and macrophages. There was unaltered secretion of cytokines following exposure of peptide-conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5β1) were more effective in targeting of activated....... Therefore, this study demonstrates the feasibility of constructing a peptide-conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells....

  13. Microtubules Growth Rate Alteration in Human Endothelial Cells

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    Irina B. Alieva

    2010-01-01

    Full Text Available To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with “normal” (similar to those in monolayer EC and “fast” (three times as much growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.

  14. Metabolic Responses in Endothelial Cells Following Exposure to Ketone Bodies

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    Erika Meroni

    2018-02-01

    Full Text Available The ketogenic diet (KD is a high-fat, low-carbohydrate diet based on the induction of the synthesis of ketone bodies (KB. Despite its widespread use, the systemic impact of KD is not completely understood. The purpose of this study was to evaluate the effects of physiological levels of KB on HMEC-1 endothelial cells. To this aim, DNA oxidative damage and the activation of Nrf2, a known transcriptional factor involved in cell responses to oxidative stress, were assessed. The exposure of cells to KB exerted a moderate genotoxic effect, measured by a significant increase in DNA oxidative damage. However, cells pre-treated with KB for 48 h and subjected to a secondary oxidative insult (H2O2, significantly decreased DNA damage compared to control oxidized cells. This protection occurred by the activation of Nrf2 pathway. In KB-treated cells, we found increased levels of Nrf2 in nuclear extracts and higher gene expression of HO-1, a target gene of Nrf2, compared to control cells. These results suggest that KB, by inducing moderate oxidative stress, activate the transcription factor Nrf2, which induces the transcription of target genes involved in the cellular antioxidant defense system.

  15. Metabolic Responses in Endothelial Cells Following Exposure to Ketone Bodies.

    Science.gov (United States)

    Meroni, Erika; Papini, Nadia; Criscuoli, Franca; Casiraghi, Maria C; Massaccesi, Luca; Basilico, Nicoletta; Erba, Daniela

    2018-02-22

    The ketogenic diet (KD) is a high-fat, low-carbohydrate diet based on the induction of the synthesis of ketone bodies (KB). Despite its widespread use, the systemic impact of KD is not completely understood. The purpose of this study was to evaluate the effects of physiological levels of KB on HMEC-1 endothelial cells. To this aim, DNA oxidative damage and the activation of Nrf2, a known transcriptional factor involved in cell responses to oxidative stress, were assessed. The exposure of cells to KB exerted a moderate genotoxic effect, measured by a significant increase in DNA oxidative damage. However, cells pre-treated with KB for 48 h and subjected to a secondary oxidative insult (H₂O₂), significantly decreased DNA damage compared to control oxidized cells. This protection occurred by the activation of Nrf2 pathway. In KB-treated cells, we found increased levels of Nrf2 in nuclear extracts and higher gene expression of HO-1, a target gene of Nrf2, compared to control cells. These results suggest that KB, by inducing moderate oxidative stress, activate the transcription factor Nrf2, which induces the transcription of target genes involved in the cellular antioxidant defense system.

  16. Endovascular treatment of unruptured intracranial aneurysms and circulating endothelial cells.

    Science.gov (United States)

    Vendrell, Jean-François; Cezar, Renaud; Kuster, Nils; Lobotesis, Kyriakos; Costalat, Vincent; Machi, Paolo; Bonafe, Alain; Vendrell, Jean-Pierre

    2013-04-01

    To evaluate the potential implication of circulating endothelial cells (CECs) in complications following endovascular treatment (EVT) of unruptured intracranial aneurysms. CECs characterized as CD146(+)/CD105(+)/CD45(-)/DAPI(+) were considered to originate from an altered endothelial cell layer of the vessel wall. In 15 patients, CECs were characterized and enumerated by the CellTracks(®) System in blood samples from: (1) femoral artery (FA), (2) internal carotid artery (ICA) before (ICA1) and after procedure (ICA2), and (3) a peripheral vein before (PV1) and after EVT (PV2). Ischemic brain events were assessed using diffusion weighted imaging (DWI-MRI) before and 24h after EVT. In ICA1, the median number of single CECs and clusters of 2-5 CECs were higher than in FA, ICA2, PV1 and PV2 samples (P5 cells, sometimes >50μm, were mainly observed in ICA1 and never in PV1, PV2 or PV samples from ten healthy subjects. This distribution of CECs suggested femoral and ICA injury by the devices used, leading to endothelium shearing and desquamation of CECs. All patients discharged on day two (NIHSS score=0), however silent ischemic brain lesions were observed in 9/15 (60%). EVT detaches single and clusters of CECs from wall arteries that may be implicated in silent ischemic brain lesions genesis. Enumeration of CECs associated with DWI-MRI might represent an interesting strategy for monitoring and optimizing endovascular devices, and further limit EVT-related complications. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. Endothelial progenitor cells display clonal restriction in multiple myeloma

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    Dai Kezhi

    2006-06-01

    Full Text Available Abstract Background In multiple myeloma (MM, increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI patterns in female patients by a human androgen receptor assay (HUMARA. In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. Methods A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (VH. Results In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele in 64% (n = 7. In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele. In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with VH primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5 of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status

  18. Endothelial progenitor cells display clonal restriction in multiple myeloma

    International Nuclear Information System (INIS)

    Braunstein, Marc; Özçelik, Tayfun; Bağişlar, Sevgi; Vakil, Varsha; Smith, Eric LP; Dai, Kezhi; Akyerli, Cemaliye B; Batuman, Olcay A

    2006-01-01

    In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (V H ). In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (≥ 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (≥ 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with V H primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI

  19. Delta- and gamma-tocotrienol isomers are potent in inhibiting inflammation and endothelial activation in stimulated human endothelial cells

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    Suhaila Muid

    2016-07-01

    Full Text Available Background: Tocotrienols (TCTs are more potent antioxidants than α-tocopherol (TOC. However, the effectiveness and mechanism of the action of TCT isomers as anti-atherosclerotic agents in stimulated human endothelial cells under inflammatory conditions are not well established. Aims: 1 To compare the effects of different TCT isomers on inflammation, endothelial activation, and endothelial nitric oxide synthase (eNOS. 2 To identify the two most potent TCT isomers in stimulated human endothelial cells. 3 To investigate the effects of TCT isomers on NFκB activation, and protein and gene expression levels in stimulated human endothelial cells. Methods: Human umbilical vein endothelial cells were incubated with various concentrations of TCT isomers or α-TOC (0.3–10 µM, together with lipopolysaccharides for 16 h. Supernatant cells were collected and measured for protein and gene expression of cytokines (interleukin-6, or IL-6; tumor necrosis factor-alpha, or TNF-α, adhesion molecules (intercellular cell adhesion molecule-1, or ICAM-1; vascular cell adhesion molecule-1, or VCAM-1; and e-selectin, eNOS, and NFκB. Results: δ-TCT is the most potent TCT isomer in the inhibition of IL-6, ICAM-1, VCAM-1, and NFκB, and it is the second potent in inhibiting e-selectin and eNOS. γ-TCT isomer is the most potent isomer in inhibiting e-selectin and eNOS, and it is the second most potent in inhibiting is IL-6, VCAM-1, and NFκB. For ICAM-1 protein expression, the most potent is δ-TCT followed by α-TCT. α- and β-TCT inhibit IL-6 at the highest concentration (10 µM but enhance IL-6 at lower concentrations. γ-TCT markedly increases eNOS expression by 8–11-fold at higher concentrations (5–10 µM but exhibits neutral effects at lower concentrations. Conclusion: δ- and γ-TCT are the two most potent TCT isomers in terms of the inhibition of inflammation and endothelial activation whilst enhancing eNOS, possibly mediated via the NFκB pathway. Hence

  20. Effects of amelogenins on angiogenesis-associated processes of endothelial cells

    DEFF Research Database (Denmark)

    Almqvist, S; Kleinman, H K; Werthén, M

    2011-01-01

    To study the effects of an amelogenin mixture on integrin-dependent adhesion, DNA synthesis and apoptosis of cultured human dermal microvascular endothelial cells and angiogenesis in an organotypic assay.......To study the effects of an amelogenin mixture on integrin-dependent adhesion, DNA synthesis and apoptosis of cultured human dermal microvascular endothelial cells and angiogenesis in an organotypic assay....

  1. Na(+), K(+)-ATPase dysfunction causes cerebrovascular endothelial cell degeneration in rat prefrontal cortex slice cultures.

    Science.gov (United States)

    Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Katsuki, Hiroshi

    2016-08-01

    Cerebrovascular endothelial cell dysfunction resulting in imbalance of cerebral blood flow contributes to the onset of psychiatric disorders such as depression, schizophrenia and bipolar disorder. Although decrease in Na(+), K(+)-ATPase activity has been reported in the patients with schizophrenia and bipolar disorder, the contribution of Na(+), K(+)-ATPase to endothelial cell dysfunction remains poorly understood. Here, by using rat neonatal prefrontal cortex slice cultures, we demonstrated that pharmacological inhibition of Na(+), K(+)-ATPase by ouabain induced endothelial cell injury. Treatment with ouabain significantly decreased immunoreactive area of rat endothelial cell antigen-1 (RECA-1), a marker of endothelial cells, in a time-dependent manner. Ouabain also decreased Bcl-2/Bax ratio and phosphorylation level of glycogen synthase kinase 3β (GSK3β) (Ser9), which were prevented by lithium carbonate. On the other hand, ouabain-induced endothelial cell injury was exacerbated by concomitant treatment with LY294002, an inhibitor of phosphoinositide 3- (PI3-) kinase. We also found that xestospongin C, an inhibitor of inositol triphosphate (IP3) receptor, but not SEA0400, an inhibitor of Na(+), Ca(2+) exchanger (NCX), protected endothelial cells from cytotoxicity of ouabain. These results suggest that cerebrovascular endothelial cell degeneration induced by Na(+), K(+)-ATPase inhibition resulting in Ca(2+) release from endoplasmic reticulum (ER) and activation of GSK3β signaling underlies pathogenesis of these psychiatric disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Recent advances in understanding the roles of vascular endothelial cells in allergic inflammation.

    Science.gov (United States)

    Shoda, Tetsuo; Futamura, Kyoko; Orihara, Kanami; Emi-Sugie, Maiko; Saito, Hirohisa; Matsumoto, Kenji; Matsuda, Akio

    2016-01-01

    Allergic disorders commonly involve both chronic tissue inflammation and remodeling caused by immunological reactions to various antigens on tissue surfaces. Due to their anatomical location, vascular endothelial cells are the final responders to interact with various exogenous factors that come into contact with the epithelial surface, such as pathogen-associated molecular patterns (PAMPs) and antigens. Recent studies have shed light on the important roles of endothelial cells in the development and exacerbation of allergic disorders. For instance, endothelial cells have the greatest potential to produce several key molecules that are deeply involved in allergic inflammation, such as periostin and thymus and activation-regulated chemokine (TARC/CCL17). Additionally, endothelial cells were recently shown to be important functional targets for IL-33--an essential regulator of allergic inflammation. Notably, almost all endothelial cell responses and functions involved in allergic inflammation are not suppressed by corticosteroids. These corticosteroid-refractory endothelial cell responses and functions include TNF-α-associated angiogenesis, leukocyte adhesion, IL-33-mediated responses and periostin and TARC production. Therefore, these unique responses and functions of endothelial cells may be critically involved in the pathogenesis of various allergic disorders, especially their refractory processes. Here, we review recent studies, including ours, which have elucidated previously unknown pathophysiological roles of vascular endothelial cells in allergic inflammation and discuss the possibility of endothelium-targeted therapy for allergic disorders. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  3. Complement Activation Induces Neutrophil Adhesion and Neutrophil-Platelet Aggregate Formation on Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Magdalena Riedl

    2017-01-01

    Discussion: Therefore, our findings of (i neutrophils adhering to complement-activated endothelial cells, (ii the formation of neutrophil-platelet aggregates on endothelial cells, and (iii the ability of aHUS serum to induce similar effects identify a possible role for neutrophils in aHUS manifestation.

  4. Corneal endothelial cell changes associated with cataract surgery in patients with type 2 diabetes mellitus

    DEFF Research Database (Denmark)

    Hugod, Mikkel; Storr-Paulsen, Allan; Norregaard, Jens Christian

    2011-01-01

    To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation.......To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation....

  5. In vivo transluminal microvascular endothelial cell seeding on balloon injured rabbit arteries.

    NARCIS (Netherlands)

    Arts, C.H.; Groot, P.G. de; Attevelt, N.; Heijnen-Snyder, G.J.; Verhagen, H.J.; Eikelboom, B.C.; Blankensteijn, J.D.

    2004-01-01

    AIM: Seeding venous endothelial cells (EC) onto damaged vascular surfaces attenuates the development of intimal hyperplasia. Unlike venous EC, fat derived microvascular endothelial cells (MVEC) do not require a culture step to increase the yield. The authors investigated whether fat derived MVEC are

  6. Coniferyl aldehyde attenuates radiation enteropathy by inhibiting cell death and promoting endothelial cell function.

    Science.gov (United States)

    Jeong, Ye-Ji; Jung, Myung Gu; Son, Yeonghoon; Jang, Jun-Ho; Lee, Yoon-Jin; Kim, Sung-Ho; Ko, Young-Gyo; Lee, Yun-Sil; Lee, Hae-June

    2015-01-01

    Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function.

  7. Haematopoietic stem cells and endothelial progenitor cells in healthy men: effect of aging and training.

    NARCIS (Netherlands)

    Thijssen, D.H.J.; Vos, J.B.; Verseyden, C.; Zonneveld, A.J. van; Smits, P.; Sweep, C.G.J.; Hopman, M.T.E.; Boer, H.C. de

    2006-01-01

    The number of hematopoietic stem cells (HSC) and endothelial progenitor cells (EPC) is thought to be a marker for neovascularization and vascular repair. Because physical inactivity and aging are risk factors for cardiovascular diseases, these factors may influence the numbers of HSCs and EPCs.

  8. Endothelial induced EMT in breast epithelial cells with stem cell properties

    DEFF Research Database (Denmark)

    Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla

    2011-01-01

    of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D...

  9. Effects of Nebivolol on Endothelial Gene Expression during Oxidative Stress in Human Umbilical Vein Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ulisse Garbin

    2008-01-01

    Full Text Available The endothelium plays a key role in the development of atherogenesis and its inflammatory and proliferative status influences the progression of atherosclerosis. The aim of this study is to compare the effects of two beta blockers such as nebivolol and atenolol on gene expression in human umbilical vein endothelial cells (HUVECs following an oxidant stimulus. HUVECs were incubated with nebivolol or atenolol (10 micromol/L for 24 hours and oxidative stress was induced by the addition of oxidized (ox-LDL. Ox-LDL upregulated adhesion molecules (ICAM-1, ICAM-2, ICAM-3, E-selectin, and P-selectin; proteins linked to inflammation (IL-6 and TNFalpha, thrombotic state (tissue factor, PAI-1 and uPA, hypertension such as endothelin-1 (ET-1, and vascular remodeling such as metalloproteinases (MMP-2, MMP-9 and protease inhibitor (TIMP-1. The exposure of HUVECs to nebivolol, but not to atenolol, reduced these genes upregulated by oxidative stress both in terms of protein and RNA expression. The known antioxidant properties of the third generation beta blocker nebivolol seem to account to the observed differences seen when compared to atenolol and support the specific potential protective role of this beta blocker on the expression of a number of genes involved in the initiation and progression of atherosclerosis.

  10. Cell-cell interactions mediate cytoskeleton organization and collective endothelial cell chemotaxis.

    Science.gov (United States)

    Shamloo, Amir

    2014-09-01

    This study investigates the role of cell-cell and cell-ligand interactions in cytoskeleton organization of endothelial cells (ECs) and their directional migration within a microfluidic device. The migration of ECs in response to a biochemical factor was studied. Mathematical analysis of the cell migration pathways and cellular cytoskeleton revealed that directional migration, migration persistence length, migration speed, and cytoskeletal stress fiber alignment can be mediated by the level of cell contacts as well as the presence or absence of a biochemical polarizing factor. It was shown that in the presence of a biochemical polarizing factor, higher cell density and more frequent cell contacts has a reinforcing effect on collective cell chemotaxis. In contrast, in the absence of a polarizing factor, high cell density can decrease or suppress the ability of the cells to migrate. Also, the correlation of actin stress fiber organization and alignment with directional migration of ECs was investigated. It was shown that in the presence of a biochemical polarizing factor, stress fibers within the cytoskeleton of ECs can be significantly aligned parallel to the gradient direction when the cells have higher level of contacts. The results also show that the organization and alignment of actin stress fibers is mediated by cell adhesion junctions during collective cell migration and introduce cell-cell interactions as a key factor during collective cell chemotaxis. © 2014 Wiley Periodicals, Inc.

  11. Endothelial Progenitor Cells in Diabetic Microvascular Complications: Friends or Foes?

    Directory of Open Access Journals (Sweden)

    Cai-Guo Yu

    2016-01-01

    Full Text Available Despite being featured as metabolic disorder, diabetic patients are largely affected by hyperglycemia-induced vascular abnormality. Accumulated evidence has confirmed the beneficial effect of endothelial progenitor cells (EPCs in coronary heart disease. However, antivascular endothelial growth factor (anti-VEGF treatment is the main therapy for diabetic retinopathy and nephropathy, indicating the uncertain role of EPCs in the pathogenesis of diabetic microvascular disease. In this review, we first illustrate how hyperglycemia induces metabolic and epigenetic changes in EPCs, which exerts deleterious impact on their number and function. We then discuss how abnormal angiogenesis develops in eyes and kidneys under diabetes condition, focusing on “VEGF uncoupling with nitric oxide” and “competitive angiopoietin 1/angiopoietin 2” mechanisms that are shared in both organs. Next, we dissect the nature of EPCs in diabetic microvascular complications. After we overview the current EPCs-related strategies, we point out new EPCs-associated options for future exploration. Ultimately, we hope that this review would uncover the mysterious nature of EPCs in diabetic microvascular disease for therapeutics.

  12. Endothelial Progenitor Cells in Diabetic Microvascular Complications: Friends or Foes?

    Science.gov (United States)

    Yu, Cai-Guo; Zhang, Ning; Yuan, Sha-Sha; Ma, Yan; Yang, Long-Yan; Feng, Ying-Mei; Zhao, Dong

    2016-01-01

    Despite being featured as metabolic disorder, diabetic patients are largely affected by hyperglycemia-induced vascular abnormality. Accumulated evidence has confirmed the beneficial effect of endothelial progenitor cells (EPCs) in coronary heart disease. However, antivascular endothelial growth factor (anti-VEGF) treatment is the main therapy for diabetic retinopathy and nephropathy, indicating the uncertain role of EPCs in the pathogenesis of diabetic microvascular disease. In this review, we first illustrate how hyperglycemia induces metabolic and epigenetic changes in EPCs, which exerts deleterious impact on their number and function. We then discuss how abnormal angiogenesis develops in eyes and kidneys under diabetes condition, focusing on "VEGF uncoupling with nitric oxide" and "competitive angiopoietin 1/angiopoietin 2" mechanisms that are shared in both organs. Next, we dissect the nature of EPCs in diabetic microvascular complications. After we overview the current EPCs-related strategies, we point out new EPCs-associated options for future exploration. Ultimately, we hope that this review would uncover the mysterious nature of EPCs in diabetic microvascular disease for therapeutics.

  13. Late Release of Circulating Endothelial Cells and Endothelial Progenitor Cells after Chemotherapy Predicts Response and Survival in Cancer Patients

    Directory of Open Access Journals (Sweden)

    Jeanine M. Roodhart

    2010-01-01

    Full Text Available We and others have previously demonstrated that the acute release of progenitor cells in response to chemotherapy actually reduces the efficacy of the chemotherapy. Here, we take these data further and investigate the clinical relevance of circulating endothelial (progenitor cells (CE(PCs and modulatory cytokines in patients after chemotherapy with relation to progression-free and overall survival (PFS/OS. Patients treated with various chemotherapeutics were included. Blood sampling was performed at baseline, 4 hours, and 7 and 21 days after chemotherapy. The mononuclear cell fraction was analyzed for CE(PC by FACS analysis. Plasma was analyzed for cytokines by ELISA or Luminex technique. CE(PCs were correlated with response and PFS/OS using Cox proportional hazard regression analysis. We measured CE(PCs and cytokines in 71 patients. Only patients treated with paclitaxel showed an immediate increase in endothelial progenitor cell 4 hours after start of treatment. These immediate changes did not correlate with response or survival. After 7 and 21 days of chemotherapy, a large and consistent increase in CE(PC was found (P < .01, independent of the type of chemotherapy. Changes in CE(PC levels at day 7 correlated with an increase in tumor volume after three cycles of chemotherapy and predicted PFS/OS, regardless of the tumor type or chemotherapy. These findings indicate that the late release of CE(PC is a common phenomenon after chemotherapeutic treatment. The correlation with a clinical response and survival provides further support for the biologic relevance of these cells in patients' prognosis and stresses their possible use as a therapeutic target.

  14. Catalase and superoxide dismutase conjugated with platelet-endothelial cell adhesion molecule antibody distinctly alleviate abnormal endothelial permeability caused by exogenous reactive oxygen species and vascular endothelial growth factor.

    Science.gov (United States)

    Han, Jingyan; Shuvaev, Vladimir V; Muzykantov, Vladimir R

    2011-07-01

    Reactive oxygen species (ROS) superoxide anion (O(2)()) and hydrogen peroxide (H(2)O(2)) produced by activated leukocytes and endothelial cells in sites of inflammation or ischemia cause endothelial barrier dysfunction that may lead to tissue edema. Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically bind to endothelium, quench the corresponding ROS, and alleviate vascular oxidative stress and inflammation. In the present work, we studied the effects of anti-PECAM/catalase and anti-PECAM/SOD conjugates on the abnormal permeability manifested by transendothelial electrical resistance decline, increased fluorescein isothiocyanate-dextran influx, and redistribution of vascular endothelial-cadherin in human umbilical vein endothelial cell (HUVEC) monolayers. Anti-PECAM/catalase protected HUVEC monolayers against H(2)O(2)-induced endothelial barrier dysfunction. Polyethylene glycol-conjugated catalase exerted orders of magnitude lower endothelial uptake and no protective effect, similarly to IgG/catalase. Anti-PECAM/catalase, but not anti-PECAM/SOD, alleviated endothelial hyperpermeability caused by exposure to hypoxanthine/xanthine oxidase, implicating primarily H(2)O(2) in the disruption of the endothelial barrier in this model. Thrombin-induced endothelial permeability was not affected by treatment with anti-PECAM/AOEs or the NADPH oxidase inhibitor apocynin or overexpression of AOEs, indicating that the endogenous ROS play no key role in thrombin-mediated endothelial barrier dysfunction. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase, inhibited a vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability, identifying a key role of endogenous O(2)() in the VEGF-mediated regulation of endothelial barrier function. Therefore, AOEs targeted to endothelial cells provide versatile molecular tools for testing the roles of

  15. A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

    Science.gov (United States)

    Ribera, Jordi; Pauta, Montse; Melgar-Lesmes, Pedro; Córdoba, Bernat; Bosch, Anna; Calvo, Maria; Rodrigo-Torres, Daniel; Sancho-Bru, Pau; Mira, Aurea; Jiménez, Wladimiro; Morales-Ruiz, Manuel

    2017-11-01

    Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl 4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl 4 -treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-β1 (TGF-β1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P livers correlated with a significant decrease in liver fibrosis ( P livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization. NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-β1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization. Copyright © 2017 the American

  16. Activation of the canonical Wnt/β-catenin pathway enhances monocyte adhesion to endothelial cells

    International Nuclear Information System (INIS)

    Lee, Dong Kun; Nathan Grantham, R.; Trachte, Aaron L.; Mannion, John D.; Wilson, Colleen L.

    2006-01-01

    Monocyte adhesion to vascular endothelium has been reported to be one of the early processes in the development of atherosclerosis. In an attempt to develop strategies to prevent or delay atherosclerosis progression, we analyzed effects of the Wnt/β-catenin signaling pathway on monocyte adhesion to various human endothelial cells. Adhesion of fluorescein-labeled monocytes to various human endothelial cells was analyzed under a fluorescent microscope. Unlike sodium chloride, lithium chloride enhanced monocyte adhesion to endothelial cells in a dose-dependent manner. We further demonstrated that inhibitors for glycogen synthase kinase (GSK)-3β or proteosome enhanced monocyte-endothelial cell adhesion. Results of semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) indicated that activation of Wnt/β-catenin pathway did not change expression levels of mRNA for adhesion molecules. In conclusion, the canonical Wnt/β-catenin pathway enhanced monocyte-endothelial cell adhesion without changing expression levels of adhesion molecules

  17. Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions.

    Science.gov (United States)

    Zayed, S A; Gaafar, T M; Samy, R M; Sabry, D; Nasr, A S; Maksoud, Fa Abdel

    2016-11-01

    Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.

  18. Equine Endothelial Cells Support Productive Infection of Equine Infectious Anemia Virus

    OpenAIRE

    Maury, Wendy; Oaks, J. Lindsay; Bradley, Sarahann

    1998-01-01

    Previous cell infectivity studies have demonstrated that the lentivirus equine infectious anemia virus (EIAV) infects tissue macrophages in vivo and in vitro. In addition, some strains of EIAV replicate to high titer in vitro in equine fibroblasts and fibroblast cell lines. Here we report a new cell type, macrovascular endothelial cells, that is infectible with EIAV. We tested the ability of EIAV to infect purified endothelial cells isolated from equine umbilical cords and renal arteries. Inf...

  19. Interaction between Pasteurella multocida B:2 and its derivatives with bovine aortic endothelial cell (BAEC).

    Science.gov (United States)

    Kamal, Nuriqmaliza M; Zamri-Saad, M; Masarudin, Mas Jaffri; Othman, Sarah

    2017-06-19

    Pasteurella multocida B:2 causes bovine haemorrhagic septicaemia (HS), leading to rapid fatalities in cattle and buffaloes. An attenuated derivative of P. multocida B:2 GDH7, was previously constructed through mutation of the gdhA gene and proved to be an effective live attenuated vaccine for HS. Currently, only two potential live attenuated vaccine candidates for HS are being reported; P. multocida B:2 GDH7 and P. multocida B:2 JRMT12. This study primarily aims to investigate the potential of P. multocida B:2 GDH7 strain as a delivery vehicle for DNA vaccine for future multivalent applications. An investigation on the adherence, invasion and intracellular survival of bacterial strains within the bovine aortic endothelial cell line (BAEC) were carried out. The potential vaccine strain, P. multocida B:2 GDH7, was significantly better (p ≤ 0.05) at adhering to and invading BAEC compared to its parent strain and to P. multocida B:2 JRMT12 and survived intracellularly 7 h post treatment, with a steady decline over time. A dual reporter plasmid, pSRGM, which enabled tracking of bacterial movement from the extracellular environment into the intracellular compartment of the mammalian cells, was subsequently transformed into P. multocida B:2 GDH7. Intracellular trafficking of the vaccine strain, P. multocida B:2 GDH7 was subsequently visualized by tracking the reporter proteins via confocal laser scanning microscopy (CLSM). The ability of P. multocida B:2 GDH7 to model bactofection represents a possibility for this vaccine strain to be used as a delivery vehicle for DNA vaccine for future multivalent protection in cattle and buffaloes.

  20. Effects of Neospora caninum infection on brain microvascular endothelial cells bioenergetics

    Directory of Open Access Journals (Sweden)

    Elsheikha Hany M

    2013-01-01

    Full Text Available Abstract Background The brain is the most commonly affected organ during Neospora caninum infection but the mechanisms utilized by this protozoan parasite for traversal of the blood–brain barrier (BBB are not yet understood. Herein, we investigate the cellular pathogenicity of N. caninum infection on bioenergetics of human brain microvascular endothelial cells (HBMECs, a fundamental component of the BBB. Methods We tracked the growth kinetics of N. caninum in HBMECs. Focusing on cell bioenergetics, oxygen consumption rate (OCR was determined using Clark electrode system and mitochondrial membrane potential (ΔΨm was evaluated using DePsipher staining by fluorescence microscopy in the presence and absence of infection. Results HBMECs provided a receptive environment for parasite proliferation. N. caninum tachyzoites were able to invade and replicate within HBMECs without significantly altering cell proliferation rate, as measured with the MTT assay, up to 24 hr post infection (pi. The oxygen consumption rate (OCR was significantly inhibited (p 6 cell min-1 and from −0.29±0.09 to −0.16±0.1 nmol 106 cell min-1 for uninfected HBMECs and free N. Caninum tachyzoites, respectively]. After normalization for DNA content the basal OCR did not differ between two host cell types: HBMECs and K562. The OCR of HBMECs was significantly elevated 24 hr pi in the absence of substrate, in 10 mM glucose and in the presence of a tetramethyl-p-phenylenediamine (TMPD/ascorbate redox shuttle. Although quantitatively similar results were observed for uninfected K562 cells, there was no effect on their OCR 24 hr pi with N. caninum under any of the above substrate conditions. 6mM azide abolished OCR in all situations. Mitochondrial staining with DePsipher indicated no change in their membrane potential (Δψm up to 24 hr pi. Conclusions N. caninum is able to grow in HBMECs without markedly disrupting their normal proliferation or mitochondrial integrity. However

  1. Time-lapse microscopy of lung endothelial cells under hypoxia

    Science.gov (United States)

    Mehrvar, Shima; Ghanian, Zahra; Kondouri, Ganesh; Camara, Amadou S.; Ranji, Mahsa

    2017-02-01

    Objective: This study utilizes fluorescence microscopy to assess the effect of the oxygen tension on the production of reactive oxygen species (ROS) in mitochondria of fetal pulmonary artery endothelial cells (FPAECs). Introduction: Hypoxia is a severe oxygen stress, which mostly causes irreversible injury in lung cells. However, in some studies, it is reported that hypoxia decreases the severity of injuries. In this study, ROS production level was examined in hypoxic FPAECs treated with pentachlorophenol (PCP, uncoupler). This work was accomplished by monitoring and quantifying the changes in the level of the produced ROS in hypoxic cells before and after PCP treatment. Materials and methods: The dynamic of the mitochondrial ROS production in two groups of FPAECs was measured over time using time-lapse microscopy. For the first group, cells were incubated in 3% hypoxic condition for 2 hours and then continuously were exposed to hypoxic condition for imaging as well. For the second group, cells were incubated in normal oxygen condition. Time lapse images of the cells loaded with Mito-SOX (ROS indicator) were acquired, and the red fluorescence intensity profile of the cells was calculated. Changes in the level of the fluorescence intensity profile while they are treated with PCP indicates the dynamics of the ROS level. Results: The intensity profiles of the PCP-treated cells in the first group showed 47% lower ROS production rate than the PCP-treated cells in the second group. Conclusion: Time lapse microscopy revealed that hypoxic cells have lower ROS generation while treated with PCP. Therefore, this result suggests that hypoxia decreased electron transport chain activity in uncoupled chain.

  2. Effect of laser modified surface microtopochemistry on endothelial cell growth.

    Science.gov (United States)

    Duncan, A C; Rouais, F; Lazare, S; Bordenave, L; Baquey, Ch

    2007-02-15

    The introduction of microelectronics technology in the area of biological sciences has brought forth previously unforeseeable applications such as DNA or protein biochips, miniaturized, multiparametric biosensors for high performance multianalyte assays, DNA sequencing, biocomputers, and substrates for controlled cell growth (i.e. tissue engineering). We developed and investigated a new method using "cold" excimer laser beam technology combined with microlithographical techniques to create surfaces with well defined 3D microdomains in order to delineate critical microscopic surface features governing cell-material interactions. Microfabricated surfaces with microgrooves 30-3 microm deep, 10 - 1 microm wide spaced 30 microm apart were obtained with micron resolution, by "microsculpturing" polymer model surfaces using a computer controlled laser KrF excimer beam coupled with a microlithographic projection technique. The laser beam after exiting a mask was focused onto the polymer target surface via an optical setup allowing for a 10-fold reduction of the mask pattern. Various 3D micropatterned features were obtained at the micron level. Reproducible submicron features could also be obtained using this method. Subsequently, model human umbilical endothelial cells (HUVEC) were cultured on the laser microfabricated surfaces in order to study the effects of specific microscopic surface features on cell deposition and orientation. Cell deposition patterns were found to be microstructure dependant, and showed cell orientation dependency for features in the cell range dimension, a behaviour significantly different from that of a previously studied cell model (osteoprogenitor cell). This model may be a promising in so far as it is very rapid (a time frame less than a second per square centimeter of micropatterned surface) and provides further insights into the effects of surface microtopography on cell response with possible applications in the field of biosensors

  3. Do Neural Cells Communicate with Endothelial Cells via Secretory Exosomes and Microvesicles?

    Directory of Open Access Journals (Sweden)

    Neil R. Smalheiser

    2009-01-01

    Full Text Available Neurons, glial, cells, and brain tumor cells tissues release small vesicles (secretory exosomes and microvesicles, which may represent a novel mechanism by which neuronal activity could influence angiogenesis within the embryonic and mature brain. If CNS-derived vesicles can enter the bloodstream as well, they may communicate with endothelial cells in the peripheral circulation and with cells concerned with immune surveillance.

  4. Single-Cell Analysis Identifies Distinct Stages of Human Endothelial-to-Hematopoietic Transition

    Directory of Open Access Journals (Sweden)

    Carolina Guibentif

    2017-04-01

    Full Text Available During development, hematopoietic cells originate from endothelium in a process known as endothelial-to-hematopoietic transition (EHT. To study human EHT, we coupled flow cytometry and single-cell transcriptional analyses of human pluripotent stem cell-derived CD34+ cells. The resulting transcriptional hierarchy showed a continuum of endothelial and hematopoietic signatures. At the interface of these two signatures, a unique group of cells displayed both an endothelial signature and high levels of key hematopoietic stem cell-associated genes. This interphase group was validated via sort and subculture as an immediate precursor to hematopoietic cells. Differential expression analyses further divided this population into subgroups, which, upon subculture, showed distinct hematopoietic lineage differentiation potentials. We therefore propose that immediate precursors to hematopoietic cells already have their hematopoietic lineage restrictions defined prior to complete downregulation of the endothelial signature. These findings increase our understanding of the processes of de novo hematopoietic cell generation in the human developmental context.

  5. Arterial endothelial cytokines guide extravillous trophoblast invasion towards spiral arteries; an in-vitro study with the trophoblast cell line ACH-3P and female non-uterine endothelial cells.

    Science.gov (United States)

    Weiss, Gregor; Huppertz, Berthold; Siwetz, Monika; Lang, Ingrid; Moser, Gerit

    2016-02-01

    Invasion of extravillous trophoblasts (EVT) is tightly linked to appropriate cell to cell contact as well as paracrine guidance of EVT by maternal uterine cells, conducted by a variety of locally expressed cytokines. Here we investigated the interaction of the first trimester trophoblast cell line ACH-3P with adult iliac arterial (AEC) and venous endothelial cells (VEC). The impact of ACH-3P conditioned medium (Cdm), obtained at 2.5% and 21% oxygen, on endothelial cell viability (LDH-Assay) and network formation (Matrigel-Assay) was tested. We investigated cytokine expression of AEC- and VEC-Cdm and confirmed results with ELISA analysis, and investigated the influence of Cdm on ACH-3P proliferation and invasion. Additionally, direct co-culture experiments with ACH-3P and AEC on Matrigel were performed. A subset of experiments was verified with primary trophoblasts as well as with first trimester placenta in situ specimens. ACH-3P-Cdm significantly enhanced cell viability of AEC and VEC after 72 h. ACH-3P-Cdm at 2.5% oxygen stabilized endothelial network structures in Matrigel up to 24 h, similar to the effect of a direct co-culture of AEC and ACH-3P. AEC and VEC showed a similar pattern of secreted cytokines. However, elevated levels of cytokines secreted by AEC were found for GRO, IL-6, MMP-1 and uPAR. ELISA confirmed elevated concentrations of IL-6 and uPAR in AEC compared to VEC. ACH-3P and primary trophoblasts more likely invaded towards AEC-Cdm than towards VEC-Cdm. Addition of IL-6 to Cdm increased the invasion potential of both cell types. AEC- and VEC-Cdm reduced ACH-3P cell proliferation after 24 h of culture. IL-6 was highly expressed in uterine AEC compared to VEC as visualized by immunohistochemistry. The presented results clearly demonstrate that cytokines of both cell types, AEC and trophoblasts, differentially contribute to successful guidance and interaction in the process of trophoblast invasion. Copyright © 2015 Elsevier Ltd. All rights

  6. Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144 during endothelial differentiation

    Directory of Open Access Journals (Sweden)

    Libermann Towia

    2008-05-01

    Full Text Available Abstract Background Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation. Methods Mouse embryonic stem (ES cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2+ cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2+ stem cells expressing the endothelial-specific marker CD144 (VE-cadherin was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages. Results Microarray analysis of >45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2+, CD41+, and CD144+ and VEGF-R2-, CD41-, and CD144-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation. Conclusion The isolation of CD144+ cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that

  7. An In Vitro Study of Differentiation of Hematopoietic Cells to Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Qi Ru Wang

    2011-01-01

    medium (ECCM. BM-EPCs were characterized in terms of phenotype, lineage potential, and their functional properties. Endothelial cell colonies derived from BM-EPC were cultured with ECCM for 3 months. Cultured EPC colony cells expressed endothelial cell markers and formed the capillary-like network in vitro. EPC colony cells expressed differential proliferative capacity; some of the colonies exhibited a high proliferative potential (HPP capacity up to 20 population doublings. More importantly, these HPP-EPCs expressed hematopoietic marker CD45, exhibited endocytic activities, and preserved some of the myeloid cell activity. In addition, the HPP-EPCs secrete various growth factors including VEGF and GM-CSF into the culture medium. The results demonstrate that these EPCs were primarily derived from hematopoietic origin of early precursor cells and maintained high proliferative potential capacity, a feature with a significant potential in the application of cell therapy in ischemic diseases.

  8. RhoA GTPase regulates radiation-induced alterations in endothelial cell adhesion and migration

    International Nuclear Information System (INIS)

    Rousseau, Matthieu; Gaugler, Marie-Hélène; Rodallec, Audrey; Bonnaud, Stéphanie; Paris, François; Corre, Isabelle

    2011-01-01

    Highlights: ► We explore the role of RhoA in endothelial cell response to ionizing radiation. ► RhoA is rapidly activated by single high-dose of radiation. ► Radiation leads to RhoA/ROCK-dependent actin cytoskeleton remodeling. ► Radiation-induced apoptosis does not require the RhoA/ROCK pathway. ► Radiation-induced alteration of endothelial adhesion and migration requires RhoA/ROCK. -- Abstract: Endothelial cells of the microvasculature are major target of ionizing radiation, responsible of the radiation-induced vascular early dysfunctions. Molecular signaling pathways involved in endothelial responses to ionizing radiation, despite being increasingly investigated, still need precise characterization. Small GTPase RhoA and its effector ROCK are crucial signaling molecules involved in many endothelial cellular functions. Recent studies identified implication of RhoA/ROCK in radiation-induced increase in endothelial permeability but other endothelial functions altered by radiation might also require RhoA proteins. Human microvascular endothelial cells HMEC-1, either treated with Y-27632 (inhibitor of ROCK) or invalidated for RhoA by RNA interference were exposed to 15 Gy. We showed a rapid radiation-induced activation of RhoA, leading to a deep reorganisation of actin cytoskeleton with rapid formation of stress fibers. Endothelial early apoptosis induced by ionizing radiation was not affected by Y-27632 pre-treatment or RhoA depletion. Endothelial adhesion to fibronectin and formation of focal adhesions increased in response to radiation in a RhoA/ROCK-dependent manner. Consistent with its pro-adhesive role, ionizing radiation also decreased endothelial cells migration and RhoA was required for this inhibition. These results highlight the role of RhoA GTPase in ionizing radiation-induced deregulation of essential endothelial functions linked to actin cytoskeleton.

  9. Shear Stress Inhibits Apoptosis of Ischemic Brain Microvascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Xiafeng Shen

    2013-01-01

    Full Text Available As a therapeutic strategy for ischemic stroke, to restore or increase cerebral blood flow (CBF is the most fundamental option. Laminar shear stress (LS, as an important force generated by CBF, mainly acts on brain microvascular endothelial cells (BMECs. In order to study whether LS was a protective factor in stroke, we investigated LS-intervented ischemic apoptosis of rat BMECs (rBMECs through PE Annexin V/7-AAD, JC-1 and Hoechst 33258 staining to observe the membranous, mitochondrial and nuclear dysfunction. Real-time PCR and western blot were also used to test the gene and protein expressions of Tie-2, Bcl-2 and Akt, which were respectively related to maintain membranous, mitochondrial and nuclear norm. The results showed that LS could be a helpful stimulus for ischemic rBMECs survival. Simultaneously, membranous, mitochondrial and nuclear regulation played an important role in this process.

  10. Endothelialization of a non-woven silk fibroin net for use in tissue engineering: growth and gene regulation of human endothelial cells.

    Science.gov (United States)

    Unger, R E; Peters, K; Wolf, M; Motta, A; Migliaresi, C; Kirkpatrick, C J

    2004-09-01

    We have previously shown that a biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons is an excellent scaffolding material for a wide variety of human cells of different tissue types. Endothelialization must take place for a biomaterial to be successful after implantation. Therefore, primary human endothelial cells and the human endothelial cell lines, HPMEC-ST1.6R and ISO-HAS-1, were examined for adherence and growth patterns on the fibroin nets by confocal laser scanning microscopy after vital staining of the cells and by electron microscopy. Endothelial cells adhered and spread along individual fibers of the nets and did not fill the gaps between individual fibers. Higher attachment and growth coverage was obtained if nets were first coated with gelatin, fibronectin or collagen type I. Proinflammatory markers of endothelial cells on the fibers exhibited a non-activated state and LPS-stimulated cells exhibited activation of these markers. Furthermore, a typical PECAM-1 localization at cell-cell contacts was observed. Scanning electron microscopic examination of fibroin nets after removal of cells did not demonstrate any changes to the fibroin structure. HUVEC and HDMEC on fibroin nets embedded in collagen type I gels formed microvessel-like structures. Thus, silk fibroin nets are a highly endothelial cell-compatible scaffolding material that support the growth, normal and inducible cell functions and angiogenesis potential of human endothelial cells in vitro similar to that observed in vivo.

  11. Evidence for endothelial cell origin of vinyl chloride-induced hepatic angiosarcoma.

    Science.gov (United States)

    Fortwengler, H P; Jones, D; Espinosa, E; Tamburro, C H

    1981-06-01

    Previous reports of hepatic angiosarcoma have not clearly defined the cellular type from which this tumor arises, as evidenced by the terminology of endothelioma, Kupffer cell sarcoma, endothelial cell sarcoma, and hemangioendothelial sarcoma, etc., which have been used interchangeably. In addition, there has been no consensus on the separate entity of Kupffer and sinusoidal endothelial cells. In the work presented here, evidence for the endothelial cell origin of this tumor is provided by the demonstration of factor VIII, a known endothelial cell marker, in the tumor cells. Fluorescence due to the presence of factor VIII appeared intense in the tumor sinusoidal cells of all four vinyl chloride-associated angiosarcomas studied, whereas normal liver sinusoidal lining cells showed negligible fluorescence.

  12. The Cytoskeleton and ATP in Sulfur Mustard-Medicated Injury to Endothelial Cells and Keratinocytes

    National Research Council Canada - National Science Library

    Hinshaw, Daniel

    1998-01-01

    The major goal of this project is to test the hypothesis that sulfur mustard (SM)-mediated cell death in keratinocytes and endothelial cells is primarily apoptotic in nature, and that several factors...

  13. Hypertonic saline impedes tumor cell-endothelial cell interaction by reducing adhesion molecule and laminin expression.

    LENUS (Irish Health Repository)

    Shields, Conor J

    2012-02-03

    BACKGROUND: Hypertonic saline infusion dampens inflammatory responses and suppresses neutrophil-endothelial interaction by reducing adhesion molecule expression. This study tested the hypothesis that hypertonic saline attenuates tumor cell adhesion to the endothelium through a similar mechanism. METHODS: Human colon cancer cells (LS174T) were transfected with green fluorescent protein and exposed to lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6 under hypertonic and isotonic conditions for 1 and 4 hours. Confluent human umbilical vein endothelial cells were similarly exposed. Cellular apoptosis and expression of adhesion molecules and laminin were measured by flow cytometry. Tumor cell adhesion to endothelium and laminin was assessed with fluorescence microscopy. Data are represented as mean +\\/- standard error of mean, and an ANOVA test was performed to gauge statistical significance, with P <.05 considered significant. RESULTS: Hypertonic exposure significantly reduced tumor cell adhesion despite the presence of the perioperative cell stressors (42 +\\/- 2.9 vs 172.5 +\\/- 12.4, P <.05), attenuated tumor cell beta-1 integrin (14.43 vs 23.84, P <.05), and endothelial cell laminin expression (22.78 +\\/- 2.2 vs 33.74 +\\/- 2.4, P <.05), but did not significantly alter cell viability. CONCLUSION: Hypertonic saline significantly attenuates tumor cell adhesion to endothelium by inhibiting adhesion molecule and laminin expression. This may halt the metastatic behavior of tumor cells shed at surgery.

  14. File list: InP.CDV.10.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: Unc.CDV.20.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  17. File list: InP.CDV.05.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  18. File list: Unc.CDV.50.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  1. File list: Unc.CDV.10.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  2. File list: DNS.CDV.05.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  6. File list: Unc.CDV.05.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  7. File list: Oth.CDV.20.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  8. File list: Oth.CDV.10.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  9. File list: DNS.CDV.20.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  10. File list: Oth.CDV.50.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  11. File list: InP.CDV.20.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  12. File list: DNS.CDV.10.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  13. File list: Pol.CDV.20.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  14. File list: Pol.CDV.10.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  15. File list: DNS.CDV.50.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

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  16. File list: InP.CDV.50.AllAg.Coronary_artery_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.CDV.50.AllAg.Coronary_artery_endothelial_cells hg19 Input control Cardiovascular Coronary arte...ry endothelial cells http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.CDV.50.AllAg.Coronary_artery_endothelial_cells.bed ...

  17. Angiogenic sprouting is regulated by endothelial cell expression of Slug.

    Science.gov (United States)

    Welch-Reardon, Katrina M; Ehsan, Seema M; Wang, Kehui; Wu, Nan; Newman, Andrew C; Romero-Lopez, Monica; Fong, Ashley H; George, Steven C; Edwards, Robert A; Hughes, Christopher C W

    2014-05-01

    The Snail family of zinc-finger transcription factors are evolutionarily conserved proteins that control processes requiring cell movement. Specifically, they regulate epithelial-to-mesenchymal transitions (EMT) where an epithelial cell severs intercellular junctions, degrades basement membrane and becomes a migratory, mesenchymal-like cell. Interestingly, Slug expression has been observed in angiogenic endothelial cells (EC) in vivo, suggesting that angiogenic sprouting may share common attributes with EMT. Here, we demonstrate that sprouting EC in vitro express both Slug and Snail, and that siRNA-mediated knockdown of either inhibits sprouting and migration in multiple in vitro angiogenesis assays. We find that expression of MT1-MMP, but not of VE-Cadherin, is regulated by Slug and that loss of sprouting as a consequence of reduced Slug expression can be reversed by lentiviral-mediated re-expression of MT1-MMP. Activity of MMP2 and MMP9 are also affected by Slug expression, likely through MT1-MMP. Importantly, we find enhanced expression of Slug in EC in human colorectal cancer samples compared with normal colon tissue, suggesting a role for Slug in pathological angiogenesis. In summary, these data implicate Slug as an important regulator of sprouting angiogenesis, particularly in pathological settings.

  18. Bortezomib induces autophagic death in proliferating human endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Belloni, Daniela; Veschini, Lorenzo [Myeloma Unit, Department of Oncology, IRCCS H San Raffaele, Milan (Italy); Foglieni, Chiara [Department of Cardiology, IRCCS H San Raffaele, Milan (Italy); Dell' Antonio, Giacomo [Department of Pathology, IRCCS H San Raffaele, Milan (Italy); Caligaris-Cappio, Federico [Myeloma Unit, Department of Oncology, IRCCS H San Raffaele, Milan (Italy); Universita Vita-Salute IRCCS H San Raffaele, Milan (Italy); Ferrarini, Marina [Myeloma Unit, Department of Oncology, IRCCS H San Raffaele, Milan (Italy); Ferrero, Elisabetta, E-mail: elisabetta.ferrero@hsr.it [Myeloma Unit, Department of Oncology, IRCCS H San Raffaele, Milan (Italy)

    2010-04-01

    The proteasome inhibitor Bortezomib has been approved for the treatment of relapsed/refractory multiple myeloma (MM), thanks to its ability to induce MM cell apoptosis. Moreover, Bortezomib has antiangiogenic properties. We report that endothelial cells (EC) exposed to Bortezomib undergo death to an extent that depends strictly on their activation state. Indeed, while quiescent EC are resistant to Bortezomib, the drug results maximally toxic in EC switched toward angiogenesis with FGF, and exerts a moderate effect on subconfluent HUVEC. Moreover, EC activation state deeply influences the death pathway elicited by Bortezomib: after treatment, angiogenesis-triggered EC display typical features of apoptosis. Conversely, death of subconfluent EC is preceded by ROS generation and signs typical of autophagy, including intense cytoplasmic vacuolization with evidence of autophagosomes at electron microscopy, and conversion of the cytosolic MAP LC3 I form toward the autophagosome-associated LC3 II form. Treatment with the specific autophagy inhibitor 3-MA prevents both LC3 I/LC3 II conversion and HUVEC cell death. Finally, early removal of Bortezomib is accompanied by the recovery of cell shape and viability. These findings strongly suggest that Bortezomib induces either apoptosis or autophagy in EC; interfering with the autophagic response may potentiate the antiangiogenic effect of the drug.

  19. The role of shear stress and altered tissue properties on endothelial to mesenchymal transformation and tumor-endothelial cell interaction.

    Science.gov (United States)

    Mina, Sara G; Huang, Peter; Murray, Bruce T; Mahler, Gretchen J

    2017-07-01

    Tumor development is influenced by stromal cells in aspects including invasion, growth, angiogenesis, and metastasis. Activated fibroblasts are one group of stromal cells involved in cancer metastasis, and one source of activated fibroblasts is endothelial to mesenchymal transformation (EndMT). EndMT begins when the endothelial cells delaminate from the cell monolayer, lose cell-cell contacts, lose endothelial markers such as vascular endothelial-cadherin (VE-cadherin), gain mesenchymal markers like alpha-smooth muscle actin (α-SMA), and acquire mesenchymal cell-like properties. A three-dimensional (3D) culture microfluidic device was developed for investigating the role of steady low shear stress (1 dyne/cm 2 ) and altered extracellular matrix (ECM) composition and stiffness on EndMT. Shear stresses resulting from fluid flow within tumor tissue are relevant to both cancer metastasis and treatment effectiveness. Low and oscillatory shear stress rates have been shown to enhance the invasion of metastatic cancer cells through specific changes in actin and tubulin remodeling. The 3D ECM within the device was composed of type I collagen and glycosaminoglycans (GAGs), hyaluronic acid and chondroitin sulfate. An increase in collagen and GAGs has been observed in the solid tumor microenvironment and has been correlated with poor prognosis in many different cancer types. In this study, it was found that ECM composition and low shear stress upregulated EndMT, including upregulation of mesenchymal-like markers (α-SMA and Snail) and downregulated endothelial marker protein and gene expression (VE-cadherin). Furthermore, this novel model was utilized to investigate the role of EndMT in breast cancer cell proliferation and migration. Cancer cell spheroids were embedded within the 3D ECM of the microfluidic device. The results using this device show for the first time that the breast cancer spheroid size is dependent on shear stress and that the cancer cell migration rate

  20. Smooth muscle progenitor cells from peripheral blood promote the neovascularization of endothelial colony-forming cells

    Energy Technology Data Exchange (ETDEWEB)

    Joo, Hyung Joon; Seo, Ha-Rim [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of); Jeong, Hyo Eun [Department of Mechanical Engineering, Korea University, Seoul (Korea, Republic of); Choi, Seung-Cheol; Park, Jae Hyung; Yu, Cheol Woong; Hong, Soon Jun [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of); Chung, Seok [Department of Mechanical Engineering, Korea University, Seoul (Korea, Republic of); Lim, Do-Sun, E-mail: dslmd@kumc.or.kr [Department of Cardiology, Cardiovascular Center, College of Medicine, Korea University, Seoul (Korea, Republic of)

    2014-07-11

    Highlights: • Two distinct vascular progenitor cells are induced from adult peripheral blood. • ECFCs induce vascular structures in vitro and in vivo. • SMPCs augment the in vitro and in vivo angiogenic potential of ECFCs. • Both cell types have synergistic therapeutic potential in ischemic hindlimb model. - Abstract: Proangiogenic cell therapy using autologous progenitors is a promising strategy for treating ischemic disease. Considering that neovascularization is a harmonized cellular process that involves both endothelial cells and vascular smooth muscle cells, peripheral blood-originating endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SMPCs), which are similar to mature endothelial cells and vascular smooth muscle cells, could be attractive cellular candidates to achieve therapeutic neovascularization. We successfully induced populations of two different vascular progenitor cells (ECFCs and SMPCs) from adult peripheral blood. Both progenitor cell types expressed endothelial-specific or smooth muscle-specific genes and markers, respectively. In a protein array focused on angiogenic cytokines, SMPCs demonstrated significantly higher expression of bFGF, EGF, TIMP2, ENA78, and TIMP1 compared to ECFCs. Conditioned medium from SMPCs and co-culture with SMPCs revealed that SMPCs promoted cell proliferation, migration, and the in vitro angiogenesis of ECFCs. Finally, co-transplantation of ECFCs and SMPCs induced robust in vivo neovascularization, as well as improved blood perfusion and tissue repair, in a mouse ischemic hindlimb model. Taken together, we have provided the first evidence of a cell therapy strategy for therapeutic neovascularization using two different types of autologous progenitors (ECFCs and SMPCs) derived from adult peripheral blood.

  1. Interactions of Pseudomonas aeruginosa and Corynebacterium spp. with non-phagocytic brain microvascular endothelial cells and phagocytic Acanthamoeba castellanii.

    Science.gov (United States)

    Siddiqui, Ruqaiyyah; Lakhundi, Sahreena; Khan, Naveed Ahmed

    2015-06-01

    Several lines of evidence suggest that Acanthamoeba interact with bacteria, which may aid in pathogenic bacterial transmission to susceptible hosts, and these interactions may have influenced evolution of bacterial pathogenicity. In this study, we tested if Gram-negative Pseudomonas aeruginosa and Gram-positive Corynebacterium spp. can associate/invade and survive inside Acanthamoeba castellanii trophozoites and cysts, as well as non-phagocytic human brain microvascular endothelial cells. The results revealed that both Corynebacterium spp. and P. aeruginosa were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. In contrast, P. aeruginosa exhibited higher association as well as invasion of non-phagocytic HBMEC compared with Corynebacterium spp. Notably, P. aeruginosa remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (74.54 bacteria per amoebae) compared with Corynebacterium spp. (2.69 bacteria per amoeba) (P Acanthamoeba cysts can be airborne, these findings suggest that Acanthamoeba is a potential vector in the transmission of P. aeruginosa to susceptible hosts. When bacterial-ridden amoebae were exposed to favourable (nutrient-rich) conditions, A. castellanii emerged as vegetative trophozoites and remained viable, and likewise viable P. aeruginosa were also observed but rarely any Corynebacterium spp. were observed. Correspondingly, P. aeruginosa but not Corynebacterium spp. exhibited higher cytotoxicity to non-phagocytic HBMEC, producing more than 75% cell death in 24 h, compared to 20% cell death observed with Corynebacterium spp. Additionally, it was observed that the bacterial conditioned medium had no negative effect on A. castellanii growth. Further characterization of amoebal and bacterial interactions will assist in identifying the role of Acanthamoeba in the transmission and evolution of pathogenic bacteria.

  2. Manipulating Endothelial Progenitor Cell Homing with Sphingosine-1-Phosphate for Terapeutic Angiogenesis

    Science.gov (United States)

    Williams, Priscilla Anne

    Ischemic vascular diseases are the main cause of mortality worldwide and yet current therapies only delay disease progression and improve quality of life without addressing the fundamental problem of tissue loss. Within the field of tissue engineering, therapeutic angiogenesis provides a promising approach to alternatively provide new blood vessel formation via spatiotemporally controlled delivery of proangiogenic agents. Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid that is upregulated under ischemic conditions, has recently gained great enthusiasm as a potential mediator in neovascularization strategies given its essential roles in promoting both neovessel formation and stabilization, and cellular trafficking along highly regulated endogenous gradients. Herein, the governing hypothesis guiding this dissertation is that local biomaterial-controlled delivery of S1P may be used to enhance migration and recruitment of vascular progenitor cells for enhanced therapeutic angiogenesis within ischemic tissue. The initial work in this dissertation investigated the effect of hypoxia on the angiogenic response of both mature and progenitor endothelial cells to S1P stimulation in vitro. Outgrowth endothelial cells (OECs) were isolated from human umbilical cord blood to provide a clinically relevant source of vascular progenitor cells for the studies conducted within this dissertation. S1P stimulation promoted angiogenic activity of both ECs and OECs under both ambient and hypoxic (1%) oxygen tensions. Furthermore, dual therapy with the combination of S1P and vascular endothelial growth factor (VEGF) further enhanced cellular responses. Interestingly, hypoxia substantially augmented the functional response of OECs to S1P, resulting in 25-fold and 6.5-fold increases in directed migration and sprouting, respectively. Thus, these studies highlighted the potential for S1P as a therapeutic agent for treatment of ischemic diseases. An injectable biomaterial system

  3. Impact of diabetic serum on endothelial cells: An in-vitro-analysis of endothelial dysfunction in diabetes mellitus type 2

    International Nuclear Information System (INIS)

    Muenzel, Daniela; Lehle, Karla; Haubner, Frank; Schmid, Christof; Birnbaum, Dietrich E.; Preuner, Juergen G.

    2007-01-01

    Diabetic endothelial dysfunction was characterized by altered levels of adhesion molecules and cytokines. Aim of our study was to evaluate the effects of diabetic serum on cell-growth and proinflammatory markers in human saphenous vein endothelial cells (HSVEC) from diabetic and non-diabetic patients. Diabetic serum showed (1) complementary proliferative activity for non-diabetic and diabetic HSVEC, (2) unchanged surface expression of adhesion molecules, and (3) elevated levels of sICAM-1 in HSVEC of all donors. The concentration of sVCAM-1 was increased only in diabetic cells. The proinflammatory state of diabetic HSVEC characterized by increased levels of cytokines was compensated. We concluded that even under normoglycemic conditions the serum itself contains critical factors leading to abnormal regulation of inflammation in diabetics. We introduced an in vitro model of diabetes representing the endothelial situation at the beginning of diabetes (non-diabetic cells/diabetic serum) as well as the diabetic chronic state (diabetic cells/diabetic serum)

  4. N-Isopropylacrylamide-co-glycidylmethacrylate as a Thermoresponsive Substrate for Corneal Endothelial Cell Sheet Engineering

    Directory of Open Access Journals (Sweden)

    Bernadette K. Madathil

    2014-01-01

    Full Text Available Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.

  5. Biophysical Assessment of Single Cell Cytotoxicity: Diesel Exhaust Particle-Treated Human Aortic Endothelial Cells

    OpenAIRE

    Wu, Yangzhe; Yu, Tian; Gilbertson, Timothy A.; Zhou, Anhong; Xu, Hao; Nguyen, Kytai Truong

    2012-01-01

    Exposure to diesel exhaust particles (DEPs), a major source of traffic-related air pollution, has become a serious health concern due to its adverse influences on human health including cardiovascular and respiratory disorders. To elucidate the relationship between biophysical properties (cell topography, cytoskeleton organizations, and cell mechanics) and functions of endothelial cells exposed to DEPs, atomic force microscope (AFM) was applied to analyze the toxic effects of DEPs on a model ...

  6. Density-gradient centrifugation enables the purification of cultured corneal endothelial cells for cell therapy by eliminating senescent cells

    Science.gov (United States)

    Okumura, Naoki; Kusakabe, Ayaka; Hirano, Hiroatsu; Inoue, Ryota; Okazaki, Yugo; Nakano, Shinichiro; Kinoshita, Shigeru; Koizumi, Noriko

    2015-01-01

    The corneal endothelium is essential for maintaining corneal transparency; therefore, corneal endothelial dysfunction causes serious vision loss. Tissue engineering-based therapy is potentially a less invasive and more effective therapeutic modality. We recently started a first-in-man clinical trial of cell-based therapy for treating corneal endothelial dysfunction in Japan. However, the senescence of corneal endothelial cells (CECs) during the serial passage culture needed to obtain massive quantities of cells for clinical use is a serious technical obstacle preventing the push of this regenerative therapy to clinical settings. Here, we show evidence from an animal model confirming that senescent cells are less effective in cell therapy. In addition, we propose that density-gradient centrifugation can eliminate the senescent cells and purify high potency CECs for clinical use. This simple technique might be applicable for other types of cells in the settings of regenerative medicine. PMID:26443440

  7. Enhancement of endothelial cell migration by constitutively active LPA{sub 1}-expressing tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Kitayoshi, Misaho; Kato, Kohei; Tanabe, Eriko; Yoshikawa, Kyohei; Fukui, Rie [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Mutated LPA{sub 1} stimulates cell migration of endothelial cells. Black-Right-Pointing-Pointer VEGF expressions are increased by mutated LPA{sub 1}. Black-Right-Pointing-Pointer LPA signaling via mutated LPA{sub 1} is involved in angiogenesis. Black-Right-Pointing-Pointer Mutated LPA{sub 1} promotes cancer cell progression. -- Abstract: Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA{sub 1} to LPA{sub 6}). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA{sub 1} induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA{sub 1} on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA{sub 1} than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA{sub 1}. These results suggest that activation of LPA signaling via mutated LPA{sub 1} may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

  8. Role of the endothelin axis in astrocyte- and endothelial cell-mediated chemoprotection of cancer cells

    Science.gov (United States)

    Kim, Seung Wook; Choi, Hyun Jin; Lee, Ho-Jeong; He, Junqin; Wu, Qiuyu; Langley, Robert R.; Fidler, Isaiah J.; Kim, Sun-Jin

    2014-01-01

    Background Recent evidence suggests that astrocytes protect cancer cells from chemotherapy by stimulating upregulation of anti-apoptotic genes in those cells. We investigated the possibility that activation of the endothelin axis orchestrates survival gene expression and chemoprotection in MDA-MB-231 breast cancer cells and H226 lung cancer cells. Methods Cancer cells, murine astrocytes, and murine fibroblasts were grown in isolation, and expression of endothelin (ET) peptides and ET receptors (ETAR and ETBR) compared with expression on cancer cells and astrocytes (or cancer cells and fibroblasts) that were co-incubated for 48 hours. Type-specific endothelin receptor antagonists were used to evaluate the contribution of ETAR and ETBR to astrocyte-induced activation of the protein kinase B (AKT)/mitogen-activated protein kinase (MAPK) signal transduction pathways, anti-apoptotic gene expression, and chemoprotection of cancer cells. We also investigated the chemoprotective potential of brain endothelial cells and microglial cells. Results Gap junction signaling between MDA-MB-231 cancer cells and astrocytes stimulates upregulation of interleukin 6 (IL-6) and IL-8 expression in cancer cells, which increases ET-1 production from astrocytes and ET receptor expression on cancer cells. ET-1 signals for activation of AKT/MAPK and upregulation of survival proteins that protect cancer cells from taxol. Brain endothelial cell-mediated chemoprotection of cancer cells also involves endothelin signaling. Dual antagonism of ETAR and ETBR is required to abolish astrocyte- and endothelial cell-mediated chemoprotection. Conclusions Bidirectional signaling between astrocytes and cancer cells involves upregulation and activation of the endothelin axis, which protects cancer cells from cytotoxicity induced by chemotherapeutic drugs. PMID:25008093

  9. JNK2 promotes endothelial cell alignment under flow.

    Directory of Open Access Journals (Sweden)

    Cornelia Hahn

    Full Text Available Endothelial cells in straight, unbranched segments of arteries elongate and align in the direction of flow, a feature which is highly correlated with reduced atherosclerosis in these regions. The mitogen-activated protein kinase c-Jun N-terminal kinase (JNK is activated by flow and is linked to inflammatory gene expression and apoptosis. We previously showed that JNK activation by flow is mediated by integrins and is observed in cells plated on fibronectin but not on collagen or basement membrane proteins. We now show thatJNK2 activation in response to laminar shear stress is biphasic, with an early peak and a later peak. Activated JNK localizes to focal adhesions at the ends of actin stress fibers, correlates with integrin activation and requires integrin binding to the extracellular matrix. Reducing JNK2 activation by siRNA inhibits alignment in response to shear stress. Cells on collagen, where JNK activity is low, align slowly. These data show that an inflammatory pathway facilitates adaptation to laminar flow, thereby revealing an unexpected connection between adaptation and inflammatory pathways.

  10. Cheiradone: a vascular endothelial cell growth factor receptor antagonist

    Directory of Open Access Journals (Sweden)

    Ahmed Nessar

    2008-01-01

    Full Text Available Abstract Background Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with physiological (for example wound healing and pathological conditions (tumour development. Vascular endothelial growth factor (VEGF, fibroblast growth factor-2 (FGF-2 and epidermal growth factor (EGF are the major angiogenic regulators. We have identified a natural product (cheiradone isolated from a Euphorbia species which inhibited in vivo and in vitro VEGF- stimulated angiogenesis but had no effect on FGF-2 or EGF activity. Two primary cultures, bovine aortic and human dermal endothelial cells were used in in vitro (proliferation, wound healing, invasion in Matrigel and tube formation and in vivo (the chick chorioallantoic membrane models of angiogenesis in the presence of growth factors and cheiradone. In all cases, the concentration of cheiradone which caused 50% inhibition (IC50 was determined. The effect of cheiradone on the binding of growth factors to their receptors was also investigated. Results Cheiradone inhibited all stages of VEGF-induced angiogenesis with IC50 values in the range 5.20–7.50 μM but did not inhibit FGF-2 or EGF-induced angiogenesis. It also inhibited VEGF binding to VEGF receptor-1 and 2 with IC50 values of 2.9 and 0.61 μM respectively. Conclusion Cheiradone inhibited VEGF-induced angiogenesis by binding to VEGF receptors -1 and -2 and may be a useful investigative tool to study the specific contribution of VEGF to angiogenesis and may have therapeutic potential.

  11. Proteolytic cleavage of vascular adhesion protein-1 induced by vascular endothelial growth factor in retinal capillary endothelial cells.

    Science.gov (United States)

    Yoshida, Shiho; Murata, Miyuki; Noda, Kousuke; Matsuda, Takashi; Saito, Michiyuki; Saito, Wataru; Kanda, Atsuhiro; Ishida, Susumu

    2018-02-01

    To investigate the mechanism of soluble vascular adhesion protein-1 (sVAP-1) accumulation induced by vascular endothelial growth factor (VEGF) in the vitreous of patients with diabetic retinopathy (DR). Experimental. Protein levels of sVAP-1 and N epsilon-(hexanoyl)lysine (HEL), an oxidative stress marker, in the vitreous samples from patients with proliferative diabetic retinopathy (PDR) with or without intravitreal bevacizumab (IVB) injection were determined by ELISA. The effect of VEGF on both mRNA expression of Vap-1 and secretion of sVAP-1 in rat retinal capillary endothelial cells (TR-iBRB2) was analyzed by real-time PCR and western blotting, respectively. In addition, the impact of VEGF on production and activation ratios of matrix metalloproteinase (MMP)-2 and MMP-9 was examined by gelatin zymography. Hydrogen peroxide production and reactive oxygen species (ROS) levels were assessed in the supernatants of TR-iBRB2 cells treated with VEGF. IVB injection decreased vitreous levels of sVAP-1 and HEL in patients with PDR. VEGF stimulation released sVAP-1 protein from TR-iBRB2 cells as a consequence of membrane-anchored VAP-1 shedding by MMP-2 and MMP-9. In addition, VEGF increased hydrogen peroxide generation and ROS augmentation through spermine oxidation by sVAP-1 as semicarbazide-sensitive amine oxidase (SSAO) in the supernatant of cultured endothelial cells. The current data demonstrate that proangiogenic factor VEGF induces sVAP-1 release from retinal capillary endothelial cells and facilitates hydrogen peroxide generation via enzymatic property of sVAP-1, followed by the increase of oxidative stress, one of the crucial factors in the pathogenesis of DR.

  12. Adhesion of endothelial cells and adsorption of serum proteins on gas-plasma treated polytetrafluoroethylene

    NARCIS (Netherlands)

    Dekker, A.; Dekker, A.; Reitsma, K.; Beugeling, T.; Beugeling, T.; Bantjes, A.; Bantjes, A.; Feijen, Jan; van Aken, W.G.

    1991-01-01

    From in vitro experiments it is known that human endothelial cells show poor adhesion to hydrophobic polymers. The hydrophobicity of vascular prostheses manufactured from Teflon® or Dacron® may be the reason why endothelialization of these grafts does not occur after implantation in humans. We

  13. A microfluidic wound-healing assay for quantifying endothelial cell migration

    NARCIS (Netherlands)

    van der Meer, Andries Dirk; Vermeul, Kim; Poot, Andreas A.; Feijen, Jan; Vermes, I.

    A microfluidic wound-healing assay for quantifying endothelial cell migration. Am J Physiol Heart Circ Physiol 298: H719–H725, 2010. First published November 20, 2009; doi:10.1152/ajpheart.00933.2009.—Endothelial migration is an important process in the formation of blood vessels and the repair of

  14. The Level of Circulating Endothelial Progenitor Cell Is Associated with Cerebral Vasoreactivity: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Chih-Ping Chung

    2015-01-01

    Full Text Available Endothelial progenitor cell is known to be able to repair injured vessels. We assessed the hypothesis that endothelial progenitor cell also modulates cerebral endothelial function in healthy status. We used transcranial color-coded sonography to measure middle cerebral arterial vasoreactivity to CO2 (breath-holding index in healthy subjects and observed its relationship with the number of circulating CD34CD133+ cells. To detect significant correlations between each characteristic and breath-holding index of middle cerebral artery, we used univariate and multivariate regression analyses. 22 young healthy subjects were included in the present study (6 men, 16 women; mean age: 28.45 ± 3.98 years, range: 22–34 years. The mean breath-holding index and CD45lowCD34+CD133+ cells number were 0.95 ± 0.48% and 0.52 ± 0.26, respectively. The level of CD34CD133+ cells was independently associated with middle cerebral artery’s vasoreactivity (r=0.439,P=0.04. Our results suggest that endothelial progenitor cell also modulates healthy cerebral vessels’ endothelial function. This ability of endothelial progenitor cell could be potentially applied therapeutically and for prevention in conditions with cerebral endothelial dysfunction and cerebral ischemia.

  15. Endothelial Cells in Antibody-Mediated Rejection of Kidney Transplantation: Pathogenesis Mechanisms and Therapeutic Implications

    Directory of Open Access Journals (Sweden)

    Shuo Wang

    2017-01-01

    Full Text Available Antibody-mediated rejection (AMR has been identified as a main obstacle for stable immune tolerance and long survival of kidney allografts. In spite of new insights into the underlying mechanisms of AMR, accurate diagnosis and efficient treatment are still challenges in clinical practice. Endothelium is the first barrier between recipients’ immune systems and grafts in vascularized organ transplants. Considering that endothelial cells express a number of antigens that can be attacked by various allo- and autoantibodies, endothelial cells act as main targets for the recipients’ humoral immune responses. Importantly, emerging evidence has shown that endothelial cells in transplants could also initiate protective mechanisms in response to immune injuries. A better understanding of the role of endothelial cells during the pathogenesis of AMR might provide novel therapeutic targets. In the present review, we summarize the antigens expressed by endothelial cells and also discuss the activation and accommodation of endothelial cells as well as their clinical implications. Collectively, the progress discussed in this review indicates endothelial cells as promising targets to improve current diagnosis and therapeutic regimens for AMR.

  16. IL-20 activates human lymphatic endothelial cells causing cell signalling and tube formation

    DEFF Research Database (Denmark)

    Hammer, Troels; Tritsaris, Katerina; Hübschmann, Martin V

    2009-01-01

    IL-20 is an arteriogenic cytokine that remodels collateral networks in vivo, and plays a role in cellular organization. Here, we investigate its role in lymphangiogenesis using a lymphatic endothelial cell line, hTERT-HDLEC, which expresses the lymphatic markers LYVE-1 and podoplanin. Upon...

  17. Endothelial cell death and intimal foam cell accumulation in the coronary artery of infected hypercholesterolemic minipigs

    DEFF Research Database (Denmark)

    Birck, Malene Muusfeldt; Saraste, Antti; Hyttel, Poul

    2013-01-01

    Apoptosis of endothelial cells (ECs) has been suggested to play a role in atherosclerosis. We studied the synergism of hypercholesterolemia with Chlamydia pneumoniae and influenza virus infections on EC morphology and intimal changes in a minipig model. The coronary artery was excised at euthanasia...

  18. Epac1 increases migration of endothelial cells and melanoma cells via FGF2-mediated paracrine signaling

    DEFF Research Database (Denmark)

    Baljinnyam, Erdene; Umemura, Masanari; Chuang, Christine

    2014-01-01

    Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N-sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N-sulfation of HS in melanoma. Ther...

  19. Extracellular Vesicles from Vascular Endothelial Cells Promote Survival, Proliferation and Motility of Oligodendrocyte Precursor Cells.

    Directory of Open Access Journals (Sweden)

    Masashi Kurachi

    Full Text Available We previously examined the effect of brain microvascular endothelial cell (MVEC transplantation on rat white matter infarction, and found that MVEC transplantation promoted remyelination of demyelinated axons in the infarct region and reduced apoptotic death of oligodendrocyte precursor cells (OPCs. We also found that the conditioned medium (CM from cultured MVECs inhibited apoptosis of cultured OPCs. In this study, we examined contribution of extracellular vesicles (EVs contained in the CM to its inhibitory effect on OPC apoptosis. Removal of EVs from the CM by ultracentrifugation reduced its inhibitory effect on OPC apoptosis. To confirm whether EVs derived from MVECs are taken up by cultured OPCs, we labeled EVs with PKH67, a fluorescent dye, and added them to OPC cultures. Many vesicular structures labeled with PKH67 were found within OPCs immediately after their addition. Next we examined the effect of MVEC-derived EVs on OPC behaviors. After 2 days in culture with EVs, there was significantly less pyknotic and more BrdU-positive OPCs when compared to control. We also examined the effect of EVs on motility of OPCs. OPCs migrated longer in the presence of EVs when compared to control. To examine whether these effects on cultured OPCs are shared by EVs from endothelial cells, we prepared EVs from conditioned media of several types of endothelial cells, and tested their effects on cultured OPCs. EVs from all types of endothelial cells we examined reduced apoptosis of OPCs and promoted their motility. Identification of the molecules contained in EVs from endothelial cells may prove helpful for establishment of effective therapies for demyelinating diseases.

  20. CD13 is a novel mediator of monocytic/endothelial cell adhesion

    DEFF Research Database (Denmark)

    Mina-Osorio, Paola; Winnicka, Beata; O'Conor, Catherine

    2008-01-01

    During inflammation, cell surface adhesion molecules guide the adhesion and migration of circulating leukocytes across the endothelial cells lining the blood vessels to access the site of injury. The transmembrane molecule CD13 is expressed on monocytes and endothelial cells and has been shown...... to mediate homotypic cell adhesion, which may imply a role for CD13 in inflammatory monocyte trafficking. Here, we show that ligation and clustering of CD13 by mAb or viral ligands potently induce myeloid cell/endothelial adhesion in a signal transduction-dependent manner involving monocytic cytoskeletal...... rearrangement and filopodia formation. Treatment with soluble recombinant (r)CD13 blocks this CD13-dependent adhesion, and CD13 molecules from monocytic and endothelial cells are present in the same immunocomplex, suggesting a direct participation of CD13 in the adhesive interaction. This concept...

  1. Homocysteine pre-treatment increases redox capacity in both endothelial and tumor cells.

    Science.gov (United States)

    Díaz-Santiago, Elena; Rodríguez-Caso, Luis; Cárdenas, Casimiro; Serrano, José J; Quesada, Ana R; Medina, Miguel Ángel

    2017-07-01

    We studied the modulatory effects of homocysteine pre-treatment on the disulfide reduction capacity of tumor and endothelial cells. Human MDA-MB-231 breast carcinoma and bovine aorta endothelial cells were pre-treated for 1-24 hours with 0.5-5 mM homocysteine or homocysteine thiolactone. After washing to eliminate any rest of homocysteine or homocysteine thiolactone, cell redox capacity was determined by using a method for measuring disulfide reduction. Homocysteine pre-treatments for 1-4 hours at a concentration of 0.5-5 mM increase the disulfide reduction capacity of both tumor and endothelial cells. This effect cannot be fully mimicked by either cysteine or homocysteine thiolactone pre-treatments of tumor cells. Taken together, our data suggest that homocysteine can behave as an anti-oxidant agent by increasing the anti-oxidant capacity of tumor and endothelial cells.

  2. Endothelial and beta cell composite aggregates for improved function of a bioartificial pancreas encapsulation device.

    Science.gov (United States)

    Skrzypek, Katarzyna; Barrera, Yazmin Brito; Groth, Thomas; Stamatialis, Dimitrios

    2018-03-01

    Encapsulation of pancreatic islets or beta cells is a promising strategy for treatment of type 1 diabetes by providing an immune isolated environment and allowing for transplantation in a different location than the liver. However, islets used for encapsulation often show lower functionality due to the damaging of islet endothelial cells during the isolation procedure. Factors produced by endothelial cells have great impact on beta cell insulin secretion. Therefore, mutual signaling between endothelial cells and beta cells should be considered for the development of encapsulation systems to achieve high insulin secretion and maintain beta cell viability. Here, we investigate whether co-culture of beta cells with endothelial cells could improve beta cell function within encapsulation devices. Mouse insulinoma MIN6 cells and human umbilical vein endothelial cells were used for creating composite aggregates on agarose microwell platform. The composite aggregates were encapsulated within flat poly(ether sulfone)/polyvinylpyrrolidone device. Their functionality was assessed by glucose-induced insulin secretion test and compared to non-encapsulated free-floating aggregates. We created composite aggregates of 80-100 µm in diameter, closely mimicking pancreatic islets. Upon glucose stimulation, their insulin secretion is improved in comparison to aggregates consisting of only MIN6 cells. Moreover, the composite aggregates encapsulated within a device secrete more insulin than aggregates consisting of only MIN6 cells. Composite aggregates of MIN6 cells with human umbilical vein endothelial cells have improved insulin secretion in comparison to MIN6 aggregates showing that the interaction of beta cell and endothelial cell is crucial for a functional encapsulation system.

  3. Posttranscriptional stimulation of endothelial cell matrix metalloproteinases 2 and 1 by endothelioma cells.

    Science.gov (United States)

    Taraboletti, G; Sonzogni, L; Vergani, V; Hosseini, G; Ceruti, R; Ghilardi, C; Bastone, A; Toschi, E; Borsotti, P; Scanziani, E; Giavazzi, R; Pepper, M S; Stetler-Stevenson, W G; Bani, M R

    2000-08-01

    Matrix metalloproteinases (MMPs) play a critical role in the development of hemangioma-like vascular tumors in mice injected with murine eEnd.1 endothelioma cells. The current study was designed to (a) characterize the presence of MMPs in the vascular tumor, (b) define whether these MMPs originate from the transformed cells or from the recruited stromal cells and (c) study the stimulatory effect of eEnd.1 cells on the production of MMPs by endothelial cells. Several gelatinases were present in the eEnd.1 tumor extract, including latent and activated MMP-2 (72-kDa gelatinase A, EC 3.4.24. 24) and MMP-9 (92-kDa gelatinase B, EC 3.4.24.35). Immunohistochemical analysis of the tumor revealed focal reactivity for MMP-2. No gelatinase was produced by cultured eEnd.1 cells, or by six of nine related endothelioma cell lines, suggesting that stroma cells, particularly endothelial cells recruited by the tumor cells, rather than eEnd.1 cells themselves, are the source of the gelatinases observed in the tumors in vivo. The conditioned medium of eEnd.1 cells stimulated the release of MMP-2 and MMP-1 (interstitial collagenase, EC 3.4.24.7) by endothelial cells, but not of the inhibitor TIMP-2. The increased production of MMP-2 and MMP-1, observed at the protein level (zymogram and Western blot analysis), occurred through a posttranscriptional mechanism, since no increase in mRNA was observed and the stimulation was not prevented by inhibitors of protein synthesis. The inhibitory effects of monensin and brefeldin A, inhibitors of protein secretion, and the decrease in cell-associated MMP-2 in stimulated endothelial cells indicated that regulation occurred mostly at the level of protease secretion. MMPs are known to be regulated at different levels; this study indicates that, in endothelial cells, the stimulation of MMPs can also occur at the level of secretion, a mechanism that provides a rapid mobilization of these crucial enzymes in the early phases of angiogenesis. Copyright

  4. Pathogen sensing pathways in human embryonic stem cell derived-endothelial cells: role of NOD1 receptors.

    Directory of Open Access Journals (Sweden)

    Daniel M Reed

    Full Text Available Human embryonic stem cell-derived endothelial cells (hESC-EC, as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR Toll-like receptor (TLR-4 and nucleotide-binding oligomerisation domain-containing protein (NOD-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC. HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC, and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.

  5. Micromanipulation of endothelial cells: Ultrasound-microbubble-cell interaction

    NARCIS (Netherlands)

    van Wamel, Annemieke; Bouakaz, Ayache; Versluis, Michel; de Jong, N.

    2004-01-01

    Ultrasound (US) in combination with contrast microbubbles has been shown to alter the permeability of cell membranes without affecting cell viability. This permeabilisation feature is used to design new drug delivery systems using US and contrast agents. The underlying mechanisms are still unknown.

  6. The effect of uric acid on homocysteine-induced endothelial dysfunction in bovine aortic endothelial cells

    Czech Academy of Sciences Publication Activity Database

    Papežíková, Ivana; Pekarová, Michaela; Lojek, Antonín; Kubala, Lukáš

    2009-01-01

    Roč. 30, č. 1 (2009), s. 112-115 ISSN 0172-780X R&D Projects: GA ČR(CZ) GP204/07/P539 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : uric acid * homocysteine * endothelial dysfunction Subject RIV: BO - Biophysics Impact factor: 1.047, year: 2009

  7. Rapamycin Inhibits Proliferation of Hemangioma Endothelial Cells by Reducing HIF-1-Dependent Expression of VEGF

    Science.gov (United States)

    Medici, Damian; Olsen, Bjorn R.

    2012-01-01

    Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas. PMID:22900063

  8. Sphingosine 1-Phosphate Induces Platelet/Endothelial Cell Adhesion Molecule-1 Tyrosine Phosphorylation in Bovine Aortic Endothelial Cells through a PP2-Inhibitable Mechanism

    Directory of Open Access Journals (Sweden)

    Yu-Ting Huang

    2007-12-01

    Full Text Available Sphingosine-1-phosphate (S1P is a low-molecular-weight phospholipid derivative released by activated platelets. S1P transduces signals through a family of G protein-coupled receptors to modulate various physiological behaviors of endothelial cells. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31 is a 130-kDa protein expressed on the surfaces of leukocytes, platelets, and endothelial cells. Upon PECAM-1 activation, its cytoplasmic tyrosine residues become phosphorylated and bind with SH2 domain-containing proteins, thus leading to the downstream functions mediated by PECAM-1. In the present study, we found that S1P induced PECAM-1 tyrosine phosphorylation and SHP-2 association in bovine aortic endothelial cells (BAECs by immunoprecipitation and western blotting. The pretreatment of BAECs with a series of chemical inhibitors to determine the signaling pathway showed that the PECAM-1 phosphorylation was inhibited by PP2, indicating the participation of Src family kinases. These results demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in BAECs through mediation of Src family kinases, and this may regulate the physiological behaviors of endothelial cells.

  9. An optimized method for mouse liver sinusoidal endothelial cell isolation

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, Jeremy, E-mail: jeremy.meyer@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Lacotte, Stéphanie, E-mail: stephanie.lacotte@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Morel, Philippe, E-mail: philippe.morel@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Gonelle-Gispert, Carmen, E-mail: carmen.gonelle@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Bühler, Léo, E-mail: leo.buhler@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland)

    2016-12-10

    The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic

  10. Size and Dynamics of Caveolae Studied Using Nanoparticles in Living Endothelial Cells

    OpenAIRE

    Wang, Zhenjia; Tiruppathi, Chinnaswamy; Minshall, Richard D.; Malik, Asrar B.

    2009-01-01

    Caveolae are plasma membrane invaginations prominent in all endothelial cells lining blood vessels. Caveolae characteristically bud to form free cytoplasmic vesicles capable of transporting carrier proteins such as albumin through the cell. However, caveolae size distribution and dynamics in living endothelial cells and ability of caveolae to internalize nanoparticles are not well understood. We demonstrate here the design of a dual-color nanoparticle pair to measure non-invasively caveolae s...

  11. Interaction of human endothelial cells and nickel-titanium materials modified with silicon ions

    Energy Technology Data Exchange (ETDEWEB)

    Lotkov, Aleksandr I., E-mail: lotkov@ispms.tsc.ru; Kashin, Oleg A., E-mail: okashin@ispms.tsc.ru [Institute of Strength Physics and Materials Science SB RAS, Tomsk, 634055 (Russian Federation); Kudryavtseva, Yuliya A., E-mail: yulia-k1970@mail.ru; Antonova, Larisa V., E-mail: antonova.la@mail.ru; Matveeva, Vera G., E-mail: matveeva-vg@mail.ru; Sergeeva, Evgeniya A., E-mail: sergeewa.ew@yandex.ru [Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, 650002 (Russian Federation); Kudryashov, Andrey N., E-mail: kudryashov@angioline.ru [Angioline Interventional Device Ltd, Novosibirsk, 630090 (Russian Federation)

    2015-10-27

    The paper studies the influence of chemical and phase compositions of NiTi surface layers modified with Si ions by plasma immersion implantation on their interaction with endothelial cells. It is shown that certain technological modes of Si ion implantation enhance the adhesion, proliferation, and viability of endothelial cells. It is found that the Si-modified NiTi surface is capable of stimulating the formation of capillary-like structures in the cell culture.

  12. Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis

    Science.gov (United States)

    Lerner, Thomas R.; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R.G.; Borel, Sophie; Diedrich, Collin R.; Rohde, Manfred; Wainwright, Helen; Collinson, Lucy M.; Wilkinson, Robert J.; Griffiths, Gareth; Gutierrez, Maximiliano G.

    2016-01-01

    In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes. PMID:26901813

  13. Gold nanoparticle interactions with endothelial cells cultured under physiological conditions.

    Science.gov (United States)

    Freese, C; Anspach, L; Deller, R C; Richards, S-J; Gibson, M I; Kirkpatrick, C J; Unger, R E

    2017-03-28

    PEGylated gold nanoparticles (AuNPs) have an extended circulation time after intravenous injection in vivo and exhibit favorable properties for biosensing, diagnostic imaging, and cancer treatment. No impact of PEGylated AuNPs on the barrier forming properties of endothelial cells (ECs) has been reported, but recent studies demonstrated that unexpected effects on erythrocytes are observed. Almost all studies to date have been with static-cultured ECs. Herein, ECs maintained under physiological cyclic stretch and flow conditions and used to generate a blood-brain barrier model were exposed to 20 nm PEGylated AuNPs. An evaluation of toxic effects, cell stress, the release profile of pro-inflammatory cytokines, and blood-brain barrier properties showed that even under physiological conditions no obvious effects of PEGylated AuNPs on ECs were observed. These findings suggest that 20 nm-sized, PEGylated AuNPs may be a useful tool for biomedical applications, as they do not affect the normal function of healthy ECs after entering the blood stream.

  14. A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

    Science.gov (United States)

    Bartakova, Alena; Kuzmenko, Olga; Alvarez-Delfin, Karen; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

    2018-01-01

    Purpose Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors (“proliferative media”) to media without those factors (“stabilizing media”). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies. PMID:29625488

  15. Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells.

    Directory of Open Access Journals (Sweden)

    Dilyana Todorova

    Full Text Available BACKGROUND: Circulating CD34(+ cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN could target progenitor cell injury by Natural Killer (NK cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+-derived Endothelial Colony Forming Cells (ECFC can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+ progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+ cells expressing FKN was identified as an independent variable inversely correlated to CD34(+ progenitor cell count. We further showed that treatment of CD34(+ circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+ progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.

  16. Human breast microvascular endothelial cells retain phenotypic traits in long-term finite life span culture

    DEFF Research Database (Denmark)

    Sigurdsson, Valgardur; Fridriksdottir, Agla J R; Kjartansson, Jens

    2007-01-01

    Attempts to study endothelial-epithelial interactions in the human breast have been hampered by lack of protocols for long-term cultivation of breast endothelial cells (BRENCs). The aim of this study was to establish long-term cultures of BRENCs and to compare their phenotypic traits with the tis......Attempts to study endothelial-epithelial interactions in the human breast have been hampered by lack of protocols for long-term cultivation of breast endothelial cells (BRENCs). The aim of this study was to establish long-term cultures of BRENCs and to compare their phenotypic traits...... with the tissue of origin. Microvasculature was localized in situ by immunohistochemistry in breast samples. From this tissue, collagen-rich stroma and adipose tissue were dissected mechanically and further disaggregated to release microvessel organoids. BRENCs were cultured from these organoids in endothelial...

  17. Comparison of corneal endothelial cell measurements by two non-contact specular microscopes.

    Science.gov (United States)

    Gasser, Laura; Reinhard, Thomas; Böhringer, Daniel

    2015-07-29

    Measurement of corneal endothelial cell density is important both for clinical diagnosis as well as clinical studies. Since endothelial cell loss is considered irreversible in humans, even small changes in endothelial cell density are relevant. Therefore it is important to know whether different instruments for endothelial cell density measurements give the same results and can thus be used interchangeably. In this study we compare corneal endothelial cell density and morphometry measurements from two widely used non-contact specular microscopes, the Topcon SP3000P and Konan Noncon Robo SP8000. Endothelial cell measurements were performed with both the Topcon SP3000P and Konan Noncon Robo SP8000 on 34 eyes of 18 consecutive patients of our cornea clinics with poor image quality being the only exclusion criterion. Images were obtained using the auto-focussing method and manual cell selection. Endothelial cell density (ECD), hexagonal cell ratio (HEX) and coefficient of value (CV) of the endothelial cell layer were calculated by the instruments' built-in software. ECD values calculated by the Konan were systematically higher than Topcon values: in 94 % of eyes Konan gave a higher value than Topcon, leading to a mean difference in ECD between the instruments of 187 cells/mm(2) (P < 0.001 in paired Wilcoxon test). HEX showed a broad range of values and differed greatly with only weak correlation between the two instruments. CV values for Konan mostly exceeded Topcon values, and only showed a weak correlation between the two instruments as well. Values for ECD between the Konan and the Topcon do correlate well, but the ECDs calculated by the Konan are systematically higher than Topcon values. Both HEX and CV vary greatly and do not correlate sufficiently. Thus we recommend not to use the Konan and the Topcon instrument interchangeably.

  18. IL-27 inhibits lymphatic endothelial cell proliferation by STAT1-regulated gene expression

    DEFF Research Database (Denmark)

    Nielsen, Sebastian Rune; Hammer, Troels; Gibson, Josefine

    2013-01-01

    OBJECTIVE: IL-27 belongs to the IL-12 family of cytokines and is recognized for its role in Th cell differentiation and as an inhibitor of tumor-angiogenesis. The purpose of this study was to investigate the effect of IL-27 on proliferation of lymphatic endothelial cells to gain insight...... into the interplay between the immune system and development of the lymphatic system. METHODS: IL-27-stimulated signal transduction in human dermal lymphatic endothelial cells was measured by western blotting and synthesis of CXCL10 and CXCL11 by use of RT-PCR and ELISA. Proliferation was measured using MTT and Brd......U kits and the role of STAT1 and chemokines was determined by use of siRNA and recombinant proteins. RESULTS: Stimulation of lymphatic endothelial cell cultures with IL-27 induced JAK dependent phosphorylation of STAT1 and STAT3 and inhibited lymphatic endothelial cell proliferation and migration...

  19. Regulation of staphylococcal enterotoxin B-elicited nitric oxide production by endothelial cells.

    Science.gov (United States)

    LeClaire, R D; Kell, W M; Sadik, R A; Downs, M B; Parker, G W

    1995-01-01

    The effect of staphylococcal enterotoxin B (SEB)-elicited inducible nitric oxide synthase (iNOS) in mouse endothelial cells was investigated. Results showed that SEB stimulated the same level of NO production in gamma interferon (IFN-gamma)-primed cells as did trichloroacetic acid-extracted lipopolysaccharide. The kinetics of induced NO production and expression of mRNA for iNOS differed markedly in endothelial and macrophage cells. Induced endothelial nitrite production was transient and was 15 to 20% of that generated by macrophage cells; mRNA levels peaked by 2 h and then steadily declined, whereas macrophage message levels continually increased. The ability of endothelial cells to produce SEB-induced NO depended on priming with IFN-gamma, although detectable mRNA could be elicited by SEB alone. Induction of endothelial iNOS mRNA was inhibited by cycloheximide, which indicated a requirement for de novo protein synthesis. Niacinamide and interleukin-10 significantly reduced SEB-induced endothelial NO production. Both are reported to affect IFN-gamma-induced class II major histocompatibility complex (MHC) expression on antigen-presenting cells. Niacinamide reduced iNOS mRNA levels and markedly reduced IFN-gamma induction of endothelial class II MHC surface antigen. Interleukin-10 did not consistently reduce iNOS mRNA expression and had no effect on IFN-gamma induction of endothelial class II MHC surface antigen. These results suggest that SEB interacts with IFN-gamma-primed endothelial cells to elicit induced NO and that this induction can be effectively modulated at the receptor or transcriptional level. PMID:7529748

  20. Microparticles of pregnant women and preeclamptic patients activate endothelial cells in the presence of monocytes.

    Science.gov (United States)

    Lok, Christine A R; Snijder, Karin S; Nieuwland, Rienk; Van Der Post, Joris A M; de Vos, Paul; Faas, Marijke M

    2012-03-01

    Preeclampsia is a pregnancy-specific disorder that may result from an adverse maternal response to circulating placenta-derived factors, causing a systemic inflammation including endothelial activation. Plasma from preeclamptic patients was shown to induce endothelial activation in the presence of monocytes. We investigated whether microparticles (MP) are the plasma factors causing this activation of endothelial cells. Monocultures and co-cultures of monocytes and endothelial cells were incubated with plasma, MP-poor plasma or isolated MP from non-pregnant and pregnant women and preeclamptic patients (each n = 8). ICAM-1 expression was analyzed with flow cytometry. The expression of ICAM-1 was significantly increased in monocytes and endothelial cells in co-cultures after the addition of isolated MP from preeclamptic patients (P = 0.017) and to a lesser extent in pregnant women (P = 0.012) compared to non-pregnant controls. Microparticles from preeclamptic patients activate endothelial cells in the presence of monocytes. Whether all MP have the same effect on monocytes and endothelial cells or only a specific subgroup is the focus of future research. © 2011 John Wiley & Sons A/S.

  1. Do endothelial cells belong to the primitive stem leukemic clone in CML? Role of extracellular vesicles.

    Science.gov (United States)

    Ramos, Teresa L; Sánchez-Abarca, Luis Ignacio; López-Ruano, Guillermo; Muntión, Sandra; Preciado, Silvia; Hernández-Ruano, Montserrat; Rosado, Belén; de las Heras, Natalia; Chillón, M Carmen; Hernández-Hernández, Ángel; González, Marcos; Sánchez-Guijo, Fermín; Del Cañizo, Consuelo

    2015-08-01

    The expression of BCR-ABL in hematopoietic stem cells is a well-defined primary event in chronic myeloid leukemia (CML). Some reports have described the presence of BCR-ABL on endothelial cells from CML patients, suggesting the origin of the disease in a primitive hemangioblastic cell. On the other hand, extracellular vesicles (EVs) released by CML leukemic cells are involved in the angiogenesis modulation process. In the current work we hypothesized that EVs released from BCR-ABL(+) cells may carry inside the oncogene that can be transferred to endothelial cells leading to the expression of both BCR-ABL transcript and the oncoprotein. EVs from K562 cells and plasma of newly diagnosed CML patients were isolated by ultracentrifugation. RT-PCR analysis detected the presence of BCR-ABL RNA in the EVs isolated from both K562 cells and plasma of CML patients. The incorporation of these EVs into endothelial cells was demonstrated by flow cytometry and fluorescence microscopy showed that after 24h of incubation most EVs were incorporated. BCR-ABL transcripts were detected in all experiments on endothelial cells incubated with EVs from both sources. The presence of BCR-ABL on endothelial cells incubated with Philadelphia(+) EVs was also confirmed by Western blot assays. In summary, endothelial cells acquire BCR-ABL RNA and the oncoprotein after incubation with EVs released from Ph(+) positive cells (either from K562 cells or from plasma of newly diagnosed CML patients). This results challenge the hypothesis that endothelial cells may be part of the Philadelphia(+) clone in CML. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells.

    Science.gov (United States)

    Alghanem, Ahmad F; Wilkinson, Emma L; Emmett, Maxine S; Aljasir, Mohammad A; Holmes, Katherine; Rothermel, Beverley A; Simms, Victoria A; Heath, Victoria L; Cross, Michael J

    2017-08-01

    Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF.

  3. Generation and characterization of a spontaneously immortalized endothelial cell line from mice microcirculation.

    Science.gov (United States)

    Loiola, Rodrigo A; Torres, Tathiany C; Aburaya, Carla M; Landgraf, Maristella A; Landgraf, Richardt G; Bosco Pesquero, João; Fernandes, Liliam

    2013-05-01

    Endothelial cells from microvasculature are directly involved in a large number of vascular diseases; however, culture of these cells is problematic, since most methodologies employ proteolytic enzymes or mechanical techniques, leading to cell damage and contamination of endothelial cultures with other cellular types. Besides, primary cultured cells have a short life span in vitro and undergo replicative senescence after 3-4 passages, limiting long-term studies. In the present work we report the generation of a spontaneously immortalized endothelial culture obtained from mice pulmonary capillaries. Firstly, primary (third passage) and immortalized (100th) cultures were established. Further, monoclonal populations were obtained by serial dilutions from immortalized cultures. Cells were analyzed according to: (1) morphological appearance, (2) expression of specific endothelial markers by fluorescent staining [von Willebrand Factor (vWF), endothelial nitric oxide synthase (eNOS), angiotensin converting enzyme (ACE) and Ulex europaeus (UEA-1)] and by flow cytometry (endoglin, VE-cadherin and VCAM-1), and (3) release of nitric oxide (NO), assessed by the specific fluorescent dye DAF-2 DA, and prostacyclin (PGI2), quantified by enzyme immune assay. In both cultures cells grew in monolayers and presented cobblestone appearance at confluence. Positive staining for vWF, eNOS, ACE and UEA-1 was detected in cloned as well as in early-passage cultured cells. Similarly, cultures presented equal expressions of endoglin, VE-cadherin and VCAM-1. Values of NO and PGI2 levels did not differ between cultures. From these results we confirm that the described spontaneously immortalized endothelial cell line is capable of unlimited growth and retains typical morphological and functional properties exhibited by primary cultured cells. Therefore, the endothelial cell line described in the present study can become a suitable tool in the field of endothelium research and can be useful for

  4. Hemeoxygenase-1 Mediates an Adaptive Response to Spermidine-Induced Cell Death in Human Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Hana Yang

    2013-01-01

    Full Text Available Spermidine (SPD is a ubiquitous polycation that is commonly distributed in living organisms. Intracellular levels of SPD are tightly regulated, and SPD controls cell proliferation and death. However, SPD undergoes oxidation in the presence of serum, producing aldehydes, hydrogen peroxide, and ammonia, which exert cytotoxic effect on cells. Hemeoxygenase-1 (HO-1 is thought to have a protective effect against oxidative stress. Upregulation of HO-1 in endothelial cells is considered to be beneficial in the cardiovascular disease. In the present study, we demonstrate that the ubiquitous polyamine, SPD, induces HO-1 in human umbilical vein endothelial cells (HUVECs. SPD-induced HO-1 expression was examined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR. Involvement of reactive oxygen species, serum amine oxidase, PI3K/Akt signaling pathway, and transcription factor Nrf2 in the induction of HO-1 by SPD was also investigated. Furthermore, small interfering RNA knockdown of Nrf2 or HO-1 and treatment with the specific HO-1 inhibitor ZnPP exhibited a noteworthy increase of death of SPD-stimulated HUVECs. In conclusion, these results suggest that SPD induces PI3K/Akt-Nrf2-mediated HO-1 expression in human endothelial cells, which may have a role in cytoprotection of the cells against oxidative stress-induced death.

  5. Distribution of a 69-kD laminin-binding protein in aortic and microvascular endothelial cells: modulation during cell attachment, spreading, and migration

    DEFF Research Database (Denmark)

    Yannariello-Brown, J; Wewer, U; Liotta, L

    1988-01-01

    Affinity chromatography and immunolocalization techniques were used to investigate the mechanism(s) by which endothelial cells interact with the basement membrane component laminin. Bovine aortic endothelial cells (BAEC) membranes were solubilized and incubated with a laminin-Sepharose affinity...

  6. Chorein Sensitivity of Actin Polymerization, Cell Shape and Mechanical Stiffness of Vascular Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Ioana Alesutan

    2013-09-01

    Full Text Available Background/Aims: Endothelial cell stiffness plays a key role in endothelium-dependent control of vascular tone and arterial blood pressure. Actin polymerization and distribution of microfilaments is essential for mechanical cell stiffness. Chorein, a protein encoded by the VPS13A gene, defective in chorea-acanthocytosis (ChAc, is involved in neuronal cell survival as well as cortical actin polymerization of erythrocytes and blood platelets. Chorein is expressed in a wide variety of further cells, yet nothing is known about the impact of chorein on cells other than neurons, erythrocytes and platelets. The present study explored whether chorein is expressed in human umbilical vein endothelial cells (HUVECs and addressed the putative role of chorein in the regulation of cytoskeletal architecture, stiffness and survival of those cells. Methods: In HUVECs with or without silencing of the VPS13A gene, VPS13A mRNA expression was determined utilizing quantitative RT-PCR, cytoskeletal organization visualized by confocal microscopy, G/F actin ratio and phosphorylation status of focal adhesion kinase quantified by western blotting, cell death determined by flow cytometry, mechanical properties studied by atomic force microscopy (AFM and cell morphology analysed by scanning ion conductance microscopy (SICM. Results: VPS13A mRNA expression was detectable in HUVECs. Silencing of the VPS13A gene attenuated the filamentous actin network, decreased the ratio of soluble G-actin over filamentous F-actin, reduced cell stiffness and changed cell morphology as compared to HUVECs silenced with negative control siRNA. These effects were paralleled by a significant decrease in FAK phosphorylation following VPS13A silencing. Moreover, silencing of the VPS13A gene increased caspase 3 activity and induced necrosis in HUVECs. Conclusions: Chorein is a novel regulator of cytoskeletal architecture, cell shape, mechanical stiffness and survival of vascular endothelial cells.

  7. Wnt5a-mediated non-canonical Wnt signalling regulates human endothelial cell proliferation and migration

    International Nuclear Information System (INIS)

    Cheng Chingwen; Yeh Juching; Fan Taiping; Smith, Stephen K.; Charnock-Jones, D. Stephen

    2008-01-01

    Cell to cell interaction is one of the key processes effecting angiogenesis and endothelial cell function. Wnt signalling is mediated through cell-cell interaction and is involved in many developmental processes and cellular functions. In this study, we investigated the possible function of Wnt5a and the non-canonical Wnt pathway in human endothelial cells. We found that Wnt5a-mediated non-canonical Wnt signalling regulated endothelial cell proliferation. Blocking this pathway using antibody, siRNA or a down-stream inhibitor led to suppression of endothelial cell proliferation, migration, and monolayer wound closure. We also found that the mRNA level of Wnt5a is up-regulated when endothelial cells are treated with a cocktail of inflammatory cytokines. Our findings suggest non-canonical Wnt signalling plays a role in regulating endothelial cell growth and possibly in angiogenesis

  8. Hypoxia and the Presence of Human Vascular Endothelial Cells Affect Prostate Cancer Cell Invasion and Metabolism

    Directory of Open Access Journals (Sweden)

    Ellen Ackerstaff

    2007-12-01

    Full Text Available Tumor progression and metastasis are influenced by hypoxia, as well as by interactions between cancer cells and components of the stroma, such as endothelial cells. Here, we have used a magnetic resonance (MRcompatible invasion assay to further understand the effects of hypoxia on human prostate cancer cell invasion and metabolism in the presence and absence of human umbilical vein endothelial cells (HUVECs. Additionally, we compared endogenous activities of selected proteases related to invasion in PC-3 cells and HUVECs, profiled gene expression of PC-3 cells by microarray, evaluated cell proliferation of PC-3 cells and HUVECs by flow cytometry, under hypoxic and oxygenated conditions. The invasion of less-invasive DU-145 cells was not affected by either hypoxia or the presence of HUVECs. However, hypoxia significantly decreased the invasion of PC-3 cells. This hypoxia-induced decrease was attenuated by the presence of HUVECs, whereas under oxygenated conditions, HUVECs did not alter the invasion of PC-3 cells. Cell metabolism changed distinctly with hypoxia and invasion. The endogenous activity of selected extracellular proteases, although altered by hypoxia, did not fully explain the hypoxia-induced changes in invasion. Gene expression profiling indicated that hypoxia affects multiple cellular functions and pathways.

  9. Pathological aging of the vascular endothelium: are endothelial progenitor cells the sentinels of the cardiovascular system?

    Science.gov (United States)

    Thorin-Trescases, Nathalie; Voghel, Guillaume; Gendron, Marie-Eve; Krummen, Stephane; Farhat, Nada; Drouin, Annick; Perrault, Louis P; Thorin, Eric

    2005-10-01

    Aging is associated with vascular endothelial dysfunction, which ultimately leads to atherosclerosis. On the other hand, it is clear that in young patients with risk factors for cardiovascular diseases (CVD), endothelial dysfunction is an early marker of the ongoing atherogenic process. It is therefore tempting to speculate that risk factors for CVD accelerate the aging process. The aging of an endothelial cell (EC) is not chronological but rather dependent on its replication rate. ECs have a finite number of divisions and enter replicative senescence after exhaustion of this potential. Telomere attrition is believed to be responsible for this phenomenon. Upon reaching a critical minimal telomere length, ECs enter a nondividing state of replicative senescence. Recently, endothelial progenitor cells originating from the bone marrow have been isolated from the circulation. They integrate into the endothelial layer of the vessel and contribute to healing, ischemic repair and angiogenesis. A completely new field of investigation is now open. Are endothelial progenitor cells sensitive to the aging process? Do they prevent endothelial dysfunction? Are they the ultimate shield against the damages induced by risk factors for CVD? There are no definite answers to these questions, but the potential of these cells is tremendous and understanding their physiology is essential.

  10. Fusobacterium nucleatum adhesin FadA binds vascular endothelial cadherin and alters endothelial integrity.

    Science.gov (United States)

    Fardini, Yann; Wang, Xiaowei; Témoin, Stéphanie; Nithianantham, Stanley; Lee, David; Shoham, Menachem; Han, Yiping W

    2011-12-01

    Fusobacterium nucleatum is a Gram-negative oral anaerobe, capable of systemic dissemination causing infections and abscesses, often in mixed-species, at different body sites. We have shown previously that F. nucleatum adheres to and invades host epithelial and endothelial cells via a novel FadA adhesin. In this study, vascular endothelial (VE)-cadherin, a member of the cadherin family and a cell-cell junction molecule, was identified as the endothelial receptor for FadA, required for F. nucleatum binding to the cells. FadA colocalized with VE-cadherin on endothelial cells, causing relocation of VE-cadherin away from the cell-cell junctions. As a result, the endothelial permeability was increased, allowing the bacteria to cross the endothelium through loosened junctions. This crossing mechanism may explain why the organism is able to disseminate systemically to colonize in different body sites and even overcome the placental and blood-brain barriers. Co-incubation of F. nucleatum and Escherichia coli enhanced penetration of the endothelial cells by the latter in the transwell assays, suggesting F. nucleatum may serve as an 'enabler' for other microorganisms to spread systemically. This may explain why F. nucleatum is often found in mixed infections. This study reveals a possible novel dissemination mechanism utilized by pathogens. © 2011 Blackwell Publishing Ltd.

  11. Redox modulation of tyrosine phosphorylation-dependent neutrophil adherence to endothelial cells

    International Nuclear Information System (INIS)

    Thibodeau, Paul A.; Gozin, Alexia; Gougerot-Pocidalo, Marie-Anne; Pasquier, Catherine

    2005-01-01

    Reactive oxygen species (ROS) are now well known to be involved in an increased interaction between neutrophils and endothelial cells. Previously, we have shown that the increased adhesion of neutrophils to ROS-stimulated endothelial cells involves an increase in tyrosine phosphorylation of the focal adhesion kinase, p125 FAK , and several cytoskeleton proteins. This review article focuses on the involvement of adhesion molecules in the increased adhesion of neutrophils to ROS-stimulated endothelial cells, on the oxygen species responsible for this adhesion, and on the intracellular signaling pathway leading to the modification of the cytoskeleton by ROS. The evidence from our laboratory and others describing these events is summarized. Finally, the future perspectives that need to be explored in order to inhibit or reduce the ROS-mediated adhesion of neutrophils to endothelial cells are addressed

  12. Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells

    DEFF Research Database (Denmark)

    Høgh, Mette; Tannetta, D; Sargent, I

    2006-01-01

    Objective Syncytiotrophoblast membrane fragments (STBM) exist in the peripheral circulation in pregnant women and it has been shown that the level of circulating STBM is significantly increased with pre-eclampsia compared with uncomplicated pregnancies. STBM could be one of the factors which...... directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. Design Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from...... results. Results Overall, the results do not show any great changes in gene expression in endothelial cells after STBM treatment (28 genes changed two-fold or more out of approximately 10 000 genes examined by microarray). In general, the changes observed are consistent with inhibition of proliferation...

  13. Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells

    DEFF Research Database (Denmark)

    Hoegh, A M; Tannetta, D; Sargent, I

    2006-01-01

    OBJECTIVE: Syncytiotrophoblast membrane fragments (STBM) exist in the peripheral circulation in pregnant women and it has been shown that the level of circulating STBM is significantly increased with pre-eclampsia compared with uncomplicated pregnancies. STBM could be one of the factors which...... directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. DESIGN: Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from...... results. RESULTS: Overall, the results do not show any great changes in gene expression in endothelial cells after STBM treatment (28 genes changed two-fold or more out of approximately 10,000 genes examined by microarray). In general, the changes observed are consistent with inhibition of proliferation...

  14. ROCK inhibitor enhances adhesion and wound healing of human corneal endothelial cells.

    Directory of Open Access Journals (Sweden)

    Aurélien Pipparelli

    Full Text Available Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and "pump" functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34, the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.

  15. Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells.

    Science.gov (United States)

    Olszewska-Pazdrak, Barbara; Hein, Travis W; Olszewska, Paulina; Carney, Darrell H

    2009-05-01

    Coronary artery disease results in progressive vascular stenosis associated with chronic myocardial ischemia. Vascular endothelial growth factor (VEGF) stimulates endothelial cell angiogenic responses to revascularize ischemic tissues; however, the effect of chronic hypoxia on the responsiveness of endothelial cells to VEGF remains unclear. We, therefore, investigated whether hypoxia alters VEGF-stimulated signaling and angiogenic responses in primary human coronary artery endothelial (HCAE) cells. Exposure of HCAE cells to hypoxia (1% O(2)) for 24 h decreased VEGF-stimulated endothelial cell migration ( approximately 82%), proliferation ( approximately 30%), and tube formation. Hypoxia attenuated VEGF-stimulated activation of endothelial nitric oxide (NO) synthase (eNOS) ( approximately 72%) and reduced NO production in VEGF-stimulated cells from 237 +/- 38.8 to 61.3 +/- 28.4 nmol/l. Moreover, hypoxia also decreased the ratio of phosphorylated eNOS to total eNOS in VEGF-stimulated cells by approximately 50%. This effect was not observed in thrombin-stimulated cells, suggesting that hypoxia specifically inhibited VEGF signaling upstream of eNOS phosphorylation. VEGF-induced activation of Akt, ERK1/2, p38, p70S6 kinases, and S6 ribosomal protein was also attenuated in hypoxic cells. Moreover, VEGF-stimulated phosphorylation of VEGF receptor-2 (KDR) at Y996 and Y1175 was decreased by hypoxia. This decrease correlated with a 70 +/- 12% decrease in KDR protein expression. Analysis of mRNA from these cells showed that hypoxia reduced steady-state levels of KDR mRNA by 52 +/- 16% and decreased mRNA stability relative to normoxic cells. Our findings demonstrate that chronic hypoxia attenuates VEGF-stimulated signaling in HCAE cells by specific downregulation of KDR expression. These data provide a novel explanation for the impaired angiogenic responses to VEGF in endothelial cells exposed to chronic hypoxia.

  16. The coffee diterpene kahweol inhibits tumor necrosis factor-α-induced expression of cell adhesion molecules in human endothelial cells

    International Nuclear Information System (INIS)

    Kim, Hyung Gyun; Kim, Ji Young; Hwang, Yong Pil; Lee, Kyung Jin; Lee, Kwang Youl; Kim, Dong Hee; Kim, Dong Hyun; Jeong, Hye Gwang

    2006-01-01

    Endothelial cells produce adhesion molecules after being stimulated with various inflammatory cytokines. These adhesion molecules play an important role in the development of atherogenesis. Recent studies have highlighted the chemoprotective and anti-inflammatory effects of kahweol, a coffee-specific diterpene. This study examined the effects of kahweol on the cytokine-induced monocyte/human endothelial cell interaction, which is a crucial early event in atherogenesis. Kahweol inhibited the adhesion of TNFα-induced monocytes to endothelial cells and suppressed the TNFα-induced protein and mRNA expression of the cell adhesion molecules, VCAM-1 and ICAM-1. Furthermore, kahweol inhibited the TNFα-induced JAK2-PI3K/Akt-NF-κB activation pathway in these cells. Overall, kahweol has anti-inflammatory and anti-atherosclerotic activities, which occurs partly by down-regulating the pathway that affects the expression and interaction of the cell adhesion molecules on endothelial cells

  17. Endothelial Cell Migration and Vascular Endothelial Growth Factor Expression Are the Result of Loss of Breast Tissue Polarity

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Amy; Cuevas, Ileana; Kenny, Paraic A; Miyake, Hiroshi; Mace, Kimberley; Ghajar, Cyrus; Boudreau, Aaron; Bissell, Mina; Boudreau, Nancy

    2009-05-26

    Recruiting a new blood supply is a rate-limiting step in tumor progression. In a three-dimensional model of breast carcinogenesis, disorganized, proliferative transformed breast epithelial cells express significantly higher expression of angiogenic genes compared with their polarized, growth-arrested nonmalignant counterparts. Elevated vascular endothelial growth factor (VEGF) secretion by malignant cells enhanced recruitment of endothelial cells (EC) in heterotypic cocultures. Significantly, phenotypic reversion of malignant cells via reexpression of HoxD10, which is lost in malignant progression, significantly attenuated VEGF expression in a hypoxia-inducible factor 1{alpha}-independent fashion and reduced EC migration. This was due primarily to restoring polarity: forced proliferation of polarized, nonmalignant cells did not induce VEGF expression and EC recruitment, whereas disrupting the architecture of growth-arrested, reverted cells did. These data show that disrupting cytostructure activates the angiogenic switch even in the absence of proliferation and/or hypoxia and restoring organization of malignant clusters reduces VEGF expression and EC activation to levels found in quiescent nonmalignant epithelium. These data confirm the importance of tissue architecture and polarity in malignant progression.

  18. IMPACT OF MECHANICAL MYOCARDIAL INJURY PRODUCTS, LPS AND THEIR COMBINATION ON HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

    Directory of Open Access Journals (Sweden)

    V. G. Matveeva

    2014-01-01

    Full Text Available Complicated systemic inflammatory response (SIR often determines the outcome in patients after cardiac surgery. Systemic endothelial activation plays the most important role in SIR pathogenesis. We have studied the impact of mechanical myocardial injury products, LPS and their combination on human umbilical vein endothelial cells (HUVEC. We have found that HUVEC increase the production of proinflammatory cytokines in response tocardiomyocyte cytosolic fraction responsible for mechanical injury modeling. 2% cytosolic fraction containing 0.204 ng/mL of Hsp70 was a greater stimulus for endothelial cells to produce IL-6 and IL8 than moderateendotoxin concentrations.

  19. Glial cell ceruloplasmin and hepcidin differentially regulate iron efflux from brain microvascular endothelial cells.

    Science.gov (United States)

    McCarthy, Ryan C; Kosman, Daniel J

    2014-01-01

    We have used an in vitro model system to probe the iron transport pathway across the brain microvascular endothelial cells (BMVEC) of the blood-brain barrier (BBB). This model consists of human BMVEC (hBMVEC) and C6 glioma cells (as an astrocytic cell line) grown in a transwell, a cell culture system commonly used to quantify metabolite flux across a cell-derived barrier. We found that iron efflux from hBMVEC through the ferrous iron permease ferroportin (Fpn) was stimulated by secretion of the soluble form of the multi-copper ferroxidase, ceruloplasmin (sCp) from the co-cultured C6 cells. Reciprocally, expression of sCp mRNA in the C6 cells was increased by neighboring hBMVEC. In addition, data indicate that C6 cell-secreted hepcidin stimulates internalization of hBMVEC Fpn but only when the end-feet projections characteristic of this glia-derived cell line are proximal to the endothelial cells. This hepcidin-dependent loss of Fpn correlated with knock-down of iron efflux from the hBMVEC; this result was consistent with the mechanism by which hepcidin regulates iron efflux in mammalian cells. In summary, the data support a model of iron trafficking across the BBB in which the capillary endothelium induce the underlying astrocytes to produce the ferroxidase activity needed to support Fpn-mediated iron efflux. Reciprocally, astrocyte proximity modulates the effective concentration of hepcidin at the endothelial cell membrane and thus the surface expression of hBMVEC Fpn. These results are independent of the source of hBMVEC iron (transferrin or non-transferrin bound) indicating that the model developed here is broadly applicable to brain iron homeostasis.

  20. Differential effects of corticosteroids and theophylline on the adhesive interaction between eosinophils and endothelial cells

    Directory of Open Access Journals (Sweden)

    Makoto Nagata

    2004-01-01

    Full Text Available Corticosteroids and theophylline have been used widely for the treatment of asthma. These two classes of drugs appear to reduce the tissue infiltration of eosinophils, predominant inflammatory cells in the airways of asthmatic patients. Corticosteroids inhibit the generation of both endothelial-activating Th2 cytokines (e.g. interleukin (IL-4/IL-13 and eosinophil growth factors (e.g. IL-5/granulocyte-macrophage colony stimulating factor and also attenuate the effects of eosinophil growth factors on the differentiation and prolonged survival of eosinophils. However, corticosteroids modulate directly neither eosinophil adhesiveness nor the expression of adhesion proteins on endothelial cells in vitro. Therefore, it is likely that the inhibitory effect of corticosteroids on the tissue infiltration of eosinophils is the consequence of indirect mechanisms, mainly via the inhibition of cytokines. Interestingly, theophylline, which is generally accepted as a bronchodilator, attenuates eosinophil adhesion to endothelial cells in vitro at a clinically therapeutic concentration. Furthermore, theophylline inhibits the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 on endothelial cells that had been stimulated with IL-4 plus tumor necrosis factor-a. Thus, theophylline possibly exerts an inhibitory effect on both the adhesive property of eosinophils and the expression of adhesion molecules on endothelial cells. These findings possibly indicate that theophylline would be adequate to supplement the actions of corticosteroids in asthmatic airway inflammation, partly via its inhibitory effect on the interaction between blood eosinophils and endothelial cells.

  1. Effect of pore size and interpore distance on endothelial cell growth on polymers.

    Science.gov (United States)

    Narayan, D; Venkatraman, S S

    2008-12-01

    The endothelization of polymers using surface modification has received great attention. In particular, creation of physical surface features such as craters or pores has been an active area of research. However, there have been no reported studies of the effects of pore sizes (wide range) and interpore distance on endothelial cell growth. This report details the study done on endothelial cell attachment on the surfaces of polymers modified by porogen leaching. The polymeric system studied includes PLLA and PLGA (80/20). Factors such as porogen type, pore size, and interpore distance were varied, and the surface was evaluated for its influence on endothelial cell growth. Three groups of pore sizes were evaluated: small (5-20 mum), medium (20-45 mum), and large pores (45-90 mum). Two porogens were evaluated: sugar and gelatin. In addition to counting the attached endothelial cells, their proliferation was also quantified. Pore size and interpore distances were evaluated using scanning electron microscopy (SEM), and cell morphology was studied by staining with crystal violet. Analysis of variance demonstrated that the main parameters, pore size and interpore distance were significant in endothelial cell growth. In PLGA (80/20), it was found that endothelial cell growth was enhanced by smaller pore size and lower interpore distance, whereas the growth was poor on PLLA regardless of pore features. (c) 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.

  2. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    International Nuclear Information System (INIS)

    Highsmith, R.F.; Gallaher, M.J.

    1986-01-01

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive 125 I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface

  3. Thrombin-specific inactivation of endothelial cell derived plasminogen activator

    Energy Technology Data Exchange (ETDEWEB)

    Highsmith, R.F.; Gallaher, M.J.

    1986-03-05

    Although thrombin (T) has diverse functions in the overall hemostatic mechanism, relatively little is known about its direct effect on components of the fibrinolytic enzyme system. The authors have investigated the interaction of T with plasminogen activators (PA) derived from bovine aortic endothelial cells (EC) in culture (2-5th passage, preconfluent monolayers). Varying concentrations of purified bovine or human thrombin were added to EC-conditioned media (CM). CM + T mixtures were assayed at various times for PA activity using purified plasminogen and a sensitive /sup 125/I-fibrinogenolytic or caseinolytic assay. T (5 nM), but not plasmin or trypsin at equivalent concentrations, resulted in a time-dependent inhibition of the PA activity in CM. T had no effect on the PA activity of urokinase, streptokinase or preformed plasmin. The ability of T to inactivate the EC-derived PA was abolished by prior treatment of T with active site-directed reagents. SDS-PAGE and zymography with copolymerized fibrinogen and plasminogen revealed further specificity in that only one of the multiple-molecular weight forms of PA present in EC-CM was inactivated by T. The authors conclude that in a highly specific fashion, T inactivates the predominant PA present in EC-CM by limited proteolysis. Thus, another potentially important function of T is suggested which may have particular significance in the temporal regulation of coagulation and fibrinolysis at the blood-endothelium interface.

  4. Adhesion Molecule Expression in Human Endothelial Cells under Simulated Microgravity

    Science.gov (United States)

    Rudimov, E. G.; Andreeva, E. R.; Buravkova, L. B.

    2013-02-01

    High gravisensitivity of endothelium is now well recognized. Therefore, the microgravity can be one of the main factors affecting the endothelium in space flight. In this work we studied the effects of gravity vector randomization (3D-clinorotation in RPM) on the viability of endothelial cells from human umbilical vein (HUVEC) and the expression of adhesion molecules on its surface. After RPM exposure, HUVEC conditioning medium was collected for cytokines evaluation, a part of vials was used for immunocytochemistry and other one - for cytofluorimetric analysis of ICAM-I, VCAM-I, PECAM-I, E-selectin, Endoglin, VE-cadherin expression. The viability of HUVEC and constitutive expression of EC marker molecules PECAM-I and Endoglin were similar in all experimental groups both after 6 and 24 hrs of exposure. There were no differences in ICAM-I and E-selectin expression on HUVEC in 3 groups after 6 hrs of exposure. 24 hrs incubation has provoked decrease in ICAM-I and E-selectin expression. Thus, gravity vector randomization can lead to the disruption of ECs monolayer.

  5. Vascular endothelial growth factor attachment to hydroxyapatite via self-assembled monolayers promotes angiogenic activity of endothelial cells

    International Nuclear Information System (INIS)

    Solomon, Kimberly D.; Ong, Joo L.

    2013-01-01

    Currently, tissue engineered constructs for critical sized bone defects are non-vascularized. There are many strategies used in order to promote vascularization, including delivery of growth factors such as vascular endothelial growth factor (VEGF). In this study, hydroxyapatite (HA) was coated with self-assembled monolayers (SAMs). The SAMs were in turn used to covalently bind VEGF to the surface of HA. The different SAM chain length ratios (phosphonoundecanoic acid (11-PUDA):16-phosphonohexadecanoic acid (16-PHDA) utilized in this study were 0:100, 25:75, 50:50, 75:25, and 100:0. Surfaces were characterized by contact angle (CA) and atomic force microscopy, and an in vitro VEGF release study was performed. It was observed that CA and root-mean-squared roughness were not significantly affected by the addition of SAMs, but that CA was significantly lowered with the addition of VEGF. VEGF release profiles of bound VEGF groups all demonstrated less initial burst release than adsorbed control, indicating that VEGF was retained on the HA surface when bound by SAMs. An in vitro study using human aortic endothelial cells (HAECs) demonstrated that bound VEGF increased metabolic activity and caused sustained production of angiopoietin-2, an angiogenic marker, over 28 days. In conclusion, SAMs provide a feasible option for growth factor delivery from HA surfaces, enhancing angiogenic activity of HAECs in vitro. - Highlights: • Vascular endothelial growth factor (VEGF) is attached to hydroxyapatite (HA). • Self-assembled monolayers (SAMs) delay the release of VEGF from hydroxyapatite. • SAM chain length ratio affects the total mass of VEGF released. • VEGF on HA up-regulates proliferation and angiogenic activity of endothelial cells

  6. Inhibition of the proliferation and acceleration of migration of vascular endothelial cells by increased cysteine-rich motor neuron 1

    International Nuclear Information System (INIS)

    Nakashima, Yukiko; Morimoto, Mayuka; Toda, Ken-ichi; Shinya, Tomohiro; Sato, Keizo; Takahashi, Satoru

    2015-01-01

    Cysteine-rich motor neuron 1 (CRIM1) is upregulated only in extracellular matrix gels by angiogenic factors such as vascular endothelial growth factor (VEGF). It then plays a critical role in the tube formation of endothelial cells. In the present study, we investigated the effects of increased CRIM1 on other endothelial functions such as proliferation and migration. Knock down of CRIM1 had no effect on VEGF-induced proliferation or migration of human umbilical vein endothelial cells (HUVECs), indicating that basal CRIM1 is not involved in the proliferation or migration of endothelial cells. Stable CRIM1-overexpressing endothelial F-2 cells, termed CR1 and CR2, were constructed, because it was difficult to prepare monolayer HUVECs that expressed high levels of CRIM1. Proliferation was reduced and migration was accelerated in both CR1 and CR2 cells, compared with normal F-2 cells. Furthermore, the transient overexpression of CRIM1 resulted in decreased proliferation and increased migration of bovine aortic endothelial cells. In contrast, neither proliferation nor migration of COS-7 cells were changed by the overexpression of CRIM1. These results demonstrate that increased CRIM1 reduces the proliferation and accelerates the migration of endothelial cells. These CRIM1 effects might contribute to tube formation of endothelial cells. CRIM1 induced by angiogenic factors may serve as a regulator in endothelial cells to switch from proliferating cells to morphological differentiation. - Highlights: • CRIM1 was upregulated only in tubular endothelial cells, but not in monolayers. • Increased CRIM1 reduced the proliferation of endothelial cells. • Increased CRIM1 accelerated the migration of endothelial cells. • Increased CRIM1 had no effect on the proliferation or migration of COS-7 cells

  7. Inhibition of the proliferation and acceleration of migration of vascular endothelial cells by increased cysteine-rich motor neuron 1

    Energy Technology Data Exchange (ETDEWEB)

    Nakashima, Yukiko; Morimoto, Mayuka [Department of Immunobiology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women' s University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179 (Japan); Toda, Ken-ichi [Department of Dermatology, Kitano Hospital, The Tazuke Kofukai Nedical Institute, 2-4-20 Ohgimachi, Kita-ku, Osaka 530-8480 (Japan); Shinya, Tomohiro; Sato, Keizo [Department of Clinical Biochemistry, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, Nobeoka, Miyazaki 882-8508 (Japan); Takahashi, Satoru, E-mail: imwalrus@mukogawa-u.ac.jp [Department of Immunobiology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women' s University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179 (Japan); Institute for Biosciences, Mukogawa Women' s University, 11-68 Koshien Kyuban-cho, Nishinomiya, Hyogo 663-8179 (Japan)

    2015-07-03

    Cysteine-rich motor neuron 1 (CRIM1) is upregulated only in extracellular matrix gels by angiogenic factors such as vascular endothelial growth factor (VEGF). It then plays a critical role in the tube formation of endothelial cells. In the present study, we investigated the effects of increased CRIM1 on other endothelial functions such as proliferation and migration. Knock down of CRIM1 had no effect on VEGF-induced proliferation or migration of human umbilical vein endothelial cells (HUVECs), indicating that basal CRIM1 is not involved in the proliferation or migration of endothelial cells. Stable CRIM1-overexpressing endothelial F-2 cells, termed CR1 and CR2, were constructed, because it was difficult to prepare monolayer HUVECs that expressed high levels of CRIM1. Proliferation was reduced and migration was accelerated in both CR1 and CR2 cells, compared with normal F-2 cells. Furthermore, the transient overexpression of CRIM1 resulted in decreased proliferation and increased migration of bovine aortic endothelial cells. In contrast, neither proliferation nor migration of COS-7 cells were changed by the overexpression of CRIM1. These results demonstrate that increased CRIM1 reduces the proliferation and accelerates the migration of endothelial cells. These CRIM1 effects might contribute to tube formation of endothelial cells. CRIM1 induced by angiogenic factors may serve as a regulator in endothelial cells to switch from proliferating cells to morphological differentiation. - Highlights: • CRIM1 was upregulated only in tubular endothelial cells, but not in monolayers. • Increased CRIM1 reduced the proliferation of endothelial cells. • Increased CRIM1 accelerated the migration of endothelial cells. • Increased CRIM1 had no effect on the proliferation or migration of COS-7 cells.

  8. Characterization of novel NADPH oxidases in endothelial cells under basal and stress conditions

    OpenAIRE

    Petry, Andreas

    2009-01-01

    Increased levels of reactive oxygen species (ROS) contribute to vascular diseases like pulmonary hypertension and atherosclerosis. Although a NOX2-containing NADPH oxidase similar to the neutrophil one has been described to be active in endothelial cells, the contribution of newly discovered NOX homologues (NOX1-NOX5) was still unclear. Therefore, the overall aim of this study was to better characterize the expression, regulation and function of NOX homologues in different endothelial cell mo...

  9. FK506 BINDING PROTEIN 12 DEFICIENCY IN ENDOTHELIAL AND HEMATOPOIETIC CELLS DECREASES REGULATORY T CELLS AND CAUSES HYPERTENSION

    Science.gov (United States)

    Chiasson, Valorie L.; Talreja, Deepa; Young, Kristina J.; Chatterjee, Piyali; Banes-Berceli, Amy K.; Mitchell, Brett M.

    2011-01-01

    Patients treated with the immunosuppressive drug tacrolimus (FK506), which binds FK506 Binding Protein 12 (FKBP12) then inhibits the calcium-dependent phosphatase calcineurin, exhibit decreased regulatory T cells, endothelial dysfunction, and hypertension; however the mechanisms and whether altered T cell polarization play a role are unknown. Tacrolimus treatment of mice for 1 week dose-dependently decreased CD4+/FoxP3+ (regulatory T cells) and increased CD4+/IL-17+ (T helper 17) cells in the spleen, and caused endothelial dysfunction and hypertension. To determine the mechanisms, we crossed floxed FKBP12 mice with Tie2-Cre mice to generate offspring lacking FKBP12 in endothelial and hematopoietic cells only (FKBP12EC KO). Given FKBP12’s role in inhibiting TGF-β receptor activation, Tie2-Cre-mediated deletion of FKBP12 increased TGF-β receptor activation and SMAD2/3 signaling. FKBP12EC KO mice exhibited increased vascular expression of genes and proteins related to endothelial cell activation and inflammation. Serum levels of the pro-inflammatory cytokines IL-2, IL-6, IFNγ, IL-17a, IL-21, and IL-23 were increased significantly suggesting a Th17 cell-mediated inflammatory state. Flow cytometry studies confirmed this as splenocyte levels of CD4+/IL-17+ cells were increased significantly while CD4+/FoxP3+ cells were decreased in FKBP12EC KO mice. Furthermore, spleens from FKBP12EC KO mice showed increased STAT3 activation, involved in Th17 cell induction, and decreased STAT5 activation, involved in regulatory T cell induction. FKBP12EC KO mice also exhibited endothelial dysfunction and hypertension. These data suggest that tacrolimus, through its activation of TGF-β receptors in endothelial and hematopoietic cells, may cause endothelial dysfunction and hypertension by activating endothelial cells, reducing Tregs, and increasing Th17 cell polarization and inflammation. PMID:21518963

  10. Construction of tissue-engineered heart valves by using decellularized scaffolds and endothelial progenitor cells.

    Science.gov (United States)

    Fang, Ning-Tao; Xie, Shang-Zhe; Wang, Song-Mei; Gao, Hong-Yang; Wu, Chun-Gen; Pan, Luan-Feng

    2007-04-20

    Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds. Decellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 x 10(6) cells/cm(2) and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA). EPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer

  11. Sparstolonin B inhibits pro-angiogenic functions and blocks cell cycle progression in endothelial cells.

    Science.gov (United States)

    Bateman, Henry R; Liang, Qiaoli; Fan, Daping; Rodriguez, Vanessa; Lessner, Susan M

    2013-01-01

    Sparstolonin B (SsnB) is a novel bioactive compound isolated from Sparganium stoloniferum, an herb historically used in Traditional Chinese Medicine as an anti-tumor agent. Angiogenesis, the process of new capillary formation from existing blood vessels, is dysregulated in many pathological disorders, including diabetic retinopathy, tumor growth, and atherosclerosis. In functional assays, SsnB inhibited endothelial cell tube formation (Matrigel method) and cell migration (Transwell method) in a dose-dependent manner. Microarray experiments with human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) demonstrated differential expression of several hundred genes in response to SsnB exposure (916 and 356 genes, respectively, with fold change ≥2, pcell types showed significant overlap, including genes associated with cell proliferation and cell cycle. Flow cytometric cell cycle analysis of HUVECs treated with SsnB showed an increase of cells in the G1 phase and a decrease of cells in the S phase. Cyclin E2 (CCNE2) and Cell division cycle 6 (CDC6) are regulatory proteins that control cell cycle progression through the G1/S checkpoint. Both CCNE2 and CDC6 were downregulated in the microarray data. Real Time quantitative PCR confirmed that gene expression of CCNE2 and CDC6 in HUVECs was downregulated after SsnB exposure, to 64% and 35% of controls, respectively. The data suggest that SsnB may exert its anti-angiogenic properties in part by downregulating CCNE2 and CDC6, halting progression through the G1/S checkpoint. In the chick chorioallantoic membrane (CAM) assay, SsnB caused significant reduction in capillary length and branching number relative to the vehicle control group. Overall, SsnB caused a significant reduction in angiogenesis (ANOVA, p<0.05), demonstrating its ex vivo efficacy.

  12. Podocalyxin regulates murine lung vascular permeability by altering endothelial cell adhesion.

    Directory of Open Access Journals (Sweden)

    Erin J Debruin

    Full Text Available Despite the widespread use of CD34-family sialomucins (CD34, podocalyxin and endoglycan as vascular endothelial cell markers, there is remarkably little known of their vascular function. Podocalyxin (gene name Podxl, in particular, has been difficult to study in adult vasculature as germ-line deletion of podocalyxin in mice leads to kidney malformations and perinatal death. We generated mice that conditionally delete podocalyxin in vascular endothelial cells (Podxl(ΔEC mice to study the homeostatic role of podocalyxin in adult mouse vessels. Although Podxl(ΔEC adult mice are viable, their lungs display increased lung volume and changes to the matrix composition. Intriguingly, this was associated with increased basal and inflammation-induced pulmonary vascular permeability. To further investigate the etiology of these defects, we isolated mouse pulmonary endothelial cells. Podxl(ΔEC endothelial cells display mildly enhanced static adhesion to fibronectin but spread normally when plated on fibronectin-coated transwells. In contrast, Podxl(ΔEC endothelial cells exhibit a severely impaired ability to spread on laminin and, to a lesser extent, collagen I coated transwells. The data suggest that, in endothelial cells, podocalyxin plays a previously unrecognized role in maintaining vascular integrity, likely through orchestrating interactions with extracellular matrix components and basement membranes, and that this influences downstream epithelial architecture.

  13. Size and dynamics of caveolae studied using nanoparticles in living endothelial cells.

    Science.gov (United States)

    Wang, Zhenjia; Tiruppathi, Chinnaswamy; Minshall, Richard D; Malik, Asrar B

    2009-12-22

    Caveolae are plasma membrane invaginations prominent in all endothelial cells lining blood vessels. Caveolae characteristically bud to form free cytoplasmic vesicles capable of transporting carrier proteins such as albumin through the cell. However, caveolae size distribution and dynamics in living endothelial cells and ability of caveolae to internalize nanoparticles are not well understood. We demonstrate here the design of a dual-color nanoparticle pair to measure noninvasively caveolae size and dynamics. First, we coated nanoparticles with BSA (bovine serum albumin) to address whether albumin promoted their delivery. Albumin has been shown to bind to protein on endothelial cell surface localized in caveolae and activate albumin endocytosis. Imaging of BSA-coated nanoparticles varying from 20 to 100 nm in diameter in endothelial cells demonstrated that caveolae-mediated nanoparticle uptake was dependent on albumin coating of particles. We also showed that caveolae could accommodate up to 100 nm diameter nanoparticles, a size larger than the diameter of typical caveolae, suggesting compliant property of caveolae. Together, our results show the feasibility of tracking multicolored nanoparticles in living endothelial cells and potential usefulness for designing therapeutic nanoparticle cargo to cross the limiting vessel wall endothelial barrier.

  14. Combinatorial Extracellular Matrix Microenvironments for Probing Endothelial Differentiation of Human Pluripotent Stem Cells.

    Science.gov (United States)

    Hou, Luqia; Kim, Joseph J; Wanjare, Maureen; Patlolla, Bhagat; Coller, John; Natu, Vanita; Hastie, Trevor J; Huang, Ngan F

    2017-07-26

    Endothelial cells derived from human pluripotent stem cells are a promising cell type for enhancing angiogenesis in ischemic cardiovascular tissues. However, our understanding of microenvironmental factors that modulate the process of endothelial differentiation is limited. We examined the role of combinatorial extracellular matrix (ECM) proteins on endothelial differentiation systematically using an arrayed microscale platform. Human pluripotent stem cells were differentiated on the arrayed ECM microenvironments for 5 days. Combinatorial ECMs composed of collagen IV + heparan sulfate + laminin (CHL) or collagen IV + gelatin + heparan sulfate (CGH) demonstrated significantly higher expression of CD31, compared to single-factor ECMs. These results were corroborated by fluorescence activated cell sorting showing a 48% yield of CD31 + /VE-cadherin + cells on CHL, compared to 27% on matrigel. To elucidate the signaling mechanism, a gene expression time course revealed that VE-cadherin and FLK1 were upregulated in a dynamically similar manner as integrin subunit β3 (>50 fold). To demonstrate the functional importance of integrin β3 in promoting endothelial differentiation, the addition of neutralization antibody inhibited endothelial differentiation on CHL-modified dishes by >50%. These data suggest that optimal combinatorial ECMs enhance endothelial differentiation, compared to many single-factor ECMs, in part through an integrin β3-mediated pathway.

  15. Circulating endothelial cells and procoagulant microparticles in patients with glioblastoma: prognostic value.

    Directory of Open Access Journals (Sweden)

    Gaspar Reynés

    Full Text Available AIM: Circulating endothelial cells and microparticles are prognostic factors in cancer. However, their prognostic and predictive value in patients with glioblastoma is unclear. The objective of this study was to investigate the potential prognostic value of circulating endothelial cells and microparticles in patients with newly diagnosed glioblastoma treated with standard radiotherapy and concomitant temozolomide. In addition, we have analyzed the methylation status of the MGMT promoter. METHODS: Peripheral blood samples were obtained before and at the end of the concomitant treatment. Blood samples from healthy volunteers were also obtained as controls. Endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Microparticles were quantified by flow cytometry. Microparticle-mediated procoagulant activity was measured by endogen thrombin generation and by phospholipid-dependent clotting time. Methylation status of MGMT promoter was determined by multiplex ligation-dependent probe amplification. RESULTS: Pretreatment levels of circulating endothelial cells and microparticles were higher in patients than in controls (p<0.001. After treatment, levels of microparticles and thrombin generation decreased, and phospholipid-dependent clotting time increased significantly. A high pretreatment endothelial cell count, corresponding to the 99(th percentile in controls, was associated with poor overall survival. MGMT promoter methylation was present in 27% of tumor samples and was associated to a higher overall survival (66 weeks vs 30 weeks, p<0.004. CONCLUSION: Levels of circulating endothelial cells may have prognostic value in patients with glioblastoma.

  16. Design optimization of a helical endothelial cell culture device.

    Science.gov (United States)

    Van Doormaal, Mark A; Ethier, C Ross

    2010-10-01

    The specific roles of mass transfer and fluid dynamic stresses on endothelial function, important in atherogenesis, are not known. Further, the effects of mass transfer and fluid dynamic stresses are difficult to separate because areas of "abnormal" mass transfer and "abnormal" wall shear stress tend to co-localize (where abnormal is defined as any deviation from the mass transfer rate or wall shear stress present in a long straight artery with the same flow rate and diameter). Our goal was to design a cell culture device which gives maximum separation between areas of abnormal shear stress and areas of abnormal mass transfer. We used design optimization principles to design a helical cell culture device. Using shear stress and mass transfer fields predicted by solving the governing equations, the area of the device which was exposed to low rates of mass transfer and normal levels of wall shear stress was determined. The design optimization method then maximized this area by varying the design variables, resulting in the optimum design. The optimum design had Reynolds number = 50, helical radius = 3.23 and helical pitch = 3.82. The area of the device which was exposed to low rates of mass transfer and regular levels of wall shear stress was about 4.5 times the inlet cross-sectional area of the device or about 5% of the device total internal surface area. An optimum design was successfully determined and the methodology used was shown to be robust. The area of the device which was exposed to low rates of mass transfer and regular levels of wall shear stress occurred in a defined region which should aid further experimental work.

  17. Pulmonary endothelial cell activation during experimental acute kidney injury.

    Science.gov (United States)

    Feltes, Carolyn M; Hassoun, Heitham T; Lie, Mihaela L; Cheadle, Chris; Rabb, Hamid

    2011-08-01

    Acute kidney injury (AKI) leads to increased lung microvascular permeability, leukocyte infiltration, and upregulation of soluble inflammatory proteins in rodents. Most work investigating connections between AKI and pulmonary dysfunction, however, has focused on characterizing whole lung tissue changes associated with AKI. Studies at the cellular level are essential to understanding the molecular basis of lung changes during AKI. Given that the pulmonary microvascular barrier is functionally abnormal during AKI, we hypothesized that AKI induces a specific proinflammatory and proapoptotic lung endothelial cell (EC) response. Four and 24 h after kidney ischemia/reperfusion injury or bilateral nephrectomy, murine pulmonary ECs were isolated via tissue digestion followed by magnetic bead sorting. Purified lung ECs were analyzed for changes in mRNA expression using real-time SuperArray polymerase chain reaction analysis of genes related to EC function. In parallel experiments, confluent rat pulmonary microvascular ECs were treated with AKI or control serum to evaluate functional cellular alterations. Immunocytochemistry and FACS analysis of Annexin V/propidium iodide staining were used to evaluate cytoskeletal changes and promotion of apoptosis. Isolated murine pulmonary ECs exhibited significant changes in the expression of gene products related to inflammation, vascular reactivity, and programmed cell death. Further experiments using an in vitro rat pulmonary microvascular EC system revealed that AKI serum induced functional cellular changes related to apoptosis, including structural actin alterations and phosphatidylserine translocation. Analysis and segregation of both upregulated and downregulated genes into functional roles suggest that these transcriptional events likely participate in the transition to an activated proinflammatory and proapoptotic EC phenotype during AKI. Further mechanistic analysis of EC-specific events in the lung during AKI might reveal

  18. Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Askari, Ara A. [Barts and the London, Queen Mary University, London (United Kingdom); Thomson, Scott [Comparative Biomedical Sciences, Royal Veterinary College, London (United Kingdom); Edin, Matthew L.; Lih, Fred B.; Zeldin, Darryl C. [Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (United States); Bishop-Bailey, David, E-mail: dbishopbailey@rvc.ac.uk [Comparative Biomedical Sciences, Royal Veterinary College, London (United Kingdom)

    2014-04-04

    Highlights: • We examined epoxygenase product formation and regulation in endothelial cells. • The epoxygenase CYP2J2 is an LPS (TLR-4) inducible enzyme in endothelial cells. • The endothelial cell line EA.Hy926 synthesises epoxygenase products. • Inhibition of endothelial epoxygenases increases TNFα secretion. • Soluble epoxide hydrolase inhibitors reduce inflammation-induced TNFα and NFκB. - Abstract: The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity.

  19. Priming Mesenchymal Stem Cells with Endothelial Growth Medium Boosts Stem Cell Therapy for Systemic Arterial Hypertension

    Directory of Open Access Journals (Sweden)

    Lucas Felipe de Oliveira

    2015-01-01

    Full Text Available Systemic arterial hypertension (SAH, a clinical syndrome characterized by persistent elevation of arterial pressure, is often associated with abnormalities such as microvascular rarefaction, defective angiogenesis, and endothelial dysfunction. Mesenchymal stem cells (MSCs, which normally induce angiogenesis and improve endothelial function, are defective in SAH. The central aim of this study was to evaluate whether priming of MSCs with endothelial growth medium (EGM-2 increases their therapeutic effects in spontaneously hypertensive rats (SHRs. Adult female SHRs were administered an intraperitoneal injection of vehicle solution n=10, MSCs cultured in conventional medium (DMEM plus 10% FBS, n=11, or MSCs cultured in conventional medium followed by 72 hours in EGM-2 (pMSC, n=10. Priming of the MSCs reduced the basal cell death rate in vitro. The administration of pMSCs significantly induced a prolonged reduction (10 days in arterial pressure, a decrease in cardiac hypertrophy, an improvement in endothelium-dependent vasodilation response to acetylcholine, and an increase in skeletal muscle microvascular density compared to the vehicle and MSC groups. The transplanted cells were rarely found in the hearts and kidneys. Taken together, our findings indicate that priming of MSCs boosts stem cell therapy for the treatment of SAH.

  20. Engraftment of donor mesenchymal stem cells in chimeric BXSB includes vascular endothelial cells and hepatocytes

    Directory of Open Access Journals (Sweden)

    Jones OY

    2011-12-01

    Full Text Available Olcay Y Jones1, Faysal Gok2, Elisabeth J Rushing3, Iren Horkayne-Szakaly4, Atif A Ahmed51Department of Pediatrics, Walter Reed National Military Medical Center, Bethesda, MD, USA; 2Department of Pediatrics, Gulhane Military Medical Academy, Ankara, Turkey; 3Institut für Neuropathologie, Universitäts Spital Zürich, Zürich, Switzerland; 4Department of Neuropathology, Armed Forces Institute of Pathology, Washington, DC, USA; 5Department of Pathology and Laboratory Medicine, Children's Mercy Hospitals and Clinics, Kansas City, MO, USAAbstract: Somatic tissue engraftment was studied in BXSB mice treated with mesenchymal stem cell transplantation. Hosts were conditioned with nonlethal radiation prior to introducing donor cells from major histocompatibility complex-matched green fluorescent protein transgenic mice. Transplant protocols differed for route of injection, ie, intravenous (i.v. versus intraperitoneal (i.p., and source of mesenchymal stem cells, ie, unfractionated bone marrow cells, ex vivo expanded mesenchymal stem cells, or bone chips. Tissue chimerism was determined after short (10–12 weeks or long (62 weeks posttransplant follow-up by immunohistochemistry for green fluorescent protein. Engraftment of endothelial cells was seen in several organs including liver sinusoidal cells in i.v. treated mice with ex vivo expanded mesenchymal stem cells or with unfractionated bone marrow cells. Periportal engraftment of liver hepatocytes, but not engraftment of endothelial cells, was found in mice injected i.p. with bone chips. Engraftment of adipocytes was a common denominator in both i.v. and i.p. routes and occurred during early phases post-transplant. Disease control was more robust in mice that received both i.v. bone marrow and i.p. bone chips compared to mice that received i.v. bone marrow alone. Thus, the data support potential use of mesenchymal stem cell transplant for treatment of severe lupus. Future studies are needed to optimize

  1. Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease.

    Science.gov (United States)

    Camus, Stéphane M; De Moraes, João A; Bonnin, Philippe; Abbyad, Paul; Le Jeune, Sylvain; Lionnet, François; Loufrani, Laurent; Grimaud, Linda; Lambry, Jean-Christophe; Charue, Dominique; Kiger, Laurent; Renard, Jean-Marie; Larroque, Claire; Le Clésiau, Hervé; Tedgui, Alain; Bruneval, Patrick; Barja-Fidalgo, Christina; Alexandrou, Antigoni; Tharaux, Pierre-Louis; Boulanger, Chantal M; Blanc-Brude, Olivier P

    2015-06-11

    Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD. © 2015 by The American Society of Hematology.

  2. Capillary network formation from dispersed endothelial cells: Influence of cell traction, cell adhesion, and extracellular matrix rigidity

    Science.gov (United States)

    Ramos, João R. D.; Travasso, Rui; Carvalho, João

    2018-01-01

    The formation of a functional vascular network depends on biological, chemical, and physical processes being extremely well coordinated. Among them, the mechanical properties of the extracellular matrix and cell adhesion are fundamental to achieve a functional network of endothelial cells, able to fully cover a required domain. By the use of a Cellular Potts Model and Finite Element Method it is shown that there exists a range of values of endothelial traction forces, cell-cell adhesion, and matrix rigidities where the network can spontaneously be formed, and its properties are characterized. We obtain the analytical relation that the minimum traction force required for cell network formation must obey. This minimum value for the traction force is approximately independent on the considered cell number and cell-cell adhesion. We quantify how these two parameters influence the morphology of the resulting networks (size and number of meshes).

  3. Simultaneous harvesting of endothelial progenitor cells and mesenchymal stem cells from the human umbilical cord.

    Science.gov (United States)

    Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu

    2018-01-01

    The human umbilical cord (UC) is usually discarded as biological waste. However, it has attracted interest as a source of cells including endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs), which have demonstrated enormous potential in regenerative medicine. The present study describes a convenient protocol that has been developed to sequentially extract these two cell types from a single UC. EPCs which had properties of progenitor cells were successfully isolated from the UC vein. These cells had cobble-shaped morphology and expressed Flt-1, KDR, VE-cadherin, von Willebrand factor and CD31 mRNA, in addition to CD73, CD105 and vascular endothelial growth factor receptor-2. In addition to absorbing fluorescent-labeled acetylated low density protein and binding to fluorescein isothiocyanate-UEA-l, they were able to form vascular tube-like structures on Matrigel. Typical fibroblast-like cells, which were isolated from the Wharton's jelly, were confirmed to be MSCs by their expression of CD73, CD90 and CD105, and their ability to differentiate into adipocytes and osteoblasts. Thus, the human UC-derived cells may be suitable for use in tissue engineering and cell therapy.

  4. Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury

    Science.gov (United States)

    Mitra, Srabani

    2015-01-01

    Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS) and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC) apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1) induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control) nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury. PMID:26710067

  5. Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury.

    Science.gov (United States)

    Mitra, Srabani; Wewers, Mark D; Sarkar, Anasuya

    2015-01-01

    Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS) and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC) apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1) induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control) nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury.

  6. Mononuclear Phagocyte-Derived Microparticulate Caspase-1 Induces Pulmonary Vascular Endothelial Cell Injury.

    Directory of Open Access Journals (Sweden)

    Srabani Mitra

    Full Text Available Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS and impacts disease progression. Lung endothelial injury has traditionally been focused on the role of neutrophil trafficking to lung vascular integrin receptors induced by proinflammatory cytokine expression. Although much is known about the pathogenesis of cell injury and death in ALI/ARDS, gaps remain in our knowledge; as a result of which there is currently no effective pharmacologic therapy. Enzymes known as caspases are essential for completion of the apoptotic program and secretion of pro-inflammatory cytokines. We hypothesized that caspase-1 may serve as a key regulator of human pulmonary microvascular endothelial cell (HPMVEC apoptosis in ALI/ARDS. Our recent experiments confirm that microparticles released from stimulated monocytic cells (THP1 induce lung endothelial cell apoptosis. Microparticles pretreated with the caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, were unable to induce cell death of HPMVEC, suggesting the role of caspase-1 or its substrate in the induction of HPMVEC cell death. Neither un-induced microparticles (control nor direct treatment with LPS induced apoptosis of HPMVEC. Further experiments showed that caspase-1 uptake into HPMVEC and the induction of HPMVEC apoptosis was facilitated by caspase-1 interactions with microparticulate vesicles. Altering vesicle integrity completely abrogated apoptosis of HPMVEC suggesting an encapsulation requirement for target cell uptake of active caspase-1. Taken together, we confirm that microparticle centered caspase-1 can play a regulator role in endothelial cell injury.

  7. Fulvic Acid Attenuates Resistin-Induced Adhesion of HCT-116 Colorectal Cancer Cells to Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Wen-Shih Huang

    2015-12-01

    Full Text Available A high level of serum resistin has recently been found in patients with a number of cancers, including colorectal cancer (CRC. Hence, resistin may play a role in CRC development. Fulvic acid (FA, a class of humic substances, possesses pharmacological properties. However, the effect of FA on cancer pathophysiology remains unclear. The aim of this study was to investigate the effect of resistin on the endothelial adhesion of CRC and to determine whether FA elicits an antagonistic mechanism to neutralize this resistin effect. Human HCT-116 (p53-negative and SW-48 (p53-positive CRC cells and human umbilical vein endothelial cells (HUVECs were used in the experiments. Treatment of both HCT-116 and SW-48 cells with resistin increases the adhesion of both cells to HUVECs. This result indicated that p53 may not regulate this resistin effect. A mechanistic study in HCT-116 cells further showed that this resistin effect occurs via the activation of NF-κB and the expression of intercellular adhesion molecule-1 (ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1. Co-treating cells with both FA and resistin revealed that FA significantly attenuated the resistin-increased NF-κB activation and ICAM-1/VCAM-1 expression and the consequent adhesion of HCT-116 cells to HUVECs. These results demonstrate the role of resistin in promoting HCT-116 cell adhesion to HUVECs and indicate that FA might be a potential candidate for the inhibition of the endothelial adhesion of CRC in response to resistin.

  8. Oxidative Stress Induces Endothelial Cell Senescence via Downregulation of Sirt6

    Directory of Open Access Journals (Sweden)

    Rong Liu

    2014-01-01

    Full Text Available Accumulating evidence has shown that diabetes accelerates aging and endothelial cell senescence is involved in the pathogenesis of diabetic vascular complications, including diabetic retinopathy. Oxidative stress is recognized as a key factor in the induction of endothelial senescence and diabetic retinopathy. However, specific mechanisms involved in oxidative stress-induced endothelial senescence have not been elucidated. We hypothesized that Sirt6, which is a nuclear, chromatin-bound protein critically involved in many pathophysiologic processes such as aging and inflammation, may have a role in oxidative stress-induced vascular cell senescence. Measurement of Sirt6 expression in human endothelial cells revealed that H2O2 treatment significantly reduced Sirt6 protein. The loss of Sirt6 was associated with an induction of a senescence phenotype in endothelial cells, including decreased cell growth, proliferation and angiogenic ability, and increased expression of senescence-associated β-galactosidase activity. Additionally, H2O2 treatment reduced eNOS expression, enhanced p21 expression, and dephosphorylated (activated retinoblastoma (Rb protein. All of these alternations were attenuated by overexpression of Sirt6, while partial knockdown of Sirt6 expression by siRNA mimicked the effect of H2O2. In conclusion, these results suggest that Sirt6 is a critical regulator of endothelial senescence and oxidative stress-induced downregulation of Sirt6 is likely involved in the pathogenesis of diabetic retinopathy.

  9. Increased affinity of endothelial cells to NiTi using ultraviolet irradiation: An in vitro study.

    Science.gov (United States)

    Tateshima, Satoshi; Kaneko, Naoki; Yamada, Masahiro; Duckwiler, Gary; Vinuela, Fernando; Ogawa, Takahiro

    2018-04-01

    Nickel-titanium alloy (NiTi) is one of the most popular materials used endovascularly because of its shape memory and superelasticity. The NiTi device needs to be covered by endothelial cells after being placed in the blood vessel to reduce ischemic complications. The objective of this study was to examine the impact of ultraviolet (UV) irradiation on the biocompatibility of NiTi surfaces with endothelial cells. NiTi sheets were treated with UV irradiation for 48 h and human aorta derived endothelial cells were used in this study. UV irradiation converted the NiTi surface to hydrophilic state and increased albumin adsorption. The number of endothelial cell migration, attachment, proliferation as well as their metabolic activity were significantly increased on UV treated NiTi. This study provides the first evidence of the photoactivation of NiTi surfaces by UV irradiation and demonstrates improved biocompatibility of UV-treated NiTi surfaces with vascular endothelial cells. These results suggest that UV irradiation may promote endothelialization of NiTi devices in blood vessels. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1034-1038, 2018. © 2017 Wiley Periodicals, Inc.

  10. Involvement of RhoA/Rho kinase signaling in VEGF-induced endothelial cell migration and angiogenesis in vitro

    NARCIS (Netherlands)

    Nieuw Amerongen, G.P. van; Koolwijk, P.; Versteilen, A.; Hinsbergh, V.W.M. van

    2003-01-01

    Objective - Growth factor-induced angiogenesis involves migration of endothelial cells (ECs) into perivascular areas and requires active remodeling of the endothelial F-actin cytoskeleton. The small GTPase RhoA previously has been implicated in vascular endothelial growth factor (VEGF)-induced

  11. NOVEL CHARACTERIZATION OF bEnd.3 CELLS THAT EXPRESS LYMPHATIC VESSEL ENDOTHELIAL HYALURONAN RECEPTOR-1

    OpenAIRE

    Yuen, D.; Leu, R.; Tse, J.; Wang, S.; Chen, L.L.; Chen, L.

    2014-01-01

    Murine bEnd.3 endothelioma cell line has been widely used in vascular research and here we report the novel finding that bEnd.3 cells express lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and vascular endothelial growth factor receptor-3 (VEGFR-3). Moreover, these cells express progenitor cell markers of Sca-1 and CD133. Upon stimulation with tumor necrosis factor-alpha (TNF-α), the bEnd.3 cells demonstrate enhanced formation of capillary-type tubes, which express LYVE-1. As the...

  12. Oral cancer/endothelial cell fusion experiences nuclear fusion and acquisition of enhanced survival potential

    Energy Technology Data Exchange (ETDEWEB)

    Song, Kai [Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Qingdao University, Shandong Province (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Song, Yong [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Department of Stomatology, Liu Zhou People' s Hospital, Guangxi (China); Zhao, Xiao-Ping; Shen, Hui; Wang, Meng; Yan, Ting-lin [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Liu, Ke, E-mail: liuke.1999@aliyun.com [Department of Oral and Maxillofacial-Head and Neck oncology, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Shang, Zheng-jun, E-mail: shangzhengjun@hotmail.com [Department of Oral and Maxillofacial-Head and Neck oncology, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China)

    2014-10-15

    Most previous studies have linked cancer–macrophage fusion with tumor progression and metastasis. However, the characteristics of hybrid cells derived from oral cancer and endothelial cells and their involvement in cancer remained unknown. Double-immunofluorescent staining and fluorescent in situ hybridization (FISH) were performed to confirm spontaneous cell fusion between eGFP-labeled human umbilical vein endothelial cells (HUVECs) and RFP-labeled SCC9, and to detect the expression of vementin and cytokeratin 18 in the hybrids. The property of chemo-resistance of such hybrids was examined by TUNEL assay. The hybrid cells in xenografted tumor were identified by FISH and GFP/RFP dual-immunofluoresence staining. We showed that SCC9 cells spontaneously fused with cocultured endothelial cells, and the resultant hybrid cells maintained the division and proliferation activity after re-plating and thawing. Such hybrids expressed markers of both parental cells and became more resistant to chemotherapeutic drug cisplatin as compared to the parental SCC9 cells. Our in vivo data indicated that the hybrid cells contributed to tumor composition by using of immunostaining and FISH analysis, even though the hybrid cells and SCC9 cells were mixed with 1:10,000, according to the FACS data. Our study suggested that the fusion events between oral cancer and endothelial cells undergo nuclear fusion and acquire a new property of drug resistance and consequently enhanced survival potential. These experimental findings provide further supportive evidence for the theory that cell fusion is involved in cancer progression. - Highlights: • The fusion events between oral cancer and endothelial cells undergo nuclear fusion. • The resulting hybrid cells acquire a new property of drug resistance. • The resulting hybrid cells express the markers of both parental cells (i.e. vimentin and cytokeratin 18). • The hybrid cells contribute to tumor repopulation in vivo.

  13. Listeria monocytogenes activated p38 MAPK and induced IL-8 secretion in a nucleotide-binding oligomerization domain 1-dependent manner in endothelial cells.

    Science.gov (United States)

    Opitz, Bastian; Püschel, Anja; Beermann, Wiebke; Hocke, Andreas C; Förster, Stefanie; Schmeck, Bernd; van Laak, Vincent; Chakraborty, Trinad; Suttorp, Norbert; Hippenstiel, Stefan

    2006-01-01

    Nucleotide-binding oligomerization domain (Nod) proteins serve as intracellular pattern recognition molecules recognizing peptidoglycans. To further examine intracellular immune recognition, we used Listeria monocytogenes as an organism particularly amenable for studying innate immunity to intracellular pathogens. In contrast to wild-type L. monocytogenes, the nonpathogenic Listeria innocua, or L. monocytogenes mutants lacking internalin B or listeriolysin O, poorly invaded host cells and escaped into host cell cytoplasm, respectively, and were therefore used as controls. In this study, we show that only the invasive wild-type L. monocytogenes, but not the listeriolysin O- or internalin B-negative L. monocytogenes mutants or L. innocua, substantially induced IL-8 production in HUVEC. RNA interference and Nod1-overexpression experiments demonstrated that Nod1 is critically involved in chemokine secretion and NF-kappaB activation initiated by L. monocytogenes in human endothelial cells. Moreover, we show for the first time that Nod1 mediated activation of p38 MAPK signaling induced by L. monocytogenes. Finally, L. monocytogenes- and Nod1-induced IL-8 production was blocked by a specific p38 inhibitor. In conclusion, L. monocytogenes induced a Nod1-dependent activation of p38 MAPK signaling and NF-kappaB which resulted in IL-8 production in endothelial cells. Thus, Nod1 is an important component of a cytoplasmic surveillance pathway.

  14. Norepinephrine stimulates mobilization of endothelial progenitor cells after limb ischemia.

    Directory of Open Access Journals (Sweden)

    Qijun Jiang

    Full Text Available OBJECTIVE: During several pathological processes such as cancer progression, thermal injury, wound healing and hindlimb ischemia, the mobilization of endothelial progenitor cells (EPCs mobilization was enhanced with an increase of sympathetic nerve activity and norepinephrine (NE secretion, yet the cellular and molecular mechanisms involved in the effects of NE on EPCs has less been investigated. METHODS AND RESULTS: EPCs from BMs, peripheral circulation and spleens, the VEGF concentration in BM, skeletal muscle, peripheral circulation and spleen and angiogenesis in ischemic gastrocnemius were quantified in mice with hindlimbs ischemia. Systemic treatment of NE significantly increased EPCs number in BM, peripheral circulation and spleen, VEGF concentration in BM and skeletal muscle and angiogenesis in ischemic gastrocnemius in mice with hind limb ischemia, but did not affair VEGF concentration in peripheral circulation and spleen. EPCs isolated from healthy adults were cultured with NE in vitro to evaluate proliferation potential, migration capacity and phosphorylations of Akt and eNOS signal moleculars. Treatment of NE induced a significant increase in number of EPCs in the S-phase in a dose-dependent manner, as well as migrative activity of EPCs in vitro (p<0.05. The co-treatment of Phentolamine, I127, LY294002 and L-NAME with NE blocked the effects of NE on EPCs proliferation and migration. Treatment with NE significantly increased phosphorylation of Akt and eNOS of EPCs. Addition of phentolamine and I127 attenuated the activation of Akt/eNOS pathway, but metoprolol could not. Pretreatment of mice with either Phentolamine or I127 significantly attenuated the effects of NE on EPCs in vivo, VEGF concentration in BM, skeletal muscle and angiogenesis in ischemic gastrocnemius, but Metoprolol did not. CONCLUSION: These results unravel that sympathetic nervous system regulate EPCs mobilization and their pro-angiogenic capacity via α adrenoceptor

  15. Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

    DEFF Research Database (Denmark)

    Gustafsson, E; Brakebusch, C; Hietanen, K

    2001-01-01

    germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth......Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid...... the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate...

  16. Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

    DEFF Research Database (Denmark)

    Gustafsson, E; Brakebusch, C; Hietanen, K

    2001-01-01

    Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid...... the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate...... germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth...

  17. Nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, protects against Shiga toxin cytotoxicity in human microvascular endothelial cells.

    Science.gov (United States)

    Ohmi, K; Kiyokawa, N; Sekino, T; Suzuki, T; Mimori, K; Taguchi, T; Nakajima, H; Katagiri, Y U; Fujimoto, J; Nakao, H; Takeda, T

    2001-01-01

    Infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) cause microvascular endothelial cell damage, resulting in hemorrhagic colitis and hemolytic uremic syndrome. The prevention of endothelial cell damage is therefore a crucial step in overcoming this disorder. Here, we report that nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, has a protective effect against the cytotoxicity of Stxs in human microvascular endothelial cells (HMVECs). The relative viability of cells treated with 1.5-15 pM of Stx1 was reduced to 10-20% of that without Stx1. However, the viability of cells treated with NBT (10-100 microM) remained higher than 80%, even in the presence of Stx1. NBT also protected against Stx1 cytotoxicity in sodium butyrate-treated hypersensitive HMVECs. The protective effect of NBT against Stx cytotoxicity may be due to the depletion of ATP in the cells, thereby inhibiting the entry of Stx1.

  18. Human endothelial progenitor cells internalize high-density lipoprotein.

    Directory of Open Access Journals (Sweden)

    Kaemisa Srisen

    Full Text Available Endothelial progenitor cells (EPCs originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL, and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate, cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal

  19. Cell-Autonomous Defects in Thymic Epithelial Cells Disrupt Endothelial-Perivascular Cell Interactions in the Mouse Thymus

    Science.gov (United States)

    Bryson, Jerrod L.; Griffith, Ann V.; Hughes III, Bernard; Saito, Fumi; Takahama, Yousuke; Richie, Ellen R.; Manley, Nancy R.

    2013-01-01

    The thymus is composed of multiple stromal elements comprising specialized stromal microenvironments responsible for the development of self-tolerant and self-restricted T cells. Here, we investigated the ontogeny and maturation of the thymic vasculature. We show that endothelial cells initially enter the thymus at E13.5, with PDGFR-β+ mesenchymal cells following at E14.5. Using an allelic series of the thymic epithelial cell (TEC) specific transcription factor Foxn1, we showed that these events are delayed by 1–2 days in Foxn1 Δ/Δ mice, and this phenotype was exacerbated with reduced Foxn1 dosage. At subsequent stages there were fewer capillaries, leaky blood vessels, disrupted endothelium - perivascular cell interactions, endothelial cell vacuolization, and an overall failure of vascular organization. The expression of both VEGF-A and PDGF-B, which are both primarily expressed in vasculature-associated mesenchyme or endothelium in the thymus, were reduced at E13.5 and E15.5 in Foxn1 Δ/Δ mice compared with controls. These data suggest that Foxn1 is required in TECs both to recruit endothelial cells and for endothelial cells to communicate with thymic mesenchyme, and for the differentiation of vascular-associated mesenchymal cells. These data show that Foxn1 function in TECs is required for normal thymus size and to generate the cellular and molecular environment needed for normal thymic vascularization. These data further demonstrate a novel TEC-mesenchyme-endothelial interaction required for proper fetal thymus organogenesis. PMID:23750244

  20. Cell-autonomous defects in thymic epithelial cells disrupt endothelial-perivascular cell interactions in the mouse thymus.

    Directory of Open Access Journals (Sweden)

    Jerrod L Bryson

    Full Text Available The thymus is composed of multiple stromal elements comprising specialized stromal microenvironments responsible for the development of self-tolerant and self-restricted T cells. Here, we investigated the ontogeny and maturation of the thymic vasculature. We show that endothelial cells initially enter the thymus at E13.5, with PDGFR-β(+ mesenchymal cells following at E14.5. Using an allelic series of the thymic epithelial cell (TEC specific transcription factor Foxn1, we showed that these events are delayed by 1-2 days in Foxn1 (Δ/Δ mice, and this phenotype was exacerbated with reduced Foxn1 dosage. At subsequent stages there were fewer capillaries, leaky blood vessels, disrupted endothelium - perivascular cell interactions, endothelial cell vacuolization, and an overall failure of vascular organization. The expression of both VEGF-A and PDGF-B, which are both primarily expressed in vasculature-associated mesenchyme or endothelium in the thymus, were reduced at E13.5 and E15.5 in Foxn1 (Δ/Δ mice compared with controls. These data suggest that Foxn1 is required in TECs both to recruit endothelial cells and for endothelial cells to communicate with thymic mesenchyme, and for the differentiation of vascular-associated mesenchymal cells. These data show that Foxn1 function in TECs is required for normal thymus size and to generate the cellular and molecular environment needed for normal thymic vascularization. These data further demonstrate a novel TEC-mesenchyme-endothelial interaction required for proper fetal thymus organogenesis.

  1. Microparticles from apoptotic monocytes enhance nitrosative stress in human endothelial cells.

    Science.gov (United States)

    Mastronardi, Maria Letizia; Mostefai, Hadj Ahmed; Soleti, Raffaella; Agouni, Abdelali; Martínez, Maria Carmen; Andriantsitohaina, Ramaroson

    2011-12-01

    Microparticles are membrane vesicles with procoagulant and proinflammatory properties released during cell activation or apoptosis. Microparticles from monocytes have been implicated in atherosclerosis and vascular inflammation, but their direct effects on endothelial cells are not completely elucidated. The present study was designed to dissect the signaling pathways of monocytic microparticles in endothelial cells with respect to both NO pathway and reactive oxygen species. Microparticles were produced by treatment of human monocytic cell line THP-1 with the apoptotic agent VP-16. Human endothelial cells were treated with monocytic microparticles and then, we studied their effects on nitrosative and oxidative stresses. Incubation of human endothelial cells with microparticles enhanced the production of NO without affecting superoxide anions generation. Microparticles did not affect endothelial NO synthase expression and its phosphorylation. Interestingly, microparticles decreased caveolin-1 expression and increased its phosphorylation. Inhibition of PI-3-kinase or MEK1/2 reversed the effects of microparticles on caveolin-1 expression but not its phosphorylation. Moreover, microparticles increased nitration of several proteins, reflecting peroxynitrite production, which was prevented by blockade of PI-3-kinase pathway. In summary, monocyte microparticles active multiple pathways related to nitrosative stress in endothelial cells including both PI-3-kinase and ERK1/2 in the regulation of caveolin-1 expression. These data underscore the pleiotropic effect of microparticles on endothelial cells and suggest that they probably play a critical role on vascular function. 2010 The Authors Fundamental and Clinical Pharmacology. 2010 Société Française de Pharmacologie et de Thérapeutique.

  2. Long noncoding RNA MALAT1 regulates endothelial cell function and vessel growth.

    Science.gov (United States)

    Michalik, Katharina M; You, Xintian; Manavski, Yosif; Doddaballapur, Anuradha; Zörnig, Martin; Braun, Thomas; John, David; Ponomareva, Yuliya; Chen, Wei; Uchida, Shizuka; Boon, Reinier A; Dimmeler, Stefanie

    2014-04-25

    The human genome harbors a large number of sequences encoding for RNAs that are not translated but control cellular functions by distinct mechanisms. The expression and function of the longer transcripts namely the long noncoding RNAs in the vasculature are largely unknown. Here, we characterized the expression of long noncoding RNAs in human endothelial cells and elucidated the function of the highly expressed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Endothelial cells of different origin express relative high levels of the conserved long noncoding RNAs MALAT1, taurine upregulated gene 1 (TUG1), maternally expressed 3 (MEG3), linc00657, and linc00493. MALAT1 was significantly increased by hypoxia and controls a phenotypic switch in endothelial cells. Silencing of MALAT1 by small interfering RNAs or GapmeRs induced a promigratory response and increased basal sprouting and migration, whereas proliferation of endothelial cells was inhibited. When angiogenesis was further stimulated by vascular endothelial growth factor, MALAT1 small interfering RNAs induced discontinuous sprouts indicative of defective proliferation of stalk cells. In vivo studies confirmed that genetic ablation of MALAT1 inhibited proliferation of endothelial cells and reduced neonatal retina vascularization. Pharmacological inhibition of MALAT1 by GapmeRs reduced blood flow recovery and capillary density after hindlimb ischemia. Gene expression profiling followed by confirmatory quantitative reverse transcriptase-polymerase chain reaction demonstrated that silencing of MALAT1 impaired the expression of various cell cycle regulators. Silencing of MALAT1 tips the balance from a proliferative to a migratory endothelial cell phenotype in vitro, and its genetic deletion or pharmacological inhibition reduces vascular growth in vivo.

  3. Hyaluronan protection of corneal endothelial cells against extracellular histones after phacoemulsification.

    Science.gov (United States)

    Kawano, Hiroki; Sakamoto, Taiji; Ito, Takashi; Miyata, Kazunori; Hashiguchi, Teruto; Maruyama, Ikuro

    2014-11-01

    To determine the effect of histones on corneal endothelial cells generated during cataract surgery. Kagoshima University Hospital, Kagoshima, Japan. Experimental study. Standard phacoemulsification was performed on enucleated pig eyes. Histones in the anterior segment of the eye were determined by immunohistochemistry. Cultured human corneal endothelial cells were exposed to histones for 18 hours, and cell viability was determined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt assay. The concentration of interleukin-6 (IL-6) in the culture medium of human corneal endothelial cells was measured using enzyme-linked immunosorbent assay. The effects of signal inhibitors U0126, SB203580, and SP600125 were evaluated. The protective effect of hyaluronan against histones was evaluated in human corneal endothelial cells with and without hyaluronan. Cellular debris containing histones was observed in the anterior chamber of pig eyes after phacoemulsification. Exposure of human corneal endothelial cells to 50 μg/mL of histones or more led to cytotoxic effects. The IL-6 concentration was significantly increased dose dependently after exposure of human corneal endothelial cells to histones (Phistone-induced IL-6 production was significantly decreased by extracellular signal-regulated kinases 1/2 and p-38 mitogen-activated protein kinase inhibitors (Phistones caused formation of histone aggregates, decreased the cytotoxic effects of the histones, and blocked the increase in IL-6 (PHistones were released extracellularly during phacoemulsification and exposure of human corneal endothelial cells to histones increased the IL-6 secretion. The intraoperative use of hyaluronan may decrease the cytotoxic effects of histones released during cataract surgery. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  4. Tpl2 Inhibitors Thwart Endothelial Cell Function in Angiogenesis and Peritoneal Dissemination

    Directory of Open Access Journals (Sweden)

    Wen-Jane Lee

    2013-09-01

    Full Text Available Angiogenesis is critical in the development of cancer, which involves several angiogenic factors in its peritoneal dissemination. The role of protein tumor progression locus 2 (Tpl2 in angiogenic factor-related endothelial cell angiogenesis is still unclear. To understand the precise mechanism(s of Tpl2 inhibition in endothelial cells, this study investigated the role of Tpl2 in mediating angiogenic signals using in vitro, in vivo, and ex vivo models. Results showed that inhibition of Tpl2 inhibitor significantly reduced peritoneal dissemination in a mouse model by positron emission tomography/computed tomography imaging. Simultaneously, inhibiting Tpl2 blocked angiogenesis in tumor nodules and prevented angiogenic factor-induced proliferating cell nuclear antigen (PCNA in endothelial cells. Vascular endothelial growth factor (VEGF or chemokine (C-X-C motif ligand 1 (CXCL1 increased Tpl2 kinase activity and phosphorylation in a dose- and time-dependent manner. Furthermore, Tpl2 inhibition or ablation by siRNA prevented the angiogenic signal-induced tube formation in Matrigel plug assay or aortic ring assay. Inhibiting Tpl2 also prevented the angiogenic factor-induced chemotactic motility and migration of endothelial cells. Tpl2 inhibition by CXCL1 or epidermal growth factor in endothelial cells was associated with inactivation of CCAAT/enhancer binding protein β, nuclear factor κ light-chain enhancer of activated B cells, and activating protein 1 and suppression of VEGF expression. Thus, Tpl2 inhibitors thwart Tpl2-regulated VEGF by inactivating transcription factors involved in angiogenic factor-triggered endothelial cell angiogenesis. These results suggest that the therapeutic inhibition of Tpl2 may extend beyond cancer and include the treatment of other diseases involving pathologic angiogenesis.

  5. Rescue of Brain Function Using Tunneling Nanotubes Between Neural Stem Cells and Brain Microvascular Endothelial Cells.

    Science.gov (United States)

    Wang, Xiaoqing; Yu, Xiaowen; Xie, Chong; Tan, Zijian; Tian, Qi; Zhu, Desheng; Liu, Mingyuan; Guan, Yangtai

    2016-05-01

    Evidence indicates that neural stem cells (NSCs) can ameliorate cerebral ischemia in animal models. In this study, we investigated the mechanism underlying one of the neuroprotective effects of NSCs: tunneling nanotube (TNT) formation. We addressed whether the control of cell-to-cell communication processes between NSCs and brain microvascular endothelial cells (BMECs) and, particularly, the control of TNT formation could influence the rescue function of stem cells. In an attempt to mimic the cellular microenvironment in vitro, a co-culture system consisting of terminally differentiated BMECs from mice in a distressed state and NSCs was constructed. Additionally, engraftment experiments with infarcted mouse brains revealed that control of TNT formation influenced the effects of stem cell transplantation in vivo. In conclusion, our findings provide the first evidence that TNTs exist between NSCs and BMECs and that regulation of TNT formation alters cell function.

  6. Derivation of corneal endothelial cell-like cells from rat neural crest cells in vitro.

    Directory of Open Access Journals (Sweden)

    Chengqun Ju

    Full Text Available The aim of this study was to investigate the feasibility of inducing rat neural crest cells (NCC to differentiate to functional corneal endothelial cell (CEC-like cells in vitro. Rat NCC were induced with adult CEC-derived conditioned medium. Immunofluorescence, flow cytometry and real time RT-PCR assay were used to detect expression of the corneal endothelium differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2. CFDA SE-labeled CEC-like cells were transplanted to the corneal endothelium of a rat corneal endothelium deficiency model, and an eye-down position was maintained for 24 hours to allow cell attachment. The animals were observed for as long as 2 months after surgery and underwent clinical and histological examination. Spindle-like NCC turned to polygonal CEC-like after induction and expressed N-cadherin, FoxC1, Pitx2, zonula occludens-1 and sodium-potassium pump Na(+/K(+ ATPase. The corneas of the experimental group were much clearer than those of the control group and the mean corneal thickness in the experimental group was significantly less than in the control group7, 14, 21 and 28 days after surgery. Confocal microscopy through focusing and histological analysis confirmed that green fluorescence-positive CEC-like cells formed a monolayer covering the Descemet's membrane in the experimental group. In conclusion, CEC-like cells derived from NCCs displayed characters of native CEC, and the induction protocol provides guidance for future human CEC induction from NCC.

  7. Nitric oxide modulates the expression of endothelial cell adhesion molecules involved in angiogenesis and leukocyte recruitment.

    Science.gov (United States)

    Carreau, Aude; Kieda, Claudine; Grillon, Catherine

    2011-01-01

    Tumor angiogenesis and immune response have in common to be cell recognition mechanisms, which are based on specific adhesion molecules and dependent on nitric oxide (NO(•)). The aim of the present study is to deepen the mechanisms of angiogenesis and inflammation regulation by NO(•) to find out the molecular regulation processes that govern endothelial cell permeability and leukocyte transmigration. Effects of NO(•), either exogenous or produced in hypoxic conditions, were studied on microvascular endothelial cells from skin and lymph node because of their strong involvement in melanoma progression. We found that NO(•) down-regulation of pseudo-vessel formation was linked to a decrease in endothelial cell ability to adhere to each other which can be explain, in part, by the inhibition of PECAM-1/CD31 expression. On the other hand, NO(•) was shown to be able to decrease leukocyte adhesion on an endothelial monolayer, performed either in static or in rolling conditions, and to modulate differentially CD34, ICAM-1/CD54, ICAM-2/CD102 and VCAM-1/CD106 expression. In conclusion, during angiogenesis and leukocyte recruitment, NO(•) regulates cell interactions by controlling adhesion molecule expression and subsequently cell adhesion. Moreover, each endothelial cell type presents its own organospecific response to NO(•), reflecting the functions of the tissue they originate from. Copyright © 2010 Elsevier Inc. All rights reserved.

  8. Behaviour of human umbilical vein endothelial cells (HUVEC) cultivated in microfluidic channels

    NARCIS (Netherlands)

    Mulder, Patty P. M. F. A.; Molema, Grietje; Koster, Sander; van der Linden, Heiko J.; Verpoorte, Elisabeth

    2006-01-01

    Our long-term goal is to develop advanced tools for cell studies and analysis based on microfluidic systems. In this paper, we report on endothelial cell cultivation in microchannels and 96-well tissue plates, and compare cell phenotype and cellular status in the two enviroments. This was done under

  9. Stereological analysis of neuron, glial and endothelial cell numbers in the human amygdaloid complex.

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    María García-Amado

    Full Text Available Cell number alterations in the amygdaloid complex (AC might coincide with neurological and psychiatric pathologies with anxiety imbalances as well as with changes in brain functionality during aging. This stereological study focused on estimating, in samples from 7 control individuals aged 20 to 75 years old, the number and density of neurons, glia and endothelial cells in the entire AC and in its 5 nuclear groups (including the basolateral (BL, corticomedial and central groups, 5 nuclei and 13 nuclear subdivisions. The volume and total cell number in these territories were determined on Nissl-stained sections with the Cavalieri principle and the optical fractionator. The AC mean volume was 956 mm(3 and mean cell numbers (x10(6 were: 15.3 neurons, 60 glial cells and 16.8 endothelial cells. The numbers of endothelial cells and neurons were similar in each AC region and were one fourth the number of glial cells. Analysis of the influence of the individuals' age at death on volume, cell number and density in each of these 24 AC regions suggested that aging does not affect regional size or the amount of glial cells, but that neuron and endothelial cell numbers respectively tended to decrease and increase in territories such as AC or BL. These accurate stereological measures of volume and total cell numbers and densities in the AC of control individuals could serve as appropriate reference values to evaluate subtle alterations in this structure in pathological conditions.

  10. Stereological analysis of neuron, glial and endothelial cell numbers in the human amygdaloid complex.

    Science.gov (United States)

    García-Amado, María; Prensa, Lucía

    2012-01-01

    Cell number alterations in the amygdaloid complex (AC) might coincide with neurological and psychiatric pathologies with anxiety imbalances as well as with changes in brain functionality during aging. This stereological study focused on estimating, in samples from 7 control individuals aged 20 to 75 years old, the number and density of neurons, glia and endothelial cells in the entire AC and in its 5 nuclear groups (including the basolateral (BL), corticomedial and central groups), 5 nuclei and 13 nuclear subdivisions. The volume and total cell number in these territories were determined on Nissl-stained sections with the Cavalieri principle and the optical fractionator. The AC mean volume was 956 mm(3) and mean cell numbers (x10(6)) were: 15.3 neurons, 60 glial cells and 16.8 endothelial cells. The numbers of endothelial cells and neurons were similar in each AC region and were one fourth the number of glial cells. Analysis of the influence of the individuals' age at death on volume, cell number and density in each of these 24 AC regions suggested that aging does not affect regional size or the amount of glial cells, but that neuron and endothelial cell numbers respectively tended to decrease and increase in territories such as AC or BL. These accurate stereological measures of volume and total cell numbers and densities in the AC of control individuals could serve as appropriate reference values to evaluate subtle alterations in this structure in pathological conditions.

  11. Endothelial cells assemble into a 3-dimensional prevascular network in a bone tissue engineering construct

    NARCIS (Netherlands)

    Rouwkema, Jeroen; de Boer, Jan; van Blitterswijk, Clemens

    2006-01-01

    To engineer tissues with clinically relevant dimensions, one must overcome the challenge of rapidly creating functional blood vessels to supply cells with oxygen and nutrients and to remove waste products. We tested the hypothesis that endothelial cells, cocultured with osteoprogenitor cells, can

  12. Propionyl-L-Carnitine Enhances Wound Healing and Counteracts Microvascular Endothelial Cell Dysfunction.

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    Maria Giovanna Scioli

    Full Text Available Impaired wound healing represents a high cost for health care systems. Endothelial dysfunction characterizes dermal microangiopathy and contributes to delayed wound healing and chronic ulcers. Endothelial dysfunction impairs cutaneous microvascular blood flow by inducing an imbalance between vasorelaxation and vasoconstriction as a consequence of reduced nitric oxide (NO production and the increase of oxidative stress and inflammation. Propionyl-L-carnitine (PLC is a natural derivative of carnitine that has been reported to ameliorate post-ischemic blood flow recovery.We investigated the effects of PLC in rat skin flap and cutaneous wound healing. A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS reduction, inducible nitric oxide synthase (iNOS and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF, placental growth factor (PlGF and reduction of NADPH-oxidase 4 (Nox4 expression. In serum-deprived human dermal microvascular endothelial cell cultures, PLC ameliorated endothelial dysfunction by increasing iNOS, PlGF, VEGF receptors 1 and 2 expression and NO level. In addition, PLC counteracted serum deprivation-induced impairment of mitochondrial β-oxidation, Nox4 and cellular adhesion molecule (CAM expression, ROS generation and leukocyte adhesion. Moreover, dermal microvascular endothelial cell dysfunction was prevented by Nox4 inhibition. Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction.PLC treatment improved rat skin flap viability, accelerated wound healing and dermal angiogenesis. The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction. Antioxidant therapy and

  13. Propionyl-L-Carnitine Enhances Wound Healing and Counteracts Microvascular Endothelial Cell Dysfunction.

    Science.gov (United States)

    Scioli, Maria Giovanna; Lo Giudice, Pietro; Bielli, Alessandra; Tarallo, Valeria; De Rosa, Alfonso; De Falco, Sandro; Orlandi, Augusto

    2015-01-01

    Impaired wound healing represents a high cost for health care systems. Endothelial dysfunction characterizes dermal microangiopathy and contributes to delayed wound healing and chronic ulcers. Endothelial dysfunction impairs cutaneous microvascular blood flow by inducing an imbalance between vasorelaxation and vasoconstriction as a consequence of reduced nitric oxide (NO) production and the increase of oxidative stress and inflammation. Propionyl-L-carnitine (PLC) is a natural derivative of carnitine that has been reported to ameliorate post-ischemic blood flow recovery. We investigated the effects of PLC in rat skin flap and cutaneous wound healing. A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS) reduction, inducible nitric oxide synthase (iNOS) and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and reduction of NADPH-oxidase 4 (Nox4) expression. In serum-deprived human dermal microvascular endothelial cell cultures, PLC ameliorated endothelial dysfunction by increasing iNOS, PlGF, VEGF receptors 1 and 2 expression and NO level. In addition, PLC counteracted serum deprivation-induced impairment of mitochondrial β-oxidation, Nox4 and cellular adhesion molecule (CAM) expression, ROS generation and leukocyte adhesion. Moreover, dermal microvascular endothelial cell dysfunction was prevented by Nox4 inhibition. Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction. PLC treatment improved rat skin flap viability, accelerated wound healing and dermal angiogenesis. The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction. Antioxidant therapy and pharmacological

  14. Mechanism of the toxicity induced by natural humic acid on human vascular endothelial cells

    OpenAIRE

    Kihara, Yusuke; Yustiawati; Tanaka, Masato; Gumiri, Sulmin; Ardianor; Hosokawa, Toshiyuki; Tanaka, Shunitz; Saito, Takeshi; Kurasaki, Masaaki

    2012-01-01

    Humic acid (HA), a group of high-molecular weight organic compounds characterized by an ability to bind heavy metals, is normally found in natural water. Although the impairment of vascular endothelial cells in the presence of humic substances has been reported to be involved in some diseases, the mechanisms responsible for this involvement remain unclear. In this study, we examined the cytotoxicity of HA obtained from peatland in Central Kalimantan, Indonesia, to human vascular endothelial c...

  15. Endothelial Differentiation of Human Adipose-Derived Stem Cells on Polyglycolic Acid/Polylactic Acid Mesh

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    Meng Deng

    2015-01-01

    Full Text Available Adipose-derived stem cell (ADSC is considered as a cell source potentially useful for angiogenesis in tissue engineering and regenerative medicine. This study investigated the growth and endothelial differentiation of human ADSCs on polyglycolic acid/polylactic acid (PGA/PLA mesh compared to 2D plastic. Cell adhesion, viability, and distribution of hADSCs on PGA/PLA mesh were observed by CM-Dil labeling, live/dead staining, and SEM examination while endothelial differentiation was evaluated by flow cytometry, Ac-LDL/UEA-1 uptake assay, immunofluorescence stainings, and gene expression analysis of endothelial related markers. Results showed hADSCs gained a mature endothelial phenotype with a positive ratio of 21.4 ± 3.7% for CD31+/CD34− when induced in 3D mesh after 21 days, which was further verified by the expressions of a comprehensive range of endothelial related markers, whereas hADSCs in 2D induced and 2D/3D noninduced groups all failed to differentiate into endothelial cells. Moreover, compared to 2D groups, the expression for α-SMA was markedly suppressed in 3D cultured hADSCs. This study first demonstrated the endothelial differentiation of hADSCs on the PGA/PLA mesh and pointed out the synergistic effect of PGA/PLA 3D culture and growth factors on the acquisition of mature characteristic endothelial phenotype. We believed this study would be the initial step towards the generation of prevascularized tissue engineered constructs.

  16. Microcapsules functionalized with neuraminidase can enter vascular endothelial cells in vitro.

    Science.gov (United States)

    Liu, Weizhi; Wang, Xiaocong; Bai, Ke; Lin, Miao; Sukhorukov, Gleb; Wang, Wen

    2014-12-06

    Microcapsules made of polyelectrolyte multilayers exhibit no or low toxicity, appropriate mechanical stability, variable controllable degradation and can incorporate remote release mechanisms triggered by various stimuli, making them well suited for targeted drug delivery to live cells. This study investigates interactions between microcapsules made of synthetic (i.e. polystyrenesulfonate sodium salt/polyallylamine hydrochloride) or natural (i.e. dextran sulfate/poly-L-arginine) polyelectrolyte and human umbilical vein endothelial cells with particular focus on the effect of the glycocalyx layer on the intake of microcapsules by endothelial cells. Neuraminidase cleaves N-acetyl neuraminic acid residues of glycoproteins and targets the sialic acid component of the glycocalyx on the cell membrane. Three-dimensional confocal images reveal that microcapsules, functionalized with neuraminidase, can be internalized by endothelial cells. Capsules without neuraminidase are blocked by the glycocalyx layer. Uptake of the microcapsules is most significant in the first 2 h. Following their internalization by endothelial cells, biodegradable DS/PArg capsules rupture by day 5; however, there is no obvious change in the shape and integrity of PSS/PAH capsules within the period of observation. Results from the study support our hypothesis that the glycocalyx functions as an endothelial barrier to cross-membrane movement of microcapsules. Neuraminidase-loaded microcapsules can enter endothelial cells by localized cleavage of glycocalyx components with minimum disruption of the glycocalyx layer and therefore have high potential to act as drug delivery vehicles to reach tissues beyond the endothelial barrier of blood vessels. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  17. Characterization of immortalized human dermal microvascular endothelial cells (HMEC-1) for the study of HDL functionality.

    Science.gov (United States)

    Muñoz-Vega, Mónica; Massó, Felipe; Páez, Araceli; Carreón-Torres, Elizabeth; Cabrera-Fuentes, Hector A; Fragoso, José Manuel; Pérez-Hernández, Nonanzit; Martinez, Laurent O; Najib, Souad; Vargas-Alarcón, Gilberto; Pérez-Méndez, Óscar

    2018-03-09

    Primary cultures endothelial cells have been used as models of endothelial related diseases such atherosclerosis. Biological behavior of primary cultures is donor-dependent and data could not be easily reproducible; endothelial cell lines are emerging options, particularly, human dermal microvascular endothelial cells (HMEC-1), that should be validated to substitute primary cultures for the study of HDL functions. Morphology, size and granularity of cells were assessed by phase contrast microscopy and flow cytometry of HMEC-1. The adhesion molecules, ICAM-1and VCAM-1 after TNF-α stimulation, and endothelial markers CD105 endoglin, as well as HDL receptor SR-BI were determined by flow cytometry. Internalization of HDL protein was demonstrated by confocal microscopy using HDL labeled with Alexa Fluor 488. HUVECs were used as reference to compared the characteristics with HMEC-1. HMEC-1 and HUVEC had similar morphologies, size and granularity. HMEC-1 expressed endothelial markers as HUVECs, as well as functional SR-B1 receptor since the cell line was able to internalize HDL particles. HMEC-1 effectively increased ICAM-1 and VCAM-1 expression after TNF-α stimulation. HUVECs showed more sensibility to TNF-α stimulus but the range of ICAM-1 and VCAM-1 expression was less homogeneous than in HMEC-1, probably due to biological variation of the former. Finally, the expression of adhesion molecules in HMEC-1 was attenuated by co-incubation with HDL. HMEC-1 possess characteristics of endothelial cells, similar to HUVECs, being a cell line suitable to evaluate the functionality of HDL vis-à-vis the endothelium.

  18. Middle T antigen-transformed endothelial cells exhibit an increased activity of nitric oxide synthase

    OpenAIRE

    1995-01-01

    Endothelioma cell lines transformed by polyoma virus middle T antigen (mTa) cause cavernous hemangiomas in syngeneic mice by recruitment of host cells. The production of nitric oxide (NO), as measured by nitrite and citrulline production, was significantly higher in mTa-transformed endothelial cells in comparison with nontransformed control cells. The maximal activity of NO synthase (NOS) was about 200-fold higher in cell lysates from the tEnd.1 endothelioma cell line than in lysates from non...

  19. Endothelial Cell Culture Under Perfusion On A Polyester-Toner Microfluidic Device.

    Science.gov (United States)

    Urbaczek, Ana Carolina; Leão, Paulo Augusto Gomes Carneiro; Souza, Fayene Zeferino Ribeiro de; Afonso, Ana; Vieira Alberice, Juliana; Cappelini, Luciana Teresa Dias; Carlos, Iracilda Zeppone; Carrilho, Emanuel

    2017-09-05

    This study presents an inexpensive and easy way to produce a microfluidic device that mimics a blood vessel, serving as a start point for cell culture under perfusion, cardiovascular research, and toxicological studies. Endpoint assays (i.e., MTT reduction and NO assays) were used and revealed that the components making up the microchip, which is made of polyester and toner (PT), did not induce cell death or nitric oxide (NO) production. Applying oxygen plasma and fibronectin improved the adhesion and proliferation endothelial cell along the microchannel. As expected, these treatments showed an increase in vascular endothelial growth factor (VEGF-A) concentration profiles, which is correlated with adherence and cell proliferation, thus promoting endothelialization of the device for neovascularization. Regardless the simplicity of the device, our "vein-on-a-chip" mimetic has a potential to serve as a powerful tool for those that demand a rapid microfabrication method in cell biology or organ-on-a-chip research.

  20. Immunoglobulin G (IgG) Expression in Human Umbilical Cord Endothelial Cells

    Science.gov (United States)

    Zhao, Yingying; Liu, Yuxuan; Chen, Zhengshan; Korteweg, Christine; Gu, Jiang

    2011-01-01

    Traditional views hold that immunoglobulin G (IgG) in the human umbilical cord is internalized by human umbilical endothelial cells for passive immunity. In this study, the protein and mRNA transcripts of IgG were found in the cytoplasm of human umbilical endothelial cells by immunohistochemistry, in situ hybridization, and reverse transcription PCR (RT-PCR). The essential enzymes for IgG synthesis and assembling, RAG1 (recombination activating gene 1), RAG2, and variable (V), diversity (D), and joining (J) segments for recombination of IgG, were also found in these cells by RT-PCR and real-time PCR. These results indicate that umbilical endothelial cells are capable of synthesizing IgG with properties similar to those of immune cells and that they may play additional roles besides lining the vessels and transporting IgG. PMID:21430258

  1. Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells.

    Science.gov (United States)

    Hamuro, Junji; Toda, Munetoyo; Asada, Kazuko; Hiraga, Asako; Schlötzer-Schrehardt, Ursula; Montoya, Monty; Sotozono, Chie; Ueno, Morio; Kinoshita, Shigeru

    2016-09-01

    To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy. Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na+/K+ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. Flow cytometry analysis identified the effector cell expressing CD166+CD105-CD44-∼+/-CD26-CD24-, but CD200-, and the presence of other SPs with CD166+ CD105-CD44+++ (CD26 and CD24, either + or -) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Na+/K+ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200∼220 μm2, and the density of cHCECs exceeded 2500 cells/mm2. A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.

  2. Mycoplasma suis infection results endothelial cell damage and activation: new insight into the cell tropism and pathogenicity of hemotrophic mycoplasma

    Directory of Open Access Journals (Sweden)

    Sokoli Albina

    2013-02-01

    Full Text Available Abstract Hemotrophic mycoplasmas (HM are highly specialized red blood cell parasites that cause infectious anemia in a variety of mammals, including humans. To date, no in vitro cultivation systems for HM have been available, resulting in relatively little information about the pathogenesis of HM infection. In pigs, Mycoplasma suis-induced infectious anemia is associated with hemorrhagic diathesis, and coagulation dysfunction. However, intravasal coagulation and subsequent consumption coagulopathy can only partly explain the sequence of events leading to hemorrhagic diathesis manifesting as cyanosis, petechial bleeding, and ecchymosis, and to disseminated coagulation. The involvement of endothelial activation and damage in M. suis-associated pathogenesis was investigated using light and electron microscopy, immunohistochemistry, and cell sorting. M. suis interacted directly with endothelial cells in vitro and in vivo. Endothelial activation, widespread endothelial damage, and adherence of red blood cells to the endothelium were evident in M. suis-infected pigs. These alterations of the endothelium were accompanied by hemorrhage, intravascular coagulation, vascular occlusion, and massive morphological changes within the parenchyma. M. suis biofilm-like microcolonies formed on the surface of endothelial cells, and may represent a putative persistence mechanism of M. suis. In vitro analysis demonstrated that M. suis interacted with the endothelial cytoskeletal protein actin, and induced actin condensation and activation of endothelial cells, as determined by the up-regulation of ICAM, PECAM, E-selectin, and P-selectin. These findings demonstrate an additional cell tropism of HM for endothelial cells and suggest that M. suis interferes with the protective function of the endothelium, resulting in hemorrhagic diathesis.

  3. The acute exposure effects of inhaled nickel nanoparticles on murine endothelial progenitor cells.

    Science.gov (United States)

    Liberda, Eric N; Cuevas, Azita K; Qu, Qingshan; Chen, Lung Chi

    2014-08-01

    The discovery of endothelial progenitor cells (EPCs) may help to explain observed cardiovascular effects associated with inhaled nickel nanoparticle exposures, such as increases in vascular inflammation, generation of reactive oxygen species, altered vasomotor tone and potentiated atherosclerosis in murine species. Following an acute whole body inhalation exposure to 500 µg/m(3) of nickel nanoparticles for 5 h, bone marrow EPCs from C57BL/6 mice were isolated. EPCs were harvested for their RNA or used in a variety of assays including chemotaxis, tube formation and proliferation. Gene expression was assessed for important receptors involved in EPC mobilization and homing using RT-PCR methods. EPCs, circulating endothelial progenitor cells (CEPCs), circulating endothelial cells (CECs) and endothelial microparticles (EMPs) were quantified on a BD FACSCalibur to examine endothelial damage and repair associated with the exposure. Acute exposure to inhaled nickel nanoparticles significantly increased both bone marrow EPCs as well as their levels in circulation (CEPCs). CECs were significantly elevated indicating that endothelial damage occurred due to the exposure. There was no significant difference in EMPs between the two groups. Tube formation and chemotaxis, but not proliferation, of bone marrow EPCs was impaired in the nickel nanoparticle exposed group. These results coincided with a decrease in the mRNA of receptors involved in EPC mobilization and homing. These data provide new insight into how an acute nickel nanoparticle exposure to half of the current Occupational Safety & Health Administration (OSHA) permissible exposure limit may adversely affect EPCs and exacerbate cardiovascular disease states.

  4. Oral Mucosa Harbors a High Frequency of Endothelial Cells: A Novel Postnatal Cell Source for Angiogenic Regeneration.

    Science.gov (United States)

    Zhou, Jian; Rogers, Jason H; Lee, Scott H; Sun, DongMing; Yao, Hai; Mao, Jeremy J; Kong, Kimi Y

    2017-01-15

    Endothelial progenitor cells/endothelial cells (EPCs/ECs) have great potential to treat pathological conditions such as cardiac infarction, muscle ischemia, and bone fractures, but isolation of EPC/ECs from existing cell sources is challenging due to their low EC frequency. We have isolated endothelial progenitor (EP)-like cells from rat oral mucosa and characterized their yield, immunophenotype, growth, and in vivo angiogenic potential. The frequency of EP-like cells derived from oral mucosa is thousands of folds higher than EPCs derived from donor-match bone marrow samples. EP-like cells from oral mucosa were positive for EC markers CD31, VE-Cadherin, and VEGFR2. Oral mucosa-derived EP-like cells displayed robust uptake of acetylated low-density lipoprotein and formed stable capillary networks in Matrigel. Subcutaneously implanted oral mucosa-derived EP-like cells anastomosed with host blood vessels, implicating their ability to elicit angiogenesis. Similar to endothelial colony-forming cells, EP-like cells from oral mucosa have a significantly higher proliferative rate than human umbilical vein endothelial cells. These findings identify a putative EPC source that is easily accessible in the oral cavity, potentially from discarded tissue specimens, and yet with robust yield and potency for angiogenesis in tissue and organ regeneration.

  5. Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea.

    Science.gov (United States)

    Lopes, Flavia C M; Traina, Fabiola; Almeida, Camila B; Leonardo, Flavia C; Franco-Penteado, Carla F; Garrido, Vanessa T; Colella, Marina P; Soares, Raquel; Olalla-Saad, Sara T; Costa, Fernando F; Conran, Nicola

    2015-06-01

    As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the

  6. Cannabidiol attenuates high glucose-induced endothelial cell inflammatory response and barrier disruption

    Science.gov (United States)

    Rajesh, Mohanraj; Mukhopadhyay, Partha; Bátkai, Sándor; Haskó, György; Liaudet, Lucas; Drel, Viktor R.; Obrosova, Irina G.; Pacher, Pál

    2008-01-01

    A nonpsychoactive cannabinoid cannabidiol (CBD) has been shown to exert potent anti-inflammatory and antioxidant effects and has recently been reported to lower the incidence of diabetes in nonobese diabetic mice and to preserve the blood-retinal barrier in experimental diabetes. In this study we have investigated the effects of CBD on high glucose (HG)-induced, mitochondrial superoxide generation, NF-κB activation, nitrotyrosine formation, inducible nitric oxide synthase (iNOS) and adhesion molecules ICAM-1 and VCAM-1 expression, monocyte-endothelial adhesion, transendothelial migration of monocytes, and disruption of endothelial barrier function in human coronary artery endothelial cells (HCAECs). HG markedly increased mitochondrial superoxide generation (measured by flow cytometry using MitoSOX), NF-κB activation, nitrotyrosine formation, upregulation of iNOS and adhesion molecules ICAM-1 and VCAM-1, transendothelial migration of monocytes, and monocyte-endothelial adhesion in HCAECs. HG also decreased endothelial barrier function measured by increased permeability and diminished expression of vascular endothelial cadherin in HCAECs. Remarkably, all the above mentioned effects of HG were attenuated by CBD pretreatment. Since a disruption of the endothelial function and integrity by HG is a crucial early event underlying the development of various diabetic complications, our results suggest that CBD, which has recently been approved for the treatment of inflammation, pain, and spasticity associated with multiple sclerosis in humans, may have significant therapeutic benefits against diabetic complications and atherosclerosis. PMID:17384130

  7. [Effects of glucose concentration fluctuation on function of cultured bovine arterial endothelial cells].

    Science.gov (United States)

    Ye, Xi-yun; Tu, Qian; Tong, Zhi; Weng, Yu-jing; Wang, Yao-fa

    2010-03-01

    To explore the effects of glucose concentration fluctuation on function of cultured bovine arterial endothelial cells and underlying mechanism. The thoracic aorta of newborn calf was used for primary endothelial cells culture. Cells were divided into 3 groups and cultured for 48 h: control group (C, 5.5 mmol/L), constant high glucose group (HG, 30 mmol/L) and glucose fluctuation (GF, three circles of 2 h 30 mmol/L followed by 3 h 5.5 mmol/L, 30 mmol/L overnight, repeat the whole procedure on the following day) groups. The membranes fluidity of endothelial cells was detected by fluorescence polarization method. The contents of sorbierite, aldose reductase (AR), sorbitol dehydrogenase (SDH) and advanced glycation end products (AGEs) were measured. RAGE, eNOS and ET-1 mRNA expressions were detected by semi-quantitative RT-PCR. The membranes fluidity of endothelial cells in HG or GF group were significantly decreased compared with the control group (all P RAGE, eNOS and ET-1 were significantly upregulated compared with the control group (all P bovine arterial endothelial cells dysfunction than high glucose via activating polyols metabolic pathways, upregulating the expression of AGEs, eNOS and ET-1. Therefore, glucose concentration fluctuation might play a crucial role on macrovascular complications of diabetes.

  8. Regional heterogeneity of endothelial cells in the porcine vortex vein system.

    Science.gov (United States)

    Tan, Priscilla Ern Zhi; Yu, Paula K; Cringle, Stephen J; Morgan, William H; Yu, Dao-Yi

    2013-09-01

    The aim of this study was to investigate whether region-dependent endothelial heterogeneity is present within the porcine vortex vein system. The superior temporal vortex vein in young adult pig eyes were dissected out and cannulated. The intact vortex vein system down to the choroidal veins was then perfused with labels for f-actin and nucleic acid. The endothelial cells within the choroidal veins, pre-ampulla, anterior portion of the ampulla, mid-ampulla, posterior portion of the ampulla, post-ampulla, intra-scleral canal and the extra-ocular vortex vein regions were studied in detail using a confocal microscopy technique. The endothelial cell and nuclei length, width, area and perimeter were measured and compared between the different regions. Significant regional differences in the endothelial cell and nuclei length, width, area and perimeter were observed throughout the porcine vortex vein system. Most notably, very narrow and elongated endothelia were found in the post-ampulla region. A lack of smooth muscle cells was noted in the ampulla region compared to other regions. Heterogeneity in endothelial cell morphology is present throughout the porcine vortex vein system and there is a lack of smooth muscle cells in the ampulla region. This likely reflects the highly varied haemodynamic conditions and potential blood flow control mechanisms in different regions of the vortex vein system. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles

    Directory of Open Access Journals (Sweden)

    Tudor Braniste

    2016-09-01

    Full Text Available Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle–cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN.

  10. Endothelial cell tropism is a determinant of H5N1 pathogenesis in mammalian species.

    Directory of Open Access Journals (Sweden)

    Smanla Tundup

    2017-03-01

    Full Text Available The cellular and molecular mechanisms underpinning the unusually high virulence of highly pathogenic avian influenza H5N1 viruses in mammalian species remains unknown. Here, we investigated if the cell tropism of H5N1 virus is a determinant of enhanced virulence in mammalian species. We engineered H5N1 viruses with restricted cell tropism through the exploitation of cell type-specific microRNA expression by incorporating microRNA target sites into the viral genome. Restriction of H5N1 replication in endothelial cells via miR-126 ameliorated disease symptoms, prevented systemic viral spread and limited mortality, despite showing similar levels of peak viral replication in the lungs as compared to control virus-infected mice. Similarly, restriction of H5N1 replication in endothelial cells resulted in ameliorated disease symptoms and decreased viral spread in ferrets. Our studies demonstrate that H5N1 infection of endothelial cells results in excessive production of cytokines and reduces endothelial barrier integrity in the lungs, which culminates in vascular leakage and viral pneumonia. Importantly, our studies suggest a need for a combinational therapy that targets viral components, suppresses host immune responses, and improves endothelial barrier integrity for the treatment of highly pathogenic H5N1 virus infections.

  11. Long-term corneal endothelial cell changes in pediatric intraocular lens reposition and exchange cases.

    Science.gov (United States)

    Wang, Yan; Wu, Mingxing; Zhu, Liyuan; Liu, Yizhi

    2012-04-01

    To evaluate long-term corneal endothelial cell changes of intraocular lens (IOL) reposition and exchange in children. State key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China In this retrospective study, all IOL reposition and exchange procedures performed in patients under 14 years old between January 1999 and April 2009 were included. Follow-up outcomes included corneal endothelial cell density, hexagonality, coefficient of variance, average cell size. IOL reposition procedures in 12 eyes (12 cases) (reposition group, RPG), and IOL exchanges in eight eyes (eight cases) (exchange group, EXG) were performed because of IOL pupillary capture or IOL dislocation. Median of follow-up was 44.5 months in RPG and 66.2 months in EXG. The density of corneal endothelial cells in RPG (2,053 ± 493/mm(2)) and EXG (2,100 ± 758/mm(2)) was significantly decreased in comparison to the control eyes (3,116 ± 335/mm(2)). Hexagonality of corneal endothelial cells and coefficient of variance showed no difference among the control group, RPG and EXG (P > 0.05). The density of corneal endothelial cells was conspicuously decreased after IOL reposition or exchange procedures in childhood cases. Longer follow-up must be conducted in these cases.

  12. Glycocalyx Degradation Induces a Proinflammatory Phenotype and Increased Leukocyte Adhesion in Cultured Endothelial Cells under Flow.

    Directory of Open Access Journals (Sweden)

    Karli K McDonald

    Full Text Available Leukocyte adhesion to the endothelium is an early step in the pathogenesis of atherosclerosis. Effective adhesion requires the binding of leukocytes to their cognate receptors on the surface of endothelial cells. The glycocalyx covers the surface of endothelial cells and is important in the mechanotransduction of shear stress. This study aimed to identify the molecular mechanisms underlying the role of the glycocalyx in leukocyte adhesion under flow. We performed experiments using 3-D cell culture models, exposing human abdominal aortic endothelial cells to steady laminar shear stress (10 dynes/cm2 for 24 hours. We found that with the enzymatic degradation of the glycocalyx, endothelial cells developed a proinflammatory phenotype when exposed to uniform steady shear stress leading to an increase in leukocyte adhesion. Our results show an up-regulation of ICAM-1 with degradation compared to non-degraded controls (3-fold increase, p<0.05 and we attribute this effect to a de-regulation in NF-κB activity in response to flow. These results suggest that the glycocalyx is not solely a physical barrier to adhesion but rather plays an important role in governing the phenotype of endothelial cells, a key determinant in leukocyte adhesion. We provide evidence for how the destabilization of this structure may be an early and defining feature in the initiation of atherosclerosis.

  13. Reactive Oxygen Species, Mitochondria, and Endothelial Cell Death during In Vitro Simulated Dives.

    Science.gov (United States)

    Wang, Qiong; Guerrero, François; Mazur, Aleksandra; Lambrechts, Kate; Buzzacott, Peter; Belhomme, Marac; Theron, Michaël

    2015-07-01

    Excessive reactive oxygen species (ROS) is considered a consequence of hyperoxia and a major contributor to diving-derived vascular endothelial damage and decompression sickness. The aims of this work were: 1) to directly observe endothelial ROS production during simulated air dives as well as its relation with both mitochondrial activity and cell survival; and 2) to determine which ambient factor during air diving (hydrostatic pressure or oxygen and/or nitrogen partial pressure) is responsible for the observed modifications. In vitro diving simulation was performed with bovine arterial endothelial cells under real-time observation. The effects of air diving, hydrostatic, oxygen and nitrogen pressures, and N-acetylcysteine (NAC) treatment on mitochondrial ROS generation, mitochondrial membrane potential and cellular survival during simulation were investigated. Vascular endothelial cells performing air diving simulation suffered excessive mitochondrial ROS, mitochondrial depolarization, and cell death. These effects were prevented by NAC: after NAC treatment, the cells presented no difference in damage from nondiving cells. Oxygen diving showed a higher effect on ROS generation but lower impacts on mitochondrial depolarization and cell death than hydrostatic or nitrogen diving. Nitrogen diving had no effect on the inductions of ROS, mito-depolarization, or cell death. This study is the first direct observation of mitochondrial ROS production, mitochondrial membrane potential and cell survival during diving. Simulated air SCUBA diving induces excessive ROS production, which leads to mitochondrial depolarization and endothelial cell death. Oxygen partial pressure plays a crucial role in the production of ROS. Deleterious effects of hyperoxia-induced ROS are potentiated by hydrostatic pressure. These findings hold new implications for the pathogenesis of diving-derived endothelial dysfunction.

  14. Necklace-like detachment of endothelial cell layer from arterial wall under low-calcium condition.

    Science.gov (United States)

    Yamaguchi, H; Morisada, M; Kaku, H; Onodera, T; Kurokawa, R

    1994-10-01

    The diversified morphological manifestations in various tissues and organs obtained by administration of differing amounts of calcium chelating agents were reported in previous papers (Yamaguchi et al 1981 a & b; 1982; 1990; 1993). In our recent research described here, administration of a moderate dose of Na2EDTA over the short term demonstrated necklace-like detachment from the arterial wall without disruption of the endothelial cell chain. Intercellular spaces in the media just beneath the detached endothelial cell layer was stained strongly with colloidal iron staining. Electron microscopic observation revealed that the detached endothelial cells showed a lot of elongated anchoring villi from the basal surface, usually seen at the luminal surface, adhered to the degenerative and thin flattened internal elastic lamellae. The alteration of the colloidal iron staining of the vascular wall under the low-calcium condition is suggested to be induced by loosening of the molecular structure of glycosaminoglycans (GAGs) as well as glycoproteins (GPs), comprising the important component of the intercellular matrix and elastic lamellae, which would induce a change in their pasty or viscous character. This would be an accelerative factor for detachment of endothelial cells. Moreover, the lack of the waving of the internal elastic lamellae, trapping of endothelial cytoplasmic processes among them, would play the decisive role in the total detachment of the endothelial cell layer. On the other hand, the low-calcium condition did not adversely influence the joining of endothelial cells. The pathognomatic mechanism will be discussed, with a comparison made to the angiolytic changes provoked by a large amount of Na2EDTA.

  15. Effects of intraocular irrigating solutions on the spreading of rabbit corneal endothelial cells on extracellular matrices.

    Science.gov (United States)

    Nishida, T; Otori, T

    1991-01-01

    The effects of four commercially available irrigating solutions on the spreading of rabbit corneal endothelial cells on various extracellular matrices were studied. Cultured rabbit corneal endothelial cells, suspended in one of the following intraocular irrigating solutions, Opeguard MA, BSS, BSS Plus, lactated Ringer solution (Lactec) or physiological saline, were placed on uncoated tissue culture plates or on plates coated with extracellular matrices (fibronectin, laminin, collagen type I, or collagen type IV). The cell area was measured after 45 minutes' incubation. The cells spread on all of the extracellular matrices examined but not on the uncoated tissue culture plates. On the fibronectin or laminin matrix, the cell area was significantly greater with Opeguard MA or BSS Plus. On laminin and collagen type IV, the cell area was the greatest with Opeguard MA. On collagen type I, the cell area was significantly greater with Opeguard MA, BSS, or BSS Plus. These results demonstrated that the rabbit corneal endothelial cells responded to the extracellular matrices, and that Opeguard MA or BSS Plus provided more favorable conditions for the spreading of these cells. These results indicated that both Opeguard MA and BSS Plus might aid the spreading of corneal endothelial cells during wound-healing immediately after intraocular surgery.

  16. Effects of energy deprivation induced by fluorocitrate in immortalised rat brain microvessel endothelial cells.

    Science.gov (United States)

    Rist, R J; Romero, I A; Chan, M W; Abbott, N J

    1996-08-19

    The effects of the mitochondrial aconitase inhibitor, fluorocitrate on the immortalised rat brain endothelial cell line (RBE4) were investigated. Treatment with different concentrations of fluorocitrate (0-1 mM) for 24 h induced a significant, concentration-dependent decrease in the MTT reduction (an index of mitochondrial function), intracellular ATP content, glucose consumption and lactate production by RBE4 cell monolayers but did not alter the glucose to lactate ratio at concentrations lower than 0.5 mM. At all concentrations, fluorocitrate induced a significant decrease in the protein content per well. Fluorocitrate treatment of confluent RBE4 cells induced a marked redistribution of the F-actin cytoskeleton from a characteristic marginal band to a more diffuse cytosolic pattern. This redistribution of the cytoskeleton coincided with a reduction in the total cellular F-actin content of the RBE4 cells at fluorocitrate concentrations greater than 0.5 mM. Treatment of confluent RBE4 cells with fluorocitrate had no significant effect on RBE4 cell monolayer permeability measured by FITC-dextran or [14C]sucrose. These results show that whilst energy deprivation following fluorocitrate treatment induces significant changes in the RBE4 cell F-actin cytoskeleton and cellular metabolism, it does not have any significant effect on endothelial cell monolayer permeability. These results demonstrate that profound toxic effects on endothelial cell structure and metabolism are not necessarily accompanied by changes in endothelial cell monolayer permeability.

  17. Biocompatibility of Poly-ε-caprolactone-hydroxyapatite composite on mouse bone marrow-derived osteoblasts and endothelial cells

    Directory of Open Access Journals (Sweden)

    Wooley Paul H

    2009-02-01

    Full Text Available Abstract Background Tissue-engineered bone may be developed by seeding the cells capable of both osteogenesis and vascularization on biocompatible composite scaffolds. The current study investigated the performance of mice bone marrow-derived osteogenic cells and endothelial cells as seeded on hydroxyapatite (HA and poly-ε-caprolactone (PCL composite scaffolds. Methods Mononuclear cells were induced to osteoblasts and endothelial cells respectively, which were defined by the expression of osteocalcin, alkaline phosphatase (ALP, and deposits of calcium-containing crystal for osteoblasts, or by the expression of vascular endothelial growth factor receptor-2 (VEGFR-2 and von Willebrand factor (vWF, and the formation of a capillary network in Matrigel™ for endothelial cells. Both types of cell were seeded respectively on PCL-HA scaffolds at HA to PCL weight ratio of 1:1, 1:4, or 0:1 and were evaluated using scanning electron microscopy, ALP activity (of osteoblasts and nitric oxide production (of endothelial cells plus the assessment of cell viability. Results The results indicated that HA led to a positive stimulation of osteoblasts viability and ALP activity, while HA showed less influence on endothelial cells viability. An elevated nitric oxide production of endothelial cells was observed in HA-containing group. Conclusion Supplement of HA into PCL improved biocompatible for bone marrow-derived osteoblasts and endothelial cells. The PCL-HA composite integrating with two types of cells may provide a useful system for tissue-engineered bone grafts with vascularization.

  18. GLP-1 inhibits VEGFA-mediated signaling in isolated human endothelial cells and VEGFA-induced dilation of rat mesenteric arteries

    DEFF Research Database (Denmark)

    Rotbøl, Cecilie Egholm; Khammy, Makhala Michell; Dalsgaard, Thomas

    2016-01-01

    to PLCγ activation, Src, and endothelial NOS (eNOS) signaling, thereby controlling endothelial vessel tone. By using RT-PCR analysis, we found mRNA for the GLP-1 receptor (GLP-1R) in human dermal microvascular endothelial cells (HDMEC), human retinal microvascular endothelial cells, and rat arteries...

  19. RON kinase inhibition reduces renal endothelial injury in sickle cell disease mice.

    Science.gov (United States)

    Khaibullina, Alfia; Adjei, Elena A; Afangbedji, Nowah; Ivanov, Andrey; Kumari, Namita; Almeida, Luis E F; Quezado, Zenaide M N; Nekhai, Sergei; Jerebtsova, Marina

    2018-03-08

    Sickle cell disease patients are at increased risk of developing a chronic kidney disease. Endothelial dysfunction and inflammation associated with hemolysis lead to vasculopathy and contribute to the development of renal disease. Here we used a Townes sickle cell disease mouse model to examine renal endothelial injury. Renal disease in Townes mice was associated with glomerular hypertrophy, capillary dilation and congestion, and significant endothelial injury. We also detected substantial renal macrophage infiltration, and accumulation of macrophage stimulating protein 1 in glomerular capillary. Treatment of human cultured macrophages with hemin or red blood cell lysates significantly increased expression of macrophage membrane-associated protease that might cleave and activate circulating macrophage stimulating protein 1 precursor. Macrophage stimulating protein 1 binds to and activates RON kinase, a cell surface receptor tyrosine kinase. In cultured human renal glomerular endothelial cells, macrophage stimulating protein 1 induced RON downstream signaling, resulting in increased phosphorylation of ERK and AKT kinases, expression Von Willebrand factor, increased cell motility, and re-organization of F-actin. Specificity of macrophage stimulating protein 1 function was confirmed by treatment with RON kinase inhibitor BMS-777607 that significantly reduced downstream signaling. Moreover, treatment of sickle cell mice with BMS-777607 significantly reduced glomerular hypertrophy, capillary dilation and congestion, and endothelial injury. Taken together, our findings demonstrated that RON kinase is involved in the induction of renal endothelial injury in sickle cell mice. Inhibition of RON kinase activation may provide a novel approach for prevention of renal disease development in sickle cell disease. Copyright © 2018, Ferrata Storti Foundation.

  20. Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Matveeva, V. G., E-mail: matveeva-vg@mail.ru; Antonova, L. V., E-mail: antonova.la@mail.ru; Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S. [Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, 650002 (Russian Federation)

    2015-10-27

    We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwoven scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.

  1. MiR-492 impairs the angiogenic potential of endothelial cells

    DEFF Research Database (Denmark)

    Patella, Francesca; Leucci, Eleonora; Evangelista, Monica

    2013-01-01

    Endothelial cells growing in high glucose-containing medium show reduced cell proliferation and in vitro angiogenesis. Evidence suggests that the molecular pathways leading to these cellular responses are controlled by microRNAs, endogenous post-transcriptional regulators of gene expression....... To identify the microRNAs and their targeted genes involved in the glucose responses, we performed the miRNA signature of Human Umbelical Vein Endothelial Cells (HUVECs) exposed and unexposed to high glucose. Among differentially expressed microRNAs, we analysed miR-492 and showed that its overexpression...... was able to reduce proliferation, migration and tube formation of HUVEC. These effects were accompanied by the down-regulation of eNOS, a key regulator of the endothelial cell function. We showed that eNOS was indirectly down-regulated by miR-492 and we discovered that miR-492 was able to bind m...

  2. Angiogenesis gene expression in murine endothelial cells during post-pneumonectomy lung growth

    Directory of Open Access Journals (Sweden)

    Konerding Moritz A

    2011-07-01

    Full Text Available Abstract Although blood vessel growth occurs readily in the systemic bronchial circulation, angiogenesis in the pulmonary circulation is rare. Compensatory lung growth after pneumonectomy is an experimental model with presumed alveolar capillary angiogenesis. To investigate the genes participating in murine neoalveolarization, we studied the expression of angiogenesis genes in lung endothelial cells. After left pneumonectomy, the remaining right lung was examined on days 3, 6, 14 and 21days after surgery and compared to both no surgery and sham thoracotomy controls. The lungs were enzymatically digested and CD31+ endothelial cells were isolated using flow cytometry cell sorting. The transcriptional profile of the CD31+ endothelial cells was assessed using quantitative real-time polymerase chain reaction (PCR arrays. Focusing on 84 angiogenesis-associated genes, we identified 22 genes with greater than 4-fold regulation and significantly enhanced transcription (p

  3. Arachidonic metabolism and radiation toxicity in cultures of vascular endothelial cells

    International Nuclear Information System (INIS)

    Eldor, A.; Vlodavsky, I.; Fuks, Z.; Matzner, Y.; Rubin, D.B.

    1989-01-01

    The authors conclude that the observed changes in eicosanoid production by vascular endothelial cells exposed to ionizing irradiation may be relevant to the pathogenesis of post-radiation injury in small and large blood vessels. Anomalies of PGI 2 production may lead to thrombosis and accelerated arteriosclerosis which are observed in irradiated vessels. The generation of potent cells may greatly facilitate inflammation in irradiated vessels. The model of irradiated cultured endothelial cells may also be useful for the study of various methods and agents aimed at reducing the radiation induced damage to blood vessels. Evaluation of the capacity of cultured endothelial cells to produce eicosanoids may serve as an appropriate index for the metabolic damage induced by radiation. (author)

  4. Arteries provide essential guidance cues for lymphatic endothelial cells in the zebrafish trunk.

    Science.gov (United States)

    Bussmann, Jeroen; Bos, Frank L; Urasaki, Akihiro; Kawakami, Koichi; Duckers, Henricus J; Schulte-Merker, Stefan

    2010-08-01

    The endothelial cells of the vertebrate lymphatic system assemble into complex networks, but local cues that guide the migration of this distinct set of cells are currently unknown. As a model for lymphatic patterning, we have studied the simple vascular network of the zebrafish trunk consisting of three types of lymphatic vessels that develop in close connection with the blood vasculature. We have generated transgenic lines that allow us to distinguish between arterial, venous and lymphatic endothelial cells (LECs) within a single zebrafish embryo. We found that LECs migrate exclusively along arteries in a manner that suggests that arterial endothelial cells serve as the LEC migratory substrate. In the absence of intersegmental arteries, LEC migration in the trunk is blocked. Our data therefore demonstrate a crucial role for arteries in LEC guidance.

  5. Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

    Science.gov (United States)

    Matveeva, V. G.; Antonova, L. V.; Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S.

    2015-10-01

    We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwoven scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.

  6. Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

    International Nuclear Information System (INIS)

    Matveeva, V. G.; Antonova, L. V.; Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S.

    2015-01-01

    We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwoven scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds

  7. A novel immunotoxin reveals a new role for CD321 in endothelial cells.

    Science.gov (United States)

    Fukuhara, Takeshi; Kim, Jia; Hokaiwado, Shintaro; Nawa, Makiko; Okamoto, Hayato; Kogiso, Tomohiko; Watabe, Tetsuro; Hattori, Nobutaka

    2017-01-01

    There are currently several antibody therapies that directly target tumors, and antibody-drug conjugates represent a novel moiety as next generation therapeutics. Here, we used a unique screening probe, DT3C, to identify functional antibodies that recognized surface molecules and functional epitopes, and which provided toxin delivery capability. Accordingly, we generated the 90G4 antibody, which induced DT3C-dependent cytotoxicity in endothelial cells. Molecular analysis revealed that 90G4 recognized CD321, a protein localized at tight junctions. Although CD321 plays a pivotal role in inflammation and lymphocyte trans-endothelial migration, little is known about its mechanism of action in endothelial cells. Targeting of CD321 by the 90G4 immunotoxin induced cell death. Moreover, 90G4 immunotoxin caused cytotoxicity primarily in migratory endothelial cells, but not in those forming sheets, suggesting a critical role for CD321 in tumor angiogenesis. We also found that hypoxia triggered redistribution of CD321 to a punctate localization on the basal side of cells, resulting in functional impairment of tight junctions and increased motility. Thus, our findings raise the intriguing possibility that endothelial CD321 presented cellular localization in tight junction as well as multifunctional dynamics in several conditions, leading to illuminate the importance of widely-expressed CD321 as a potential target for antitumor therapy.

  8. A novel immunotoxin reveals a new role for CD321 in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Takeshi Fukuhara

    Full Text Available There are currently several antibody therapies that directly target tumors, and antibody-drug conjugates represent a novel moiety as next generation therapeutics. Here, we used a unique screening probe, DT3C, to identify functional antibodies that recognized surface molecules and functional epitopes, and which provided toxin delivery capability. Accordingly, we generated the 90G4 antibody, which induced DT3C-dependent cytotoxicity in endothelial cells. Molecular analysis revealed that 90G4 recognized CD321, a protein localized at tight junctions. Although CD321 plays a pivotal role in inflammation and lymphocyte trans-endothelial migration, little is known about its mechanism of action in endothelial cells. Targeting of CD321 by the 90G4 immunotoxin induced cell death. Moreover, 90G4 immunotoxin caused cytotoxicity primarily in migratory endothelial cells, but not in those forming sheets, suggesting a critical role for CD321 in tumor angiogenesis. We also found that hypoxia triggered redistribution of CD321 to a punctate localization on the basal side of cells, resulting in functional impairment of tight junctions and increased motility. Thus, our findings raise the intriguing possibility that endothelial CD321 presented cellular localization in tight junction as well as multifunctional dynamics in several conditions, leading to illuminate the importance of widely-expressed CD321 as a potential target for antitumor therapy.

  9. MicroRNA-1185 Induces Endothelial Cell Apoptosis by Targeting UVRAG and KRIT1

    Directory of Open Access Journals (Sweden)

    Haoyuan Deng

    2017-04-01

    Full Text Available Background/Aims: Atherosclerosis is a multifactorial chronic disease and is the main cause of death and impairment in the world. Endothelial injury and apoptosis play a crucial role in the onset and development of atherosclerosis. MicroRNAs (miRNAs have been proven to be involved in the pathogenesis of atherosclerosis. However, studies of the functional role of apoptosis-related miRNAs in the endothelium during atherogenesis are limited. Methods: Cell injury and apoptosis were measured in five types of cells transfected with miR-1185 or co-transfected with miR-1185 and its inhibitor. Bioinformatics analysis and a luciferase reporter assay were used to confirm the targets of miR-1185. The effects of the targets of miR-1185 on endothelial apoptosis were determined using small-interfering RNA. Results: In this study, we first report that miR-1185 significantly promoted apoptosis in endothelial cells but not in vascular smooth muscle cells and macrophages. A mechanistic analysis showed that ultraviolet irradiation resistance-associated gene (UVRAG and krev1 interaction trapped gene 1 (KRIT1, targets of miR-1185, mediated miR-1185-induced endothelial cell apoptosis. Conclusion: The results revealed the impact of miR-1185 on endothelial apoptosis, suggesting that miR-1185 may be a potential target for the prevention and treatment of atherosclerosis.

  10. Effect of glaucoma tube shunt parameters on cornea endothelial cells in patients with Ahmed valve implants.<